Combination Therapies and Related Methods

Abstract

Provided herein are the use of KRAS (e.g., hKRAS) inhibitors in combination with agents that specifically bind to EGFR (e.g., hEGFR) and TGFβ (e.g., hTGFβ), as well as compositions and kits comprising them. The methods provided herein are useful in methods for treating cancer (e.g., KRAS (e.g., hKRAS) variant cancer). Further provided herein are combination regimens, combination compositions, pharmaceutical compositions, and kits comprising KRAS (e.g., hKRAS) inhibitors and agents that specifically bind to EGFR (e.g., hEGFR) and TGFβ (e.g., hTGFβ).

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Application No. 63 / 483,399, filed February 6, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to methods involving the use of KRAS (e.g., hKRAS) inhibitors in combination with agents that specifically bind to EGFR (e.g., hEGFR) and TGFβ (e.g., hTGFβ). The present disclosure further relates to compositions and kits comprising same. [Background technology]

[0003] KRAS is a small GTPase that regulates diverse cellular processes, including cell proliferation, survival, differentiation, and migration. KRAS cycles between a GTP-bound active state and a GDP-bound inactive state, and this cycling is primarily regulated by guanine nucleotide exchange factors. Active KRAS mediates its functional effects through downstream signaling pathways, such as MAPK and PI3K kinases. KRAS variants containing one or more amino acid alterations (e.g., amino acid substitutions (e.g., single amino acid substitutions)) that impair GTP hydrolysis to GDP and thus permanently lock KRAS in an active state have been reported in various cancers, including various solid tumors (e.g., pancreatic, lung, colon, rectal, and colorectal cancers). These KRAS variant cancers are typically associated with increased tumorigenicity, aggressive disease, and poor prognosis. Summary of the Invention

[0004] Provided herein, inter alia, are methods utilizing KRAS (e.g., hKRAS) inhibitors in combination with agents that specifically bind EGFR (e.g., hEGFR) and TGFβ (e.g., hTGFβ), including, e.g., methods of treating cancer, inhibiting the KRAS (e.g., hKRAS) pathway and the TGFβ (e.g., hTGFβ) pathway, restoring cancer sensitivity to KRAS (e.g., hKRAS) inhibitors, and suppressing or preventing cancer resistance to KRAS (e.g., hKRAS) inhibitors, along with, e.g., diagnostic methods, combination regimens, combination compositions, pharmaceutical compositions, and kits.

[0005] In one aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject (a) a Kirsten rat sarcoma virus homolog (KRAS) (e.g., human KRAS (hKRAS)) inhibitor in combination with (b) a fusion protein comprising (i) a first partial structure that specifically binds epidermal growth factor receptor (EGFR) (e.g., human EGFR (hEGFR)), the first partial structure being operably linked to (ii) a second partial structure that specifically binds transforming growth factor beta (TGFβ) (e.g., human TGFβ (hTGFβ)), thereby treating cancer in a subject in need thereof.

[0006] In one aspect, provided herein is a method of treating a KRAS (e.g., hKRAS) variant cancer in a subject in need thereof, comprising: (a) receiving test results indicating that the KRAS (e.g., hKRAS) variant cancer exhibits resistance to a KRAS inhibitor, in combination with: (b) administering to the subject a KRAS (e.g., hKRAS) inhibitor and a fusion protein, wherein the fusion protein comprises (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), and the first partial structure is operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ), thereby treating the KRAS (e.g., hKRAS) variant cancer in the subject.

[0007] In one aspect, provided herein is a method of treating a KRAS (e.g., hKRAS) variant cancer in a subject in need thereof, comprising: (a) administering a KRAS (e.g., hKRAS) inhibitor to the subject; (b) receiving test results indicating that the KRAS (e.g., hKRAS) variant cancer has developed resistance to the KRAS (e.g., hKRAS) inhibitor; and (c) administering the KRAS (e.g., hKRAS) inhibitor in combination with a fusion protein to the subject, the fusion protein comprising (i) a first partial structure that specifically binds to EGFR (e.g., hEGFR), the first partial structure operably linked to (ii) a second partial structure that specifically binds to TGFβ (e.g., hTGFβ), thereby treating a KRAS (e.g., hKRAS) variant cancer in the subject.

[0008] In one aspect, provided herein is a method of treating a KRAS (e.g., hKRAS) variant cancer in a subject in need thereof, comprising: (a) determining that the KRAS (e.g., hKRAS) variant cancer is resistant to a KRAS (e.g., hKRAS) inhibitor; and (b) administering to the subject a KRAS (e.g., hKRAS) inhibitor in combination with a fusion protein comprising (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), the first partial structure operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ), thereby treating the KRAS (e.g., hKRAS) variant cancer in the subject.

[0009] In one aspect, provided herein is a method of delivering a KRAS (e.g., hKRAS) inhibitor and a fusion protein to a subject in need thereof, the method comprising administering (a) a KRAS (e.g., hKRAS) inhibitor and (b) a fusion protein comprising (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ), thereby delivering the KRAS (e.g., hKRAS) inhibitor and the fusion protein to the subject.

[0010] In one aspect, provided herein is a method of inhibiting the KRAS (e.g., hKRAS) pathway and the TGFβ (e.g., hTGFβ) pathway in a subject in need thereof, comprising administering (a) a KRAS (e.g., hKRAS) inhibitor in combination with (b) a fusion protein comprising (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ), thereby inhibiting the KRAS (e.g., hKRAS) pathway and the TGFβ (e.g., hTGFβ) pathway in the subject.

[0011] In one aspect, provided herein is a method of restoring sensitivity to a KRAS (e.g., hKRAS) inhibitor in a subject in need thereof, the method comprising administering to the subject a fusion protein comprising (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ), thereby restoring sensitivity to a KRAS (e.g., hKRAS) inhibitor in the subject.

[0012] In one aspect, provided herein is a method for suppressing or preventing resistance to a KRAS (e.g., hKRAS) inhibitor in a subject in need thereof, comprising administering to the subject (a) a KRAS (e.g., hKRAS) inhibitor in combination with (b) a fusion protein comprising (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ), thereby suppressing or preventing resistance to a KRAS (e.g., hKRAS) inhibitor in the subject.

[0013] In one aspect, provided herein is a pharmaceutical composition comprising: (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein); (b) a fusion protein (e.g., a fusion protein described herein) comprising a first partial structure that specifically binds (i) EGFR (e.g., hEGFR), the first partial structure being operably linked to a second partial structure that specifically binds (ii) TGFβ (e.g., hTGFβ); and (c) a pharmaceutically acceptable excipient.

[0014] In one aspect, provided herein is (ii) a kit comprising: (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein), or a pharmaceutical composition thereof; and (b) a fusion protein (e.g., a fusion protein described herein), or a pharmaceutical composition thereof, the fusion protein comprising (i) a first partial structure that specifically binds to EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds to TGFβ (e.g., hTGFβ); (ii) a pharmaceutical composition described herein; (iii) a combination regimen described herein; or (iv) a combination composition described herein.

[0015] For clarity, the following exemplary embodiments apply to each of the above aspects as if individually set forth after each aspect.

[0016] In some embodiments, the KRAS (eg, hKRAS) inhibitors and fusion proteins are administered in an amount and for a time sufficient to treat or prevent cancer in the subject.

[0017] In some embodiments, the cancer is a KRAS (e.g., hKRAS) variant cancer. In some embodiments, the cancer has been determined to contain a KRAS (e.g., hKRAS) variant. In some embodiments, the KRAS (e.g., hKRAS) variant comprises a KRAS (e.g., hKRAS)-activating amino acid modification (e.g., substitution). In some embodiments, the KRAS (e.g., hKRAS) variant comprises an amino acid modification (e.g., substitution) at amino acid position G12, G13, or Q61, numbered relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the KRAS (e.g., hKRAS) variant comprises an amino acid modification (e.g., substitution) at amino acid position G12 or G13, numbered relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, G12S, G13D, or Q61H. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, G12S, or G13D. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12A, G12S, or G12D. In some embodiments, the KRAS (eg, hKRAS) variant comprises a G12C amino acid substitution, numbered based on the amino acid sequence of SEQ ID NO:3.

[0018] In some embodiments, the cancer is resistant to a KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer has been determined to be resistant to a KRAS (e.g., hKRAS) inhibitor.

[0019] In some embodiments, the KRAS (e.g., hKRAS) inhibitor has been previously administered to the subject. In some embodiments, the KRAS (e.g., hKRAS) inhibitor has been previously administered to the subject in the absence of the fusion protein.

[0020] In some embodiments, the cancer cells overexpress EGFR (e.g., hEGFR) relative to non-cancer cells. In some embodiments, the cancer cells have been determined to overexpress EGFR (e.g., hEGFR) relative to non-cancer cells. In some embodiments, the cancer cells express an EGFR (e.g., hEGFR) variant. In some embodiments, the cancer cells have been determined to express an EGFR (e.g., hEGFR) variant. In some embodiments, the cancer cells overexpress hTGFβ (e.g., hTGFβ) relative to non-cancer cells. In some embodiments, the cancer cells have been determined to overexpress TGFβ (e.g., hTGFβ) relative to non-cancer cells.

[0021] In some embodiments, the cancer is localized, locally advanced, or metastatic. In some embodiments, the cancer is a carcinoma (e.g., adenocarcinoma, squamous cell carcinoma). In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma), colon cancer (e.g., colon adenocarcinoma), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), breast cancer (e.g., invasive ductal carcinoma), gastric cancer (e.g., gastric adenocarcinoma), endometrial cancer (e.g., undifferentiated endometrial carcinoma), uterine cancer (e.g., endometrial carcinoma), testicular cancer (e.g., testicular germ cell carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), bile duct cancer (e.g., cholangiocarcinoma), myelodysplastic carcinoma, or esophageal cancer (e.g., esophageal adenocarcinoma, gastroesophageal junction carcinoma). In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma), colon cancer (e.g., colon adenocarcinoma), or pancreatic cancer (e.g., pancreatic ductal adenocarcinoma). In some embodiments, the cancer is non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, colon adenocarcinoma, or pancreatic ductal adenocarcinoma.

[0022] In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor). In some embodiments, a KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor.

[0023] In some embodiments, the cancer in the subject is resistant to a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0024] In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit KRAS (e.g., hKRAS) variants. In some embodiments, the KRAS (e.g., hKRAS) variants comprise KRAS (e.g., hKRAS)-activating amino acid modifications. In some embodiments, the KRAS (e.g., hKRAS) variants comprise amino acid modifications at amino acid positions G12, G13, or Q61, numbered relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the KRAS (e.g., hKRAS) variants comprise amino acid modifications at amino acid positions G12 or G13, numbered relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the KRAS (e.g., hKRAS) variants comprise any one or more of the following amino acid substitutions, numbered relative to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, G13D, or Q61H. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, or G13D. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12A, or G12D. In some embodiments, the KRAS (e.g., hKRAS) variant comprises a G12C amino acid substitution, numbered with reference to the amino acid sequence of SEQ ID NO: 3.

[0025] In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises or consists of a small molecule, a protein (e.g., an antibody or functional fragment or variant thereof), a nucleic acid, a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises or consists of a small molecule. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor in Table 2. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises sotorasib (AMG-510).

[0026] In some embodiments, the first substructure comprises an antibody, or a functional fragment or functional variant thereof. In some embodiments, the first substructure comprises a full-length antibody, a single-chain variable fragment (scFv), a Fab, or a single-domain antibody (sdAb). In some embodiments, the first substructure comprises a full-length antibody.

[0027] In some embodiments, the first partial structure comprises a variable heavy chain (VH) region comprising three complementarity determining regions: VH CDR1, VH CDR2, and VH CDR3, and a variable light chain (VL) region comprising three complementarity determining regions: VL CDR1, VL CDR2, and VL CDR3.

[0028] In some embodiments, (a) the amino acid sequence of the VH CDR1 comprises the amino acid sequence SEQ ID NO:39, or the amino acid sequence of SEQ ID NO:39 with one, two, or three amino acid modifications; (b) the amino acid sequence of the VH CDR2 comprises the amino acid sequence SEQ ID NO:40, or the amino acid sequence of SEQ ID NO:40 with one, two, or three amino acid modifications; (c) the amino acid sequence of the VH CDR3 comprises the amino acid sequence SEQ ID NO:41, or the amino acid sequence of SEQ ID NO:41 with one, two, or three amino acid modifications; (d) the amino acid sequence of the VL CDR1 comprises the amino acid sequence SEQ ID NO:42, or the amino acid sequence of SEQ ID NO:42 with one, two, or three amino acid modifications; (e) the amino acid sequence of the VL CDR2 comprises the amino acid sequence SEQ ID NO:43, or the amino acid sequence of SEQ ID NO:43 with one, two, or three amino acid modifications; and (f) the amino acid sequence of the VL CDR3 comprises the amino acid sequence SEQ ID NO:44, or the amino acid sequence of SEQ ID NO:44 with one, two, or three amino acid modifications.

[0029] In some embodiments, the VH region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:45; and the VL region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:46.

[0030] In some embodiments, the first partial structure comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of the HC is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48; and the amino acid sequence of the LC is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49.

[0031] In some embodiments, the second substructure comprises an antibody, or a functional fragment or functional variant thereof, hi some embodiments, the second substructure comprises a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

[0032] In some embodiments, the second substructure comprises or consists of at least a portion of the extracellular domain (ECD) of a transforming growth factor-beta receptor (TGFβR) (e.g., hTGFβR). In some embodiments, the second substructure comprises or consists of at least a portion of the ECD of a transforming growth factor-beta receptor II (TGFβRII) (e.g., hTGFβRII). In some embodiments, the second substructure comprises or consists of the ECD of a TGFβRII (e.g., hTGFβRII). In some embodiments, the amino acid sequence of the second substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73.

[0033] In some embodiments, the fusion protein includes (iii) a third partial structure that specifically binds to TGFβ (e.g., hTGFβ), the third partial structure being operably linked to the first partial structure.

[0034] In some embodiments, the third substructure comprises an antibody, or a functional fragment or functional variant thereof, hi some embodiments, the third substructure comprises a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

[0035] In some embodiments, the third substructure comprises or consists of at least a portion of the extracellular domain (ECD) of TGFβR (e.g., hTGFβR). In some embodiments, the third substructure comprises or consists of at least a portion of the ECD of TGFβRII (e.g., hTGFβRII). In some embodiments, the third substructure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII). In some embodiments, the amino acid sequence of the third substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73. In some embodiments, the amino acid sequence of the second substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73; and the amino acid sequence of the third substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73.

[0036] In some embodiments, the amino acid sequence of the second substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the third substructure.

[0037] In some embodiments, the first substructure comprises a full-length antibody and the N-terminus of the second substructure is operably connected (e.g., optionally via a linker) to the C-terminus of the first light chain.

[0038] In some embodiments, the first substructure comprises a full-length antibody, and the N-terminus of the second substructure is operably connected (e.g., optionally via a linker) to the C-terminus of the first heavy chain.

[0039] In some embodiments, the first substructure comprises a full-length antibody, the N-terminus of the second substructure is operably connected (e.g., optionally via a linker) to the C-terminus of the first light chain, and the N-terminus of the third substructure is operably connected (e.g., optionally via a linker) to the C-terminus of the second light chain.

[0040] In some embodiments, the first substructure comprises a full-length antibody, the N-terminus of the second substructure is operably connected to the C-terminus of the first heavy chain (e.g., optionally via a linker), and the N-terminus of the third substructure is operably connected to the C-terminus of the second heavy chain (e.g., optionally via a linker).

[0041] In some embodiments, the first substructure is operably connected directly to the second substructure. In some embodiments, the first substructure is operably connected indirectly to the second substructure. In some embodiments, the first substructure is operably connected indirectly to the second substructure via a first peptide linker. In some embodiments, the first peptide linker comprises or consists of glycine or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs: 105-124; or (b) the amino acid sequence of any one of SEQ ID NOs: 105-124 containing or consisting of one, two, or three amino acid modifications. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of SEQ ID NO: 111; or (b) the amino acid sequence of SEQ ID NO: 111 containing or consisting of one, two, or three amino acid modifications.

[0042] In some embodiments, the first substructure is operably connected directly to the third substructure. In some embodiments, the first substructure is operably connected indirectly to the third substructure. In some embodiments, the first substructure is operably connected indirectly to the third substructure via a second peptide linker. In some embodiments, the second peptide linker comprises or consists of glycine or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the second peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NO: 111; or (b) the amino acid sequence of any one of SEQ ID NO: 111 comprising or consisting of one, two, or three amino acid modifications. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of SEQ ID NO: 111; or (b) the amino acid sequence of SEQ ID NO: 111 comprising or consisting of one, two, or three amino acid modifications. In some embodiments, the amino acid sequence of the first peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second peptide linker.

[0043] In some embodiments, the amino acid sequence of the first peptide linker is 100% identical to the amino acid sequence of the second peptide linker.

[0044] In some embodiments, (a) the first substructure comprises or consists of a full-length antibody; (b) the second substructure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII); (c) the third substructure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII); (d) the second substructure is operably connected to the first substructure by a first peptide linker; and (e) the third substructure is operably connected to the first substructure by a second peptide linker.

[0045] In some embodiments, the N-terminus of the second substructure is operably connected to the C-terminus of a first light chain of the full-length antibody, and the N-terminus of the third substructure is operably connected to the C-terminus of a second light chain of the full-length antibody.

[0046] In some embodiments, (a)(i) the amino acid sequence of the VH CDR1 comprises the amino acid sequence SEQ ID NO:39, or the amino acid sequence of SEQ ID NO:39 with one, two, or three amino acid modifications; the amino acid sequence of the VH CDR2 comprises the amino acid sequence SEQ ID NO:40, or the amino acid sequence of SEQ ID NO:40 with one, two, or three amino acid modifications; the amino acid sequence of the VH CDR3 comprises the amino acid sequence SEQ ID NO:41, or the amino acid sequence of SEQ ID NO:41 with one, two, or three amino acid modifications; the amino acid sequence of the VL CDR1 comprises the amino acid sequence SEQ ID NO:42, or the amino acid sequence of SEQ ID NO:42 with one, two, or three amino acid modifications; the amino acid sequence of the VL CDR2 comprises the amino acid sequence SEQ ID NO:43, or the amino acid sequence of SEQ ID NO:43 with one, two, or three amino acid modifications; the amino acid sequence of the CDR3 comprises the amino acid sequence SEQ ID NO:44, or the amino acid sequence of SEQ ID NO:44 containing one, two, or three amino acid modifications; (ii) the VH region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:45; the VL region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:45; and / or (iii) the amino acid sequence of the heavy chain of the full-length antibody is SEQ ID NO: the amino acid sequence of the light chain of the full-length antibody is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:49; (b) the amino acid sequence of the second substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73;(c) the amino acid sequence of the third substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73; and (d) the amino acid sequence of the first peptide linker is at least 85%, 86%, 87%, 88%, or 100% identical to the amino acid sequence of SEQ ID NO: 111. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; (e) the amino acid sequence of the second peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 111;

[0047] In some embodiments, the fusion protein comprises: (a) a first polypeptide, wherein the amino acid sequence of the first polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125; and (b) a second polypeptide, wherein the amino acid sequence of the second polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125. (c) a third polypeptide, wherein the amino acid sequence of the third polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126; and (d) a fourth polypeptide, wherein the amino acid sequence of the fourth polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126.

[0048] In one aspect, provided herein is a method of treating a KRAS (e.g., hKRAS) variant cancer in a subject in need thereof, comprising: (a) receiving test results indicating that the KRAS (e.g., hKRAS) variant cancer exhibits resistance to a KRAS inhibitor, in combination with: (b) administering to the subject a KRAS (e.g., hKRAS) inhibitor and a fusion protein, wherein the fusion protein comprises (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), and the first partial structure is operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ), thereby treating the KRAS (e.g., hKRAS) variant cancer in the subject.

[0049] In some embodiments, a KRAS (eg, hKRAS) inhibitor has been previously administered to the subject.

[0050] In some embodiments, the KRAS (e.g., hKRAS) inhibitor and fusion protein are administered in an amount and for a time sufficient to treat a KRAS (e.g., hKRAS) variant cancer in the subject.

[0051] In some embodiments, the cancer is a KRAS (e.g., hKRAS) variant cancer.

[0052] In some embodiments, the cancer has been determined to contain a KRAS (e.g., hKRAS) variant.

[0053] In some embodiments, the KRAS (eg, hKRAS) variant comprises a KRAS (eg, hKRAS)-activating amino acid modification (eg, substitution).

[0054] In some embodiments, the KRAS (eg, hKRAS) variant comprises an amino acid modification (eg, a substitution) at amino acid position G12, G13, or Q61, numbered relative to the amino acid sequence of SEQ ID NO:3.

[0055] In some embodiments, the KRAS (eg, hKRAS) variant comprises an amino acid modification (eg, a substitution) at amino acid position G12 or G13, numbered relative to the amino acid sequence of SEQ ID NO:3.

[0056] In some embodiments, the KRAS (e.g., hKRAS) variant includes any one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, G12S, G13D, or Q61H.

[0057] In some embodiments, the KRAS (e.g., hKRAS) variant includes any one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, G12S, or G13D.

[0058] In some embodiments, the KRAS (e.g., hKRAS) variant includes any one of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12A, G12S, or G12D.

[0059] In some embodiments, the KRAS (eg, hKRAS) variant comprises a G12C amino acid substitution, numbered based on the amino acid sequence of SEQ ID NO:3.

[0060] In some embodiments, the cancer is resistant to a KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer has been determined to be resistant to a KRAS (e.g., hKRAS) inhibitor.

[0061] In some embodiments, the KRAS (e.g., hKRAS) inhibitor has been previously administered to the subject. In some embodiments, the KRAS (e.g., hKRAS) inhibitor has been previously administered to the subject in the absence of the fusion protein.

[0062] In some embodiments, the cancer cells overexpress EGFR (e.g., hEGFR) relative to non-cancer cells. In some embodiments, the cancer cells have been determined to overexpress EGFR (e.g., hEGFR) relative to non-cancer cells. In some embodiments, the cancer cells express an EGFR (e.g., hEGFR) variant. In some embodiments, the cancer cells have been determined to express an EGFR (e.g., hEGFR) variant. In some embodiments, the cancer cells overexpress hTGFβ (e.g., hTGFβ) relative to non-cancer cells. In some embodiments, the cancer cells have been determined to overexpress TGFβ (e.g., hTGFβ) relative to non-cancer cells.

[0063] In some embodiments, the cancer is localized, locally advanced, or metastatic.

[0064] In some embodiments, the cancer is a carcinoma (e.g., adenocarcinoma, squamous cell carcinoma).

[0065] In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma), colon cancer (e.g., colon adenocarcinoma), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), breast cancer (e.g., invasive ductal carcinoma), gastric cancer (e.g., gastric adenocarcinoma), endometrial cancer (e.g., undifferentiated endometrial carcinoma), uterine cancer (e.g., endometrial carcinoma), testicular cancer (e.g., testicular germ cell carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), bile duct cancer (e.g., cholangiocarcinoma), myelodysplastic carcinoma, or esophageal cancer (e.g., esophageal adenocarcinoma, gastroesophageal junction carcinoma).

[0066] In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma), colon cancer (e.g., colon adenocarcinoma), or pancreatic cancer (e.g., pancreatic ductal adenocarcinoma).

[0067] In some embodiments, the cancer is non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, colorectal adenocarcinoma, or pancreatic ductal adenocarcinoma.

[0068] In some embodiments, the subject has previously received a KRAS (eg, hKRAS) inhibitor (eg, a KRAS (eg, hKRAS) inhibitor).

[0069] In some embodiments, the subject has not previously been administered a KRAS (e.g., hKRAS) inhibitor. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor.

[0070] In some embodiments, the cancer in the subject is resistant to a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0071] In some embodiments, the KRAS (eg, hKRAS) inhibitor can selectively inhibit a KRAS (eg, hKRAS) variant.

[0072] In some embodiments, the KRAS (e.g., hKRAS) variant comprises a KRAS (e.g., hKRAS)-activating amino acid modification. In some embodiments, the KRAS (e.g., hKRAS) variant comprises an amino acid modification at amino acid position G12, G13, or Q61, numbered relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the KRAS (e.g., hKRAS) variant comprises an amino acid modification at amino acid position G12 or G13, numbered relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered relative to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, G13D, or Q61H. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, or G13D. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12A, or G12D. In some embodiments, the KRAS (e.g., hKRAS) variant comprises a G12C amino acid substitution, numbered with reference to the amino acid sequence of SEQ ID NO: 3.

[0073] In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises or consists of a small molecule, a protein (e.g., an antibody or functional fragment or variant thereof), a nucleic acid, a carbohydrate, a lipid, a metal, or a toxin. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises or consists of a small molecule. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor in Table 2. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises sotorasib (AMG-510).

[0074] In some embodiments, the first substructure comprises an antibody, or a functional fragment or functional variant thereof. In some embodiments, the first substructure comprises a full-length antibody, a single-chain variable fragment (scFv), a Fab, or a single-domain antibody (sdAb). In some embodiments, the first substructure comprises a full-length antibody.

[0075] In some embodiments, the first partial structure comprises a variable heavy chain (VH) region comprising three complementarity determining regions: VH CDR1, VH CDR2, and VH CDR3, and a variable light chain (VL) region comprising three complementarity determining regions: VL CDR1, VL CDR2, and VL CDR3.

[0076] In some embodiments, (a) the amino acid sequence of the VH CDR1 comprises the amino acid sequence SEQ ID NO:39, or the amino acid sequence of SEQ ID NO:39 with one, two, or three amino acid modifications; (b) the amino acid sequence of the VH CDR2 comprises the amino acid sequence SEQ ID NO:40, or the amino acid sequence of SEQ ID NO:40 with one, two, or three amino acid modifications; (c) the amino acid sequence of the VH CDR3 comprises the amino acid sequence SEQ ID NO:41, or the amino acid sequence of SEQ ID NO:41 with one, two, or three amino acid modifications; (d) the amino acid sequence of the VL CDR1 comprises the amino acid sequence SEQ ID NO:42, or the amino acid sequence of SEQ ID NO:42 with one, two, or three amino acid modifications; (e) the amino acid sequence of the VL CDR2 comprises the amino acid sequence SEQ ID NO:43, or the amino acid sequence of SEQ ID NO:43 with one, two, or three amino acid modifications; and (f) the amino acid sequence of the VL CDR3 comprises the amino acid sequence SEQ ID NO:44, or the amino acid sequence of SEQ ID NO:44 with one, two, or three amino acid modifications.

[0077] In some embodiments, the VH region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:45; and the VL region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:46.

[0078] In some embodiments, the first partial structure comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of the HC is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48; and the amino acid sequence of the LC is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49.

[0079] In some embodiments, the second substructure comprises an antibody, or a functional fragment or functional variant thereof.

[0080] In some embodiments, the second substructure comprises a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

[0081] In some embodiments, the second substructure comprises or consists of at least a portion of the extracellular domain (ECD) of a transforming growth factor-beta receptor (TGFβR) (e.g., hTGFβR). In some embodiments, the second substructure comprises or consists of at least a portion of the ECD of a transforming growth factor-beta receptor II (TGFβRII) (e.g., hTGFβRII). In some embodiments, the second substructure comprises or consists of the ECD of a TGFβRII (e.g., hTGFβRII). In some embodiments, the amino acid sequence of the second substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73.

[0082] In some embodiments, the fusion protein includes (iii) a third partial structure that specifically binds to TGFβ (e.g., hTGFβ), the third partial structure being operably linked to the first partial structure.

[0083] In some embodiments, the third substructure comprises an antibody, or a functional fragment or functional variant thereof.

[0084] In some embodiments, the third substructure comprises a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

[0085] In some embodiments, the third substructure comprises or consists of at least a portion of the extracellular domain (ECD) of TGFβR (e.g., hTGFβR). In some embodiments, the third substructure comprises or consists of at least a portion of the ECD of TGFβRII (e.g., hTGFβRII). In some embodiments, the third substructure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII). In some embodiments, the amino acid sequence of the third substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73. In some embodiments, the amino acid sequence of the second substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73; and the amino acid sequence of the third substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73.

[0086] In some embodiments, the amino acid sequence of the second substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the third substructure.

[0087] In some embodiments, the first substructure comprises a full-length antibody and the N-terminus of the second substructure is operably connected (e.g., optionally via a linker) to the C-terminus of the first light chain.

[0088] In some embodiments, the first substructure comprises a full-length antibody, and the N-terminus of the second substructure is operably connected (e.g., optionally via a linker) to the C-terminus of the first heavy chain.

[0089] In some embodiments, the first substructure comprises a full-length antibody, the N-terminus of the second substructure is operably connected (e.g., optionally via a linker) to the C-terminus of the first light chain, and the N-terminus of the third substructure is operably connected (e.g., optionally via a linker) to the C-terminus of the second light chain.

[0090] In some embodiments, the first substructure comprises a full-length antibody, the N-terminus of the second substructure is operably connected to the C-terminus of the first heavy chain (e.g., optionally via a linker), and the N-terminus of the third substructure is operably connected to the C-terminus of the second heavy chain (e.g., optionally via a linker).

[0091] In some embodiments, the first substructure is operably connected directly to the second substructure. In some embodiments, the first substructure is operably connected indirectly to the second substructure. In some embodiments, the first substructure is operably connected indirectly to the second substructure via a first peptide linker. In some embodiments, the first peptide linker comprises or consists of glycine or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NOs: 105-124; or (b) the amino acid sequence of any one of SEQ ID NOs: 105-124 containing or consisting of one, two, or three amino acid modifications. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of SEQ ID NO: 111; or (b) the amino acid sequence of SEQ ID NO: 111 containing or consisting of one, two, or three amino acid modifications.

[0092] In some embodiments, the first substructure is operably connected directly to the third substructure.

[0093] In some embodiments, the first substructure is operably connected indirectly to the third substructure. In some embodiments, the first substructure is operably connected indirectly to the third substructure by a second peptide linker. In some embodiments, the second peptide linker comprises or consists of glycine or glycine and serine amino acid residues. In some embodiments, the amino acid sequence of the second peptide linker comprises or consists of (a) the amino acid sequence of any one of SEQ ID NO: 111; or (b) the amino acid sequence of any one of SEQ ID NO: 111 comprising or consisting of one, two, or three amino acid modifications. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of (a) the amino acid sequence of SEQ ID NO: 111; or (b) the amino acid sequence of SEQ ID NO: 111 comprising or consisting of one, two, or three amino acid modifications. In some embodiments, the amino acid sequence of the first peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second peptide linker.

[0094] In some embodiments, the amino acid sequence of the first peptide linker is 100% identical to the amino acid sequence of the second peptide linker.

[0095] In some embodiments, (a) the first substructure comprises or consists of a full-length antibody; (b) the second substructure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII); (c) the third substructure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII); (d) the second substructure is operably connected to the first substructure by a first peptide linker; and (e) the third substructure is operably connected to the first substructure by a second peptide linker.

[0096] In some embodiments, the N-terminus of the second substructure is operably connected to the C-terminus of a first light chain of the full-length antibody, and the N-terminus of the third substructure is operably connected to the C-terminus of a second light chain of the full-length antibody.

[0097] In some embodiments, (a)(i) the amino acid sequence of the VH CDR1 comprises the amino acid sequence SEQ ID NO:39, or the amino acid sequence of SEQ ID NO:39 with one, two, or three amino acid modifications; the amino acid sequence of the VH CDR2 comprises the amino acid sequence SEQ ID NO:40, or the amino acid sequence of SEQ ID NO:40 with one, two, or three amino acid modifications; the amino acid sequence of the VH CDR3 comprises the amino acid sequence SEQ ID NO:41, or the amino acid sequence of SEQ ID NO:41 with one, two, or three amino acid modifications; the amino acid sequence of the VL CDR1 comprises the amino acid sequence SEQ ID NO:42, or the amino acid sequence of SEQ ID NO:42 with one, two, or three amino acid modifications; the amino acid sequence of the VL CDR2 comprises the amino acid sequence SEQ ID NO:43, or the amino acid sequence of SEQ ID NO:43 with one, two, or three amino acid modifications; the amino acid sequence of the CDR3 comprises the amino acid sequence SEQ ID NO:44, or the amino acid sequence of SEQ ID NO:44 containing one, two, or three amino acid modifications; (ii) the VH region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:45; the VL region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:46; and / or (iii) the amino acid sequence of the heavy chain of the full-length antibody comprises the sequence the amino acid sequence of the light chain of the full-length antibody is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:49; (b) the amino acid sequence of the second substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72;(c) the amino acid sequence of the third substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72; and (d) the amino acid sequence of the first peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 111. (e) the amino acid sequence of the second peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 111;

[0098] In some embodiments, the fusion protein comprises: (a) a first polypeptide, wherein the amino acid sequence of the first polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125; and (b) a second polypeptide, wherein the amino acid sequence of the second polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125. (c) a third polypeptide, wherein the amino acid sequence of the third polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126; and (d) a fourth polypeptide, wherein the amino acid sequence of the fourth polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126. [Brief explanation of the drawings]

[0099] [Figure 1-1] Figure 1A is a line graph showing the relative cytotoxicity for each of the indicated treatments (AMG510 + BCA101 (10 μg / mL)); AMG510; and BCA101 (10 μg / mL)) in an H358 in vitro lung cancer model. Synergistic activity was calculated using the Bliss model as indicated in the line graph. Figure 1B is a line graph showing the relative cytotoxicity for each of the indicated treatments (AMG510 + BCA101 (50 μg / mL)); AMG510; and BCA101 (50 μg / mL)) in an H1792 in vitro lung cancer model. Synergistic activity was calculated using the Bliss model as indicated in the line graph. [Figure 1-2] Figure 1C is a line graph showing the relative cytotoxicity for each of the indicated treatments (AMG510 + BCA101 (50 μg / mL)); AMG510; and BCA101 (50 μg / mL)) in the SW837 in vitro colon cancer model. Synergistic activity was calculated using the Bliss model as indicated in the line graph. Figure 1D is a line graph showing the relative cytotoxicity for each of the indicated treatments (AMG510 + BCA101 (50 μg / mL)); AMG510; and BCA101 (50 μg / mL)) in the SW1463 in vitro colon cancer model. Synergistic activity was calculated using the Bliss model as indicated in the line graph. [Figure 2] 1 is a bar graph showing percent viability of H358 lung cancer cells in vitro for the indicated treatments (control; AMG510, BCA101, AMG510+BCA101; cetuximab; AMG510+cetuximab; TGFβRIIFC; AMG510+TGFβRIIFC; cetuximab+TGFβRIIFC; and AMG510+cetuximab+TGFβRIIFC). [Figure 3]1 is a line graph showing percent viability for each of the indicated in vitro H1792 lung cancer cell cultures in the indicated treatments (H1792wt; H1792-TGFβ1; H1792-TGFβ1+cetuximab; and H1792-TGFβ1+BCA101). [Figure 4] 1 is a bar graph showing percent viability for each of the indicated in vitro H358 lung cancer cell cultures (H358wt; H358 AMG510-resistant) in the indicated treatment group (AMG510 at 0 nM, 1.28 nM, 6.4 nM, 32 nM, or 160 nM). [Figure 5-1] Figure 5A is a series of histograms showing the mean fluorescence intensity (MFI) of EGFR cell surface expression assessed by flow cytometry in KRASG12C mutant lung and colon cancer cell lines: NCI-H1792 (G12C homozygous), NCI-H358 (G12C heterozygous), and SW1463 (G12C homozygous). [Figure 5-2] Figure 5B is a bar graph presenting the MFI of cell surface expression of EGFR data assessed by flow cytometry presented in Figure 5A for KRASG12C mutant lung and colon cancer cell lines: NCI-H1792 (G12C homozygous), NCI-H358 (G12C heterozygous), and SW1463 (G12C homozygous). [Figure 6] 1 is a bar graph showing expression levels of TGF-β from KRASG12C mutant lung and colon cancer cell lines: NCI-H1792 (G12C homozygous) (lung cancer), NCI-H358 (G12C heterozygous) (lung cancer), and SW1463 (G12C homozygous) (colon cancer) as assessed by ELISA. [Figure 7] 1 is a bar graph showing the levels of TGF-β1 (as measured by ELISA) expressed by KRASG12C inhibitor (KRASG12Ci)-resistant NCI-H358 (shown as H358) and NCI-H1792 (shown as H1792) treated with KRASi or untreated controls. [Figure 8]1 is a bar graph showing the fold change in the levels of TGF-β1 expressed by SW1463 KRASG12Ci-resistant cells / SW1463 parental cells. [Figure 9] 1 is a bar graph showing the fold change in the level of cell surface EGFR expressed by H1792 KRASG12Ci-resistant cells / the level of cell surface EGFR expressed by the parental cells, and the level of cell surface EGFR expressed by SW1463 KRASG12Ci-resistant cells / the level of cell surface EGFR expressed by the parental cells. The graph is plotted as the fold change in median EGFR fluorescence intensity between KRASG12Ci-resistant and parental cells. [Figure 10] 1 is a line graph showing percent viability for each of the indicated in vitro H1792 lung cancer cell cultures in the indicated treatment groups (H1792, H1792 KRASG12Ci-Resistant, H1792 KRASG12Ci-Resistant+Cetuximab; and H1792 KRASG12Ci-Resistant+BCA101). [Figure 11] 1 is a line graph showing percent viability for each of the indicated in vitro SW1463 colon cancer cell cultures in the indicated treatment groups (SW1463, SW1463 KRASG12Ci-resistant; and SW1463 KRASG12Ci-resistant+BCA101). [Figure 12] 1 is a line graph showing the mean tumor volume ± SEM (mm3) of NCI-H358 xenograft tumors in nude mice treated with the indicated agent (BCA101 (5 mg / kg) alone, AMG510 (5 mg / kg) alone, and AMG510 (5 mg / kg) + BCA101 (5 mg / kg)) or placebo. n = 10 mice per group. Statistical significance was calculated by two-way ANOVA with Tukey's multiple comparison test comparing the mean tumor volume per treatment with every other treatment. *p ≤ 0.05, **p ≤ 0.01. [Figure 13] FIG. 13 is a magnified image of the boxed area in FIG. 12, excluding the placebo group. DETAILED DESCRIPTION OF THE INVENTION

[0100] KRAS (e.g., hKRAS) inhibitors have been evaluated and, in some cases, approved for the treatment of certain KRAS (e.g., hKRAS) variant cancers. However, a significant number of subjects treated with KRAS (e.g., hKRAS) inhibitors develop resistance to the KRAS (e.g., hKRAS) inhibitors, ultimately limiting the clinical use of the treatment. The present inventors have specifically identified that administration of a drug (e.g., fusion protein) that specifically binds to EGFR (e.g., hEGFR) and TGFβ synergistically improves the cytotoxicity of KRAS (e.g., hKRAS) inhibitors and rescues TGFβ-induced KRAS (e.g., hKRAS) inhibitor resistance. Therefore, the methods, pharmaceutical compositions, and kits described herein are good candidates for the treatment of cancers (e.g., KRAS (e.g., hKRAS) variant cancers, EGFR (e.g., hEGFR)-expressing cancers). Thus, the present disclosure provides, inter alia, novel methods, pharmaceutical compositions and kits for use, for example, in the treatment of cancer (e.g., KRAS (e.g., hKRAS) variant cancers, EGFR (e.g., hEGFR) expressing cancers).

[0101] table of contents 5.1 Definition 5.2 KRAS inhibitors 5.3 EGFR binding substructure 5.4 TGFβ binding substructure 5.5 Fusion Proteins and Polypeptides 5.5.1 Ig constant region 5.5.2 Linker 5.5.3 Orientation 5.5.3.1 Homodimeric Full-Length Antibody-C-Terminal Light Chain Fusion Proteins 5.5.3.2 Homodimeric Full-Length Antibody-C-Terminal Heavy Chain Fusion Proteins 5.5.4 Exemplary Fusion Proteins 5.5.5 Affinities of Fusion Proteins and Their Antigen-Binding Substructures 5.6 Methods for Producing Fusion Proteins and Their Components 5.7 Combination regimens 5.8 Combination Compositions 5.9 Polynucleotides, Vectors, Carriers, and Host Cells 5.10 Pharmaceutical Compositions 5.11 How to use 5.11.1 Method of Delivery 5.11.2 Methods of Inhibiting the KRAS and TGFβ Pathways 5.11.3 Methods of Restoring Sensitivity to KRAS Inhibitors 5.11.4 Methods of Suppressing or Preventing Resistance to KRAS Inhibitors 5.11.5 Methods of Treating Cancer 5.11.5.1 Cancer 5.12 Kit 5.13 Exemplary Embodiments

[0102] 5.1 Definition The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0103] 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.

[0104] In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents 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.

[0105] Whenever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and "consisting essentially of" are also provided, and conversely, whenever an embodiment is described herein using the words "consisting of" and "consisting essentially of," it is understood that other similar embodiments described with the term "comprising" are also provided.

[0106] The term "and / or," as used herein, should be construed as a specific disclosure of each of the two specified features or components, with or without the other. Thus, when used herein in a phrase such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when used in a phrase such as "A, B, and / or C," the term "and / or" is intended to encompass each of the following aspects: 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).

[0107] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range and, where applicable, fractions thereof (e.g., tenths and hundredths of an integer), unless otherwise indicated.

[0108] The term "about" refers to a value or composition that is within an acceptable error range for the 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. When a particular value or composition is provided in this application and claims, unless otherwise stated, the meaning of "about" should be considered to be within the acceptable error range for that particular value or composition.

[0109] Where proteins and / or polypeptides are described herein, it is understood that the polynucleotides (e.g., RNA (e.g., mRNA) or DNA polynucleotides) that encode the proteins or polypeptides are also provided herein.

[0110] Where proteins, polypeptides, polynucleotides, cells, expression vectors, and the like are described herein, it is understood that isolated forms of the proteins, polypeptides, polynucleotides, cells, expression vectors, and the like are also provided herein.

[0111] Where a protein, polypeptide, polynucleotide, or the like is described herein, it is understood that recombinant forms of the protein, polypeptide, polynucleotide, or the like are also provided herein.

[0112] It is understood that where a polypeptide or set of polypeptides is described herein, a protein comprising the polypeptide or set of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) is also provided herein, and conversely, where a protein comprising a polypeptide or set of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) is described herein, the polypeptide or set of polypeptides is also provided herein.

[0113] As used herein, the term "administration" refers to the physical introduction of an agent, e.g., a therapeutic agent (or a precursor of a therapeutic agent that is metabolized or modified 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 of skill in the art. Administration may occur, for example, once, multiple times, and / or over one or more extended periods of time.

[0114] As used herein, the term "affinity" refers to the strength of binding of one protein (e.g., an antibody) to another protein (e.g., an antigen). Protein affinity is measured by the dissociation constant, Kd, ​​defined as [antibody] x [antigen] / [antibody-antigen], where [antibody-antigen] is the molar concentration of the antibody-antigen complex, [antibody] is the molar concentration of unbound antibody, and [antigen] is the molar concentration of unbound antigen. The affinity constant, Ka, is defined by 1 / Kd. Standard methods for measuring affinity are known to those of skill in the art. Exemplary methods for measuring affinity are described herein, see, e.g., § 5.5.5.

[0115] As used herein, the terms "antibody" (singular) or "antibodies" (plural) are used in the broadest sense and encompass a variety of immunoglobulin (Ig) (e.g., human Ig (hIg)) structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific, trispecific) antibodies, and antigen fragments (i.e., antigen-binding fragments or variants) that exhibit the desired antigen-binding activity. Thus, the term antibody includes, for example, full-length antibodies; antigen-binding fragments of full-length antibodies; molecules comprising antibody CDRs, VH regions, and / or VL regions; and antibody-like scaffolds (e.g., fibronectin). Examples of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelized antibodies, intrabodies, affibodies, diabodies, tribodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies (e.g., VHH, (VHH)2), single chain antibodies, single chain Fvs (scFv; (scFv)2), Fab fragments (e.g., Fab, single chain Fab (scFab), F(ab')2 fragments), disulfide-linked Fvs (sdFv), Fc fusions (e.g., Fab-Fc, scFv-Fc, VHH-Fc, (scFv)2-Fc, (VHH)2-Fc), and antigen-binding fragments of any of the foregoing, as well as conjugates or derived proteins comprising any of the foregoing. Antibodies may be of any Ig isotype (e.g., IgG, IgE, IgM, IgD, or IgA), any class of Ig (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b). In certain embodiments, the antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In some embodiments, the antibody is a human, humanized, or chimeric IgG1 or IgG4 monoclonal antibody. In some embodiments, the term antibody refers to a monoclonal or polyclonal antibody population. The antibodies described herein can be produced by any standard method known in the art, for example, by recombinant production in a host cell, see e.g., §5.6, or by synthetic production.

[0116] As used herein, with respect to a polynucleotide, the term "derived from" refers to a polynucleotide or fragment thereof having at least 70% sequence identity to a reference polynucleotide (e.g., a naturally occurring polynucleotide). With respect to a polypeptide, the term "derived from" refers to a polypeptide or protein comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of a reference polypeptide or protein (e.g., a naturally occurring polypeptide or protein). The term "derived from," as used herein, does not refer to any particular process or method for obtaining the polynucleotide, polypeptide, or protein. For example, a polynucleotide, polypeptide, or protein may be recombinantly produced or chemically synthesized.

[0117] As used herein, the term "disease" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, disorder, pathology, condition, or symptom in which physiological function is impaired, regardless of the nature of the etiology.

[0118] The terms "DNA" and "polydeoxyribonucleotide" are used interchangeably herein to refer to a macromolecule comprising multiple deoxyribonucleotides polymerized through phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0119] As used herein, the term "Fc region" refers to the C-terminal region of an Ig heavy chain, including, from N-terminus to C-terminus, at least the CH2 region and the CH3 region to which it is operably connected. In some embodiments, the Fc region includes an Ig hinge region or at least a portion of an Ig hinge region operably connected to the N-terminus of the CH2 region. In some embodiments, the Fc region is engineered relative to a reference Fc region. Additional examples of proteins with engineered Fc regions can be found in Saunders 2019 (KO Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life," 2019, Frontiers in Immunology, Vol. 10, Art. 1296, pp. 1-20, the entire contents of which are incorporated herein by reference for all purposes).

[0120] As used herein, the term "EGFR" or "epidermal growth factor receptor" refers to a transmembrane receptor member of the ErbB family of receptors that serves as a receptor for the EGF family of extracellular proteins. An exemplary reference amino acid sequence of mature human EGFR (hEGFR) protein is set forth in SEQ ID NO:37.

[0121] As used herein, the term "EGFR variant" or "variant EGFR" refers to an EGFR protein or polypeptide that includes at least one amino acid modification (e.g., an amino acid substitution) compared to a reference (e.g., a wild-type (e.g., SEQ ID NO: 37)) EGFR protein or polypeptide. In some embodiments, the at least one amino acid modification is an amino acid substitution. In some embodiments, the EGFR variant exhibits increased activity.

[0122] As used herein, the term "full-length antibody" refers to a full-length antibody having a structure substantially similar to a native antibody structure: (i) a first immunoglobulin (Ig) light chain comprising, from N-terminus to C-terminus, a light chain variable region (VL) region and a light chain constant region (CL) region; (ii) a first Ig heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable region (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (iii) a first Ig heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. (iv) a second Ig heavy chain comprising, from N-terminus to C-terminus, a VL region and a VH region, wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and the first heavy chain and the second heavy chain associate to form a dimer. In some embodiments, the two heavy chains comprise substantially identical amino acid sequences, and the two light chains comprise substantially identical amino acid sequences. In some embodiments, the two heavy chains comprise substantially identical amino acid sequences, except for one or more amino acid modifications (e.g., as described herein) that promote proper heavy chain heterodimerization, and the two light chains comprise substantially identical amino acid sequences. Antibody chains may be substantially identical, although they may not be completely identical if they differ due to post-translational modifications such as C-terminal truncation of lysine residues, alternative glycosylation patterns, etc.

[0123] The term "functional variant," as used herein with respect to a polypeptide or protein, refers to a polypeptide or protein that contains at least one, but not more than 15%, not more than 12%, not more than 10%, or not more than 8% amino acid alterations (e.g., substitutions, deletions, additions) compared to the amino acid sequence of a reference polypeptide or protein, and that retains at least one specific function of the reference polypeptide or protein. Not all functions of the reference polypeptide or protein (e.g., wild-type) need be retained by a functional variant of a protein. In some cases, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein. For example, a functional variant of an antibody that specifically binds to EGFR can refer to an antibody that specifically binds to EGFR that contains one or more amino acid substitutions compared to the reference antibody that retains the ability to specifically bind to EGFR.

[0124] The term "functional fragment," as used herein with respect to a polypeptide or protein, refers to a fragment of a reference polypeptide or protein that retains at least one specific function. Not all functions of a reference polypeptide or protein need be retained by a functional fragment of a polypeptide or protein. In some cases, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein. In some embodiments, a functional fragment polypeptide or protein contains at least one, but not more than 15%, not more than 12%, not more than 10%, or not more than 8% amino acid deletion compared to the amino acid sequence of the reference polypeptide or protein. For example, a functional fragment of an antibody that specifically binds to EGFR can refer to a fragment of the antibody that retains the ability to specifically bind to EGFR.

[0125] As used herein, the term "fused" and its grammatical equivalents refer to the operative connection of at least a first polypeptide (or protein) to a second polypeptide (or protein), where the first and second polypeptides (or proteins) are not naturally found operatively connected to each other. For example, the first and second polypeptides (or proteins) may be derived from different proteins. The term fused encompasses both the direct connection of at least two polypeptides by a peptide bond and the indirect connection by a linker (e.g., a peptide linker).

[0126] As used herein, the terms "fusion polypeptide" or "fusion protein" and their grammatical equivalents refer to a polypeptide or protein comprising at least one polypeptide (or protein) operably linked to another polypeptide (or protein), wherein the first and second polypeptides (or proteins) are not naturally found operably linked to each other. For example, in some embodiments, the first and second polypeptides of a fusion protein are each derived from different proteins. The at least two polypeptides of a fusion protein may be operably linked directly by a peptide bond or indirectly by a linker (e.g., a peptide linker). Thus, for example, the term "fusion polypeptide" encompasses embodiments in which polypeptide A is operably linked directly to polypeptide B by a peptide bond (polypeptide A-polypeptide B) and embodiments in which polypeptide A is operably linked to polypeptide B by a peptide linker (polypeptide A-peptide linker-polypeptide B).

[0127] As used herein, the term "in combination" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen specifies the dosage and periodicity of administration of each agent so that the effects of the separate agents on the subject overlap. In some embodiments, delivery of two or more agents is simultaneous or parallel, and the agents may be combined. In other embodiments, two or more agents are not combined but are administered sequentially as part of a prescribed regimen (e.g., a prime-boost vaccine regimen). In some embodiments, administering two or more agents or treatments in combination means administering them such that the reduction in symptoms or other parameters associated with the condition is greater than that observed with one agent or treatment delivered alone or in the absence of the other agent. The effect of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent can be accomplished by any suitable route, including, but not limited to, oral, intravenous, subcutaneous, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes.

[0128] As used herein, the term "KRAS" or "KRAS" or "Kirsten rat sarcoma viral oncogene homolog" collectively refers to a group of KRAS human GTPases, including KRAS isoform 2A and KRAS isoform 2B. The term also includes naturally occurring and engineered variants of KRAS, such as variants described herein, such as KRAS G12C, G12A, etc. The amino acid sequence of an exemplary reference mature human KRAS (hKRAS) isoform 2A protein is set forth in SEQ ID NO: 1, and the amino acid sequence of an exemplary reference mature hKRAS isoform 2B protein is set forth in SEQ ID NO: 3.

[0129] As used herein, the term "KRAS inhibitor" or "KRASi" refers to any substructure (e.g., small molecule compound, protein, polypeptide, nucleic acid, etc.) that can negatively modulate or completely inhibit all or a portion of the enzymatic activity of KRAS (e.g., hKRAS). Exemplary KRAS inhibitors include, but are not limited to, any one or more of the compounds disclosed herein (see, e.g., §5.2), pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, polymorphs thereof, and stereochemical isomers thereof. The term "KRAS inhibitor" includes both specific KRAS inhibitors and pan-KRAS inhibitors.

[0130] As used herein, the term "specifically inhibit" when used in reference to a KRAS inhibitor refers to the ability of the KRAS inhibitor to preferentially, negatively modulate or completely inhibit all or a portion of the enzymatic activity of one or more defined KRAS (e.g., hKRAS) proteins (e.g., one or more defined KRAS variants (e.g., KRAS variant G12C)) compared to other KRAS proteins (e.g., reference KRAS (e.g., wild-type KRAS), other KRAS variants). For example, a KRAS inhibitor that can specifically inhibit KRAS G12C refers to an hKRAS inhibitor that can preferentially, negatively modulate or completely inhibit all or a portion of the enzymatic activity of hKRAS G12C compared to, for example, a reference KRAS (e.g., wild-type KRAS (e.g., hKRAS comprising the amino acid sequence set set forth in SEQ ID NO: 1 or 3)). In some embodiments, a particular KRAS inhibitor may be designated by KRAS, followed by the variant that the KRAS inhibitor specifically inhibits, followed by "inhibitor" or "i." For example, a KRAS inhibitor that is selective for KRAS variants containing a G12C substitution may be referred to herein as a "KRAS-G12Ci" or "KRAS-G12C inhibitor," etc. (See, e.g., Examples 9-11).

[0131] As used herein, the term "KRAS pathway" refers to the signaling pathway and downstream effects initiated by active KRAS (eg, GTP-bound KRAS).

[0132] As used herein, the term "isolated" with respect to a polypeptide, protein, or polynucleotide refers to a polypeptide, protein, or polynucleotide that is substantially free from other cellular components with which it is naturally associated.

[0133] As used herein, the term "KRAS variant" or "variant KRAS" refers to a KRAS protein or polypeptide that comprises at least one amino acid modification (e.g., amino acid substitution) compared to a reference (e.g., wild-type (e.g., SEQ ID NO: 1 or 3)) KRAS protein or polypeptide. In some embodiments, the at least one amino acid modification is an amino acid substitution. In some embodiments, the KRAS variant exhibits increased activity. In some embodiments, the KRAS variant is constitutively active. Exemplary KRAS variants include KRAS proteins or polypeptides that comprise the amino acid sequence of SEQ ID NO: 1 or 3 and also contain a G12C, G12V, G12R, G12D, G12A, G13D, or Q61H amino acid substitution (amino acid numbering relative to SEQ ID NO: 1).

[0134] As used herein, the term "KRAS-activating amino acid modification" refers to an amino acid modification in the amino acid sequence of a KRAS protein relative to the amino acid sequence of a reference (e.g., wild-type (e.g., SEQ ID NO: 1 or 3)) KRAS protein or polypeptide, which amino acid modification locks the KRAS protein in a constitutively active state.

[0135] As used herein, the term "KRAS variant cancer" refers to a cancer that is determined to involve, be mediated by, or express a KRAS variant.A non-limiting example of a KRAS variant cancer is a cancer that is determined to express KRAS G12C (i.e., a cancer that is determined to express a KRAS protein or polypeptide that comprises the amino acid sequence of SEQ ID NO: 1 or 3 and also contains a G12C amino acid substitution (amino acid numbering based on SEQ ID NO: 1)).

[0136] As used herein, with respect to a polynucleotide, the term "modified" refers to a polynucleotide that contains at least one substitution, alteration, inversion, addition, or deletion of a nucleotide (e.g., one or more amino acid substitutions) compared to a reference polynucleotide. A modification may include the incorporation of a non-naturally occurring nucleotide residue. As used herein, with respect to an amino acid sequence, the term "modified" refers to an amino acid sequence that contains at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference amino acid sequence. A modification may include the incorporation of a non-naturally occurring amino acid residue. Naturally occurring amino acid derivatives are not considered modified amino acids for the 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 would not be considered an amino acid modification for the 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.

[0137] As used herein, the term "substructure" is used generally to describe any macromolecule or small molecule that can be incorporated into the fusion proteins or polypeptides described herein. Substructures of a fusion protein or polypeptide are operably connected. Exemplary substructures include, but are not limited to, proteins, polypeptides, polynucleotides (e.g., DNA, RNA), small molecules, carbohydrates, lipids, and synthetic polymers (e.g., polymers of PEG). In some embodiments, the substructure is a polypeptide. In some embodiments, the substructure is a protein.

[0138] As used herein, the term "operably connected" refers to the linkage of two moieties in a functional relationship. For example, a polypeptide is operably connected to another polypeptide when they are linked in-frame (directly or indirectly via a peptide linker) such that both polypeptides are functional (e.g., a fusion protein or polypeptide described herein). Or, for example, a transcriptional regulatory polynucleotide, such as a promoter, enhancer, or other expression control element, is operably linked to a polynucleotide encoding a protein if it affects the transcription of the polynucleotide encoding the protein. The term "operably connected" can also refer to the conjugation of a moiety, for example, to a polynucleotide or polypeptide (e.g., the conjugation of a PEG polymer to a protein or polypeptide).

[0139] The determination of "percent identity" between two sequences (e.g., peptides or proteins (amino acid sequences) or polynucleotides (nucleic acid sequences)) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which are incorporated herein by reference in their entirety. BLAST nucleotide searches can be performed with NBLAST nucleotide program parameters set to, for example, score=100 and word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with XBLAST program parameters set to, for example, score=50 and word length=3 to obtain amino acid sequences homologous to the protein molecules described herein. To achieve gapped alignments for comparison, gapped BLAST can be used, as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, the entire contents of which are incorporated herein by reference. Alternatively, PSI BLAST can be used to perform an iterated search to detect distant relationships between molecules (ibid.). When using BLAST, gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 residue mass table, a gap length penalty of 12, and a gap penalty of 4 can be used. 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 exact matches are counted.

[0140] As used herein, the term "pharmaceutical composition" refers to a composition suitable for administration to an animal, e.g., a human subject, comprising a therapeutic agent and a pharmaceutically acceptable carrier or diluent. A "pharmaceutically acceptable carrier or diluent" refers to a substance intended for use in contact with the tissues of humans and / or non-human animals, without undue toxicity, irritation, allergic response, or other problem or complication, consistent with a reasonable therapeutic benefit / risk ratio.

[0141] As used herein, the term "pharmaceutically acceptable salt" generally has its art-recognized meaning and refers to a derivative of a compound provided herein in which the parent compound has been modified by converting an existing acid or basic moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts of the compounds provided herein include conventional non-toxic salts of the parent compound, formed, for example, from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the compounds provided herein can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by combining the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water, an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), the entire contents of each of these references being incorporated herein by reference for all purposes.

[0142] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of DNA or RNA. Nucleic acid molecules can be single- or double-stranded; can contain natural, non-natural, or modified nucleotides; and can contain natural internucleotide linkages, including non-natural or modified internucleotide linkages, e.g., phosphoramidate or phosphorothioate linkages, rather than the phosphodiesters found between nucleotides in unmodified nucleic acid molecules. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules obtained by any means available in the art, including, but not limited to, recombinant means using conventional cloning techniques and the polymerase chain reaction, such as cloning nucleic acid molecules from recombinant libraries or cellular genomes, as well as by synthetic means. Unless otherwise noted, nucleic acid sequences presented herein will reference thymidine (T) for representative DNA sequences; however, those skilled in the art will understand that thymidine (T) will be substituted for uracil (U) when the sequences represent RNA (e.g., mRNA). Thus, any RNA polynucleotide encoded by a DNA identified by a particular sequence identification number may also include the corresponding RNA (e.g., mRNA) sequence encoded by that DNA in which each thymidine (T) in the DNA sequence is replaced with uracil (U).

[0143] As used herein, the term "polypeptide" refers to a polymer of at least two (e.g., at least five) amino acids linked by peptide bonds. The term "polypeptide" does not indicate a specific length of the polymer chain of amino acids. It is common in the art to refer to shorter polymers of amino acids (e.g., approximately 2 to 50 amino acids) as peptides and longer polymers of amino acids (e.g., approximately more than 50 amino acids) as polypeptides. However, the terms "peptide" and "polypeptide" are used interchangeably herein.

[0144] As used herein, the term "protein" refers to a polypeptide or a set (i.e., at least two) polypeptides. In embodiments where a protein comprises a set of polypeptides, the set of polypeptides associate to form a functional unit (i.e., a quaternary structure). In some embodiments, a polypeptide or set of polypeptides folds into their three-dimensional structure (i.e., a tertiary or quaternary structure). It should be understood that where a polypeptide or set of polypeptides is contemplated herein, a protein comprising the polypeptide or set of polypeptides folded into their three-dimensional structure (i.e., a tertiary or quaternary structure) is also provided herein, and conversely, where a protein comprising a polypeptide or set of polypeptides folded into their three-dimensional structure (i.e., a tertiary or quaternary structure) is contemplated herein, the polypeptide or set of polypeptides is also provided herein.

[0145] The terms "RNA" and "polyribonucleotide" are used interchangeably herein and refer to a macromolecule that comprises multiple ribonucleotides polymerized through phosphodiester bonds.Ribonucleotide is a nucleotide whose sugar is ribose.RNA may contain modified nucleotides, may contain natural internucleotide linkages, and may contain non-natural or modified internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of the phosphodiester found between the nucleotides of unmodified nucleic acid molecules.

[0146] As used herein, the term "collecting a sample" refers to obtaining a sample. This term includes direct obtaining from a subject and indirect obtaining by one or more third parties, one of whom directly obtains the sample from the subject.

[0147] As used herein, the term "sample" encompasses various biological specimens obtained from a subject. Exemplary sample types include, for example, solid tissue samples, such as biopsies (or cells obtained therefrom and the progeny of these cells), tissue cultures (or cells obtained therefrom and the progeny of these cells), and cell cultures (or cells obtained therefrom and the progeny of these cells); blood and other liquid samples of biological origin (including, but not limited to, whole blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, saliva, amniotic fluid, feces, synovial fluid, etc.); and nasopharyngeal swabs. The term also includes samples that have been manipulated in some way after receipt from a subject, for example, by centrifugation, filtration, washing, precipitation, dialysis, chromatography, lysis, treatment with reagents, enrichment for certain cell populations, cooling, freezing, staining, etc. In some embodiments, the sample contains cancer cells. In some embodiments, the sample is a tissue sample containing cancer cells.

[0148] The term "scFv" or "single-chain variable fragment," as used herein, refers to an antigen-binding protein or polypeptide comprising a VH operably connected to a VL (e.g., via a peptide linker). In some embodiments, the VH is operably connected to the VL via a peptide linker. The components of an scFv can be in either orientation; for example, an scFv can comprise, from N-terminus to C-terminus, a VH, a peptide linker, and a VL, or can comprise, from N-terminus to C-terminus, a VL, a peptide linker, and a VH.

[0149] The term "(scFv)2" as used herein refers to an antibody comprising a first and a second scFv that are operably connected (e.g., via a peptide linker). The first and second scFvs may specifically bind to the same or different antigens. In some embodiments, the first and second scFvs are operably connected by a peptide linker.

[0150] The term "scFv-Fc," as used herein, refers to an antibody comprising an scFv operably linked to an Fc domain or to a subunit of an Fc domain (e.g., via a peptide linker). In some embodiments, the scFv is operably connected to only the first Fc domain of a first and second Fc domain pair. In some embodiments, the first scFv is operably connected to the first Fc domain and the second scFv is operably connected to the second Fc domain of a first and second Fc domain pair.

[0151] The term "(scFv)2-Fc," as used herein, refers to an (scFv)2 operably linked to an Fc domain or to a subunit of an Fc domain (e.g., via a peptide linker). In some embodiments, the (scFv)2 is operably connected to only the first Fc domain of a first and second Fc domain pair. In some embodiments, the first (scFv)2 is operably connected to the first Fc domain and the second (scFv)2 is operably connected to the second Fc domain of a first and second Fc domain pair.

[0152] As used herein, the term "single domain antibody" or "sdAb" refers to an antibody having a single monomeric variable antibody domain. An sdAb can specifically bind to a particular antigen. A VHH (as defined herein) is an example of an sdAb.

[0153] As used herein, the term "specifically binds" refers to a preferential interaction, i.e., a significantly higher binding affinity, between a first protein (e.g., a ligand) and a second protein (e.g., the ligand's cognate receptor) compared to other amino acid sequences. As used herein, when a first protein or polypeptide is said to "specifically bind" to a second protein or polypeptide, it is understood that the first protein or polypeptide specifically binds to an epitope of the second protein or polypeptide. The term "epitope" refers to the portion of the second protein or polypeptide that the first protein or polypeptide specifically recognizes. The term "specifically binds" includes molecules that are cross-reactive with the same epitope in different species. For example, an antibody or protein that specifically binds to human EGFR may be cross-reactive with EGFR from another species (e.g., cynomolgus monkey, murine, etc.) and still be considered herein to specifically bind to human EGFR.

[0154] As used herein, the term "subject" includes any animal, e.g., a human or other animal. In some embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In some embodiments, the subject is a human. In some embodiments, the method subject is a non-human mammal. In some embodiments, the subject is a non-human mammal, e.g., a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In some embodiments, the subject is a bird, e.g., a member of the avian taxonomic group Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleaognathae (e.g., ostrich, emu), Columbiformes (e.g., pigeon, dove), or Psittaciformes (e.g., parrot).

[0155] As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to the amount of therapeutic agent, when used alone or in combination with another therapeutic agent, that ameliorates a condition, e.g., prevents a subject from developing a disease (or infection); ameliorates symptoms of a disease or infection, e.g., reduces the severity of symptoms of a disease or infection, reduces the frequency or duration of symptoms of a disease or infection, or prolongs the symptom-free period of a disease or infection; prevents or reduces functional impairment or disability caused by a disease or infection; or promotes the regression of a disease (or infection). The ability of a therapeutic agent to ameliorate a condition can be evaluated using various methods known to those skilled in the art, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0156] As used herein, the term "TGFβ" or "transforming growth factor β" refers to a multifunctional 25 kDa dimeric cytokine belonging to the transforming growth factor superfamily. The amino acid sequence of an exemplary reference mature human TGFβ1 (hTGFβ1) protein is set forth in SEQ ID NO: 61, the amino acid sequence of an exemplary reference mature human TGFβ-2 (hTGFβ2) protein is set forth in SEQ ID NO: 63, and the amino acid sequence of an exemplary reference mature human TGFβ-3 (hTGFβ3) protein is set forth in SEQ ID NO: 65.

[0157] As used herein, the term "TGFβRI" or "transforming growth factor beta receptor I" refers to a transmembrane protein having a protein kinase domain that forms a heterodimeric complex with TGFβRII and binds to TGFβ. An exemplary reference amino acid sequence of mature human TGFβRI (hTGFβRI) protein is set forth in SEQ ID NO:67.

[0158] As used herein, the term "TGFβRII" or "transforming growth factor beta receptor II" refers to a transmembrane protein having a protein kinase domain that forms a heterodimeric complex with TGFβRI and binds to TGFβ. An exemplary reference amino acid sequence of mature human TGFβRII (hTGFβRII) protein is set forth in SEQ ID NO:70.

[0159] As used herein, the term "TGFβ pathway" refers to the signaling pathway and downstream effects initiated by the binding of TGFβ to TGFβR (a heterotetramer composed of two TβRI:TβRII heterodimers (e.g., two hTβRI:hTβRII heterodimers)).

[0160] As used herein, the terms "treat," "treating," "treatment," and the like refer to alleviating or ameliorating a disease or infection and / or its associated symptoms, or achieving a desired pharmacological and / or physiological effect. It will be understood that treating a disease or infection does not require, but does not exclude, complete elimination of the disease or its associated symptoms. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely relieves, reduces, eliminates, ameliorates, alleviates, reduces the intensity of, or cures the disease and / or adverse symptoms resulting from the disease. In some embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the occurrence or recurrence of the disease. To accomplish this, the methods of the present disclosure comprise administering a therapeutically effective amount of a composition described herein.

[0161] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0162] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0163] The term "VHH" as used herein refers to a type of single domain antibody (sdAb) having a single monomeric heavy chain variable antibody domain (VH). Such antibodies can be found in or produced by camelid mammals (e.g., camels, llamas) that naturally lack light chains, or can be produced synthetically.

[0164] The term "(VHH)2" as used herein refers to an antibody comprising a first and a second VHH that are operably connected (e.g., via a peptide linker). The first and second VHHs may specifically bind to the same or different antigens. In some embodiments, the first and second VHHs are operably connected by a peptide linker.

[0165] The term "VHH-Fc," as used herein, refers to an antibody comprising a VHH operably linked to an Fc domain or to a subunit of an Fc domain (e.g., via a peptide linker). In some embodiments, the VHH is operably connected to only the first Fc domain of a first and second Fc domain pair. In some embodiments, a first VHH is operably connected to the first Fc domain and a second VHH is operably connected to the second Fc domain of a first Fc and second Fc pair.

[0166] The term "(VHH)2-Fc," as used herein, refers to a (VHH)2 operably linked to an Fc domain or to a subunit of an Fc domain (e.g., via a peptide linker). In some embodiments, the (VHH)2 is operably connected to only the first Fc domain of a first and second Fc domain pair. In some embodiments, the first (VHH)2 is operably connected to the first Fc domain and the second (VHH)2 is operably connected to the second Fc domain of a first Fc and second Fc pair.

[0167] 5.2 KRAS inhibitors Kirsten rat sarcoma viral oncogene homolog (KRAS) is a small guanosine triphosphatase (GTPase) that functions primarily by linking membrane growth factor receptors to intracellular signaling pathways and transcription factors.

[0168] The KRAS protein exists in an inactive conformation primarily bound to guanosine diphosphate (GDP) and maintained by its guanosine triphosphate (GTP) hydrolysis activity. KRAS interacts with GTPase-activating proteins (GAPs), which accelerate GTP conversion to GDP, whereas the binding of guanosine nucleotide exchange factors (GEFs) to KRAS passively loads KRAS with GTP. GTP binding to KRAS shifts the active site from an open to a closed conformation, allowing the interaction and activation of multiple downstream effector pathways, including the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Accumulation of activated KRAS in vivo leads to the activation of downstream signaling pathways and is associated with tumorigenesis, aggressive disease, and poor prognosis. The upstream signaling pathway of KRAS primarily involves cell surface receptors such as EGFR (ERBB1), human epidermal growth factor receptor 2 (HER2 (ERBB2)), HER3 (ERBB3), and ERBB4.

[0169] The KRAS variant that fixes protein in activated state is found in different types of cancer, including but not limited to colon, colon, rectum, lung and pancreatic cancer.The activated state KRAS mutation is more commonly found at G12 and G13 positions (numbering based on SEQ ID NO: 3).Additional KRAS variants associated with cancer are known in the art and are also described herein. For example, Adachi Y, Ito K, Hayashi Y, et al. Epithelial-to-Mesenchymal Transition is a Cause of Both Intrinsic and Acquired Resistance to KRAS G12C Inhibitor in KRAS G12C-Mutant Non-Small Cell Lung Cancer. Clin Cancer Res. 2020;26(22):5962-5973. doi:10.1158 / 1078-0432.CCR-20-2077;Huang, L., Guo, Z., Wang, F. et al. KRAS mutation: from undruggable to druggable in cancer. Sig Transduct Target Ther 6, 386 (2021). https: / / doi.org / 10.1038 / s41392-021-00780-4; and Ryan MB, Coker O, Sorokin A, et al. KRASG12C-independent feedback activation of wild-type RAS constrains KRASG12C inhibitor efficacy. Cell Rep. 2022;39(12):110993. doi:10.1016 / j.celrep.2022.110993, the entire contents of each of which are incorporated herein by reference for all purposes.

[0170] There are two splice isoforms of human KRAS mRNA, resulting in two variants of the KRAS protein. The variant commonly referred to as KRAS isoform B is the major isoform. The amino acid sequences of the reference hKRAS isoform 2A polypeptide and the reference hKRAS isoform 2B are shown in SEQ ID NOs: 1 and 3, respectively. The amino acid sequences of exemplary hKRAS 2A variants G12C, G12V, G12R, G12D, G12A, G12S, G13D, and Q61H are also provided in SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12, respectively. The amino acid sequences of exemplary hKRAS 2B variants G12C, G12V, G12R, G12D, G12A, G12S, G13D, and Q61H are also provided in SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, and 20, respectively. See Table 1 herein.

[0171] Both isoforms of KRAS contain an N-terminal initiation methionine. It is known in the art that KRAS can be further processed by cells, resulting in the cleavage of the N-terminal initiation methionine. Dharmaiah, S., Tran, TH, Messing, S. et al. Structures of N-terminally processed KRAS provide insight into the role of N-acetylation. Sci Rep 9, 10512 (2019). https: / / doi.org / 10.1038 / s41598-019-46846-w, the entire contents of which are incorporated herein by reference for all purposes. The amino acid sequences of the reference hKRAS isoform 2A polypeptide without an initiation methionine and the reference hKRAS isoform 2B polypeptide without an initiation methionine are set forth in SEQ ID NOs: 2 and 4, respectively. The amino acid sequences of exemplary hKRAS 2A variants G12C, G12V, G12R, G12D, G12A, G12S, G13D, and Q61H, which do not have a starting methionine, are provided in SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, and 28, respectively. The amino acid sequences of exemplary hKRAS 2B variants G12C, G12V, G12R, G12D, G12A, G12S, G13D, and Q61H, which do not have a starting methionine, are provided in SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, and 36, respectively. See Table 1 herein.

[0172] Unless otherwise noted, amino acid numbering in KRAS refers to the KRAS isoform containing the initiating methionine (ie, SEQ ID NO: 1 - hKRAS isoform 2A; SEQ ID NO: 3 - hKRAS isoform 2B).

[0173] [Table 1-1]

[0174] [Table 1-2]

[0175] [Table 1-3]

[0176] [Table 1-4]

[0177] [Table 1-5]

[0178] In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit a KRAS (e.g., hKRAS) variant. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit more than one KRAS (e.g., hKRAS) variant. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit at least one KRAS (e.g., hKRAS) variant. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit multiple KRAS variants.

[0179] In some embodiments, a KRAS (eg, hKRAS) inhibitor can inhibit a KRAS variant that includes a KRAS-activating amino acid modification.

[0180] In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit a KRAS (e.g., hKRAS) variant comprising an amino acid substitution at amino acid position G12, G13, or Q61, numbered relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit a KRAS (e.g., hKRAS) variant comprising an amino acid substitution at amino acid position G12 or G13, numbered relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit a KRAS (e.g., hKRAS) variant comprising an amino acid substitution at amino acid position G12, numbered relative to the amino acid sequence of SEQ ID NO: 1.

[0181] In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit KRAS (e.g., hKRAS) variants that include one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 1: G12C, G12V, G12R, G12D, G12A, G12S, G13D, or Q61H. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit KRAS (e.g., hKRAS) variants that include one of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 1: G12C, G12V, G12R, G12D, G12S, G12A, or G13D. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit a KRAS (e.g., hKRAS) variant that includes one of the following amino acid substitutions, G12C, G12V, G12R, G12D, G12S, or G12A, numbered relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can inhibit a KRAS (e.g., hKRAS) variant that includes a G12C amino acid substitution, numbered relative to the amino acid sequence of SEQ ID NO: 1.

[0182] In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit a KRAS (e.g., hKRAS) variant. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit more than one KRAS (e.g., hKRAS) variant. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit at least one KRAS (e.g., hKRAS) variant. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit multiple KRAS variants.

[0183] In some embodiments, a KRAS (eg, hKRAS) inhibitor can selectively inhibit KRAS variants that contain KRAS-activating amino acid modifications.

[0184] In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit KRAS (e.g., hKRAS) variants comprising an amino acid substitution at amino acid position G12, G13, or Q61, numbered relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit KRAS (e.g., hKRAS) variants comprising an amino acid substitution at amino acid position G12 or G13, numbered relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit KRAS (e.g., hKRAS) variants comprising an amino acid substitution at amino acid position G12, numbered relative to the amino acid sequence of SEQ ID NO: 1.

[0185] In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit KRAS (e.g., hKRAS) variants that include one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 1: G12C, G12V, G12R, G12D, G12A, G12S, G13D, or Q61H. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit KRAS (e.g., hKRAS) variants that include one of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 1: G12C, G12V, G12R, G12D, G12S, G12A, or G13D. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit KRAS (e.g., hKRAS) variants that include one of the following amino acid substitutions, G12C, G12V, G12R, G12D, G12S, or G12A, numbered relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the KRAS (e.g., hKRAS) inhibitor can selectively inhibit KRAS (e.g., hKRAS) variants that include a G12C amino acid substitution, numbered relative to the amino acid sequence of SEQ ID NO: 1.

[0186] In some embodiments, the KRAS (eg, hKRAS) variant comprises a KRAS (eg, hKRAS)-activating amino acid modification.

[0187] In some embodiments, the KRAS (e.g., hKRAS) variant comprises an amino acid modification at amino acid positions G12, G13, or Q61, numbered relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the KRAS (e.g., hKRAS) variant comprises an amino acid modification at amino acid positions G12 or G13, numbered relative to the amino acid sequence of SEQ ID NO: 1.

[0188] In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 1: G12C, G12V, G12R, G12D, G12A, G12S, G13D, or Q61H. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 1: G12C, G12V, G12R, G12D, G12A, G12S, or G13D. In some embodiments, the KRAS (e.g., hKRAS) variant comprises any one of the following amino acid substitutions, numbered with reference to the amino acid sequence of SEQ ID NO: 1: G12C, G12V, G12R, G12A, G12S, or G12D. In some embodiments, the KRAS (eg, hKRAS) variant comprises a G12C amino acid substitution, numbered based on the amino acid sequence of SEQ ID NO:1.

[0189] In some embodiments, the methods, pharmaceutical compositions, kits, etc. described herein utilize or include a KRAS (e.g., hKRAS) inhibitor. KRAS (e.g., hKRAS) inhibitors include, but are not limited to, small molecules, proteins (e.g., antibodies and functional fragments or variants thereof, such as full-length antibodies, single-chain variable fragments (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v)), nucleic acids, carbohydrates, lipids, metals, or toxins. KRAS inhibitors are known in the art, and any suitable KRAS (e.g., hKRAS) inhibitor may be utilized in the aspects and embodiments described herein. In some embodiments, the KRAS (e.g., hKRAS) inhibitor is a small molecule.

[0190] In some embodiments, the KRAS (e.g., hKRAS) inhibitor is / includes sotorasib (AMG-510), MRTX849, ARS-1620, GDC-6036, LY3499446, JAB-21822, GFH925, JDQ443, HBI-2438, or YL-15293. In some embodiments, the KRAS inhibitor comprises sotorasib (AMG-510), GDC-6036, LY3499446, JAB-21822, GFH925, JDQ443, HBI-2438, YL-15293, sotorasib (AMG-510), adagrasib (MRTX849), garsolasib, MRTX-1257, D-1553, BI-1823911, ARS-853, ARS-1620, HS-10370, MK-1084, BPI-421286, or GH35. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises sotorasib (AMG-510), adagrasib (MRTX849), or ARS-1620.

[0191] In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises sotorasib (AMG-510), whose chemical name is 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-(1M)-1-[4-methyl-2-(propan-2-yl)pyridin-3-yl]-4-[(2S)-2-methyl-4-(prop-2enoyl)piperazin-1-yl]pyrido[2,3-d]pyrimidin-2(1H)-one.

[0192] Additional exemplary KRAS (e.g., hKRAS) inhibitors include JNJ-74699157 (ARS-3248) (Janssen Research & Development, LLC), LY3537982 (Eli Lilly and Company), D-1553 (InventisBio Inc.), BI 1823911 (Boehringer Ingelheim), JAB21822 (Jacobio Pharmaceuticals Co., Ltd.), GFH925 (Genfleet Therapeutics Co., Ltd.), HS-10370 (Jiangsu Hansoh Pharmaceutical Co., Ltd.), MK-1084 (Merck Sharp & Dohme LLC), BPI-421286 (Betta Pharmaceuticals Co., Ltd.), GH35 (Suzhou Genhouse Bio Co., Ltd.), D3S-001 (D3 Bio(Wuxi) Co., Ltd.), IBI-351 (Innovent Biologics, Inc.), RMC-6291 (Revolution Medicines), HBI2438 (Huyabio International, LLC), YL15293 (YingLi Pharmaceutical Co. Ltd.), JAB-22000 (Jacobio Pharmaceuticals Co., Ltd.), HRS-4642 (Jiangsu Hengrui Medicine), ASP-3082 (Astellas Pharma), RMC-6236 (Revolution Medicines), or BI-1701963 (Boehringer Ingelheim).

[0193] Exemplary hKRAS inhibitors are provided in Table 2. The hKRAS inhibitors are intended to be merely exemplary and not limiting. See Table 2 herein.

[0194] [Table 2-1]

[0195] Table 2-2

[0196] Table 2-3

[0197] Table 2-4

[0198] Table 2-5

[0199] Table 2-6

[0200] Table 2-7

[0201] Table 2-8

[0202] Table 2-9

[0203] Table 2-10

[0204] Table 2-11

[0205] Table 2-12

[0206] Table 2-13

[0207] Table 2-14

[0208] Table 2-15

[0209] Table 2-16

[0210] Table 2-17

[0211] Table 2-18

[0212] Table 2-19

[0213] Table 2-20

[0214] Table 2-21

[0215] Table 2-22

[0216] Table 2-23

[0217] Table 2-24

[0218] Table 2-25

[0219] Table 2-26

[0220] Table 2-27

[0221] Table 2-28

[0222] Table 2-29

[0223] Table 2-30

[0224] Table 2-31

[0225] Table 2-32

[0226] Table 2-33

[0227] Table 2-34

[0228] Table 2-35

[0229] In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that is capable of inhibiting the KRAS (e.g., hKRAS) variant G12C. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that is capable of inhibiting the KRAS (e.g., hKRAS) variant G12D. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that is capable of inhibiting the KRAS (e.g., hKRAS) variant G12S. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that is capable of inhibiting the KRAS (e.g., hKRAS) variant G12V. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that is capable of inhibiting the KRAS (e.g., hKRAS) variant G12R. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that is capable of inhibiting the KRAS (e.g., hKRAS) variant Q61H. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that is capable of inhibiting SOS1.

[0230] In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that can selectively inhibit the KRAS (e.g., hKRAS) variant G12C. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that can selectively inhibit the KRAS (e.g., hKRAS) variant G12D. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that can selectively inhibit the KRAS (e.g., hKRAS) variant G12S. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that can selectively inhibit the KRAS (e.g., hKRAS) variant G12V. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that can selectively inhibit the KRAS (e.g., hKRAS) variant G12R. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that can selectively inhibit the KRAS (e.g., hKRAS) variant Q61H. In some embodiments, the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor shown in Table 2 that can selectively inhibit SOS1.

[0231] In some embodiments, the KRAS (e.g., hKRAS) inhibitor is any one or more of the compounds disclosed herein (see, e.g., §5.2), their pharmaceutically acceptable salts, their hydrates, their solvates, their polymorphs, and their stereochemical isomers.

[0232] Exemplary salts include, but are not limited to, salts derived from such inorganic bases, such as aluminum, ammonium, calcium, copper (II and I), ferric, ferrous, lithium, magnesium, manganese (II and I), potassium, sodium, zinc, and the like. Organic bases include salts of primary, secondary, and tertiary amines derived from both naturally occurring and synthetic sources. Exemplary organic bases include ammonia, methylamine, ethylamine, propylamine, isopropylamine, butylamine isomers, betaine, caffeine, choline, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, N,N'-dibenzylethylenediamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, tromethamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, quinuclidine, pyridine, quinoline, and isoquinoline; benzathine, N-methyl-D-glucamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, hydrabamine salts, and salts with amino acids such as histidine, arginine, and lysine.

[0233] In some embodiments, KRAS (e.g., hKRAS) inhibitors exist as solvates. In some embodiments, the solvent used to prepare solvates is an aqueous solution, and therefore solvates are often referred to as hydrates. KRAS (e.g., hKRAS) inhibitors can exist as hydrates, and this hydrate can be obtained, for example, by crystallization from a solvent or from an aqueous solution. In this regard, one, two, three, or any number of solvent or water molecules can be combined with KRAS (e.g., hKRAS) inhibitors to form solvates and hydrates.

[0234] 5.3 EGFR binding substructure Epidermal growth factor receptor (EGFR) is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands.Activation of EGFR (e.g., human EGFR (hEGFR)) leads to the activation of several downstream signaling proteins, which initiate various signaling cascades, including MAPK, Akt and JNK pathways, leading to DNA synthesis, cell proliferation, cell migration and adhesion.Some cancers (e.g., human cancers) are accompanied by overexpression of EGFR (e.g., hEGFR) compared with non-cancer cells, resulting in, for example, overactivation of EGFR (e.g., hEGFR), and increased cell proliferation and growth.

[0235] The amino acid sequences of the reference mature and immature hEGFR polypeptides are set forth in SEQ ID NOs: 37 and 38, respectively. See Table 3 herein.

[0236] [Table 3]

[0237] Some aspects and embodiments described herein utilize a substructure that specifically binds to EGFR (e.g., hEGFR) (also referred to herein as an EGFR-binding substructure, an EGFR-binding domain, or an anti-EGFR substructure or domain). For example, in some aspects and embodiments, a fusion protein (e.g., a fusion protein described herein) includes a substructure that specifically binds to EGFR (e.g., hEGFR).

[0238] In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises an antibody, or a functional fragment or functional variant thereof. In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises a full-length antibody, scFv, (scFv)2, scFv-Fc, (scFv)2-Fc, Fab, Fab', F(ab')2, F(v), a single domain antibody, a single-chain antibody, VHH, (VHH)2, VHH-Fc, or (VHH)2-Fc. In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises a full-length antibody, scFv, Fab, or VHH. In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises a full-length antibody, a single-chain variable fragment (scFv), Fab, or a single-domain antibody (sdAb). In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises a full-length antibody.

[0239] In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises cetuximab (or a functional variant or fragment thereof). In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises panitumumab (or a functional variant or fragment thereof). In some embodiments, the EGFR (e.g., hEGFR) binding substructure cross-competes with cetuximab. In some embodiments, the EGFR (e.g., hEGFR) binding substructure specifically binds to the same epitope as cetuximab. In some embodiments, the amino acid sequences of the VH and VL CDRs of the EGFR (e.g., hEGFR) binding substructure are identical to the amino acid sequences of the VH and VL CDRs of cetuximab; or one or more of the VH and VL CDRs of the EGFR (e.g., hEGFR) binding substructure comprise or consist of one, two, or three amino acid modifications compared to the VH and VL CDRs of cetuximab.

[0240] In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises panitumumab (or a functional variant or fragment thereof). In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises panitumumab (or a functional variant or fragment thereof). In some embodiments, the EGFR (e.g., hEGFR) binding substructure cross-competes with panitumumab. In some embodiments, the EGFR (e.g., hEGFR) binding substructure specifically binds to the same epitope as panitumumab. In some embodiments, the amino acid sequences of the VH and VL CDRs of the EGFR (e.g., hEGFR) binding substructure are identical to the amino acid sequences of the VH and VL CDRs of panitumumab; or one or more of the VH and VL CDRs of the EGFR (e.g., hEGFR) binding substructure comprise or consist of one, two, or three amino acid modifications compared to the VH and VL CDRs of panitumumab.

[0241] The amino acid sequences of exemplary EGFR (eg, hEGFR) binding substructures (and their components) are provided in Table 4. CDRs are defined according to Kabat.

[0242] [Table 4-1]

[0243] [Table 4-2]

[0244] In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises an EGFR (e.g., hEGFR) binding domain provided in Table 4. In some embodiments, the amino acid sequence of the EGFR (e.g., hEGFR) binding substructure comprises the amino acid sequence of one or more EGFR (e.g., hEGFR) binding substructure components provided in Table 4. In some embodiments, the amino acid sequence of one or more components of the EGFR (e.g., hEGFR) binding substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of one or more components of the EGFR (e.g., hEGFR) binding substructure in Table 4.

[0245] In some embodiments, the EGFR binding substructure comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3.

[0246] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 of the VH shown in Table 4, or the amino acid sequence of VH CDR1 of the VH shown in Table 4 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 of the VH shown in Table 4, or the amino acid sequence of VH CDR2 of the VH shown in Table 4 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 of the VH shown in Table 4, or the amino acid sequence of VH CDR3 of the VH shown in Table 4 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0247] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 of the VH shown in Table 4, or the amino acid sequence of VH CDR1 of the VH shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 of the VH shown in Table 4, or the amino acid sequence of VH CDR2 of the VH shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 of the VH shown in Table 4, or the amino acid sequence of VH CDR3 of the VH shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0248] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0249] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0250] In some embodiments, the EGFR-binding substructure comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0251] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 of the VL shown in Table 4, or the amino acid sequence of the VL CDR1 of the VL shown in Table 4 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of the VL shown in Table 4, or the amino acid sequence of the VL CDR2 of the VL shown in Table 4 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of the VL shown in Table 4, or the amino acid sequence of the VL CDR3 of the VL shown in Table 4 comprising or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0252] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 of the VL shown in Table 4, or the amino acid sequence of the VL CDR1 of the VL shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of the VL shown in Table 4, or the amino acid sequence of the VL CDR2 of the VL shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of the VL shown in Table 4, or the amino acid sequence of the VL CDR3 of the VL shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0253] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 shown in Table 4, or the amino acid sequence of the VL CDR1 shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 shown in Table 4, or the amino acid sequence of the VL CDR2 shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 shown in Table 4, or the amino acid sequence of the VL CDR3 shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0254] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 shown in Table 4, or the amino acid sequence of the VL CDR1 shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 shown in Table 4, or the amino acid sequence of the VL CDR2 shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 shown in Table 4, or the amino acid sequence of the VL CDR3 shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0255] In some embodiments, the EGFR binding substructure comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0256] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 of the VH shown in Table 4, or the amino acid sequence of VH CDR1 of the VH shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 of the VH shown in Table 4, or the amino acid sequence of VH CDR2 of the VH shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 of the VH shown in Table 4, or the amino acid sequence of VH CDR3 of the VH shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of VL CDR1 of the VL shown in Table 4. the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence of the VL CDR2 of the VL shown in Table 4 or the amino acid sequence of the VL CDR2 of the VL shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitution, deletion, addition, etc.); the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence of the VL CDR3 of the VL shown in Table 4 or the amino acid sequence of the VL CDR3 of the VL shown in Table 4 containing or consisting of one, two or three amino acid modifications (e.g., substitution, deletion, addition, etc.).

[0257] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence of VH CDR1 shown in Table 4, or the amino acid sequence of VH CDR1 shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence of VH CDR2 shown in Table 4, or the amino acid sequence of VH CDR2 shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence of VH CDR3 shown in Table 4, or the amino acid sequence of VH CDR3 shown in Table 4 containing or consisting of one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises or consists of the amino acid sequence of VL CDR1 shown in Table 4. the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence of the VL CDR1 shown in Table 4 or the amino acid sequence of the VL CDR2 shown in Table 4 or the amino acid sequence of the VL CDR2 shown in Table 4 or the amino acid sequence of the VL CDR2 shown in Table 4 or the amino acid sequence of the VL CDR2 shown in Table 4 or the amino acid sequence of the VL CDR2 shown in Table 4 or the amino acid sequence of the VL CDR3 shown in Table 4 or the amino acid sequence of the VL CDR3 shown in Table 4 or the amino acid sequence of the VL CDR3 shown in Table 4 or the amino acid sequence of the VL CDR3 shown in Table 4 or the amino acid sequence of the VL CDR3 shown in Table 4 or the amino acid sequence of the VL CDR3 shown in Table 4

[0258] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of a VH shown in Table 4. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of a VL shown in Table 4. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the VH shown in Table 4; and the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of the VL shown in Table 4.

[0259] In some embodiments, an EGFR (e.g., hEGFR) binding substructure comprises a heavy chain (HC) comprising or 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 a heavy chain in Table 4. In some embodiments, an EGFR (e.g., hEGFR) binding substructure comprises a light chain (LC) comprising or 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 a heavy chain in Table 4. In some embodiments, the EGFR (e.g., hEGFR) binding substructure comprises a heavy chain (HC) comprising or 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 the heavy chain in Table 4; and a light chain comprising or 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 the heavy chain in Table 4.

[0260] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 39, or the amino acid sequence set forth in SEQ ID NO: 39 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 40, or the amino acid sequence set forth in SEQ ID NO: 40 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 41, or the amino acid sequence set forth in SEQ ID NO: 41 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0261] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 39, or the amino acid sequence set forth in SEQ ID NO: 39 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 40, or the amino acid sequence set forth in SEQ ID NO: 40 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 41, or the amino acid sequence set forth in SEQ ID NO: 41 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0262] In some embodiments, the amino acid sequence of the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 42, or the amino acid sequence set forth in SEQ ID NO: 42 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 43, or the amino acid sequence set forth in SEQ ID NO: 43 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 44, or the amino acid sequence set forth in SEQ ID NO: 44 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0263] In some embodiments, the amino acid sequence of the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 42, or the amino acid sequence set forth in SEQ ID NO: 42 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 43, or the amino acid sequence set forth in SEQ ID NO: 43 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 44, or the amino acid sequence set forth in SEQ ID NO: 44 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0264] In some embodiments, the amino acid sequence of the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 39, or the amino acid sequence set forth in SEQ ID NO: 39 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 40, or the amino acid sequence set forth in SEQ ID NO: 40 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 41, or the amino acid sequence set forth in SEQ ID NO: 41 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 42, or the amino acid sequence set forth in SEQ ID NO: 42 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 43, or the amino acid sequence set forth in SEQ ID NO: 43 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 44, or the amino acid sequence set forth in SEQ ID NO: 44 containing one, two or three amino acid alterations (eg, substitutions, deletions, additions, etc.).

[0265] In some embodiments, the amino acid sequence of the VH comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the amino acid sequence of the VL comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the amino acid sequence of the VH comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:45; and the amino acid sequence of the VL comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:46.

[0266] In some embodiments, the amino acid sequence of the heavy chain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the amino acid sequence of the heavy chain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the amino acid sequence of the light chain comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the amino acid sequence of the heavy chain comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:47; and the amino acid sequence of the light chain comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the amino acid sequence of the heavy chain comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:48; and the amino acid sequence of the light chain comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:49.

[0267] In some embodiments, the amino acid sequence of VH CDR1 consists of the amino acid sequence set forth in SEQ ID NO: 39, or the amino acid sequence set forth in SEQ ID NO: 39 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 consists of the amino acid sequence set forth in SEQ ID NO: 40, or the amino acid sequence set forth in SEQ ID NO: 40 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 consists of the amino acid sequence set forth in SEQ ID NO: 41, or the amino acid sequence set forth in SEQ ID NO: 41 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0268] In some embodiments, the amino acid sequence of VH CDR1 consists of the amino acid sequence set forth in SEQ ID NO: 39, or the amino acid sequence set forth in SEQ ID NO: 39 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 consists of the amino acid sequence set forth in SEQ ID NO: 40, or the amino acid sequence set forth in SEQ ID NO: 40 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 consists of the amino acid sequence set forth in SEQ ID NO: 41, or the amino acid sequence set forth in SEQ ID NO: 41 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0269] In some embodiments, the amino acid sequence of the VL CDR1 consists of the amino acid sequence set forth in SEQ ID NO: 42, or the amino acid sequence set forth in SEQ ID NO: 42 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 consists of the amino acid sequence set forth in SEQ ID NO: 43, or the amino acid sequence set forth in SEQ ID NO: 43 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 consists of the amino acid sequence set forth in SEQ ID NO: 44, or the amino acid sequence set forth in SEQ ID NO: 44 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0270] In some embodiments, the amino acid sequence of the VL CDR1 consists of the amino acid sequence set forth in SEQ ID NO: 42, or the amino acid sequence set forth in SEQ ID NO: 42 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 consists of the amino acid sequence set forth in SEQ ID NO: 43, or the amino acid sequence set forth in SEQ ID NO: 43 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 consists of the amino acid sequence set forth in SEQ ID NO: 44, or the amino acid sequence set forth in SEQ ID NO: 44 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0271] In some embodiments, the amino acid sequence of the VH CDR1 consists of the amino acid sequence set forth in SEQ ID NO: 39, or the amino acid sequence set forth in SEQ ID NO: 39 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 consists of the amino acid sequence set forth in SEQ ID NO: 40, or the amino acid sequence set forth in SEQ ID NO: 40 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR3 consists of the amino acid sequence set forth in SEQ ID NO: 41, or the amino acid sequence set forth in SEQ ID NO: 41 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR1 consists of the amino acid sequence set forth in SEQ ID NO: 42, or the amino acid sequence set forth in SEQ ID NO: 42 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR2 consists of the amino acid sequence set forth in SEQ ID NO: 43 or the amino acid sequence set forth in SEQ ID NO: 43 containing one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR3 consists of the amino acid sequence set forth in SEQ ID NO: 44 or the amino acid sequence set forth in SEQ ID NO: 44 containing one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0272] In some embodiments, the amino acid sequence of the VH consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the amino acid sequence of the VL consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the amino acid sequence of the VH consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:45; and the amino acid sequence of the VL consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:46.

[0273] In some embodiments, the amino acid sequence of the heavy chain consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the amino acid sequence of the heavy chain consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the amino acid sequence of the light chain consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the amino acid sequence of the heavy chain consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:47; and the amino acid sequence of the light chain consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the amino acid sequence of the heavy chain consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:48; and the amino acid sequence of the light chain consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:49.

[0274] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50, or the amino acid sequence set forth in SEQ ID NO: 50 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 51, or the amino acid sequence set forth in SEQ ID NO: 51 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 52, or the amino acid sequence set forth in SEQ ID NO: 52 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0275] In some embodiments, the amino acid sequence of VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50, or the amino acid sequence set forth in SEQ ID NO: 50 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 51, or the amino acid sequence set forth in SEQ ID NO: 51 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 52, or the amino acid sequence set forth in SEQ ID NO: 52 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0276] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 53, or the amino acid sequence set forth in SEQ ID NO: 53 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, or the amino acid sequence set forth in SEQ ID NO: 54 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.). In some embodiments, the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55, or the amino acid sequence set forth in SEQ ID NO: 55 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0277] In some embodiments, the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 53, or the amino acid sequence set forth in SEQ ID NO: 53 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, or the amino acid sequence set forth in SEQ ID NO: 54 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of the VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55, or the amino acid sequence set forth in SEQ ID NO: 55 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0278] In some embodiments, the amino acid sequence of the VH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:50, or the amino acid sequence set forth in SEQ ID NO:50 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:51, or the amino acid sequence set forth in SEQ ID NO:51 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:52, or the amino acid sequence set forth in SEQ ID NO:52 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of the VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:53, or the amino acid sequence set forth in SEQ ID NO:53 with one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, or the amino acid sequence set forth in SEQ ID NO: 54 containing one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55, or the amino acid sequence set forth in SEQ ID NO: 55 containing one, two or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0279] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:57. In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:56; and the amino acid sequence of the VL comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:57.

[0280] In some embodiments, the amino acid sequence of the heavy chain comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the amino acid sequence of the heavy chain comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:59. In some embodiments, the amino acid sequence of the light chain comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:60. In some embodiments, the amino acid sequence of the heavy chain comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:58; and the amino acid sequence of the light chain comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:60. In some embodiments, the amino acid sequence of the heavy chain comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:59; and the amino acid sequence of the light chain comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:60.

[0281] 5.4 TGFβ binding substructure Transforming growth factor beta (TGFβ) is a multifunctional dimeric cytokine belonging to the transforming growth factor superfamily. There are three mammalian forms of human TGFβ (hTGFβ), namely, hTGFβ-1, hTGFβ-2, and hTGFβ-3, and each biologically active form is a dimer. hTGFβ signaling is initiated by binding of hTGFβ to its serine and threonine kinase receptors, type I receptor (TGFβRI) and type II receptor (TGFβRII), on the cell membrane. It is known in the art that, in certain circumstances, TGFβ is pro-tumorigenic, for example, playing a role in stimulating matrix deposition, disrupting immune function, and inducing epithelial-mesenchymal transition (EMT).

[0282] The amino acid sequences of the reference mature hTGFβ-1 polypeptide and immature hTGFβ-1 polypeptide are set forth in SEQ ID NOs: 61 and 62, respectively. The amino acid sequences of the reference mature hTGFβ-2 polypeptide and immature hTGFβ-2 polypeptide are set forth in SEQ ID NOs: 63 and 64, respectively. The amino acid sequences of the reference mature hTGFβ-3 polypeptide and immature hTGFβ-3 polypeptide are set forth in SEQ ID NOs: 65 and 66, respectively. The amino acid sequences of the reference mature human TGFβRI (hTGFβRI) polypeptide, immature hTGFβRI polypeptide, and the extracellular domain (ECD) of the reference hTGFβRI polypeptide are set forth in SEQ ID NOs: 67, 68, and 69, respectively. The amino acid sequences of the reference mature human TGFβRII (hTGFβRII) polypeptide, immature hTGFβRII polypeptide, and the ECD of the reference hTGFβRII polypeptide are set forth in SEQ ID NOs: 70, 71, and 72, respectively. See Table 5 herein. In some embodiments, the extracellular domain of hTGFβRII does not include the N-terminal threonine residue (SEQ ID NO: 73).

[0283] [Table 5-1]

[0284] [Table 5-2]

[0285] [Table 5-3]

[0286] Some aspects and embodiments described herein utilize a substructure that specifically binds to TGFβ (e.g., hTGFβ) (also referred to herein as a TGFβ-binding substructure, a TGFβ-binding domain, or an anti-TGFβ-binding domain or substructure). For example, in some aspects and embodiments, a fusion protein (e.g., a fusion protein described herein) includes a substructure that specifically binds to TGFβ (e.g., hTGFβ).

[0287] In some embodiments, the hTGFβ binding substructure specifically binds to at least one of hTGFβ1, hTGFβ2, and hTGFβ3. In some embodiments, the hTGFβ binding substructure specifically binds to hTGFβ1. In some embodiments, the hTGFβ binding substructure specifically binds to hTGFβ2. In some embodiments, the hTGFβ binding substructure specifically binds to hTGFβ3. In some embodiments, the hTGFβ binding substructure specifically binds to hTGFβ1 and hTGFβ2. In some embodiments, the hTGFβ binding substructure specifically binds to hTGFβ1 and hTGFβ3. In some embodiments, the hTGFβ binding substructure specifically binds to hTGFβ2 and hTGFβ3. In some embodiments, the hTGFβ binding substructure specifically binds to hTGFβ1, hTGFβ2, and hTGFβ2.

[0288] In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises an antibody, or a functional fragment or functional variant thereof. In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises a full-length antibody, scFv, (scFv)2, scFv-Fc, (scFv)2-Fc, Fab, Fab', F(ab')2, F(v), a single domain antibody, a single chain antibody, VHH, (VHH)2, VHH-Fc, (VHH)2-Fc. In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises a full-length antibody, scFv, Fab, or VHH. In some embodiments, the EGFR binding substructure comprises a full-length antibody, a single chain variable fragment (scFv), Fab, or single domain antibody (sdAb). In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises a full-length antibody.

[0289] In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of at least a portion of the extracellular domain (ECD) of a transforming growth factor-beta receptor (TGFβR) (e.g., hTGFβR). In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of at least a portion of the ECD of a TGFβR (e.g., hTGFβR), and the TGFβ (e.g., hTGFβ) binding substructure retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of at least a portion of the ECD of a TGFβRII (e.g., hTGFβRII). In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of at least a portion of the ECD of TGFβRII (e.g., hTGFβRII), and the TGFβ (e.g., hTGFβ) binding substructure retains the ability to specifically bind to TGFβ (e.g., hTGFβ).

[0290] In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of a portion of the ECD of a TGFβR (e.g., hTGFβR) sufficient to mediate binding to TGFβ (e.g., hTGFβ). In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of a portion of the ECD of a TGFβRII (e.g., hTGFβRII) sufficient to mediate binding to TGFβ (e.g., hTGFβ). In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of a portion of the ECD of a naturally occurring TGFβR (e.g., hTGFβR) sufficient to mediate binding to TGFβ (e.g., hTGFβ). In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of a portion of the ECD of naturally occurring TGFβRII (e.g., hTGFβRII) sufficient to mediate binding to TGFβ (e.g., hTGFβ).

[0291] In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of at least a portion of the ECD of TGFβRII (e.g., hTGFβRII). In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of at least a portion of the ECD of TGFβRII (e.g., hTGFβRII), and the TGFβ (e.g., hTGFβ) binding substructure retains the ability to specifically bind to TGFβ. In some embodiments, the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of at least a portion of the ECD of TGFβRII (e.g., hTGFβRII).

[0292] In some embodiments, the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) shown in Table 5. In some embodiments, the amino acid sequence of the ECD of a TGFβRII (e.g., hTGFβRII) polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the ECD of a TGFβRII (e.g., hTGFβRII) shown in Table 5. In some embodiments, the ECD of a TGFβR (e.g., hTGFβR) polypeptide comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) shown in Table 5. In some embodiments, the ECD of a TGFβRII (e.g., hTGFβRII) polypeptide comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the ECD of a TGFβRII (e.g., hTGFβRII) shown in Table 5. In some embodiments, the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) polypeptide comprises or consists of the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) shown in Table 5. In some embodiments, the amino acid sequence of the ECD of a TGFβRII (e.g., hTGFβRII) polypeptide comprises or consists of the amino acid sequence of the ECD of a TGFβRII (e.g., hTGFβRII) shown in Table 5.

[0293] In some embodiments, the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the amino acid sequence of the ECD of a TGFβRII (e.g., hTGFβRII) polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the ECD of a TGFβR (e.g., hTGFβR) polypeptide comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the ECD of a TGFβRII (e.g., hTGFβRII) polypeptide comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73.

[0294] In some embodiments, the ECD of a TGFβR (e.g., hTGFβR) polypeptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the ECD of a TGFβRII (e.g., hTGFβRII) polypeptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73.

[0295] In some embodiments, the ECD of a TGFβR (e.g., hTGFβR) polypeptide consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the ECD of a TGFβRII (e.g., hTGFβRII) polypeptide consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73.

[0296] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) shown in Table 5. In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the ECD of a TGFβRII (e.g., hTGFβRII) shown in Table 5.

[0297] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of one or more amino acids (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD, and retains the ability to specifically bind to TGFβ. In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of a TGFβR shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ).

[0298] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of one or more amino acids (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids at the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of a TGFβR (e.g., hTGFβR) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid to the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the additional amino acids correspond to the amino acid sequence of a naturally occurring TGFβR (e.g., hTGFβR).

[0299] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of TGFβRII (e.g., hTGFβRII) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of one or more amino acids (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD, and retains the ability to specifically bind to TGFβ. In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of TGFβRII shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD and retains the ability to specifically bind TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of TGFβRII (e.g., hTGFβRII) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ).

[0300] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of TGFβRII (e.g., hTGFβRII) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of one or more amino acids (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of TGFβRII (e.g., hTGFβRII) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids at the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of the ECD of TGFβRII (e.g., hTGFβRII) shown in Table 5, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid to the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the additional amino acids correspond to the amino acid sequence of a naturally occurring TGFβRII (e.g., hTGFβRII).

[0301] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73.

[0302] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73.

[0303] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73.

[0304] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 73.

[0305] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 72, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of one or more amino acids (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 73, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of one or more amino acids (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ).

[0306] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 72, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acids at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 73, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acids at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ).

[0307] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 72, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD, and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 73, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises a deletion of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the ECD, and retains the ability to specifically bind to TGFβ (e.g., hTGFβ).

[0308] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 72, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of one or more amino acids (e.g., from 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 amino acids) at the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 73, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of one or more amino acids (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ).

[0309] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 72, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids to the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 73, and the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids to the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ).

[0310] In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 72, wherein the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid to the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ). In some embodiments, the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the amino acid sequence of SEQ ID NO: 73, wherein the amino acid sequence of the TGFβ (e.g., hTGFβ) binding substructure comprises the addition of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid to the C-terminus of the ECD and retains the ability to specifically bind to TGFβ (e.g., hTGFβ).

[0311] 5.5 Fusion Proteins and Polypeptides Some aspects and embodiments described herein utilize fusion proteins. The fusion proteins described herein include at least a first substructure (e.g., an EGFR (e.g., an hEGFR)-binding substructure (e.g., an EGFR (e.g., an hEGFR)-binding substructure described herein)) operably linked to a second substructure (e.g., a TGFβ (e.g., an hTGFβ)-binding substructure (e.g., an hTGFβ)-binding substructure described herein)), wherein the second substructure (e.g., a TGFβ (e.g., an hTGFβ)-binding substructure) is heterologous to the first substructure (e.g., an EGFR (e.g., hEGFR)-binding substructure). In some embodiments, the fusion protein comprises at least one EGFR (e.g., hEGFR) binding substructure (e.g., an EGFR (e.g., hEGFR) binding substructure described herein, see e.g., §5.3) operably linked to at least one TGFβ (e.g., hTGFβ) binding substructure (e.g., a TGFβ (e.g., hTGFβ) binding substructure described herein, see e.g., §5.4).

[0312] In some embodiments, the fusion protein comprises or consists of two EGFR (e.g., hEGFR)-binding substructures. In some embodiments, the fusion protein comprises or consists of two TGFβ (e.g., hTGFβ)-binding substructures. In some embodiments, the fusion protein comprises or consists of two EGFR (e.g., hEGFR)-binding substructures and two TGFβ (e.g., hTGFβ)-binding substructures.

[0313] In some embodiments, the fusion protein comprises or consists of one EGFR (e.g., hEGFR)-binding substructure. In some embodiments, the fusion protein comprises or consists of one TGFβ (e.g., hTGFβ)-binding substructure. In some embodiments, the fusion protein comprises or consists of one EGFR (e.g., hEGFR)-binding substructure and one TGFβ (e.g., hTGFβ)-binding substructure.

[0314] In some embodiments, the fusion protein comprises or consists of two EGFR (e.g., hEGFR)-binding substructures. In some embodiments, the fusion protein comprises or consists of two TGFβ (e.g., hTGFβ)-binding substructures. In some embodiments, the fusion protein comprises or consists of two EGFR (e.g., hEGFR)-binding substructures and two TGFβ (e.g., hTGFβ)-binding substructures.

[0315] In some embodiments, the fusion protein comprises or consists of one EGFR (e.g., hEGFR)-binding substructure and two TGFβ (e.g., hTGFβ)-binding substructures. In some embodiments, the fusion protein comprises or consists of two EGFR (e.g., hEGFR)-binding substructures and one TGFβ (e.g., hTGFβ)-binding substructure. In some embodiments, the fusion protein comprises two EGFR (e.g., hEGFR)-binding substructures and two TGFβ (e.g., hTGFβ)-binding substructures.

[0316] In some embodiments, the fusion protein comprises an EGFR-binding substructure that is a full-length antibody and two TGFβ (e.g., hTGFβ)-binding substructures. In some embodiments, the fusion protein comprises an EGFR (e.g., hEGFR)-binding substructure that is a full-length antibody and two TGFβ (e.g., hTGFβ)-binding substructures, wherein one of the two TGFβ (e.g., hTGFβ)-binding substructures is operably linked to one light chain of the full-length antibody and a second TGFβ (e.g., hTGFβ)-binding substructure is operably linked to a second light chain of the full-length antibody.

[0317] In some embodiments, the fusion protein comprises: (a) a first partial structure comprising or consisting of a full-length antibody that specifically binds to EGFR (e.g., hEGFR); (b) a second partial structure comprising or consisting of the ECD of TGFβRII (e.g., hTGFβRII); (c) a third partial structure comprising or consisting of the ECD of TGFβRII (e.g., hTGFβRII), wherein the second partial structure is operably connected to the first partial structure by a first peptide linker; and (e) the third partial structure is operably connected to the first partial structure by a second peptide linker.

[0318] 5.5.1 Ig constant region In some embodiments, the fusion protein (or one or more substructures thereof) described herein comprises one or more Ig (e.g., hIg) heavy chain constant regions (e.g., CH1 region, hinge region, CH2 region, CH3 region, Fc region). In some embodiments, the hIg is human IgG (hIgG). In some embodiments, the hIgG is hIgG1, IgG2, IgG3, or IgG4. In some embodiments, the hIgG is IgG1 or IgG4. In some embodiments, the hIgG is hIgG1. In some embodiments, the hIgG is hIgG4.

[0319] In some embodiments, the fusion protein (or one or more substructures thereof) comprises an Fc region. In some embodiments, the Fc region is a portion of a full-length antibody. In some embodiments, the Fc region comprises, or consists of, at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Fc region comprises, or consists of, a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Fc region comprises, or consists of, at least a portion of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the Fc region comprises, or consists of, an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the Fc region comprises, or consists of, at least a portion of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the Fc region comprises, or consists of, an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the Fc region comprises or consists of at least a portion of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region. In some embodiments, the Fc region comprises or consists of a hIgG4 hinge region, a hIgG4 CH2 region, and a hIgG4 CH3 region.

[0320] Exemplary reference hIgG1 and hIgG4 heavy chain constant region amino acid sequences that may be incorporated into one or more of the embodiments described herein (e.g., multispecific proteins (or one or more polypeptides thereof)) are provided in Table 6.

[0321] [Table 6-1]

[0322] [Table 6-2]

[0323] [Table 6-3]

[0324] [Table 6-4]

[0325] In some embodiments, the fusion protein (or one or more polypeptides thereof) comprises one or more hIg constant regions, wherein the amino acid sequence of the one or more hIg constant regions comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the one or more hIg constant regions comprises or consists of an amino acid sequence set forth in Table 6.

[0326] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence set forth in Table 6 and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence set forth in Table 6 that contains or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence set forth in Table 6 that contains or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence shown in Table 6, which contains or consists of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions).

[0327] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence set forth in Table 6 and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence set forth in Table 6 containing or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence set forth in Table 6 containing or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence set forth in Table 6 containing or consisting of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0328] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 86-89. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 86-89.

[0329] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 86-89, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 86-89, containing or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 86-89, containing or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 86 to 89, which contains or consists of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications (e.g., amino acid substitutions, deletions or additions).

[0330] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 86-89, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 86-89, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 86-89, comprising or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 86 to 89, which contains or consists of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.

[0331] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 103-104. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 103-104.

[0332] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 103-104, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 103-104, containing or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 103-104, containing or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 103-104, which contains or consists of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications (e.g., amino acid substitutions, deletions or additions).

[0333] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 103-104, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 103-104, comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 103-104, comprising or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 103 to 104, containing or consisting of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.

[0334] 5.5.2 Linker The substructures of the fusion proteins described herein (e.g., an EGFR (e.g., hEGFR)-binding substructure and a TGFβ (e.g., hTGFβ)-binding substructure) can be operably connected directly, e.g., by a peptide bond, or indirectly, e.g., by a linker (e.g., a peptide linker). In some embodiments, one or more of the substructures of the fusion protein are operably connected directly by a peptide bond. In some embodiments, one or more of the substructures of the fusion protein are operably connected indirectly by a peptide linker.

[0335] In some embodiments, the peptide linker is one or any combination of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and / or a non-helical linker.

[0336] In some embodiments, the peptide linker comprises 2 to 30, 5 to 30, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 2 to 25, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 2 to 20, 5 to 20, 10 to 20, 15 to 20, 2 to 15, 5 to 15, 10 to 15, 2 to 10, or 5 to 10 amino acid residues, or a range of such amino acid residues. In some embodiments, the peptide linker comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In some embodiments, the linker comprises or consists of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In some embodiments, the linker comprises or consists of at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues.

[0337] In some embodiments, the amino acid sequence of the peptide linker comprises or consists of glycine, serine, or both glycine and serine amino acid residues, hi some embodiments, the amino acid sequence of the peptide linker comprises or consists of glycine, serine, and proline amino acid residues.

[0338] The amino acid sequences of exemplary peptide linkers that may be incorporated into one or more of the embodiments (eg, fusion proteins and polypeptides) described herein are set out / provided in Table 7.

[0339] [Table 7]

[0340] In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers shown in Table 7. In some embodiments, the amino acid sequence of the peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a peptide linker shown in Table 7.

[0341] In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers shown in Table 7, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers shown in Table 7 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions). In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers shown in Table 7, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of the linkers shown in Table 7 with one, two, or three amino acid substitutions.

[0342] In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 105-124. In some embodiments, the amino acid sequence of the peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 105-124. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 111. In some embodiments, the amino acid sequence of the peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 111. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of SEQ ID NO: 111. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of any one of SEQ ID NO: 111. In some embodiments, the amino acid sequence of the peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 111.

[0343] In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 105-124, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 105-124 with one, two, or three amino acid modifications (e.g., substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 105-124, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 105-124 with one, two, or three amino acid substitutions.

[0344] In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 111, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 111. In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 111 with one, two, or three amino acid modifications (e.g., substitutions, additions, deletions). In some embodiments, the amino acid sequence of the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 111 with one, two, or three amino acid substitutions.

[0345] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of SEQ ID NO: 111, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of SEQ ID NO: 111. In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of SEQ ID NO: 111 with one, two, or three amino acid modifications (e.g., substitutions, additions, deletions). In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of SEQ ID NO: 111 with one, two, or three amino acid substitutions.

[0346] In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 111, and further comprises one or more, but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 111. In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 111 with one, two, or three amino acid modifications (e.g., substitutions, additions, deletions). In some embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 111 with one, two, or three amino acid substitutions.

[0347] 5.5.3 Orientation The substructures (e.g., EGFR (e.g., hEGFR) binding substructures (e.g., EGFR (e.g., hEGFR) binding substructures described herein), TGFβ (e.g., TGFβ (e.g., hTGFβ) binding substructures described herein) of a fusion protein (e.g., a fusion protein described herein) can be arranged in any configuration or order, so long as each substructure maintains the ability to mediate its function (e.g., an EGFR (e.g., hEGFR) binding substructure maintains the ability to bind to EGFR (e.g., hEGFR), and a TGFβ (e.g., hTGFβ) binding substructure maintains the ability to bind to TGFβ (e.g., hTGFβ)).

[0348] Exemplary orientations of the fusion proteins described herein are provided below. The exemplary orientations are intended to be illustrative only, not limiting.

[0349] 5.5.3.1 Homodimeric Full-Length Antibody-C-Terminal Light Chain Fusion Proteins In some embodiments, the fusion protein comprises an EGFR (e.g., hEGFR)-binding substructure, a first TGFβ (e.g., hTGFβ)-binding substructure, and a second TGFβ (e.g., hTGFβ)-binding substructure, wherein (a) the EGFR (e.g., hEGFR)-binding substructure comprises: (i) a first Ig light chain comprising, from N-terminus to C-terminus, a light chain variable region (VL) region and a light chain constant region (CL) region; (ii) a first Ig heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable region (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second Ig heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (iv) a second Ig light chain comprising, from N-terminus to C-terminus, a VL region and a VH region. The antibody is a full-length antibody, wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and the first heavy chain and the second heavy chain associate to form a dimer; (b) the N-terminus of a first TGFβ (e.g., hTGFβ) binding substructure (e.g., the ECD of TGFβR (e.g., TGFβRII (e.g., hTGFβRII))) is operably connected to the C-terminus of a first Ig light chain of the full-length antibody; and (c) the N-terminus of a second TGFβ binding substructure (e.g., the ECD of TGFβR (e.g., TGFβRII (e.g., hTGFβRII))) is operably connected to the C-terminus of a second Ig light chain of the full-length antibody.

[0350] In some embodiments, a first TGFβ (e.g., hTGFβ) binding substructure is operably connected to the C-terminus of a first Ig light chain of the full-length antibody by a first peptide linker (e.g., a peptide linker described herein, e.g., SEQ ID NO: 111); and a second TGFβ (e.g., hTGFβ) binding substructure is operably connected to the C-terminus of a second Ig light chain of the full-length antibody by a second peptide linker (e.g., a peptide linker described herein, e.g., SEQ ID NO: 111). In some embodiments, the amino acid sequence of the first peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second peptide linker. In some embodiments, the amino acid sequence of the first peptide linker is 100% identical to the amino acid sequence of the second peptide linker.

[0351] In some embodiments, the amino acid sequence of a TGFβ (e.g., hTGFβ) binding substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a second TGFβ (e.g., hTGFβ) binding substructure. In some embodiments, the amino acid sequence of a TGFβ (e.g., hTGFβ) binding substructure is 100% identical to the amino acid sequence of a second TGFβ (e.g., hTGFβ) binding substructure. In some embodiments, the first TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the ECD of TGFβR (e.g., TGFβRII (e.g., hTGFβRII)); and the second TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the ECD of TGFβR (e.g., TGFβRII (e.g., hTGFβRII)).

[0352] In some embodiments, the fusion protein comprises: (a) a first polypeptide comprising, from N-terminus to C-terminus, a first light chain variable region (VL), a first light chain constant region (CL), a first optional peptide linker, and a first TGFβ binding substructure (e.g., the ECD of a TGFβR (e.g., TGFβRII (e.g., hTGFβRII))); (b) a second polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable region (VH) and a first heavy chain constant region (CH); and (c) a second polypeptide comprising, from N-terminus to C-terminus, a second VH and a second CH. and (d) a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL, a second CL, a second optional peptide linker, and a second TGFβ-binding substructure (e.g., the ECD of a TGFβR (e.g., TGFβRII (e.g., hTGFβRII))); the first VL and the first VH form a first antigen-binding domain that specifically binds to EGFR (e.g., hEGFR); and the second VL and the second VH form a second antigen-binding domain that specifically binds to EGFR (e.g., hEGFR). In some embodiments, the first polypeptide 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 the fourth polypeptide. In some embodiments, the second polypeptide 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 the third polypeptide. In some embodiments, the amino acid sequence of the first TGFβ binding substructure (e.g., the ECD of a TGFβR (e.g., TGFβRII (e.g., hTGFβRII))) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a second TGFβ binding substructure (e.g., the ECD of a TGFβR (e.g., TGFβRII (e.g., hTGFβRII))). In some embodiments, the first CH region comprises, from N-terminus to C-terminus, a first CH1 domain, a first hinge domain, a first CH2 domain, and a first CH3 domain.In some embodiments, the second CH region comprises, from N-terminus to C-terminus, a second CH1 domain, a second hinge domain, a second CH2 domain, and a second CH3 domain.

[0353] In some embodiments, the fusion protein comprises: (a) a first substructure comprising or consisting of a full-length antibody that specifically binds to EGFR (e.g., hEGFR); (b) a second substructure comprising or consisting of the ECD of TGFβRII (e.g., hTGFβRII); and (c) a third substructure comprising or consisting of the ECD of TGFβRII (e.g., hTGFβRII); the second substructure is operably connected to the first substructure by a first peptide linker, the third substructure is operably connected to the first substructure by a second peptide linker, the N-terminus of the second substructure is operably connected to the C-terminus of a first light chain of the full-length antibody, and the N-terminus of the third substructure is operably connected to the C-terminus of a second light chain of the full-length antibody.

[0354] 5.5.3.2 Homodimeric Full-Length Antibody-C-Terminal Heavy Chain Fusion Proteins In some embodiments, a fusion protein (e.g., a fusion protein described herein) comprises an EGFR (e.g., hEGFR)-binding substructure, a first TGFβ (e.g., hTGFβ)-binding substructure, and a second TGFβ (e.g., hTGFβ)-binding substructure, wherein (a) the EGFR (e.g., hEGFR)-binding substructure comprises: (i) a first Ig light chain comprising, from N-terminus to C-terminus, a light chain variable region (VL) region and a light chain constant region (CL) region; (ii) a first Ig heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable region (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second Ig heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (iv) a second Ig heavy chain comprising, from N-terminus to C-terminus, a VL region and a VH region. wherein the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and the first heavy chain and the second heavy chain associate to form a dimer; (b) the N-terminus of a first TGFβ binding substructure (e.g., an ECD of a TGFβR (e.g., TGFβRII (e.g., hTGFβRII))) is operably connected to the C-terminus of the first Ig heavy chain of the full-length antibody; and (c) the N-terminus of a second TGFβ (e.g., hTGFβ) binding substructure (e.g., an ECD of a TGFβR (e.g., TGFβRII (e.g., hTGFβRII))) is operably connected to the C-terminus of the second Ig heavy chain of the full-length antibody.

[0355] In some embodiments, a first TGFβ (e.g., hTGFβ) binding substructure is operably connected to the C-terminus of a first Ig heavy chain of the full-length antibody by a first peptide linker (e.g., a peptide linker described herein, e.g., SEQ ID NO: 111); and a second TGFβ (e.g., hTGFβ) binding substructure is operably connected to the C-terminus of a second Ig heavy chain of the full-length antibody by a second peptide linker (e.g., a peptide linker described herein, e.g., SEQ ID NO: 111). In some embodiments, the amino acid sequence of the first peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second peptide linker. In some embodiments, the amino acid sequence of the first peptide linker is 100% identical to the amino acid sequence of the second peptide linker.

[0356] In some embodiments, the amino acid sequence of a TGFβ (e.g., hTGFβ) binding substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a second TGFβ (e.g., hTGFβ) binding substructure. In some embodiments, the amino acid sequence of a TGFβ (e.g., hTGFβ) binding substructure is 100% identical to the amino acid sequence of a second TGFβ (e.g., hTGFβ) binding substructure. In some embodiments, the first TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the ECD of TGFβR (e.g., TGFβRII (e.g., hTGFβRII)); and the second TGFβ (e.g., hTGFβ) binding substructure comprises or consists of the ECD of TGFβR (e.g., TGFβRII (e.g., hTGFβRII)).

[0357] In some embodiments, the fusion protein comprises: (a) a first polypeptide comprising, from N-terminus to C-terminus, a first light chain variable region (VL) and a first light chain constant region (CL); (b) a second polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable region (VH), a first heavy chain constant region (CH), a first optional peptide linker, and a first TGFβ (e.g., hTGFβ) binding substructure (e.g., the ECD of a TGFβR (e.g., TGFβRII (e.g., hTGFβRII))); and (c) a second VH, a second CH, a second optional and (d) a third polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL; the first VL and the first VH form a first antigen-binding domain that specifically binds to EGFR (e.g., hEGFR); and the second VL and the second VH form a second antigen-binding domain that specifically binds to EGFR (e.g., hEGFR). In some embodiments, the first polypeptide 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 the fourth polypeptide. In some embodiments, the second polypeptide 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 the third polypeptide. In some embodiments, the amino acid sequence of the first TGFβ (e.g., hTGFβ) binding substructure (e.g., the ECD of a TGFβR (e.g., TGFβRII (e.g., hTGFβRII))) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a second TGFβ (e.g., hTGFβ) binding substructure (e.g., the ECD of a TGFβR (e.g., TGFβRII (e.g., hTGFβRII))).In some embodiments, the first CH region comprises, from N- to C-terminus, a first CH1 domain, a first hinge domain, a first CH2 domain, and a first CH3 domain, hi some embodiments, the second CH region comprises, from N- to C-terminus, a second CH1 domain, a second hinge domain, a second CH2 domain, and a second CH3 domain.

[0358] 5.5.4 Exemplary Fusion Proteins The amino acid sequences of exemplary fusion proteins described herein are provided in Table 8. Each of the fusion proteins comprises an EGFR (e.g., hEGFR)-binding substructure and two TGFβ-binding substructures, where the EGFR (e.g., hEGFR)-binding substructure comprises a full-length antibody comprising two EGFR (e.g., hEGFR)-binding domains, and each TGFβ (e.g., hTGFβ)-binding substructure comprises the ECD of TGFβRII (e.g., hTGFβRII). The fusion proteins provided in Table 8 are merely exemplary and are not intended to be limiting.

[0359] [Table 8-1]

[0360] [Table 8-2]

[0361] [Table 8-3]

[0362] [Table 8-4]

[0363] In some embodiments, the fusion protein comprises a heavy chain (HC) comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HC shown in Table 8; and a light chain (LC) comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the LC shown in Table 8.

[0364] In some embodiments, the fusion protein comprises a HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125. In some embodiments, the fusion protein comprises a HC consisting of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125.

[0365] In some embodiments, the fusion protein comprises an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126. In some embodiments, the fusion protein comprises an LC consisting of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126.

[0366] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125; and an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126.

[0367] In some embodiments, the fusion protein comprises an HC consisting of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125; and an LC consisting of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126.

[0368] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 127.

[0369] In some embodiments, the fusion protein comprises an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 128.

[0370] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 127; and an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 128.

[0371] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 129.

[0372] In some embodiments, the fusion protein comprises an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 130.

[0373] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 129; and an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 130.

[0374] In some embodiments, the fusion protein comprises a HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:131.

[0375] In some embodiments, the fusion protein comprises an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 132.

[0376] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 131; and an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 132.

[0377] In some embodiments, the fusion protein comprises a HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 133.

[0378] In some embodiments, the fusion protein comprises an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 134.

[0379] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 133; and an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 134.

[0380] In some embodiments, the fusion protein comprises a HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 135.

[0381] In some embodiments, the fusion protein comprises an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 136.

[0382] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 135; and an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 136.

[0383] In some embodiments, the fusion protein comprises a HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137.

[0384] In some embodiments, the fusion protein comprises an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 138.

[0385] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 137; and an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 138.

[0386] In some embodiments, the fusion protein comprises a HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 139.

[0387] In some embodiments, the fusion protein comprises an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 140.

[0388] In some embodiments, the fusion protein comprises an HC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 139; and an LC comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 140.

[0389] 5.5.5 Affinities of Fusion Proteins and Their Antigen-Binding Substructures The binding affinity of any of the fusion proteins or components thereof (e.g., EGFR (e.g., hEGFR)-binding substructures, TGFβ (e.g., hTGFβ)-binding substructures) described herein can be measured by standard assays known in the art and described herein. For example, binding affinity can be measured by ELISA. Binding affinity can also be measured by surface plasmon resonance (SPR) (e.g., BIAcore®-based assays), a common method known in the art (see, e.g., Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 55:2560, 1993; and U.S. Pat. Nos. 5,283,173 and 5,468,614, the entire contents of each of which are incorporated herein by reference for all purposes). SPR measures the change in the concentration of a molecule on a sensor surface when the molecule binds to or dissociates from the surface. The change in SPR signal is directly proportional to the change in mass concentration near the surface, allowing the measurement of the binding kinetics between two molecules (e.g., proteins). The dissociation constant of the complex can be determined by monitoring the change in refractive index versus time as a buffer solution passes over the chip.

[0390] Other suitable assays for measuring binding of a multispecific protein described herein to one or more of its cognate antigens include, for example, immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs); or determining binding by monitoring changes in the spectroscopic or optical properties of the protein by fluorescence, UV absorbance, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blots, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, and other methods for detecting protein binding.

[0391] As described above, the functionality of a fusion protein can be tested by any method known in the art, such as ELISA. For fusion proteins with more than one functionality, each functionality can be measured in a separate assay, for example, TGFβ (e.g., hTGFβ) binding and EGFR (e.g., hEGFR) binding can be measured in two separate ELISAs. For example, an ELISA plate can be coated with an EGFR (e.g., hEGFR) Fc chimera to assess EGFR (e.g., hEGFR) binding; another ELISA plate can be coated with TGFβ (e.g., TGFβ1 (e.g., hTGFβ1)) to assess TGFβ (e.g., TGFβ1 (e.g., hTGFβ1)) binding.

[0392] Both functionalities can also be evaluated by bifunctional ELISA. For example, an anti-idiotypic monoclonal antibody against an EGFR (e.g., hEGFR) antibody (e.g., cetuximab or its variant) can be used to capture the EGFR antibody portion of the fusion protein (e.g., BCA101), and the bound fusion protein (e.g., BCA101) can be detected by an enzyme-labeled polyclonal antibody against a TGFβ (e.g., hTGFβ) binding moiety (e.g., the ECD of TGFβRII (e.g., hTGFβRII)). The concentration of the fusion protein (e.g., BCA101) in the sample can be back-calculated from the fusion protein calibration curve.

[0393] Target binding can also be assessed by Biacore, in which EGFR (e.g., hEGFR) and TGFβ (e.g., TGFβ1 (e.g., hTGFβ1)) targets are immobilized on an activated CM5 chip and then incubated with a range of concentrations of the fusion protein (e.g., BCA101). Additional in vitro functional assays can also be performed to assess the fusion protein, including, for example, cell surface binding by flow cytometry, inhibition of cell proliferation, ADCC assay; neutralization of TGFβ (e.g., TGFβ1 (e.g., hTGFβ1))-induced IL-11 release; and neutralization of TGFβ (e.g., TGFβ1 (e.g., hTGFβ1))-induced SMAD signaling.

[0394] 5.6 Methods for Producing Fusion Proteins and Their Components The fusion proteins (and their polypeptides) and their components (e.g., their substructures) described herein can be produced using standard methods known in the art. For example, each can be produced by recombinant techniques in host cells (e.g., insect cells, mammalian cells, bacteria) transfected or transduced with a nucleic acid expression vector (e.g., a plasmid, a viral vector (e.g., a baculovirus expression vector)) encoding the fusion protein (or one or more polypeptides thereof). Such general methods are common knowledge in the art. Expression vectors typically contain an expression cassette comprising nucleic acid sequences capable of causing expression of a nucleic acid molecule encoding a protein or polypeptide of interest, such as a promoter, enhancer, polyadenylation signal, etc. Those skilled in the art will recognize that various promoter and enhancer elements can be used to achieve expression of nucleic acid molecules in host cells. For example, promoters can be constitutive or regulated promoters, and they can be obtained from various sources, e.g., viral, prokaryotic, or eukaryotic sources, or can be artificially designed. After transfection or transduction, host cells containing an expression vector encoding a protein or polypeptide of interest are cultured under conditions that promote the expression of the nucleic acid molecule encoding the antigenic peptide or protein. Culture media are available from various commercial sources, and a suitable medium can be routinely selected to allow the host cells to express the protein or polypeptide of interest. Host cells can be in adherent or suspension culture, and those skilled in the art can optimize the culture method for the particular host cells selected. For example, suspension cells can be cultured, for example, in a bioreactor, e.g., in a batch or fed-batch process. The produced protein or polypeptide can be isolated from the cell culture, e.g., by column chromatography in either a flow-through mode or a bind-and-elute mode.Examples include, but are not limited to, ion exchange resins and affinity resins, such as lentil lectin sepharose, and mixed-mode cation exchange-hydrophobic interaction columns (CEX-HIC). Proteins or polypeptides can be concentrated and buffer-exchanged by ultrafiltration, and the retentate from ultrafiltration can be filtered through an appropriate filter, such as a 0.22 μm filter. See, for example, Hacker, David (Ed.), Recombinant Protein Expression in Mammalian Cells: Methods and Protocols (Methods in Molecular Biology), Humana Press (2018); and McPherson et al., "Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant," Chapter 4, in Sunil Thomas (ed.), Vaccine Design: Methods and Protocols: Volume 1: Vaccines for Human Diseases, Methods in Molecular Biology, Springer, New York, 2016. See also U.S. Patent No. 5,762,939, the entire contents of each of which are incorporated herein by reference for all purposes. The fusion proteins (and polypeptides thereof) and components thereof (e.g., substructures thereof) described herein can be produced synthetically.

[0395] In embodiments, the disclosure features methods of making the fusion proteins (and polypeptides thereof) and components thereof (e.g., substructures thereof) described herein. The methods include: (a) recombinantly expressing a fusion protein (or polypeptide thereof) or component thereof (e.g., substructure thereof) described herein; (b) enriching, e.g., purifying, the fusion protein (or polypeptide thereof) or component thereof (e.g., substructure thereof) described herein; (c) evaluating the fusion protein (or polypeptide thereof) or component thereof (e.g., substructure thereof) described herein for the presence of process impurities or contaminants; and (d) formulating the fusion protein (or polypeptide thereof) or component thereof (e.g., substructure thereof) described herein as a pharmaceutical composition if the fusion protein (or polypeptide thereof) or component thereof (e.g., substructure thereof) described herein meets the threshold specification for the process impurity or contaminant. The process impurities or contaminants evaluated can be, for example, one or more of process-related impurities, such as host cell proteins, host cell DNA, or cell culture components (e.g., inducers, antibiotics, or media components); product-related impurities (e.g., precursors, fragments, aggregates, degradation products); or contaminants, such as endotoxins, bacteria, or viral contaminants.

[0396] 5.7 Combination regimens In one aspect, the present invention provides a method for the treatment of cancer, comprising: (i) (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein) (e.g., a pharmaceutical composition comprising a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein); (ii) (a) a fusion protein (or polypeptide thereof) described herein, (b) a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, (c) a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), (d) a host cell described herein (e.g., a host cell comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)), (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), (f) a vector comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), (g) a vector comprising a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide) described herein), (h ...i) ) a carrier described herein (e.g., a carrier comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)), or (f) a pharmaceutical composition described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), a host cell described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g.,Provided herein are combination regimens comprising a host cell comprising a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), or a carrier described herein (e.g., a pharmaceutical composition comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)).

[0397] Components (i) and (ii) set forth above for the combination regimen may or may not be combined in a single dosage form. In some embodiments, components (i) and (ii) set forth above for the combination regimen are not combined in a single dosage form. In some embodiments, components (i) and (ii) set forth above for the combination regimen are combined in a single dosage form.

[0398] The components (i) and (ii) shown above for the combination regimen can be formulated in separate formulations or in a single formulation. In some embodiments, the components (i) and (ii) shown above for the combination regimen are formulated in separate formulations. The components (i) and (ii) shown above for the combination regimen are formulated in a single formulation.

[0399] The components (i) and (ii) shown above for the combination regimen can be formulated in separate pharmaceutical formulations or in a single pharmaceutical formulation. In some embodiments, the components (i) and (ii) shown above for the combination regimen are formulated in separate pharmaceutical formulations. The components (i) and (ii) shown above for the combination regimen are formulated in a single pharmaceutical formulation.

[0400] The combination regimens described herein can be utilized, for example, in any of the combination compositions described herein (see, e.g., §5.8), in any of the pharmaceutical compositions described herein (see, e.g., §5.10), in any of the methods of use described herein (see, e.g., §5.11), or in any of the kits described herein (see, e.g., §5.12).

[0401] 5.8 Combination Compositions In one aspect, the present invention relates to a pharmaceutical composition comprising: (i) (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein) (e.g., a pharmaceutical composition comprising a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein); (ii) (a) a fusion protein (or polypeptide thereof) described herein, (b) a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, (c) a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), (d) a host cell described herein (e.g., a host cell comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)), (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), (f) a vector comprising a vector comprising a vector encoding a vector encoding a vector encoding a fusion protein (or polypeptide) described herein), (g) a vector comprising a vector encoding a vector encoding a vector encoding a vector encoding a vector), (h) a vector comprising a vector encoding a vector), (i) a vector comprising a vector encoding a vector), (j) a vector comprising a vector encoding a vector), (j) a vector comprising a vector encoding a vector), (j) a vector comprising a vector encoding a vector), (i) a vector comprising a vector), (j) a vector comprising a vector), (i ... ) a carrier described herein (e.g., a carrier comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)), or (f) a pharmaceutical composition described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), a host cell described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g.,Provided herein are combination compositions comprising a host cell comprising a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), or a carrier described herein (e.g., a pharmaceutical composition comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)).

[0402] The combination compositions described herein can be utilized, for example, in any of the combination compositions described herein (see, e.g., §5.8), in any of the pharmaceutical compositions described herein (see, e.g., §5.10), in any of the methods of use described herein (see, e.g., §5.11), or in any of the kits described herein (see, e.g., §5.12).

[0403] 5.9 Polynucleotides, Vectors, Carriers, and Host Cells In one aspect, provided herein are polynucleotides (e.g., DNA, RNA) that encode the fusion proteins described herein (or any (e.g., one or more) polypeptides thereof) and components thereof described herein (e.g., substructures thereof (e.g., EGFR-binding substructures, TGFβ-binding substructures)). In some embodiments, the polynucleotide is a DNA polynucleotide or an RNA polynucleotide. In some embodiments, the polynucleotide is an mRNA polynucleotide. In some embodiments, the polynucleotide is codon-optimized. Codon optimization can be used to match the codon frequency in the target with that in the host organism to ensure correct folding; bias the guanosine (G) and / or cytosine I content to increase nucleic acid stability; minimize tandem repeat codons or base runs that may impair gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translation rates to allow correct folding of various domains of the protein; or reduce or eliminate problematic secondary structures within the polynucleotide. In some embodiments, the codon-optimized nucleic acid sequence exhibits one or more of the above (compared to a reference nucleic acid sequence). In some embodiments, the codon-optimized nucleic acid sequence exhibits one or more of increased resistance to in vivo degradation, increased in vivo stability, reduced secondary structure, and / or increased in vivo translatability compared to a reference nucleic acid sequence. Codon optimization methods, tools, algorithms, and services are known in the art; non-limiting examples include services from GeneArt (Life Technologies) and DNA2.0 (Menlo Park, Calif.).In some embodiments, the open reading frame (ORF) sequence is optimized using an optimization algorithm. In some embodiments, the nucleic acid sequence is modified to optimize the number of G and / or C nucleotides compared to a reference nucleic acid sequence. The increase in the number of G and C nucleotides can be achieved by substituting codons containing adenosine (T) or thymine (T) (or uracil (U)) nucleotides with codons containing G or C nucleotides.

[0404] In one aspect, provided herein is a vector comprising a polynucleotide (e.g., DNA, RNA) described herein (e.g., a polynucleotide encoding a fusion protein (or one or more polypeptides thereof) described herein) or a component thereof (e.g., a substructure thereof (e.g., an EGFR-binding substructure, a TGFβ-binding substructure)) described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector (e.g., a plasmid).

[0405] In one aspect, provided herein is a carrier comprising: (a) a fusion protein (or one or more polypeptides thereof) described herein or a component thereof described herein (e.g., a substructure thereof (e.g., an EGFR-binding substructure, a TGFβ-binding substructure)), a polynucleotide described herein (e.g., a polynucleotide encoding / comprising a fusion protein (or any (e.g., one or more) polypeptides thereof) described herein and a component thereof described herein (e.g., a substructure thereof (e.g., an EGFR-binding substructure, a TGFβ-binding substructure)), or a vector described herein (e.g., a vector comprising a polynucleotide described herein); and / or (b) a KRAS inhibitor (e.g., a KRAS inhibitor described herein). Carriers include, but are not limited to, lipid-based carriers, such as lipid nanoparticles (LNPs), liposomes, lipoplexes, or nanoliposomes. In some embodiments, the carrier is an LNP, e.g., an LNP described herein.

[0406] In one aspect, provided herein is a host cell comprising: (a) a fusion protein (or one or more polypeptides thereof) described herein or a component thereof described herein (e.g., a substructure thereof (e.g., an EGFR-binding substructure, a TGFβ-binding substructure)), a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein (or any (e.g., one or more) polypeptides thereof and a component thereof described herein (e.g., a substructure thereof (e.g., an EGFR-binding substructure, a TGFβ-binding substructure))), a vector described herein (e.g., a vector comprising a polynucleotide described herein), or a carrier described herein (e.g., a carrier comprising a fusion protein (or any (e.g., one or more) polypeptides thereof) described herein and a component thereof described herein (e.g., a substructure thereof (e.g., an EGFR-binding substructure, a TGFβ-binding substructure)); and / or (b) a KRAS inhibitor (e.g., a KRAS inhibitor described herein).

[0407] 5.10 Pharmaceutical Compositions In one aspect, provided herein is a pharmaceutical composition comprising a fusion protein described herein, a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), a vector described herein (e.g., a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein)), a host cell described herein (e.g., a fusion protein described herein, a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), a vector described herein (e.g., a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein)), or a carrier described herein (e.g., a host cell comprising a fusion protein described herein, a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), a vector described herein (e.g., a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein)), or a host cell described herein); and (b) a pharmaceutically acceptable excipient (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, the entire contents of which are incorporated herein by reference for all purposes).

[0408] In one aspect, provided herein is a pharmaceutical composition comprising: (a) a KRAS inhibitor (e.g., a KRAS inhibitor described herein); and (b) a pharmaceutically acceptable excipient (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, the entire contents of which are incorporated herein by reference for all purposes).

[0409] In one aspect, a composition comprising (a) a fusion protein described herein, a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), a vector described herein (e.g., a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein)), a host cell described herein (e.g., a fusion protein described herein, a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), a vector described herein (e.g., a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein)), or a carrier described herein (e.g., a host cell comprising a fusion protein described herein, a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), a vector described herein (e.g., a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein)), or a host cell described herein); (b) a KRAS inhibitor (e.g., a KRAS inhibitor described herein); and (c) a pharmaceutically acceptable excipient (e.g., a carrier described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, See, e.g., PA, the entire contents of which are incorporated herein by reference for all purposes).

[0410] Also provided herein are pharmaceutical compositions comprising a fusion protein (or polypeptide thereof) described herein or a component thereof (e.g., substructure thereof) described herein, a polynucleotide described herein, a vector described herein, a host cell described herein, or a carrier described herein, which lack a predetermined threshold amount or a detectable amount of process impurities or contaminants, e.g., lacking a predetermined threshold amount or a detectable amount of process-related impurities, e.g., host cell proteins, host cell DNA, or cell culture components (e.g., inducers, antibiotics, or media components); product-related impurities (e.g., precursors, fragments, aggregates, degradation products); or contaminants, e.g., endotoxins, bacteria, viral contaminants.

[0411] In one aspect, also provided herein is a method of making a pharmaceutical composition described herein, the method comprising providing (a) a fusion protein described herein (or one or more polypeptides thereof), a polynucleotide described herein, a vector described herein, a host cell described herein, or a carrier described herein, and (b) a KRAS inhibitor (e.g., a KRAS inhibitor described herein), and formulating (a) and (b) into a pharmaceutically acceptable composition by the addition of one or more pharmaceutically acceptable excipients.

[0412] Acceptable excipients (e.g., carriers and stabilizers) are preferably nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers, e.g., phosphate, citrate, or other organic acids; antioxidants, including ascorbic acid or methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; or m-cresol); low molecular weight (less than about 10 residues) polypeptides; tannins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, or other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0413] The pharmaceutical composition can be formulated for any route of administration to a subject. Non-limiting embodiments include parenteral administration, such as intramuscular, intradermal, subcutaneous, transdermal, or mucosal administration, such as inhalation, intranasal, oral, etc. In one embodiment, the pharmaceutical composition is formulated for administration by intramuscular, intradermal, or subcutaneous injection. In one embodiment, the pharmaceutical composition is formulated for administration by intramuscular injection. In one embodiment, the pharmaceutical composition is formulated for administration by intradermal injection. In one embodiment, the pharmaceutical composition is formulated for administration by subcutaneous injection. Injections can be prepared in conventional forms, either as a solution or a suspension. Injections may contain one or more excipients. Exemplary excipients include, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins. In some embodiments, the pharmaceutical composition is formulated in a single dose. In some embodiments, the pharmaceutical composition is formulated in multiple doses.

[0414] Pharmaceutically acceptable excipients used in the parenteral preparations described herein include, for example, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, or other pharmaceutically acceptable substances. Examples of aqueous vehicles that can be incorporated into one or more of the formulations described herein include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose or lactated Ringer's injection. Non-aqueous parenteral vehicles that can be incorporated into one or more of the formulations described herein include vegetable fixed oil, cottonseed oil, corn oil, sesame oil, or peanut oil. Antibacterial agents in bacteriostatic or fungistatic concentrates, including phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, or benzethonium chloride, can be added to the parenteral preparations described herein and packaged in multi-dose containers. Isotonicity agents that may be incorporated into one or more of the formulations described herein include sodium chloride or dextrose. Buffers that may be incorporated into one or more of the formulations described herein include phosphate or citrate. Antioxidants that may be incorporated into one or more of the formulations described herein include sodium bisulfate. Local anesthetics that may be incorporated into one or more of the formulations described herein include procaine hydrochloride. Suspending and dispersing agents that may be incorporated into one or more of the formulations described herein include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone. Emulsifiers that may be incorporated into one or more of the formulations described herein include polysorbate 80 (TWEEN® 80). The sequestering or chelating agent of metal ion that can be incorporated into one or more of the formulations described herein is EDTA.The pharmaceutical carrier that can be incorporated into one or more of the formulations described herein also includes ethyl alcohol, polyethylene glycol or propylene glycol for water-miscible vehicle; or sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0415] The exact dosage that will be used in pharmaceutical compositions will also depend on the route of administration and the severity of the condition caused by it, and should be determined according to the judgment of the practitioner and the circumstances of each patient.For example, effective dosage can also vary depending on the means of administration, target site, patient's physiological condition (including age, weight and health condition), other medications administered, or whether treatment is preventive or therapeutic.Treatment dosage is preferably adjusted to optimize safety and effectiveness.

[0416] In some embodiments, the fusion protein (e.g., BCA101) is formulated according to any of the aspects and embodiments described in International Publication No. 2022132201 (PCT / US2021010066), the entire contents of which are incorporated herein by reference for all purposes.

[0417] For example, in some embodiments, the fusion protein (e.g., BCA101) is formulated into a liquid formulation comprising a buffering agent present at a concentration of about 5 mM to about 30 mM and a tonicity 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.

[0418] 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 comprises about 10 mM citrate phosphate.

[0419] In some embodiments, the tonicity agent is a saccharide. In some embodiments, the tonicity agent is a disaccharide. In some embodiments, the tonicity agent is sucrose or trehalose. In some embodiments, the tonicity agent is sucrose. In some embodiments, the tonicity agent is present at a concentration of about 5% w / v to about 10% w / v, 6% w / v to about 10% w / v, 7% w / v to about 10% w / v, 8% w / v to about 10% w / v, 5% w / v to about 9% w / v, 5% w / v to about 8% w / v, 6% w / v to about 9% w / v, 6% w / v to about 8% w / v, 7% w / v to about 9% w / v, or 7% w / v to about 8% w / v. In some embodiments, the tonicity agent is present at a concentration of about 5% w / v to about 8% w / v. In some embodiments, the tonicity agent is present at a concentration of about 5% w / v, 6% w / v, 7% w / v, 8% w / v, 9% w / v, or 10% w / v. In some embodiments, the tonicity agent is present at a concentration of about 8% w / v. In some embodiments, the tonicity agent is sucrose and is present at a concentration of about 8% w / v.

[0420] 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 comprises polysorbate 80. 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 0.01-0.1% w / v, 0.02-0.1% w / v, 0.01-0.9% w / v, 0.01-0.8% w / v, 0.01-0.7% w / v, 0.01-0.6% w / v, 0.01-0.5% w / v, 0.01-0.4% w / v, 0.01-0.3% w / v, 0.01-0.2% w / v, 0.01-0.1% w / v, 0.02-0.9% w / v, 0.02-0.8% w / v, 0.02-0.7% w / v, 0.02-0. Present at concentrations of 6% w / v, 0.02-0.5% w / v, 0.02-0.4% w / v, 0.02-0.3% w / v, 0.02-0.2% w / v, 0.02-0.1% w / v, 0.005-0.9% w / v, 0.005-0.8% w / v, 0.005-0.7% w / v, 0.005-0.6% w / v, 0.005-0.5% w / v, 0.005-0.4% w / v, 0.005-0.3% w / v, 0.005-0.2% w / v, or 0.005-0.1% w / v. 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.

[0421] 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.

[0422] In some embodiments, the liquid pharmaceutical composition has an osmolality of about 150 mOsmol / kg to about 400 mOsmol / kg. In some embodiments, the liquid pharmaceutical composition has an osmolality of about 150 mOsmol / kg to about 350 mOsmol / kg, 150 mOsmol / kg to about 300 mOsmol / kg, 200 mOsmol / kg to about 400 mOsmol / kg, 250 mOsmol / kg to about 400 mOsmol / kg, 300 mOsmol / kg to about 400 mOsmol / kg, 300 mOsmol / kg to about 350 mOsmol / kg, 250 mOsmol / kg to about 350 mOsmol / kg, or 250 mOsmol / kg to about 300 mOsmol / kg. In some embodiments, the liquid pharmaceutical composition has 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.

[0423] In some embodiments, the liquid pharmaceutical composition is stable for at least 12, 18, or 24 months when stored at −20° C. In some embodiments, the liquid pharmaceutical composition is stable for at least 12, 18, or 24 months when stored at 2-8° C.

[0424] In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition remains substantially the same for at least 12, 18, 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, 18, 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, 18, or 24 months when stored at 2-8° C.

[0425] In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition does not decrease by more than 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% after storage at -80°C for 12, 18, 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%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% after storage at -20°C for 12, 18, 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%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% after storage at 2-8°C for 12, 18, or 24 months.

[0426] In some embodiments, the liquid pharmaceutical composition is stable upon 1, 2, 3, 4, or 5 cycles of freezing and thawing.

[0427] In some embodiments, the fusion protein retains bifunctional activity for at least 12, 18, or 24 months when stored at −20° C. as measured by bifunctional enzyme-linked immunosorbent assay (ELISA). In some embodiments, the fusion protein retains bifunctional activity for at least 12, 18, or 24 months when stored at −20° C. as measured by bifunctional ELISA. In some embodiments, the fusion protein retains bifunctional activity for at least 12, 18, or 24 months when stored at 2-8° C. as measured by bifunctional ELISA.

[0428] In some embodiments, the liquid pharmaceutical composition comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the fusion protein in aggregated form.

[0429] In some embodiments, the liquid pharmaceutical composition has at least one characteristic selected from the group consisting of: (a) increased shelf life, (b) increased temperature stability, (c) reduced aggregate formation, (d) increased chemical stability, (e) reduced fragmentation, and / or (I) reduced viscosity, compared to a reference formulation after 12, 18, or 24 months of storage at -20°C or 2-8°C.

[0430] In some embodiments, the liquid pharmaceutical composition has at least one characteristic 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 12, 18, or 24 months of storage at -20°C or 2-8°C.

[0431] 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. In some embodiments, the fusion protein is present at a concentration of about 50 mg / ml.

[0432] In some embodiments, the fusion protein (e.g., BCA101) is formulated as follows: a liquid formulation comprising or consisting of 50 mg / ml of the fusion protein (e.g., BCA101), 8.0% w / v sucrose, 0.02% w / v polysorbate 80, and 10 mM citrate phosphate buffer at pH 6.0.

[0433] In some embodiments, where the KRAS (e.g., hKRAS) inhibitor is sotorasib, the sotorasib is in the form of a tablet. In some embodiments, where the KRAS (e.g., hKRAS) inhibitor is sotorasib, the sotorasib is in the form of a tablet for oral administration. In some embodiments, where the KRAS (e.g., hKRAS) inhibitor is sotorasib, the sotorasib is in the form of a film-coated tablet. In some embodiments, the tablet core comprises or consists of any one, two, three, or four of the following: microcrystalline cellulose, lactose monohydrate, croscarmellose sodium, and magnesium stearate. In some embodiments, the tablet core comprises or consists of any one, two, three, or four of the following, in addition to the active ingredient (sotorasib): microcrystalline cellulose, lactose monohydrate, croscarmellose sodium, and magnesium stearate. In some embodiments, the film coating material comprises or consists of any one, two, three, four, or five of the following: polyvinyl alcohol, titanium dioxide, polyethylene glycol, talc, and iron oxide yellow. In some embodiments, the KRAS (e.g., hKRAS) inhibitor is sotorasib, which is orally administered at 960 mg once daily. In some embodiments, sotorasib is orally administered as a whole tablet with or without food.

[0434] 5.11 How to use Provided herein are various methods utilizing KRAS (e.g., hKRAS) inhibitors (e.g., KRAS inhibitors described herein), fusion proteins (and polypeptides thereof) described herein, substructures described herein (e.g., EGFR (e.g., hEGFR) binding substructures, TGFβ (e.g., hTGFβ) binding substructures), polynucleotides described herein, vectors described herein, host cells described herein, carriers described herein, and pharmaceutical compositions described herein. In some embodiments, the method includes administering to a subject (i) a fusion protein described herein (or a polypeptide thereof), a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), a vector described herein (e.g., a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein)), a host cell described herein (e.g., a host cell comprising a fusion protein described herein, a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), a vector described herein (e.g., a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein))), or a carrier described herein (e.g., a carrier comprising a fusion protein described herein, a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), a vector described herein (e.g., a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein)), or a host cell described herein); and (ii) a KRAS inhibitor (e.g., a KRAS inhibitor described herein), or a pharmaceutical composition comprising a KRAS inhibitor; or a pharmaceutical composition comprising (i) and (ii). Exemplary subjects include mammals, e.g., humans, and non-human mammals, e.g., non-human primates. In some embodiments, the subject is a human.

[0435] The dosage of a KRAS inhibitor, a fusion protein or polypeptide described herein, a polynucleotide described herein, a vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein to be administered to a subject according to any of the methods described herein can be determined according to standard techniques known to those skilled in the art, including the route of administration, the age and weight of the subject, and the type of adjuvant (if any) to be used. In some embodiments, each agent is administered in an amount (e.g., an effective amount) effective to mediate the method.

[0436] 5.11.1 Method of Delivery In one aspect, a method for delivering a KRAS (e.g., hKRAS) inhibitor and a fusion protein to a subject includes: (i) administering to a subject a pharmaceutical composition comprising (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein) or (b) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein);(ii) (a) a fusion protein described herein, (b) a polynucleotide encoding a fusion protein described herein, (c) a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), (d) a host cell described herein (e.g., a host cell comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), (e) a carrier described herein (e.g., a carrier comprising a fusion protein described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), or (f) a pharmaceutical composition described herein (e.g., a fusion protein described herein). Provided herein are methods for delivering a KRAS (e.g., hKRAS) inhibitor and a fusion protein to a subject, comprising administering to the subject a pharmaceutical composition comprising a fusion protein described herein, a polynucleotide encoding a fusion protein described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein), a host cell described herein (e.g., a host cell comprising a fusion protein described herein, a polynucleotide encoding a fusion protein described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), or a carrier described herein (e.g., a carrier comprising a fusion protein described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), thereby delivering a KRAS (e.g., hKRAS) inhibitor and a fusion protein to a subject;

[0437] In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor). In some embodiments, a KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer in the subject is resistant to the KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0438] In one aspect, provided herein is a method of delivering a KRAS (e.g., hKRAS) inhibitor and a fusion protein to a subject, the method comprising administering to the subject: (i) a pharmaceutical composition comprising: (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein); and (ii) (a) a fusion protein described herein, (b) a polynucleotide encoding a fusion protein described herein, (c) a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), (d) a host cell described herein (e.g., a host cell comprising a fusion protein described herein, a polynucleotide encoding a fusion protein described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), or (e) a carrier described herein (e.g., a carrier comprising a fusion protein described herein, a polynucleotide encoding a fusion protein described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), thereby delivering a KRAS (e.g., hKRAS) inhibitor and a fusion protein to a subject.

[0439] In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor). In some embodiments, a KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer in the subject is resistant to the KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0440] In one aspect, provided herein is a method of delivering a KRAS (e.g., hKRAS) inhibitor and a fusion protein to a subject, the method comprising administering to the subject a combination composition described herein (see, e.g., §5.8) or a combination regimen described herein (see, e.g., §5.7), thereby delivering a KRAS (e.g., hKRAS) inhibitor and a fusion protein.

[0441] In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor). In some embodiments, a KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer in the subject is resistant to the KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0442] 5.11.2 Methods of Inhibiting the KRAS and TGFβ Pathways

[0010] In one aspect, a method of inhibiting the KRAS (e.g., hKRAS) and TGFβ pathway in a subject in need thereof includes administering to a subject a pharmaceutical composition comprising: (i) (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein) or (b) a KRAS (e.g., hKRAS) inhibitor described herein;(ii) (a) a fusion protein (or polypeptide thereof) described herein, (b) a polynucleotide encoding a fusion protein described herein, (c) a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), (d) a host cell described herein (e.g., a host cell comprising a fusion protein described herein, a polynucleotide encoding a fusion protein described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), (e) a carrier described herein (e.g., a carrier comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), or (f) a pharmaceutical composition described herein (e.g., a fusion protein described herein, a polynucleotide encoding a fusion protein described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)). Provided herein are methods for inhibiting the KRAS (e.g., hKRAS) pathway and the TGFβ pathway in a subject, thereby comprising administering to a subject a polynucleotide encoding a fusion protein described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein), a host cell described herein (e.g., a host cell comprising a fusion protein described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), or a carrier described herein (e.g., a pharmaceutical composition comprising a fusion protein described herein, a polynucleotide encoding a fusion protein described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)).

[0443] In some embodiments, the KRAS (eg, hKRAS) inhibitor and fusion protein are administered in an amount and for a sufficient time to inhibit the KRAS (eg, hKRAS) pathway and the TGFβ pathway in the subject.

[0444] As described herein, (i) and (ii) can be administered to a subject concurrently or sequentially. In some embodiments, (i) and (ii) are administered concurrently. In some embodiments, (i) and (ii) are administered sequentially. In some embodiments, (i) is administered before (ii). In some embodiments, (ii) is administered before (i).

[0445] In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor). In some embodiments, a KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer in the subject is resistant to the KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0446] In one aspect, provided herein is a method of inhibiting the KRAS (e.g., hKRAS) and TGFβ pathways in a subject in need thereof, comprising delivering to the subject a combination composition described herein (see, e.g., §5.8) or a combination regimen described herein (see, e.g., §5.7), thereby inhibiting the KRAS (e.g., hKRAS) and TGFβ pathways in the subject.

[0447] In some embodiments, the KRAS (eg, hKRAS) inhibitor and fusion protein are administered in an amount and for a sufficient time to inhibit the KRAS (eg, hKRAS) pathway and the TGFβ pathway in the subject.

[0448] In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor). In some embodiments, a KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer in the subject is resistant to the KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0449]

[0013] In one aspect, a method for inhibiting the KRAS (e.g., hKRAS) and TGFβ pathway in a subject in need thereof is provided, comprising administering to a subject a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein); (ii) (a) a fusion protein (or polypeptide thereof) described herein, (b) a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein, (c) a vector comprising the polynucleotide described herein (e.g., a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein), (d) a host cell described herein (e.g., a vector comprising the fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein), (e.g., a vector comprising the fusion protein (or polypeptide thereof) described herein), (f) a vector comprising the fusion protein (or polypeptide thereof) described herein, (g) a vector comprising the fusion protein (or polypeptide thereof) described herein, (h) a vector comprising the fusion protein (or polypeptide thereof) described herein, (i) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein); Provided herein are methods that include administering to a subject a pharmaceutical composition comprising (e) a polynucleotide encoding a fusion protein (or polypeptide thereof), a vector comprising the polynucleotide described herein (e.g., a host cell comprising a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein) or (e) a carrier described herein (e.g., a carrier comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)).

[0450] In some embodiments, the KRAS (eg, hKRAS) inhibitor and fusion protein are administered in an amount and for a sufficient time to inhibit the KRAS (eg, hKRAS) pathway and the TGFβ pathway in the subject.

[0451] As described herein, (i) and (ii) can be administered to a subject concurrently or sequentially. In some embodiments, (i) and (ii) are administered concurrently. In some embodiments, (i) and (ii) are administered sequentially. In some embodiments, (i) is administered before (ii). In some embodiments, (ii) is administered before (i).

[0452] In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor). In some embodiments, a KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer in the subject is resistant to the KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0453] In one aspect, the present invention relates to a combination therapy for inhibiting the KRAS (e.g., hKRAS) and TGFβ pathways in a subject in need thereof, comprising administering the combination therapy to the subject; and (i) a pharmaceutical composition comprising: (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein) or (b) a KRAS (e.g., hKRAS) inhibitor described herein; and (ii) (a) a fusion protein (or polypeptide thereof) described herein, (b) a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein, (c) a vector comprising the polynucleotide described herein (e.g., a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein), (d) a host cell described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein, a vector comprising the polynucleotide described herein (e.g., a fusion protein (or polypeptide thereof) described herein), or (e.g., a vector comprising the polynucleotide described herein (e.g., a fusion protein (or polypeptide thereof) described herein). (e) a carrier described herein (e.g., a carrier comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)), or (f) a pharmaceutical composition described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), a host cell described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein,Provided herein are combination therapies comprising a host cell comprising a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein) or a carrier described herein (e.g., a pharmaceutical composition comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, or a carrier comprising a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)).

[0454] In some embodiments, the KRAS (eg, hKRAS) inhibitor and fusion protein are administered in an amount and for a sufficient time to inhibit the KRAS (eg, hKRAS) pathway and the TGFβ pathway in the subject.

[0455] As described herein, (i) and (ii) can be administered to a subject concurrently or sequentially. In some embodiments, (i) and (ii) are administered concurrently. In some embodiments, (i) and (ii) are administered sequentially. In some embodiments, (i) is administered before (ii). In some embodiments, (ii) is administered before (i).

[0456] In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor). In some embodiments, a KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer in the subject is resistant to the KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0457] In one aspect, a combination therapy for use in a method of inhibiting the KRAS (e.g., hKRAS) and TGFβ pathways in a subject in need thereof, comprising: (i) a pharmaceutical composition comprising (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein) or (b) a KRAS (e.g., hKRAS) inhibitor described herein; and (ii) (a) a fusion protein (or polypeptide thereof) described herein, (b) a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein, (c) a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein), (d) a host cell described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding the fusion protein (or polypeptide thereof) described herein). (e) a carrier described herein (e.g., a carrier comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)), or (f) a pharmaceutical composition described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), a host cell described herein (e.g., a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein),Provided herein is a combination therapy comprising a host cell comprising a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein), or a carrier described herein (e.g., a pharmaceutical composition comprising a fusion protein (or polypeptide thereof) described herein, a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein, a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein (or polypeptide thereof) described herein)), wherein the method comprises administering the combination therapy to a subject.

[0458] In some embodiments, the KRAS (eg, hKRAS) inhibitor and fusion protein are administered in an amount and for a sufficient time to inhibit the KRAS (eg, hKRAS) pathway and the TGFβ pathway in the subject.

[0459] As described herein, (i) and (ii) can be administered to a subject concurrently or sequentially. In some embodiments, (i) and (ii) are administered concurrently. In some embodiments, (i) and (ii) are administered sequentially. In some embodiments, (i) is administered before (ii). In some embodiments, (ii) is administered before (i).

[0460] In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor). In some embodiments, a KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject. In some embodiments, the subject has previously been administered a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor), and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor. In some embodiments, the cancer in the subject is resistant to the KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor).

[0461] In one aspect, (i) (a) a fusion protein (or polypeptide thereof) described herein, (b) a polynucleotide encoding a fusion protein described herein, (c) a vector comprising a polynucleotide described herein (e.g., a polynucleotide encoding a fusion protein described herein), (d) a host cell described herein (e.g., a host cell comprising a fusion protein described herein, a polynucleotide encoding a fusion protein described herein, or a vector comprising a polynucleotide described herein (e.g., a vector comprising a polynucleotide encoding a fusion protein described herein)), (e) a carrier described herein (e.g., a carrier comprising a fusion protein (or polypeptide thereof) described herein...

Claims

1. 1. A method of treating cancer in a subject in need thereof, comprising: (a) a Kirsten rat sarcoma virus homolog (KRAS) (e.g., a human KRAS (hKRAS)) inhibitor, (b) in combination with a fusion protein comprising (i) a first partial structure that specifically binds to epidermal growth factor receptor (EGFR) (e.g., human EGFR (hEGFR)), said first partial structure operably linked to (ii) a second partial structure that specifically binds to transforming growth factor β (TGFβ) (e.g., human TGFβ (hTGFβ)); administering to said subject thereby treating cancer in said subject.

2. 10. The method of claim 1, wherein the KRAS (e.g., hKRAS) inhibitor and the fusion protein are administered in an amount and for a time sufficient to treat or prevent the cancer in the subject.

3. 1. A method of treating a KRAS (e.g., hKRAS) variant cancer in a subject in need thereof, comprising: (a) receiving test results indicating that the KRAS (e.g., hKRAS) variant cancer exhibits resistance to a KRAS inhibitor; and (b) administering to the subject the KRAS (e.g., hKRAS) inhibitor in combination with a fusion protein comprising (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), the first partial structure operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ). thereby treating said KRAS (e.g., hKRAS) variant cancer in said subject.

4. 4. The method of claim 3, wherein the KRAS (e.g., hKRAS) inhibitor has been previously administered to the subject.

5. 5. The method of claim 3 or 4, wherein the KRAS (e.g., hKRAS) inhibitor and the fusion protein are administered in an amount and for a time sufficient to treat the KRAS (e.g., hKRAS) variant cancer in the subject.

6. 1. A method of treating a KRAS (e.g., hKRAS) variant cancer in a subject in need thereof, comprising: (a) administering to the subject a KRAS (e.g., hKRAS) inhibitor; (b) receiving test results indicating that the KRAS (e.g., hKRAS) variant cancer has developed resistance to the KRAS (e.g., hKRAS) inhibitor; and (c) administering to the subject the KRAS (e.g., hKRAS) inhibitor in combination with a fusion protein comprising (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), the first partial structure operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ). thereby treating said KRAS (e.g., hKRAS) variant cancer in said subject.

7. 7. The method of claim 6, wherein the KRAS (e.g., hKRAS) inhibitor and the fusion protein are administered in an amount and for a time sufficient to treat the KRAS (e.g., hKRAS) variant cancer in the subject.

8. 1. A method of treating a KRAS (e.g., hKRAS) variant cancer in a subject in need thereof, comprising: (a) determining that the KRAS (e.g., hKRAS) variant cancer is resistant to a KRAS (e.g., hKRAS) inhibitor; and (b) administering to the subject a KRAS (e.g., hKRAS) inhibitor in combination with a fusion protein comprising (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), said first partial structure operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ). thereby treating said KRAS (e.g., hKRAS) variant cancer in said subject.

9. 9. The method of claim 8, wherein the subject has previously received treatment with the KRAS (e.g., hKRAS) inhibitor.

10. 10. The method of claim 8 or 9, wherein the KRAS (e.g., hKRAS) inhibitor and the fusion protein are administered in an amount and for a time sufficient to treat the KRAS (e.g., hKRAS) variant cancer in the subject.

11. 10. The method of any one of the preceding claims, wherein the cancer is a KRAS (e.g., hKRAS) variant cancer.

12. 10. The method of any one of the preceding claims, wherein the cancer is determined to contain a KRAS (e.g., hKRAS) variant.

13. 13. The method of claim 11 or 12, wherein the KRAS (e.g., hKRAS) variant comprises a KRAS (e.g., hKRAS)-activating amino acid modification (e.g., substitution).

14. 14. The method of any one of claims 11 to 13, wherein the KRAS (e.g., hKRAS) variant comprises an amino acid modification (e.g., substitution) at amino acid positions G12, G13, or Q61, numbered based on the amino acid sequence of SEQ ID NO:

3.

15. 15. The method of any one of claims 11 to 14, wherein the KRAS (e.g., hKRAS) variant comprises an amino acid modification (e.g., substitution) at amino acid position G12 or G13, numbered based on the amino acid sequence of SEQ ID NO:

3.

16. 16. The method of any one of claims 11 to 15, wherein the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered based on the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, G12S, G13D, or Q61H.

17. 17. The method of any one of claims 11 to 16, wherein the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered based on the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, G12S, or G13D.

18. 18. The method of any one of claims 11 to 17, wherein the KRAS (e.g., hKRAS) variant comprises any one of the following amino acid substitutions, numbered based on the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12A, G12S, or G12D.

19. 19. The method of any one of claims 11 to 18, wherein the KRAS (e.g., hKRAS) variant comprises a G12C amino acid substitution, numbered based on the amino acid sequence of SEQ ID NO:

3.

20. 10. The method of any one of the preceding claims, wherein the cancer is resistant to the KRAS (e.g., hKRAS) inhibitor.

21. 10. The method of any one of the preceding claims, wherein the cancer has been determined to be resistant to the KRAS (e.g., hKRAS) inhibitor.

22. 2. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) inhibitor has been previously administered to the subject.

23. 10. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) inhibitor has previously been administered to the subject in the absence of the fusion protein.

24. 2. The method of any one of the preceding claims, wherein the cancer cells overexpress EGFR (e.g., hEGFR) compared to non-cancer cells.

25. 2. The method of any one of the preceding claims, wherein the cancer cells have been determined to overexpress EGFR (e.g., hEGFR) compared to non-cancer cells.

26. 2. The method of any one of the preceding claims, wherein the cancer cells express an EGFR (e.g., hEGFR) variant.

27. 2. The method of any one of the preceding claims, wherein the cancer cells are determined to express an EGFR (e.g., hEGFR) variant.

28. 2. The method of any one of the preceding claims, wherein the cancer cells overexpress hTGFβ (e.g., hTGFβ) compared to non-cancerous cells.

29. 2. The method of any one of the preceding claims, wherein the cancer cells have been determined to overexpress TGFβ (e.g., hTGFβ) compared to non-cancerous cells.

30. 10. The method of any one of the preceding claims, wherein the cancer is localized, locally advanced, or metastatic.

31. 10. The method of any one of the preceding claims, wherein the cancer is a carcinoma (e.g., adenocarcinoma, squamous cell carcinoma).

32. 2. The method of any one of the preceding claims, wherein the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma), colorectal cancer (e.g., colorectal adenocarcinoma), colon cancer (e.g., colon adenocarcinoma), rectal cancer (e.g., rectal adenocarcinoma), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), breast cancer (e.g., invasive ductal carcinoma), gastric cancer (e.g., gastric adenocarcinoma), endometrial cancer (e.g., undifferentiated endometrial carcinoma), uterine cancer (e.g., endometrial carcinoma), testicular cancer (e.g., testicular germ cell carcinoma), cervical cancer (e.g., cervical squamous cell carcinoma), bile duct cancer (e.g., cholangiocarcinoma), myelodysplastic cancer, or esophageal cancer (e.g., esophageal adenocarcinoma, gastroesophageal junction cancer).

33. 2. The method of any one of the preceding claims, wherein the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma), colorectal cancer (e.g., colorectal adenocarcinoma), colon cancer (e.g., colon adenocarcinoma), rectal cancer (e.g., rectal adenocarcinoma), or pancreatic cancer (e.g., pancreatic ductal adenocarcinoma).

34. 10. The method of any one of the preceding claims, wherein the cancer is non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, colorectal adenocarcinoma, or pancreatic ductal adenocarcinoma.

35. 1. A method for delivering a KRAS (e.g., hKRAS) inhibitor and a fusion protein to a subject in need thereof, comprising: (a) administering a KRAS (e.g., hKRAS) inhibitor to (b) a fusion protein comprising (i) a first partial structure that specifically binds to EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds to TGFβ (e.g., hTGFβ). administering in combination with thereby delivering said KRAS (e.g., hKRAS) inhibitor and fusion protein to a subject.

36. 1. A method of inhibiting the KRAS (e.g., hKRAS) pathway and the TGFβ (e.g., hTGFβ) pathway in a subject in need thereof, comprising: (a) administering a KRAS (e.g., hKRAS) inhibitor to (b) a fusion protein comprising (i) a first partial structure that specifically binds to EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds to TGFβ (e.g., hTGFβ). administering in combination with thereby inhibiting the KRAS (e.g., hKRAS) pathway and the TGFβ (e.g., hTGFβ) pathway in the subject.

37. The method of claim 36, wherein the KRAS (e.g., hKRAS) inhibitor and the fusion protein are administered in an amount and for a time sufficient to inhibit the KRAS (e.g., hKRAS) pathway and the TGFβ (e.g., hTGFβ) pathway in the subject.

38. A method for restoring sensitivity to a KRAS (e.g., hKRAS) inhibitor in a subject in need thereof, comprising administering to the subject a fusion protein comprising (i) a first partial structure that specifically binds to EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds to TGFβ (e.g., hTGFβ), thereby restoring sensitivity to the KRAS (e.g., hKRAS) inhibitor in the subject.

39. 39. The method of claim 38, wherein the fusion protein is administered in an amount and for a time sufficient to restore sensitivity to the KRAS (e.g., hKRAS) inhibitor in the subject.

40. 40. The method of any one of claims 38 to 39, wherein the cancer in the subject is resistant to the KRAS (e.g., hKRAS) inhibitor.

41. 41. The method of any one of claims 38 to 40, wherein the cancer has been determined to be resistant to the KRAS (e.g., hKRAS) inhibitor.

42. 42. The method of any one of claims 38 to 41, further comprising administering to the subject the KRAS (e.g., hKRAS) inhibitor in combination with the fusion protein.

43. 1. A method of suppressing or preventing resistance to a KRAS (e.g., hKRAS) inhibitor in a subject in need thereof, comprising: (a) administering a KRAS (e.g., hKRAS) inhibitor to (b) a fusion protein comprising (i) a first partial structure that specifically binds to EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds to TGFβ (e.g., hTGFβ). administering to said subject in combination with thereby suppressing or preventing resistance to said KRAS (e.g., hKRAS) inhibitor in said subject.

44. 44. The method of claim 43, wherein the fusion protein is administered in an amount and for a time sufficient to inhibit the development of resistance to the KRAS (e.g., hKRAS) inhibitor in the subject.

45. 45. The method of any one of claims 35 to 44, wherein the KRAS (e.g., hKRAS) inhibitor has previously been administered to the subject.

46. The method of any one of the preceding claims, wherein the subject has previously received a KRAS (e.g., hKRAS) inhibitor (e.g., the KRAS (e.g., hKRAS) inhibitor).

47. 10. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) inhibitor has not been previously administered to the subject.

48. The method of any one of the preceding claims, wherein the subject has previously received a KRAS (e.g., hKRAS) inhibitor (e.g., the KRAS (e.g., hKRAS) inhibitor) and the cancer in the subject has developed resistance to the KRAS (e.g., hKRAS) inhibitor.

49. The method of any one of the preceding claims, wherein the cancer in the subject is resistant to a KRAS (e.g., hKRAS) inhibitor (e.g., the KRAS (e.g., hKRAS) inhibitor).

50. 2. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) inhibitor is capable of selectively inhibiting KRAS (e.g., hKRAS) variants.

51. 2. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) variant comprises a KRAS (e.g., hKRAS)-activating amino acid modification.

52. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) variant comprises an amino acid modification at amino acid positions G12, G13, or Q61, numbered based on the amino acid sequence of SEQ ID NO:

3.

53. 2. The method of claim 1, wherein the KRAS (e.g., hKRAS) variant comprises an amino acid modification at amino acid position G12 or G13, numbered based on the amino acid sequence of SEQ ID NO:

3.

54. 10. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered based on the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, G13D, or Q61H.

55. 3. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) variant comprises any one or more of the following amino acid substitutions, numbered based on the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12D, G12A, or G13D.

56. 3. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) variant comprises any one of the following amino acid substitutions, numbered based on the amino acid sequence of SEQ ID NO: 3: G12C, G12V, G12R, G12A, or G12D.

57. 10. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) variant comprises a G12C amino acid substitution, numbered based on the amino acid sequence of SEQ ID NO:

3.

58. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) inhibitor comprises or consists of a small molecule, a protein (e.g., an antibody or a functional fragment or variant thereof), a nucleic acid, a carbohydrate, a lipid, a metal, or a toxin.

59. 10. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) inhibitor comprises or consists of a small molecule.

60. 2. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) inhibitor comprises a KRAS (e.g., hKRAS) inhibitor in Table 2.

61. 10. The method of any one of the preceding claims, wherein the KRAS (e.g., hKRAS) inhibitor comprises sotorasib (AMG-510).

62. 10. The method of any one of the preceding claims, wherein the first substructure comprises an antibody, or a functional fragment or variant thereof.

63. 10. The method of any one of the preceding claims, wherein the first substructure comprises a full-length antibody, a single-chain variable fragment (scFv), a Fab, or a single-domain antibody (sdAb).

64. 10. The method of claim 1, wherein the first substructure comprises a full-length antibody.

65. 10. The method of claim 9, wherein the first partial structure comprises a variable heavy chain (VH) region comprising three complementarity determining regions: VH CDR1, VH CDR2, and VH CDR3, and a variable light chain (VL) region comprising three complementarity determining regions: VL CDR1, VL CDR2, and VL CDR3.

66. (a) the amino acid sequence of the VH CDR1 comprises the amino acid sequence SEQ ID NO:39, or the amino acid sequence of SEQ ID NO:39 containing one, two, or three amino acid modifications; (b) the amino acid sequence of VH CDR2 comprises the amino acid sequence SEQ ID NO:40, or the amino acid sequence of SEQ ID NO:40 containing one, two, or three amino acid modifications; (c) the amino acid sequence of the VH CDR3 comprises the amino acid sequence SEQ ID NO:41, or the amino acid sequence of SEQ ID NO:41 containing one, two, or three amino acid modifications; (d) the amino acid sequence of the VL CDR1 comprises the amino acid sequence SEQ ID NO:42, or the amino acid sequence of SEQ ID NO:42 containing one, two, or three amino acid modifications; (e) the amino acid sequence of the VL CDR2 comprises the amino acid sequence SEQ ID NO:43, or the amino acid sequence of SEQ ID NO:43 containing one, two, or three amino acid modifications; (f) the amino acid sequence of the VL CDR3 comprises the amino acid sequence SEQ ID NO:44, or the amino acid sequence of SEQ ID NO:44 containing one, two, or three amino acid modifications; 10. A method according to any one of the preceding claims.

67. 10. The method of claim 1, wherein the VH region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45; and the VL region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

46.

68. 10. The method of claim 1, wherein the first partial structure comprises a heavy chain (HC) and a light chain (LC), and the amino acid sequence of the HC is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48; and the amino acid sequence of the LC is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

49.

69. 10. The method of any one of the preceding claims, wherein the second substructure comprises an antibody, or a functional fragment or variant thereof.

70. 2. The method of any one of the preceding claims, wherein the second substructure comprises a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

71. 10. The method of any one of the preceding claims, wherein the second substructure comprises or consists of at least a portion of the extracellular domain (ECD) of a transforming growth factor-beta receptor (TGFβR) (e.g., hTGFβR).

72. 2. The method of any one of the preceding claims, wherein the second substructure comprises or consists of at least a portion of the ECD of transforming growth factor-beta receptor II (TGFβRII) (e.g., hTGFβRII).

73. 2. The method of claim 1, wherein the second partial structure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII).

74. 10. The method of any one of the preceding claims, wherein the amino acid sequence of the second substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73.

75. 10. The method of claim 1, wherein the fusion protein comprises (iii) a third partial structure that specifically binds to TGFβ (e.g., hTGFβ), the third partial structure being operably connected to the first partial structure.

76. 10. The method of claim 1, wherein the third substructure comprises an antibody, or a functional fragment or variant thereof.

77. 2. The method of claim 1, wherein the third substructure comprises a full-length antibody, a single-chain variable fragment (scFv), a scFv2, a scFv-Fc, a Fab, a Fab', a F(ab')2, or an F(v).

78. 2. The method of claim 1, wherein the third substructure comprises or consists of at least a portion of the extracellular domain (ECD) of a TGFβR (e.g., hTGFβR).

79. 2. The method of claim 1, wherein the third partial structure comprises or consists of at least a portion of the ECD of TGFβRII (e.g., hTGFβRII).

80. 2. The method of claim 1, wherein the third partial structure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII).

81. 10. The method of any one of the preceding claims, wherein the amino acid sequence of the third substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73.

82. 7. The method of any one of the preceding claims, wherein the amino acid sequence of the second substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73; and the amino acid sequence of the third substructure comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72 or 73.

83. 10. The method of any one of the preceding claims, wherein the amino acid sequence of the second substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the third substructure.

84. 10. The method of any one of the preceding claims, wherein the first substructure comprises a full-length antibody and the N-terminus of the second substructure is operably connected (e.g., optionally via a linker) to the C-terminus of a first light chain.

85. 10. The method of any one of the preceding claims, wherein the first substructure comprises a full-length antibody and the N-terminus of the second substructure is operably connected (e.g., optionally via a linker) to the C-terminus of the first heavy chain.

86. 10. The method of claim 1, wherein the first partial structure comprises a full-length antibody, the N-terminus of the second partial structure is operably connected to the C-terminus of the first light chain (e.g., optionally via a linker), and the N-terminus of the third partial structure is operably connected to the C-terminus of the second light chain (e.g., optionally via a linker).

87. 10. The method of claim 1, wherein the first partial structure comprises a full-length antibody, the N-terminus of the second partial structure is operably connected to the C-terminus of the first heavy chain (e.g., optionally via a linker), and the N-terminus of the third partial structure is operably connected to the C-terminus of the second heavy chain (e.g., optionally via a linker).

88. 10. A method according to any one of the preceding claims, wherein the first sub-structure is operatively connected directly to the second sub-structure.

89. 10. A method according to any one of the preceding claims, wherein the first sub-structure is indirectly operably connected to the second sub-structure.

90. 2. The method of claim 1, wherein the first substructure is operably connected to the second substructure indirectly by a first peptide linker.

91. 10. The method of claim 1, wherein the first peptide linker comprises or consists of glycine or glycine and serine amino acid residues.

92. 10. The method of any one of the preceding claims, wherein the amino acid sequence of the first peptide linker comprises or consists of: (a) the amino acid sequence of any one of SEQ ID NOs: 105-124; or (b) the amino acid sequence of any one of SEQ ID NOs: 105-124 comprising or consisting of one, two or three amino acid modifications.

93. 10. The method of any one of the preceding claims, wherein the amino acid sequence of the first peptide linker comprises or consists of: (a) the amino acid sequence of SEQ ID NO: 111; or (b) the amino acid sequence of SEQ ID NO: 111 containing or consisting of one, two or three amino acid modifications.

94. 10. The method of claim 1, wherein the first substructure is operatively connected directly to the third substructure.

95. 10. A method according to any one of the preceding claims, wherein the first sub-structure is indirectly operably connected to the third sub-structure.

96. 2. The method of claim 1, wherein the first substructure is operably connected to the third substructure indirectly by a second peptide linker.

97. 97. The method of claim 96, wherein the second peptide linker comprises or consists of glycine or glycine and serine amino acid residues.

98. 98. The method of claim 96 or 97, wherein the amino acid sequence of the second peptide linker comprises or consists of: (a) the amino acid sequence of any one of SEQ ID NOs: 111; or (b) the amino acid sequence of any one of SEQ ID NOs: 111 containing or consisting of one, two, or three amino acid modifications.

99. 99. The method of any one of claims 96 to 98, wherein the amino acid sequence of the first peptide linker comprises or consists of: (a) the amino acid sequence of SEQ ID NO: 111; or (b) the amino acid sequence of SEQ ID NO: 111 containing or consisting of one, two or three amino acid modifications.

100. 100. The method of any one of claims 96 to 99, wherein the amino acid sequence of the first peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the second peptide linker.

101. 101. The method of any one of claims 96 to 100, wherein the amino acid sequence of the first peptide linker is 100% identical to the amino acid sequence of the second peptide linker.

102. (a) the first substructure comprises or consists of a full-length antibody; (b) the second substructure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII); (c) the third substructure comprises or consists of the ECD of TGFβRII (e.g., hTGFβRII); (d) the second substructure is operably connected to the first substructure by a first peptide linker; (e) the third substructure is operably connected to the first substructure by a second peptide linker; 10. A method according to any one of the preceding claims.

103. 103. The method of claim 102, wherein the N-terminus of the second partial structure is operably connected to the C-terminus of the first light chain of the full-length antibody, and the N-terminus of the third partial structure is operably connected to the C-terminus of the second light chain of the full-length antibody.

104. (a)(i) the amino acid sequence of a VH CDR1 comprises the amino acid sequence SEQ ID NO:39, or the amino acid sequence of SEQ ID NO:39 with one, two or three amino acid modifications; the amino acid sequence of a VH CDR2 comprises the amino acid sequence SEQ ID NO:40, or the amino acid sequence of SEQ ID NO:40 with one, two or three amino acid modifications; the amino acid sequence of a VH CDR3 comprises the amino acid sequence SEQ ID NO:41, or the amino acid sequence of SEQ ID NO:41 with one, two or three amino acid modifications; the amino acid sequence of a VL CDR1 comprises the amino acid sequence SEQ ID NO:42, or the amino acid sequence of SEQ ID NO:42 with one, two or three amino acid modifications; and the amino acid sequence of a VL CDR2 comprises the amino acid sequence SEQ ID NO:43, or the amino acid sequence of SEQ ID NO:43 with one, two or three amino acid modifications; (ii) the amino acid sequence of the CDR3 comprises the amino acid sequence SEQ ID NO:44, or the amino acid sequence of SEQ ID NO:44 containing one, two, or three amino acid modifications; (ii) the VH region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:45, and the VL region comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

46. and / or (iii) the amino acid sequence of the heavy chain of the full-length antibody is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:48 and the amino acid sequence of the light chain of the full-length antibody is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:49; (b) the amino acid sequence of the second substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:72; (c) the amino acid sequence of the third substructure is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 72; (d) the amino acid sequence of the first peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:111; (e) the amino acid sequence of the second peptide linker is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:111; 104. The method of any one of claims 102 to 103.

105. the fusion protein (a) a first polypeptide, wherein the amino acid sequence of the first polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125; (b) a second polypeptide, wherein the amino acid sequence of the second polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125; (c) a third polypeptide, wherein the amino acid sequence of the third polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126; (d) a fourth polypeptide, wherein the amino acid sequence of the fourth polypeptide comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 126; and 10. The method of any one of the preceding claims, comprising:

106. (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein); (b) a fusion protein (e.g., a fusion protein described herein) comprising (i) a first partial structure that specifically binds to EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds to TGFβ (e.g., hTGFβ); A combination regimen including:

107. (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein); (b) a fusion protein (e.g., a fusion protein described herein) comprising (i) a first partial structure that specifically binds EGFR (e.g., hEGFR), said first partial structure operably linked to (ii) a second partial structure that specifically binds TGFβ (e.g., hTGFβ); (c) a pharmaceutically acceptable excipient; and 10. A pharmaceutical composition comprising:

108. (i) (a) a KRAS (e.g., hKRAS) inhibitor (e.g., a KRAS (e.g., hKRAS) inhibitor described herein), or a pharmaceutical composition thereof, and (b) a fusion protein (e.g., a fusion protein described herein), comprising (i) a first partial structure that specifically binds to EGFR (e.g., hEGFR), the first partial structure being operably linked to (ii) a second partial structure that specifically binds to TGFβ (e.g., hTGFβ), or a pharmaceutical composition thereof; (ii) a combination therapy according to claim 106; or (iii) The pharmaceutical composition according to claim 107. Kit including:

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