Pharmaceutical compositions comprising Anti-191p4d12 antibody drug conjugates and methods of use thereof

Abstract

To provide pharmaceutical compositions of ANTI-191P4D12 antibody drug conjugates.SOLUTION: A pharmaceutical composition comprises an antibody drug conjugate comprising an antibody or antigen binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethyl auristatin E (MMAE) and a pharmaceutically acceptable excipient comprising L-histidine, polysorbate-20 (TWEEN-20(R)), and at least one of trehalose dihydrate and sucrose.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 774,819, filed December 3, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] 1. Field Provided herein are pharmaceutical compositions comprising anti-191P4D12 antibody drug conjugates. Also provided herein are methods of using the pharmaceutical compositions. [Background technology]

[0003] 2. background A drug substance is usually administered as part of a formulation in combination with one or more other agents that perform various specialized pharmaceutical functions. Pharmaceutical excipients have various functions and contribute to pharmaceutical formulations in many different ways, such as solubilizing, diluting, thickening, stabilizing, preserving, coloring, flavoring, etc. Properties that can be considered when formulating an active drug substance include bioavailability, ease of manufacture, ease of administration, and dosage form stability. Due to the various properties of the active drug substance to be formulated, dosage forms typically require pharmaceutical excipients that are uniquely tailored to the active drug substance to achieve advantageous physical and pharmaceutical properties.

[0004] Thus, a need exists for pharmaceutical compositions of anti-191P4D12 antibody drug conjugates that have advantageous physical and pharmaceutical properties. The present invention fulfills this need and provides related advantages. Summary of the Invention

[0005] 3. overview In one aspect, provided herein is a pharmaceutical composition comprising: (a) an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethyl auristatin E (MMAE), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:7, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:8; and (b) a pharmaceutically acceptable excipient comprising L-histidine, polysorbate 20 (TWEEN-20®), and at least one of trehalose dihydrate and sucrose.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR H1 comprising the amino acid sequence of SEQ ID NO:9, CDR H2 comprising the amino acid sequence of SEQ ID NO:10, CDR H3 comprising the amino acid sequence of SEQ ID NO:11; CDR L1 comprising the amino acid sequence of SEQ ID NO:12, CDR L2 comprising the amino acid sequence of SEQ ID NO:13, and CDR L3 comprising the amino acid sequence of SEQ ID NO:14.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (arginine) of SEQ ID NO:8.

[0008] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain comprising the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8.

[0009] In some embodiments, the antigen-binding fragment is a Fab, F(ab')2, Fv, or scFv fragment.

[0010] In some embodiments, the antibody is a fully human antibody.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof is recombinantly produced.

[0012] In some embodiments, the antibody drug conjugate has the following structure: TIFF2025170007000002.tif36160, where L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10.

[0013] In some embodiments, p is 2-8.

[0014] In some embodiments, the antibody or antigen-binding fragment is linked to each unit of monomethyl auristatin E (MMAE) via a linker.

[0015] In some embodiments, the linker is an enzyme-cleavable linker, and in one embodiment, the linker forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof.

[0016] In some embodiments, the linker is -A a -W w -Y y -A- is an extender unit, a is 0 or 1, -W- is an amino acid unit, w is an integer ranging from 0 to 12, -Y- is a spacer unit, and y is 0, 1, or 2.

[0017] In some embodiments, the extender unit has the structure of formula (1) below, the amino acid unit is valine citrulline, and the spacer unit is a PAB group having the structure of formula (2) below. TIFF2025170007000003.tif32128TIFF2025170007000004.tif35128

[0018] In some embodiments, the extender unit forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof, and the spacer unit is linked to the MMAE via a carbamate group.

[0019] In some embodiments, the antibody drug conjugate comprises 1 unit to 10 units of MMAE per antibody or antigen-binding fragment thereof.

[0020] In some embodiments, the antibody drug conjugate comprises 2 to 8 units of MMAE per antibody or antigen-binding fragment thereof.

[0021] In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 1 mg / mL to about 20 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 5 mg / mL to about 15 mg / mL. In other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 8 mg / mL to about 12 mg / mL. In still other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10 mg / mL.

[0022] In some embodiments, L-histidine is present in the range of about 5 mM to about 50 mM. In other embodiments, L-histidine is present in the range of about 10 mM to about 40 mM. In other embodiments, L-histidine is present in the range of about 15 mM to about 35 mM. In other embodiments, L-histidine is present in the range of about 15 mM to about 30 mM. In other embodiments, L-histidine is present in the range of about 15 mM to about 25 mM. In still other embodiments, L-histidine is present at about 20 mM.

[0023] In some embodiments, the concentration of TWEEN-20 ranges from about 0.001% to about 0.1% (v / v). In other embodiments, the concentration of TWEEN-20 ranges from about 0.0025% to about 0.075% (v / v). In other embodiments, the concentration of TWEEN-20 ranges from about 0.005% to about 0.05% (v / v). In other embodiments, the concentration of TWEEN-20 ranges from about 0.01% to about 0.03% (v / v). In still other embodiments, the concentration of TWEEN-20 ranges from about 0.02% (v / v).

[0024] In some embodiments, the pharmaceutical compositions provided herein comprise trehalose dihydrate. In some embodiments, trehalose dihydrate is present in the range of about 1% to about 20% (w / v). In some embodiments, trehalose dihydrate is present in the range of about 2% to about 15% (w / v). In other embodiments, trehalose dihydrate is present in the range of about 3% to about 10% (w / v). In still other embodiments, trehalose dihydrate is present in the range of about 4% to about 6% (w / v). In still other embodiments, trehalose dihydrate is present at about 5.5% (w / v).

[0025] In some embodiments, trehalose dihydrate is present in a range of about 50 mM to about 300 mM. In some embodiments, trehalose dihydrate is present in a range of about 75 mM to about 250 mM. In other embodiments, trehalose dihydrate is present in a range of about 100 mM to about 200 mM. In still other embodiments, trehalose dihydrate is present in a range of about 130 mM to about 150 mM. In still other embodiments, trehalose dihydrate is present at about 146 mM.

[0026] In some embodiments, the pharmaceutical composition comprises sucrose. In some embodiments, sucrose is present in the range of about 1% to about 20% (w / v). In some embodiments, sucrose is present in the range of about 2% to about 15% (w / v). In other embodiments, sucrose is present in the range of about 3% to about 10% (w / v). In other embodiments, sucrose is present in the range of about 4% to about 6% (w / v). In still other embodiments, sucrose is present at about 5.5% (w / v).

[0027] In some embodiments, sucrose is present in a range of about 50 mM to about 300 mM. In other embodiments, sucrose is present in a range of about 75 mM to about 250 mM. In other embodiments, sucrose is present in a range of about 100 mM to about 200 mM. In still other embodiments, sucrose is present in a range of about 130 mM to about 150 mM. In still other embodiments, sucrose is present at about 146 mM.

[0028] In some embodiments, the pharmaceutical composition has a pH ranging from about 5.5 to about 6.5. In some embodiments, the pharmaceutical composition has a pH ranging from about 5.7 to about 6.3. In other embodiments, the pharmaceutical composition has a pH of about 6.0.

[0029] In some embodiments, the pH is measured at room temperature. In some embodiments, the pH is measured at about 15°C to about 27°C. In other embodiments, the pH is measured at about 4°C. In other embodiments, the pH is measured at about 25°C.

[0030] In some embodiments, the pharmaceutical compositions provided herein comprise hydrochloric acid (HCl). In some embodiments, the pH is adjusted with HCl.

[0031] In other embodiments, the pharmaceutical compositions provided herein comprise succinic acid. In some embodiments, the pH is adjusted with succinic acid.

[0032] In some embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, and at least one of about 5.5% (w / v) trehalose dihydrate or about 5% (w / v) sucrose. In some embodiments, the pharmaceutical compositions provided herein further comprise HCl or succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In other embodiments, the pH is about 6.0 at 25°C.

[0033] In some specific aspects, the pharmaceutical compositions provided herein comprise: (a) the structure: TIFF2025170007000005.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and HCl. and has a pH of about 6.0 at 25°C.

[0034] In some embodiments, the antibody drug conjugate is at a concentration of about 10 mg / mL.

[0035] In other specific aspects, the pharmaceutical compositions provided herein comprise: (a) the structure: TIFF2025170007000006.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and succinic acid. and has a pH of about 6.0 at 25°C.

[0036] In some embodiments, the antibody drug conjugate is at a concentration of about 10 mg / mL in the pharmaceutical compositions provided herein.

[0037] In yet another specific aspect, the pharmaceutical compositions provided herein comprise: (a) the structure: TIFF2025170007000007.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.0% (w / v) sucrose, and HCl. and has a pH of about 6.0 at 25°C.

[0038] In some embodiments, the antibody drug conjugate is at a concentration of about 10 mg / mL in the pharmaceutical compositions provided herein.

[0039] In some embodiments, the pharmaceutical compositions provided herein are in liquid form.

[0040] In other aspects, the pharmaceutical compositions provided herein are lyophilized.

[0041] In another aspect, provided herein is a lyophilized composition made by lyophilizing a pharmaceutical composition provided herein.

[0042] In some embodiments, the pharmaceutical composition is stored at -80°C, 4°C, 25°C, or 37°C.

[0043] In another aspect, provided herein is a method of preventing or treating a disease or disorder in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition provided herein.

[0044] In some embodiments, the subject is a human subject.

[0045] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is colon cancer, pancreatic cancer, ovarian cancer, lung cancer, bladder cancer, breast cancer, esophageal cancer, head cancer, or neck cancer.

[0046] In specific embodiments, the cancer is colon cancer. In specific embodiments, the cancer is pancreatic cancer. In specific embodiments, the cancer is ovarian cancer. In specific embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In specific embodiments, the cancer is bladder cancer. In specific embodiments, the cancer is advanced bladder cancer. In specific embodiments, the cancer is metastatic bladder cancer. In specific embodiments, the cancer is breast cancer. In specific embodiments, the cancer is esophageal cancer. In specific embodiments, the cancer is head cancer. In specific embodiments, the cancer is neck cancer. In specific embodiments, the cancer has tumor cells that express 191P4D12.

[0047] In some embodiments, the methods provided herein further comprise administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In other embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In still other embodiments, the PD-1 inhibitor is pembrolizumab or nivolumab. In other embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In other embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0048] In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 10 mg / kg of subject body weight. In other embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 5 mg / kg of subject body weight. In still other embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 2.5 mg / kg of subject body weight. In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 1.25 mg / kg of subject body weight. In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of about 1 mg / kg of subject body weight. In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of about 1.25 mg / kg of subject body weight.

[0049] In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion.

[0050] In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes in two three-weekly cycles. In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1 and 8 of every three-weekly cycle. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion on day 1 of every three-weekly cycle. In some embodiments, the immune checkpoint inhibitor is pembrolizumab, and the pembrolizumab is administered in an amount of about 200 mg over about 30 minutes. In other embodiments, the immune checkpoint inhibitor is atezolizumab, and the atezolizumab is administered in an amount of about 1200 mg over about 60 minutes or about 30 minutes.

[0051] In other embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes, three times a four-week cycle. In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1, 8, and 15 of each four-week cycle. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In other embodiments, the immune checkpoint inhibitor is atezolizumab. [The present invention 1001] (a) an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethyl auristatin E (MMAE), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:7, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:8; and (b) a pharmaceutically acceptable excipient comprising L-histidine, polysorbate 20 (TWEEN-20®), and at least one of trehalose dihydrate and sucrose; 10. A pharmaceutical composition comprising: [The present invention 1002] 1001. A pharmaceutical composition of the present invention, wherein the antibody or antigen-binding fragment thereof comprises CDR H1 having the amino acid sequence of SEQ ID NO:9, CDR H2 having the amino acid sequence of SEQ ID NO:10, CDR H3 having the amino acid sequence of SEQ ID NO:11, CDR L1 having the amino acid sequence of SEQ ID NO:12, CDR L2 having the amino acid sequence of SEQ ID NO:13, and CDR L3 having the amino acid sequence of SEQ ID NO:14. [The present invention 1003] 1001. The pharmaceutical composition of the present invention, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 136th amino acid (serine) of SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) of SEQ ID NO:8. [The present invention 1004] 1001. The pharmaceutical composition of the present invention, wherein the antibody comprises a heavy chain comprising an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO:7, and a light chain comprising an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO:8. [The present invention 1005] The pharmaceutical composition of any of claims 1001 to 1004, wherein the antigen-binding fragment is a Fab, F(ab')2, Fv or scFv fragment. [The present invention 1006] 6. The pharmaceutical composition of any one of claims 1001 to 1005, wherein the antibody is a fully human antibody. [The present invention 1007] 1007. The pharmaceutical composition of any of claims 1001 to 1006, wherein the antibody or antigen-binding fragment thereof is recombinantly produced. [The present invention 1008] 1. The antibody drug conjugate of claim 1, wherein the antibody drug conjugate has the following structure: TIFF2025170007000008.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; The pharmaceutical composition of any one of 1001 to 1007 of the present invention. [The present invention 1009] The pharmaceutical composition of the present invention 1008, wherein p is 2 to 8. [The present invention 1010] 1001. The pharmaceutical composition of the present invention, wherein the antibody or antigen-binding fragment is linked to each unit of monomethyl auristatin E (MMAE) via a linker. [The present invention 1011] The pharmaceutical composition of the present invention 1010, wherein the linker is an enzyme-cleavable linker and forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof. [The present invention 1012] Linker is -A a -W w -Y y -having the formula wherein -A- is an extender unit, a is 0 or 1, -W- is an amino acid unit, w is an integer ranging from 0 to 12, -Y- is a spacer unit, and y is 0, 1, or 2. A pharmaceutical composition of the present invention. [The present invention 1013] 1012. The pharmaceutical composition of the present invention, wherein the extender unit has the structure of the following formula (1), the amino acid unit is valine citrulline, and the spacer unit is a PAB group having the structure of the following formula (2): TIFF2025170007000009.tif78128. [The present invention 1014] 1012. The pharmaceutical composition of the present invention, wherein the extender unit forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof, and the spacer unit is linked to MMAE via a carbamate group. [The present invention 1015] 1001. The pharmaceutical composition of the present invention, wherein the antibody drug conjugate comprises 1 to 10 units of MMAE per antibody or antigen-binding fragment thereof. [The present invention 1016] 1015. The pharmaceutical composition of the present invention, wherein the antibody drug conjugate comprises 2 to 8 units of MMAE per antibody or antigen-binding fragment thereof. [The present invention 1017] 1016. The pharmaceutical composition of any of claims 1001 to 1016, comprising the antibody-drug conjugate at a concentration of 1 to 20 mg / mL. [The present invention 1018] 1017. The pharmaceutical composition of the present invention, comprising an antibody-drug conjugate at a concentration of 5 to 15 mg / mL. [The present invention 1019] 1017. The pharmaceutical composition of the present invention, comprising an antibody-drug conjugate at a concentration of 8 to 12 mg / mL. [The present invention 1020] 1017. The pharmaceutical composition of the present invention, comprising an antibody drug conjugate at a concentration of about 10 mg / mL. [The present invention 1021] The pharmaceutical composition of any of claims 1001 to 1020, wherein L-histidine is present in the range of 5 to 50 mM. [The present invention 1022] The pharmaceutical composition of any one of claims 1001 to 1020, wherein L-histidine is present in the range of 10 to 40 mM. [The present invention 1023] The pharmaceutical composition of any of claims 1001 to 1020, wherein L-histidine is present in the range of 15 to 35 mM. [The present invention 1024] The pharmaceutical composition of any of claims 1001 to 1020, wherein L-histidine is present in the range of 15 to 30 mM. [The present invention 1025] The pharmaceutical composition of any one of claims 1001 to 1020, wherein L-histidine is present in the range of 15 to 25 mM. [The present invention 1026] 1020. The pharmaceutical composition of any of claims 1001 to 1020, wherein L-histidine is present at about 20 mM. [The present invention 1027] The pharmaceutical composition of any one of claims 1001 to 1026, wherein the concentration of TWEEN-20 is in the range of 0.001 to 0.1% (v / v). [The present invention 1028] The pharmaceutical composition of any one of claims 1001 to 1026, wherein the concentration of TWEEN-20 is in the range of 0.0025 to 0.075% (v / v). [The present invention 1029] The pharmaceutical composition of any one of claims 1001 to 1026, wherein the concentration of TWEEN-20 is in the range of 0.005 to 0.05% (v / v). [The present invention 1030] The pharmaceutical composition of any one of claims 1001 to 1026, wherein the concentration of TWEEN-20 is in the range of 0.01 to 0.03% (v / v). [The present invention 1031] 1027. The pharmaceutical composition of any one of claims 1001 to 1026, wherein the concentration of TWEEN-20 is in the range of about 0.02% (v / v). [The present invention 1032] Any of the pharmaceutical compositions of 1001 to 1031 of the present invention, which comprises trehalose dihydrate. [The present invention 1033] 1032. The pharmaceutical composition of the present invention, wherein trehalose dihydrate is present in the range of 1 to 20% (w / v). [The present invention 1034] 1032. The pharmaceutical composition of the present invention, wherein trehalose dihydrate is present in the range of 2 to 15% (w / v). [This invention 1035] 1032. The pharmaceutical composition of the present invention, wherein trehalose dihydrate is present in the range of 3 to 10% (w / v). [The present invention 1036] 1032. The pharmaceutical composition of the present invention, wherein trehalose dihydrate is present in the range of 4-6% (w / v). [This invention 1037] 1032. The pharmaceutical composition of claim 1032, wherein trehalose dihydrate is present at about 5.5% (w / v). [The present invention 1038] 1032. The pharmaceutical composition of the present invention, wherein trehalose dihydrate is present in the range of 50 mM to 300 mM. [This invention 1039] 1032. The pharmaceutical composition of the present invention, wherein trehalose dihydrate is present in the range of 75 mM to 250 mM. [The present invention 1040] 1032. The pharmaceutical composition of the present invention, wherein trehalose dihydrate is present in the range of 100 mM to 200 mM. [This invention 1041] 1032. The pharmaceutical composition of the present invention, wherein trehalose dihydrate is present in the range of 130 mM to 150 mM. [The present invention 1042] 1032. The pharmaceutical composition of claim 1032, wherein the trehalose dihydrate is present at about 146 mM. [This invention 1043] Any of the pharmaceutical compositions of 1001 to 1031 of the present invention, which contains sucrose. [This invention 1044] 1043. The pharmaceutical composition of the present invention, wherein sucrose is present in the range of 1 to 20% (w / v). [This invention 1045] 1043. The pharmaceutical composition of the present invention, wherein sucrose is present in the range of 2 to 15% (w / v). [The present invention 1046] 1043. The pharmaceutical composition of the present invention, wherein sucrose is present in the range of 3 to 10% (w / v). [This invention 1047] 1043. The pharmaceutical composition of the present invention, wherein sucrose is present in the range of 4-6% (w / v). [This invention 1048] 1043. The pharmaceutical composition of invention 1043, wherein sucrose is present at about 5.5% (w / v). [This invention 1049] 1043. The pharmaceutical composition of claim 1043, wherein sucrose is present in the range of 50 mM to 300 mM. [The present invention 1050] 1043. The pharmaceutical composition of claim 1043, wherein sucrose is present in the range of 75 mM to 250 mM. [This invention 1051] 1043. The pharmaceutical composition of the present invention, wherein sucrose is present in the range of 100 mM to 200 mM. [This invention 1052] 1043. The pharmaceutical composition of claim 1043, wherein sucrose is present in the range of 130 mM to 150 mM. [This invention 1053] 1043. The pharmaceutical composition of claim 1043, wherein sucrose is present at about 146 mM. [This invention 1054] 4. The pharmaceutical composition of any one of claims 1001 to 1053, having a pH in the range of 5.5 to 6.5. [This invention 1055] 4. The pharmaceutical composition of any one of claims 1001 to 1053, having a pH in the range of 5.7 to 6.3. [This invention 1056] 1001-1053. The pharmaceutical composition of any one of claims 1001-1053, having a pH of about 6.0. [This invention 1057] 1054-1056. The pharmaceutical composition of any one of claims 1054-1056, wherein the pH is measured at room temperature. [This invention 1058] Any of the pharmaceutical compositions of 1054 to 1056, wherein the pH is measured at 15°C to 27°C. [This invention 1059] Any of the pharmaceutical compositions of 1054 to 1056, wherein the pH is measured at 4°C. [The present invention 1060] Any of the pharmaceutical compositions of 1054 to 1056, wherein the pH is measured at 25°C. [This invention 1061] The pharmaceutical composition of any one of claims 1001 to 1060, comprising hydrochloric acid (HCl). [This invention 1062] 10. The pharmaceutical composition of any one of claims 1001 to 1060, wherein the pH is adjusted with HCl. [This invention 1063] Any of the pharmaceutical compositions of 1001 to 1060 of the present inventions, comprising succinic acid. [This invention 1064] The pharmaceutical composition of any one of claims 1001 to 1060, wherein the pH is adjusted with succinic acid. [This invention 1065] Any of the pharmaceutical compositions of claims 1001 to 1016, comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, and at least one of about 5.5% (w / v) trehalose dihydrate or about 5% (w / v) sucrose. [The present invention 1066] The pharmaceutical composition of invention 1065, further comprising HCl or succinic acid. [This invention 1067] The pharmaceutical composition of invention 1065 or invention 1066, having a pH of 6.0 at room temperature. [The present invention 1068] The pharmaceutical composition of invention 1065 or invention 1066, having a pH of 6.0 at 25°C. [The present invention 1069] (a) the structure: TIFF2025170007000010.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20®, about 5.5% (w / v) trehalose dihydrate, and HCl. and having a pH of about 6.0 at 25°C. Pharmaceutical compositions. [The present invention 1070] The pharmaceutical composition of the present invention 1069, wherein the antibody drug conjugate is at a concentration of about 10 mg / mL. [This invention 1071] (a) the structure: TIFF2025170007000011.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20®, about 5.5% (w / v) trehalose dihydrate, and succinic acid. and having a pH of about 6.0 at 25°C. Pharmaceutical compositions. [This invention 1072] 1071. The pharmaceutical composition of invention 1071, wherein the antibody drug conjugate is at a concentration of about 10 mg / mL. [This invention 1073] (a) the structure: TIFF2025170007000012.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20®, about 5.0% (w / v) sucrose, and HCl. and having a pH of about 6.0 at 25°C. Pharmaceutical compositions. [This invention 1074] The pharmaceutical composition of the present invention 1073, wherein the antibody drug conjugate is at a concentration of about 10 mg / mL. [This invention 1075] The pharmaceutical composition of any one of claims 1001 to 1074, which is in liquid form. [This invention 1076] The pharmaceutical composition of any one of claims 1001 to 1074, which is freeze-dried. [This invention 1077] A freeze-dried composition produced by freeze-drying any one of the pharmaceutical compositions of the present inventions 1001 to 1074. [This invention 1078] The pharmaceutical composition of any one of claims 1001 to 1074, which is stored at -80°C, 4°C, 25°C or 37°C. [This invention 1079] A method for preventing or treating a disease or disorder in a subject, comprising the step of administering to the subject an effective amount of the pharmaceutical composition of any one of the present inventions 1001 to 1078. [The present invention 1080] The method of claim 1079, wherein the subject is a human subject. [This invention 1081] The method of claim 1080, wherein the cancer is colon cancer, pancreatic cancer, ovarian cancer, lung cancer, bladder cancer, urothelial cancer, breast cancer, esophageal cancer, head cancer, or neck cancer. [This invention 1082] The method of claim 1081, wherein the cancer is colon cancer. [This invention 1083] The method of claim 1081, wherein the cancer is pancreatic cancer. [This invention 1084] The method of claim 1081, wherein the cancer is ovarian cancer. [This invention 1085] The method of claim 1081, wherein the cancer is lung cancer, and optionally the lung cancer is non-small cell lung cancer. [The present invention 1086] The method of claim 1081, wherein the cancer is bladder cancer or urothelial cancer. [This invention 1087] The method of claim 1086, wherein the bladder cancer is advanced bladder cancer or advanced urothelial carcinoma. [This invention 1088] The method of claim 1086, wherein the bladder cancer is metastatic bladder cancer or metastatic urothelial carcinoma. [This invention 1089] The method of claim 1081, wherein the cancer is breast cancer. [The present invention 1090] The method of claim 1081, wherein the cancer is esophageal cancer. [This invention 1091] The method of claim 1081, wherein the cancer is head and neck cancer. [This invention 1092] The method of claim 1081, wherein the cancer is cervical cancer. [This invention 1093] The method of claim 1080, wherein the cancer has tumor cells that express 191P4D12. [This invention 1094] The method of any of claims 1079 to 1093, further comprising the step of administering a second therapeutic agent to the subject. [This invention 1095] The method of claim 1094, wherein the second therapeutic agent is an immune checkpoint inhibitor. [This invention 1096] The method of claim 1095, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. [This invention 1097] The method of claim 1096, wherein the immune checkpoint inhibitor is a PD-1 inhibitor. [This invention 1098] The method of claim 1097, wherein the PD-1 inhibitor is nivolumab. [This invention 1099] The method of claim 1096, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor. [The present invention 1100] 1099. The method of claim 1099, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [The present invention 1101] The method of any of claims 1079 to 1100, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 10 mg / kg of subject body weight. [The present invention 1102] 1101. The method of claim 1101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 5 mg / kg of subject body weight. [The present invention 1103] 1101. The method of claim 1101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 2.5 mg / kg of subject body weight. [The present invention 1104] 1101. The method of claim 1101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 1.25 mg / kg of subject body weight. [This invention 1105] 1101. The method of claim 1101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of about 1 mg / kg of subject body weight. [The present invention 1106] 1101. The method of claim 1101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of about 1.25 mg / kg of subject body weight. [This invention 1107] The method of any of claims 1101 to 1106, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion. [This invention 1108] The method of claim 1107, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes in two cycles every three weeks. [This invention 1109] The method of claim 1108, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over approximately 30 minutes on days 1 and 8 of every three-week cycle. [The present invention 1110] 1109. The method of claim 1109, further comprising administering the immune checkpoint inhibitor by intravenous (IV) injection or infusion on day 1 of every three-week cycle. [The present invention 1111] The method of claim 1110, wherein the immune checkpoint inhibitor is administered in an amount of about 100 mg to about 1500 mg over about 30 or 60 minutes. [The present invention 1112] The method of claim 1107, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of three times every four weeks. [The present invention 1113] The method of claim 1112, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over approximately 30 minutes on days 1, 8, and 15 of every four-week cycle. [This invention 1114] The method of claim 1113, further comprising administering the immune checkpoint inhibitor by intravenous (IV) injection or infusion. [Brief explanation of the drawings]

[0052] [Figure 1A] 1 shows the results of SDS-PAGE analysis of a 14-day stability test of formulations F1 to F14 at 40° C. [Figure 1B] 1 shows the results of SDS-PAGE analysis of a 14-day stability test of formulations F1 to F14 at 40° C. [Figure 1C] 1 shows the results of SDS-PAGE analysis of a 14-day stability test of formulations F1 to F14 at 40° C. [Figure 1D] 1 shows the results of SDS-PAGE analysis of a 14-day stability test of formulations F1 to F14 at 40° C. [Figure 1E] An overview of the PR-HPLC studies described in Section 6.1 is shown. Figures 1F, 1G, and 1H show the results of SE-HPLC analysis of formulations F1-F14 at 40°C. [Figure 1F] 1 shows the results of SE-HPLC analysis of formulations F1 to F14 at 40°C. [Figure 1G] 1 shows the results of SE-HPLC analysis of formulations F1 to F14 at 40°C. [Figure 1H]1 shows the results of SE-HPLC analysis of formulations F1 to F14 at 40°C. [Figure 2A] 1 shows the results of the shaking test of formulations F4, F9 and F14 at TO. [Figure 2B] 1 shows SDS-PAGE results of cyclic freeze-thaw testing of formulations F4, F9, and F14. [Figure 2C] The total cumulative counts per mL measured by HIAC for formulations F4, F9, and F14 are shown. [Figure 3A] 1 shows the results of residual moisture analysis of formulations F4, F9, and F14. [Figure 3B] 1 shows the A280 (concentration) results for a 12-week simultaneous BDS and DP formulation study. [Figure 3C] 1 shows the A330 (turbidity) results for a 12-week simultaneous BDS and DP formulation study. [Figure 3D] The results of SDS-PAGE analysis of BDS (before lyophilization) at TO are shown. [Figure 3E] The results of SDS-PAGE analysis of BDS stored at -70°C or 2 to 8°C for 12 weeks are shown. [Figure 3F] 1 shows the results of SDS-PAGE analysis of DP after lyophilization and reconstitution at TO. [Figure 3G] 1 shows the results of SDS-PAGE analysis of DP (after lyophilization and reconstitution) stored at 25° C. or 40° C. for 12 weeks. [Figure 3H] The results of SDS-PAGE analysis of DP (after lyophilization and reconstitution) stored at 2-8°C for 12 weeks are shown. [Figure 3I] The results of SE-HPLC analysis of AGS-22M6E BDS stored at 2-8°C and -70°C for 12 weeks, and lyophilized AGS-22M6E stored at 2-8°C, 25°C / 60% RH, and 40°C / 75% RH for 12 weeks are shown. [Figure 4] 1 shows the results of SDS-PAGE analysis of lyophilized formulations of F4 at both 3.0 mL and 1.5 mL fill volumes. [Figure 5A-1]The nucleotide and amino acid sequences of the 191P4D12 protein are shown. [Figure 5A-2] The nucleotide and amino acid sequences of the 191P4D12 protein are shown. [Figure 5B] The nucleotide and amino acid sequences of the heavy and light chains of Ha22-2(2.4)6.1 are shown. [Figure 5C] The amino acid sequences of the heavy and light chains of Ha22-2(2.4)6.1 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0053] 5. Detailed Description Before the present disclosure is further described, it is to be understood that the disclosure is not limited to particular embodiments described herein, and that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0054] 5.1 Definition Techniques and procedures described or referenced herein include, for example, those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies :Methods and Protocols (Albitar ed.2010); and Antibody Engineering These include those that are generally well understood and / or commonly employed by those skilled in the art using conventional methodology, such as the widely used methods described in Vols 1 and 2 (Kontermann and Dubel eds., 2d ed. 2010).

[0055] Unless otherwise defined herein, the technical and scientific terms used in this description have the meanings that are commonly understood by those skilled in the art. For the purpose of interpreting this specification, the following explanations of terms shall apply, and where necessary, terms used in the singular shall also include the plural, and vice versa. If the explanations of terms provided contradict the documents incorporated herein by reference, the explanations of terms provided below shall prevail.

[0056] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonist, antagonist, neutralizing, full-length, or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof, as described below. Antibodies may be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies from other species, such as mice and rabbits. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides that are capable of binding to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region. For example, Antibody Engineering (Borrebaeck ed.,2d ed.1995); and Kuby, Immunology(3d ed. 1997). In specific embodiments, specific molecular antigens can be bound by the antibodies provided herein, including polypeptides or epitopes. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, where a functional fragment refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that contain an antigen-binding domain or site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol.Biology and Biotechnology:A Comprehensive Desk Reference (Myers ed.,1995);Huston et al.,1993,Cell Biophysics 22:189-224;Pluckthun and Skerra,1989,Meth.Enzymol.178:497-515;and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibodies can be agonist or antagonist antibodies.

[0057] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which may include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0058] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other natural or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell, e.g., on or within a cancer cell.

[0059] An "intact" antibody is one that comprises an antigen-binding site and a CL and at least a heavy chain constant region, CH1, CH2, and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0060] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to a portion of an antibody (e.g., a CDR) that contains amino acid residues that interact with an antigen and confer specificity and affinity for the antigen to the binding agent. As used herein, "antigen-binding fragment" includes an "antibody fragment" that contains a portion of an intact antibody, e.g., the antigen-binding region or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies and di-diabodies (e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. 90:6444-48; Lu et al., 2005, J. Biol. Chem. 280:19665-72; Hudson ...). al., 2003, Nat. Med. 9:129-34; WO 93 / 11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single-chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domain antibodies (sdAbs) (see, e.g., Woolven et al., 1999, Immunogenetics 50:98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49); and multispecific antibodies formed from antibody fragments.

[0061] The terms "bind" or "binding" refer to interactions between molecules, including, for example, forming a complex. The interaction can be a non-covalent interaction, including, for example, hydrogen bonding interactions, ionic bonding interactions, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of all non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k off ) and association rate (k on ) ratio (k off / k on ) is the dissociation constant K D which is inversely proportional to the affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies for different antibody-antigen complexes. on and k off The dissociation constant K of the antibodies provided herein depends on both D The affinity of an antibody can be determined using any method provided herein or any other method known to those skilled in the art. The affinity at one binding site does not necessarily reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeat antigenic determinants, such as a multivalent antigen, contacts an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site increases the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0062] In connection with the antibodies or antigen-binding fragments thereof described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein to refer to binding molecules of an antigen-binding domain, such as a polypeptide, that specifically bind to an antigen. Antibodies or antigen-binding fragments that bind to or specifically bind to an antigen may be cross-reactive with related antigens. In certain embodiments, antibodies or antigen-binding fragments that bind to or specifically bind to an antigen do not cross-react with other antigens. Antibodies or antigen-binding fragments that bind to or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those skilled in the art. In some embodiments, an antibody or antigen-binding fragment binds to or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigens as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least twice the background signal or noise, and may be greater than 10 times the background. For discussions regarding binding specificity, see, e.g., Fundamental Immunology332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent of binding of an antibody or antigen-binding fragment to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. Terms such as "specific binding," "specifically binds to," or "specific for" refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, such as excess unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. Antibody or antigen-binding fragments that bind to an antigen include those that can bind to the antigen with sufficient affinity so that the binding molecule is useful, for example, as a diagnostic agent for targeting the antigen. In certain embodiments, antibodies or antigen-binding fragments that bind to an antigen have a molecular weight of less than 1000 nM, less than 800 nM, less than 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM a dissociation constant (K) of less than 1000 nM, 800 nM, 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM D In certain embodiments, the antibody or antigen-binding fragment binds to an epitope of an antigen that is conserved among antigens from different species (e.g., between the human species and the cynomolgus monkey species).

[0063] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific exemplary embodiments include the following. In one embodiment, "K D " or "K D The "K value" can be measured by assays known in the art, for example, binding assays. D can be measured, for example, in an RIA performed using the Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). D or K D Values ​​can also be measured by using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays, for example by Octet® using an Octet® QK384 system, or by Biacore® using, for example, a Biacore® TM-2000 or Biacore® TM-3000. "On-rate" or "rate of association" or "association rate" or "k" can also be determined using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, for example, using an Octet® QK384, Biacore® TM-2000, or Biacore® TM-3000 system.

[0064] In certain embodiments, antibodies or antigen-binding fragments can include "chimeric" sequences in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55).

[0065] In certain embodiments, an antibody or antigen-binding fragment can comprise a portion of a "humanized" form of a non-human (e.g., murine) antibody, which is a chimeric antibody comprising a human immunoglobulin (e.g., recipient antibody) in which native CDR residues are replaced by residues from a corresponding CDR (e.g., donor antibody) of a non-human species, such as mouse, rat, rabbit, or non-human primate, containing the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. The heavy or light chain of a humanized antibody can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-29; Presta, 1992, Curr. Op. Struct. Biol. 2:593-96; Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; U.S. Patent Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0066] In certain embodiments, an antibody or antigen-binding fragment can comprise a portion of a "fully human antibody" or "human antibody," which terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. In specific embodiments, these terms refer to an antibody that comprises variable and constant regions of human origin. A "fully human" antibody, in certain embodiments, can also encompass an antibody that binds a polypeptide and that is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" is used in conjunction with the term "human antibody" as defined by Kabat et al. (Kabat et al. (1991) Sequences of Proteins of Immunological Interest "Human antibodies" include antibodies containing variable and constant regions corresponding to human germline immunoglobulin sequences as described by the National Institute of Health and Human Services, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. A "human antibody" is one that has an amino acid sequence corresponding to that of an antibody produced by a human and / or is produced using any of the techniques for producing human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1:755-68). Cole et al., Monoclonal Antibodies and Cancer Therapy77 (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5:368-74 are also available for preparing human monoclonal antibodies. Human antibodies can be prepared by administering antigen to transgenic animals, such as mice, that have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., 2006, Proc. Natl. Acad. Sci. USA 103:3557-62 concerning human antibodies generated via human B cell hybridoma technology.

[0067] In certain embodiments, an antibody or antigen-binding fragment can comprise a portion of a "recombinant human antibody," which term includes human antibodies prepared, expressed, generated, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or a cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest(See, e.g., US Department of Health and Human Services, NIH Publication No. 91-3242.) However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0068] In certain embodiments, an antibody or antigen-binding fragment can comprise a portion of a "monoclonal antibody," a term used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations, and each monoclonal antibody typically recognizes a single epitope on an antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to a particular method for producing the antibody. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or can be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol. 222:581-97. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. For example, Short Protocols in Molecular Biology See (Ausubel et al. eds., 5th ed. 2002).

[0069] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. The pairing of a VH and VL forms a single antigen-binding site. The structure and properties of different classes of antibodies are described in detail in, e.g., Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds.,5 th ed. 2001).

[0070] The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgGs typically contain two Fab regions, each present in one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region from each of the heavy and light chains. More specifically, the heavy chain variable and constant regions in the Fab region are the VH and CH1 regions, and the light chain variable and constant regions in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions within the Fab region can be arranged in various ways to confer antigen-binding capability according to the present disclosure. For example, as with the Fab region of a conventional IgG, the VH and CH1 regions can be on one polypeptide, while the VL and CL regions can be on separate polypeptides. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the following sections.

[0071] The terms "variable region," "variable domain," "V region," or "V domain" refer to the portion of an antibody light or heavy chain, generally located at the amino terminus of the light or heavy chain, approximately 120 to 130 amino acids in length for heavy chains and approximately 100 to 110 amino acids in length for light chains, that is used for binding and specificity of each particular antibody to its specific antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that certain segments of the variable region vary significantly in sequence among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for a particular antigen. However, variability is not uniformly distributed across the 110-amino acid span of the variable region. Instead, the V region consists of less variable (e.g., relatively invariant) stretches of approximately 15 to 30 amino acids called framework regions (FRs) separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions," each approximately 9 to 12 amino acids in length. The variable regions of the heavy and light chains each contain four FRs that adopt a primarily β-sheet configuration, connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991). The constant region is not directly involved in binding of the antibody to an antigen, but exhibits various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region varies significantly in sequence among different antibodies. In a specific embodiment, the variable region is a human variable region.

[0072] The terms "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertions into, the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbered sequence at the regions of homology. The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is commonly used when referring to residues within the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of a human IgG1 EU antibody. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0073] The term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 50 to 70 kDa, the amino-terminal portion of which contains a variable region of approximately 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five different types (e.g., isotypes) designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the heavy chain constant region. Different heavy chains vary in size, with α, δ, and γ containing approximately 450 amino acids, and μ and ε containing approximately 550 amino acids. When combined with light chains, these different types of heavy chains give rise to five well-known classes (e.g., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM, each of which contains four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4.

[0074] The term "light chain," when used in reference to an antibody, refers to a polypeptide chain of about 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain.

[0075] As used herein, the terms "hypervariable region," "HVR," "complementarity-determining region," and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VL β-sheet framework. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0076] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al. supra). Instead, Chothia refers to the position of structural loops (see, for example, Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). When numbered using Kabat's numbering rules, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (e.g., Antibody EngineeringVol. 2 (Kontermann and Dubel eds., 2nd ed. 2010). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system specialized for immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to both in terms of amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides within structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from one species of immunoglobulin onto an acceptor framework, typically from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309:657-70. For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT-specific numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are shown below.

[0077] (Table 30) TIFF2025170007000013.tif77137

[0078] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementarity-determining region" of a given antibody or region thereof, such as a variable region, and the individual CDRs of an antibody or region thereof (e.g., "CDR-H1, CDR-H2"), should be understood to encompass the complementarity-determining regions defined by any of the known schemes described hereinabove. In some cases, a scheme for identifying a particular CDR or CDRs is specified, such as CDRs defined by the Kabat, Chothia, or Contact methods. In other cases, the specific amino acid sequences of the CDRs are given.

[0079] The hypervariable regions may include "extended hypervariable regions" such as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0080] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as interacting with Fc receptors. This term refers to the portion of an immunoglobulin molecule that contains a more conserved amino acid sequence than the other portions of the immunoglobulin, including the antigen-binding site, the variable region. The constant region may include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0081] The term "framework" or "FR" refers to variable region residues adjacent to the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region or CDR residues.

[0082] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226 or the amino acid residue at position Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations containing a mixture of antibodies with and without the K447 residue. A "functional Fc region" possesses the "effector functions" of a native-sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those skilled in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the native-sequence Fc region or the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0083] As used herein, "epitope" is a term of art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope can be a linear epitope, a conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a "linear" epitope), or it can include amino acids from two or more discontinuous regions of the polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). In general, those skilled in the art will understand that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids regardless of whether they are folded into a native three-dimensional protein structure. In other embodiments, the binding molecule requires the amino acid residues that make up the epitope to adopt a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0084] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified, either naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Polypeptides containing one or more analogs of an amino acid, including, but not limited to, unnatural amino acids, as well as other modifications known in the art, are also included within the definition. Because the polypeptides of the present disclosure can be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, the "polypeptide" can exist as a single chain or as two or more associated chains.

[0085] The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) described herein, to introduce the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can contain a selection sequence or marker operable for stable integration into a host cell chromosome. Furthermore, the vector can contain one or more selection marker genes and appropriate expression control sequences. Selection marker genes that can be included provide, for example, resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not present in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both the heavy and light chains of an antibody, or the VH and VL of an antibody), both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. In the case of single vector expression, the encoding nucleic acids may be operably linked to one common expression control sequence, or may be linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecule will be expressed in an amount sufficient to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0086] As used herein, the term "host" refers to an animal, such as a mammal (e.g., a human).

[0087] As used herein, the term "host cell" refers to a particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations, or due to integration of the nucleic acid molecule into the host cell genome.

[0088] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the nucleic acid molecule's natural source. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In specific embodiments, one or more nucleic acid molecules encoding the antibodies described herein are isolated or purified. This term encompasses nucleic acid sequences that have been removed from their naturally occurring environment, including recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule can include isolated forms of the molecule.

[0089] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, generally single-stranded, synthetic polynucleotide, generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells that produce the binding molecules of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is referred to as the transcription direction, and the region of the DNA strand that contains the same sequence as the RNA transcript 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence," and the region of the DNA strand that contains the same sequence as the RNA transcript 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0090] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or otherwise approved for use in animals, and more particularly, in humans. United States Pharmacopoeia , European Pharmacopoeia , or other generally recognized pharmacopoeias.

[0091] "Excipient" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizing agents, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0092] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as l-histidine, glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, sucrose, trehalose dihydrate, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0093] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0094] In some embodiments, excipients are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like.

[0095] A composition containing a pharmaceutical compound can include, for example, a binding molecule (eg, an antibody) in isolated or purified form, together with an appropriate amount of an excipient.

[0096] The abbreviation "MMAE" refers to monomethyl auristatin E.

[0097] Unless otherwise specified, the term "alkyl" refers to a saturated straight or branched chain hydrocarbon containing from about 1 to about 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 1 to about 8 carbon atoms being preferred. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.Alkyl groups, whether alone or as part of another group, include: -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S3R', -S(O)2R', -S(O)R', -OH, ═O, -N3, -NH2, -N and -C(R')2, ... The groups -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl are -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C It may be further substituted with one or more groups including, but not limited to, (O)N(R'')2, -NHC(O)R'', -SR'', -S03R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0098] Unless otherwise specified, the terms "alkenyl" and "alkynyl" refer to straight and branched carbon chains containing from about 2 to about 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 2 to about 8 carbon atoms being preferred. Alkenyl chains have at least one double bond in the chain, and alkynyl chains have at least one triple bond in the chain. Examples of alkenyl groups include, but are not limited to, ethylene or vinyl, allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and -2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but are not limited to, acetylene, propargyl, acetylenyl, propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1 butynyl.Alkenyl and alkynyl groups, whether alone or as part of another group, include: -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S3R', -S(O)2R', -S(O)R', -OH, =O, -N3, and -C-C alkyl, -C-C alkenyl, -C-C alkynyl, or -aryl, and optionally substituted with one or more groups, preferably 1 to 3 groups (and optional further substituents selected from halogen), including but not limited to -NH, -NH(R'), -N(R') and -CN, wherein each R' is independently selected from -H, ... The -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups include -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', - It may be further substituted with one or more substituents including, but not limited to, C(O)N(R'')2, -NHC(O)R'', -SR'', -S03R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0099] Unless otherwise specified, the term "alkylene" refers to a saturated branched or straight-chain hydrocarbon radical containing from about 1 to about 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 1 to about 8 carbon atoms being preferred, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylenes include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decalene, 1,4-cyclohexylene, and the like.Alkylene groups, whether alone or as part of another group, include: -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S3R', -S(O)2R', -S(O)R', -OH, ═O, -N3, -NH2, -N and -C(R')2, ... The -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups include -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C( and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0100] Unless otherwise specified, the term "alkenylene" refers to an optionally substituted alkylene group containing at least one carbon-carbon double bond. Exemplary alkenylene groups include, for example, ethenylene (-CH=CH-) and propenylene (-CH=CHCH-).

[0101] Unless otherwise specified, the term "alkynylene" refers to an optionally substituted alkylene group containing at least one carbon-carbon triple bond. Exemplary alkynylene groups include, for example, acetylene (-C≡C-), propargyl (-CHC≡C-), and 4-pentynyl (-CHCHCHC≡CH-).

[0102] Unless otherwise specified, the term "aryl" refers to a monovalent aromatic hydrocarbon radical of 6 to 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein) derived by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented as "Ar" in the exemplary structures. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, and the like.

[0103] Aryl groups, whether alone or as part of another group, include -halogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S3R', -S(O) and -CN, wherein each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl; The C8 alkynyl, O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), and -aryl groups include —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl, -halogen, —O—(C1-C8 alkyl), —O—(C2-C8 alkenyl), —O—(C2-C8 alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH2, —C(O)NHR″ , -C(O)N(R'')2, -NHC(O)R'', -SR'', -S03R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0104] Unless otherwise specified, the term "arylene" refers to an optionally substituted aryl group that is divalent (i.e., derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent aromatic ring system) and can be in the ortho, meta, or para configuration, as shown in the following structure with phenyl as an exemplary aryl group: TIFF2025170007000014.tif22128 Typical "-(C1-C8 alkylene)aryl", "-(C2-C8 alkenylene)aryl", "and -(C2-C8 alkynylene)aryl" groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like.

[0105] Unless otherwise specified, the term "heterocycle" refers to a monocyclic, bicyclic, or polycyclic ring system having 3 to 14 ring atoms (also referred to as ring members), wherein at least one ring atom of at least one ring is a heteroatom selected from N, O, P, or S (and all combinations and subcombinations of ranges and specific numbers of carbon atoms and heteroatoms therein). A heterocycle can have 1 to 4 ring heteroatoms independently selected from N, O, P, or S. One or more N, C, or S atoms in a heterocycle can be oxidized. Monocyclic heterocycles preferably have 3 to 7 ring members (e.g., 2 to 6 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S), and bicyclic heterocycles preferably have 5 to 10 ring members (e.g., 4 to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S). The heteroatom-containing ring can be aromatic or non-aromatic. Unless otherwise specified, a heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. A heterocycle is defined as a ring having a heteroatom or carbon atom that results in a stable structure. Principles of Modern Heterocyclic Chemistry "(WA Benjamin, New York, 1968), especially chapters 1, 3, 4, 6, 7, and 9;" The Chemistry of Heterocyclic Compounds,A series of Monographs" (John Wiley & Sons, New York, 1950-present), especially volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. 82:5566 (1960). Examples of "heterocyclic" groups include, by way of example and without limitation, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl. , quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl , thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl, β-carbolinyl, phenazinyl Examples of alkyl groups include benzotriazolyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and isatinoyl.Preferred "heterocycle" groups include, but are not limited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, and tetrazolyl.Heterocyclic groups, whether alone or as part of another group, include -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S03R', -S( and -CN, wherein each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and ... (C2-C8 alkynyl), -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, and -aryl groups include -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C It may be further substituted with one or more substituents including, but not limited to, (O)N(R'')2, -NHC(O)R'', -SR'', -S03R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or aryl.

[0106] By way of example and not limitation, the carbon-bonded heterocycle can be bonded at the following positions: 2-, 3-, 4-, 5-, or 6-position of pyridine; 3-, 4-, 5-, or 6-position of pyridazine; 2-, 4-, 5-, or 6-position of pyrimidine; 2-, 3-, 5-, or 6-position of pyrazine; 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole; 2-, 4-, or 5-position of oxazole, imidazole, or thiazole; 3-, 4-, or 5-position of isoxazole, pyrazole, or isothiazole; 2- or 3-position of aziridine; 2-, 3-, or 4-position of azetidine; 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline; or 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of isoquinoline. Even more typically, the carbon-linked heterocycle includes 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

[0107] By way of example and not limitation, nitrogen-bonded heterocycles can be bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole; the 2-position of isoindole or isoindoline; the 4-position of morpholine; and the 9-position of carbazole or β-carboline. Even more typically, nitrogen-bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0108] Unless otherwise specified, the term "carbocycle" refers to a saturated or unsaturated non-aromatic monocyclic, bicyclic, or polycyclic ring system having 3 to 14 ring atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), all of which are carbon atoms. Monocyclic carbocycles preferably have 3 to 6 ring atoms, and even more preferably 5 or 6 ring atoms. Bicyclic carbocycles preferably have 7 to 12 ring atoms, for example, arranged as a bicyclo[4,5], [5,5], [5,6], or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclo[5,6] or [6,6] system. The term "carbocycle" includes, for example, a monocyclic carbocycle fused to an aryl ring (e.g., a monocyclic carbocycle fused to a benzene ring). Carbocycles preferably have 3 to 8 carbon ring atoms.Carbocyclic groups, either alone or as part of another group, include, for example, -halogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S3R', -S(O)2R' , -S(O)R', -OH, ═O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, and preferably one or two groups (and optional further substituents selected from halogen), wherein each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and wherein said -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl are independently substituted with one or more groups, preferably one or two groups (and optional further substituents selected from halogen), including but not limited to -S(O)R', -OH, ═O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, Alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), and -aryl groups include -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O) It may be further substituted with one or more substituents including, but not limited to, NHR", -C(O)N(R"), -NHC(O)R", -SR", -SOR", -S(O)R", -S(O)R", -OH, -N, -NH, -NH(R"), -N(R") and -CN, where each R" is independently selected from -H, -C-C alkyl, -C-C alkenyl, -C-C alkynyl, or -aryl.

[0109] Examples of monocyclic carbocyclic substituents include -cyclopropyl, -cyclobutyl, -cyclopentyl, -1-cyclopent-1-enyl, -1-cyclopent-2-enyl, -1-cyclopent-3-enyl, cyclohexyl, -1-cyclohex-1-enyl, -1-cyclohex-2-enyl, -1-cyclohex-3-enyl, -cycloheptyl, -cyclooctyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, and -cyclooctadienyl.

[0110] "Carbocyclo," whether used alone or as part of another group, refers to an optionally substituted carbocyclic group, as defined above, that is divalent (i.e., derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent carbocyclic ring system).

[0111] Unless the context dictates otherwise, a hyphen (-) designates the point of attachment to the pendant molecule. Thus, the terms "-(C1-C8 alkylene)aryl" or "-C1-C8 alkylene(aryl)" refer to a C1-C8 alkylene radical, as defined herein, where the alkylene radical is attached to a pendant molecule at any of the alkylene radical's carbon atoms, and one of the hydrogen atoms attached to the alkylene radical's carbon atom is replaced with an aryl radical, as defined herein.

[0112] When a particular group is "substituted," the group can have one or more substituents independently selected from the list of substituents, preferably 1 to 5 substituents, more preferably 1 to 3 substituents, and most preferably 1 to 2 substituents. However, the group can have any number of substituents, generally selected from halogen. Multiple groups that are substituted are also designated as such. It is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definition elsewhere in that molecule. It is understood that the substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and can be readily synthesized by techniques known in the art and the methods described herein.

[0113] As used herein, a protecting group refers to a group that selectively blocks one reactive site in a multifunctional compound, either temporarily or permanently.Suitable hydroxy protecting groups for use in the present invention are pharmaceutically acceptable, and may or may not need to be cleaved from the parent compound after administration to a subject in order for the compound to be active.Cleavage is due to normal metabolic processes in the body.Hydroxy protecting groups are well known in the art, and the entire contents of which are incorporated herein by reference for all purposes. Protective Groups in Organic Synthesis by TWGreene and PGMWuts(John Wiley&sons,3 rdSee, for example, the "Ethylenediaminetetraacetic Acid (Ethylene Diamine ... 2-chloroethoxy)methyl ether, 2-(trimethylsilyl)ethoxymethyl ether, menthoxymethyl ether, tetrahydropyranyl ether, 1-methoxycyclohexyl ether, 4-methoxytetrahydrothiopyranyl ether, 4-methoxytetrahydrothiopyranyl ether S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl ether, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl ether, 1,4-dioxan-2-yl ether, tetrahydrofuranyl ether, tetrahydrothiofuranyl ether;Substituted ethyl ethers, for example, 1-ethoxyethyl ether, 1-(2-chloroethoxy)ethyl ether, 1-[2-(trimethylsilyl)ethoxy]ethyl ether, 1-methyl-1-methoxyethyl ether, 1-methyl-1-benzyloxyethyl ether, 1-methyl-1-benzyloxy-2-fluoroethyl ether, 1-methyl-1-phenoxyethyl ether, 2-trimethylsilyl ether, t-butyl ether, allyl ether, propargyl ether, p-chlorophenyl ether, p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, tripropylsilyl ether, dimethylisopropylsilyl ether, diethylisopropylsilyl ether, dimethylhexylsilyl ether, t-butyl ether

[0033] Preferred protecting groups include, but are not limited to, alkyl dimethylsilyl ether, diphenylmethylsilyl ether, benzoyl formate ester, acetate ester, chloroacetate ester, dichloroacetate ester, trichloroacetate ester, trifluoroacetate ester, methoxyacetate ester, triphenylmethoxyacetate ester, phenylacetate ester, benzoate ester, alkyl methyl carbonate, alkyl 9-fluorenylmethyl carbonate, alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate, 1,1-dimethyl-2,2,2-trichloroethyl carbonate, alkyl sulfonate, methanesulfonate, benzylsulfonate, tosylate, methylene acetal, ethylidene acetal, and t-butyl methylidene ketal. Preferred protecting groups are of the formula -R; a ,Si(R a )(R a )(R a ), -C(O)R a , -C(O)OR a , -C(O)NH(R a ), -S(O)R a , -S(O)2OH, P(O)(OH)2, and -P(O)(OH)OR a where R a is C1~C20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, -C1-C 20 Alkylene (Carbocyclic), -C2~C 20 Alkenylene (carbocyclic ring), -C2~C 20 Alkynylene (Carbocyclic), -C6~C 10 Aryl, -C1-C 20 Alkylene (aryl), -C2~C 20 Alkenylene (aryl), -C2~C 20 Alkynylene (aryl), -C1-C 20 Alkylene (heterocycle), -C2~C 20 Alkenylene (heterocycle), or -C2~C 20 Alkynylene (heterocycle), wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocycle radicals, either alone or as part of another group, may be substituted.

[0114] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a binding molecule (e.g., an antibody) or pharmaceutical composition provided herein sufficient to effect a desired result.

[0115] The terms "subject" and "patient" can be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, diagnosed with a condition or disorder. In another embodiment, a subject is a mammal, e.g., a human, at risk of developing a condition or disorder.

[0116] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0117] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment can be determined by assessing whether there has been a reduction, alleviation, and / or reduction of one or more symptoms associated with the underlying disease, such that an improvement is observed for the patient, even though the patient may still be suffering from the underlying disease. The term "treating" includes both management and amelioration of disease. The terms "manage," "managing," and "management" refer to the beneficial effects a subject derives from a therapy that does not necessarily result in a cure of the disease.

[0118] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or one or more associated symptoms (e.g., cancer).

[0119] The terms "cancer" or "cancer cell" are used herein to refer to tissue or cells found in neoplasms that have characteristics that distinguish them from normal tissue or tissue cells. Such characteristics include, but are not limited to, the degree of anaplasia, irregular shape, unclear cell outlines, nuclear size, changes in nuclear or cytoplasmic structure, other phenotypic changes, the presence of cellular proteins indicative of cancer or a precancerous state, an increased number of mitoses, and the ability to metastasize. Words related to "cancer" include carcinoma, sarcoma, tumor, epithelioma, leukemia, lymphoma, polyp, and scirrhous carcinoma, transformation, neoplasia, and the like.

[0120] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0121] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0122] Whenever an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, whenever an embodiment is described herein using the phrase "consisting essentially of," it is understood that other similar embodiments described in terms of "consisting of" are also provided.

[0123] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0124] 5.2 Pharmaceutical Compositions In one aspect, provided herein is a "pharmaceutical composition" comprising an antibody-drug conjugate provided herein and one or more pharmaceutically or physiologically acceptable excipients. In certain embodiments, the antibody-drug conjugate is provided in combination with or separately from one or more additional agents. Also provided are compositions comprising such one or more additional agents and one or more pharmaceutically or physiologically acceptable excipients. In certain embodiments, the antibody-drug conjugate and the additional agent(s) are present in therapeutically acceptable amounts. The pharmaceutical composition can be used in accordance with the methods and uses provided herein. Thus, for example, the pharmaceutical composition can be administered ex vivo or in vivo to a subject to carry out the therapeutic methods and uses provided herein. The pharmaceutical compositions provided herein can be formulated to be compatible with the intended method or route of administration, and exemplary routes of administration are described herein.

[0125] In some aspects, pharmaceutical compositions of cancer or tumor modulating antibody drug conjugates are provided.

[0126] In some aspects, the pharmaceutical composition may further comprise other therapeutically active agents or compounds disclosed herein or known to those skilled in the art, which can be used in the treatment or prevention of various diseases and disorders (e.g., cancer) described herein. As noted above, the additional therapeutically active agents or compounds may be present in a separate pharmaceutical composition(s).

[0127] Pharmaceutical compositions typically comprise a therapeutically effective amount of at least one of the antibody-drug conjugates provided herein and one or more pharmaceutically acceptable formulation agents. In certain embodiments, the pharmaceutical composition further comprises one or more additional agents described herein.

[0128] In one aspect, a pharmaceutical composition comprises an antibody drug conjugate provided herein. In some aspects, the pharmaceutical composition comprises a therapeutically effective amount of an antibody drug conjugate provided herein. In certain aspects, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0129] In some embodiments, the antibody drug conjugate in the pharmaceutical compositions provided herein is selected from the antibody drug conjugates described in Section 5.3, below.

[0130] In certain embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of 0.1 mg / mL to 100 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of 1 mg / mL to 20 mg / mL. In other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of 5 mg / mL to 15 mg / mL. In other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of 8 mg / mL to 12 mg / mL. In other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of 9 mg / mL to 11 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 9.5 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 9.6 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 9.7 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 9.8 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 9.9 mg / mL. In yet other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10 mg / mL. In still other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.1 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.2 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.3 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.3 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.4 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.5 mg / mL.

[0131] In some embodiments, the pharmaceutical compositions provided herein comprise L-histidine, TWEEN-20, and at least one of trehalose dihydrate or sucrose. In some embodiments, the pharmaceutical compositions provided herein further comprise hydrochloric acid (HCl) or succinic acid.

[0132] In some embodiments, the concentration of L-histidine useful in the pharmaceutical compositions provided herein ranges from 5 mM to 50 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 10 mM to 40 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 35 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 30 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 25 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 35 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 16 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 17 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 18 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 19 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 20 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 21 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 22 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 23 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 24 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 25 mM.

[0133] In some embodiments, the concentration of TWEEN-20 useful in the pharmaceutical compositions provided herein ranges from 0.001% to 0.1% (v / v). In other embodiments, the concentration of TWEEN-20 ranges from 0.0025% to 0.075% (v / v). In one embodiment, the concentration of TWEEN-20 ranges from 0.005% to 0.05% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.0075% to 0.025% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.0075% to 0.05% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.01% to 0.03% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.01% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.015% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.016% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.017% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.018% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.019% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.02% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.021% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.022% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.023% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.024% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.025% (v / v).

[0134] In one embodiment, the concentration of trehalose dihydrate useful in the pharmaceutical compositions provided herein ranges from 1% to 20% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 2% to 15% (w / v). In one embodiment, the concentration of trehalose dihydrate ranges from 3% to 10% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4% to 9% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4% to 8% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4% to 7% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4% to 6% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4.5% to 6% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 4.6% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 4.7% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 4.8% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 4.9% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.0% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.1% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.2% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.3% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.4% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.5% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.6% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.7% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.8% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.9% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 6.0% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 6.1% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 6.2% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 6.3% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 6.4% (w / v).In another embodiment, the concentration of trehalose dihydrate is about 6.5% (w / v).

[0135] In certain embodiments, the molar concentration of trehalose dihydrate is 50 mM to 300 mM. In other embodiments, the molar concentration of trehalose dihydrate is 75 mM to 250 mM. In some embodiments, the molar concentration of trehalose dihydrate is 100 mM to 200 mM. In other embodiments, the molar concentration of trehalose dihydrate is 130 mM to 150 mM. In some embodiments, the molar concentration of trehalose dihydrate is 135 mM to 150 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 135 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 136 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 137 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 138 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 139 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 140 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 141 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 142 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 143 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 144 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 145 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 146 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 150 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 151 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 151 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 152 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 153 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 154 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 155 mM.

[0136] In one embodiment, the concentration of sucrose useful in the pharmaceutical compositions provided herein ranges from 1% to 20% (w / v). In another embodiment, the concentration of sucrose ranges from 2% to 15% (w / v). In one embodiment, the concentration of sucrose ranges from 3% to 10% (w / v). In another embodiment, the concentration of sucrose ranges from 4% to 9% (w / v). In another embodiment, the concentration of sucrose ranges from 4% to 8% (w / v). In another embodiment, the concentration of sucrose ranges from 4% to 7% (w / v). In another embodiment, the concentration of sucrose ranges from 4% to 6% (w / v). In another embodiment, the concentration of sucrose ranges from 4.5% to 6% (w / v). In another embodiment, the concentration of sucrose is about 4.6% (w / v). In another embodiment, the concentration of sucrose is about 4.7% (w / v). In another embodiment, the sucrose concentration is about 4.8% (w / v). In another embodiment, the sucrose concentration is about 4.9% (w / v). In another embodiment, the sucrose concentration is about 5.0% (w / v). In another embodiment, the sucrose concentration is about 5.1% (w / v). In another embodiment, the sucrose concentration is about 5.2% (w / v). In another embodiment, the sucrose concentration is about 5.3% (w / v). In another embodiment, the sucrose concentration is about 5.4% (w / v). In another embodiment, the sucrose concentration is about 5.5% (w / v). In another embodiment, the sucrose concentration is about 5.6% (w / v). In another embodiment, the sucrose concentration is about 5.7% (w / v). In another embodiment, the sucrose concentration is about 5.8% (w / v). In another embodiment, the sucrose concentration is about 5.9% (w / v). In another embodiment, the sucrose concentration is about 6.0% (w / v). In another embodiment, the sucrose concentration is about 6.1% (w / v). In another embodiment, the sucrose concentration is about 6.2% (w / v). In another embodiment, the sucrose concentration is about 6.3% (w / v). In another embodiment, the sucrose concentration is about 6.4% (w / v). In another embodiment, the sucrose concentration is about 6.5% (w / v).

[0137] In certain embodiments, the molar concentration of sucrose is 50 mM to 300 mM. In other embodiments, the molar concentration of sucrose is 75 mM to 250 mM. In some embodiments, the molar concentration of sucrose is 100 mM to 200 mM. In other embodiments, the molar concentration of sucrose is 130 mM to 150 mM. In some embodiments, the molar concentration of sucrose is 135 mM to 150 mM. In certain embodiments, the molar concentration of sucrose is about 135 mM. In certain embodiments, the molar concentration of sucrose is about 136 mM. In certain embodiments, the molar concentration of sucrose is about 137 mM. In certain embodiments, the molar concentration of sucrose is about 138 mM. In certain embodiments, the molar concentration of sucrose is about 139 mM. In certain embodiments, the molar concentration of sucrose is about 140 mM. In certain embodiments, the molar concentration of sucrose is about 141 mM. In certain embodiments, the molar concentration of sucrose is about 142 mM. In certain embodiments, the molar concentration of sucrose is about 143 mM. In certain embodiments, the molar concentration of sucrose is about 144 mM. In certain embodiments, the molar concentration of sucrose is about 145 mM. In certain embodiments, the molar concentration of sucrose is about 146 mM. In certain embodiments, the molar concentration of sucrose is about 150 mM. In certain embodiments, the molar concentration of sucrose is about 151 mM. In certain embodiments, the molar concentration of sucrose is about 151 mM. In certain embodiments, the molar concentration of sucrose is about 152 mM. In certain embodiments, the molar concentration of sucrose is about 153 mM. In certain embodiments, the molar concentration of sucrose is about 154 mM. In certain embodiments, the molar concentration of sucrose is about 155 mM.

[0138] In some embodiments, the pharmaceutical compositions provided herein comprise HCl. In other embodiments, the pharmaceutical compositions provided herein comprise succinic acid.

[0139] In some embodiments, the pharmaceutical compositions provided herein have a pH in the range of 5.5 to 6.5. In other embodiments, the pharmaceutical compositions provided herein have a pH in the range of 5.7 to 6.3. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 5.7. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 5.8. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 5.9. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.0. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.1. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.2. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.3.

[0140] In some embodiments, the pH is measured at room temperature. In other embodiments, the pH is measured between 15°C and 27°C. In still other embodiments, the pH is measured at 4°C. In still other embodiments, the pH is measured at 25°C.

[0141] In some embodiments, the pH is adjusted with HCl. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.5 to 6.5 at room temperature. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.7 to 6.3 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.7 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.8 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.9 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.0 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.1 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.2 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.3 at room temperature.

[0142] In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH ranging from 5.5 to 6.5 at 15°C to 27°C. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH ranging from 5.7 to 6.3 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.2 at 15° C. to 27° C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.3 at 15° C. to 27° C.

[0143] In some embodiments, the pH is adjusted with succinic acid. In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH in the range of 5.5 to 6.5 at room temperature. In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH in the range of 5.7 to 6.3 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.7 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.8 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.9 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.0 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.1 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.2 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.3 at room temperature.

[0144] In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH ranging from 5.5 to 6.5 at 15°C to 27°C. In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH ranging from 5.7 to 6.3 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.2 at 15° C. to 27° C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.3 at 15° C. to 27° C.

[0145] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, and at least one of about 5.5% (w / v) trehalose dihydrate or about 5% (w / v) sucrose. In some embodiments, the pharmaceutical compositions provided herein further comprise HCl or succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25°C.

[0146] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and HCl. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25°C.

[0147] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5% (w / v) sucrose, and HCl. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25°C.

[0148] In other specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25°C.

[0149] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5% (w / v) sucrose and succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25°C.

[0150] In a specific aspect, provided herein is a method for treating a pulmonary arthritis, comprising: (a) the structure: TIFF2025170007000015.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and HCl. wherein the antibody drug conjugate is at a concentration of about 10 mg / mL and the pH is about 6.0 at 25° C.

[0151] In another specific aspect, the pharmaceutical compositions provided herein comprise: (a) the structure: TIFF2025170007000016.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and succinic acid. wherein the antibody drug conjugate is at a concentration of about 10 mg / mL and the pH is about 6.0 at 25° C.

[0152] In yet another specific aspect, the pharmaceutical compositions provided herein comprise: (a) the structure: TIFF2025170007000017.tif36160, wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.0% (w / v) sucrose, and HCl. wherein the antibody drug conjugate is at a concentration of about 10 mg / mL and the pH is about 6.0 at 25° C.

[0153] Although specific numbers (and numerical ranges thereof) are provided, it is understood that in certain embodiments, numbers within, for example, 2%, 5%, 10%, 15%, or 20% of said numbers (or numerical ranges) are also contemplated. Other exemplary pharmaceutical compositions are provided in the experimental section below.

[0154] The primary solvent in the vehicle can be either aqueous or non-aqueous in nature. In addition, the vehicle can contain other pharmaceutically acceptable excipients to modify or maintain the pH, osmolality, viscosity, sterility or stability of the pharmaceutical composition. In certain embodiments, the pharmaceutically acceptable vehicle is an aqueous buffer solution. In other embodiments, the vehicle contains, for example, sodium chloride and / or sodium citrate.

[0155] The pharmaceutical compositions provided herein may further comprise other pharmaceutically acceptable formulating agents for modifying or maintaining the release rate of the antibody drug conjugate and / or additional agent as described herein. Such formulating agents include substances known to those skilled in the art for preparing sustained-release formulations. For further references regarding pharmaceutically and physiologically acceptable formulating agents, see, for example, Remington's Pharmaceutical Sciences ,18th Ed.(1990,Mack Publishing Co.,Easton,Pa.18042)pages 1435-1712, The Merck Index , 12th Ed. (1996, Merck Publishing Group, Whitehouse, NJ); and Pharmaceutical Principles of Solid Dosage Forms (1993, Technonic Publishing Co., Inc., Lancaster, Pa.) Additional pharmaceutical compositions suitable for administration are known in the art and are applicable to the methods and compositions provided herein.

[0156] In some embodiments, the pharmaceutical compositions provided herein are in liquid form. In other embodiments, the pharmaceutical compositions provided herein are lyophilized.

[0157] Pharmaceutical compositions may be stored in sterile vials as solutions, suspensions, gels, emulsions, solids, or dehydrated or lyophilized powders. Such compositions may be stored in a ready-to-use form, a lyophilized form requiring reconstitution before use, a liquid form requiring dilution before use, or any other acceptable form. In some embodiments, pharmaceutical compositions are provided in single-use containers (e.g., single-use vials, ampoules, syringes, or auto-injectors (e.g., similar to EpiPen®)), while in other embodiments, they are provided in multi-use containers (e.g., multi-use vials). Any drug delivery device may be used to deliver the peptides and other agents described herein, including implants (e.g., implantable pumps) and catheter systems, both of which are known to those skilled in the art. Depot injections, typically administered subcutaneously or intramuscularly, may also be utilized to release the peptides and / or other agents described herein over a defined period of time. Depot injections are typically either solid or oil-based and generally contain at least one of the formulation components described herein. Those skilled in the art are familiar with the possible formulations and uses of depot injections. Certain embodiments contemplate the use of Nano Precision Medical's depot delivery technology (Nano Precision Medical; Emeryville, CA). This technology utilizes titania nanotube membranes that produce zero-order release rates for macromolecules, such as protein and peptide therapeutics. The biocompatible membrane is housed in a small subcutaneous implant that provides long-term (e.g., up to one year) constant-rate delivery of therapeutic macromolecules.

[0158] A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Thus, the pharmaceutical composition contains excipients suitable for administration by routes including parenteral (e.g., subcutaneous (sc), intravenous, intramuscular, or intraperitoneal), intradermal, oral (e.g., ingestion), inhalation, intracavity, intracranial, and transdermal (topical). Other exemplary routes of administration are described herein.

[0159] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. The suspension may be formulated using suitable dispersing or wetting agents and suspending agents disclosed herein or known to those skilled in the art. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable diluents, solvents, and dispersion media that may be used include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS), ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Additionally, sterile fixed oils are conventionally used as solvents or suspending media. Any bland fixed oil, including synthetic mono- or diglycerides, may be used for this purpose. Additionally, fatty acids, such as oleic acid, are used in the preparation of injectables. Prolonged absorption of certain injectable formulations can be achieved by including an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0160] In one aspect, the pharmaceutical compositions provided herein can be administered parenterally by injection, infusion, or implantation for local or systemic administration. As used herein, parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.

[0161] In one aspect, the pharmaceutical compositions provided herein can be formulated into any dosage form suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solution or suspension in liquid prior to injection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmacy (see, e.g., Remington, The Science and Practice of Pharmacy, supra).

[0162] In one aspect, a pharmaceutical composition intended for parenteral administration may comprise one or more pharmaceutically acceptable excipients including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against microbial growth, stabilizers, solubility enhancers, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.

[0163] In one embodiment, suitable aqueous vehicles include, but are not limited to, water, saline, saline or phosphate-buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, medium-chain triglycerides of coconut oil, and palm seed oil. Water-miscible vehicles include, but are not limited to, ethanol, 1,3-butanediol, liquid polyethylene glycols (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl-2-pyrrolidone, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0164] In one embodiment, suitable antimicrobial agents or preservatives include, but are not limited to, phenol, cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl- and propyl-paraben, and sorbic acid. Suitable isotonicity agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffers include, but are not limited to, phosphates and citrates. Suitable antioxidants include those described herein, including bisulfites and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents include those described herein, including sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agents include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable sequestering or chelating agents include, but are not limited to, EDTA. Suitable pH adjusters include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, such as α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, and sulfobutylether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, KS).

[0165] In one aspect, the pharmaceutical compositions provided herein can be formulated for single or multiple doses.Single dose formulations are packaged in ampoules, vials, or syringes.Multiple dose parenteral formulations can contain antibacterial agents at bacteriostatic or fungistatic concentrations.All parenteral formulations must be sterile, as is known and practiced in the art.

[0166] In one aspect, the pharmaceutical composition is provided as a ready-to-use sterile solution. In another aspect, the pharmaceutical composition is provided as a sterile dry soluble product, including lyophilized powders and hypodermic tablets, which are reconstituted with a vehicle before use. In yet another aspect, the pharmaceutical composition is provided as a ready-to-use sterile suspension. In yet another aspect, the pharmaceutical composition is provided as a sterile dry insoluble product, which is reconstituted with a vehicle before use. In yet another aspect, the pharmaceutical composition is provided as a ready-to-use sterile emulsion.

[0167] In one aspect, the pharmaceutical compositions provided herein can be formulated as immediate or modified release dosage forms, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release forms.

[0168] In one aspect, the pharmaceutical composition can be formulated as a suspension, solid, semi-solid, or thixotropic liquid for administration as an implanted depot. In one aspect, the pharmaceutical compositions provided herein are dispersed in a solid internal matrix surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredients in the pharmaceutical composition to diffuse through.

[0169] In one aspect, suitable inner matrices include polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, silicone carbonate copolymer, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate.

[0170] In one aspect, suitable outer polymeric membranes include polyethylene, polypropylene, ethylene / propylene copolymers, ethylene / ethyl acrylate copolymers, ethylene / vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomeric polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol copolymers, ethylene / vinyloxyethanol copolymers, and ethylene / vinyl acetate / vinyl alcohol terpolymers.

[0171] Aqueous suspensions contain the active material mixed with excipients suitable for their manufacture.Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersing agents or wetting agents can be natural phosphatides, for example, lecithin, or condensation products of alkylene oxides with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, for example, polyethylene sorbitan monooleate.Aqueous suspensions can also contain one or more preservatives.

[0172] Oily suspension can be prepared by suspending active ingredient in vegetable oil, such as peanut oil, olive oil, sesame oil or coconut oil, or mineral oil such as liquid paraffin.Oily suspension can contain thickening agent, such as beeswax, hard paraffin or cetyl alcohol.Sweetener and flavoring agent as mentioned above can be added to provide a pleasant oral preparation.

[0173] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

[0174] The pharmaceutical compositions provided herein can be in the form of oil-in-water emulsion.Oil phase can be vegetable oil, such as olive oil or peanut oil, or mineral oil, such as liquid paraffin, or their mixture.Suitable emulsifier can be natural gum, such as gum arabic or gum tragacanth; natural phosphatides, such as soybean, lecithin, and fatty acid-derived ester or partial ester; hexitol anhydride, such as sorbitan monooleate; and condensation product of partial ester and ethylene oxide, such as polyoxyethylene sorbitan monooleate.

[0175] Pharmaceutical compositions can also contain excipients to protect the composition from rapid degradation or excretion from the body, such as controlled-release formulations, including implants, liposomes, hydrogels, prodrugs, and microencapsulated delivery systems.For example, time-delay materials such as glyceryl monostearate or glyceryl stearate can be used alone or in combination with wax.Prolonged absorption of injectable pharmaceutical compositions can be achieved by including an absorption-delaying agent, such as aluminum monostearate or gelatin.Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.

[0176] The pharmaceutical compositions provided herein may be stored at -80°C, 4°C, 25°C, or 37°C.

[0177] Lyophilized compositions can be prepared by freeze-drying the liquid pharmaceutical compositions provided herein. In a specific embodiment, the pharmaceutical compositions provided herein are freeze-dried pharmaceutical compositions. In some embodiments, the pharmaceutical preparations are freeze-dried powders and can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels.

[0178] In some aspects, preparation of the lyophilized formulations provided herein involves batch processing of the formulated bulk solution for lyophilization, sterile filtration, filling into vials, freezing the vials in a lyophilization chamber, followed by lyophilization, stoppering, and capping.

[0179] A freeze-dryer can be used to prepare lyophilized formulations. For example, a VirTis Genesis Model EL pilot unit can be used. The unit incorporates a chamber with three working shelves (total usable shelf area approximately 0.4 square meters), an external condenser, and a mechanical vacuum pump system. Cascaded mechanical refrigeration can cool the shelves to -70°C or below and the external condenser to -90°C or below. Shelf temperature and chamber pressure were automatically controlled to + / - 0.5°C and + / - 2 microns (milliTorr), respectively. The unit was equipped with a capacitance manometer vacuum gauge, a Pirani vacuum gauge, a pressure transducer (to measure 0 to 1 atmosphere), and a relative humidity sensor.

[0180] The lyophilized powder can be prepared by dissolving the antibody-drug conjugate or its pharmaceutically acceptable derivative provided herein in a suitable solvent. In some embodiments, the lyophilized powder is sterilized. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single dose or multiple doses of the antibody-drug conjugate. The lyophilized powder can be stored under appropriate conditions, for example, at about 4°C to room temperature.

[0181] This lyophilized powder is reconstituted with water for injection to provide a formulation for parenteral administration.For reconstitution, the lyophilized powder is added to sterile water or other suitable excipients.The amount can be empirically determined and adjusted according to specific needs.

[0182] An exemplary reconstitution procedure is as follows: (1) Attach an 18- or 20-gauge needle to a 5 mL or 3 mL syringe and fill the syringe with water for injection (WFI)-grade water; (2) Measure the appropriate amount of WFI using the syringe's scale, ensuring there are no air bubbles in the syringe; (3) Insert the needle into the rubber stopper; (4) Dispense the entire contents of the syringe down the vial wall into a container, remove the syringe and needle, and place in a sharps container; (4) Carefully solubilize the entire contents of the vial by continuously swirling the vial until completely reconstituted (e.g., about 20 seconds to about 40 seconds), minimizing excessive agitation of the protein solution, which may result in foaming.

[0183] 5.3 Anti-191P4D12 Antibody Drug Conjugates The pharmaceutical compositions, formulations, and dosage forms provided herein comprise anti-191P4D12 antibody-drug conjugates. The anti-191P4D12 antibody-drug conjugates provided herein comprise an antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of a cytotoxic agent (or drug unit). The cytotoxic agent (or drug unit) can be covalently bound directly or via a linker unit (LU).

[0184] In some embodiments, the antibody drug conjugate compound has the following formula, or a pharmaceutically acceptable salt or solvate thereof: L-(LU-D) p (I) During the ceremony, L is an antibody unit, e.g., an anti-191P4D12 antibody or antigen-binding fragment thereof, provided in Section 5.3.1, infra; (LU-D) is the Linker Unit-Drug Unit moiety; where: LU- is a linker unit, D is a drug entity having cytostatic or cytotoxic activity against target cells; p is an integer from 1 to 20.

[0185] In some embodiments, p is in the range 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. In some embodiments, p is in the range of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is about 1. In other embodiments, p is about 2. In other embodiments, p is about 3. In other embodiments, p is about 4. In other embodiments, p is about 5. In other embodiments, p is about 6. In other embodiments, p is about 7. In other embodiments, p is about 8. In other embodiments, p is about 9. In other embodiments, p is about 10.

[0186] In some embodiments, the antibody drug conjugate compound has the following formula, or a pharmaceutically acceptable salt or solvate thereof: L-(A a -W w -Y y -D) p (II) During the ceremony, L is an antibody unit, e.g., an anti-191P4D12 antibody or antigen-binding fragment thereof, provided in Section 5.3.1 below; and -A a -W w -Y y - is a linker unit (LU), where -A- is an extender unit, a is 0 or 1, each -W- is independently an amino acid unit; w is an integer ranging from 0 to 12, -Y- is a self-immolative spacer unit; y is 0, 1 or 2; D is a drug entity having cytostatic or cytotoxic activity against target cells; p is an integer from 1 to 20.

[0187] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, p is in the range 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. In some embodiments, p is in the range of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4. In some embodiments, when w is not 0, y is 1 or 2. In some embodiments, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2 to 12, and y is 1 or 2. In some embodiments, a is 1, and w and y are 0.

[0188] For compositions containing multiple antibodies or antigen-binding fragments thereof, drug loading is represented by p, the average number of drug molecules per antibody unit. Drug loading can range from 1 to 20 drugs per antibody (D). The average number of drugs per antibody in a conjugation reaction preparation can be characterized by conventional means, such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of antibody-drug conjugates with respect to p can also be determined. In some cases, separation, purification, and characterization of a homogeneous antibody-drug conjugate with a specific value of p from antibody-drug conjugates with other drug loads can be achieved by means such as reverse-phase HPLC or electrophoresis. In an exemplary embodiment, p is 2 to 8.

[0189] 5.3.1 Anti-191P4D12 Antibodies or Antigen-Binding Fragments In one embodiment, the antibody or antigen-binding fragment thereof that binds to a 191P4D12-related protein is an antibody or antigen-binding fragment thereof that specifically binds to a 191P4D12 protein comprising the amino acid sequence of SEQ ID NO:2 (see Figure 5A). The corresponding cDNA encoding the 191P4D12 protein has the sequence of SEQ ID NO:1 (see Figure 5A).

[0190] Antibodies that specifically bind to a 191P4D12 protein comprising the amino acid sequence of SEQ ID NO:2 include antibodies that can bind to other 191P4D12-related proteins. For example, antibodies that bind to a 191P4D12 protein comprising the amino acid sequence of SEQ ID NO:2 can bind to 191P4D12-related proteins, such as 191P4D12 variants and homologs or analogs thereof.

[0191] In some embodiments, the anti-191P4D12 antibodies provided herein are monoclonal antibodies.

[0192] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:4 (cDNA sequence of SEQ ID NO:3) and / or a light chain comprising the amino acid sequence of SEQ ID NO:6 (cDNA sequence of SEQ ID NO:5), as shown in Figure 5B.

[0193] In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO: 7, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 8. SEQ ID NO: 7 and SEQ ID NO: 8 are shown in Figure 5C and are listed below. TIFF2025170007000018.tif99165

[0194] In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO:22 (which is the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO:7), and a light chain variable region comprising the amino acid sequence of the light chain variable region set forth in SEQ ID NO:23 (which is the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (arginine) of SEQ ID NO:8). In other embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:22 (which is the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO:7), and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:23 (which is the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (arginine) of SEQ ID NO:8). SEQ ID NO:22 and SEQ ID NO:23 are listed below. TIFF2025170007000019.tif55165

[0195] CDR sequences can be determined according to well-known numbering systems. As mentioned above, CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al. supra). Instead, Chothia refers to the position of structural loops (see, for example, Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). When numbered using Kabat's numbering rules, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (e.g., Antibody EngineeringVol. 2 (Kontermann and Dubel eds., 2nd ed. 2010). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system specialized for immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to both in terms of amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides within structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from one species of immunoglobulin onto an acceptor framework, typically from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309:657-70. For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT-specific numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are shown in Table 30 above.

[0196] In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:7 according to the Kabat numbering, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:8 according to the Kabat numbering.

[0197] In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:7 according to AbM numbering, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:8 according to AbM numbering.

[0198] In another embodiment, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:7 according to Chothia numbering, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:8 according to Chothia numbering.

[0199] In another embodiment, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) having the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:7 by Contact numbering, and a light chain variable region comprising a CDR having the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:8 by Contact numbering.

[0200] In yet another embodiment, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:7 according to the IMGT numbering system, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:8 according to the IMGT numbering system.

[0201] As noted above, CDR sequences according to different numbering systems can be readily determined using online tools such as those provided by the Antigen Receptor Numbering and Receptor Classification (ANARCI). For example, the heavy chain CDR sequences in SEQ ID NO:7 and the light chain CDR sequences in SEQ ID NO:8 according to the Kabat numbering as determined by ANARCI are listed in Table 31 below.

[0202] (Table 31) TIFF2025170007000020.tif31166

[0203] As another example, the heavy chain CDR sequences within SEQ ID NO:22 and the light chain CDR sequences within SEQ ID NO:23 according to the IMGT numbering as determined by ANARCI are listed in Table 32 below.

[0204] (Table 32) TIFF2025170007000021.tif31166

[0205] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR H1 comprising the amino acid sequence of SEQ ID NO:9, a CDR H2 comprising the amino acid sequence of SEQ ID NO:10, a CDR H3 comprising the amino acid sequence of SEQ ID NO:11, a CDR L1 comprising the amino acid sequence of SEQ ID NO:12, a CDR L2 comprising the amino acid sequence of SEQ ID NO:13, and a CDR L3 comprising the amino acid sequence of SEQ ID NO:14.

[0206] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (arginine) of SEQ ID NO:8.

[0207] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain comprising the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8.

[0208] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including, but not limited to, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the antibodies described herein, it is contemplated that antibody variants can be prepared. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art will recognize that amino acid changes can alter post-translational processes of the antibody, for example, by changing the number or position of glycosylation sites or altering membrane anchoring properties.

[0209] In some embodiments, the antibodies provided herein are chemically modified, for example, by the covalent attachment of any type of molecule to the antibody. Antibody derivatives can include antibodies chemically modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody can contain one or more non-classical amino acids.

[0210] Mutations can be substitutions, deletions, or insertions of one or more codons encoding a single domain antibody or polypeptide, resulting in a change in the amino acid sequence compared to the original antibody or polypeptide. Amino acid substitutions can be, for example, conservative amino acid substitutions, resulting from the replacement of one amino acid with another amino acid containing similar structural and / or chemical properties, such as the replacement of leucine with serine. Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Insertions or deletions can range from about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions comprise fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule. In specific embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Permissible variations can be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for the activity exhibited by the parent antibody.

[0211] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue.

[0212] Antibodies generated by conservative amino acid substitutions are included in the present disclosure. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue containing a side chain with a similar charge. As described above, families of amino acid residues containing side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed, the activity of the protein can be determined, and conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) can be made to maintain or not significantly change the properties.

[0213] Amino acids can be grouped according to similarities in the properties of their side chains (e.g., Lehninger, Biochemistry73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0214] For example, any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may be substituted with another amino acid, e.g., alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0215] Modifications can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J. 237:1-7; and Zoller et al., 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34:315-23), or other known techniques can be performed on cloned DNA to produce anti-anti-MSLN antibody mutant DNA.

[0216] Covalent modification of antibodies is included within the scope of this disclosure. Covalent modification includes reacting targeted amino acid residues of the antibody with organic derivatizing agents that can react with selected side chains or N- or C-terminal residues of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0217] Other types of covalent modifications of antibodies within the scope of the present disclosure include altering the native glycosylation pattern of the antibody or polypeptide (see, e.g., Beck et al., 2008, Curr. Pharm. Biotechnol. 9:482-501; and Walsh, 2010, Drug Discov. Today 15:773-80), and linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, e.g., by methods described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

[0218] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 70% homology to the heavy chain shown in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 75% homology to the heavy chain shown in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 80% homology to the heavy chain shown in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 85% homology to the heavy chain shown in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 90% homology to the heavy chain shown in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 95% homology to the heavy chain shown in SEQ ID NO:7.

[0219] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 70% homology to the light chain shown in SEQ ID NO:8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 75% homology to the light chain shown in SEQ ID NO:8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 80% homology to the light chain shown in SEQ ID NO:8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 85% homology to the light chain shown in SEQ ID NO:8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 90% homology to the light chain shown in SEQ ID NO:8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 95% homology to the light chain shown in SEQ ID NO:8.

[0220] In some embodiments, the anti-191P4D12 antibodies provided herein comprise the heavy and light chain CDR regions of the antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chain CDR regions comprising amino acid sequences that are homologous to the amino acid sequences of the heavy and light chain CDR regions of Ha22-2(2,4)6.1, wherein the antibody retains the desired functional properties of the anti-191P4D12 antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0221] In some aspects, the antibodies or antigen-binding fragments thereof provided herein comprise a humanized heavy chain variable region and a humanized light chain variable region, wherein: (a) the heavy chain variable region comprises a CDR comprising the amino acid sequence of the heavy chain variable region CDR shown in the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; (b) The light chain variable region comprises a CDR comprising the amino acid sequence of the light chain variable region CDR shown in the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0222] In some embodiments, the anti-191P4D12 antibodies provided herein comprise the heavy and light chain variable regions of the antibody designated Ha22-2(2,4)6.1 (see Figure 3), which is produced by the hybridoma deposited at the American Type Culture Collection (ATCC) under accession number PTA-11267, or heavy and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of the heavy and light chain variable regions of Ha22-2(2,4)6.1, wherein the antibodies retain the desired functional properties of the anti-191P4D12 antibodies provided herein. Constant regions of any subclass can be selected as the constant region of the antibodies of the present invention. In one embodiment, a human IgG1 constant region can be used as the heavy chain constant region, and a human Igκ constant region can be used as the light chain constant region.

[0223] In some embodiments, the anti-191P4D12 antibodies provided herein comprise the heavy and light chains of the antibody designated Ha22-2(2,4)6.1 (see Figure 3) produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chains comprising amino acid sequences homologous to the amino acid sequences of the heavy and light chains of Ha22-2(2,4)6.1, wherein the antibodies retain the desired functional properties of the anti-191P4D12 antibodies provided herein.

[0224] In some aspects, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; and (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to the light chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267;

[0225] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 85% identical to the heavy chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to the heavy chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 95% identical to the heavy chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain variable region can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0226] In some embodiments, the light chain variable region comprises an amino acid sequence that is at least 85% identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain variable region comprises an amino acid sequence that is at least 90% identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the light chain variable region comprises an amino acid sequence that is at least 95% identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain variable region can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the light chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0227] In other aspects, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain and a light chain, wherein: (a) the heavy chain comprises an amino acid sequence that is at least 80% identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; and (b) the light chain comprises an amino acid sequence that is at least 80% homologous to the light chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0228] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 85% identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain comprises an amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the heavy chain comprises an amino acid sequence that is at least 95% identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0229] In some embodiments, the light chain comprises an amino acid sequence that is at least 85% identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain comprises an amino acid sequence that is at least 90% identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the light chain comprises an amino acid sequence that is at least 95% identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the light chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0230] The engineered antibodies provided herein include those in which modifications have been made to framework residues within the VH and / or VL (e.g., to improve the properties of the antibody). Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. To return framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence (e.g., "backmutating" leucine to methionine), for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0231] Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T-cell epitopes and thereby reduce the potential immunogenicity of the antibody. This approach, also referred to as "deimmunization," is described in further detail in U.S. Patent Application Publication No. 2003 / 0153043 by Carr et al.

[0232] In addition to, or instead of, modifications made within the framework or CDR regions, antibodies of the invention can be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, the anti-191P4D12 antibodies provided herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, also to alter one or more functional properties of the antibody. Each of these aspects is described in further detail below.

[0233] In one embodiment, the hinge region of CH1 is modified to change, for example, increase or decrease, the number of cysteine ​​residues in the hinge region. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the anti-191P4D12 antibody.

[0234] In another embodiment, the Fc-hinge region of the antibody is mutated to decrease the biological half-life of the anti-191P4D12 antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has reduced Staphylococcus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0235] In another embodiment, the anti-191P4D12 antibody is modified to increase its biological half-life. Various approaches are possible. For example, mutations can be introduced as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase biological half-life, the antibody can be altered in the CH1 or CL region to include salvage receptor binding epitopes obtained from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al.

[0236] In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from specific amino acid residues can be replaced with a different amino acid residue so that the antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0237] The reactivity of anti-191P4D12 antibodies with 191P4D12-related proteins can be established by a number of well-known means, including Western blot, immunoprecipitation, ELISA, and FACS analysis, using 191P4D12-related proteins, 191P4D12-expressing cells, or extracts thereof, as needed. 191P4D12 antibodies or fragments thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes. Furthermore, bispecific antibodies specific for two or more 191P4D12 epitopes can be generated using methods commonly known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53:2560-2565).

[0238] In yet another specific embodiment, the anti-191P4D12 antibody provided herein comprises the heavy and light chains of the antibody designated Ha22-2(2,4)6.1. The heavy chain of Ha22-2(2,4)6.1 consists of the amino acid sequence ranging from E residue 20 to K residue 466 of SEQ ID NO:7, and the light chain of Ha22-2(2,4)6.1 consists of the amino acid sequence ranging from D residue 23 to C residue 236 of SEQ ID NO:8.

[0239] Ha22-2(2,4)6.1 Hybridomas that produce antibodies called August 18, 2010 to the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 (via Federal Express), accession no. PTA-11267 was assigned.

[0240] 5.3.2 Cytotoxic Agents (Drug Units) In some embodiments, the ADC comprises an antibody or antigen-binding fragment thereof conjugated to a dolastatin or auristatin, a peptide analog and derivative of dolostatin (U.S. Patent Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and to have anticancer (U.S. Patent No. 5,663,149) and antifungal (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965) activity. The dolastatin or auristatin drug unit can be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug unit (WO 02 / 088172).

[0241] Exemplary auristatin embodiments include the N-terminally linked monomethyl auristatin drug units DE and DF disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, published March 28, 2004, and described in U.S. Patent Application Publication No. 2005 / 0238649, the disclosure of which is expressly incorporated by reference in its entirety.

[0242] In some embodiments, the auristatin is MMAE (where the wavy line indicates the covalent attachment to the linker of the antibody drug conjugate). TIFF2025170007000022.tif24140

[0243] In some embodiments, exemplary embodiments comprising MMAE and a linker moiety (further described herein) have the following structure (where L represents an antibody and p ranges from 1 to 12): TIFF2025170007000023.tif36160

[0244] Typically, peptide-based drug units can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to solution-phase synthesis methods well known in the field of peptide chemistry (see E. Schroder and K. Lubke, "The Peptides," volume 1, pp. 76-136, 1965, Academic Press). Auristatin / dolastatin Drug Units may be prepared according to the methods of U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, GR, et al. Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

[0245] 5.3.3 Linker Typically, an antibody drug conjugate comprises a linker unit between the drug unit (e.g., MMAE) and the antibody unit (e.g., an anti-191P4D12 antibody or antigen-binding fragment thereof). In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In still other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation.

[0246] In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker can be, for example, a peptidyl linker cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug within target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyl linkers cleavable by enzymes present in 191P4D12-expressing cells. For example, a peptidyl linker cleavable by cathepsin B, a thiol-dependent protease highly expressed in cancerous tissues, can be used (e.g., a Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 15)). Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, the entire contents of which are incorporated herein by reference for all purposes. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker). One advantage of using intracellular proteolytic release of a therapeutic agent is that the agent is typically attenuated when conjugated, and the serum stability of the conjugate is typically high.

[0247] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or below pH 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (such as a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, eg, US Pat. No. 5,622,929).

[0248] In still other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), which can be formed using SPDB and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987; see also U.S. Pat. No. 4,880,935).

[0249] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0250] In still other embodiments, the linker unit is not cleavable and the drug is released by antibody degradation (see U.S. Patent Application Publication No. 2005 / 0238649, incorporated herein by reference in its entirety for all purposes).

[0251] Typically, the linker is substantially insensitive to the extracellular environment. As used herein, in the context of a linker, "substantially insensitive to the extracellular environment" means that when the antibody-drug conjugate is present in an extracellular environment (e.g., plasma), about 20% or less, typically about 15% or less, more typically about 10% or less, and even more typically about 5% or less, about 3% or less, or about 1% or less of the linkers in a sample of the antibody-drug conjugate are cleaved. Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate compound with plasma for a predetermined time (e.g., 2 hours, 4 hours, 8 hours, 16 hours, or 24 hours) and then quantifying the amount of free drug present in the plasma.

[0252] In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent portion of an antibody-drug conjugate compound described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both an auristatin compound and an anti-191P4D12 antibody or antigen-binding fragment thereof.

[0253] Various exemplary linkers that can be used with the compositions and methods of the present invention are described in WO 2004-010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 20050238649, and U.S. Patent Application Publication No. 2006 / 0024317 (each of which is incorporated by reference in its entirety for all purposes).

[0254] A "Linker Unit" (LU) is a bifunctional compound that can be used to link a Drug Unit and an Antibody Unit to form an antibody drug conjugate. In some embodiments, the Linker Unit has the formula: -A a -W w -Y y - and where -A- is an extender unit; a is 0 or 1, each -W- is independently an amino acid unit; w is an integer ranging from 0 to 12, -Y- is a self-immolative spacer unit; y is 0, 1, or 2.

[0255] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, when w is 1-12, y is 1 or 2. In some embodiments, w is 2-12, and y is 1 or 2. In some embodiments, a is 1, and w and y are 0.

[0256] 5.3.3.1 Decompression Unit The extender unit (A), when present, can link the antibody unit to the amino acid unit (-W-), if present, to the spacer unit (-Y-), if present, or to the drug unit (-D). Useful functional groups that may be present on an anti-191P4D12 antibody or antigen-binding fragment thereof (e.g., Ha22-2(2,4)6.1) naturally or through chemical manipulation include, but are not limited to, sulfhydryl, amino, hydroxyl, anomeric hydroxyl groups of carbohydrates, and carboxyl. Suitable functional groups are sulfhydryl and amino. In one example, sulfhydryl groups can be generated by reduction of intramolecular disulfide bonds in an anti-191P4D12 antibody or antigen-binding fragment thereof. In another embodiment, sulfhydryl groups can be generated by reacting amino groups of lysine moieties of an anti-191P4D12 antibody or antigen-binding fragment with 2-iminothiolane (Traut's reagent) or other sulfhydryl-generating reagents. In certain embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof is a recombinant antibody and is engineered to carry one or more lysines. In certain other embodiments, the recombinant anti-191P4D12 antibody is engineered to carry additional sulfhydryl groups, e.g., additional cysteines.

[0257] In one embodiment, the extender unit forms a bond with a sulfur atom of the antibody unit. The sulfur atom can be derived from a sulfhydryl group of the antibody. Representative extender units of this embodiment are shown in brackets in Formulas IIIa and IIIb below, where L-, -W-, -Y-, -D, w, and y are as defined above, and R 17 is -C1~C 10 Alkylene-, -C1~C 10 Alkenylene-, -C1~C 10 Alkynylene-, carbocyclo-, -O-(C1-C8 alkylene)-, O-(C1-C8 alkenylene)-, -O-(C1-C8 alkynylene)-, -arylene-, -C1-C 10 Alkylene-arylene-, -C2~C 10 Alkenylene-arylene, -C2-C 10Alkynylene-arylene, -arylene-C1-C 10 Alkylene, -arylene-C2~C 10 Alkenylene, -arylene-C2-C 10 Alkynylene-, -C1~C 10 Alkylene-(carbocyclo)-, -C2-C 10 Alkenylene-(carbocyclo)-, -C2-C 10 Alkynylene-(carbocyclo)-, -(carbocyclo)-C1-C 10 Alkylene-, -(carbocyclo)-C2-C 10 Alkenylene, -(carbocyclo)-C2-C 10 Alkynylene, -heterocyclo-, -C1-C 10 Alkylene-(heterocyclo)-, -C2-C 10 Alkenylene-(heterocyclo)-, -C2-C 10 Alkynylene-(heterocyclo)-, -(heterocyclo)-C1-C 10 Alkylene-, -(heterocyclo)-C2-C 10 Alkenylene-, -(heterocyclo)-C1-C 10 Alkynylene-, -(CH2CH2O) r - or -(CH2CH2O) r and r is an integer ranging from 1 to 10, wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclo, heterocyclo, and arylene radicals, either alone or as part of another group, are optionally substituted. In some embodiments, said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclo, heterocyclo, and arylene radicals, either alone or as part of another group, are unsubstituted.

[0258] In some embodiments, R 17 is -C1~C 10 Alkylene-, -carbocyclo-, -O-(C1-C8 alkylene)-, -arylene-, -C1-C 10Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1~C 10 Alkylene-(carbocyclo)-, -(carbocyclo)-C1-C 10 Alkylene-, -C3-C8 heterocyclo-, -C1-C 10 Alkylene-(heterocyclo)-, -(heterocyclo)-C1-C 10 Alkylene-, -(CH2CH2O) r - and -(CH2CH2O) r -CH2-, and r is an integer ranging from 1 to 10, wherein the alkylene group is unsubstituted and the remaining groups are optionally substituted.

[0259] It should be understood that from all exemplary embodiments, even if not explicitly stated, 1 to 20 drug units (p=1-20) can be linked to an antibody unit. TIFF2025170007000024.tif57128

[0260] An exemplary extender unit is R 17 is an extender unit of formula IIIa where is —(CH 2 ) 5 —. TIFF2025170007000025.tif24128

[0261] Another exemplary extender unit is R 17 -(CH2CH2O) r -CH2- and r is 2. TIFF2025170007000026.tif24128

[0262] An exemplary extender unit is R 17 is arylene- or arylene-C1~C 10 The extender unit of Formula IIIa is alkylene-. In some embodiments, the aryl group is an unsubstituted phenyl group.

[0263] Yet another exemplary extender unit is R 17 is an extender unit of formula IIIb where is —(CH 2 ) 5 —. TIFF2025170007000027.tif20128

[0264] In certain embodiments, the extender unit is linked to the antibody unit via a disulfide bond between a sulfur atom of the antibody unit and a sulfur atom of the extender unit. A representative extender unit of this embodiment is depicted within the brackets of Formula IV, where R 17 , L-, -W-, -Y-, -D, w and y are as defined above. TIFF2025170007000028.tif18128

[0265] It should be noted that throughout this application, the S moiety in the following formulae refers to the sulfur atom of the antibody unit, unless the context dictates otherwise. TIFF2025170007000029.tif12128

[0266] In certain structural descriptions of sulfur-linked ADCs herein, the antibody is represented as "L." It can also be represented as "Ab-S." The inclusion of "S" merely indicates the sulfur bond characteristic and does not indicate that a particular sulfur atom has multiple linker-drug moieties. The left parenthesis in structures using the "Ab-S" designation can also be placed to the left of the sulfur atom between Ab and S, which is an equivalent designation for the ADCs of the invention described throughout this specification.

[0267] In yet other embodiments, the extender comprises a reactive site capable of forming a bond with a primary or secondary amino group of an antibody unit. Examples of these reactive sites include, but are not limited to, activated esters, such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Representative extender units of this embodiment are depicted within square brackets in Formulas Va and Vb, where -R 17 -, L-, -W-, -Y-, -D, w and y are as defined above. TIFF2025170007000030.tif53128

[0268] In some embodiments, the extender comprises a reactive site that is reactive toward a modified carbohydrate (-CHO) group that may be present on an antibody unit. For example, the carbohydrate can be mildly oxidized using a reagent such as sodium periodate, and the resulting (-CHO) unit of the oxidized carbohydrate can be condensed with an extender containing a functional group such as a hydrazide, oxime, primary or secondary amine, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide as described in Kaneko et al., 1991, Bioconjugate Chem. 2:133-41. Representative extender units of this embodiment are depicted within square brackets in Formulas VIa, VIb, and VIc, where -R 17 -, L-, -W-, -Y-, -D, w and y are as defined above. TIFF2025170007000031.tif77128

[0269] 5.3.3.2 Amino acid units The amino acid unit (-W-), when present, links the extender unit to the spacer unit if the spacer unit is present, the extender unit to the drug unit if the spacer unit is absent, and the antibody unit to the drug unit if the extender unit and spacer unit are absent.

[0270] W w - can be, for example, a monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit. Each -W- unit independently has the formula shown below in square brackets, where w is an integer ranging from 0 to 12: TIFF2025170007000032.tif29128 in formula, R 19is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, The file is TIFF2025170007000033.tif107148.

[0271] In some embodiments, the amino acid unit can be enzymatically cleaved by one or more enzymes, including cancer or tumor-associated proteases, to liberate the drug unit (-D), which in one embodiment, upon release, is protonated in vivo to provide the drug (D).

[0272] In certain embodiments, the amino acid units comprise natural amino acids. In other embodiments, the amino acid units comprise unnatural amino acids. Exemplary Ww units are represented by Formulas VII-IX below: TIFF2025170007000034.tif21128 formula, R 20 and R 21 is as follows: TIFF2025170007000035.tif120147TIFF2025170007000036.tif21128In formula, R 20 , R 21 and R 22 is as follows: TIFF2025170007000037.tif37134TIFF2025170007000038.tif21128In formula, R 20 , R 21 , R 22 and R23 is as follows: TIFF2025170007000039.tif28137.

[0273] Exemplary amino acid units include R 20 is benzyl and R 21 is -(CH2)4NH2; R 20 is isopropyl, and R 21 is -(CH2)4NH2; or R 20 is isopropyl, and R 21 Examples include, but are not limited to, units of formula VII above, wherein is —(CH 2 ) 3 NHCONH 2 .

[0274] Another exemplary amino acid unit is R 20 is benzyl and R 21 is benzyl and R 22 is a unit of formula VIII where is —(CH2)4NH2.

[0275] -W is useful due to enzymatic cleavage by certain enzymes, e.g., tumor-associated proteases. w - units can be designed to optimize their selectivity. w -units whose cleavage is catalyzed by cathepsins B, C and D, or plasmin proteases.

[0276] In one aspect, -W w - is a dipeptide, tripeptide, tetrapeptide or pentapeptide. 19 , R 20 , R 21 , R 22 or R 23 If is other than hydrogen, R 19 , R 20 , R 21 , R 22 or R 23 The carbon atom to which is attached is chiral.

[0277] R 19 , R 20 , R21 , R 22 or R 23 Each carbon atom to which is attached is independently in the (S) or (R) configuration.

[0278] In one specific embodiment, the amino acid unit is valine-citrulline (vc or Val-Cit). In another specific embodiment, the amino acid unit is phenylalanine-lysine (i.e., fk). In yet another specific embodiment, the amino acid unit is N-methylvaline-citrulline. In yet another specific embodiment, the amino acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine, isonipecotic acid lysine, β-alanine lysine, glycine serine valine glutamine, and isonepecotic acid.

[0279] 5.3.3.3 Spacer Units The Spacer unit (-Y-), when present, links the Amino Acid unit to the Drug unit, if present. Alternatively, the Spacer unit links the Extender unit to the Drug unit, if the Amino Acid unit is absent. The Spacer unit also links the Drug unit to the Antibody unit, if both the Amino Acid unit and the Extender unit are absent.

[0280] Spacer units are of two general types: non-self-immolative or self-immolative. A non-self-immolative spacer unit is one in which some or all of the spacer unit remains attached to the Drug unit after cleavage, particularly enzymatic cleavage, of the amino acid unit from the antibody-drug conjugate. Examples of non-self-immolative spacer units include, but are not limited to, a (glycine-glycine) spacer unit and a glycine spacer unit (both depicted in Scheme 1) (below). When a conjugate comprising a glycine-glycine spacer unit or a glycine spacer unit undergoes enzymatic cleavage by an enzyme (e.g., a tumor cell-associated protease, a cancer cell-associated protease, or a lymphocyte-associated protease), the glycine-glycine-Drug unit or glycine-Drug unit is cleaved from L-Aa-Ww-. In one embodiment, an independent hydrolysis reaction occurs within the target cell, cleaving the glycine-Drug unit bond and liberating the drug. Scheme 1 TIFF2025170007000040.tif47128

[0281] In some embodiments, the non-self-immolative spacer unit (-Y-) is -Gly-. In some embodiments, the non-self-immolative spacer unit (-Y-) is -Gly-Gly-.

[0282] In one embodiment, the spacer unit is absent (-Y where y=0). y -).

[0283] Alternatively, an antibody-drug conjugate comprising a self-immolative spacer unit can release -D. As used herein, the term "self-immolative spacer" refers to a bifunctional chemical moiety that can covalently link two spaced chemical moieties together into a stable tripartite molecule, which spontaneously separates from the second chemical moiety upon cleavage of the bond to the first moiety.

[0284] In some embodiments, -Y y - is the phenylene moiety Q mp-aminobenzyl alcohol (PAB) units (see Schemes 2 and 3) substituted with Q, where Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano, and m is an integer ranging from 0 to 4. The alkyl, alkenyl, and alkynyl groups, either alone or as part of another group, can be substituted.

[0285] In some embodiments, -Y- is linked to -W through the amino nitrogen atom of the PAB group. w - is a PAB group linked to - and directly bonded to -D through a carbonate, carbamate, or ether group. Without being bound to a particular theory or mechanism, Scheme 2 displays a possible drug release mechanism for a PAB group directly bonded to -D through a carbamate or carbonate group, as described by Toki et al., 2002, J. Org. Chem. 67:1866-1872. Scheme 2 TIFF2025170007000041.tif97128

[0286] In Scheme 2, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; and p ranges from 1 to about 20. The alkyl, alkenyl, and alkynyl groups, either alone or as part of another group, can be substituted.

[0287] Without being bound by any particular theory or mechanism, Scheme 3 depicts a possible drug release mechanism for a PAB group directly attached to -D via an ether or amine bond, where D contains an oxygen or nitrogen group that is part of the drug unit. Scheme 3 TIFF2025170007000042.tif120128

[0288] In Scheme 3, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; and p ranges from 1 to about 20. The alkyl, alkenyl, and alkynyl groups, either alone or as part of another group, can be substituted.

[0289] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Spacers that undergo cyclization upon hydrolysis of the amide bond can also be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing drugs substituted at the alpha position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-immolative spacer.

[0290] In one embodiment, the spacer unit is a branched bis(hydroxymethyl)-styrene (BHMS) unit as depicted in Scheme 4, which can be used to incorporate and release multiple drugs. Scheme 4 TIFF2025170007000043.tif37128

[0291] In Scheme 4, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; n is 0 or 1; and p ranges from 1 to about 20. The alkyl, alkenyl, and alkynyl groups, either alone or as part of another group, can be substituted.

[0292] In some embodiments, the -D units are the same. In yet other embodiments, the -D moieties are different.

[0293] In one aspect, the spacer unit (-Y y -) are represented by Formulas X-XII: TIFF2025170007000044.tif28128 where Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano, and m is an integer ranging from 0 to 4. The alkyl, alkenyl, and alkynyl groups, either alone or as part of another group, can be substituted. TIFF2025170007000045.tif9128 and TIFF2025170007000046.tif15128.

[0294] Embodiments of Formulas I and II that include antibody-drug conjugate compounds can include: TIFF2025170007000047.tif19128 where w and y are 0, 1 or 2, respectively. TIFF2025170007000048.tif19128 where w and y are each 0. TIFF2025170007000049.tif180128.

[0295] 5.3.3.4 Drug burden Drug loading is represented by p and is the average number of drug units per antibody in the molecule. Drug loading can range from 1 to 20 drug units (D) per antibody. The ADCs provided herein include a collection of antibodies or antigen-binding fragments conjugated with, for example, 1 to 20 drug units. The average number of drug units per antibody in an ADC preparation from a conjugation reaction can be characterized by conventional means, such as mass spectrometry and ELISA assays. The quantitative distribution of the ADC with respect to p can also be determined. In some cases, separation, purification, and characterization of a homogeneous ADC with a particular value of p from ADCs with other drug loads can be achieved by means such as electrophoresis.

[0296] In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 20. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 18. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 15. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 12. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 10. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 9. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 8. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 7. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 6. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 5. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 4. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 3. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 12. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 10. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 9. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 8. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 7. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 6. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 5.

[0297] In certain embodiments, the drug loading of the ADCs provided herein is in the range of 1 to about 8, about 2 to about 6, about 3 to about 5, about 3 to about 4, about 3.1 to about 3.9, about 3.2 to about 3.8, about 3.2 to about 3.7, about 3.2 to about 3.6, about 3.3 to about 3.8, or about 3.3 to about 3.7.

[0298] In certain embodiments, fewer drug units than the theoretical maximum are conjugated to the antibody during the conjugation reaction. The antibody may contain, for example, lysine residues that do not react with either the drug-linker intermediate or the linker reagent. Generally, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be linked to drug units; in fact, most cysteine ​​thiol residues in antibodies exist as disulfide bridges. In certain embodiments, antibodies can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reducing conditions to generate reactive cysteine ​​thiol groups. In certain embodiments, antibodies are subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine. In some embodiments, linker units or drug units are conjugated via lysine residues on the antibody unit. In some embodiments, linker units or drug units are conjugated via cysteine ​​residues on the antibody unit.

[0299] In some embodiments, the amino acid attached to the linker unit or drug unit is in the heavy chain of the antibody or antigen-binding fragment thereof. In some embodiments, the amino acid attached to the linker unit or drug unit is in the light chain of the antibody or antigen-binding fragment thereof. In some embodiments, the amino acid attached to the linker unit or drug unit is in the hinge region of the antibody or antigen-binding fragment thereof. In some embodiments, the amino acid attached to the linker unit or drug unit is in the Fc region of the antibody or antigen-binding fragment thereof. In other embodiments, the amino acid attached to the linker unit or drug unit is in the constant region of the antibody or antigen-binding fragment thereof (e.g., CH1, CH2, or CH3 of the heavy chain, or CH1 of the light chain). In still other embodiments, the amino acid attached to the linker unit or drug unit is in the VH framework region of the antibody or antigen-binding fragment thereof. In still other embodiments, the amino acid attached to the linker unit or drug unit is in the VL framework region of the antibody or antigen-binding fragment thereof.

[0300] The loading (drug / antibody ratio) of an ADC can be controlled in various ways, for example, by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limited reduction conditions for cysteine ​​thiol modification, or (iv) recombinantly manipulating the amino acid sequence of the antibody such that the number and position of cysteine ​​residues are altered to control the number and / or position of linker-drug bonds (such as in a ThioMab or ThioFab prepared as disclosed herein and in WO 2006 / 034488, which is incorporated herein by reference in its entirety).

[0301] It should be understood that when multiple nucleophilic groups react with a drug-linker intermediate or linker reagent and subsequently with a drug unit reagent, the resulting product is a mixture of ADC compounds with a distribution of one or more drug units attached to an antibody unit. The average number of drugs per antibody can be calculated from the mixture by a dual antibody-specific and drug-specific ELISA antibody assay. Individual ADC molecules can be identified in a mixture by mass spectrometry and separated by HPLC, for example, hydrophobic interaction chromatography (e.g., Hamblett, KJ, et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, SC, et al., "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, homogeneous ADCs having a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0302] 5.3.3 Preparation of Antibody Drug Conjugates The production of the antibody-drug conjugates provided herein can be achieved by any technique known to those skilled in the art. Briefly, an antibody-drug conjugate comprises an anti-191P4D12 antibody or antigen-binding fragment thereof as an antibody unit, a drug, and optionally a linker connecting the drug and the binder. In some embodiments, the antibody is an anti-191P4D12 antibody comprising the CDR regions of the antibody designated Ha22-2(2,4)6.1 described above. In a specific embodiment, the antibody is an anti-191P4D12 antibody comprising the heavy chain variable region and light chain variable region of the antibody designated Ha22-2(2,4)6.1 described above. In a specific embodiment, the antibody is an anti-191P4D12 antibody comprising the heavy chain and light chain of the antibody designated Ha22-2(2,4)6.1 described above.

[0303] Several different reactions are available for covalently attaching drugs and / or linkers to binders. This is often achieved by reaction of amino acid residues on binders, such as antibody molecules, including the amine groups of lysine, the free carboxylic acid groups of glutamic and aspartic acids, the sulfhydryl groups of cysteine, and various moieties of aromatic amino acids. One of the most commonly used nonspecific methods of covalent attachment is the carbodiimide reaction, which links the carboxyl (or amino) group of a compound to the amino (or carboxy) group of an antibody. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino groups of compounds to the amino groups of antibody molecules. The Schiff base reaction is also available for attaching drugs to binders. This method involves periodate oxidation of drugs containing glycol or hydroxy groups, thus forming aldehydes, which are then reacted with the binder. Attachment occurs via the formation of a Schiff base with the amino group of the binder. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to binders. Other techniques are known to those skilled in the art and are within the scope of the present invention.

[0304] In certain embodiments, an intermediate that is a precursor to the linker is reacted with the drug under appropriate conditions. In certain embodiments, a reactive group is used on the drug and / or intermediate. The product of the reaction between the drug and the intermediate, or a derivatized drug, is then reacted with an anti-191P4D12 antibody under appropriate conditions.

[0305] Each of the specific units of the antibody-drug conjugate is described in more detail herein. The synthesis and structure of exemplary linker units, extender units, amino acid units, self-immolative spacer units, and drug units are also described in U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649, and 2005-0009751, each of which is incorporated herein by reference in its entirety for all purposes.

[0306] Exemplary methods for producing the antibody drug conjugates provided herein are briefly described below.

[0307] The Ha22-2(2,4)6.1 antibody was conjugated to the auristatin derivative MMAE using the vc(Val-Cit) linker described herein to generate an antibody drug conjugate (ADC) (designated AGS-22M6E) using the following protocol: Conjugation of the vc(Val-Cit) linker to MMAE (Seattle Genetics, Inc., Seattle, WA) was completed using the general method shown in Scheme 5 below to generate the cytotoxic vcMMAE (see U.S. Patent No. 7,659,241). Scheme 5 General synthesis method for vcMMAE TIFF2025170007000050.tif99161 where AA1 = amino acid 1 AA2 = amino acid 2 AA5 = amino acid 5 DIL = Dry Solo In DAP = Dolaproin Linker = Val-Cit(vc)

[0308] The antibody drug conjugate AGS-22M6E was then produced using the following protocol.

[0309] Briefly, a 15 mg / mL solution of Ha22-2(2,4)6.1 antibody in 10 mM acetate, 1% sorbitol, and 3% L-arginine at pH 5.0 was adjusted to pH 7.5, 5 mM EDTA, and 150 mM sodium chloride by adding 20% ​​volume of 0.1 M TrisCl, 25 mM EDTA, and 750 mM NaCl at pH 8.4. The antibody was then partially reduced by adding 2.3 molar equivalents of TCEP (per mole of MAb) and stirred at 37°C for 2 hours. The partially reduced antibody solution was then cooled to 5°C, and 4.4 molar equivalents of vcMMAE (per mole of antibody) were added as a 6% (v / v) solution in DMSO. The mixture was stirred at 5°C for 60 minutes, followed by the addition of 1 molar equivalent of N-acetylcysteine ​​per mole of vcMMAE, followed by further stirring for 15 minutes. Excess quenched vcMMAE and other reaction components are removed by ultrafiltration / diafiltration of the antibody drug conjugate (ADC) with 10 volumes of 20 mM histidine, pH 6.0.

[0310] The resulting antibody drug conjugate AGS-22M6E has the following formula: TIFF2025170007000051.tif36160In the formula, L is Ha22-2(2,4)6.1 and p is 1 to 20.

[0311] 5.4 Methods of Using the Pharmaceutical Compositions In one aspect, provided herein is a method for preventing or treating a disease or disorder in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition provided herein. In some embodiments, the subject is a human subject.

[0312] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer has tumor cells that express 191P4D12. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is colon cancer, pancreatic cancer, ovarian cancer, lung cancer, bladder cancer, breast cancer, esophageal cancer, head cancer, or neck cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is advanced bladder cancer. In some embodiments, the cancer is metastatic bladder cancer. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is advanced urothelial carcinoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is head cancer. In some embodiments, the cancer is neck cancer. In some embodiments, the cancer is an advanced or metastatic cancer.

[0313] In some embodiments, treatment with the pharmaceutical compositions provided herein is indicated for subjects who have undergone one or more chemotherapy regimens. Alternatively, the pharmaceutical compositions provided herein are combined with chemotherapy or radiation regimens for subjects who have not undergone chemotherapy treatment. Furthermore, in some embodiments, the use of the pharmaceutical compositions provided herein may allow for the use of reduced doses of combined chemotherapy, particularly for subjects who do not tolerate the toxicity of chemotherapy well. In some embodiments, the pharmaceutical compositions disclosed herein are administered to patients with metastatic urothelial carcinoma who have shown disease progression or recurrence during or after treatment with an immune checkpoint inhibitor.

[0314] Methods of administering the pharmaceutical compositions provided herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral) administration. In a specific embodiment, the pharmaceutical compositions provided herein are administered intranasally, intramuscularly, intravenously, or subcutaneously. The pharmaceutical compositions provided herein can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can also be used, for example, by use of an inhaler or nebulizer and a formulation containing an aerosolizing agent. See, e.g., U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated by reference herein in its entirety.

[0315] In specific embodiments, it may be desirable to administer the pharmaceutical compositions provided herein locally to the area requiring treatment. This can be achieved, for example, but not limited to, by local infusion, local administration (e.g., by intranasal spray), injection, or using an implant, which is a porous, non-porous, or gelatinous material, including a membrane, such as a silastic membrane, or a fiber. In some embodiments, when administering the pharmaceutical compositions provided herein, care must be taken to use a material to which the antibody-drug conjugates provided herein do not absorb.

[0316] In another embodiment, the pharmaceutical compositions provided herein can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0317] In another embodiment, the pharmaceutical compositions provided herein can be delivered in a controlled or sustained release system. In one embodiment, controlled or sustained release can be achieved using a pump (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., antibody-drug conjugates provided herein) or pharmaceutical compositions provided herein (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7:190). 1:105); U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication WO 99 / 15154; and PCT Publication WO 99 / 20253.Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymers used in sustained-release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, controlled-release or sustained-release systems can be placed near the therapeutic target, i.e., the nasal cavity or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Controlled-release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to those skilled in the art can be used to produce sustained-release formulations containing the antibody-drug conjugates or pharmaceutical compositions provided herein.See, for example, U.S. Pat. No. 4,526,938, PCT International Publication Nos. 91 / 05548 and 96 / 20698, Ning et al., 1996, "Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology 39:179-189, Song et al., 1995, "Antibody-Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Proc. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997, each of which is incorporated herein by reference in its entirety. al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc.Int'l.Symp.Control Rel.Bioact.Mater.24:759-760.

[0318] The amount of pharmaceutical compositions provided herein that are effective for preventing and / or treating cancer can be determined by standard clinical techniques.In addition, in vitro assays can optionally be used to help identify optimal dosage ranges.The exact dosage used also depends on the route of administration and the severity of disease or disorder, and should be determined according to the judgment of the practitioner and the individual circumstances of each subject.Effective dosages can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

[0319] In certain embodiments, the therapeutic methods provided herein contemplate the administration of a single ADC, as well as a combination or cocktail of different ADCs comprising different anti-191P4D12 antibodies or different drug units. In some embodiments, such methods have particular advantages because they include ADCs that target different epitopes, utilize different effector mechanisms, or directly combine cytotoxic antibodies with antibodies that rely on immune effector function. Such methods can exhibit synergistic therapeutic effects. Furthermore, the pharmaceutical compositions provided herein can be administered simultaneously with other therapies, including, but not limited to, various chemotherapeutic and biologic agents, androgen blockers, immunomodulators (e.g., IL-2, GM-CSF), surgery, or radiation.

[0320] In one aspect, there is a synergistic effect when tumors, including human tumors, are treated with the pharmaceutical compositions provided herein in conjunction with chemotherapeutic agents or radiation or a combination thereof.

[0321] Methods of inhibiting tumor cell growth using a combination of the pharmaceutical compositions and chemotherapy or radiation or both provided herein include administering the pharmaceutical compositions before, during, or after the initiation of chemotherapy or radiation therapy, as well as any combination thereof (i.e., before and during, before and after, during and after, or before, during and after chemotherapy and / or radiation therapy). Depending on the treatment protocol and the needs of the particular patient, the methods are implemented to provide the most effective treatment and ultimately extend the patient's lifespan.

[0322] Administration of chemotherapeutic agents can be accomplished in a variety of ways, including systemic administration by parenteral and enteral routes. In one embodiment, the chemotherapeutic agents are administered separately. Specific examples of chemotherapeutic agents or chemotherapies include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (Taxol), docetaxel (Taxotere), aldesleukin (ALD), and cyclophosphamide. , asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, taxol and combinations thereof.

[0323] The radiation source used in combination with the pharmaceutical compositions provided herein can be either external or internal to the patient being treated.When the radiation source is outside the patient's body, the treatment is known as external beam radiation therapy (EBRT).When the radiation source is inside the patient's body, the treatment is called brachytherapy (BT).

[0324] The above therapeutic regimens may be further combined with additional cancer therapeutic agents and / or regimens, such as additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful for treating abnormal cell proliferation or cancer, antibodies that inhibit tumor growth by binding to IGF-1R (e.g., anti-CTLA-4 antibodies described in WO 2005 / 092380 (Pfizer)) or other ligands, and cytokines.

[0325] When mammal undergoes additional chemotherapy, the above-mentioned chemotherapeutic agents can be used.In addition, growth factor inhibitors, biological response modifiers, antihormonal therapy, selective estrogen receptor modulators (SERM), angiogenesis inhibitors and antiandrogens can be used.For example, antihormonal agents, such as antiestrogens such as Nolvadex (tamoxifen), or antiandrogens such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionanilide) can be used.

[0326] In some embodiments, the pharmaceutical compositions provided herein are used in combination with a second therapeutic agent, for example, to treat cancer.

[0327] In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that reduces, inhibits, prevents, or regulates one or more checkpoint proteins, either in whole or in part. Without being limited to a particular theory, checkpoint proteins regulate T cell activation or function. Many checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer, 2012, 12, 252-264). These proteins appear to be involved in costimulatory or inhibitory interactions in T cell responses. Immune checkpoint proteins appear to regulate and maintain self-tolerance and the duration and magnitude of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies.

[0328] In one embodiment, the checkpoint inhibitor is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies include, but are not limited to, those described in U.S. Patent Nos. 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; ​​6,682,736; 6,984,720; and 7,605,238, all of which are incorporated herein in their entirety. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206). In another embodiment, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101). Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is commercially available under the trade name Yervoy™.

[0329] In one embodiment, the checkpoint inhibitor is a PD-1 / PD-L1 inhibitor. Examples of PD-1 / PD-L1 inhibitors include, but are not limited to, those described in U.S. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; and 8,217,149, as well as PCT Publication Nos. WO 2003042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400, and WO 2011161699, all of which are incorporated herein in their entireties.

[0330] In one embodiment, the checkpoint inhibitor is a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558, or MDX1106), or pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab). In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is commercially available under the trade name Opdivo™. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 antibody and is commercially available under the trade name Keytruda™. In yet another embodiment, the anti-PD-1 antibody is the humanized antibody CT-011. CT-011 administered alone failed to demonstrate a response in the treatment of relapsed acute myeloid leukemia (AML). In yet another embodiment, the anti-PD-1 antibody is the fusion protein AMP-224. In another embodiment, the PD-1 antibody is BGB-A317. BGB-A317 is a monoclonal antibody specifically engineered for its ability to bind to Fcγ receptor I, and possesses a unique binding signature with high affinity and excellent target specificity for PD-1.

[0331] In one embodiment, the checkpoint inhibitor is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is MEDI4736 (durvalumab). In another embodiment, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01). In yet another embodiment, the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A, and Tecentriq®).

[0332] In one embodiment, the checkpoint inhibitor is a PD-L2 inhibitor. In one embodiment, the PD-L2 inhibitor is an anti-PD-L2 antibody. In one embodiment, the anti-PD-L2 antibody is rHIgM12B7A.

[0333] In one embodiment, the checkpoint inhibitor is a lymphocyte activation gene 3 (LAG-3) inhibitor. In one embodiment, the LAG-3 inhibitor is IMP321, a soluble Ig fusion protein (Brignone et al., J. Immunol., 2007, 179, 4202-4211). In another embodiment, the LAG-3 inhibitor is BMS-986016.

[0334] In one embodiment, the checkpoint inhibitor is a B7 inhibitor. In one embodiment, the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor. In one embodiment, the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody (Loo et al., Clin. Cancer Res., 2012, 3834).

[0335] In one embodiment, the checkpoint inhibitor is a TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207, 2187-94).

[0336] In one embodiment, the checkpoint inhibitor is an OX40 (CD134) agonist. In one embodiment, the checkpoint inhibitor is an anti-OX40 antibody. In one embodiment, the anti-OX40 antibody is anti-OX-40. In another embodiment, the anti-OX40 antibody is MEDI6469.

[0337] In one embodiment, the checkpoint inhibitor is a GITR agonist. In one embodiment, the checkpoint inhibitor is an anti-GITR antibody. In one embodiment, the anti-GITR antibody is TRX518.

[0338] In one embodiment, the checkpoint inhibitor is a CD137 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD137 antibody. In one embodiment, the anti-CD137 antibody is urelumab. In another embodiment, the anti-CD137 antibody is PF-05082566.

[0339] In one embodiment, the checkpoint inhibitor is a CD40 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD40 antibody. In one embodiment, the anti-CD40 antibody is CF-870,893.

[0340] In one embodiment, the checkpoint inhibitor is recombinant human interleukin-15 (rhIL-15).

[0341] In one embodiment, the checkpoint inhibitor is an IDO inhibitor. In one embodiment, the IDO inhibitor is INCB024360. In another embodiment, the IDO inhibitor is indoximod.

[0342] In certain embodiments, the combination therapies provided herein include two or more of the checkpoint inhibitors described herein (including checkpoint inhibitors of the same or different classes). Additionally, the combination therapies described herein can be used in combination with one or more second active agents described herein, where appropriate to treat a disease described herein and as understood in the art.

[0343] In some embodiments, the checkpoint inhibitor is administered prior to administration of the pharmaceutical composition. In other embodiments, the checkpoint inhibitor is administered simultaneously (e.g., during the same administration period) with the pharmaceutical composition provided herein. In yet other embodiments, the checkpoint inhibitor is administered after administration of the pharmaceutical composition provided herein.

[0344] In some embodiments, the amount of checkpoint inhibitor can be determined by standard clinical techniques.

[0345] The dose of checkpoint inhibitor results in a serum titer of about 0.1 μg / ml to about 450 μg / ml, and in some embodiments is at least 0.1 μg / ml, at least 0.2 μg / ml, at least 0.4 μg / ml, at least 0.5 μg / ml, at least 0.6 μg / ml, at least 0.8 μg / ml, at least 1 μg / ml, at least 1.5 μg / ml, e.g., at least 2 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least At least 25 μg / ml, at least 30 μg / ml, at least 35 μg / ml, at least 40 μg / ml, at least 50 μg / ml, at least 75 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 200 μg / ml, at least 250 μg / ml, at least 300 μg / ml, at least 350 μg / ml, at least 400 μg / ml, or at least 450 μg / ml can be administered to a human for the prevention and / or treatment of cancer. It should be understood that the precise dose of checkpoint inhibitor employed will also depend on the route of administration, and the severity of the cancer in the subject, and should be decided according to the judgment of the treating physician and each patient's circumstances.

[0346] In some embodiments, the dosage of a checkpoint inhibitor (e.g., a PD-1 inhibitor or a PD-L1 inhibitor) administered to a patient is typically between 0.1 mg / kg and 100 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is between about 1 mg / kg and about 75 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is between 1 mg / kg and 20 mg / kg of the subject's body weight, e.g., between 1 mg / kg and 5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 1 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 1.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 2 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 2.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 3 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 3.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 4 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 4.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 5.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 6 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 6.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 7 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 7.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 8 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 8.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 9.0 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 10.0 mg / kg of the subject's body weight, hi some embodiments, the dosage administered to a patient is about 15.0 mg / kg of the subject's body weight.In some embodiments, the dosage administered to a patient is about 20.0 mg / kg of the subject's body weight.

[0347] In some embodiments, the pharmaceutical compositions provided herein are supplied as a dry, sterile, lyophilized powder or water-free concentrate in a sealed container, which can be reconstituted, for example, with water or saline, to the appropriate concentration for administration to a subject. In certain embodiments, the antibody-drug conjugate is supplied as a dry, sterile, lyophilized powder in a sealed container in a unit dose of at least 0.1 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, or at least 3 mg, e.g., at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg. The lyophilized antibody-drug conjugate can be stored in its original container at 2-8°C, and the antibody-drug conjugate can be administered within 12 hours, e.g., within 6 hours, within 5 hours, within 3 hours, or within 1 hour, after reconstitution. In alternative embodiments, pharmaceutical compositions comprising the antibody drug conjugates provided herein are supplied in liquid form in a sealed container indicating the amount and concentration of the antibody drug conjugate. In certain embodiments, the liquid form of the antibody drug conjugate is supplied in a sealed container at at least 0.1 mg / ml, at least 0.5 mg / ml, or at least 1 mg / ml, e.g., at least 5 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 30 mg / ml, at least 40 mg / ml, at least 50 mg / ml, at least 60 mg / ml, at least 70 mg / ml, at least 80 mg / ml, at least 90 mg / ml, or at least 100 mg / ml.

[0348] In some embodiments, the amount of a prophylactic or therapeutic agent provided herein (e.g., an antibody drug conjugate provided herein), or pharmaceutical composition that is effective in the prevention and / or treatment of cancer can be determined by standard clinical techniques.

[0349] Thus, from about 0.1 μg / ml to about 450 μg / ml, in some embodiments at least 0.1 μg / ml, at least 0.2 μg / ml, at least 0.4 μg / ml, at least 0.5 μg / ml, at least 0.6 μg / ml, at least 0.8 μg / ml, at least 1 μg / ml, at least 1.5 μg / ml, for example at least 2 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 30 μg / ml, at least A dosage of antibody drug conjugate in a pharmaceutical composition that results in a serum titer of at least 35 μg / ml, at least 40 μg / ml, at least 50 μg / ml, at least 75 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 200 μg / ml, at least 250 μg / ml, at least 300 μg / ml, at least 350 μg / ml, at least 400 μg / ml, or at least 450 μg / ml can be administered to a human for the prevention and / or treatment of cancer. It should be understood that the precise dose to be employed in the formulation will also depend on the route of administration, and the severity of the cancer in the subject, and should be decided according to the judgment of the treating physician and each patient's circumstances.

[0350] Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0351] For pharmaceutical compositions comprising an antibody drug conjugate provided herein, the dosage of the antibody drug conjugate administered to a patient is typically 0.1 mg / kg to 100 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 1 mg / kg to about 75 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is 1 mg / kg to 20 mg / kg of the subject's body weight, e.g., 1 mg / kg to 5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 1 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 1.25 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 1.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 2 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 2.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 3 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 3.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 4 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 4.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 5.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 6 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 6.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 7 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 7.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 8 mg / kg of the subject's body weight. In some embodiments, the dosage administered to a patient is about 8.5 mg / kg of the subject's body weight.

[0352] In some embodiments, the antibody drug conjugates formulated into pharmaceutical compositions provided herein are administered based on the patient's actual body weight at baseline, and the dose remains unchanged unless the patient's body weight changes by ≥ 10% from the baseline of the previous cycle or dose adjustment criteria are met. In some embodiments, actual body weight is used, except for patients weighing more than 100 kg, in which case the dose is calculated based on a body weight of 100 kg. In some embodiments, the maximum dose is 100 mg for patients receiving a dose level of 1.00 mg / kg and 125 mg for patients receiving a dose level of 1.25 mg / kg.

[0353] In one embodiment, to treat cancer, an antibody drug conjugate formulated into the pharmaceutical composition at about or less than 100 mg / kg, about or less than 75 mg / kg, about or less than 50 mg / kg, about or less than 25 mg / kg, about or less than 10 mg / kg, about or less than 5 mg / kg, about or less than 1 mg / kg, about or less than 0.5 mg / kg, or about or less than 0.1 mg / kg is administered 5, 4, 3, 2, or 1 time. In some embodiments, pharmaceutical compositions comprising an antibody drug conjugate provided herein are administered about 1 to 12 times, and the dose can be administered as needed, e.g., weekly, biweekly, monthly, bimonthly, trimonthly, etc., as determined by a physician. In some embodiments, lower doses (e.g., 0.1 mg / kg to 15 mg / kg) can be administered more frequently (e.g., 3 to 6 times), while in other embodiments, higher doses (e.g., 25 mg / kg to 100 mg / kg) can be administered less frequently (e.g., 1 to 3 times).

[0354] In some embodiments, a single dose of an antibody drug conjugate formulated into a pharmaceutical composition provided herein is administered to a patient in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 cycles every two weeks (e.g., about 14 days) over a period of time (e.g., one year) to prevent and / or treat cancer, wherein the dose is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, or combinations thereof (i.e., the monthly dose of each administration may or may not be the same).

[0355] In some embodiments, a single dose of an antibody drug conjugate formulated into a pharmaceutical composition provided herein is administered to a patient in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 cycles every 3 weeks (e.g., about 21 days) over a period of time (e.g., 1 year) to prevent and / or treat cancer, wherein the dose is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, or combinations thereof (i.e., the monthly dose of each administration may or may not be the same).

[0356] In some embodiments, a single dose of an antibody drug conjugate formulated into a pharmaceutical composition provided herein is administered to a patient in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 cycles every 4 weeks (e.g., about 28 days) over a period of time (e.g., 1 year) to prevent and / or treat cancer, with doses of about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, or combinations thereof (i.e., the monthly dose of each administration may or may not be the same).

[0357] In another aspect, a single dose of an antibody drug conjugate formulated into a pharmaceutical composition provided herein is administered to a patient 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times at approximately monthly (e.g., about 30 day) intervals over a period of time (e.g., 1 year) to prevent and / or treat cancer, and the doses are about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, or about 5 mg / kg. / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, or combinations thereof (i.e., the monthly dose of each administration may or may not be the same).

[0358] In another aspect, a single dose of an antibody drug conjugate formulated into a pharmaceutical composition provided herein is administered to a patient once, twice, three times, four times, five times, or six times at intervals of approximately every two months (e.g., about 60 days) over a period of time (e.g., one year) to prevent and / or treat cancer, and the doses are about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, or about 6 mg / kg. g, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, or combinations thereof (i.e., the monthly dose of each administration may or may not be the same).

[0359] In yet another aspect, a single dose of an antibody drug conjugate formulated into a pharmaceutical composition provided herein is administered to a patient once, twice, three times, or four times at intervals of approximately every three months (e.g., about 120 days) over a period of time (e.g., one year) to prevent and / or treat cancer, and the dose is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg , about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, about 100 mg / kg, or combinations thereof (i.e., the monthly dose of each administration may or may not be the same).

[0360] In certain embodiments, the route of administration of a dose of an antibody drug conjugate formulated into a pharmaceutical composition provided herein to a patient is intranasally, intramuscularly, intravenously, or a combination thereof, although other routes as described herein are also acceptable. Each dose may or may not be administered by the same route of administration. In some embodiments, an antibody drug conjugate formulated into a pharmaceutical composition provided herein may be administered via multiple routes of administration simultaneously with or subsequent to other doses of one or more additional therapeutic agents.

[0361] In some more specific embodiments, the antibody drug conjugates formulated into pharmaceutical compositions provided herein are administered by intravenous (IV) injection or infusion at a dose of about 1 mg / kg of subject body weight, about 1.25 mg / kg of subject body weight, or about 1.5 mg / kg of subject body weight.

[0362] In some more specific embodiments, the antibody drug conjugate formulated into a pharmaceutical composition provided herein is administered by intravenous (IV) injection or infusion over about 30 minutes at a dose of about 1 mg / kg of subject body weight, about 1.25 mg / kg of subject body weight, or about 1.5 mg / kg of subject body weight, twice every three weeks. In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1 and 8 of every three-week cycle. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion once or more times each three weeks. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion on day 1 of every three-week cycle. In some embodiments, the immune checkpoint inhibitor is pembrolizumab, and the pembrolizumab is administered in an amount of about 200 mg over about 30 minutes. In other embodiments, the immune checkpoint inhibitor is atezolizumab, and the atezolizumab is administered in an amount of about 1200 mg over about 60 minutes or about 30 minutes. In some embodiments, the antibody drug conjugate is administered to a patient with urothelial carcinoma who has shown disease progression or recurrence during or after treatment with an immune checkpoint inhibitor. In some embodiments, the antibody drug conjugate is administered to a patient with metastatic urothelial carcinoma who has shown disease progression or recurrence during or after treatment with an immune checkpoint inhibitor.

[0363] In other more specific embodiments, the antibody drug conjugate formulated into a pharmaceutical composition provided herein is administered by intravenous (IV) injection or infusion over about 30 minutes at a dose of about 1 mg / kg, about 1.25 mg / kg, or about 1.5 mg / kg of subject body weight, for three cycles every four weeks. In some embodiments, the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1, 8, and 15 of each four-week cycle. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion one or more times each four weeks. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In other embodiments, the immune checkpoint inhibitor is atezolizumab. In some embodiments, the antibody drug conjugate is administered to a patient with urothelial carcinoma who has experienced disease progression or recurrence during or after treatment with an immune checkpoint inhibitor. In some embodiments, the antibody drug conjugate is administered to patients with metastatic urothelial carcinoma who have shown disease progression or recurrence during or after treatment with an immune checkpoint inhibitor.

[0364] For the sake of brevity, certain abbreviations are used herein. One example is the one-letter abbreviation for amino acid residues. The amino acids and their corresponding three-letter and one-letter abbreviations are as follows: TIFF2025170007000052.tif143128

[0365] The present invention is generally disclosed herein using categorical language to describe numerous aspects. The present invention also specifically includes aspects in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays or analyses, is completely or partially excluded. Thus, the present invention is generally not expressed herein in terms of what the invention does not include, but aspects not expressly included in the present invention are nevertheless disclosed herein.

[0366] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. It is expected that variations of the disclosed embodiments may become apparent to those skilled in the art upon reading the foregoing description, and that such variations may be utilized appropriately by those skilled in the art. Accordingly, it is intended that the present invention be practiced otherwise than as specifically described herein, and that the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, the present invention encompasses any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

[0367] All publications, patent applications, accession numbers, and other references cited herein are incorporated by reference in their entirety, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0368] Having described several aspects of the present invention, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the description in the experimental section is intended to illustrate, but not limit, the scope of the invention as set forth in the claims. [Example]

[0369] 6. Example The following is a description of various methods and materials used in the testing, presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and is not intended to limit the scope of what the inventors regard as their invention, nor is it intended to represent that the following experiments have been performed or are all experiments that may be performed. It should be understood that exemplary descriptions written in the present tense have not necessarily been performed, but rather, descriptions may be performed to generate data and the like relevant to the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0370] 6.1 Example 1 - pH and Buffer Screening AGS-22M6E was formulated at 10 mg / mL in 14 candidate buffers (all 20 mM; detailed in Table 1 below). Formulations using 20 mM sodium citrate buffer titrated with citric acid to pH 5.2 and 5.7, and 20 mM histidine buffer titrated with HCl to pH 5.5, 6.0, and 6.5, were evaluated. Additionally, three different anions, namely chloride, phosphate, and succinate, were evaluated in the histidine buffer system. Liquid formulations were subjected to a 40°C storage temperature for two weeks, room temperature (RT) agitation for 24 hours, and freeze-thaw cycles (freezing at -70°C and thawing at 20°C to 25°C for one, three, and ten cycles).

[0371] (Table 1) TIFF2025170007000053.tif85133 NOTE: 5% sucrose (molecular weight 342) = 146 mM; 5.5% trehalose dihydrate (molecular weight 378) = 146 mM.

[0372] The formulation preparation and study design are described in more detail below.

[0373] Protein products used in formulation testing Four tubes containing approximately 50 ml each of AGS-22M6E (Lot No. AGS22M6-VCE-02) were received frozen, totaling approximately 2.5 grams. AGS-22M6E was 12.5 mg / mL in 20 mM histidine pH 6.0 buffer containing 5% sucrose and 0.02% polysorbate 20. The material was stored at -70°C until use.

[0374] Preparation of formulation buffer A stock solution containing citric acid (0.1 M), sodium citrate (0.1 M) and L-histidine (0.2 M), succinic acid (0.25 M), trehalose dihydrate (40%), sucrose (40%), hydrochloric acid (2 M) and phosphoric acid (2 M) was prepared according to Table 2 below.

[0375] (Table 2) TIFF2025170007000054.tif68152

[0376] The reagents were weighed according to the table above. An appropriate amount of Milli-Q water was added to dissolve the reagents. The solution was filtered through a 0.22 μm filter.

[0377] Preparation of dialysis formulation buffer 1.0 L of each formulation was prepared for dialysis and placebo vial filling according to Table 3 below.

[0378] (Table 3) TIFF2025170007000055.tif99167

[0379] The pH was adjusted with an appropriate acid to a target pH of ±0.1. The buffer was stored at 4°C until use.

[0380] Preparation of formulations Four tubes, each containing 50 ml of AGS-22M6E (Lot No. AGS22M6-VCE-02), were thawed in a room-temperature water bath and then combined in a 250 ml bottle. 11 ml was allocated for each formulation and added to a dialysis cassette. The cassette was placed in a beaker containing approximately 40-fold excess formulation buffer and stirred overnight at 2°C to 8°C. The buffer was discarded, and fresh buffer was added and stirred overnight at 2°C to 8°C. The material was removed from the cassette, transferred to a 50 ml tube, the concentration determined, and the volume adjusted with the corresponding formulation buffer to a final concentration of 10 mg / ml. The placebo was the corresponding buffer used to formulate the product.

[0381] Filling and stoppering of drug product vials Sterile filtration and filling were performed in a Baker SG600 laminar flow hood. The formulation and placebo were sterile filtered using aseptic technique (Millipore Millex-GV 0.22 μm PVDF syringe filter, No. SLGV033RS). Sterile stoppered vials (Hollister-Stier 2 ml sterile stoppered vials, No. 7505ZA) were decrimped in the hood and the stoppers removed using aseptic technique. 1.0 ml of the formulated product or placebo was filled into the vial and then restoppered.

[0382] Material Requirements and Sample Map The material requirements and sample map are as follows:

[0383] (Table 4) TIFF2025170007000056.tif132170

[0384] Time points and assays The time points and assays are as follows in Table 5.

[0385] (Table 5) TIFF2025170007000057.tif69167

[0386] Stability study design for liquid formulation at 40°C Formulation and placebo vials were placed upright in an incubator set at 40°C. At each time point, one active vial and one placebo vial for each formulation were removed from storage according to the sample map. Samples were frozen at -70°C and batch analyzed at the end of the study. Prior to analysis, samples were thawed at room temperature. A set of three aliquots of each sample (70 μL aliquots for each sample) was filtered through a 0.22 μm filter and then frozen at -70°C. After analytical testing, the remaining material was stored overnight at 2°C to 8°C in case retesting was required. After all assays were completed, the remaining material was stored at -70°C. Two frozen aliquots were used for cIEF and potency assays.

[0387] Freeze-thaw (-70°C) stability test design One vial (1.0 mL fill) of each formulation was frozen and placed upright in a -70°C freezer for at least 4 hours. To thaw, each vial was removed from storage and thawed at room temperature until no ice was observed, then the vial was gently swirled. This constituted one complete freeze-thaw cycle. One, three, and ten freeze-thaw cycles were completed for each test formulation sample vial. After the final freeze-thaw cycle, all samples were evaluated by analytical testing. A set of three aliquots of each sample (70 μL aliquots for each sample) was immediately frozen at -70°C. After analytical testing, the remaining material was stored overnight at 2°C to 8°C in case retesting was required. After all assays were completed, the remaining material was stored at -70°C. Two frozen aliquots were used for cIEF and potency assays.

[0388] Mixing Test Design One vial of each formulation was placed upright in a standard freezer box. The box was then placed in an IKA-VIBRAMAX-VXR orbital shaker set at 500 rpm for 24 hours at room temperature. Samples were then removed and stored at -70°C until analysis.

[0389] Formulation Standards 1.2 mL of AGS-22M6E (Lot No. AGS22M6-VCE-02) starting material (12.5 mg / mL in 20 mM histidine pH 6.0 buffer containing 5% sucrose and 0.02% polysorbate 20) was taken and aliquoted at 200 μl / vial and then stored at −70° C. as the formulation standard for this study.

[0390] Appearance, A280 (protein concentration and drug loading), A330 (turbidity), SE-HPLC, non-reducing and reducing SDS-PAGE, RP-HPLC-NPI.iCIEF and potency were used to assess the stability of AGS-22M6E.

[0391] exterior All samples were free of color, cloudiness, and particulates over the course of the test. No particulates were observed even after shaking.

[0392] A280 (protein concentration) analysis The results of the A280 analysis are shown in Table 6 below.

[0393] (Table 6) TIFF2025170007000058.tif132128TIFF2025170007000059.tif133128

[0394] As shown, no change in protein concentration was observed.

[0395] A330 (turbidity) analysis The results of the A330 analysis are shown in Table 7 below.

[0396] (Table 7) TIFF2025170007000060.tif150128

[0397] As shown, formulations F1 and F6 showed the most significant increase in turbidity over time. At T=0, a higher turbidity was observed for formulation F6 that was not observed in the other formulations.

[0398] SDS-PAGE analysisThe results of SDS-PAGE analysis are shown in Figures 1A, 1B, 1C, and 1D. A small low-molecular-weight (LMW) band (approximately 35 kD) was observed by reducing SDS-PAGE in F1 and F6 after 14 days. In non-reducing SDS-PAGE analysis, F1, F2, F6, and F7 also showed a small high-molecular-weight (HMW) band (approximately 200 kD) that was not previously present at T=0.

[0399] RP-HPLC analysis Table 8 and Figure 1E show the results of RP-HPLC analysis. For either formulation, free SGD1010 (trace cleavage of the drug MMAE) was not detected by RP-HPLC at t = 0, but after 14 days at 40 °C, SGD1010 (ranging from 0.17 µM to 1.59 µM) was observed, with the histidine increasing slightly faster at higher pH than the citrate formulation, and histidine / succinate performing slightly better than histidine / phosphate and histidine / HCl.

[0400] (Table 8) TIFF2025170007000061.tif134159

[0401] SE-HPLC analysis As shown in Table 9 below and Figures 1F, 1G, and 1H, increased levels of HMW aggregates were evident by SE-HPLC for all formulations from pH 5.2 to pH 5.7, with citrate formulations showing more aggregates than histidine at the corresponding pH. At similar pHs, citrate showed more aggregates than histidine. The histidine formulation at pH 6.0 showed better stability than those at pH 5.5 and pH 6.5. No differences were observed between trehalose and sucrose.

[0402] (Table 9) TIFF2025170007000062.tif170143TIFF2025170007000063.tif75144

[0403] No significant changes were observed between any of the formulations after 24 hours of shaking at room temperature or after 1, 3, and 10 freeze-thaw cycles, as demonstrated by A330, SDS-PAGE, and SE-HPLC (data not shown). This provided assurance that formulation test samples could be withdrawn at different time points and stored at -70°C.

[0404] Based on the results obtained from this study, formulations F4, F9 and F14 were selected as the best among the 14 formulations tested and were therefore selected as the three formulations to be further evaluated in subsequent studies.

[0405] 6.2 Example 2 - Bulk Drug Substance (BDS) Freeze-Thaw and Shaking Test Formulations F4, F9, and F14 were prepared as described in Section 6.1 above. Formulations F4, F9, and F14 were subjected to 1, 3, and 10 cycles of freezing at both -20°C and -70°C, respectively, followed by thawing at 20°C to 25°C. Samples were analyzed by visual inspection, concentration (A280) measurement, turbidity (A330) measurement, SE-HPLC, and SDS-PAGE (R&NR). For the 10-cycle freeze-thaw test, samples were also analyzed by RP-HPLC NPI.

[0406] The material requirements and sample map are shown in Table 10 below.

[0407] (Table 10) TIFF2025170007000064.tif78128Note: Vials: 5 mL sterile screw-cap polycarbonate bottles (Nalgene 5 mL, No. 3500-05) BDS Freeze / Thaw: Fill 1 mL into a 5 mL polycarbonate bottle. · BDS Shaking: Fill 3.5mL into a 5mL polycarbonate bottle.

[0408] Table 11 below lists the assays and time points.

[0409] (Table 11) TIFF2025170007000065.tif74153

[0410] A 24-hour room temperature agitation test was also conducted for each formulation, and all test samples were analyzed for appearance, concentration (A280), turbidity (A330), SE-HPLC, and HIAC. Selected samples from the above tests were also used for iCIEF and potency testing.

[0411] The formulation vial filling and stoppering, agitation test design, freeze-thaw test design, and formulation standards are as follows:

[0412] Filling and stoppering of drug product vials Sterile filtration and filling were performed in a Baker SG600 laminar flow hood. The formulation and placebo were sterile filtered using aseptic technique (Millipore Millex-GV 0.22 μm PVDF syringe filter, No. SLGV033RS). A 5 mL electronic pipette with a sterile tip was used to transfer the filtered AGS-22M6E and filtered formulation buffer (placebo) into sterile screw-cap polycarbonate bottles (Nalgene 5 mL, No. 3500-05).

[0413] Mixing Test Design One vial per formulation was secured upright in a standard freezer box. The box was then mounted on an IKA-VIBRAMAX-VXR orbital shaker set at 500 rpm for 24 hours at room temperature. Samples were then removed and stored at 70°C until analysis. A set of three aliquots of each sample (70 μL aliquots for each sample) was filtered through a 0.22 μm filter and then frozen at -70°C. After analytical testing, the remaining material was stored overnight at 2°C to 8°C in case retesting was required. After all assays were completed, the remaining material was stored at -70°C. Two frozen aliquots were used for cIEF and potency assays.

[0414] Freeze-thaw (-70°C and -20°C) stability study design One vial (1 ml filled into a 5 ml polycarbonate bottle) per formulation was placed upright in a -70°C and -20°C freezer for at least 4 hours. To thaw, each vial was removed from storage and thawed at room temperature (20-25°C) until no ice was observed. The vial was then gently swirled. This constituted one complete freeze-thaw cycle. Ten freeze-thaw cycles were completed for each test formulation sample vial. After the final freeze-thaw cycle, all samples were evaluated by analytical testing. Samples were analyzed by the following methods: appearance, A280 / A248, turbidity (A330), SE-HPLC, RP-HPLC-NPI, and SDS-PAGE (R&NR). A set of three aliquots of each sample (70 μL aliquots for each sample) was filtered through a 0.22 μm filter and then frozen at -70°C. After analytical testing, the remaining material was stored overnight at 2°C to 8°C in case retesting was required. After all assays were completed, the remaining material was stored at -70°C. Two frozen aliquots were used for cIEF and potency assays.

[0415] Formulation Standards 15 mL of 12.8 mg / mL AGS-22M6E starting material in 5.0% sucrose, 0.02% Tween 20, pH 6.0 was taken and aliquoted at 500 μl / vial and then stored at −70° C. as the formulation standard for this study.

[0416] result exterior : All samples were visually analyzed in this test and no particulates were observed even after shaking.

[0417] A280 and A330 analysis The A280 and A330 data for the formulations subjected to different conditions in this study are summarized in Table 12 below. As shown, there was no change in protein concentration under either shaking or freeze-thawing conditions. Additionally, there was no increase in turbidity upon freeze-thawing or shaking.

[0418] (Table 12) TIFF2025170007000066.tif126163

[0419] SDS-PAGE analysis The results of the SDS-PAGE analysis are shown in Figures 2A and 2B. As shown, no changes are observed in the SDS-PAGE for the shaken test samples and the freeze-thawed samples. Both the reduced and non-reduced gels are comparable to the formulation standard.

[0420] RP-HPLC analysis RP-HPLC analysis was performed on the 10 cycle freeze-thaw samples. No evidence of an SGD1010 peak was seen in any of the formulations when analyzed by RP-HPLC (data not shown here).

[0421] SE-HPLC analysis The results of the SE-HPLC analysis are summarized below in Table 13. As shown, no differences were observed between TO and the shaken or freeze-thawed samples for any of the three formulations (F4, F9, and 14) or the placebo.

[0422] (Table 13) TIFF2025170007000067.tif117170

[0423] The SEC profiles obtained for each of the BDS formulations at each test condition were analyzed (data not shown). No differences were observed for any formulation at any condition compared to BDS at T=0.

[0424] Table 14 below summarizes the HIAC data for the BDS samples for each of the three formulations tested. Comparing the results for samples before and after shaking for 24 hours at room temperature reveals that fewer than 100 particles are in the 10µm to 25µm range, and fewer than 2 particles are in the 25µm range for all formulations.

[0425] (Table 14) TIFF2025170007000068.tif72162

[0426] Formulations F9 and F14 showed a slight increase in cumulative counts after agitation, while F4 showed equivalence before and after agitation (all 10 μm and 25 μm counts below the USP limit) as shown graphically in Figure 2C.

[0427] Overall, the results demonstrated that all three BDS formulations tested exhibited excellent stability under freeze-thaw cycling and agitation, with no changes observed in any of the samples compared to T=0 by any analytical method.

[0428] 6.3 Example 3 - Simultaneous BDS and Drug Product (DP) Formulation Study This study was conducted in conjunction with the study described above in Section 6.2. The formulation composition and materials used in this study were the same as those described in Section 6.2.

[0429] The material requirements and sample map are shown in the table below.

[0430] (Table 15) TIFF2025170007000069.tif77162

[0431] Time points and assays are as described in the table below.

[0432] (Table 16) TIFF2025170007000070.tif105147 NOTE: Samples in the liquid arm of the study were frozen at -70°C until samples from the lyophilized arm were prepared. The frozen liquid samples were then conditioned at the same time as the lyophilized samples so that t = 0 was the same for both the liquid and lyophilized arms of the study.

[0433] Specific lyophilization cycle parameters are outlined in the table below. After lyophilization was completed, the vials were stoppered under 50 mT vacuum.

[0434] (Table 17) TIFF2025170007000071.tif90143

[0435] Stability study design for liquid formulations at 2°C to 8°C and -70°C Formulation and placebo vials were placed upright in a freezer set at -70°C and an incubator set at 2-8°C. At each time point, one active vial and one placebo vial for each formulation were removed from storage conditions according to the sample map for analytical testing. After analytical testing, the remaining material was stored at 2-8°C in case retesting was required. Aliquots were stored at -70°C and used for cIEF and activity testing.

[0436] Stability test design for lyophilized formulation at 2°C-8°C, 25°C, and 40°C Formulation and placebo vials were placed upright in an incubator set at 2°C-8°C, an incubator set at 25°C / 60% RH, and an incubator set at 40°C / 75% RH. At each time point, one active vial and one placebo vial for each formulation were removed from storage according to the sample map for analytical testing. After analytical testing, the remaining material was stored at 2°C-8°C in case retesting was required. Aliquots were stored at -70°C and used for cIEF and activit...

Claims

1. (a) an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethyl auristatin E (MMAE), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:7, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:8; and (b) a pharmaceutically acceptable excipient comprising L-histidine, polysorbate 20 (TWEEN-20®), and at least one of trehalose dihydrate and sucrose; 10. A pharmaceutical composition comprising:

2. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises CDR H1 having the amino acid sequence of SEQ ID NO:9, CDR H2 having the amino acid sequence of SEQ ID NO:10, CDR H3 having the amino acid sequence of SEQ ID NO:11, CDR L1 having the amino acid sequence of SEQ ID NO:12, CDR L2 having the amino acid sequence of SEQ ID NO:13, and CDR L3 having the amino acid sequence of SEQ ID NO:

14.

3. 2. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 136th amino acid (serine) of SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) of SEQ ID NO:

8.

4. 2. The pharmaceutical composition of claim 1, wherein the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7 and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:

8.

5. Antigen-binding fragments are Fab and F(ab'). 2 5. The pharmaceutical composition of any one of claims 1 to 4, which is an Fv or scFv fragment.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the antibody is a fully human antibody.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

8. 1. The antibody drug conjugate of claim 1, wherein the antibody drug conjugate has the following structure: wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; 8. The pharmaceutical composition of any one of claims 1 to 7.

9. 9. The pharmaceutical composition of claim 8, wherein p is 2 to 8.

10. 2. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment is linked to each unit of monomethyl auristatin E (MMAE) via a linker.

11. 11. The pharmaceutical composition of claim 10, wherein the linker is an enzyme-cleavable linker and forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof.

12. Linker is -A a -W w -Y y -having the formula wherein -A- is an extender unit, a is 0 or 1, -W- is an amino acid unit, w is an integer ranging from 0 to 12, -Y- is a spacer unit, and y is 0, 1, or 2; 11. The pharmaceutical composition of claim 10.

13. 13. The pharmaceutical composition of claim 12, wherein the extender unit has the structure of formula (1), the amino acid unit is valine citrulline, and the spacer unit is a PAB group having the structure of formula (2): 。

14. 13. The pharmaceutical composition of claim 12, wherein the extender unit forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof, and the spacer unit is linked to MMAE via a carbamate group.

15. 10. The pharmaceutical composition of claim 1, wherein the antibody drug conjugate comprises 1 to 10 units of MMAE per antibody or antigen-binding fragment thereof.

16. 16. The pharmaceutical composition of claim 15, wherein the antibody drug conjugate comprises 2 to 8 units of MMAE per antibody or antigen-binding fragment thereof.

17. 17. The pharmaceutical composition of any one of claims 1 to 16, comprising the antibody drug conjugate at a concentration of 1 to 20 mg / mL.

18. 18. The pharmaceutical composition of claim 17, comprising the antibody drug conjugate at a concentration of 5 to 15 mg / mL.

19. 18. The pharmaceutical composition of claim 17, comprising the antibody drug conjugate at a concentration of 8 to 12 mg / mL.

20. 18. The pharmaceutical composition of claim 17, comprising the antibody drug conjugate at a concentration of about 10 mg / mL.

21. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein L-histidine is present in the range of 5 to 50 mM.

22. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein L-histidine is present in the range of 10 to 40 mM.

23. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein L-histidine is present in the range of 15 to 35 mM.

24. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein L-histidine is present in the range of 15 to 30 mM.

25. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein L-histidine is present in the range of 15 to 25 mM.

26. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein L-histidine is present at about 20 mM.

27. 27. The pharmaceutical composition of any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of 0.001 to 0.1% (v / v).

28. 27. The pharmaceutical composition of any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of 0.0025 to 0.075% (v / v).

29. 27. The pharmaceutical composition of any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of 0.005 to 0.05% (v / v).

30. 27. The pharmaceutical composition of any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of 0.01 to 0.03% (v / v).

31. 27. The pharmaceutical composition of any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of about 0.02% (v / v).

32. 32. The pharmaceutical composition of any one of claims 1 to 31, comprising trehalose dihydrate.

33. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present in the range of 1 to 20% (w / v).

34. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present in the range of 2 to 15% (w / v).

35. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present in the range of 3 to 10% (w / v).

36. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present in the range of 4-6% (w / v).

37. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present at about 5.5% (w / v).

38. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present in the range of 50 mM to 300 mM.

39. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present in the range of 75 mM to 250 mM.

40. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present in the range of 100 mM to 200 mM.

41. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present in the range of 130 mM to 150 mM.

42. 33. The pharmaceutical composition of claim 32, wherein the trehalose dihydrate is present at about 146 mM.

43. 32. The pharmaceutical composition of any one of claims 1 to 31, comprising sucrose.

44. 44. The pharmaceutical composition of claim 43, wherein the sucrose is present in the range of 1-20% (w / v).

45. 44. The pharmaceutical composition of claim 43, wherein the sucrose is present in the range of 2-15% (w / v).

46. 44. The pharmaceutical composition of claim 43, wherein the sucrose is present in the range of 3-10% (w / v).

47. 44. The pharmaceutical composition of claim 43, wherein the sucrose is present in the range of 4-6% (w / v).

48. 44. The pharmaceutical composition of claim 43, wherein the sucrose is present at about 5.5% (w / v).

49. 44. The pharmaceutical composition of claim 43, wherein sucrose is present in the range of 50 mM to 300 mM.

50. 44. The pharmaceutical composition of claim 43, wherein sucrose is present in the range of 75 mM to 250 mM.

51. 44. The pharmaceutical composition of claim 43, wherein sucrose is present in the range of 100 mM to 200 mM.

52. 44. The pharmaceutical composition of claim 43, wherein sucrose is present in the range of 130 mM to 150 mM.

53. 44. The pharmaceutical composition of claim 43, wherein the sucrose is present at about 146 mM.

54. 54. The pharmaceutical composition of any one of claims 1 to 53, having a pH in the range of 5.5 to 6.

5.

55. 54. The pharmaceutical composition of any one of claims 1 to 53, having a pH in the range of 5.7 to 6.

3.

56. 54. The pharmaceutical composition of any one of claims 1-53, having a pH of about 6.

0.

57. 57. The pharmaceutical composition of any one of claims 54 to 56, wherein the pH is measured at room temperature.

58. 57. The pharmaceutical composition of any one of claims 54 to 56, wherein the pH is measured at 15°C to 27°C.

59. 57. The pharmaceutical composition of any one of claims 54 to 56, wherein the pH is measured at 4°C.

60. 57. The pharmaceutical composition of any one of claims 54 to 56, wherein the pH is measured at 25°C.

61. 61. The pharmaceutical composition of any one of claims 1 to 60, comprising hydrochloric acid (HCl).

62. 61. The pharmaceutical composition of any one of claims 1-60, wherein the pH is adjusted with HCl.

63. 61. The pharmaceutical composition of any one of claims 1 to 60, comprising succinic acid.

64. 61. The pharmaceutical composition of any one of claims 1-60, wherein the pH is adjusted with succinic acid.

65. 17. The pharmaceutical composition of any one of claims 1-16, comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, and at least one of about 5.5% (w / v) trehalose dihydrate or about 5% (w / v) sucrose.

66. 66. The pharmaceutical composition of claim 65, further comprising HCl or succinic acid.

67. 67. The pharmaceutical composition of claim 65 or claim 66, wherein the pH is 6.0 at room temperature.

68. 67. The pharmaceutical composition of claim 65 or claim 66, having a pH of 6.0 at 25°C.

69. (a) a molecule having the following structure: wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20®, about 5.5% (w / v) trehalose dihydrate, and HCl. and having a pH of about 6.0 at 25°C. Pharmaceutical compositions.

70. 70. The pharmaceutical composition of claim 69, wherein the antibody drug conjugate is at a concentration of about 10 mg / mL.

71. (a) a molecule having the following structure: wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20®, about 5.5% (w / v) trehalose dihydrate, and succinic acid. and having a pH of about 6.0 at 25°C. Pharmaceutical compositions.

72. 72. The pharmaceutical composition of claim 71, wherein the antibody drug conjugate is at a concentration of about 10 mg / mL.

73. (a) a molecule having the following structure: wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20®, about 5.0% (w / v) sucrose, and HCl. and having a pH of about 6.0 at 25°C. Pharmaceutical compositions.

74. 74. The pharmaceutical composition of claim 73, wherein the antibody drug conjugate is at a concentration of about 10 mg / mL.

75. 75. The pharmaceutical composition of any one of claims 1 to 74, in liquid form.

76. 75. The pharmaceutical composition of any one of claims 1 to 74, which is lyophilized.

77. 75. A lyophilized composition made by lyophilizing the pharmaceutical composition of any one of claims 1-74.

78. 75. The pharmaceutical composition of any one of claims 1-74, stored at -80°C, 4°C, 25°C or 37°C.

79. 80. A method of preventing or treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 1-78.

80. 80. The method of claim 79, wherein the subject is a human subject.

81. 81. The method of claim 80, wherein the cancer is colon cancer, pancreatic cancer, ovarian cancer, lung cancer, bladder cancer, urothelial cancer, breast cancer, esophageal cancer, head cancer, or neck cancer.

82. 82. The method of claim 81, wherein the cancer is colon cancer.

83. 82. The method of claim 81, wherein the cancer is pancreatic cancer.

84. 82. The method of claim 81, wherein the cancer is ovarian cancer.

85. 82. The method of claim 81, wherein the cancer is lung cancer, and optionally the lung cancer is non-small cell lung cancer.

86. 82. The method of claim 81, wherein the cancer is bladder cancer or urothelial cancer.

87. 87. The method of claim 86, wherein the bladder cancer is advanced bladder cancer or advanced urothelial carcinoma.

88. 87. The method of claim 86, wherein the bladder cancer is metastatic bladder cancer or metastatic urothelial carcinoma.

89. 82. The method of claim 81, wherein the cancer is breast cancer.

90. 82. The method of claim 81, wherein the cancer is esophageal cancer.

91. 82. The method of claim 81, wherein the cancer is head cancer.

92. 82. The method of claim 81, wherein the cancer is cervical cancer.

93. 81. The method of claim 80, wherein the cancer has tumor cells that express 191P4D12.

94. 94. The method of any one of claims 79-93, further comprising administering to the subject a second therapeutic agent.

95. 95. The method of claim 94, wherein the second therapeutic agent is an immune checkpoint inhibitor.

96. 96. The method of claim 95, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

97. 97. The method of claim 96, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

98. 98. The method of claim 97, wherein the PD-1 inhibitor is nivolumab.

99. 97. The method of claim 96, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor.

100. 100. The method of claim 99, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

101. 101. The method of any one of claims 79-100, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1-10 mg / kg of subject body weight.

102. 102. The method of claim 101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 5 mg / kg of subject body weight.

103. 102. The method of claim 101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 2.5 mg / kg of subject body weight.

104. 102. The method of claim 101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of 1 to 1.25 mg / kg of subject body weight.

105. 102. The method of claim 101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of about 1 mg / kg of subject body weight.

106. 102. The method of claim 101, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered at a dose of about 1.25 mg / kg of subject body weight.

107. 107. The method of any one of claims 101-106, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion.

108. 108. The method of claim 107, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes in two cycles every three weeks.

109. The method of claim 108, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1 and 8 of every three-week cycle.

110. 110. The method of claim 109, further comprising administering the immune checkpoint inhibitor by intravenous (IV) injection or infusion on day 1 of every three week cycle.

111. 111. The method of claim 110, wherein the immune checkpoint inhibitor is administered in an amount of about 100 mg to about 1500 mg over about 30 or 60 minutes.

112. 108. The method of claim 107, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of three times every four weeks.

113. The method of claim 112, wherein the antibody drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1, 8, and 15 of every four-week cycle.

114. 114. The method of claim 113, further comprising administering the immune checkpoint inhibitor by intravenous (IV) injection or infusion.

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