JPWO2022032020A5 - - Google Patents

Description

Cross-Reference This application claims benefit from U.S. Provisional Patent Application No. 63/062,740, filed August 7, 2020, which is incorporated herein by reference in its entirety. Ru.

CD46 CD46, also known as complement regulatory protein, cluster of differentiation 46, and membrane cofactor protein, is an inhibitory complement receptor. Overexpression of CD46 has been observed in several cancers such as breast, colon, liver, lung, or prostate cancer. In some cases, overexpression of CD46 has been characterized as a negative prognostic factor. For example, overexpression of CD46 correlates with shorter progression-free time and shorter overall survival in breast and ovarian cancer patients. Novel therapeutic methods and regimens targeting CD46 are needed for cancer treatment.

The present disclosure provides immunoconjugates for the treatment of diseases characterized by CD46 expression on the cell surface, such as metastatic castration-resistant prostate cancer and multiple myeloma.

In some embodiments, the immunoconjugate is about 1.0 to about 4.5 mg/kg, about 1.0 to about 4.0 mg/kg, about 1.0 to about 3.5 mg/kg, about 1 .0 to about 3.0 mg/kg, about 1.0 to about 2.57 mg/kg, about 1.0 to about 2.5 mg/kg, about 1.0 to about 2.4 mg/kg, about 1.5 ～about 4.5 mg/kg, about 1.5 to about 4.0 mg/kg, about 1.5 to about 3.5 mg/kg, about 1.5 to about 3.0 mg/kg, about 1.5 to about 2.57 mg/kg, about 1.5 to about 2.5 mg/kg, about 1.5 to about 2.4 mg/kg, about 1.5 to about 2.0 mg/kg, about 1.8 to about 4. 5 mg/kg, about 1.8 to about 4.0 mg/kg, about 1.8 to about 3.5 mg/kg, about 1.8 to about 3.0 mg/kg, about 1.8 to about 2.5, or 7mg/kg, about 1.8-2.0. It is administered to a human subject at a dose of about 2.5 mg/kg, about 1.8 to about 2.4 mg/kg, or about 1.8 to about 2.0 mg/kg. In some embodiments, the immunoconjugate is administered to a human subject at a dose of about 1.5 to about 2.5 mg/kg. In some embodiments, the immunoconjugate is about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1. 7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2. 7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3. 7, about 3.8, about 3.9, or about 4.0 mg/kg to human subjects. In some embodiments, the immunoconjugate is administered to a human subject at a dose of about 1.8, about 2.4, or about 3.2 mg/kg. In some embodiments, the immunoconjugate is administered to a human subject at a dose of about 1.8 mg/kg. In some embodiments, the immunoconjugate is administered to a human subject at a dose of about 2.4 mg/kg. In some embodiments, the immunoconjugate is administered to a human subject at a dose of about 3.2 mg/kg.

In some embodiments, the immunoconjugate is administered to a human subject via intravenous infusion. In some embodiments, the immune complex is administered to a human subject every 7 days, every 14 days, every 18 days, every 21 days, or every 30 days. In some embodiments, the immunoconjugate is administered to a human subject every 21 days.

In some embodiments, the recombinant antibody is conjugated to an effector agent, the effector agent being a drug (or prodrug thereof), a peptide, a protein, a detectable label, a liposome containing the drug (or prodrug thereof). , radionuclides, viral particles, or chelates. In some embodiments, the effector includes a drug. In some embodiments, the drug is an anti-cancer agent. In some embodiments, the drug is a chemotherapeutic agent. In some embodiments, the drug is a microtubule inhibitor, a DNA damaging agent, or a polymerase inhibitor. In some embodiments, the drug is a microtubule inhibitor. In some embodiments, the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). In some embodiments, the microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE), valine-citrulline MMAE (vcMMAE), or valine-citrulline MMAF ( vcMMAF). In some embodiments, the microtubule inhibitor is monomethyl auristatin E (MMAE).

In some embodiments, the ratio of effector agent to recombinant antibody is about 3 to about 5. In some embodiments, the ratio of effector agent to recombinant antibody is about 4.

In some embodiments, the effector agent is conjugated to the recombinant antibody via a linker. In some embodiments, the linker is a peptide linker, a small molecule linker, or a linker that includes a peptide and a small molecule. In some embodiments, the linker comprises maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB).

In some embodiments, the cancer is prostate cancer. In some embodiments, the prostate cancer is castration-resistant prostate cancer. In some embodiments, the cancer is multiple myeloma. In some embodiments, the multiple myeloma is relapsed or refractory multiple myeloma.

In some embodiments, the immune complex binds to CD46 expressed on the surface of the cell and is internalized within the cell. In some embodiments, the immune complex is internalized into the cell via macropinocytosis.

In another aspect, the present disclosure provides a method of treating cancer in a human subject in need of cancer treatment, the method comprising: (a) a heavy chain (HC) comprising three complementarity determining regions (CDRs); A set that specifically binds to CD46, comprising a heavy chain variable region comprising CDR1, HC CDR2, and HC CDR3, and a light chain variable region comprising three CDRs, light chain (LC) CDR1, LC CDR2, and LC CDR3. A recombinant antibody, wherein HC CDR1, HC CDR2, and HC CDR3 each contain the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequence of SEQ ID NO: 4. , SEQ ID NO: 5, and SEQ ID NO: 6, and (b) recombinant through a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. administering an immunoconjugate comprising monomethyl auristatin E (MMAE) conjugated to an antibody, the immunoconjugate being administered at a dose of about 1.0 to about 4.0 mg/kg. provide.

In another aspect, the present disclosure provides a pharmaceutical composition comprising (a) an immunoconjugate at a concentration of about 10.0±5.0 mg/ml; and (b) a histidine buffer, wherein the immunoconjugate is , (a) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, , and a recombinant antibody that specifically binds to CD46 comprising a light chain variable region comprising LC CDR3, wherein HC CDR1, HC CDR2, and HC CDR3 are of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. (b) a recombinant antibody comprising an amino acid sequence, wherein LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and effector agents.

In some embodiments, the pharmaceutical composition comprises about 10 to about 30 mM histidine buffer. In some embodiments, the pharmaceutical composition comprises about 10 to about 20 mM histidine buffer. In some embodiments, the pharmaceutical composition further comprises a cryoprotectant. In some embodiments, the cryoprotectant is a sugar. In some embodiments, it is sucrose or trehalose. In some embodiments, the pharmaceutical composition further comprises a stabilizing agent. In some embodiments, the stabilizing agent prevents denaturation of recombinant antibodies, prevents aggregation of immune complexes, or both. In some embodiments, the stabilizing agent is a polysorbate. In some embodiments, the stabilizing agent is polysorbate 80. In some embodiments, the pharmaceutical composition has a pH of about 5.0 to about 7.0.

In some embodiments, the stabilizer is a polymer. In some embodiments, the polymer is a synthetic or semi-synthetic polymer. The polymer may be a linear polymer such as povidone or polyvinyl alcohol. The polymer may be a copolymer, such as a PVA-PEG graft copolymer. The polymer may be ionic, such as sodium carboxymethyl cellulose, sodium alginate, chitosan, or polyethylene glycol. The semi-synthetic polymer may be a nonionic polymer such as HPMC, HPC, or HEC. In some embodiments, the stabilizing agent is a surfactant. The surfactant is an ionic surfactant such as docusate sodium, sodium lauryl sulfate, or polyethyleneimine, or Tween, poloxamer, D-α-tocopheryl, polyethylene glycol succinate, polyethylene oxide-polyethylene oxide-polyethylene oxide. may be a nonionic surfactant such as a block copolymer of In some embodiments, the stabilizer is a food protein, an amino acid, or a copolymer. In some embodiments, the stabilizing agent is captisol, Monosteol, microcrystalline cellulose and corboxymethylcellulose, sorbitol, or cellulose gel.

In some embodiments, the pharmaceutical composition includes a buffer. Buffers may be selected from acetate, citrate, tartrate, histidine, glutamate, phosphate, Tris, glycine, bicarbonate, succinate, sulfate, or nitrate. In some embodiments, the pharmaceutical composition includes a tonicity adjusting agent. Tonicity agents may be selected from mannitol, sorbitol, lactose, dextrose, trehalose, sodium chloride, potassium chloride, glycerol, and glycerin. In some embodiments, the pharmaceutical composition includes a filler. The bulking agent may be a sugar or polyol selected from sucrose, trehalose, glucose, lactose, sorbitol, mannitol, and glycerol. The bulking agent may be an amino acid selected from arginine, aspartic acid, glutamic acid, lysine, proline, glycine, histidine, methionine, and alanine. The bulking agent may be a polymer or protein selected from gelatin, PVP, PLGA, PEG, dextran, cyclodextrin and derivatives, starch derivatives, HSA, and BSA. In some embodiments, the pharmaceutical composition includes an antioxidant. Antioxidants may be selected from histamine, methionine, ascorbic acid, glutathione, vitamin E, or poly(ethyleneimine). In some embodiments, the pharmaceutical composition includes an antimicrobial preservative. Pharmaceutical preservatives may be selected from benzyl alcohol, metacresol, phenol, and 2-phenoxyethanol. In some embodiments, pharmaceutical compositions may include chelating and/or complexing agents. The chelating agent may be disodium edetate, diethylenetriaminepentaacetic acid, citric acid, hexaphosphate, thioglycolic acid, or zinc.

In some embodiments, the recombinant antibody is conjugated to an effector agent, the effector agent being a drug (or prodrug thereof), a peptide, a protein, a detectable label, a liposome containing the drug (or prodrug thereof). , radionuclides, viral particles, or chelates. In some embodiments, the effector includes a drug. In some embodiments, the drug is an anti-cancer agent. In some embodiments, the drug is a chemotherapeutic agent. In some embodiments, the drug is a microtubule inhibitor, a DNA damaging agent, or a polymerase inhibitor. In some embodiments, the drug is a microtubule inhibitor. In some embodiments, the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). In some embodiments, the microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE), valine-citrulline MMAE (vcMMAE), or valine-citrulline MMAF ( vcMMAF). In some embodiments, the microtubule inhibitor is monomethyl auristatin E (MMAE). In some embodiments, the ratio of effector agent to recombinant antibody in the population of immune complexes is about 3 to about 5. In some embodiments, the ratio of effector agent to recombinant antibody in the population of immune complexes is about 4.

In some embodiments, the effector agent is conjugated to the recombinant antibody via a linker. In some embodiments, the linker is a peptide linker, a small molecule linker, or a linker that includes a peptide and a small molecule. In some embodiments, the linker comprises maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB).

In another aspect, the present disclosure provides an immunoconjugate at a concentration of about 10.0±1.0 mg/ml, about 20 mM histidine buffer, about 8.0% sucrose, and about 0.01% polysorbate 80, wherein the immunoconjugate comprises: (a) a heavy chain variable region comprising three complementarity determining regions (CDRs): CDR1, HC CDR2, and HC CDR3; , and a light chain variable region comprising three CDRs, HC CDR1, HC CDR2, HC CDR3. contains the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LC CDR1, LC CDR2, and LC CDR3 contain the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. , a recombinant antibody, and (b) monomethyl auristatin E (MMAE) conjugated to the recombinant antibody via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. include.

In another aspect, the disclosure provides a method of treating relapsed or refractory multiple myeloma (RRMM) in a human subject in need of treatment for relapsed or refractory multiple myeloma (RRMM), comprising: The heavy chain variable region, which includes three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and the three CDRs, light chain (LC) CDR1, LC CDR2, and LC CDR3. HC CDR1, HC CDR2, and HC CDR3 each have 0 to 3 amino acid modifications. , SEQ ID NO: 2, and SEQ ID NO: 3, with LC CDR1, LC CDR2, and LC CDR3 having 0 to 3 amino acid modifications, respectively, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 A method is provided, including an array.

In another aspect, the disclosure provides a method of treating relapsed or refractory multiple myeloma (RRMM) in a human subject in need of treatment for relapsed or refractory multiple myeloma (RRMM), comprising: The heavy chain variable region, which includes three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and the three CDRs, light chain (LC) CDR1, LC CDR2, and LC CDR3. administering to the subject a recombinant antibody that specifically binds to CD46 comprising a light chain variable region comprising HC CDR1, HC CDR2, and HC CDR3, respectively SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. wherein LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

In another aspect, the disclosure provides a method of treating metastatic castration-resistant prostate cancer in a human subject in need of treatment, the method comprising: CD46 contains a heavy chain variable region that includes one heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain variable region that includes three CDRs, light chain (LC) CDR1, LC CDR2, and LC CDR3. HC CDR1, HC CDR2, and HC CDR3 have 0 to 3 amino acid modifications, respectively, of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 with 0 to 3 amino acid modifications.

In another aspect, the present disclosure provides a method of treating metastatic castration-resistant prostate cancer in a human subject in need of treatment, the method comprising: CD46 contains a heavy chain variable region that includes one heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain variable region that includes three CDRs, light chain (LC) CDR1, LC CDR2, and LC CDR3. administering to the subject a recombinant antibody that specifically binds to HC CDR1, HC CDR2, HC CDR3 comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LC CDR1, LC A method is provided, wherein CDR2 and LC CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

In some embodiments, a recombinant antibody for a method of treating relapsed or refractory multiple myeloma or castration-resistant prostate cancer is conjugated to an effector agent, the effector agent being a drug (or protein thereof). drugs), peptides, proteins, detectable labels, liposomes containing drugs (or prodrugs thereof), radionuclides, viral particles, or chelates. In some embodiments, the effector includes a drug. In some embodiments, the drug is an anti-cancer agent. In some embodiments, the drug is a chemotherapeutic agent. In some embodiments, the drug is a microtubule inhibitor, a DNA damaging agent, or a polymerase inhibitor. In some embodiments, the drug is a microtubule inhibitor. In some embodiments, the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). In some embodiments, the microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE), valine-citrulline MMAE (vcMMAE), or valine-citrulline MMAF ( vcMMAF). In some embodiments, the microtubule inhibitor is monomethyl auristatin E (MMAE). In some embodiments, the ratio of effector agent to recombinant antibody is about 3 to about 5. In some embodiments, the ratio of effector agent to recombinant antibody is about 4.

In some embodiments, an effector agent for a method of treating relapsed or refractory multiple myeloma or castration-resistant prostate cancer is conjugated to the recombinant antibody via a linker. In some embodiments, the linker is a peptide linker, a small molecule linker, or a linker that includes a peptide and a small molecule. In some embodiments, the linker comprises maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB).

In some embodiments, the recombinant antibody for the method of treating relapsed or refractory multiple myeloma or castration-resistant prostate cancer is about 1.0 to about 4.5 mg/kg, about 1.0 ～about 4.0 mg/kg, about 1.0 to about 3.5 mg/kg, about 1.0 to about 3.0 mg/kg, about 1.0 to about 2.7 mg/kg, about 1.0 to about 2.5 mg/kg, about 1.0 to about 2.4 mg/kg, about 1.5 to about 4.5 mg/kg, about 1.5 to about 4.0 mg/kg, about 1.5 to about 3. 5mg/kg, about 1.5 to about 3.0mg/kg, about 1.5 to 2. about 2.7 mg/kg, about 1.5 mg/kg, 1 to about 2.5 mg/kg, about 1.5 to about 2.4 mg/kg, about 1.5 to about 2.0 mg/kg, about 1. 8 to about 4.5 mg/kg, about 1.8 to about 4.0 mg/kg, about 1.8 to about 3.5 mg/kg, about 1.8 to about 3.0 mg/kg, about 1.8 to about about 2.5 or 7 mg/kg, about 1.8 to 2.0. Administered at a dose of about 2.5 mg/kg, about 1.8 to about 2.4 mg/kg, or about 1.8 to about 2.0 mg/kg. In some embodiments, the recombinant antibody is administered at a dose of about 1.5 to about 2.5 mg/kg. In some embodiments, the recombinant antibody has a molecular weight of about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1. 7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2. 7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3. 7, about 3.8, about 3.9, or about 4.0 mg/kg. In some embodiments, the recombinant antibody is administered at a dose of about 1.8, about 2.4, or about 3.2 mg/kg. In some embodiments, the recombinant antibody is administered at a dose of about 1.8 mg/kg. In some embodiments, the recombinant antibody is administered at a dose of about 2.4 mg/kg. In some embodiments, the recombinant antibody is administered at a dose of about 3.2 mg/kg.

In some embodiments, recombinant antibodies for methods of treating relapsed or refractory multiple myeloma or castration-resistant prostate cancer are administered to the human subject via intravenous infusion. In some embodiments, the recombinant antibody is administered to the human subject every 7 days, every 14 days, every 18 days, every 21 days, every 28 days, or every month. In some embodiments, the recombinant antibody is administered to the human subject every 21 days.

In another aspect, the disclosure provides a method of treating relapsed or refractory multiple myeloma (RRMM) in a human subject in need of treatment for relapsed or refractory multiple myeloma (RRMM), comprising: administering to a subject an immune complex, the immune complex comprising: (i) a heavy chain variable comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3; A recombinant antibody that specifically binds to CD46, comprising a light chain (LC) region and a light chain variable region comprising three CDRs, HC CDR1, LC CDR2, and LC CDR3. , HC CDR2, and HC CDR3 comprise the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:5, respectively. a recombinant antibody comprising an amino acid sequence of 6 and conjugated to (ii) via a linker comprising (ii) maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB). monomethyl auristatin E (MMAE).

In another aspect, the present disclosure provides a first heavy chain comprising SEQ ID NO: 9, a first light chain comprising SEQ ID NO: 10, a second heavy chain comprising SEQ ID NO: 9, and a second heavy chain comprising SEQ ID NO: 10. providing an immunoconjugate comprising a recombinant antibody comprising a light chain of 1, 2, 3, or 4 pairs of adducts; Each adduct contains monomethyl auristatin E (MMAE) conjugated to a recombinant antibody via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker, and one pair of , 2 pairs, 3 pairs, or 4 pairs of adducts, each of which is conjugated to a pair of cysteine residues of the recombinant antibody, the pair of cysteine residues being C219 of the first heavy chain and C219 of the first light chain. C214 of the second heavy chain and C214 of the second light chain, C225 of the first heavy chain and C225 of the second light chain, and C228 of the first heavy chain and C228 of the second light chain. Selected from C228. In some embodiments, the immunoconjugate comprises two pairs of adducts.

In another aspect, the present disclosure provides a first heavy chain comprising SEQ ID NO: 9, a first light chain comprising SEQ ID NO: 10, a second heavy chain comprising SEQ ID NO: 9, and a second heavy chain comprising SEQ ID NO: 10. an immunoconjugate comprising a recombinant antibody comprising a light chain of Each adduct of pair, two, three, or four pairs of adducts is conjugated to the recombinant antibody via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. containing monomethyl auristatin E (MMAE), each of which has one, two, three, or four pairs of adducts conjugated to a pair of cysteine residues of the recombinant antibody, and wherein the pairs of cysteine residues are , C219 of the first heavy chain and C214 of the first light chain, C219 of the second heavy chain and C214 of the second light chain, C225 of the first heavy chain and C225 of the second light chain, and an immune complex selected from C228 of a first heavy chain and C228 of a second light chain; about 20 mM histidine buffer at pH 6.0; about 8.0% sucrose; % of polysorbate.

In another aspect, the disclosure provides an immunoconjugate at a concentration of about 10.0±1.0 mg/ml, about 20 mM histidine buffer, about 8.0% sucrose, and about 0.01% polysorbate 80, wherein the immunoconjugate comprises (a) a heavy chain variable region comprising three complementarity determining regions (CDRs): CDR1, HC CDR2, and HC CDR3; , and a light chain variable region comprising three CDRs, HC CDR1, HC CDR2, HC CDR3. contains the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LC CDR1, LC CDR2, and LC CDR3 contain the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. , a recombinant antibody, and (b) monomethyl auristatin E (MMAE) conjugated to the recombinant antibody via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. include.

In another aspect, the present disclosure provides a pharmaceutical composition comprising an immunoconjugate, a pharmaceutically acceptable buffer, and a pharmaceutically acceptable stabilizer, wherein the immunoconjugate comprises (a ) the heavy chain variable region, which includes three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and the three CDRs, light chain (LC) CDR1, LC CDR2, and LC A recombinant antibody that specifically binds to CD46 comprising a light chain variable region comprising CDR3, wherein HC CDR1, HC CDR2, and HC CDR3 have the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. (b) an effector agent conjugated to the recombinant antibody; and (b) an effector agent conjugated to the recombinant antibody. Provided is a pharmaceutical composition comprising: In some embodiments, the pharmaceutical composition has a pH of about 5.0 to about 7.0. In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable buffer, the buffer comprising citrate, phosphate, acetate, tromethamine, histidine, succinate, maleate, or alpha-ketoglutarate. In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable buffer, the buffer comprising about 10 mM to about 30 mM histidine and a pH of about 5 to about 7. In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable buffer, the buffer comprising citrate, phosphate, acetate, tromethamine, histidine, succinate, maleate, or alpha-ketoglutarate; The buffer contains approximately 20mM histidine. In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable stabilizing agent, wherein the stabilizing agent prevents denaturation of recombinant antibodies, prevents aggregation of immune complexes, Or both. In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable stabilizer, the stabilizer comprising a nonionic surfactant. In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable stabilizer, the stabilizer comprising polysorbate. In some embodiments, a pharmaceutical composition comprising a pharmaceutically acceptable stabilizer, the stabilizer comprising about 0.01% polysorbate 80. In some embodiments, a pharmaceutical composition comprising an immunoconjugate, a pharmaceutically acceptable buffer, and a pharmaceutically acceptable stabilizer, further comprising a pharmaceutically acceptable cryoprotectant. It is. In some embodiments, a pharmaceutical composition comprising an immunoconjugate, a pharmaceutically acceptable buffer, and a pharmaceutically acceptable stabilizer, further comprising a pharmaceutically acceptable cryoprotectant. The cryoprotectant contains sugars. In some embodiments, a pharmaceutical composition comprising an immunoconjugate, a pharmaceutically acceptable buffer, and a pharmaceutically acceptable stabilizer, further comprising a pharmaceutically acceptable cryoprotectant. The cryoprotectant comprises sugars, including about 6% to about 10% sucrose or trehalose. In some embodiments, a pharmaceutical composition comprising an immunoconjugate, a pharmaceutically acceptable buffer, and a pharmaceutically acceptable stabilizer, further comprising a pharmaceutically acceptable cryoprotectant. The cryoprotectant contains about 8.0% sucrose.

In some embodiments, a pharmaceutical composition comprising an immunoconjugate, a pharmaceutically acceptable buffer, and a pharmaceutically acceptable stabilizer, wherein the immunoconjugate comprises (a) 3 The heavy chain variable region contains three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and the three CDRs, light chain (LC) CDR1, LC CDR2, and LC CDR3. A recombinant antibody that specifically binds to CD46 comprising a light chain variable region comprising, HC CDR1, HC CDR2, and HC CDR3 comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, LC CDR1, LC CDR2, and LC CDR3 comprise a recombinant antibody comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and (b) an effector agent conjugated to the recombinant antibody. , the recombinant antibody is conjugated to an effector agent, which can be a drug (or prodrug thereof), a peptide, a protein, a detectable label, a liposome containing the drug (or prodrug thereof), a radionuclide, a viral particle, or containing chelates. In some embodiments, a pharmaceutical composition comprising an effector agent, where the effector agent is a drug. In some embodiments, a pharmaceutical composition comprising an effector agent, where the effector agent is an anti-cancer agent. In some embodiments, a pharmaceutical composition comprising a drug, where the drug is a chemotherapeutic agent. In some embodiments, a pharmaceutical composition comprising a drug, where the drug is a microtubule inhibitor, a DNA damaging agent, or a polymerase inhibitor. In some embodiments, a pharmaceutical composition comprising a drug that is a microtubule inhibitor, wherein the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or derivatives thereof). In some embodiments, a pharmaceutical composition comprising a drug that is a microtubule inhibitor, wherein the microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE). ), valine-citrulline MMAE (vcMMAE), or valine-citrulline MMAF (vcMMAF). In some embodiments, a pharmaceutical composition comprising an immune complex comprising a recombinant antibody and an effector agent as described above, wherein the ratio of effector agent to recombinant antibody in the population of immune complexes is about 3 to It is about 5. In some embodiments, a pharmaceutical composition comprising an immune complex comprising a recombinant antibody and an effector agent as described above, wherein the ratio of effector agent to recombinant antibody in the population of immune complexes is about 4. be. In some embodiments, a pharmaceutical composition comprising an immunoconjugate comprising a recombinant antibody as described above and an effector agent, wherein the effector agent is conjugated to the recombinant antibody via a linker. In some embodiments, a pharmaceutical composition comprising an effector agent conjugated to a recombinant antibody via a linker as described above, wherein the linker is a peptide linker, a small molecule linker, or a linker comprising a peptide and a small molecule. be. In some embodiments, a pharmaceutical composition comprising an effector agent conjugated to a recombinant antibody via a linker as described above, wherein the linker is maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc -vc-PAB).
In another aspect, the present disclosure provides a method of treating cancer in a human subject in need of cancer treatment, the method comprising: a first heavy chain comprising SEQ ID NO: 9; a recombinant antibody comprising a first light chain comprising a first light chain comprising SEQ ID NO:9, a second heavy chain comprising SEQ ID NO:9, and a second light chain comprising SEQ ID NO:10; administering to the subject an immunoconjugate comprising adducts, each adduct of the one, two, three, or four pairs of adducts being maleimidocaproyl-valine-citrulline-para-aminobenzyl. monomethyl auristatin E (MMAE) conjugated to a recombinant antibody via an oxycarbonyl (mc-vc-PAB) linker, each of 1, 2, 3, or 4 pairs of adducts conjugated to a pair of cysteine residues of the engineered antibody, the pair of cysteine residues being C219 of the first heavy chain and C214 of the first light chain, C219 of the second heavy chain and C214 of the second light chain. , C225 of the first heavy chain and C225 of the second light chain, and C228 of the first heavy chain and C228 of the second light chain. In some embodiments, a method of treating cancer, wherein the cancer is relapsed or refractory multiple myeloma (RRMM). In some embodiments, a method of treating cancer, wherein the cancer is metastatic castration-resistant prostate cancer (mCRPC). In some embodiments, a method of treating cancer comprises administering to a subject an immunoconjugate, the immunoconjugate comprising two pairs of adducts. In some embodiments, the method of treating cancer described above further comprises detecting CD46 in the cell. In some embodiments, the method of treating cancer described above further comprises detecting CD46 in the cells, where the detecting step comprises immunofluorescence microscopy or immunohistochemical staining. In some embodiments, the method of treating cancer described above further comprises detecting CD46 in the cell, wherein the detecting step comprises flow cytometry. In some embodiments, the method of treating cancer described above further comprises detecting CD46 in the cell, wherein the detecting step includes detecting amplification of chromosomal location 1q21. In some embodiments, a method of treating a cancer as described above, wherein the immune complex is administered to a human subject by intravenous infusion. In some embodiments, a method of treating a cancer as described above, wherein the immunoconjugate is administered to a human subject every 7 days, every 14 days, every 18 days, every 21 days, or every 30 days. . In some embodiments, a method of treating a cancer as described above, wherein the immunoconjugate is administered to a human subject every 21 days for at least three cycles. In some embodiments, the method of treating cancer described above, wherein the immunoconjugate is administered at a dose of about 1.2 to about 3.0 mg/kg. In some embodiments, a method of treating a cancer as described above, wherein the recombinant antibody is administered at a dose of about 1.8, about 2.4, about 2.7, or about 3.0 mg/kg. . In some embodiments, the method of treating cancer described above, wherein the body weight in kg of the human subject in need of treatment for cancer is such that the human subject's actual body weight is less than the subject's adjusted body weight. is the human subject's actual body weight, and is the human subject's adjusted body weight if the human subject's actual body weight is greater than or equal to the subject's adjusted body weight, and the human subject's adjusted body weight is less than 100 kg, or If the adjusted body weight of is 100 kg or more, it is 100 kg. In some embodiments, in the method of treating cancer described above, the body weight in kg of the human subject in need of treatment for cancer is the actual body weight. In some embodiments, in the method of treating cancer described above, the body weight in kg of the human subject in need of treatment for cancer is the adjusted body weight.

In another aspect, the disclosure provides a method of treating metastatic castration-resistant prostate cancer in a human subject in need of treatment, the disclosure comprising: administering to the subject an immune complex. and the immune complex comprises (i) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain, which comprises three CDRs; (LC) A recombinant antibody that specifically binds to CD46, comprising a light chain comprising a light chain variable region comprising CDR1, LC CDR2, and LC CDR3, wherein each of the HC CDR1, HC CDR2, and HC CDR3 has the sequence 1, SEQ ID NO: 2, and SEQ ID NO: 3, and LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and the following (ii ), and (ii) monomethyl auristatin E (MMAE) via a linker comprising maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB). provides a method in which the immunoconjugate is administered at a dose of about 1.2 to about 3.0 mg/kg.

In another aspect, the disclosure provides a method of treating relapsed or refractory multiple myeloma in a human subject in need of treatment, the disclosure comprising: administering an immune complex to the subject; administering, wherein the immune complex comprises: (i) a heavy chain variable region comprising three complementarity determining regions (CDRs) heavy chain (HC) CDR1, HC CDR2, and HC CDR3; A recombinant antibody that specifically binds to CD46 comprising a light chain (LC) variable region comprising a light chain (LC) CDR1, LC CDR2, and LC CDR3, wherein the HC CDR1, HC CDR2, and HC CDR3 are , comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LC CDR1, LC CDR2, and LC CDR3 comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; A recombinant antibody conjugated to (ii) monomethyl auristatin E (MMAE) via a linker comprising (ii) maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB). and wherein the immunoconjugate is administered at a dose of about 1.8 to about 3.0 mg/kg. In some embodiments, a method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject, wherein the human subject's body weight, calculated in kg, is The actual weight of the human subject if the actual weight is less than the subject's adjusted weight, and if the human subject's actual weight is greater than or equal to the subject's adjusted weight, and the human subject's adjusted weight is less than 100 kg. is the human subject's adjusted body weight, or 100 kg if the human subject's adjusted body weight is 100 kg or more. In some embodiments, a method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject, wherein the human subject's body weight calculated in kg is adjusted body weight. be. In some embodiments, the method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject, wherein the human subject's weight in kg is actual body weight.

In some embodiments, the method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject further comprises detecting CD46 in the cells. In some embodiments, a method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject, further comprising detecting CD46 in the cell, the method comprising: includes immunofluorescence microscopy or immunohistochemical staining. In some embodiments, a method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject, further comprising detecting CD46 in the cell, the method comprising: includes flow cytometry. In some embodiments, a method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject, further comprising detecting CD46 in the cell, the method comprising: comprising detecting amplification of chromosomal location 1q21. In some embodiments, a method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject as described above, wherein the immune conjugate is administered to the human subject via intravenous infusion. administered to In some embodiments, a method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject as described above, wherein the immune complex is administered every 7 days, every 14 days, It is administered to human subjects every 18 days, every 21 days, or every 30 days. In some embodiments, a method of treating metastatic castration-resistant prostate cancer or relapsed or refractory multiple myeloma in a human subject as described above, wherein the immune complex is administered every 21 days for at least three cycles. administered to human subjects.

In another aspect, the present disclosure provides a method of treating cancer in a human subject in need of cancer treatment, comprising: treating the human subject with three complementarity determining regions (CDRs): heavy chain (HC) CDR1; A recombinant antibody that specifically binds to CD46, comprising a heavy chain variable region comprising HC CDR2, and HC CDR3, and a light chain variable region comprising three CDRs, light chain (LC) CDR1, LC CDR2, and LC CDR3. wherein HC CDR1, HC CDR2, and HC CDR3 comprise the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 4, and SEQ ID NO: 3, respectively. No. 5 and SEQ ID No. 6, and an effector agent conjugated to the recombinant antibody; Provided are methods in which the method is administered at a dose of about 5.0 mg/kg, or from about 1.0 to about 4.0 mg/kg. In some embodiments, a method of treating cancer, wherein the cancer is prostate cancer. In some embodiments, a method of treating prostate cancer, wherein the prostate cancer is metastatic castration-resistant prostate cancer. In some embodiments, a method of treating cancer, wherein the cancer is multiple myeloma. In some embodiments, a method of treating multiple myeloma, wherein the cancer is relapsed or refractory multiple myeloma. In some embodiments, the method of treating cancer described above further comprises detecting CD46 expression in cancer cells. In some embodiments, the method of treating cancer described above further comprises detecting CD46 expression in cancer cells, wherein the detecting step comprises immunofluorescence microscopy or immunohistochemical staining. In some embodiments, the method of treating cancer described above further comprises detecting CD46 expression in cancer cells, wherein the detecting step comprises flow cytometry. In some embodiments, the method of treating cancer described above further comprises detecting CD46 expression in the cancer cell, wherein the detecting step includes detecting amplification of chromosomal location 1q21. In some embodiments, a method of treating a cancer as described above, wherein the cancer has higher CD46 expression than non-cancerous tissue in the same tissue type of the subject or healthy individual. In some embodiments, a method of treating a cancer as described above, wherein the cancer comprises an increased copy number of chromosome band 1q21. In some embodiments, a method of treating a cancer as described above, wherein the immunoconjugate is administered at about 1.0 to about 4.5 mg/kg, about 1.0 to about 4.0 mg/kg, about 1.0 to about 4.5 mg/kg, about 1.0 to about 4.0 mg/kg, about 1. 0 to about 3.5 mg/kg, about 1.0 to about 3.0 mg/kg, about 1.0 to about 2.7 mg/kg, about 1.0 to about 2.5 mg/kg, about 1.0 to about About 2.4 mg/kg, about 1.5 to about 4.5 mg/kg, about 1.5 to about 4.0 mg/kg, about 1.5 to about 3.5 mg/kg, about 1.5 to about 3 .0mg/kg, about 1.5 to about 2.7mg/kg, about 1.5 to about 2.5mg/kg, about 1.5 to about 2.4mg/kg, about 1.5 to about 2.0mg /kg, about 1.8 to about 4.5 mg/kg, about 1.8 to about 4.0 mg/kg, about 1.8 to about 3.5 mg/kg, about 1.8 to about 3.0 mg/kg , about 1.8 to about 2.7 mg/kg, about 1.8 to about 2.5 mg/kg, about 1.8 to about 2.4 mg/kg, or about 1.8 to about 2.0 mg/kg. Administered in doses. In some embodiments, the method of treating cancer described above, wherein the immunoconjugate is administered at a dose of about 1.2 to about 3.0 mg/kg. In some embodiments, a method of treating a cancer as described above, wherein the immunoconjugate has a molecular weight of about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1 .5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2 .5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3 .5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0 mg/kg. In some embodiments, a method of treating a cancer as described above, wherein the immunoconjugate is administered at a dose of about 1.8, about 2.4, about 2.7, or about 3.0 mg/kg. . In some embodiments, a method of treating a cancer as described above, wherein the immunoconjugate is administered at a dose of about 1.8 mg/kg. In some embodiments, a method of treating a cancer as described above, wherein the immunoconjugate is administered at a dose of about 2.4 mg/kg. In some embodiments, a method of treating a cancer as described above, wherein the immunoconjugate is administered at a dose of about 2.7 mg/kg. In some embodiments, in the method of treating cancer described above, the immunoconjugate is administered at a dose of about 3.0 mg/kg. In some embodiments, the method of treating cancer in a human subject as described above, wherein the human subject's body weight in kg is less than the subject's adjusted body weight if the human subject's actual body weight is less than the subject's adjusted body weight. actual body weight, and is the human subject's adjusted body weight if the human subject's actual body weight is greater than or equal to the subject's adjusted body weight, and the human subject's adjusted body weight is less than 100 kg, or the human subject's adjusted body weight is If it is 100 kg or more, it is 100 kg. In some embodiments, in the method of treating cancer in a human subject described above, the human subject's body weight in kg is actual body weight. In some embodiments, in the method of treating cancer in a human subject described above, the human subject's body weight in kg is an adjusted body weight. In some embodiments, a method of treating cancer in a human subject comprising administering to the human subject an immune complex comprising the recombinant antibody described above, wherein the recombinant antibody is administered via intravenous infusion. Administered to human subjects. In some embodiments, a method of treating cancer in a human subject comprising administering to the human subject an immune complex comprising the recombinant antibody described above, wherein the recombinant antibody is administered to the human subject every 7 days, every 14 days. administered to human subjects every 18 days, every 21 days, or every 30 days. In some embodiments, a method of treating cancer in a human subject comprising administering to the human subject an immune complex comprising the recombinant antibody described above, wherein the recombinant antibody is administered to human subjects on a daily basis. In some embodiments, a method of treating cancer in a human subject comprising administering to the human subject an immune complex comprising an effector agent as described above, wherein the effector agent is a drug (or prodrug thereof), Includes peptides, proteins, detectable labels, liposomes containing drugs (or prodrugs thereof), radionuclides, viral particles, or chelates. In some embodiments, a method of treating cancer in a human subject comprises administering to the human subject an immune complex comprising an effector agent as described above, the effector agent comprising a drug. In some embodiments, a method of treating cancer in a human subject as described above, wherein the effector agent comprises an anti-cancer agent. In some embodiments, a method of treating cancer in a human subject as described above, wherein the effector agent comprises a drug, and the drug is a chemotherapeutic agent. In some embodiments, a method of treating cancer in a human subject as described above, wherein the effector agent comprises a drug, the drug being a microtubule inhibitor, a DNA damaging agent, or a polymerase inhibitor. In some embodiments, a method of treating cancer in a human subject as described above, wherein the effector agent comprises a drug, and the drug is a microtubule inhibitor. In some embodiments, a method of treating cancer in a human subject as described above, wherein the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). ). In some embodiments, a method of treating cancer in a human subject as described above, wherein the microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE), valine-citrulline MMAE (vcMMAE) or valine-citrulline MMAF (vcMMAF). In some embodiments, the method of treating cancer in a human subject described above, wherein the microtubule inhibitor is monomethyl auristatin E (MMAE). In some embodiments, a method of treating cancer in a human subject comprising administering to the human subject an immune complex comprising an effector agent and a recombinant antibody as described above, the method comprising: The ratio is about 3 to about 5. In some embodiments, the method of treating cancer in a human subject described above, wherein the ratio of effector agent to recombinant antibody is about 4. In some embodiments, in the method of treating cancer in a human subject described above, the effector agent is conjugated to the recombinant antibody via a linker. In some embodiments, in the method of treating cancer in a human subject described above, the linker is a peptide linker, a small molecule linker, or a linker comprising a peptide and a small molecule. In some embodiments, a method of treating a cancer in a human subject as described above, wherein the linker comprises several types of cancer, including maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB). In an embodiment, a method of treating cancer in a human subject as described above, wherein the immune complex binds to CD46 expressed on the cell surface and is internalized by the cell. In some embodiments, a method of treating cancer in a human subject as described above, wherein the immune complex is internalized into the cell via macropinocytosis.

In another aspect, the present disclosure provides a first heavy chain comprising SEQ ID NO: 9, a first light chain comprising SEQ ID NO: 10, a second heavy chain comprising SEQ ID NO: 9, and a second heavy chain comprising SEQ ID NO: 10. an immunoconjugate for use in the treatment of cancer in a human subject comprising a cell expressing CD46, comprising a recombinant antibody comprising a light chain of and one, two, three, or four pairs of adducts. and each adduct of 1, 2, 3, or 4 pairs of adducts is linked via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. includes monomethyl auristatin E (MMAE) conjugated to a recombinant antibody, each of 1, 2, 3, or 4 pairs of adducts being conjugated to a pair of cysteine residues of the recombinant antibody; The pairs of residues are C219 of the first heavy chain and C214 of the first light chain, C219 of the second heavy chain and C214 of the second light chain, C225 of the first heavy chain and C214 of the second light chain. C225 of the chain, and C228 of the first heavy chain and C228 of the second light chain. In some embodiments, an immunoconjugate for use in treating cancer in a human subject as described above, wherein the cancer is relapsed or refractory multiple myeloma (RRMM). In some embodiments, an immunoconjugate for use in treating cancer in a human subject as described above, wherein the cancer is metastatic castration-resistant prostate cancer (mCRPC). In some embodiments, an immunoconjugate for use in treating cancer in a human subject as described above, wherein the immunoconjugate comprises two pairs of adducts. In some embodiments, an immunoconjugate for use in treating cancer in a human subject comprising a cell expressing CD46 as described above, wherein the cell is determined by immunofluorescence microscopy or immunohistochemical staining. Contains CD46. In some embodiments, an immunoconjugate for use in treating cancer in a human subject comprising a cell expressing CD46 as described above, wherein the cell comprises CD46 as determined by flow cytometry. In some embodiments, an immunoconjugate for use in treating cancer in a human subject comprising a cell expressing CD46 as described above, wherein the cell comprises an amplification of chromosomal location 1q21. In some embodiments, the immunoconjugate for use in treating cancer in a human subject as described above, wherein the immunoconjugate is formulated for intravenous infusion. In some embodiments, an immunoconjugate for use in treating cancer in a human subject as described above, wherein the immunoconjugate is administered every 7 days, every 14 days, every 18 days, every 21 days, every 28 days. administered to human subjects every month or every month. In some embodiments, the immunoconjugate for use in treating cancer in a human subject as described above, wherein the immunoconjugate is administered to the human subject every 21 days. In some embodiments, the immunoconjugate for use in treating cancer in a human subject as described above, wherein the immunoconjugate is administered at a dose of about 1.2 to about 3.0 m/kg. In some embodiments, an immunoconjugate comprising a recombinant antibody for use in treating a cancer in a human subject as described above, wherein the recombinant antibody has a molecular weight of about 1.8, about 2.4, about 2 .7, or approximately 3.0 m/kg. In some embodiments, an immunoconjugate for use in treating cancer in a human subject as described above, wherein the human subject's body weight in kg is such that the human subject's actual body weight is less than the subject's adjusted body weight. is the actual body weight of the human subject, and is the adjusted body weight of the human subject if the actual body weight of the human subject is greater than or equal to the adjusted body weight of the subject, and the adjusted body weight of the human subject is less than 100 kg. , or 100 kg if the human subject's adjusted body weight is 100 kg or more. In some embodiments, the immunoconjugate for use in treating cancer in a human subject as described above, wherein the human subject's body weight in kg is actual body weight. In some embodiments, the immunoconjugate for use in treating cancer in a human subject as described above, wherein the human subject's body weight in kg is the adjusted body weight.

In another aspect, the present disclosure provides an immunoconjugate for use in the treatment of metastatic castration-resistant prostate cancer in a human subject in need of treatment, comprising: (i) 3 The heavy chain variable region contains three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and the three CDRs, light chain (LC) CDR1, LC CDR2, and LC CDR3. A recombinant antibody that specifically binds to CD46, wherein the HC CDR1, HC CDR2, and HC CDR3 are of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. a recombinant antibody comprising an amino acid sequence, in which LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, and is conjugated to (ii) below; ii) monomethyl auristatin E (MMAE) via a linker comprising maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB), from about 1.2 to about 3.0 mg/kg; The immunoconjugate is administered at a dose of:

In another aspect, the present disclosure provides an immunoconjugate for use in the treatment of refractory multiple myeloma in a human subject in need of treatment, comprising: (i) three complementary The heavy chain variable region, which includes the sex-determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and the light chain (LC), which includes the three CDRs, CDR1, LC CDR2, and LC CDR3. A recombinant antibody that specifically binds to CD46, comprising a light chain comprising a chain variable region, wherein HC CDR1, HC CDR2, and HC CDR3 have the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. a recombinant antibody comprising: LC CDR1, LC CDR2, and LC CDR3 comprising the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, and conjugated to (ii); monomethyl auristatin E (MMAE) via a linker comprising maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) at a dose of about 1.8 to about 3.0 mg/kg. provides an immunoconjugate, which is administered at

In some embodiments, an immunoconjugate for the treatment of metastatic castration-resistant prostate cancer or refractory multiple myeloma in a human subject, wherein the human subject's body weight, calculated in kg, is The actual weight of the human subject if the actual weight is less than the subject's adjusted weight, and if the human subject's actual weight is greater than or equal to the subject's adjusted weight, and the human subject's adjusted weight is less than 100 kg. is the human subject's adjusted body weight, or 100 kg if the human subject's adjusted body weight is 100 kg or more. In some embodiments, the immunoconjugate for the treatment of metastatic castration-resistant prostate cancer or refractory multiple myeloma in a human subject, the human subject's body weight in kg is actual body weight. In some embodiments, the immunoconjugate for the treatment of metastatic castration-resistant prostate cancer or refractory multiple myeloma in a human subject, wherein the human subject's body weight in kg is the adjusted body weight. In some embodiments, an immunoconjugate for the treatment of metastatic castration-resistant prostate cancer or refractory multiple myeloma in a human subject comprising cells, wherein the cells are subjected to immunofluorescence microscopy or immunohistochemical staining. Contains CD46 as determined by. In some embodiments, an immunoconjugate for the treatment of metastatic castration-resistant prostate cancer or refractory multiple myeloma in a human subject comprising a cell, wherein the cell has CD46 as determined by flow cytometry. include. In some embodiments, an immunoconjugate for the treatment of metastatic castration-resistant prostate cancer or refractory multiple myeloma in a human subject comprising a cell, the cell comprising an amplification of chromosomal location 1q21. In some embodiments, an immunoconjugate for the treatment of metastatic castration-resistant prostate cancer or refractory multiple myeloma in a human subject, wherein the immunoconjugate is formulated for intravenous infusion. In some embodiments, an immunoconjugate for the treatment of metastatic castration-resistant prostate cancer or refractory multiple myeloma in a human subject, wherein the immunoconjugate is administered every 7 days, every 14 days, every 18 days. administered to human subjects every 21 days, every 28 days, or every month. In some embodiments, the immunoconjugate for the treatment of metastatic castration-resistant prostate cancer or refractory multiple myeloma in a human subject, wherein the immunoconjugate is administered to the human subject every 21 days for at least three cycles. administered to the subject.

In another aspect, the present disclosure provides an immunoconjugate for treating cancer in a human subject in need thereof, comprising: (a) a heavy chain (HC) comprising three complementarity determining regions (CDRs); ) specifically binds to CD46, which includes the heavy chain variable region including CDR1, HC CDR2, and HC CDR3, and the light chain variable region including the three CDRs, light chain (LC) CDR1, LC CDR2, and LC CDR3. A recombinant antibody, wherein HC CDR1, HC CDR2, and HC CDR3 comprise the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; 4, SEQ ID NO: 5, and (b) an effector agent conjugated to the recombinant antibody, at a dose of about 1.0 to about 5.0 mg/kg. provides an immunoconjugate, which is administered at In some embodiments, an immunoconjugate for treating cancer in a human subject, the cancer being prostate cancer. In some embodiments, an immunoconjugate for treating cancer in a human subject, wherein the prostate cancer is metastatic castration-resistant prostate cancer. In some embodiments, an immunoconjugate for treating cancer in a human subject, wherein the prostate cancer is multiple myeloma. In some embodiments, an immunoconjugate for treating multiple myeloma in a human subject, where the multiple myeloma is relapsed or refractory multiple myeloma. In some embodiments, an immunoconjugate for treating cancer in a human subject, wherein the cancer comprises cells expressing CD46 as determined by immunofluorescence microscopy or immunohistochemical staining. In some embodiments, an immunoconjugate for treating cancer in a human subject, wherein the cancer comprises cells expressing CD46 as determined by flow cytometry. In some embodiments, an immunoconjugate for treating cancer in a human subject, wherein the cancer comprises an amplification of chromosomal location 1q21. In some embodiments, an immunoconjugate for treating cancer in a human subject, wherein the cancer has higher CD46 expression than non-cancerous tissue in the same tissue type of the subject or healthy individual. In some embodiments, the immunoconjugate for treating cancer in a human subject, the immunoconjugate is about 1.0 to about 4.5 mg/kg, about 1.0 to about 4.0 mg/kg. kg, about 1.0 to about 3.5 mg/kg, about 1.0 to about 3.0 mg/kg, about 1.0 to about 2.7 mg/kg, about 1.0 to about 2.5 mg/kg, about 1.0 to about 2.4 mg/kg, about 1.5 to about 4.5 mg/kg, about 1.5 to about 4.0 mg/kg, about 1.5 to about 3.5 mg/kg, about 1 .5 to about 3.0 mg/kg, about 1.5 to about 2.7 mg/kg, about 1.5 to about 2.5 mg/kg, about 1.5 to about 2.4 mg/kg, about 1.5 - about 2.0 mg/kg, about 1.8 - about 4.5 mg/kg, about 1.8 - about 4.0 mg/kg, about 1.8 - about 3.5 mg/kg, about 1.8 - about 3.0 mg/kg, about 1.8 to about 2.7 mg/kg, about 1.8 to about 2.5 mg/kg, about 1.8 to about 2.4 mg/kg, or about 1.8 to about 2 Administered at a dose of .0 mg/kg. In some embodiments, the immunoconjugate for treating cancer in a human subject, the immunoconjugate is administered at a dose of about 1.2 to about 3.0 m/kg. In some embodiments, the immunoconjugate for treating cancer in a human subject, the immunoconjugate has a molecular weight of about 1.0, about 1.1, about 1.2, about 1.3, about 1 .4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2 .4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3 .4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0 mg/kg. In some embodiments, the immunoconjugate for treating cancer in a human subject, the immunoconjugate comprises about 1.8, about 2.4, about 2.7, or about 3.0 mg/kg. Administered in doses. In some embodiments, the immunoconjugate for treating cancer in a human subject, the immunoconjugate comprises about 1.8, about 2.4, about 2.7, or about 3.0 mg/kg. Administered in doses. In some embodiments, the immunoconjugate for treating cancer in a human subject, the immunoconjugate is administered at a dose of about 1.8 m/kg. In some embodiments, the immunoconjugate for treating cancer in a human subject, the immunoconjugate is administered at a dose of about 2.4 m/kg. In some embodiments, the immunoconjugate for treating cancer in a human subject, the immunoconjugate is administered at a dose of about 2.7 m/kg. In some embodiments, the immunoconjugate for treating cancer in a human subject, the immunoconjugate is administered at a dose of about 3.0 m/kg. In some embodiments, an immunoconjugate for treating cancer in a human subject, wherein the human subject's body weight in kg is less than the human subject's adjusted body weight if the human subject's actual body weight is less than the subject's adjusted body weight. the subject's actual body weight; if the human subject's actual body weight is greater than or equal to the subject's adjusted body weight, and the human subject's adjusted body weight is less than 100 kg, then the human subject's adjusted body weight, or the human subject's adjusted body weight; If the weight is 100 kg or more, it is 100 kg. In some embodiments, the immunoconjugate for treating cancer in a human subject, the human subject's body weight in kg is actual body weight. In some embodiments, the immunoconjugate for treating cancer in a human subject, the human subject's body weight in kg is the adjusted body weight. In some embodiments, an immunoconjugate comprising a recombinant antibody for treating cancer in a human subject, wherein the recombinant antibody is formulated for intravenous infusion. In some embodiments, an immunoconjugate comprising a recombinant antibody for treating cancer in a human subject, wherein the recombinant antibody is administered every 7 days, every 14 days, every 18 days, every 21 days, or administered to human subjects every 30 days. In some embodiments, an immunoconjugate comprising a recombinant antibody for treating cancer in a human subject, wherein the recombinant antibody is administered to the human subject every 21 days. In some embodiments, an immunoconjugate comprising an effector agent for treating cancer in a human subject, wherein the effector agent is a drug (or prodrug thereof), a peptide, a protein, a detectable label, a drug (or prodrugs thereof), radionuclides, viral particles, or chelates. In some embodiments, an immunoconjugate comprising an effector agent for treating cancer in a human subject, the effector agent comprising a drug. In some embodiments, an immunoconjugate comprising an effector agent for treating cancer in a human subject, the effector agent comprising an anti-cancer agent. In some embodiments, the immunoconjugate includes an effector agent, where the effector agent includes a chemotherapeutic agent. In some embodiments, an immunoconjugate that includes an effector agent includes a drug, where the drug is a microtubule inhibitor, a DNA damaging agent, or a polymerase inhibitor. In some embodiments, an immunoconjugate comprising a microtubule inhibitor, wherein the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). It is. In some embodiments, an immunoconjugate comprising a microtubule inhibitor, wherein the microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE), valine - citrulline MMAE (vcMMAE), or valine-citrulline MMAF (vcMMAF). In some embodiments, the immunoconjugate includes an effector agent, the effector agent includes a drug, the drug is a microtubule inhibitor, and the microtubule inhibitor is monomethyl auristatin E (MMAE). In some embodiments, an immune complex comprising an effector agent and a recombinant antibody, wherein the ratio of effector agent to recombinant antibody is about 3 to about 5. In some embodiments, the immune complex includes an effector agent and a recombinant antibody, wherein the ratio of effector agent to recombinant antibody is about 4. In some embodiments, an immunoconjugate comprising an effector agent and a recombinant antibody, wherein the effector agent is conjugated to the recombinant antibody via a linker. In some embodiments, the immunoconjugate comprises an effector agent conjugated to a recombinant antibody via a linker, where the linker is a peptide linker, a small molecule linker, or a linker comprising a peptide and a small molecule. In some embodiments, an immunoconjugate comprising an effector agent conjugated to a recombinant antibody via a linker, wherein the linker is maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc -PAB). In some embodiments, an immune complex comprising an effector agent and a recombinant antibody, the immune complex binds to CD46 expressed on the surface of a cell and is internalized by the cell. In some embodiments, an immune complex comprising an effector agent and a recombinant antibody, the immune complex being internalized by a cell via macropinocytosis.

In another aspect, the present disclosure provides a pharmaceutical formulation for the treatment of metastatic castration-resistant prostate cancer in a human subject in need of treatment, comprising: about 10.0±1.0 mg 20 mM histidine buffer, about 8.0% sucrose, and about 0.01% polysorbate 80, and the immune complex contains SEQ ID NO. a recombinant antibody comprising a first heavy chain comprising, a first light chain comprising SEQ ID NO: 10, a second heavy chain comprising SEQ ID NO: 9, and a second light chain comprising SEQ ID NO: 10; including 2, 3, or 4 pairs of adducts, each adduct of the 1, 2, 3, or 4 pairs of adducts being maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl monomethyl auristatin E (MMAE) conjugated to the recombinant antibody via a (mc-vc-PAB) linker, each of the 1, 2, 3, or 4 pairs of adducts is conjugated to a pair of cysteine residues, C219 of the first heavy chain and C214 of the first light chain, C219 of the second heavy chain and C214 of the second light chain, C219 of the first heavy chain and C214 of the second light chain, A pharmaceutical formulation is provided that is selected from C225 of one heavy chain and C225 of a second light chain, and C228 of the first heavy chain and C228 of the second light chain.

In another aspect, the present disclosure provides a pharmaceutical formulation for the treatment of refractory multiple myeloma in a human subject in need of treatment for refractory multiple myeloma, the pharmaceutical formulation comprising: about 10.0±1.0 mg/ml about 20 mM histidine buffer, about 8.0% sucrose, and about 0.01% polysorbate 80, wherein the immunoconjugate contains 1 heavy chain, a first light chain comprising SEQ ID NO: 10, a second heavy chain comprising SEQ ID NO: 9, and a second light chain comprising SEQ ID NO: 10, and one pair, two pairs. , 3 pairs, or 4 pairs of adducts, each adduct of the 1 pair, 2 pairs, 3 pairs, or 4 pairs of adducts being maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc monomethyl auristatin E (MMAE) conjugated to the recombinant antibody via a -vc-PAB) linker, with each of the 1, 2, 3, or 4 pairs of adducts linked to a cysteine of the recombinant antibody. conjugated to pairs of cysteine residues, C219 of the first heavy chain and C214 of the first light chain, C219 of the second heavy chain and C214 of the second light chain, C219 of the first heavy chain and C214 of the second light chain, A pharmaceutical formulation is provided that is selected from C225 of a heavy chain and C225 of a second light chain, and C228 of a first heavy chain and C228 of a second light chain.

FIG. 3 shows flow cytometry traces showing YS5FL binding to prostate cells. FIG. 3 shows flow cytometry traces showing YS5FL binding to multiple myeloma cells. FIG. 2 shows the structure of the FOR46 immune complex described herein. FIG. 3 is a hydrophobic interaction chromatography trace showing the conjugation stoichiometry of MMAE to YS5FL in FOR46. CT scan showing metastatic lesions of castration-resistant prostate cancer patient 001-09-28 (administered 2.7 mg/kg FOR46) on day 15 of cycle 3 and before treatment. Figure 2 is a graph illustrating the decrease in serum PSA of patient 001-09-28. Figure 2 is a swimmer plot showing patient status in the prostate cancer dose escalation study. PR: partial response, EOS: end of study, EOT: end of treatment, PD: disease progression. Figure 2 is a graph showing the response of multiple myeloma patient 006-05-008 to treatment with 1.8 mg/kg FOR46. Figure 2 is a graph showing the response of multiple myeloma patient 001-06-012 to treatment with 2.4 mg/kg FOR46. Figure 2 is a graph showing the response of multiple myeloma patient 003-06-014 to treatment with 2.4 mg/kg FOR46. Figure 2 is a swimmer plot showing patient status in the multiple myeloma dose escalation expansion study. EOS: end of study, EOT: end of treatment, PD: disease progression.

CD46 CD46, also known as complement regulatory protein, cluster of differentiation 46, and membrane cofactor protein, is an inhibitory complement receptor. Overexpression of CD46 has been observed in several cancers such as breast, colon, liver, lung, or prostate cancer. In some cases, overexpression of CD46 has been characterized as a negative prognostic factor. For example, overexpression of CD46 correlates with shorter progression-free time and shorter overall survival in breast and ovarian cancer patients. Provided herein are antibodies and immunoconjugates that target CD46 for the treatment of cancer. Further provided herein are specific dosages and administration regimens for administering CD46 targeting antibodies and immune complexes to human subjects in need thereof. Further provided herein are formulations of CD46-targeting antibodies and immunoconjugates for administration to a subject in need thereof that provide, eg, sufficient stability and cryoprotection.

DEFINITIONS Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing summary and the following detailed description are exemplary and explanatory only and are not intended to limit any claimed subject matter. The section headings used herein are for organizational purposes and are not to be construed as limitations on the subject matter described. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used in this specification and the appended claims, the singular forms "a,""an," and "the" include plural referents unless the context clearly dictates otherwise. Please note that. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "include,""includes," and "included" is not limiting.

As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes exact amounts. Therefore, "about 5 μL" means not only "about 5 μL" but also "5 μL". Generally, the term "about" includes amounts that are expected to be within experimental error.

The terms "antibody" and "immunoglobulin" are used interchangeably herein and in their broadest sense and include fully assembled antibodies, antibody fragments capable of binding antigen, e.g., Fab, F( ab')2, Fv, single chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, etc.

The terms "monoclonal antibody" and "mAb" are used interchangeably herein and refer to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each individual antibody of the population is Identical except that mutations occurring in the molecule may be present in small amounts.

The terms "natural antibody" and "natural immunoglobulin" refer to a heterotetrameric glycoprotein of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. It is. Each light chain is linked to a heavy chain by one covalent disulfide bond, but the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. The heavy and light chains each also have regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains.

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions include amino acid residues derived from "complementary determining regions" or "CDRs" (i.e., residues 24-34 (L1), 50-56 (L2), and 89-97 () in the light chain variable domain). L3), and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; "Sequences of Proteins of Immunological Interest First Edition" by Kabat et al. (1991), U. S. Department of Health and Human Services, NIH Publication No. 91-3242 (referred to herein as "Kabat et al."), and/or residues from "hypervariable loops" (i.e., residues in the light chain variable domain). groups 26-32 (L1), 50-52 (L2), and 91-96 (L3), and (H1), 53-55 (H2), and 96-101 (13) in the heavy chain variable domain; Chothia and "Framework" or "FR" residues include variable domain residues other than the hypervariable region residues recognized herein. It is the basis.

In some examples, the CDRs of the antibody are determined by (i) the Kabat numbering system (Kabat et al. (1991), "Sequences of Proteins of Immunological Interest First Edition", U.S. Department of Health and Human Services, NIH Publication No. 91-3242), or (ii) the Chothia numbering scheme, referred to herein as "Chothia CDR" (e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997). , J. Mol. Biol., 273: 927-948; Chothia et al. 1992, J. Mol. Biol., 227: 799-817; Tramontano A et al. 1990, J. Mol. Biol. 215(1): 175 -82; and US Pat. No. 7,709,226), or (iii) see, for example, Lefranc, M.; -P. 1999, The Immunologist, 7:132-136 and Lefranc, M. -P. 1999, Nucleic Acids Res. , 27:209-212 (“IMGT CDR”), or (iv) the ImMunoGeneTics (IMGT) numbering system as described by MacCallum et al., 1996, J. Mol. Biol. , 262:732-745. For example, Martin, A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, edited by Konterman and Diibel, Chap. ter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

With respect to the Kabat numbering system, CDRs within an antibody heavy chain molecule typically reside at amino acid positions 31-35, which can optionally include one or two additional amino acids, to which 35 (Kabat 35A and 35B in the numbering scheme) (CDR1), followed by amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acids 50-56 (CDR2), and amino acids 89-97 (CDR3). As is well known to those skilled in the art, using the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or additional amino acids due to FR and/or CDR shortening or lengthening. Therefore, the Kabat number of an amino acid is not necessarily the same as its linear amino acid number.

As used herein, the term "antigen-binding site" refers to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen binding site" refers to the portion of an antibody that includes a region that specifically binds and is complementary to part or all of an antigen. When an antigen is large, an antigen-binding molecule may bind only to a specific part of the antigen, called an epitope. An antigen binding site can be provided, for example, by one or more variable domains (also called variable regions). Preferably, the antigen binding site comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

"Specific binding" means that the binding is selective for the antigen and distinguishable from unwanted or non-specific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed with a BIAcore instrument) (Liljeblad Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the binding of the antigen-binding molecule to the antigen, as measured, for example, by SPR. In certain embodiments, molecules that bind to antigens have a dissociation constant (Kd) of less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10-7M or less, for example 10-7M to 10-13M, for example 10-9M to 10-13M).

Depending on the amino acid sequence of the constant domain of the immunoglobulin heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, IgM, and IgY, and some of these have further subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, human IgG1 and IgG3 isotypes have ADCC (antibody-dependent cell-mediated cytotoxicity) activity. The light chains of antibodies (immunoglobulins) from all vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains. I can do it.

As used herein, the term "chimeric antibody" means that a portion of the heavy and/or light chain is derived from a particular source (e.g., protein) or species, while the heavy and/or light chain refers to an antibody in which the remainder is derived from a different source (e.g., protein) or species.

As used herein, the term "recombinant human antibody" refers to antibodies isolated from host cells, such as NSO or CHO cells, or from animals (e.g., mice) that are transgenic for human immunoglobulin genes. It is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions in rearranged form. Optionally, the recombinant human antibody has been subjected to in vivo somatic hypermutation. Therefore, the amino acid sequences of the VH and VL regions of the recombinant antibody, although derived from human germline VH and VL sequences, are sequences that cannot normally be present in the human antibody germline repertoire in vivo.

The term "valence" as used herein refers to the presence of a specific number of binding sites in an antigen-binding molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. Bispecific antibodies according to the invention are at least "bivalent" and may be "trivalent" or "multivalent" (eg, "tetravalent" or "hexavalent"). In certain embodiments, antibodies of the invention have more than one binding site and are bispecific. That is, an antibody can be bispecific even when more than two binding sites are present (ie, the antibody is trivalent or multivalent). Specifically, the invention relates to bispecific, bivalent antibodies, which have one binding site for each antigen that they specifically bind.

As used herein, the term "monospecific" antibody refers to an antibody that has one or more binding sites, each binding to the same epitope of the same antigen.

The terms "individual," "subject," and "patient" are used interchangeably herein to refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. Both of these terms refer to situations characterized by the supervision (e.g., regular or intermittent) of a health care professional (e.g., physician, registered nurse, clinical nurse, physician's assistant, clerical worker, or hospice worker). is not required or limited to that situation.

As used herein, the term "percent amino acid sequence identity (%)" with respect to sequences refers to the sequence obtained by aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular sequence, but does not consider any conservative substitutions as part of sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be performed using EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNA STAR) software, etc., for example, This can be accomplished in a variety of ways within the art using available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared.

The terms "cancer" and "tumor" are used interchangeably herein and encompass all types of carcinogenic processes and/or cancerous growths. In embodiments, the cancer includes a primary tumor as well as metastatic tissue or malignantly transformed cells, tissues, or organs. In embodiments, cancer encompasses all histopathologies and stages, eg, stages of aggressiveness/severity of cancer. In embodiments, the cancer includes relapsed and/or resistant cancer.

As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to the natural history of the individual being treated. refers to a clinical intervention that seeks to change the clinical condition and can be carried out for prevention or during the course of clinical pathology. Desired effects of treatment include prevention of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, and attenuation or mitigation of the disease state. , and remission or improved prognosis. In some embodiments, the molecules of the invention are used to delay the onset of a disease or slow the progression of a disease.

As used herein, "ideal body weight" ("IBW") is 50 kg + 2.3 kg x (actual height - 60 inches) for men and 45.5 kg + 2.3 kg x (actual height - 60 inches) for women. 60 inches).

As used herein, "adjusted body weight" ("AJBW") is IBW + 0.4 x (actual body weight - IBW).

Anti-CD46 Recombinant Antibodies In some embodiments, disclosed herein are recombinant antibodies (or antigen-binding fragments thereof) that specifically bind to CD46. In some embodiments, the antibody or antigen-binding fragment or variant thereof is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment or variant thereof is a human, murine, humanized, or chimeric antibody. In some embodiments, the antibody is a functional fragment of a full-length antibody, such as a monovalent Fab, a bivalent Fab'2, a single chain variable fragment (scFv), or a functional fragment or variant thereof (e.g., a full-length (antigen-binding fragments of antibodies). In some embodiments, the recombinant antibody (or antigen-binding fragment thereof) comprises an immunoglobulin variable heavy chain domain (VH). In some embodiments, the recombinant antibody (or antigen-binding fragment thereof) comprises an immunoglobulin variable light chain domain (VL). In some embodiments, the recombinant antibody (or antigen-binding fragment thereof) comprises a VH and a VL.

In some embodiments, the recombinant antibody (or antigen-binding fragment thereof) includes an Fc region. In some embodiments, the recombinant antibody (or antigen-binding fragment thereof) is a full-length antibody. In some embodiments, the recombinant antibody (or antigen-binding fragment thereof) comprises a first light chain comprising a light chain variable region and a light chain constant region, and a first light chain comprising a heavy chain variable region and a heavy chain constant region. a second light chain comprising a light chain variable region and a light chain constant region, and a second heavy chain comprising a heavy chain variable region and a heavy chain constant region. In some embodiments, the first and second light chains have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the first and second light chains bind the same epitope. In some embodiments, the first and second heavy chains have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the first and second heavy chains bind to the same epitope.

In some embodiments, the recombinant antibody (or antigen-binding fragment thereof) is derived from a non-human (eg, rabbit or mouse) antibody. In some instances, a humanized form of a non-human antibody contains minimal non-human sequences to maintain the original antigen specificity. Optionally, the humanized antibody is a human immunoglobulin (acceptor antibody), and the CDRs of the acceptor antibody have the desired specificity, affinity, avidity, binding kinetics, and/or capacity. Residues of the CDRs of a non-human immunoglobulin (donor antibody), such as a mouse donor, are substituted. Optionally, one or more framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues of the donor antibody.

Complementarity determining region (CDR)
In some embodiments, the CD46-binding recombinant antibody comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, or sequences substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

In some embodiments, the CD46-binding recombinant antibody comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 2, or sequences substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

In some embodiments, the CD46-binding recombinant antibody comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, or sequences substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) with at least one, two, or three complementarity of the VH disclosed in Table 2. determining regions (CDRs), or sequences substantially identical thereto (e.g., sequences with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). include.

In some embodiments, the CD46-binding recombinant antibody comprises a VH that includes CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3.

In some embodiments, the CD46-binding recombinant antibody comprises a VL that includes CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6.

In some embodiments, the CD46-binding recombinant antibody has a VH comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, and CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and VL containing CDR3 of SEQ ID NO: 6.

In some embodiments, the CDRs described herein include one, two, or three amino acid modifications. In some embodiments, the modification is a substitution, addition, or deletion. In some embodiments, the CDRs described herein include one, two, or three conservative amino acid substitutions. In some embodiments, the one, two, or three amino acid modifications do not substantially modify binding to human CD46. In some embodiments, one, two, or three amino acid modifications modify binding to human CD46. In some embodiments, VH-CDR3 and/or VL-CDR3 contain amino acid substitutions that modify binding to human CD46, immunogenicity, or some other characteristic. In some embodiments, the amino acid substitution is alanine (A).

Variable Heavy Chain Region and Variable Light Chain Region In some embodiments, the CD46-binding recombinant antibody has the amino acid sequence disclosed in Table 3 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96% %, 97%, 98%, or 99% sequence identity).

In some embodiments, the CD46-binding recombinant antibody has an amino acid sequence disclosed in Table 4 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or a sequence with 99% sequence identity).

In some embodiments, the CD46-binding recombinant antibody has an amino acid sequence disclosed in Table 3 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or a VH comprising an amino acid sequence disclosed in Table 4 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%) , or sequences with 99% sequence identity).

In some embodiments, the CD46-binding recombinant antibody has the amino acid sequence of SEQ ID NO: 7 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% (sequences with sequence identity).

In some embodiments, the CD46-binding recombinant antibody has the amino acid sequence of SEQ ID NO: 8 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% (sequences with sequence identity).

In some embodiments, the CD46-binding recombinant antibody has the amino acid sequence of SEQ ID NO: 7 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% a VH comprising the amino acid sequence of SEQ ID NO: 8 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) (sequences with sequence identity).

Heavy and Light Chains In some embodiments, the CD46-binding recombinant antibody has an amino acid sequence disclosed in Table 5 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97% , 98%, or 99% sequence identity).

In some embodiments, the CD46-binding recombinant antibody has an amino acid sequence disclosed in Table 6 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

In some embodiments, the CD46-binding recombinant antibody has an amino acid sequence disclosed in Table 5 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or a heavy chain comprising an amino acid sequence disclosed in Table 6 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%); % or 99% sequence identity).

In some embodiments, the CD46-binding recombinant antibody has the amino acid sequence of SEQ ID NO: 9 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% (sequences with sequence identity).

In some embodiments, the CD46-binding recombinant antibody has the amino acid sequence of SEQ ID NO: 10 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% (sequences with sequence identity).

In some embodiments, the CD46-binding recombinant antibody has the amino acid sequence of SEQ ID NO: 9 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% 10 or a sequence substantially identical thereto (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%); % sequence identity).

In some embodiments, the anti-CD46 antibodies disclosed herein include an immunoglobulin constant region (eg, an Fc region). Exemplary Fc regions can be selected from IgG1, IgG2, IgG3, or IgG4 heavy chain constant regions, more specifically human IgG1 or IgG4 heavy chain constant regions. In some embodiments, the immunoglobulin constant region (e.g., Fc region) enhances one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function. Modified, eg mutated, to increase or decrease.

Effector Agents In some embodiments, disclosed herein are immunoconjugates comprising an anti-CD46 antibody conjugated to an effector agent (or a prodrug thereof). In some embodiments, the effector agent is a drug (or prodrug thereof), small molecule, protein, peptide, antibody, ligand, receptor, cytotoxic agent, cytostatic agent, liposome, nanoparticle, radionuclide, A cytokine, chemokine, toxin, detectable label, viral particle, or chelate.

In some embodiments, the effector agent is a drug (or a prodrug thereof). In some embodiments, the effector agent is an anti-cancer drug (or a prodrug thereof). In some embodiments, the effector agent is a chemotherapeutic agent (or a prodrug thereof). In some embodiments, the effector agent is a microtubule inhibitor (or a prodrug thereof), a DNA damaging agent (or a prodrug thereof), or a polymerase inhibitor (or a prodrug thereof).

In some embodiments, the effector agent is a microtubule inhibitor (or a prodrug thereof). In some embodiments, the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). In some embodiments, the microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE), valine-citrulline MMAE (vcMMAE), or valine-citrulline MMAF ( vcMMAF). In some embodiments, the microtubule inhibitor is monomethyl auristatin E (MMAE).

In some embodiments, the effector agent has formula A:

comprises or consists of a compound of

In certain embodiments, the effector includes a detectable label. Suitable detectable labels include, but are not limited to, radiopaque labels, nanoparticles, PET labels, MRI labels, radioactive labels, and the like. Among the radionuclides useful in various embodiments of the invention, gamma emitters, positron emitters, x-ray emitters, and fluorescent emitters are useful for localization, diagnosis and/or staging, and/or treatment. Beta and alpha emitters, as well as electron and neutron capture agents such as boron and uranium, can also be used in therapy.

Immunoconjugates In one aspect, an immunoconjugate is provided that includes an anti-CD46 antibody and an effector agent. In some embodiments, the methods described herein utilize these immunoconjugates.

In some embodiments, the immune complex comprises an anti-CD46 antibody (or antigen-binding fragment thereof) described herein. In some embodiments, the immune complex comprises a YS5FL antibody (or antigen-binding fragment thereof).

In some embodiments, the effector agent is conjugated to an anti-CD46 antibody. In some embodiments, the effector agent is attached to the anti-CD46 antibody via a linker. In some embodiments, the linker is a peptide linker, a small molecule linker, or a linker that includes a peptide and a small molecule. Exemplary peptide linkers include, but are not limited to, peptide linkers that include glycine, serine, or glycine and serine.

In some embodiments, the linker is cleavable. In some embodiments, the linker is cleaved only upon internalization into the cell. In some embodiments, the cleavable linker is cleavable only upon internalization into a cancer cell. In some embodiments, the cleavable portion of the linker is a peptide (eg, a dipeptide, eg, ValCit). In some embodiments, the cleavable linker is cleavable by a cathepsin. In some embodiments, the linker comprises a maleimide. In some embodiments, the linker comprises caproic acid. In some embodiments, the linker includes maleimide and caproic acid. In some embodiments, the linker includes a maleimide, caproic acid, and a cleavable dipeptide.

In some embodiments, the linker comprises or consists of maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB).

In some embodiments, the linker has the formula B:

comprising or consisting of a compound of

In some embodiments, the effector agent is attached to the light chain of an anti-CD46 antibody. In some embodiments, the effector agent is attached to the light chain constant region of an anti-CD46 antibody. In some embodiments, the effector agent is attached to the heavy chain of an anti-CD46 antibody. In some embodiments, the effector agent is attached to the heavy chain constant region of an anti-CD46 antibody.

In some embodiments, the effector moiety is attached to a cysteine residue of the anti-CD46 antibody. In some embodiments, the anti-CD46 antibody is partially reduced prior to conjugation to the effector moiety, such that 1-4 interchain disulfide bonds are reduced, but no intrachain disulfide bonds are reduced. Ru. Partial reduction exposes pairs of cysteine residues and makes them available for conjugation to adducts such as mc-vc-PAB-MMAE. In some embodiments, the following interchain cysteine pairs of YS5FL: C219 of the first heavy chain and C214 of the first light chain, C219 of the second heavy chain and C214 of the second light chain, C225 of the heavy chain and C225 of the second light chain, and C228 of the first heavy chain and C228 of the second light chain are exposed. In some embodiments, an effector such as mc-vc-PAB-MMAE is conjugated to 0, 1, 2, 3, or 4 pairs of cysteine residues on YS5FL.

In some embodiments, the ratio of effector agent to anti-CD46 antibody is c. In some embodiments, the ratio of effector agent to anti-CD46 antibody is 2:1, 4:1, 6:1, or 8:1. In some embodiments, the ratio of effector agent to anti-CD46 antibody is about 4:1. In some embodiments, the average ratio of effector agent to anti-CD46 antibody is about 3.7:1. In some embodiments, when the immunoconjugate includes more than one effector agent, each effector agent is the same. In some embodiments, when the immunoconjugate includes more than one effector agent, at least two effector agents are different. In some embodiments, the ratio of effector agent to anti-CD46 antibody is about 4:1, and each effector agent is the same.

Exemplary Immunoconjugates Exemplary immunoconjugates provided herein include monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB). ) comprises an anti-CD46 YS5FL antibody linked to an effector agent. In some embodiments, the ratio of MMAE to YSFL antibody is about 4:1.

In some embodiments, the immunoconjugate comprises an antibody conjugate of Formula C below, comprising a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10. This immunoconjugate, also referred to herein as FOR46, contains the YS5FL antibody linked to MMAE via an mc-vc-PAB linker.

In some embodiments, the anti-CD46 immunoconjugates described herein are produced by a process that involves reduction or partial reduction of immunoglobulin disulfide bonds. In some embodiments, the anti-CD46 immunoconjugates described herein are produced by a process that involves reduction or partial reduction of interchain disulfide bonds of immunoglobulins. In some embodiments, the reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, an effector-linker conjugate containing a maleimide reactive group is conjugated to a pair of reduced cysteines of an immunoglobulin. In some embodiments, the effector-linker conjugate is mc-vc-PAB-MMAE.

In some embodiments, the effector-linker complex is at C219, C225, or C228 of the YS5FL heavy chain (SEQ ID NO: 9) or C214 of the YS5FL light chain (SEQ ID NO: 10), or any combination thereof. is combined. In some embodiments, the effector-linker complex is conjugated to C219 of the YS5FL heavy chain and C214 of the YS5FL light chain. In some embodiments, the anti-CD46 immune complex comprises two YS5FL heavy chains and two YS5FL light chains, and the effector-linker complex comprises C219 of the first YS5FL heavy chain, C219 of the first YS5FL light chain. C214 of the second YS5FL heavy chain, and C214 of the second YS5FL light chain. In some embodiments, the anti-CD46 immune complex comprises two YS5FL heavy chains, and the effector-linker complex is conjugated to C225 of the first YS5FL heavy chain and to C225 of the second YS5FL heavy chain. Ru. In some embodiments, the anti-CD46 immunoconjugate comprises two YS5FL heavy chains, and the effector-linker conjugate is conjugated to C228 of the first YS5FL heavy chain and C228 of the second YS5FL heavy chain. Ru. In some embodiments, the immune complex comprises 2, 4, 6, or 8 effectors, where the effectors include C219 of HC1 and C214 of LC1, C219 of HC2 and C214 of LC2 as a cysteine pair. It is complexed with any one, two, three, or four of C214, C225 of HC1, C225 of HC2, C228 of HC1, and C228 of HC2, respectively.

Binding of Immune Complexes to Target Cells and Activity Against Target Cells In some embodiments, the anti-CD46 antibodies or immune complexes described herein are expressed on the surface of target cells (e.g., cancer cells). It binds to CD46 and is internalized by cells. In some embodiments, the antibody or immune complex is internalized into target cells via macropinocytosis. In some embodiments, the antibody or immune complex is targeted to the lysosomes of the cell upon internalization. In some embodiments, the antibody or immune complex induces internalization into cells without cross-linking.

In some embodiments, the anti-CD46 antibodies or immune complexes described herein mediate killing of target cells (eg, cancer cells) upon internalization. In some embodiments, the anti-CD46 antibody or immune complex induces apoptosis of target cells (eg, cancer cells) upon internalization. In some embodiments, the anti-CD46 antibody or immune complex inhibits cell division of target cells (eg, cancer cells) upon internalization. In some embodiments, the anti-CD46 antibody or immune complex selectively inhibits cell division of cancer cells upon internalization and does not inhibit cell division of non-cancerous cells upon internalization.

Production of Antibodies or Antigen-Binding Fragments Thereof In some embodiments, antibodies (and antigen-binding fragments thereof) are specifically produced using any method known in the art to be useful for the synthesis of antibodies. Produced by chemical synthesis or recombinant expression techniques.

In some embodiments, the antibody (or antigen-binding fragment thereof) is recombinantly expressed. In some embodiments, nucleic acids encoding antibodies (or antigen-binding fragments thereof) are assembled from chemically synthesized oligonucleotides. In some embodiments, antibody-encoding nucleic acid molecules are produced by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequences, or by oligonucleotide probes specific for particular gene sequences. The cloning used is generated from a suitable source, such as an antibody cDNA library or a cDNA library produced from any tissue or cell that expresses immunoglobulins.

In some embodiments, antibodies (or antigen-binding fragments thereof) are produced by immunizing animals, such as mice, to produce polyclonal or monoclonal antibodies.

In some embodiments, an expression vector comprising an antibody nucleotide sequence, or an antibody nucleotide sequence, is transferred into a host cell by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation) and then transfected. The cells are then cultured to produce antibodies using conventional techniques. In some embodiments, antibody expression is regulated by a constitutive, inducible, or tissue-specific promoter.

A variety of host expression vector systems are available to express the antibodies (or antigen-binding fragments thereof) described herein. These host expression vector systems include bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors carrying the antibody or binding fragment thereof; Yeast bacteria (e.g., Saccharomyces pichia) transformed with a recombinant yeast expression vector carrying a binding fragment coding sequence; insects infected with a recombinant viral expression vector (e.g., baculovirus) carrying an antibody or its binding fragment coding sequence; Cell lines; infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or recombinant plasmid expression vectors (e.g., Ti plasmids) with antibody or binding fragment coding sequences thereof; or containing a promoter derived from the genome of a mammalian cell (e.g. metallothionein promoter) or a promoter derived from a mammalian virus (e.g. adenovirus late promoter; vaccinia virus 7.5K promoter); Microorganisms such as, but not limited to, mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) having recombinant expression constructs for the expression of microorganisms.

Stable expression may be preferred for long-term, high-yield production of recombinant proteins. In some embodiments, cell lines are created that stably express antibodies. After introducing foreign DNA, engineered cells are allowed to grow for 1-2 days in enriched media and then switched to selective media. Selectable markers in recombinant plasmids may be used to confer resistance to selection.

In some embodiments, any method known in the art for the purification of antibodies may be used, such as chromatography (e.g., ion exchange, particularly Protein A followed by affinity for specific antigens, and sizing columns). chromatography), centrifugation, differential solubility, or any other standard technique for protein purification.

Expression Vectors The vector may include any suitable vector derived from either eukaryotic or prokaryotic sources. Optionally, vectors are obtained from bacterial (eg, E. coli), insect, yeast (eg, Pichia pastoris), algal, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A , pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2 Can be mentioned.

Exemplary insect vectors include pFastBac, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30 c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, pPolh- Examples include FLAG vectors such as FLAG1 and pPolh-MAT2, and MAT vectors such as pPolh-MAT1 and pPolh-MAT2.

Optionally, yeast vectors include Gateway pDEST 14 vector, Gateway pDEST 15 vector, Gateway pDEST 17 vector, Gateway pDEST (trademark) 24 vector, Gateway (registered trademark) pYES-DEST52 vector, pBAD-DEST49 Gateway (registered trademark) destination vector, pAO815 Pichia vector, pFLD1 Pichia pastoris vector, pGAPZ A Pichia pastoris vector, pGAPZ B Pichia - Pichia vector, pGAPZ C Pichia vector, pPIC3.5K Pichia vector, pPIC6 A Pichia vector, pPIC6 B Pichia vector, pPIC6 C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeast pYES2/NT A yeast vector, pYES2/NT B yeast vector, pYES2/NT C yeast vector, or pYES3/CT yeast vector.

Exemplary algal vectors include the pChlamy-4 vector or the MCS vector.

Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors include pRK5, p3xFLAG-CMV8, pFLAG-Myc-CMV19, pFLAG-Myc-CMV23, pFLAG-CMV2, pFLAG-CMV6a, pFLAG-CMV6b, pFLAG-CMV6c, pFLAG-CMV5.1 , pFLAG-CMV5a, pFLAG-CMV5b, pFLAG-CMV5c, p3xFLAG-CMV7.1, pFLAG-CMV20, p3xFLAG-Myc-CMV24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CM V3, or pBICEP-CMV4 can be mentioned. Mammalian stable expression vectors include pFLAG-CMV3, p3xFLAG-CMV9, p3xFLAG-CMV13, pFLAG-Myc-CMV21, p3xFLAG-Myc-CMV25, pFLAG-CMV4, p3xFLAG-CMV10, p3xFLAG-CMV14, pF LAG-Myc-CMV22, Mention may be made of p3xFLAG-Myc-CMV26, pBICEP-CMV1, or pBICEP-CMV2.

In some instances, the cell-free system is a mixture of cytoplasmic and/or nuclear components derived from cells and used for in vitro nucleic acid synthesis. In some cases, cell-free systems utilize either prokaryotic or eukaryotic components. Sometimes, nucleic acid synthesis is obtained in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include E. coli S30 Extract strain, E. Examples include, but are not limited to, coli T7S30 series, or PUREexpress (registered trademark).

Host Cell The host cell can be any suitable cell, such as a naturally occurring cell or a genetically modified cell. In some examples, the host cell is a production host cell. In some examples, the host cell is a eukaryotic cell. In other examples, the host cell is a prokaryotic cell. In some cases, eukaryotic cells include fungi (eg, yeast cells), animal cells, or plant cells. In some cases, the prokaryotic cell is a bacterial cell. Examples of bacterial cells include Gram-positive or Gram-negative bacteria. Sometimes Gram-negative bacteria are anaerobic, rod-shaped, or both.

In some examples, Gram-positive bacteria include Actinobacteria, Firmicutes, or Tenicutes. In some cases, Gram-negative bacteria include Aquifecus, Deinococcus thermus, Fibrobacterium-Chlorobium/Bacteroidetes (FCB group), Fusobacterium, Gemmatimonas, Nitrospira, Planctomycetes-Verrucomicrobium. Examples include phylum/Chlamydia (PVC group), Proteobacteria, Spirochetes, or Sinergistes. Other bacteria may be Actinobacteria, Chloroflexus, Chrysiogenes, Cyanobacteria, Deferribacter, Dictyoglobacteria, Thermodesulfobacteria, or Thermotoga. The bacterial cell can be E. coli, Clostridium botulinum, or Coli bacilli.

Exemplary prokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F' , INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2(TM), Stbl3(TM), or Stbl4(TM).

In some examples, animal cells include cells of vertebrate or invertebrate origin. Optionally, animal cells include cells from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. Optionally, fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast.

Fungi include yeasts, molds, filamentous fungi, basidiomycetes, or ascomycetes such as zygomycetes. In some examples, yeast include Ascomycota or Basidiomycota. In some cases, the Ascomycota phylum includes the subphylum Saccharomycetes (true yeasts, such as Saccharomyces cerevisiae (baker's yeast)) or the subphylum Taphrina (e.g., Schizosaccharomycetes (fission yeast)). In some cases, Basidiomycota includes the subphylum Araceae (e.g., Pyrobacteriaceae) or the subphylum Sabikine (e.g., Microbotryumycota).

Exemplary yeasts or molds include, for example, the following genera: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluiwellomyces, Zygosaccharomyces, Yarrowia, Examples include the genus Trichosporon, Rhodosporidium, Aspergillus, Fusarium, and Trichoderma. Exemplary yeasts or molds include, for example, the following species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea ( Candida stellatoidea), Candida glabrata, Candida krusei, Candida parasilosis, Candida guilliermondii, Candida viswanati, Candida lusitanie, Rhodotorula mucilaginosa, Pichia methanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Clyveromyces lactis, Chigosaccharomyces rouxi, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru, Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces baili, Cryptococcus neoformans, Cryptococcus gattii, Or Saccharomyces boulardii.

Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strains such as INVScl.

In some examples, additional animal cells include cells obtained from molluscs, arthropods, annelids, or sponges. Optionally, the additional animal cell is a mammalian cell, for example from a primate, ape, horse, cow, pig, dog, cat, or rodent. Optionally, rodents include mice, rats, hamsters, gerbils, hamsters, chinchillas, fancy rats, or guinea pigs.

Exemplary mammalian host cells include 293A cell line, 293FT cell line, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, Expi293F™ cells, Flp- In(TM) T-REx(TM) 293 Cell Line, Flp-In(TM)-293 Cell Line, Flp-In(TM)-3T3 Cell Line, Flp-In(TM)-BHK Cell Line, Flp-In (trademark) - CHO cell line, Flp-In (trademark) - CV-1 cell line, Flp-In (trademark) - Jurkat cell line, FreeStyle (trademark) 293-F cells, FreeStyle (trademark) CHO-S cells, GripTite™ 293MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per. These include, but are not limited to, C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

In some instances, the mammalian host cell is a stable cell line or cell line that has the ability to integrate the genetic material of interest into its own genome and express the genetic material product after multiple generations of cell division. . In some cases, the mammalian host cell is a transient cell line or a cell line that does not integrate the genetic material of interest into its own genome and does not have the capacity to express the genetic material product after multiple generations of cell division. be.

Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expressSF+® cells.

In some examples, plant cells include algae-derived cells. Exemplary insect cell lines include, but are not limited to, strains derived from Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC7942.

Methods of Treatment In one aspect, provided herein is a method of treating cancer by administering an anti-CD46 antibody or immune conjugate described herein.

In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is relapsed multiple myeloma. In some embodiments, the cancer is multiple myeloma in remission. In some embodiments, the cancer is relapsing or remitting multiple myeloma.

In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is castration-resistant prostate cancer. In some embodiments, the cancer is metastatic prostate cancer.

In one aspect, provided herein is an anti-CD46 antibody or immunoconjugate described herein for use as a medicament. In one aspect, provided herein is an anti-CD46 antibody or immunoconjugate as described herein for use in treating a disease, particularly for use in treating cancer. In one aspect, provided herein is an anti-CD46 antibody or immunoconjugate described herein for use in a method of treating cancer. In one aspect, provided herein is an anti-CD46 antibody or immune conjugate described herein for use in treating a disease in an individual in need thereof. In one aspect, herein provides a method of treating an individual with cancer, the method comprising administering to the individual a therapeutically effective amount of an anti-CD46 antibody or immune conjugate described herein. An anti-CD46 antibody or immunoconjugate described herein is provided for use in a CD46 antibody or immunoconjugate as described herein. In one aspect, herein an anti-CD46 antibody or immunoconjugate described herein is provided prior to the manufacture or preparation of a medicament for the treatment of a disease in an individual in need of treatment of the disease. Ru. In one aspect, provided herein is an agent for use in a method of treating cancer, comprising administering a therapeutically effective amount of the agent to an individual having cancer.

Dosing and Administration For use in therapeutic methods, the anti-CD46 antibodies or immunoconjugates described herein can be formulated, dosed, and administered consistent with principles for good medical care. . Factors considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the drug, the method of administration, the dosing schedule, and the There are other factors that are understood.

In some embodiments, the antibodies or immunoconjugates described herein are administered to a human subject by intravenous infusion. In some embodiments, the antibody or immunoconjugate is administered to a human subject every 7 days, every 14 days, every 18 days, every 21 days, or every 30 days. In some embodiments, the antibody or immunoconjugate is administered to the human subject every 21 days.

In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 1.0 to about 5.0 mg/kg. In some embodiments, the antibody or immunoconjugate is about 1.0 to about 4.5 mg/kg, about 1.0 to about 4.0 mg/kg, about 1.0 to about 3.5 mg/kg, About 1.0 to about 3.0 mg/kg, about 1.0 to about 2.7 mg/kg, about 1.0 to about 2.5 mg/kg, about 1.0 to about 2.4 mg/kg, about 1 .5 to about 4.5 mg/kg, about 1.5 to about 4.0 mg/kg, about 1.5 to about 3.5 mg/kg, about 1.5 to about 3.0 mg/kg, about 1.5 ～about 2.7 mg/kg, about 1.5 to about 2.5 mg/kg, about 1.5 to about 2.4 mg/kg, about 1.5 to about 2.0 mg/kg, about 1.8 to about 4.5 mg/kg, about 1.8 to about 4.0 mg/kg, about 1.8 to about 3.5 mg/kg, about 1.8 to about 3.0 mg/kg, about 1.8 to about 2. 7 mg/kg, about 1.8 to about 2.5 mg/kg, about 1.8 to about 2.4 mg/kg, or about 1.8 to about 2.0 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 1.5 to about 2.5 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 1.2 to about 3.0 mg/kg.

In some embodiments, the antibody or immunoconjugate is about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about administered to a human subject at a dose of 3.7, about 3.8, about 3.9, or about 4.0 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 1.8, about 2.4, about 2.7, about 3.0, or about 3.2 mg/kg. . In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 1.8 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 2.4 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 2.7 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 3.0 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 3.2 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 1.5 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 2.5 mg/kg. In some embodiments, the antibody or immunoconjugate is administered to a human subject at a dose of about 3.0 mg/kg. In some embodiments, weight is measured in kg. In some embodiments, the human subject's weight is their actual body weight. In some embodiments, weight is measured in kg. In some embodiments, the human subject's body weight is adjusted body weight (AJBW).

Determining CD46 Expression In one aspect, the present invention provides methods for (1) determining that a cancer comprises CD46; A method of treating cancer is provided. In some embodiments, cancers that express CD46 are more sensitive to treatment with anti-CD46 antibodies or immune conjugates. In some embodiments, an anti-CD46 antibody or immune conjugate is a more effective anti-cancer agent when the cancer expresses CD46 or expresses CD46 at higher levels than non-cancerous controls. In some embodiments, the non-cancer control is a matched non-cancer control tissue from a subject or individual who does not have cancer. For example, if the cancer is prostate cancer, the non-cancerous control tissue can be a healthy prostate.

In some embodiments, anti-CD46 antibodies are used to determine CD46 expression by cancer. CD46 expression by cancer (eg, cancer cells, cancerous lesions, metastatic cells) can be detected by various methods such as immunofluorescence microscopy, immunohistochemical staining, or flow cytometry.

In another embodiment, the copy number of the CD46 gene is determined in cancer. The CD46 gene is localized to band 32 (1q32) of the q-arm of chromosome 1. In some embodiments, 1q amplification indicates that CD46 is more highly expressed. In some embodiments, 1q amplification includes amplification of 1q32. In some embodiments, 1q amplification comprises 1q21 amplification and 1q32 amplification is inferred from 1q21 amplification. In some embodiments, gene amplification comprises increasing the copy number of the CD46 gene. In some embodiments, the number of copies of the CD46 gene is 3 or more. In some embodiments, the number of copies of the CD46 gene is 4, 5, 6, 7, or 8.

PHARMACEUTICAL COMPOSITIONS AND PHARMACEUTICAL FORMULATIONS In a further aspect, the invention provides a pharmaceutical composition comprising an anti-CD46 antibody or immunoconjugate as described herein, eg for use in any of the treatment methods described above. In one embodiment, a pharmaceutical composition comprises an anti-CD46 antibody or immunoconjugate provided herein and at least one pharmaceutically acceptable excipient. Preparation of pharmaceutical compositions containing anti-CD46 antibodies or immunoconjugates described herein is illustrated in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference. as known to those skilled in the art in light of this disclosure.

In some embodiments, the pharmaceutical composition includes a buffer. In some embodiments, the buffer includes histidine. In some embodiments, the pharmaceutical composition comprises about 10 to about 40 mM, about 10 to about 30 mM, or about 10 to about 20 mM histidine buffer. In some embodiments, the pharmaceutical composition comprises about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, or about 40mM histidine buffer. In some embodiments, the pharmaceutical composition comprises about 20 mM histidine buffer.

In some embodiments, the pharmaceutical composition includes a cryoprotectant. In some embodiments, the cryoprotectant includes a saccharide. In some embodiments, the cryoprotectant comprises sucrose or trehalose. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the pharmaceutical composition comprises about 4% to about 12%, about 4% to about 11%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%. %, about 5% to about 12%, about 5% to about 11%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 6% to about 12%, about 6% to about 11%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 7% to about 12%, about 7% to about 11%, about 7 % to about 10%, about 7% to about 9%, or about 7% to about 8% sucrose. In some embodiments, the pharmaceutical composition comprises about 8% sucrose.

In some embodiments, the pharmaceutical composition includes a stabilizing agent. In some embodiments, the stabilizing agent prevents denaturation of recombinant antibodies, prevents aggregation of immune complexes, or both. In some embodiments, the stabilizing agent is a polysorbate. In some embodiments, the stabilizing agent is polysorbate 20. In some embodiments, the stabilizing agent is polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.001%-0.1%, 0.001%-0.05%, 0.001%-0.04%, 0.001%-0. 0.03%, 0.001% to 0.02%, or 0.001% to 0.01% polysorbate (eg, polysorbate 80). In some embodiments, the pharmaceutical composition comprises about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0. 08%, 0.09%, or 0.1% polysorbate (eg, polysorbate 80). In some embodiments, the pharmaceutical composition comprises about 0.01% polysorbate (eg, polysorbate 80).

In some embodiments, the pharmaceutical composition has a pH of about 5.0 to about 7.0. In some embodiments, the pharmaceutical composition has a pH of about 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5. In some embodiments, the pharmaceutical composition has a pH of about 6.0.

In some embodiments, the pharmaceutical composition is about 5.0 mg/ml to 15.0 mg/ml, 5.0 mg/ml to 14.0 mg/ml, 5.0 mg/ml to 13.0 mg/ml, 5 .0mg/ml to 12.0mg/ml, 5.0mg/ml to 11.0mg/ml, 5.0mg/ml to 10.0mg/ml, 6.0mg/ml to 15.0mg/ml, 7.0mg /ml to 15.0mg/ml, 8.0mg/ml to 15.0mg/ml, 9.0mg/ml to 15.0mg/ml, or 10.0mg/ml to 15.0mg/ml. Anti-CD46 antibodies or immunoconjugates as described herein. In some embodiments, the pharmaceutical composition is about 5.0 mg/ml, 6.0 mg/ml, 7.0 mg/ml, 8.0 mg/ml, 9.0 mg/ml, 10.0 mg/ml, 11 An anti-CD46 antibody or immunoconjugate described herein at a concentration of .0 mg/ml, 12.0 mg/ml, 13.0 mg/ml, 14.0 mg/ml, or 15.0 mg/ml. In some embodiments, the pharmaceutical composition is about 5.0 mg/ml ± 1.0 mg/ml, 6.0 mg/ml ± 1.0 mg/ml, 7.0 mg/ml ± 1.0 mg/ml, 8 .0mg/ml±1.0mg/ml, 9.0mg/ml±1.0mg/ml, 10.0mg/ml±1.0mg/ml, 11.0mg/ml±1.0mg/ml, 12.0mg /ml ± 1.0 mg/ml, 13.0 mg/ml ± 1.0 mg/ml, 14.0 mg/ml ± 1.0 mg/ml, or 15.0 mg/ml ± 1.0 mg/ml. Anti-CD46 antibodies or immunoconjugates as described herein. In some embodiments, the pharmaceutical composition comprises an anti-CD46 antibody or immunoconjugate described herein at a concentration of about 10.0 mg/ml±1.0 mg/ml.

Exemplary Formulation An exemplary formulation of an anti-CD46 antibody or immunoconjugate described herein is an anti-CD46 antibody or immunoconjugate described herein at a concentration of approximately about 10.0 mg/ml ± 1.0 mg/ml. The immune complex contains about 20 mM histidine buffer, about 8.0% sucrose, about 0.01% polysorbate 80, and has a pH of 6.0.

Articles of Manufacture Another aspect of the invention provides an article of manufacture containing materials useful for treating the cancers described above. The article of manufacture includes a container and a label or insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass and plastic. The container can hold the composition by itself or in combination with another composition effective to treat the disease, and can have a sterile access port (e.g., the container can be penetrated by a hypodermic needle). (Can be an intravenous solution bag or vial with a stopper).

The label or package insert indicates that the composition is used to treat the disease of interest. Additionally, the article of manufacture comprises (a) a first container containing therein a composition comprising a bispecific antibody of the invention; and (b) a composition comprising an additional cytotoxic or other therapeutic agent. and a second container contained therein. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular disease.

Alternatively or additionally, the article of manufacture comprises a second (or The device may further include a third) container. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

Example 1: YS5FL binding to the surface of cancer cells Cell surface CD46 was detected by flow cytometry. Cells were harvested, centrifuged and resuspended in FACS buffer (PBS 2% FBS) at a concentration of 1×10 ⁶ cells/ml. 100 μL of cell suspension was dispensed into each well of a 96-well plate, and 100 μL of YS5FL at 10 μg/ml was added to the wells and incubated for 1 hour at 4°C. Cells were washed three times with FACS buffer. After the third wash, cells were resuspended in 100 μL of 1:500 diluted AlexaFluor-488 mouse anti-human IgG1 Fc secondary antibody and incubated for 1 hour at 4° C. in the dark. Cells were washed three times with 200 μL of PBS by centrifugation at 2000 RPM for 5 minutes. After the final wash, cells were resuspended in 300 μL of cold PBS and analyzed on a FACSVerse™ (BD Biosciences) flow cytometer. YS5FL specifically bound to the surface of LnCap-C4-2B, LnCap-C4, DU145, PC3-luc, and Hs27 prostate cancer cells, but not to non-tumor BPH1 cells. Figure 1. Similarly, YS5FL has RPMI8226, MM. 1S, MM. 1R, and INA6 specifically bound to the surface of multiple myeloma cells. Figure 2.

Example 2: Preparation of FOR46 immunoconjugate The structure of YS5FL conjugated to the MMAE effector via the mc-vc-PAB linker is shown in FIG. Purified YS5FL mAb (10 mg/ml) is adjusted to pH 6.8 with sodium phosphate buffer and then treated with TCEP (TCEP/mAb ratio is 2.1) for 2 hours at 22°C. The reduced mAb is reacted with mc-vc-PAB-MMAE (drug/mAb ratio of 6) in 9% dimethylacetamide for 15 minutes. The mAb was reduced a second time for 1 h, complexed a second time for 60 min, and the reaction was quenched by lowering the pH to 5.0 with 1 M acetic acid, as determined by hydrophobic interaction chromatography. Finally, a FOR46 immunoconjugate with a drug to antibody ratio of approximately 3.7 is obtained. Figure 4.

Example 3: FOR46 Drug Product A FOR46 immunoconjugate was formulated into a drug product that could be administered to human subjects. The formulation contains 10.0±1.0 mg/ml FOR46 drug substance, 20 mM L-histidine buffer, 8.0% (w/v) sucrose, and 0.01% (w/v) polysorbate 80. and the pH is 6.0. The formulation was determined to provide adequate stability (antibody denaturation and antiaggregation), buffering, and cryoprotection for storage at -20°C. After storage for 1 month at 5°C, the formulation retains >90% binding potency and cell-based activity, >90% monomer, <15 μg/ml residual MMAE, and virtually no visible particles. It wasn't included.

Example 4: Dose Escalation Study - Treatment of Metastatic Castration-Resistant Prostate Cancer with FOR46 FOR46 in human subjects with metastatic castration-resistant prostate cancer (mCRPC), including therapy-related small cell/neuroendocrine prostate cancer (tSCNC) Dose-escalation clinical trials are being conducted to determine the maximum tolerated dose (or maximum test dose) of Eligible patients have progressed on one or more androgen signaling inhibitors, have maintained castrate-state testosterone levels (<50 ng/dL), have hemoglobin (Hgb) >8 g/dL, absolute neutrophil count ( ANC) >1500/μL, platelet (Plt) >100k, aspartate transaminase to alanine transaminase ratio (ALT/AST) <2.5 times the upper limit of normal (ULN), bilirubin (Bili) <1.5 mg/dL, and Organ function was defined by less than 1.5 times the creatinine ULN. There was no history of chemotherapy for MCRPC. Eligible patients will receive or are scheduled to receive FOR46 by IV infusion every 21 days. Thirty-three subjects were enrolled at 10 dose levels ranging from 0.1 to 3.0 mg/kg. Median age was 66 (range 42-81), median baseline PSA was 41 (range 0.2-1627), and 7 subjects had visceral organ metastases. Patient demographics are shown in Table 7.

After accelerated titration, a 3+3 dose escalation design was used. Dosing was changed from actual weight to adjusted weight after excessive toxicity (neutropenia and fatigue) was observed in subjects with high body mass index (BMI). Secondary prevention of G-CSF was identified for subjects who experienced grade 3 or higher neutropenia during a previous treatment cycle. In the absence of excessive toxicity, treatment will continue if the investigator determines that potential clinical benefit exists. A 50% reduction in serum prostate-specific antigen (PSA) levels provides preliminary objective evidence of response to treatment.

The 33 subjects were grouped into 10 cohorts receiving different doses. Cohort and patient status are summarized in Table 8. The reduction in PSA and tumor burden is summarized in Table 9. At 1.2 mg/kg or higher (n=24), 9 subjects (38%) had a 50% decrease in PSA value (PSA50 response), and 15 subjects (63%) had a decrease in PSA. Three objective partial responses (PRs) were reported in eight subjects with measurable disease, and six had stable disease persistence for 9 to 39 weeks, as defined by RECIST criteria. Admitted. “New response evaluation criteria in solid tumor: Revised RECIST guideline (version 1.1)” by Eisenhauer et al., European Journal of Cancer 45 (2009) 228-249. Median treatment cycles were 6 (range 1-28) with 11 patients on study.

PSA values and RECIST results after each infusion cycle are shown in Tables 10-16 for cohorts 4-10, respectively. The results for all patients are summarized in Figure 6.

In patient 12, the tumor burden decreased most substantially. CT scan after 3 cycles of treatment at 2.7 mg/kg showed complete shrinkage of the largest tumor. Figure 5A. The sum of the maximum diameters of target lesions (SLD including pulmonary nodules and perirectal soft tissue masses) decreased from baseline 5.7 cm to 2.0 cm after cycle 6 (65% reduction). This was accompanied by a 71% reduction in serum PSA and a reduction in non-target lesions including the RP node. Figure 5B.

Neutropenia was analyzed by determining the absolute neutrophil count (ANC) as shown in Table 17. Neutropenia of grade 2 or higher was observed in 12 of 16 patients treated with at least 1.8 mg/kg FOR46.

Dose limitations in 3 of 3 high body mass index (BMI) subjects at 2.4 mg/kg dosed at actual body weight and 2 of 3 subjects at 3.0 mg/kg dosed at adjusted body weight. Toxicity was grade 4 neutropenia. The maximum tolerated dose (MTD) was 2.7 mg/kg by adjusted body weight (AJBW). The most common associated adverse events were grade 4 neutropenia in 11 of 33 subjects (33%), grade 3 neutropenia in 6 subjects (18%), and infusion in 14 subjects. related reactions (IRR) (42%), with 1 patient having grade 3 IRR. Neuropathy of any grade occurred in 7 subjects (21%) and grade 3 neuropathy occurred in 1 subject (3%). The most frequent adverse events are shown in Table 18.

We have begun a dose expansion study in patients with prostate cancer. CD46 expression is determined at the time of incorporation by immunofluorescence microscopy. Three patients with moderate or strongly positive CD46 expression were enrolled. A fourth subject with negative CD46 expression was not included.

This example demonstrates that FOR46 has an acceptable toxicity profile using weight-adjusted administration and provides promising preliminary evidence of efficacy in androgen signaling inhibitor-resistant mCRPC subjects. be done. FOR46 is currently being evaluated in two mCRPC expansion cohorts: adenocarcinoma and t-SCNC.

Example 5: Dose Escalation Study - Treatment of Relapsed or Refractory Multiple Myeloma with FOR46 A dose escalation clinical study will test human subjects with relapsed or refractory multiple myeloma as described in Example 2. Treatment with FOR46 formulation is underway. To be eligible, a patient's treatment history must include proteasome inhibitors, immunomodulatory imide drugs (ImiDs), and CD38-directed therapy. Eligible patients also have organ function indicators of hemoglobin 8 g/dL or higher, ANC 1500/pL or higher, platelets 100 k or higher, ALT/AST 2.5 times the upper limit of normal (ULN) or lower, bilirubin 1.5 mg/dL or lower, and 1.5 times or less than creatinine ULN. FOR46 was administered once every three weeks with infusion-related reaction prophylaxis by IV infusion over 30-60 minutes.

The initial protocol had a maximum dose of 2.4 mg/kg of actual body weight. If the MTD was not established using weight-adjusted dosing, escalation was done with modifications to the protocol to allow dosing at higher doses.

A dose expansion clinical trial in 10 patients receiving 2.4 mg/kg of adjusted body weight is also ongoing. Eligibility criteria for the dose expansion study were the same as for the dose escalation study, except ANC <1000/μL and platelets <75k.

The dose escalation study enrolled 15 subjects at six predefined dose levels ranging from 0.1 to 2.4 mg/kg, with one patient receiving doses of 0.1, 0.3, and 0.6 mg/kg, respectively. Dose values included 3 subjects at 1.2 and 1.8 mg/kg and 6 subjects at 2.4 mg/kg. Median age was 68 (range 33-79) for 4 women. Gain 1q was present in 9 patients (9 pts), absent in 5, and unknown in 1. Median number of previous treatment lines was 6 (range 3-17). Dosing for dose escalation and dose expansion studies is shown in Table 19. Patient characteristics are shown in Tables 20 and 21.

An accelerated titration followed by a 3+3 dose escalation design is underway in the dose escalation study. On day 1 of the 21-day cycle, the protocol-specified dose of FOR46 was infused intravenously over 30-60 minutes. Dosing was changed from actual body weight (AW) to adjusted body weight (AJBW) after excessive toxicity (neutropenia and fatigue) was observed in subjects with high body mass index (BMI). G-CSF secondary prophylaxis was administered to subjects who experienced grade 3 or higher neutropenia during a previous treatment cycle.

Safety was evaluated using the Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Only dexamethasone was allowed for infusion reaction prophylaxis. CD46 antigen density was determined on patients' MM cells by flow cytometry. Treatment efficacy was monitored by measuring immunoglobulin levels (M-protein) in serum or urine, including IgA, lambda light chain (λ), kappa light chain (κ), and M-spike protein.

The only dose-limiting toxicity was grade 4 neutropenia in one high BMI patient treated AW. This was the only dose-limiting toxicity among 6 patients at 2.4 mg/kg administered in combination AW (n=3) and ABW (n=3). At 2.4 mg/kg of AJBW, 1 of 3 patients experienced non-dose-limiting grade 4 neutropenia. The most common associated adverse event was grade 4 neutropenia in 3 patients (20%). One patient (6.7%) had grade 4 thrombocytopenia, and one patient (6.7%) each had grade 3 AST elevation, neutropenia, anemia, nausea, and peripheral neuropathy ( PN) was recognized. Adverse events are shown in Table 22.

In the preliminary evaluation, all patients receiving FOR46 at doses less than 1.8 mg/kg (i.e., 0.1 mg/kg, 0.3 mg/kg, 0.6 mg/kg, and 1.2 mg/kg) , treatment was terminated due to disease progression. Treatment was initiated for patients in the 1.8 mg/kg group. Patient 8 showed a response to FOR46 treatment with reductions in serum IgG, serum kappa light chain, serum lambda light chain, and urinary M spike protein. This response provided preliminary evidence of antitumor activity at the 1.8 mg/kg dose.

Four patients responded to FOR46 with partial response (PR) according to IMWG criteria. "International uniform response criteria for multiple myeloma. Leukemia (2006) 1-7" by BGM Durie et al. See Table 23.

Three of six response-evaluable patients in the 1.8 and 2.4 mg/kg dose escalation cohorts had partial responses (PR) lasting 21, 30, and 15 weeks, respectively. Among the PRs, 1q21 gain was not observed in one patient. In dose expansion, 3 out of 10 patients could not be evaluated. Of the 7 evaluable patients, 1 patient had a PR lasting 18 weeks and discontinued during partial response due to adverse events of peripheral neuropathy. Two patients had stable disease progression through cycles 3 and 6. Four patients had a best response with progressive disease.

Patient 006-05-008 was treated with 1.8 mg/kg FOR46. The patient is a 62-year-old Caucasian male who was diagnosed with IgG kappa MM in July 2009. The patient is 1q gain negative and has a history of treatment with (1) daratumumab, pomalidomide, and dexamethasone, (2) pomalidomide and dexamethasone, (3) lenalidomide, (4) lenalidomide and bortezomib, and (5) carfilzomib and pomalidomide. there were. The results for IgG, kappa light chain, and serum M spike are shown in Figure 7A.

Patient 001-06-012 was treated with 2.4 mg/kg FOR46. This patient is a 70-year-old Caucasian man diagnosed with IgA kappa MM in January 2013. This patient was 1q gain positive and was treated with (1) cyclophosphamide, bortezomib, and dexamethasone, (2) lenalidomide, bortezomib, and dexamethasone, (3) carfilzomib, cyclophosphamide, and dexamethasone, and (4) daratumumab. He had a history of treatment with , pomalidomide, and dexamethasone. The results for IgA, kappa light chain, and serum M spike are shown in Figure 7B.

Patient 003-06-014 was treated with FOR46 at 2.4 mg/kg (AJBW). The patient is a 56-year-old man who was diagnosed with IgA kappa myeloma in 2015. The patient was 1q21 gain positive and was treated with (1) cyclophosphamide, bortezomib, and dexamethasone, (2) carfilzomib, lenalidomide, dexamethasone, melphalan, and ASCT with maintenance ixazomib, (3) carfilzomib, daratumumab, and He had a history of treatment with dexamethasone (4) in the CAR-T clinical trial. The results for IgA, kappa light chain, and urinary M spike are shown in Figure 7C.

Results for all patients in the dose escalation study are shown in Table 24. Results for all patients in the dose expansion study are shown in Table 25. The results of both tests are summarized in Figure 8.

In summary, FOR46 demonstrates an acceptable toxicity profile with adjustable body weight dosing. There is promising evidence of efficacy against triple refractory multiple myeloma. Dose escalation studies have been extended to 2.7 mg/kg adjusted body weight.

Example 6: Formulation of FOR46 The purpose of this study was to develop an optimized formulation for FOR46. Thermal stability tests, freeze-thaw stability tests, and agitation tests were performed during the formulation development process. To select the optimal formulation, formulation stability was evaluated by overall appearance, protein concentration, pH, as well as assays including SEC-HPLC, cIEF, Caliper-SDS_R/NR, and MFI analysis.

Analytical Methods Appearance The appearance of all samples, including clarity, color, and visible particles, was examined on black and white backgrounds using a YB-2 light box.

pH
The pH of the samples was measured using a Seven Multi S4.0 pH meter equipped with an Inlab® Micro electrode. The PH meter was calibrated before each use.

Protein Concentration Protein concentration was determined by UV280 reading using a NanoDrop2000 spectrophotometer. The extinction coefficient used in all evaluation tests was 1.571 AU ^* mL ^* mg-1 ^* cm-1. All measurements were repeated twice using 2.5 μL of sample each time and average results were reported.

SEC-HPLC
Size exclusion chromatography was performed at 25°C using an Agilent 1260 Infinity system equipped with a TSKGel G3000SWXL size exclusion chromatography column (300 x 7.8 mm, 5 gm). The flow rate was set at 1.0 mL/min with a homogeneous concentration gradient. The mobile phase consisted of 50mM sodium phosphate buffer, 300mM NaCl, pH 6.8±0.1 for each sample. A loading dose of 100 μg sample was injected and detected using a UV detector at 280 nm. Data was analyzed using Waters Empower

cIEF
cIEF was performed on a ProteinSimple iCE3 instrument equipped with a FC-coated cIEF cartridge. During the formulation development stage, 50 μg of each sample was mixed with 10011 L of master mix consisting of pI markers 4.22/7.46, Servalyt 2-9, Servalyt 3-5, 1% methylcellulose solution, and 8M urea solution. After mixing, the sample was focused at 1500V for 1 minute and 3000V for 8 minutes. The detection wavelength was set at 280 nm to evaluate the charge variant distribution at different pI ranges. In the forced degradation test, the pI marker in the master mix was changed to 4.22/7.05.

Caliper-SDS_R&NR
Prior to testing the sample, pretreatment procedures such as 10 min incubation at 70°C with sample buffer, SDS, and N-ethylmalleimide (for non-reducing or NR) or dithiothreitol (for reducing or R) are performed. Treatment was necessary. A minimum volume of 42 μL (final protein concentration 0.045 mg/ml) of the loading mix was then tested on a LabChip GXII Touch at an excitation wavelength of 635 nm and an emission wavelength of 700 nm. The final results were analyzed by commercially available software: LabChip GX Reviewer.

CE-SDS_R/NR
Non-reducing CE-SDS was performed using a Beckman Coulter PA800 Enhanced or PA800Plus instrument equipped with a photodiode array detector. Samples were diluted to 4 mg/ml by dilution solution (PB-CA) and then heated for 10 min at 60 °C in the presence of 75 μl SDS sample buffer and 5 μl 100 mM NEM for non-reduced CE-SDS. did. Samples were injected for 15 seconds using +5 kV and then separated for 30 minutes at +11 kV. Detection was performed at 220 nm.

DSC Analysis DSC analysis was performed on a MicroCal™ VP-Capillary DSC system model AS12-001C by GE Healthcare. Protein samples were first diluted to 1 mg/ml with formulation buffer before analysis. 300 μL of the tested protein sample was added to a 96-well plate and 300 μL of its corresponding buffer was added as a control. The sample was heated from 10°C to 110°C in a capillary DSC system at a heating rate of 200°C/h. The samples were tested in duplicate and the DSC results (starting Tm and Tm value) were analyzed by Origin 7.0 DSC automated analysis software.

3. Excipient Screening 3.1 Study Objective This study was to evaluate the effect of NaCl, Arg-HC1, sucrose, and trehalose on the stabilization of FOR46 in selected buffers. .

3.2 Test Parameters FOR46 was formulated at a concentration of 10 mg/ml in 20 mM histidine buffer at pH 6.0. As shown in Table 1, for each formulation, 140mM NaCl, 150mM Arg-HC1, 8% (w/v) sucrose, or trehalose were added as stabilizers, respectively, and those without addition were set as blanks. .

Samples in each formulation were subjected to up to 5 cycles of freeze/thaw stress and heat stress (40°C and 25°C). The stability of FOR46 in each formulation was evaluated in various assays shown in Table 26.

3.4 Sample Preparation FOR46 was subjected to buffer exchange to 20 mM histidine at pH 6.0 by ultrafiltration. After adding the appropriate amount of sucrose, trehalose, Arg-HC1, or NaCl, the protein concentration was adjusted to 10 mg/ml, and then all samples were sterile filtered with a 0.22 μm PES membrane filter. For each formulation sample, eight 2R glass vials were filled with 1 mL of filtered DS. One vial was subjected to stress of 3 cycles of freezing and 5 cycles of thawing. For each cycle, freezing time was at least 12 hours in a -40°C freezer. Samples were thawed at room temperature. Three vials were incubated at 40°C. Two vials were incubated at 25°C. One vial from each test condition was sampled for analysis at the designated time points. One vial was designated as TO.

3.5 Results and Discussion 3.5.1 Appearance, Protein Concentration, and pH Results Immediately after short-term storage at 5 °C, obvious precipitation was observed in F1 and F2, which could be attributed to the high ionic strength in the formulation. It may be due to Therefore, F1 and F2 were excluded from the test. All remaining samples were colorless, slightly opalescent, and contained no visible particles at the beginning of the test.

After up to 4 weeks of incubation at 25° C. and 40 seconds, F5 contained no visible particles and many particles were observed in both F3 and F4. This is due to protein denaturation induced by the higher surface tension of sugar-containing formulations, and the mentioned adverse effects can be eliminated by the addition of surfactants in the final formulation.

No substantial changes in appearance were observed in F3, F4, and F5 after up to 5 cycles of freeze and thaw stress.

No substantial changes in pH and protein concentration were seen at 40°C, 25°C, and after 5 cycles of freeze-thaw.

SEC Purity SEC purity data is summarized in Table 28. No substantial changes were observed in any of the samples based on SEC data at 25° C. and after up to 5 cycles of freezing and thawing. After 4 weeks of incubation at 40°C, the SEC purity of F5 was clearly lower than F3 and F4. Therefore, it could be concluded that the stabilizing effects of sucrose and trehalose on ADC were unexpectedly substantial and comparable.

Caliper-SDS_R/NR Purity After up to 5 cycles of freezing and thawing and 4 weeks of incubation at 25°C and 40°C, no substantial change in Caliper-SDS_R/NR purity was seen in any sample.

cIEF
Based on the cIEF data, after 4 weeks of incubation at 40°C and 25°C, a substantial decrease in main peak purity was seen in all samples, with the rate of decrease being comparable between F3-F5. No substantial changes were observed after up to 5 cycles of freezing and thawing.

Drug-antibody ratio (DAR)
After 4 weeks of incubation at 40°C and 25°C and up to 5 cycles of freezing and thawing, no substantial changes in DAR were seen in all samples.

Conclusion Even though a deterioration in appearance is observed in buffers containing trehalose and sucrose, the adverse effects induced by higher surface tensions are reversible by the addition of surfactants. Surprisingly, SEC purity results showed that sucrose and trehalose exhibited significant and comparable performance in stabilizing FOR46 against heat stress. Sucrose was chosen as the excipient in the optimized formulation considering commercial cost. Surfactant screening tests are performed in 20mM histidine buffer at pH 6.0 containing 8% (w/v) sucrose (F3).

4. Surfactant Screening This study investigated the stabilization of two different surfactants (PS-80 and PS-20) at three content levels in 20mM histidine buffer containing 8% (w/v) sucrose. The purpose was to evaluate the effect. Based on the DAR data (shown in Table 34), no substantial change in DAR was seen in all samples after 4 weeks of incubation at 40°C and 25°C and up to 5 cycles of freezing and thawing. Ta.

Test Parameters FOR46 was formulated into seven formulations at 10 mg/ml in 20 mM histidine buffer, pH 6.0, containing 8% (w/v) sucrose, as shown in Table 29. PS-80 or PS-20, each with three content levels, was added to each formulation, and the formulation without surfactant served as a blank. Samples in each formulation were subjected to up to 5 cycles of freeze-thaw, heat stress (40°C), and agitation stress (300 rpm, 2 days). The stability of the ADC at specified time points was evaluated in different assays.

4.3 Drug Materials FOR46 formulated in 20mM histidine buffer at pH 6.0 containing 8% (w/v) sucrose was stored at 2-8°C prior to surfactant screening studies.

Sample Preparation After adding the designed amount of PS-80 or PS-20, the WBP2O95 ADC DS was sterile filtered with a 0.22 μm PES membrane filter. For each formulation sample, eight 2R glass vials were each filled with 1 mL of filtered DS. Two vials were subjected to 5 cycles of freeze-thaw stress. For each cycle, freezing time was at least 12 hours in a -40°C freezer. Samples were thawed at room temperature. Two vials were incubated at 40°C. Two vials were subjected to two days of stirring at ambient temperature and a speed of 300 rpm. One vial from 40°C and two vials from freeze-thaw stress and agitation stress were sampled for analysis at designated time points. Two vials were designated as TO.

4.5 Results and Discussion 4.5.1 Appearance, Protein Concentration, and pH Results After 5 freeze-thaw cycles, no substantial changes in appearance were observed in all samples. Particles and fibers were observed in F1 (no surfactant) after stirring at a speed of 300 rpm for 2 days and incubating at 40° C. for 4 weeks. It was hereby recognized that the presence of surfactants may be essential to protect ADCs in conditions of heat and agitation stress.

No substantial changes in pH and protein concentration were observed.

4.5.2 SEC Purity After 5 cycles of freeze-thaw and 2 days of agitation, no substantial change in SEC purity was observed. After 4 weeks of incubation at 40°C, a 6% decrease in main peak purity was observed for all seven formulations. Based on SEC purity data, all formulations were comparable across conditions.

4.5.3 CE-SDS_R Purity No substantial changes in CE-SDS R purity were observed under heat, freeze-thaw, and agitation stress conditions.

4.5.4 cIEF
No substantial changes in cIEF were observed after 5 cycles of freeze-thaw and 2 days of agitation. With heat stress, the main peak purity decreased substantially, but the acid peak purity increased accordingly. However, the changes between all formulations were comparable.

4.5.5 Efficacy Based on previous data, three main formulations (F2, F3, and F4) were selected for binding efficacy assays. Heat stress, agitation stress, and freeze-thaw stress did not result in substantial changes in binding potency.

4.5.6 MFI
Surprisingly, based on the MFI results, more than 10 times more particles were found in F1 compared to the remaining formulations. This confirmed that there were more particles that could not be visually observed in F1 than in the other formulations.

4.6 Conclusion Based on the appearance and MFI results, surfactants unexpectedly played an important role in protecting the ADC under conditions of heat and agitation stress. However, no differences were found between the six formulations with two different surfactants (PS-80 and PS-20) at three content levels. Lower CMC (critical micelle concentration) of PS-80 compared to PS-20, which exhibited lower effective concentration of surfactant, and possible adverse effects introduced by degradation of PS-80 at high content levels Considering this, 0.01% (w/v) PS-80 was selected for the final formulation.

FOR46 (10 mg/ml) in 20 mM histidine buffer, pH 6.0 containing 8% (w/v) sucrose and 0.01% (w/v) PS-80 was selected as the final formulation.

Claims (167)

a first heavy chain comprising SEQ ID NO: 9;
a first light chain comprising SEQ ID NO: 10;
a recombinant antibody comprising a second heavy chain comprising SEQ ID NO: 9 and a second light chain comprising SEQ ID NO: 10;
1, 2, 3, or 4 pairs of adducts, the immunoconjugate comprising:
Each adduct of the 1, 2, 3, or 4 pairs of adducts is linked to the recombinant via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. comprising monomethyl auristatin E (MMAE) conjugated to an antibody;
each of the one, two, three, or four pairs of adducts is conjugated to a pair of cysteine residues of the recombinant antibody;
The pair of cysteine residues is
C219 of the first heavy chain and C214 of the first light chain,
C219 of the second heavy chain and C214 of the second light chain,
C225 of the first heavy chain and C225 of the second light chain, and C228 of the first heavy chain and C228 of the second light chain.
An immune complex selected from. 2. The immunoconjugate of claim 1, comprising two pairs of said adducts. The immunoconjugate of claim 1 at a concentration of about 10.0±1.0 mg/ml, about 20 mM histidine buffer at pH 6.0, about 8.0% sucrose, and about 0.01% A pharmaceutical composition comprising polysorbate 80. A pharmaceutical composition comprising an immunoconjugate at a concentration of about 10.0±1.0 mg/ml, about 20 mM histidine buffer, about 8.0% sucrose, and about 0.01% polysorbate 80. and the immune complex is
(a) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and a recombinant antibody that specifically binds to CD46 comprising a light chain variable region comprising LC CDR3, wherein said HC CDR1, HC CDR2, and HC CDR3 are of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. a recombinant antibody comprising an amino acid sequence, wherein said LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;
(b) monomethyl auristatin E (MMAE) conjugated to said recombinant antibody via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. . A pharmaceutical composition comprising an immunoconjugate, a pharmaceutically acceptable buffer, and a pharmaceutically acceptable stabilizer, the composition comprising:
The immune complex is
(a) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and a recombinant antibody that specifically binds to CD46 comprising a light chain variable region comprising LC CDR3, wherein said HC CDR1, HC CDR2, and HC CDR3 are of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. a recombinant antibody comprising an amino acid sequence, wherein said LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;
(b) an effector agent conjugated to the recombinant antibody. 6. The pharmaceutical composition of claim 5, having a pH of about 5.0 to about 7.0. A pharmaceutical composition according to any one of claims 5 to 6, wherein the buffer comprises citrate, phosphate, acetate, tromethamine, histidine, succinate, maleate, or alpha-ketoglutarate. 8. The pharmaceutical composition of any one of claims 5 to 7, wherein the buffer comprises about 10 mM to about 30 mM histidine and has a pH of about 5 to about 7. 8. The pharmaceutical composition of claim 7, wherein the buffer comprises about 20 mM histidine and has a pH of about 6.0. The pharmaceutical composition according to any one of claims 5 to 9, wherein the stabilizing agent prevents denaturation of the recombinant antibody, prevents aggregation of the immune complex, or both. . 11. A pharmaceutical composition according to any one of claims 5 to 10, wherein the stabilizer comprises a nonionic surfactant. 12. The pharmaceutical composition of claim 11, wherein the stabilizer comprises polysorbate. 13. The pharmaceutical composition of claim 12, wherein the stabilizer comprises about 0.01% polysorbate 80. 14. A pharmaceutical composition according to any one of claims 5 to 13, further comprising a pharmaceutically acceptable cryoprotectant. 15. The pharmaceutical composition of claim 14, wherein the cryoprotectant comprises a saccharide. 16. The pharmaceutical composition of claim 15, wherein the sugar comprises about 6% to about 10% sucrose or trehalose. 17. The pharmaceutical composition of claim 16, wherein the cryoprotectant is about 8.0% sucrose. The recombinant antibody is conjugated to an effector agent, and the effector agent is a drug (or a prodrug thereof), a peptide, a protein, a detectable label, a liposome containing the drug (or a prodrug thereof), a radionuclide, a virus particle. 18. A pharmaceutical composition according to any one of claims 5 to 17, comprising a chelate, or a chelate. 19. The pharmaceutical composition of claim 18, wherein the effector agent comprises a drug. 20. The pharmaceutical composition according to claim 19, wherein the drug is an anticancer drug. 21. A pharmaceutical composition according to claim 19 or 20, wherein the drug is a chemotherapeutic agent. 22. The pharmaceutical composition of claim 19, 20, or 21, wherein the drug is a microtubule inhibitor, a DNA damaging agent, or a polymerase inhibitor. 23. The pharmaceutical composition of claim 22, wherein the drug is a microtubule inhibitor. 24. The pharmaceutical composition according to claim 23, wherein the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). The microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE), valine-citrulline MMAE (vcMMAE), or valine-citrulline MMAF (vcMMAF). The pharmaceutical composition according to item 22 or 23. 26. The pharmaceutical composition of claim 25, wherein the microtubule inhibitor is monomethyl auristatin E (MMAE). 27. The pharmaceutical composition of any one of claims 5-26, wherein the ratio of the effector agent to the recombinant antibody in the population of immune complexes is about 3 to about 5. 28. The pharmaceutical composition of claim 27, wherein the ratio of the effector agent to the recombinant antibody in the population of immune complexes is about 4. 29. A pharmaceutical composition according to any one of claims 5 to 28, wherein the effector agent is conjugated to the recombinant antibody via a linker. 30. The pharmaceutical composition of claim 29, wherein the linker is a peptide linker, a small molecule linker, or a linker comprising a peptide and a small molecule. 31. A pharmaceutical composition according to claim 29 or 30, wherein the linker comprises maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB). A method of treating cancer comprising cells expressing CD46 in a human subject in need of cancer treatment, the method comprising:
a first heavy chain comprising SEQ ID NO: 9;
a first light chain comprising SEQ ID NO: 10;
a recombinant antibody comprising a second heavy chain comprising SEQ ID NO: 9 and a second light chain comprising SEQ ID NO: 10;
administering an immunoconjugate comprising 1, 2, 3, or 4 pairs of adducts;
Each adduct of the 1, 2, 3, or 4 pairs of adducts is linked to the recombinant via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. comprising monomethyl auristatin E (MMAE) conjugated to an antibody;
each of the one, two, three, or four pairs of adducts is conjugated to a pair of cysteine residues of the recombinant antibody;
The pair of cysteine residues is
C219 of the first heavy chain and C214 of the first light chain,
C219 of the second heavy chain and C214 of the second light chain,
C225 of the first heavy chain and C225 of the second light chain, and C228 of the first heavy chain and C228 of the second light chain.
A method selected from. 33. The method of claim 32, wherein the cancer is relapsed or refractory multiple myeloma (RRMM). 33. The method of claim 32, wherein the cancer is metastatic castration-resistant prostate cancer (mCRPC). 35. A method according to any one of claims 32 to 34, wherein said immunoconjugate comprises two pairs of said adducts. 36. The method of any one of claims 32-35, further comprising detecting said CD46 in said cells. 37. The method of claim 36, wherein the step of detecting comprises immunofluorescence microscopy or immunohistochemical staining. 37. The method of claim 36, wherein the step of detecting comprises flow cytometry. 37. The method of claim 36, wherein the step of detecting comprises detecting amplification of chromosomal location 1q21. 40. The method of any one of claims 32-39, wherein the immune complex is administered to the human subject via intravenous infusion. 41. The method of any one of claims 32-40, wherein the immune complex is administered to the human subject every 7 days, every 14 days, every 18 days, every 21 days, or every 30 days. 42. The method of claim 41, wherein the immunoconjugate is administered to the human subject every 21 days for at least 3 cycles. 43. The method of any one of claims 32-42, wherein the immunoconjugate is administered at a dose of about 1.2 to about 3.0 mg/kg. 44. The method of claim 43, wherein the recombinant antibody is administered at a dose of about 1.8, about 2.4, about 2.7, or about 3.0 mg/kg. 45. A method according to any one of claims 43 to 44, wherein the human subject's body weight in kg is actual body weight. 45. The method of any one of claims 43-44, wherein the human subject's body weight in kg is an adjusted body weight. The body weight in kg of said human subject is
the actual weight of the human subject if the actual weight of the human subject is less than the adjusted weight of the human subject;
if the actual body weight of the human subject is greater than or equal to the adjusted body weight of the human subject, and the adjusted body weight of the human subject is less than 100 kg, then the adjusted body weight of the human subject, or the adjusted body weight of the human subject is 100 kg. If it is more than 100kg
45. A method according to any one of claims 43 to 44. A method of treating metastatic castration-resistant prostate cancer in a human subject in need of treatment for metastatic castration-resistant prostate cancer, the method comprising:
(i) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and a light chain comprising a light chain variable region comprising LC CDR3, the recombinant antibody specifically binds to CD46, wherein said HC CDR1, HC CDR2, and HC CDR3 are SEQ ID NO: 1, SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3, and the LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and are combined with the following (ii), recombinant antibody;
(ii) administering an immunoconjugate comprising monomethyl auristatin E (MMAE) via a linker comprising maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB);
The method, wherein the immunoconjugate is administered at a dose of about 1.2 to about 3.0 mg/kg. A method of treating remitting or refractory multiple myeloma in a human subject in need of treatment for remitting or refractory multiple myeloma, the method comprising:
(i) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and a light chain comprising a light chain variable region comprising LC CDR3, the recombinant antibody specifically binds to CD46, wherein said HC CDR1, HC CDR2, and HC CDR3 are SEQ ID NO: 1, SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3, and the LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and are combined with the following (ii), recombinant antibody;
(ii) administering an immunoconjugate comprising monomethyl auristatin E (MMAE) via a linker comprising maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB);
The method, wherein the immunoconjugate is administered at a dose of about 1.8 to about 3.0 mg/kg. 50. The method of any one of claims 48 to 49, wherein the human subject's body weight in kg is actual body weight. 50. The method of any one of claims 48-49, wherein the human subject's body weight calculated in kg is an adjusted body weight. The body weight calculated in kg of the human subject is
the actual weight of the human subject if the actual weight of the human subject is less than the adjusted weight of the human subject;
the actual body weight of the human subject is equal to or greater than the adjusted body weight of the human subject, and the adjusted body weight of the human subject is less than 100 kg; or the adjusted body weight of the human subject is 100 kg or more. 100kg if
50. A method according to any one of claims 48 to 49. 53. The method of any one of claims 48-52, further comprising detecting said CD46 in said cells. 54. The method of claim 53, wherein the step of detecting comprises immunofluorescence microscopy or immunohistochemical staining. 54. The method of claim 53, wherein the step of detecting comprises flow cytometry. 54. The method of claim 53, wherein the step of detecting comprises detecting amplification of chromosomal location 1q21. 57. The method of any one of claims 48-56, wherein the immune complex is administered to the human subject via intravenous infusion. 58. The method of any one of claims 48-57, wherein the immune complex is administered to the human subject every 7 days, every 14 days, every 18 days, every 21 days, or every 30 days. 59. The method of claim 58, wherein the immunoconjugate is administered to the human subject every 21 days for at least 3 cycles. A method of treating cancer in a human subject in need of cancer treatment, the method comprising:
(a) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and a recombinant antibody that specifically binds to CD46 comprising a light chain variable region comprising LC CDR3, wherein said HC CDR1, HC CDR2, and HC CDR3 are of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. a recombinant antibody comprising an amino acid sequence, wherein said LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;
(b) administering an immune complex comprising an effector agent conjugated to the recombinant antibody;
The method, wherein the immunoconjugate is administered at a dose of about 1.0 to about 5.0 mg/kg. 61. The method of claim 60, wherein the cancer is prostate cancer. 62. The method of claim 61, wherein the prostate cancer is metastatic castration-resistant prostate cancer. 61. The method of claim 60, wherein the cancer is multiple myeloma. 64. The method of claim 63, wherein the multiple myeloma is relapsed or refractory multiple myeloma. 65. The method of any one of claims 60 to 64, further comprising detecting CD46 expression in cells of the cancer. 66. The method of claim 65, wherein the step of detecting comprises immunofluorescence microscopy or immunohistochemical staining. 66. The method of claim 65, wherein the detecting step comprises flow cytometry. 66. The method of claim 65, wherein the step of detecting comprises detecting amplification of chromosomal location 1q21. 69. The method of any one of claims 60 to 68, wherein the cancer has higher CD46 expression than non-cancerous tissue of the same tissue type of the human subject or healthy individual. 69. The method of any one of claims 60-68, wherein the cancer comprises an increased copy number of chromosome band 1q21. The immune complex may be administered at about 1.0 to about 4.5 mg/kg, about 1.0 to about 4.0 mg/kg, about 1.0 to about 3.5 mg/kg, about 1.0 to about 3. 0 mg/kg, about 1.0 to about 2.7 mg/kg, about 1.0 to about 2.5 mg/kg, about 1.0 to about 2.4 mg/kg, about 1.5 to about 4.5 mg/kg kg, about 1.5 to about 4.0 mg/kg, about 1.5 to about 3.5 mg/kg, about 1.5 to about 3.0 mg/kg, about 1.5 to about 2.7 mg/kg, about 1.5 to about 2.5 mg/kg, about 1.5 to about 2.4 mg/kg, about 1.5 to about 2.0 mg/kg, about 1.8 to about 4.5 mg/kg, about 1 .8 to about 4.0 mg/kg, about 1.8 to about 3.5 mg/kg, about 1.8 to about 3.0 mg/kg, about 1.8 to about 2.7 mg/kg, about 1.8 71, administered at a dose of ~ about 2.5 mg/kg, about 1.8 to about 2.4 mg/kg, or about 1.8 to about 2.0 mg/kg. Method described. 72. The method of claim 71, wherein the immunoconjugate is administered at a dose of about 1.2 to about 3.0 mg/kg. The immune complex is about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8 , about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8 , about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8 , about 3.9, or about 4.0 mg/kg. 74. The method of claim 73, wherein the immunoconjugate is administered at a dose of about 1.8, about 2.4, about 2.7, or about 3.0 mg/kg. 75. The method of claim 74, wherein the immunoconjugate is administered at a dose of about 1.8 mg/kg. 75. The method of claim 74, wherein the immunoconjugate is administered at a dose of about 2.4 mg/kg. 75. The method of claim 74, wherein the immunoconjugate is administered at a dose of about 2.7 mg/kg. 75. The method of claim 74, wherein the immunoconjugate is administered at a dose of about 3.0 mg/kg. 79. The method of any one of claims 71 to 78, wherein the human subject's body weight in kg is actual body weight. 79. The method of any one of claims 71 to 78, wherein the human subject's body weight in kg is an adjusted body weight. The body weight in kg of said human subject is
the actual weight of the human subject if the actual weight of the human subject is less than the adjusted weight of the human subject;
the actual body weight of the human subject is equal to or greater than the adjusted body weight of the human subject, and the adjusted body weight of the human subject is less than 100 kg; or the adjusted body weight of the human subject is 100 kg or more. 100kg if
79. A method according to any one of claims 71 to 78. 82. The method of any one of claims 60-81, wherein the recombinant antibody is administered to the human subject via intravenous infusion. 83. The method of any one of claims 60-82, wherein the recombinant antibody is administered to the human subject every 7 days, every 14 days, every 18 days, every 21 days, or every 30 days. 84. The method of claim 83, wherein the recombinant antibody is administered to the human subject every 21 days for at least 3 cycles. Claims 60-84, wherein the effector agent comprises a drug (or a prodrug thereof), a peptide, a protein, a detectable label, a liposome containing a drug (or a prodrug thereof), a radionuclide, a viral particle, or a chelate. The pharmaceutical composition according to any one of . 86. The method of claim 85, wherein the effector agent comprises a drug. 87. The method of claim 86, wherein the drug is an anti-cancer drug. 88. The method of claim 86 or 87, wherein the drug is a chemotherapeutic agent. 89. The method of claim 86, 87, or 88, wherein the drug is a microtubule inhibitor, a DNA damaging agent, or a polymerase inhibitor. 90. The method of claim 89, wherein the drug is a microtubule inhibitor. 91. The method of claim 90, wherein the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). The microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE), valine-citrulline MMAE (vcMMAE), or valine-citrulline MMAF (vcMMAF). 92. The method according to item 90 or 91. 93. The method of claim 92, wherein the microtubule inhibitor is monomethyl auristatin E (MMAE). 94. The method of any one of claims 60-93, wherein the ratio of said effector agent to said recombinant antibody is about 3 to about 5. 95. The method of claim 94, wherein the ratio of said effector agent to said recombinant antibody is about 4. 96. The method of any one of claims 60-95, wherein the effector agent is conjugated to the recombinant antibody via a linker. 97. The method of claim 96, wherein the linker is a peptide linker, a small molecule linker, or a linker comprising a peptide and a small molecule. 98. The method of claim 97, wherein the linker comprises maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB). 99. The method of any one of claims 60 to 98, wherein the immune complex binds to CD46 expressed on the surface of a cell and is internalized within the cell. 100. The method of claim 99, wherein the immune complex is internalized into the cell via macropinocytosis. a first heavy chain comprising SEQ ID NO: 9;
a first light chain comprising SEQ ID NO: 10;
a recombinant antibody comprising a second heavy chain comprising SEQ ID NO: 9 and a second light chain comprising SEQ ID NO: 10;
1, 2, 3, or 4 pairs of adducts for use in the treatment of cancer in a human subject comprising cells expressing CD46, the immunoconjugate comprising:
Each adduct of the 1, 2, 3, or 4 pairs of adducts is linked to the recombinant via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. comprising monomethyl auristatin E (MMAE) conjugated to an antibody;
each of the one, two, three, or four pairs of adducts is conjugated to a pair of cysteine residues of the recombinant antibody;
The pair of cysteine residues is
C219 of the first heavy chain and C214 of the first light chain,
C219 of the second heavy chain and C214 of the second light chain,
C225 of the first heavy chain and C225 of the second light chain, and C228 of the first heavy chain and C228 of the second light chain.
An immune complex selected from. 102. The immunoconjugate of claim 101, wherein the cancer is relapsed or refractory multiple myeloma (RRMM). 102. The immunoconjugate of claim 101, wherein said cancer is metastatic castration-resistant prostate cancer (mCRPC). 104. The immunoconjugate of any one of claims 101 to 103, wherein said immunoconjugate comprises two pairs of said adducts. 105. The immunoconjugate of any one of claims 101 to 104, wherein the cells contain CD46 as determined by immunofluorescence microscopy or immunohistochemical staining. 105. The immunoconjugate of any one of claims 101 to 104, wherein the cells contain CD46 as determined by flow cytometry. 105. The immune complex of any one of claims 101 to 104, wherein the cell comprises an amplification of chromosomal location 1q21. 108. The immunoconjugate of any one of claims 101 to 107, wherein the immunoconjugate is formulated for intravenous injection. 109. Any one of claims 101-108, wherein the immune complex is administered to the human subject every 7 days, every 14 days, every 18 days, every 21 days, every 28 days, or every month. The immune complex described in. 110. The immunoconjugate of claim 109, wherein the immunoconjugate is administered to the human subject every 21 days. 111. The immunoconjugate of any one of claims 101-110, wherein the immunoconjugate is administered at a dose of about 1.2 to about 3.0 mg/kg. 112. The immunoconjugate of claim 111, wherein the recombinant antibody is administered at a dose of about 1.8, about 2.4, about 2.7, or about 3.0 mg/kg. 113. The immunoconjugate according to any one of claims 111 to 112, wherein the human subject's body weight in kg is the actual body weight. 113. The immunoconjugate of any one of claims 111-112, wherein the human subject's body weight in kg is an adjusted body weight. The body weight in kg of said human subject is
the actual weight of the human subject if the actual weight of the human subject is less than the adjusted weight of the human subject;
the actual body weight of the human subject is equal to or greater than the adjusted body weight of the human subject, and the adjusted body weight of the human subject is less than 100 kg; or the adjusted body weight of the human subject is 100 kg or more. 100kg if
113. The immunoconjugate according to any one of claims 111 to 112. An immunoconjugate for the treatment of metastatic castration-resistant prostate cancer in a human subject in need of treatment, the immunoconjugate comprising:
(i) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and a light chain comprising a light chain variable region comprising LC CDR3, the recombinant antibody specifically binds to CD46, wherein said HC CDR1, HC CDR2, and HC CDR3 are SEQ ID NO: 1, SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3, and the LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and are combined with the following (ii), recombinant antibody;
(ii) monomethyl auristatin E (MMAE) via a linker comprising maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB);
An immunoconjugate administered at a dose of about 1.2 to about 3.0 mg/kg. An immunoconjugate for the treatment of refractory multiple myeloma in a human subject in need thereof, comprising:
(i) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and a light chain comprising a light chain variable region comprising LC CDR3, the recombinant antibody specifically binds to CD46, wherein said HC CDR1, HC CDR2, and HC CDR3 are SEQ ID NO: 1, SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3, and the LC CDR1, LC CDR2, and LC CDR3 each include the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and are combined with the following (ii), recombinant antibody;
(ii) monomethyl auristatin E (MMAE) via a linker comprising maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB);
An immunoconjugate administered at a dose of about 1.8 to about 3.0 mg/kg. 118. The immunoconjugate according to any one of claims 116 to 117, wherein the body weight in kg of the human subject is the actual body weight. 118. The immunoconjugate of any one of claims 116-117, wherein the human subject's body weight calculated in kg is an adjusted body weight. The body weight calculated in kg of the human subject is
the actual weight of the human subject if the actual weight of the human subject is less than the adjusted weight of the human subject;
the actual body weight of the human subject is equal to or greater than the adjusted body weight of the human subject, and the adjusted body weight of the human subject is less than 100 kg; or the adjusted body weight of the human subject is 100 kg or more. 100kg if
118. The immunoconjugate according to any one of claims 116 to 117. 121. The immunoconjugate of any one of claims 116 to 120, wherein the cells contain CD46 as determined by immunofluorescence microscopy or immunohistochemical staining. 121. The immunoconjugate of any one of claims 116 to 120, wherein the cells contain CD46 as determined by flow cytometry. 121. The immune complex according to any one of claims 116 to 120, wherein the cell comprises an amplification of chromosomal location 1q21. 124. The immunoconjugate of any one of claims 116 to 123, wherein the immunoconjugate is formulated for intravenous injection. 125. Any one of claims 116-124, wherein the immune complex is administered to the human subject every 7 days, every 14 days, every 18 days, every 21 days, every 28 days, or every month. The immune complex described in. 126. The immunoconjugate of claim 125, wherein the immunoconjugate is administered to the human subject every 21 days for at least 3 cycles. An immunoconjugate for treating cancer in a human subject in need of cancer treatment, the immunoconjugate comprising:
(a) a heavy chain variable region comprising three complementarity determining regions (CDRs), heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and a recombinant antibody that specifically binds to CD46 comprising a light chain variable region comprising LC CDR3, wherein said HC CDR1, HC CDR2, and HC CDR3 are of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. a recombinant antibody comprising an amino acid sequence, wherein said LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;
(b) an effector agent conjugated to the recombinant antibody;
An immunoconjugate administered at a dose of about 1.0 to about 5.0 mg/kg. 128. The immunoconjugate of claim 127, wherein said cancer is prostate cancer. 129. The immunoconjugate of claim 128, wherein said prostate cancer is metastatic castration-resistant prostate cancer. 128. The immunoconjugate of claim 127, wherein said cancer is multiple myeloma. 131. The immunoconjugate of claim 130, wherein the multiple myeloma is relapsed or refractory multiple myeloma. 132. The immunoconjugate of any one of claims 127-131, wherein the cancer comprises cells expressing CD46 as determined by immunofluorescence microscopy or immunohistochemical staining. 132. The immunoconjugate of any one of claims 127-131, wherein the cancer comprises cells expressing CD46 as determined by flow cytometry. 132. The immune complex of any one of claims 127-131, wherein the cell comprises an amplification of chromosomal location 1q21. 132. The immunoconjugate of any one of claims 127 to 131, wherein the cancer has higher CD46 expression than non-cancerous tissue of the same tissue type of the human subject or healthy individual. 1.0-4.5mg/kg, 1.0-4.0mg/kg, 1.0-3.5mg/kg, 1.0-3.0mg/kg, 1.0-2.7mg/kg, 1.0-2.5mg/kg, 1.0-2.4mg/kg, 1.5-4.5mg/kg, 1.5-4.0mg/kg, 1.5-3.5mg/kg, 1.5-3.0mg/kg, 1.5-2.7mg/kg, 1.5-2.5mg/kg, 1.5-2.4mg/kg, 1.5-2.0mg/kg, 1.8-4.5mg/kg, 1.8-4.0mg/kg, 1.8-3.5mg/kg, 1.8-3.0mg/kg, 1.8-2.7mg/kg, according to any one of claims 127 to 135, administered at a dose of 1.8 to 2.5 mg/kg, 1.8 to 2.4 mg/kg, or 1.8 to 2.0 mg/kg. immune complex. 137. The immunoconjugate of claim 136, administered at a dose of about 1.2 to about 3.0 mg/kg. 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2. 2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 138. The immunoconjugate of any one of claims 127-137, administered at a dose of 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 mg/kg. 139. The immunoconjugate of claim 138, administered at a dose of 1.8, 2.4, 2.7, or 3.0 mg/kg. 140. The immunoconjugate of claim 139, administered at a dose of about 1.8 mg/kg. 140. The immunoconjugate of claim 139, administered at a dose of about 2.4 mg/kg. 140. The immunoconjugate of claim 139, administered at a dose of about 2.7 mg/kg. 140. The immunoconjugate of claim 139, administered at a dose of about 3.0 mg/kg. 144. The immunoconjugate according to any one of claims 136 to 143, wherein the body weight in kg of the human subject is the actual body weight. 144. The immunoconjugate of any one of claims 136 to 143, wherein the human subject's body weight in kg is the adjusted body weight. The body weight in kg of said human subject is
the actual weight of the human subject if the actual weight of the human subject is less than the adjusted weight of the human subject;
the actual body weight of the human subject is equal to or greater than the adjusted body weight of the human subject, and the adjusted body weight of the human subject is less than 100 kg; or the adjusted body weight of the human subject is 100 kg or more. 100kg if
144. The immunoconjugate according to any one of claims 136 to 143. 147. The immunoconjugate of any one of claims 136-146, wherein said recombinant antibody is formulated for intravenous injection. 148. The immunoconjugate of any one of claims 136-147, wherein the recombinant antibody is administered to the human subject every 7 days, every 14 days, every 18 days, every 21 days, or every 30 days. body. 149. The immunoconjugate of claim 148, wherein said recombinant antibody is administered to said human subject every 21 days. Claims 136-149 wherein the effector agent comprises a drug (or a prodrug thereof), a peptide, a protein, a detectable label, a liposome containing a drug (or a prodrug thereof), a radionuclide, a viral particle, or a chelate. The immune complex according to any one of the above. 151. The immunoconjugate of claim 150, wherein the effector agent comprises a drug. 152. The immunoconjugate of claim 151, wherein the drug is an anti-cancer drug. 152. The immunoconjugate of claim 150 or 151, wherein the drug is a chemotherapeutic agent. 153. The immunoconjugate of claim 150, 151, or 152, wherein the drug is a microtubule inhibitor, a DNA damaging agent, or a polymerase inhibitor. 151. The immunoconjugate of claim 150, wherein the drug is a microtubule inhibitor. 156. The immunoconjugate of claim 155, wherein the microtubule inhibitor is auristatin (or a derivative thereof), dolastatin-10 (or a derivative thereof), or maytansine (or a derivative thereof). The microtubule inhibitor is monomethyl auristatin F (MMAF), auristatin E (AE), monomethyl auristatin E (MMAE), valine-citrulline MMAE (vcMMAE), or valine-citrulline MMAF (vcMMAF). The immune complex according to item 155 or 156. 158. The immunoconjugate of claim 157, wherein the microtubule inhibitor is monomethyl auristatin E (MMAE). 159. The immunoconjugate of any one of claims 127-158, wherein the ratio of said effector agent to said recombinant antibody is about 3 to about 5. 160. The immunoconjugate of claim 159, wherein the ratio of said effector agent to said recombinant antibody is about 4. 161. The immunoconjugate of any one of claims 127-160, wherein the effector agent is conjugated to the recombinant antibody via a linker. 162. The immunoconjugate of claim 161, wherein the linker is a peptide linker, a small molecule linker, or a linker comprising a peptide and a small molecule. 163. The immunoconjugate of claim 162, wherein the linker comprises maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB). 164. The immune complex of any one of claims 127 to 163, wherein the immune complex binds to CD46 expressed on the surface of a cell and is internalized within the cell. 165. The immune complex of claim 164, wherein the immune complex is internalized into the cell via macropinocytosis. a first heavy chain comprising SEQ ID NO: 9;
a first light chain comprising SEQ ID NO: 10;
a recombinant antibody comprising a second heavy chain comprising SEQ ID NO:9, and a second light chain comprising SEQ ID NO:10, and one, two, three, or four pairs of adducts, about 10. an immune complex at a concentration of 0±1.0 mg/ml,
Each adduct of the 1, 2, 3, or 4 pairs of adducts is linked to the recombinant via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. comprising monomethyl auristatin E (MMAE) conjugated to an antibody;
each of the one, two, three, or four pairs of adducts is conjugated to a pair of cysteine residues of the recombinant antibody;
The pair of cysteine residues is
C219 of the first heavy chain and C214 of the first light chain,
C219 of the second heavy chain and C214 of the second light chain,
C225 of the first heavy chain and C225 of the second light chain, and C228 of the first heavy chain and C228 of the second light chain.
an immune complex selected from;
about 20 mM histidine buffer at pH 6.0;
Approximately 8.0% sucrose and
about 0.01% polysorbate 80 for the treatment of metastatic castration-resistant prostate cancer in a human subject in need thereof. a first heavy chain comprising SEQ ID NO: 9;
a first light chain comprising SEQ ID NO: 10;
a recombinant antibody comprising a second heavy chain comprising SEQ ID NO:9, and a second light chain comprising SEQ ID NO:10, and one, two, three, or four pairs of adducts, about 10. an immune complex at a concentration of 0±1.0 mg/ml,
Each adduct of the 1, 2, 3, or 4 pairs of adducts is linked to the recombinant via a maleimidocaproyl-valine-citrulline-para-aminobenzyloxycarbonyl (mc-vc-PAB) linker. comprising monomethyl auristatin E (MMAE) conjugated to an antibody;
each of the one, two, three, or four pairs of adducts is conjugated to a pair of cysteine residues of the recombinant antibody;
The pair of cysteine residues is
C219 of the first heavy chain and C214 of the first light chain,
C219 of the second heavy chain and C214 of the second light chain,
C225 of the first heavy chain and C225 of the second light chain, and C228 of the first heavy chain and C228 of the second light chain.
an immune complex selected from;
about 20 mM histidine buffer at pH 6.0;
Approximately 8.0% sucrose and
about 0.01% polysorbate 80 for the treatment of refractory multiple myeloma in a human subject in need thereof.