Anti-mesothelin antibodies and antibody-drug conjugates thereof

Abstract

The present invention discloses an antibody-drug conjugate that targets MSLN. The present invention also discloses a method for producing the antibody-drug conjugate (ADC). The present invention further discloses a novel MSLN antibody or functional fragment thereof, comprising an engineered heavy chain and a light chain.

Description

[Technical Field]

[0001] Technical Field The present invention relates to an anti-mesothelin antibody or a functional fragment thereof. The present invention also relates to an antibody-drug conjugate comprising an anti-mesothelin antibody and a small molecule drug. The present invention further relates to the use of the antibody and conjugate of the present invention in the manufacture of a medicament for the treatment of tumors. [Background technology]

[0002] Background technology MSLN (mesothelin) is an antigen recognized by the monoclonal antibody CAK1 in mesothelial cells, mesothelioma, and ovarian cancer. It is a 40 kDa cell surface glycoprotein that is highly expressed in many tumor tissues, making it an excellent target marker for therapeutic antibodies.

[0003] Although monoclonal antibodies have high therapeutic target specificity and low side effects, their effectiveness is limited when used alone. Antibody-drug conjugates, formed by linking toxins and antibodies via linkers, have both strong targeting ability and highly efficient cytotoxicity. The use of new ADC drugs has become one of the most promising immunotherapies, and cancer immunotherapy has attracted much attention.

[0004] Although several research groups have constructed antibody-drug conjugates targeting MSLN, these antibody-drug conjugates still have many drawbacks. For example, coupling through the thiol group of antibody cysteine causes the loss of the original disulfide bond between the peptide chains, making the resulting ADC unstable. Once the ADC enters the circulation, its half-life is short and its toxic side effects are severe. Coupling through the amino group of antibody lysine randomizes the coupling site, which may affect the targeting ability of the antibody. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there remains a need in the art to develop MSLN antibodies and antibody-drug conjugates comprising the antibodies with better properties. [Means for solving the problem]

[0006] Contents of the present invention The present invention provides an anti-mesothelin antibody or a functional fragment thereof, and an antibody-drug conjugate comprising the antibody or the functional fragment thereof. In particular, the antibody-drug conjugate of the present invention has high stability, few toxic side effects, and / or high affinity for mesothelin.

[0007] In detail: In one aspect, the present invention provides an antibody drug conjugate having the following structural formula: Ab-(LD) n wherein Ab is an antibody or functional fragment thereof that specifically binds to MSLN; L is empty or an optional linker; D is any therapeutic agent; Antibody drug conjugates are provided wherein n is an integer selected from 1 to 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8, or the interval between any two thereof.

[0008] Furthermore, the antibody or functional fragment thereof of the present invention comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises at least three CDR regions, and the amino acid sequence of at least one of the CDR regions has the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, or a sequence having at least 80% (preferably, 85%, 90%, 95%, 98%, or 99%) sequence identity thereto, and (ii) the light chain comprises at least three CDR regions, and the amino acid sequence of at least one of the CDR regions has the amino acid sequence set forth in SEQ ID NO: 4, 5, or 6, or a sequence having at least 80% (preferably, 85%, 90%, 95%, 98%, or 99%) sequence identity thereto. Preferably, the antibody or functional fragment thereof of the present invention comprises a heavy chain and a light chain, wherein (i) the heavy chain variable region comprises three CDR regions, and the CDR regions have the amino acid sequences set forth in SEQ ID NO: 1, 2, or 3, respectively, and / or (ii) the light chain variable region comprises three CDR regions, and the CDR regions have the amino acid sequences set forth in SEQ ID NO: 4, 5, or 6, respectively. Most preferably, the CDRs of the heavy chain of the anti-mesothelin antibody disclosed in the present invention have the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the CDRs of the light chain variable region thereof have the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0009] The present invention further provides an anti-mesothelin antibody or functional fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 or a sequence having at least 80% (preferably, 85%, 90%, 95%, 98%, or 99%) sequence identity thereto, and (ii) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 or a sequence having at least 80% (preferably, 85%, 90%, 95%, 98%, or 99%) sequence identity thereto. Preferably, the antibody comprises a heavy chain and a light chain, wherein (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 and / or (ii) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. Most preferably, the heavy chain variable region of the anti-mesothelin antibody disclosed in the present invention comprises the amino acid sequence set forth in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0010] In certain particular embodiments, the antibodies of the invention comprise a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:10 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:9.

[0011] In another aspect, the present invention provides an antibody-drug conjugate comprising an anti-mesothelin antibody or functional fragment thereof and a therapeutic agent. Preferably, the antibody-drug conjugate of the present invention further comprises a linker connecting the anti-mesothelin antibody or functional fragment thereof to the therapeutic agent.

[0012] In certain embodiments, the linker L of the present invention can be linked to the antibody by any means known in the art, preferably via a thiol group and / or an amino group. In a preferred embodiment, the antibody of the present invention is linked to the linker via a thiol group on the antibody. The linker L of the present invention can be absent (i.e., the antibody is directly linked to the therapeutic agent D) or can be any one or combination of cleavable (i.e., a linker that cleaves in an in vivo environment) or non-cleavable linkers; preferably, the linker can be selected from the linkers listed in Table 1 below.

[0013] [Table 1]

[0014] In some embodiments, the linkers of the present invention are preferably selected from the linkers listed in Table 2 below.

[0015] [Table 2]

[0016] In some embodiments of the invention, the therapeutic agent D is selected from the group consisting of maytansine compounds, V-ATPase inhibitors, pro-apoptotic agents, Be12 inhibitors, McL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTOr inhibitors, microtubule stabilizing agents, microtubule destabilizing agents, auristatins, dolastatins, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, inhibitors of mitochondrial phosphotransfer reactions, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA interfering agents, DNA minor groove binders, DHFR inhibitors and dolastatin peptides.

[0017] In some preferred embodiments of the present invention, the therapeutic agent D is a cytotoxic substance (e.g., antimetabolite, antitumor antibiotic, alkaloid), an immunopotentiator, or a radioisotope. Preferably, the therapeutic agent D can be selected from the group consisting of MMAD (monomethylauristatin D) and its derivatives, MMAE (monomethylauristatin E) and its derivatives, MMAF (monomethylauristatin F) and its derivatives, mertansine derivative M1, mertansine derivative M4, duocarmycin, calicheamicin, PBDA (pyrrolobenzodiazepine), doxorubicin, vinca alkaloids, methotrexate, vinblastine, and daunorubicin. More preferably, the therapeutic agent is selected from maytansinoids (e.g., ansamitocin or mertansine), dolastatins and their derivatives. Most preferably, the therapeutic agent is selected from the group consisting of MMAD and MMAE.

[0018] In certain embodiments, the present invention provides compounds of the general formula Ab-(LD) n wherein Ab is any anti-mesothelin antibody of the invention, L is selected from the group consisting of Py-MAA-Val-Cit-PAB, Mc-Val-Cit-PAB, D is selected from MMAD or MMAE, and n is an integer selected from 1 to 8, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or an interval between any two thereof.

[0019] In certain particular embodiments, an antibody drug conjugate of the invention has a structure shown in any of the following formulas, wherein n is 1, 2, 3, 4, 5, 6, 7, or 8:

[0020] [ka]

[0021] In particular, the present invention provides compounds of the general formula Ab-(LD) n wherein Ab is any anti-mesothelin antibody of the invention, the antibody heavy chain variable region CDRs have the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the antibody light chain variable region CDRs have the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, L is Py-MAA-Val-Cit-PAB, and D is MMAE. More specifically, the antibody drug conjugate of the invention is RC88-Py-MAA-Val-Cit-PAB-MMAE, having the structure shown by the following formula, where n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0022] [ka]

[0023] In particular, the present invention provides compounds of the general formula Ab-(LD) n wherein Ab is any anti-mesothelin antibody of the invention, the antibody heavy chain variable region CDRs have the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the antibody light chain variable region CDRs have the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, L is Py-MAA-Val-Cit-PAB, and D is MMAD. More specifically, the antibody drug conjugate of the invention is RC88-Py-MAA-Val-Cit-PAB-MMAD, having the structure shown by the following formula, where n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0024] [ka]

[0025] In particular, the present invention provides compounds of the general formula Ab-(LD) n wherein Ab is any anti-mesothelin antibody of the present invention, the antibody heavy chain variable region CDRs have the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the antibody light chain variable region CDRs have the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, L is Mc-Val-Cit-PAB, and D is MMAE. More specifically, the antibody drug conjugate of the present invention is RC88-Mc-Val-Cit-PAB-MMAE, having the structure shown by the following formula, where n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0026] [ka]

[0027] In particular, the present invention provides compounds of the general formula Ab-(LD) n wherein Ab is any anti-mesothelin antibody of the present invention, the antibody heavy chain variable region CDRs have the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the antibody light chain variable region CDRs have the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, L is Mc-Val-Cit-PAB, and D is MMAD. More specifically, the antibody drug conjugate of the present invention is RC88-Mc-Val-Cit-PAB-MMAD, having the structure shown by the following formula, where n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0028] [ka]

[0029] In particular, the present invention provides compounds of the general formula Ab-(LD) n wherein Ab is any anti-mesothelin antibody of the invention, the antibody having a heavy chain variable region sequence set forth in SEQ ID NO:7 and a light chain variable region sequence set forth in SEQ ID NO:8, L is Py-MAA-Val-Cit-PAB, and D is MMAE. More particularly, the antibody drug conjugate of the invention is RC88-Py-MAA-Val-Cit-PAB-MMAE, having the structure shown by the formula below, where n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0030] [ka]

[0031] In particular, the present invention provides compounds of the general formula Ab-(LD) n wherein Ab is any anti-mesothelin antibody of the invention, the antibody having a heavy chain variable region sequence set forth in SEQ ID NO:7 and a light chain variable region sequence set forth in SEQ ID NO:8, L is Py-MAA-Val-Cit-PAB, and D is MMAD. More particularly, the antibody drug conjugate of the invention is RC88-Py-MAA-Val-Cit-PAB-MMAD, having the structure shown by the formula below, where n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0032] [ka]

[0033] In particular, the present invention provides compounds of the general formula Ab-(LD) nwherein Ab is any anti-mesothelin antibody of the invention, the antibody having a heavy chain variable region sequence set forth in SEQ ID NO: 7 and a light chain variable region sequence set forth in SEQ ID NO: 8, L is Mc-Val-Cit-PAB, and D is MMAE. More particularly, the antibody drug conjugate of the invention is RC88-Mc-Val-Cit-PAB-MMAE, having the structure shown by the following formula, where n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0034] [ka]

[0035] In particular, the present invention provides compounds of the general formula Ab-(LD) n wherein Ab is any anti-mesothelin antibody of the invention, the antibody having a heavy chain variable region sequence set forth in SEQ ID NO:7 and a light chain variable region sequence set forth in SEQ ID NO:8, L is Mc-Val-Cit-PAB, and D is MMAD. More particularly, the antibody drug conjugate of the invention is RC88-Mc-Val-Cit-PAB-MMAD, having the structure shown by the following formula, where n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0036] [ka]

[0037] In another aspect, the present invention provides an antibody or functional fragment thereof capable of binding to mesothelin, the antibody or functional fragment thereof comprising a heavy chain and a light chain; (i) the heavy chain comprises at least three CDR regions, and the amino acid sequence of at least one of the CDR regions has the amino acid sequence set forth in SEQ ID NO: 1, 2 or 3, or a sequence having at least 80% (preferably 85%, 90%, 95%, 98% or 99%) sequence identity thereto; and / or (ii) The light chain comprises at least three CDR regions, and the amino acid sequence of at least one of the CDR regions has the amino acid sequence set forth in SEQ ID NO: 4, 5 or 6, or a sequence having at least 80% (preferably 85%, 90%, 95%, 98% or 99%) sequence identity thereto.

[0038] In certain specific embodiments, the anti-mesothelin antibodies or functional fragments thereof of the invention comprise a heavy chain and a light chain, (i) the heavy chain variable region comprises three CDR regions, each having the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3; and / or (ii) The light chain variable region comprises three CDR regions, each having the amino acid sequence set forth in SEQ ID NO: 4, 5, or 6.

[0039] In particular, the amino acid sequences of the heavy chain CDR regions of the anti-mesothelin antibodies disclosed in the present invention are set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0040] In particular, the amino acid sequences of the light chain CDR regions of the anti-mesothelin antibodies disclosed in the present invention are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0041] More specifically, in the anti-mesothelin antibodies disclosed in the present invention, the amino acid sequences of their heavy chain CDR regions are set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the amino acid sequences of their light chain variable region CDR regions are set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0042] In a further aspect, the present invention provides an anti-mesothelin antibody or functional fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain and the light chain comprise a heavy chain variable region and a light chain variable region, respectively; (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, or a sequence having at least 80% (preferably, 85%, 90%, 95%, 98% or 99%) sequence identity thereto; (ii) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 or a sequence having at least 80% (preferably 85%, 90%, 95%, 98% or 99%) sequence identity thereto;

[0043] In certain particular embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain and the light chain comprising a heavy chain variable region and a light chain variable region, respectively; (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, and / or (ii) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8;

[0044] In particular, the anti-mesothelin antibodies of the present invention comprise a heavy chain and a light chain, wherein the heavy chain and the light chain comprise the amino acid sequences set forth in SEQ ID NO:10 and SEQ ID NO:9, respectively.

[0045] In certain particular embodiments, the anti-mesothelin antibody or functional fragment thereof is isolated.

[0046] In certain specific embodiments, the anti-mesothelin antibody or functional fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a single-chain antibody (scFv) or a bispecific antibody; in certain specific embodiments, the anti-mesothelin antibody or functional fragment thereof is a monoclonal antibody; in certain specific embodiments, the anti-mesothelin antibody or functional fragment thereof is a humanized antibody; and in certain specific embodiments, the anti-mesothelin antibody or functional fragment thereof is an IgG1 kappa antibody.

[0047] In a further aspect, the invention provides an isolated polynucleotide encoding an antibody of the invention.

[0048] In a further aspect, the present invention provides a combination of isolated polynucleotides, the combination comprising a polynucleotide encoding the light chain of the antibody or functional fragment thereof of the present invention and a polynucleotide encoding the heavy chain of the antibody or functional fragment thereof of the present invention.

[0049] In a further aspect, the present invention provides an expression vector or a combination of expression vectors, comprising a polynucleotide according to the invention or a combination of polynucleotides according to the invention, wherein the polynucleotide is operably linked to control sequences in a host cell or a cell-free expression system allowing expression of a polypeptide encoded by the polynucleotide.

[0050] In a further aspect, the present invention provides a pharmaceutical composition comprising an antibody or functional fragment thereof according to the present invention and / or a conjugate according to the present invention and a pharmaceutically acceptable carrier.

[0051] In a further aspect, the present invention provides a method for treating or preventing cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, polynucleotide, polynucleotide combination, expression vector, conjugate and / or pharmaceutical composition according to the invention.

[0052] In a further aspect, the present invention provides the use of an antibody, polynucleotide, polynucleotide combination, expression vector, conjugate and / or pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment or prevention of cancer.

[0053] In a further aspect, the present invention provides an antibody, polynucleotide, polynucleotide combination, expression vector, conjugate and / or pharmaceutical composition according to the invention for use in the treatment or prevention of cancer.

[0054] In a further aspect, the invention provides the use of the antibody drug conjugate of any one of the above embodiments in the manufacture of a medicament for the treatment of cancer.

[0055] In certain particular embodiments, the cancer of the present invention is a mesothelin-positive cancer. [Brief explanation of the drawings]

[0056] [Figure 1]FIG. 1 shows SDS-PAGE analysis of RC88-PY-MAA-Val-Cit-PAB-MMAE and RC88-Mc-Val-Cit-PAB-MMAE, which are characterized by coupling of RC88 antibody with a linker to a drug conjugate. [Figure 2]

[0023] Figures showing the coupling of the conjugates of the present invention, where Figure A shows the results of the detection of the coupling of RC88-PY-MAA-Val-Cit-PAB-MMAE by hydrophobic high-performance liquid chromatography (HIC-HPLC), and Figure B shows the results of the detection of the coupling of RC88-Mc-Val-Cit-PAB-MMAE by hydrophobic high-performance liquid chromatography (HIC-HPLC). [Figure 3]

[0023] Figures showing the cytotoxic effects of the conjugates of the present invention. Figure A shows the cytotoxic effect curves of the RC88 antibody-drug conjugates of the present invention (i.e., RC88-Py-MAA-Val-Cit-PAB-MMAE, RC88-Py-MAA-Val-Cit-PAB-MMAD, RC88-Mc-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAD) in Oval-Citar-3 cells highly expressing MSLN. Figure B shows the cytotoxic effect curves of the conjugates of the present invention in Oval-Citar-3 cells highly expressing MSLN. Figure 1 shows the cytotoxicity curves of the unmodified antibody linker and cytotoxin conjugates (Py-Val-Cit-PAB-MMAE, Py-Val-Cit-PAB-MMAD, Mc-Val-Cit-PAB-MMAE, Mc-Val-Cit-PAB-MMAD). Figure 2 shows the cytotoxicity curves of the cytotoxins MMAE, MMAD, and the positive control PTX (paclitaxel) in Oval-Citar-3 cells, which highly express MSLN. The horizontal axis represents the logarithmic drug concentration, and the vertical axis represents the maximum inhibition rate at the corresponding logarithmic drug concentration. [Figure 4]This graph shows the weight of tumor-bearing mice as a function of days. Mice were administered RC88 antibody (2 mg / kg), RC88 antibody-drug conjugates (RC88-Py-MAA-Val-Cit-PAB-MMAD, RC88-Py-MAA-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAD, 2 mg / kg), and MMAE (0.0716 mg / kg) once a week for a total of three doses. The horizontal axis represents the number of days, and the vertical axis represents the weight of tumor-bearing mice after the corresponding number of days of administration. In this study, the control groups were saline (control) and MMAE. [Figure 5] Graph showing tumor volume in tumor-bearing mice as a function of days. Mice were administered RC88 antibody (2 mg / kg), RC88 antibody-drug conjugates (RC88-Py-MAA-Val-Cit-PAB-MMAD, RC88-Py-MAA-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAD, 2 mg / kg), and MMAE (0.0716 mg / kg) once a week for a total of three doses. The horizontal axis represents the number of days, and the vertical axis represents the tumor volume in tumor-bearing mice after the corresponding number of days of administration. In this study, the control groups were saline (control) and MMAE. [Figure 6] Graph showing tumor weights in tumor-bearing mice treated with RC88 antibody (2 mg / kg), RC88 antibody-drug conjugates (RC88-Py-MAA-Val-Cit-PAB-MMAD, RC88-Py-MAA-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAD, 2 mg / kg), and MMAE (0.0716 mg / kg) once a week for a total of three doses; control groups were saline (control) and MMAE. [Figure 7]This graph shows the weight of tumor-bearing mice over time. Mice were administered RC88 antibody (3 mg / kg), RC88 antibody-drug conjugates (RC88-Py-MAA-Val-Cit-PAB-MMAE, 3 mg / kg, 1.5 mg / kg, and 0.75 mg / kg), MMAE (0.06 mg / kg), RC88 antibody (3 mg / kg) + MMAE (0.06 mg / kg), IgG-MMAE (3 mg / kg), or PTX (paclitaxel, 10 mg / kg). PTX was administered twice weekly for a total of six doses, while the other drugs were administered once weekly for a total of three doses. The horizontal axis represents the number of days, and the vertical axis represents the weight of tumor-bearing mice after the corresponding number of doses. In this study, the control groups were saline (control) and MMAE. [Figure 8] Graph showing tumor volume in tumor-bearing mice as a function of days. Mice were administered RC88 antibody (3 mg / kg), RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE, 3 mg / kg, 1.5 mg / kg, 0.75 mg / kg), MMAE (0.06 mg / kg), RC88 antibody (3 mg / kg) + MMAE (0.06 mg / kg), IgG-MMAE (3 mg / kg), and PTX (paclitaxel, 10 mg / kg) (total tumor volumes in tumor-bearing mice administered drugs once a week for three doses (PTX was administered twice a week for a total of six doses, while the others were administered once a week for a total of three doses). The horizontal axis represents the number of days, and the vertical axis represents the tumor volume in tumor-bearing mice after the corresponding number of doses. In this study, the control groups were saline (control) and MMAE. [Figure 9] This shows the antitumor effect of the RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE) in a mouse model bearing Oval-Citar-3 human ovarian cancer, which highly expresses MSLN. [Figure 10] 1 shows the affinity curves of the RC88 antibody and the RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE) for MSLN-positive tumor cells as measured by ELISA. [Figure 11]Figures showing the affinity of the conjugates of the present invention for their targets. Figure A shows the affinity curves of the RC88 antibody and the RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE) for MSLN-positive tumor cells as measured by ELISA, and Figure B shows the competitive binding curves of the RC88 antibody and the RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE) for recombinant human MSLN protein. [Figure 12] 1 shows the competitive binding curves of RC88 antibody and RC88 antibody drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE) and CA125 to recombinant human MSLN protein. DETAILED DESCRIPTION OF THE INVENTION

[0057] Specific Model for Implementing the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those understood by those skilled in the art.For specific definitions and terminology in the art, practitioners can refer to Current Protocols in Molecular Biology (Ausubel).Abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art, which represent one of the 20 commonly used L-amino acids.

[0058] Although the present invention provides broad numerical ranges and approximate parameters, the numerical values in specific embodiments are described as precisely as possible. However, since standard deviations exist in each measurement, any numerical values inherently contain certain errors. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges. For example, a recited range of "1 to 10" should be considered to encompass any and all subranges between the minimum of 1 and the maximum of 10 (inclusive), i.e., all subranges starting at a minimum of 1 or more, e.g., 1 to 6.1, and ending at a maximum of 10 or less, e.g., 5.5 to 10. Furthermore, any reference that states "incorporated herein" is understood to be incorporated in its entirety.

[0059] It is further noted that, as used in the description, the singular form of a referenced object encompasses its plural form unless it is clearly and unambiguously limited to one of the objects. The term "or" can be used synonymously with the term "and / or" unless the context clearly indicates otherwise.

[0060] As used herein, the term "mesothelin," also known as MSLN, refers to any naturally occurring mature mesothelin obtained from cellular processing of the mesothelin precursor protein. The term encompasses mesothelin from any vertebrate source, including mammals such as primates (e.g., humans, apes, and monkeys) and rodents (e.g., mice and rats), unless otherwise specified, and also encompasses any naturally occurring variants, such as splice variants or allelic variants of mesothelin.

[0061] As used herein, the terms "pharmaceutical composition," "drug combination," and "drug combination" are used interchangeably and refer to at least one drug and, optionally, a pharmaceutically acceptable carrier or excipient combined together to achieve a specific purpose. In certain embodiments, a pharmaceutical composition includes combinations that are separated in time and / or space, so long as they can act together to achieve the purpose of the invention. For example, the components contained in the pharmaceutical composition (e.g., antibodies, nucleic acid molecules, nucleic acid molecule combinations, and / or conjugates according to the present invention) can be administered to a subject together or separately. When the components contained in the pharmaceutical composition are administered separately to a subject, the components can be administered to a subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffer solution), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or a polyalkylene glycol such as polypropylene glycol, a triglyceride, or the like. The type of pharmaceutically acceptable carrier used will depend inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the invention may also contain additives such as wetting agents, emulsifiers or buffer substances.

[0062] The pharmaceutical composition, vaccine or pharmaceutical preparation according to the invention may be administered via any suitable route, for example oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.

[0063] As used herein, the term "therapeutic agent" means any substance or entity that can exert a therapeutic effect (e.g., treating, preventing, ameliorating, or inhibiting any disease and / or disorder), including, but not limited to, chemotherapeutic agents, radiotherapeutic agents, immunotherapeutic agents, thermotherapeutic agents, and the like.

[0064] As used herein, "CDR region" or "CDR" refers to the hypervariable regions of immunoglobulin heavy and light chains as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th ed., USDapartment of Health and Human Services, NIH, 1991 and later versions). There are three heavy chain CDRs and three light chain CDRs. As used herein, the term CDR or CDRs is used to refer to one of these regions or even some or all of these regions, which contain most of the amino acid residues involved in affinity binding of the antibody to the antigen or its recognized epitope.

[0065] For purposes of the present invention, "identity," "identity," or "similarity" between two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or identical amino acid residues between the two sequences to be compared after optimal alignment, and this percentage is purely statistical, meaning that the differences between the two sequences are randomly distributed and throughout their length. Sequence comparison between two nucleic acid or amino acid sequences is typically performed by comparing the sequences after optimal alignment, and the comparison can be performed over a region or "comparison window." In addition to being performed manually, optimal alignment for comparing sequences can also be performed using the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], the local homology algorithm of Neddleman and Wunsch (1970) [J. MoI. Biol. 48:443], the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444], or computer software (GAP, BESTFIT, FASTA, and TFASTA, or BLAST N or BLAST P comparison software from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).

[0066] As used herein, "therapeutically effective amount" or "effective amount" refers to a dose that is sufficient to show its benefit to the subject to which it is administered. The actual amount to be administered and the rate and time course of administration depend on the condition and severity of the subject to be treated. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of general practitioners and other physicians, and is up to the physician's decision, usually taking into account the disease to be treated, the condition of each individual patient, delivery site, administration method and other known factors.

[0067] As used herein, the term "subject" refers to a mammal, such as a human, but may also be other animals, such as wild animals (e.g., herons, storks, cranes), domestic animals (e.g., ducks, geese), or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, marmots, squirrels).

[0068] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. In particular, as used herein, "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains (CH1, CH2, and CH3). Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region contains one domain (CL). The VH and VL regions can be further subdivided into multiple regions of high diversity, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain the binding domains that interact with antigens. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or elements, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system. Chimeric or humanized antibodies are also encompassed by the antibodies of the present invention.

[0069] The term "humanized antibody" refers to an antibody containing CDR regions derived from a non-human antibody, while the remaining portions of the antibody molecule are derived from one (or several) human antibodies. Furthermore, some residues in the framework region (referred to as FR) can be modified to maintain binding affinity (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al., Nature, 332:323-327, 1988). Humanized antibodies or fragments thereof according to the present invention can be prepared by techniques known to those skilled in the art (e.g., as described in the following references: Singer et al., J. Immun. 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev. 10:1-142, 1992; or Bebbington et al., Bio / Technology 10:169-175, 1992).

[0070] The term "chimeric antibody" refers to an antibody in which the variable region sequences of the antibody are derived from one species and the constant region sequences of the antibody are derived from another species, e.g., the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. Chimeric antibodies or fragments thereof according to the present invention can be prepared using genetic engineering techniques. For example, chimeric antibodies can be generated by cloning recombinant DNA containing a promoter, sequences encoding the variable regions of a non-human, particularly mouse, monoclonal antibody according to the present invention, and sequences encoding the constant regions of a human antibody. Chimeric antibodies of the present invention encoded by such recombinant genes may be, for example, mouse-human chimeras, whose specificity is determined by the variable regions derived from mouse DNA and whose isotype is determined by the constant regions derived from human DNA. For methods of preparing chimeric antibodies, see, for example, Verhoeyn et al. (BioEssays, Vol. 8:74, 1988).

[0071] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition, which displays a single binding specificity and affinity for a particular epitope.

[0072] As used herein, the term "functional fragment" refers to an antibody fragment consisting of or comprising a subsequence of the heavy or light variable chain of the antibody from which the functional fragment is derived, which subsequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which the functional fragment is derived, preferably exhibiting an affinity at least 1 / 100th that of the antibody from which the functional fragment is derived, and more preferably exhibiting an affinity at least 1 / 10th that of the antibody from which the functional fragment is derived. Such functional fragments contain a minimum of 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of the antibody sequence from which the functional fragment is derived.

[0073] As used herein, the term "DAR" refers to the drug-antibody ratio in an antibody-drug conjugate and represents the average number of drug molecules conjugated to one antibody. Preferably, the antibody-drug conjugates of the present invention have a DAR value of about 2 to about 6, for example, about 2, 2.5, 3, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 5, 5.5, 6, or any interval therebetween.

[0074] In general, to prepare monoclonal antibodies or functional fragments thereof, particularly murine monoclonal antibodies or functional fragments thereof, reference can be made to the techniques specifically described in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 726, 1988) or the technique of preparation from hybridoma cells described by Kohler and Milstein (Nature, Vol. 256: 495-497, 1975).

[0075] The monoclonal antibodies or antibody-drug conjugates of the present invention can be purified, for example, by affinity column purification, to which the MSLN antigen or one of its fragments containing the epitope specifically recognized by the antibody of the present invention has been pre-immobilized. More specifically, the monoclonal antibodies can be purified by protein A and / or G chromatography, with or without ion exchange chromatography to remove remaining protein contaminants, DNA, and LPS, with or without Sepharose gel exclusion chromatography to remove potential aggregates due to the presence of dimers or other multimers. In a more preferred embodiment, both of these techniques can be used simultaneously or sequentially.

[0076] As used herein, the term "dolastatin" refers to polypeptides isolated from the marine organism Dollabella auricularia, including, but not limited to, dolastatin 10 and dolastatin 15. Dolastatin peptides are mitotic inhibitors that exhibit potent anticancer activity and are therefore potential anticancer drugs. Researchers have further discovered and synthesized several derivatives of dolastatin peptides, such as MMAE and MMAF.

[0077] As used herein, the term "linker" refers to the portion of an antibody-drug conjugate (i.e., ADC) that connects the antibody to the drug and may be cleavable or non-cleavable. A cleavable linker (i.e., a cleavable or biodegradable linker) can be cleaved in or on a target cell to release the drug. In certain embodiments, the linkers of the present invention have excellent stability, significantly reducing drug release during delivery to the target (e.g., in the blood), thereby reducing side effects and toxicity. In some specific embodiments, the linkers of the present invention are selected from cleavable linkers such as disulfide-based linkers (which are selectively cleaved in tumor cells with a high concentration of sulfhydryl groups), peptide linkers (which are cleaved by enzymes in tumor cells), and hydrazone linkers. In other specific embodiments, the linkers of the present invention are selected from non-cleavable linkers (i.e., linkers that cannot be cleaved), such as thioether linkers. In still other embodiments, the linkers of the present invention are a combination of cleavable and non-cleavable linkers. Preferably, the linker of the present invention is selected from the group consisting of Mc-Val-Cit-PAB and Py-MAA-Val-Cit-PAB.

[0078] Anti-MSLN antibody The antibody of the antibody-drug conjugate of the present invention is characterized by specifically binding to human MSLN. Preferably, the antibody binds with high affinity, for example, at a concentration of 1×10 -7 K below M D The anti-MSLN antibody preferably exhibits one or more of the following properties: (a) 1 × 10 human MSLNs -7 M or less (e.g., 5 × 10 -8 M or less, 2×10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less) K D Combine with; (b) Oval-Citar-3 cells highly expressing MSLN are treated with an EC of, for example, 2000 ng / ml or less (e.g., 1000 ng / ml or less, 500 ng / ml or less, 400 ng / ml or less, 300 ng / ml or less, 250 ng / ml or less, 200 ng / ml or less, 150 ng / ml or less, 100 ng / ml or less, 50 ng / ml or less, 40 ng / ml or less, 30 ng / ml or less, 20 ng / ml or less, 10 ng / ml or less, or 5 ng / ml or less). 50 Preferably, EC 50 is determined by using flow cytometry or ELISA; and (c) inhibiting the in vivo proliferation of cells expressing MSLN;

[0079] Monoclonal antibody RC88 The antibody preferably used in the antibody-drug conjugate of the present invention is the human monoclonal antibody RC88. The amino acid sequences of VH and VL of RC88 are shown in SEQ ID NOs: 7 and 8, respectively.

[0080] In another embodiment, the antibody of the present invention can comprise the heavy and light chain CDR1, CDR2, and CDR3 of RC88, or a combination thereof. The amino acid sequences of VHCDR1, VHCDR2, and VHCDR3 of RC88 are set forth in SEQ ID NOS: 1 to 3, respectively. The amino acid sequences of VLCDR1, VLCDR2, and VLCDR3 of RC88 are set forth in SEQ ID NOS: 4 to 6, respectively. CDR regions are described using the Kabat system (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication NO: 91-3242; hereinafter referred to as "Kabat '3242"). [Example]

[0081] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments may be practiced in light of the above definitions and general description.

[0082] Example 1: Anti-mesothelin antibodies To generate anti-mesothelin antibodies of the present invention, immunized animals were prepared using standard methods, e.g., as described in Kohler & Milstein (1975) Nature 256:495-497, Kozbor et al. (1983) Immunol. Today 4:72, and Cole et al., in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp. 77-96.

[0083] Mesothelin was isolated from cells and purified by known techniques and used as an immunogen for animal immunization, with reference to, for example, Zola, MONOCLONAL ANTIBODIES: PREPARATION AND USE OF MONOCLONAL ANTIBODIES AND ENGINEERED ANTIBODY DERIVATIVES (BASICS: FROM BACKGROUND TO BENCH), Springer-Verlag Ltd., New York, 2000; BASIC METHODS IN ANTIBODY PRODUCTION AND CHARACTERIZATION, Chapter 11, "Antibody Purification Methods," edited by Howard and Bethell, CRC Press, 2000; ANTIBODY ENGINEERING (SPRINGER LAB MANUAL), edited by Kontermann and Dubel, Springer-Verlag, 2001.

[0084] Splenocytes were harvested from the immunized animals and fused with a myeloma cell line to generate hybridomas, and anti-MSLN antibodies with high binding affinity were then screened.

[0085] A murine anti-MSLN monoclonal antibody was humanized by grafting the light or heavy chain CDRs onto human IgG1 or heavy chain framework regions. The CDRs of the murine anti-MSLN antibody light and heavy chains were determined using the Kabat system. By aligning antibody variable region databases, human IgG1 framework regions with high homology to the murine MSLN antibody were identified. Therefore, various humanized MSLN antibody light chain variable region sequences and humanized anti-MSLN heavy chain variable region sequences were designed. Based on this design, humanized heavy and light chain variable region sequences were synthesized. To obtain a full-length humanized MSLN light chain, the humanized MSLN antibody light chain variable region was fused to a human kappa constant region by PCR, and to obtain a full-length humanized MSLN heavy chain, the humanized MSLN heavy chain variable region was fused to an IgG constant region by PCR. Various light and heavy chains were expressed together, and the purified humanized antibodies were compared to the human-mouse chimeric antibody by ELISA binding affinity, and a candidate humanized antibody (designated RC88 antibody) was obtained by screening.

[0086] Table 3 below shows the CDR amino acid sequences of the RC88 antibody light and heavy chains.

[0087] [Table 3]

[0088] Amino acid sequence of the RC88 antibody heavy chain variable region (SEQ ID NO:7):

[0089] [ka]

[0090] Amino acid sequence of the RC88 antibody light chain variable region (SEQ ID NO: 8):

[0091] [ka]

[0092] The amino acid sequences of the RC88 antibody light and heavy chains are set forth in SEQ ID NO:9 and SEQ ID NO:10.

[0093] Amino acid sequence of RC88 antibody light chain (SEQ ID NO: 9):

[0094] [ka]

[0095] Amino acid sequence of RC88 antibody heavy chain (SEQ ID NO: 10):

[0096] [ka]

[0097] Example 2: Preparation of Antibody Drug Conjugates (ADCs) Example 2a: Preparation of Linker-Drug Conjugates

[0098] [ka] TIFF0007718816000018.tif151168

[0099] (1) Synthesis of Compound 1 (Py-MAA-Val-Cit-PAB-OH) Compound Py-MAA (1,3,5-triacryloylhexahydro-1,3,5-triazine-mercaptoacetic acid, 10.00 g, 29.3 mmol) was dissolved in DMF (200 mL), HATU (16.73 g, 44.0 mmol), Val-Cit-PAB-OH (9.20 g, 23.4 mmol), and DIPEA (15.32 mL, 87.9 mmol) were added, and the mixture was stirred at room temperature for 24 hours. The reaction progress was monitored by TLC. After completion of the reaction, the solvent was evaporated under reduced pressure on a rotary evaporator. The crude product was purified by preparative high-performance liquid chromatography. The resulting solution was evaporated under reduced pressure on a rotary evaporator to give compound 1 (6.67 g, 32.4%, white solid powder).

[0100] (2) Synthesis of Compound 2 (Py-MAA-Val-Cit-PAB-PNP) Compound 1 (7.02 g, 10.0 mmol) was dissolved in DMF (200 mL), NPC (di(p-nitrophenyl)carbonate, 4.56 g, 15.0 mmol) and DIPEA (2.09 mL, 12 mmol) were added, and the reaction was carried out at room temperature for 5 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was poured into petroleum ether (1500 mL), stirred, and filtered. The resulting filter cake was washed with petroleum ether (150 mL × 3) and dried by suction to give an off-white solid powder (6.57 g, 75.7%).

[0101] (3) Synthesis of Compound 3 (Py-MAA-Val-Cit-PAB-MMAE) Compound 2 (1.74 g, 2.2 mmol) was dissolved in 20 mL of DMF, and MMAE (1.44 g, 2.0 mmol), HOBt (0.27 g, 2.0 mmol), DIPEA (0.70 mL, 4.0 mmol), and pyridine (4 mL) were added under nitrogen gas protection. The reaction was stirred at room temperature for 24 h and monitored by TLC. After completion of the reaction, purification was carried out by preparative high-performance chromatography, and the resulting solution was evaporated under reduced pressure on a rotary evaporator to give compound 3 (white solid powder, 1.35 g, 46.7%). LC-MS m / z (ES+), 1446.35 (M+H)+, IR (3334.32 cm-1, 2965.9 cm-1, 1652.70 cm-1, 1538.92 cm-1, 1436.71 cm-1).

[0102] (4) Synthesis of Compound 4 (Py-MAA-Val-Cit-PAB-MMAD) Compound 2 (0.87 g, 1.1 mmol) was dissolved in 10 mL of DMF, and MMAD (0.77 g, 1.0 mmol), HOBt (0.14 g, 1.0 mmol), DIPEA (0.35 mL, 2.0 mmol), and pyridine (2 mL) were added under nitrogen gas protection. The reaction was stirred at room temperature for 24 hours and monitored by TLC. After completion of the reaction, purification was carried out by preparative high-performance chromatography, and the resulting solution was evaporated under reduced pressure on a rotary evaporator to give compound 4 (white solid powder, 0.65 g, 43.5%). LC-MS m / z (ES+) 1499.76 (M+H)+.

[0103] (5) Synthesis of Compound 6 (Mc-Val-Cit-PAB-PNP) Compound 5 (Mc-Val-Cit-PAB, 4.58 g, 8.0 mmol) was dissolved in DMF (100 mL), NPC (di(p-nitrophenyl)carbonate, 3.65 g, 12.0 mmol) and DIPEA (1.70 mL, 9.6 mmol) were added, and the reaction was allowed to proceed at room temperature for 5 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was poured into petroleum ether (1000 mL), stirred, and filtered. The filter cake was washed with petroleum ether (60 mL × 3) and dried by suction to give an off-white solid powder (5.04 g, 85.2%).

[0104] (6) Synthesis of Compound 7 (Mc-Val-Cit-PAB-MMAE) Compound 6 (1.19 g, 1.6 mmol) was dissolved in 12 mL of DMF, and MMAE (1.08 g, 1.5 mmol), HOBt (0.21 g, 1.5 mmol), DIPEA (0.55 mL, 3.0 mmol), and pyridine (2.5 mL) were added under nitrogen gas protection. The reaction was stirred at room temperature for 24 hours and monitored by TLC. After completion of the reaction, purification was carried out by preparative high-performance chromatography, and the resulting solution was evaporated under reduced pressure on a rotary evaporator to give compound 7 (white solid powder, 0.891 g, 45.1%). LC-MS m / z (ES+) 1316.18 (M+H)+.

[0105] (7) Synthesis of compound 8 (Mc-Val-Cit-PAB-MMAD) Compound 6 (0.74 g, 1.1 mmol) was dissolved in 10 mL of DMF, and MMAD (0.77 g, 1.0 mmol), HOBt (0.14 g, 1.0 mmol), DIPEA (0.35 mL, 2.0 mmol), and pyridine (2 mL) were added under nitrogen gas protection. The reaction was stirred at room temperature for 24 hours and monitored by TLC. After completion of the reaction, purification was carried out by preparative high-performance chromatography, and the resulting solution was evaporated under reduced pressure on a rotary evaporator to give compound 8 (white solid powder, 0.59 g, 42.8%). LC-MS m / z (ES+) 1369.38 (M+H)+.

[0106] Example 2b: Preparation of antibody drug conjugates

[0107] [ka]

[0108] 10 mg / mL RC88 antibody, 10 mmol / L diethylenetriaminepentaacetic acid (DTPA), and 5 mmol / L tris-2-carboxyethylphosphine (TCEP) were added to a PCR tube and stirred at 25°C for 2 hours. Then, 25% DMSO (dimethyl sulfoxide) and 5 mmol / L drug (compounds 3, 4, 7, or 8) were added at 0°C and stirred at 25°C for 10 hours. After the reaction was completed, ultrafiltration was performed by centrifugation three times with PBS buffer to remove any remaining unreacted drug and free small molecules such as DMSO. Coupling was detected by SDS-PAGE electrophoresis and hydrophobic high-performance liquid chromatography (HIC-HPLC). RC88-PY-MAA-Val-Cit-PAB-MMAE and RC88-Mc-Val-Cit-PAB-MMAE were characterized by reduced SDS-PAGE using Novex's NuPAGE pre-made gels. The total sample volume for each sample was 10 μL, and the results are shown in Figure 1. For RC88-PY-MAA-Val-Cit-PAB-MMAE, the cross-linker covalently reconnects the reduced disulfide bond, resulting in distinct bands: 150 kDa for the intact antibody (LHHL), 125 kDa for one uncoupled light chain (LHH), 100 kDa for two coupled heavy chains (HH), 75 kDa for one coupled light chain and one coupled heavy chain (LH), and 50 kDa and 25 kDa for the heavy and light chains, respectively. For RC88-Mc-Val-Cit-PAB-MMAE, the cross-linker was absent and the disulfide bond was reduced to two sulfhydryl groups, which were then coupled to two linker-toxins, respectively; therefore, reducing SDS-PAGE showed only two bands of 50 kDa and 25 kDa.The coupling of RC88-PY-MAA-Val-Cit-PAB-MMAE and RC88-Mc-Val-Cit-PAB-MMAE was detected by hydrophobic high-performance liquid chromatography (HIC-HPLC), and the results are shown in Figure 2A and Figure 2B. The results showed that the DAR values were 3.95 (RC88-PY-MAA-Val-Cit-PAB-MMAE) and 3.9 (RC88-Mc-Val-Cit-PAB-MMAE), respectively.

[0109] Example 3: Construction of Oval-Citar-3 cells that highly express MSLN Well-grown Oval-Citar-3 cells (ATCC) were placed in a 6-well plate at 3 × 10 5 After seeding at 1 / well and allowing overnight attachment, the original medium was discarded and 400 μL of fresh medium containing 10 μg / mL polybrene (Sigma) was added. 600 μL of a lentiviral vector (pRRL-cmv) containing the human MSLN coding sequence was added at the appropriate concentration. The wells were mixed and cultured for 24 hours. After culture completion, fresh medium was replaced and cultures were expanded. Positive cells were selected using a flow cytometer. The selected positive cells were used for culture expansion, MSLN expression was analyzed by flow cytometry, and the cells with the highest MSLN expression (hereafter referred to as Oval-Citar-3-MSLN) were selected for subsequent experiments.

[0110] Example 4: Detection of cytotoxic activity of RC88 antibody and RC88 antibody drug conjugates and corresponding linker-drug and drug Oval-Citar-3-MSLN cells in good growth condition were cultured in a 96-well cell culture plate (5 × 10 4The RC88 antibody, RC88 antibody-drug conjugates (RC88-Py-MAA-Val-Cit-PAB-MMAE, RC88-Py-MAA-Val-Cit-PAB-MMAD, RC88-Mc-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAD), and the corresponding linker-drug conjugates (Py-Val-Cit-PAB-MMAE, Py-Val-Cit-PAB) were added to the wells of the cells / mL, 100 μL / well, and incubated overnight at 37°C in a CO2 incubator. -MMAD, Mc-Val-Cit-PAB-MMAE, Mc-Val-Cit-PAB-MMAD), drugs (MMAE, MMAD), and PTX (paclitaxel) were diluted in complete medium to the following concentrations: RC88 antibody, RC88 antibody-drug conjugates (RC88-Py-MAA-Val-Cit-PAB-MMAE, RC88-Py-MAA-Val-Cit-PAB-MMAD, RC88-Mc-Val-Cit- For linker-drug conjugates (Py-Val-Cit-PAB-MMAE, Py-Val-Cit-PAB-MMAD, Mc-Val-Cit-PAB-MMAE, Mc-Val-Cit-PAB-MMAD), the final concentrations were 0.32, 1.6, 8, 16, 32, 64, 128, 640, and 3200 ng / mL. The final concentrations were 0.4, 2, 10, 20, 40, 80, 160, 800, and 4000 ng / mL. For the drugs (MMAE and MMAD), the final concentrations were 0.0016, 0.008, 0.04, 0.2, 0.4, 0.8, 1.6, 8, and 40 ng / mL. For PTX (paclitaxel), the final concentrations were 0.004, 0.02, 0.098, 0.3, 0.89, 2.67, 8, 40, and 200 ng / mL. After dilution, these were added to a 96-well plate (100 μL / well). A blank group (the same volume of medium without drug) and three control groups were set up. The incubation was carried out at 37°C for 72 hours in a CO2 incubator.100 μL / well of medium (without FBS) containing 10 μL of CCK-8 was added, and the plates were incubated at 37°C in a CO2 incubator for 2 to 4 hours. The OD value at 450 nm was read using a microplate reader. The inhibition rate was calculated using the following formula: IR% = (OD. ブランク -OD 薬物 )×100 / OD ブランク Using Prism software, a four-parameter curve fit was performed using the percent inhibition as the y-value and the drug concentration as the x-value, and the drug concentration value corresponding to the value between the maximum and minimum percent inhibition was recorded (IC 50 The values were initialized by the software), and the results are shown in Figure 3 and Table 4. The results showed that RC88-Py-MAA-Val-Cit-PAB-MMAD, RC88-Mc-Val-Cit-PAB-MMAD, RC88-Py-MAA-Val-Cit-PAB-MMAE, and RC88-Mc-Val-Cit-PAB-MMAE were effective in inhibiting the proliferation of Oval-Citar-3-MSLN.

[0111] [Table 4]

[0112] Example 5: Antitumor experiments of RC88 antibody and RC88 antibody-drug conjugate in a mouse model bearing Oval-Citar-3 human ovarian cancer, which highly expresses MSLN Oval-Citar-3-MSLN cells (2 × 10 6 ) was subcutaneously inoculated into nude mice (Changzhou Cavans Laboratory Animal Co., Ltd., Certificate No.: 201611240, Registration No.: SCXK(Su)2011-0003) until the tumor volume reached approximately 100–400 mm 3After growth to 100 mg / mL, the animals were randomized. RC88 antibody (2 mg / kg), RC88 antibody-drug conjugates (RC88-Py-MAA-Val-Cit-PAB-MMAD, RC88-Py-MAA-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAD, 2 mg / kg), and MMAE (0.0716 mg / kg) were administered once a week for a total of three doses. A negative control group received an equal volume of saline at the same time. The results are shown in Figures 4, 5, and 6.

[0113] The results showed that RC88 antibody and RC88 antibody-drug conjugates did not affect the weight gain of tumor-bearing mice, and all RC88 antibody-drug conjugates (RC88-Py-MAA-Val-Cit-PAB-MMAD, RC88-Py-MAA-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAE, RC88-Mc-Val-Cit-PAB-MMAD, 2 mg / kg) showed significant inhibition of xenografts in tumor-bearing mice, while the RC88 antibody showed little antitumor effect.

[0114] Example 6: Antitumor experiment of RC88 antibody-drug conjugate in a mouse model bearing Oval-Citar-3 human ovarian cancer with high MSLN expression Oval-Citar-3-MSLN cells (2 × 10 6 ) was subcutaneously inoculated into nude mice (Changzhou Cavans Laboratory Animal Co., Ltd., Certificate No.: 201611240, Registration No.: SCXK(Su)2011-0003) until the tumor volume reached approximately 100–400 mm 3After growth to 100°C, the animals were randomized. RC88 antibody (3 mg / kg), RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE, 3 mg / kg, 1.5 mg / kg, 0.75 mg / kg), MMAE (0.06 mg / kg), RC88 antibody (3 mg / kg) + MMAE (0.06 mg / kg), human serum IgG-MMAE (3 mg / kg), and PTX (paclitaxel) (10 mg / kg) were administered once a week for a total of three doses (PTX was administered twice a week for a total of six doses). The negative control group received an equal volume of saline at the same time. The results are shown in Figures 7, 8, and 9. Results showed that for the RC88 antibody-drug conjugate, tumor-bearing mice in the 3 mg / kg and 1.5 mg / kg groups showed significant tumor reduction 7 days after the first administration; the 3 mg / kg group showed no visible tumors 10 days after administration; the 1.5 mg / kg group showed no visible tumors 17 days after administration; and the 0.75 mg / kg group showed relatively rapid tumor growth after three administrations. There was no statistically significant difference in tumor volume 21 days after administration compared with the control (saline) group (P > 0.05), with a T / C > 40%. For the paclitaxel (PTX) group, tumors were completely eliminated (CR) in two tumor-bearing mice 21 days after administration, with tumor volume and RTV statistically different from the control (saline) group (P < 0.05), with a T / C < 40%. There was no statistically significant difference between the paclitaxel (PTX) group and the RC88 antibody-drug conjugate 3 mg / kg group or 1.5 mg / kg group, and there was no significant difference between the RC88 antibody group, MMAE group, RC88 antibody + MMAE group, IgG-MMAE group, and the control (saline) group (P > 0.05).

[0115] Example 7: Detection of affinity of RC88 antibody and RC88 antibody-drug conjugate for MSLN-positive tumor cells Flow cytometry was used to detect the affinity of the RC88 antibody and the RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE) to MSLN-positive tumor cells. Logarithmically growing Oval-Citar-3-MSLN cells were cultured in 1.5 mL EP tubes (4 × 10 per group). 5The cells were centrifuged at 1500 rpm for 5 minutes in cold 1% BSA-PBS (bovine serum albumin-PBS) at 1500 rpm, washed thoroughly with PBS, and the supernatant was discarded. The cells were resuspended in 200 μL of 4% paraformaldehyde and fixed at 25°C for 15 minutes. The cells were washed once with PBS, centrifuged at 2500 rpm for 3 minutes, and the supernatant was discarded. The RC88 antibody and RC88 antibody-drug conjugate were diluted three-fold from 10,000 ng / mL to 1.52 ng / mL in cold 1% BSA-PBS (bovine serum albumin-PBS) buffer, and 200 μL of each concentration was used to resuspend the cells. Blank control cells were directly resuspended in cold 1% BSA-PBS, and negative control cells were resuspended in 5 μg / mL of hIgG (Zhongke Chenyu) prepared in 1% BSA. The incubation was carried out at 4°C for 30 minutes, with mixing by inversion every 10 minutes to ensure uniform cell incubation. After incubation, the cells were washed once with cold PBS and centrifuged at 2500 rpm for 3 minutes at 4°C. The supernatant was discarded. 200 μL of FITC (fluorescein isothiocyanate)-labeled goat anti-human IgG Fcγ (Jackson ImmunoResearch) diluted 1:200 in cold PBS was added to each tube and incubated at 4°C for 30 minutes, with mixing by inversion every 10 minutes to ensure uniform cell incubation. At the end of the incubation, the cells were washed once with cold PBS and centrifuged at 2500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were resuspended in 400 μL of PBS and transferred to a flow cytometer (BD Calibur) for detection. The results are shown in Figure 10. The results showed that both the RC88 antibody and the RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE) had strong binding affinity to MSLN-positive tumor cells with EC50 values of 153.5 ng / mL and 251.4 ng / mL, respectively.

[0116] Example 8: Detection of binding activity of RC88 antibody and RC88 antibody-drug conjugate to MSLN-positive tumor cells The binding activity of the RC88 antibody and the RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE) to MSLN-positive tumor cells was detected by ELISA. Logarithmically growing Oval-Citar-3-MSLN cells were cultured in a 96-well cell culture plate (4 × 105 cells / mL, 100 μL / well) and incubated overnight at 37°C in a CO2 incubator. The supernatant was discarded, the plate was washed three times with 0.05% PBST buffer (250 μL / well), 100 μL of 4% paraformaldehyde was added per well and fixed at 25°C for 30 minutes, the plate was washed three times with 0.05% PBST buffer (250 μL / well), and each well was filled with 3% BAS-PBST (bovine serum albumin-PBST) for 2 minutes. After incubation at room temperature for 2 hours, the plate was washed three times with 0.05% PBST buffer (250 μL / well), and samples were added. 1) Binding curve: RC88 antibody and RC88 antibody-drug conjugate were diluted in 1% BAS-PBST buffer to a 3-fold gradient from 3000 ng / mL to 0.05 ng / mL, and then added to a 96-well plate (100 μL / well). 2) Competition curve: Recombinant human MSLN protein (Yiqiao Shenzhou) was diluted from 10,000 ng / mL to 0.51 ng / mL in 1% BAS-PBST. RC88 antibody and RC88 antibody-drug conjugate were diluted to 20 ng / mL. Then, they were mixed with an equal volume of MSLN protein dilution solution and added to a 96-well plate at 100 μL / well. After incubation at 25°C for 1 hour, the plate was washed three times. HRP-labeled goat anti-human IgG Fc (Bethyl) (1:2000) diluted in 1% BAS-PBST was added to each well. After incubation at 25°C for 1 hour, the plate was washed three times. A TMB color development kit (Kangwei Century) was used for color development in the dark for 5-10 minutes, followed by 2M sulfuric acid for stopping. The plate was then read using a microplate reader. The results are shown in Figure 11. The results showed that both the RC88 antibody and the RC88 antibody-drug conjugate strongly bound to MSLN-positive tumor cells, and the RC88 antibody-drug conjugate showed a slightly decreased binding compared with the RC88 antibody, but there was no significant difference, and their EC 50The values were 11.0±0.81 ng / mL and 19.7±5.80 ng / mL, respectively. Competition experiments with recombinant human MSLN protein demonstrated that the RC88 antibody and the RC88 antibody-drug conjugate specifically bound to MSLN on the surface of Oval-Citar-3-MSLN cells.

[0117] Example 9: Competitive binding of RC88 antibody and RC88 antibody drug conjugates and CA125 to MSLN The competitive binding ability of RC88 antibody and RC88 antibody-drug conjugate (RC88-Py-MAA-Val-Cit-PAB-MMAE) to MSLN and CA125 was determined by ELISA. ELISA plates were coated with recombinant MSLN protein (Yiqiao Shenzhou, 200 ng / mL). Sample addition: RC88 antibody and RC88 antibody-drug conjugate were diluted in 1% BAS-PBST (bovine serum albumin-PBST) buffer from 20 μg / mL (50 μL / well) to 10 points. Recombinant protein CA125 (His tag, R&D) was diluted in 1% BAS-PBST buffer to 200 ng / mL (50 μL / well). The total reaction system was 100 μL / well. The secondary antibody (mouse anti-His monoclonal antibody, R&D) was diluted 5000-fold and added 100 μL / well. TMB (3,3',5,5'-tetramethylbenzidine) was used for color development for 5-7 minutes. The reaction was then stopped with 2 M sulfuric acid. The plate was read at 450 nm using a microplate reader. The results are shown in Figure 12. The results showed that the binding of recombinant CA125 protein to recombinant human MSLN protein decreased with increasing concentrations of RC88 antibody and RC88 antibody-drug conjugate, suggesting that RC88 antibody and RC88 antibody-drug conjugate can block the binding of recombinant human CA125 protein to recombinant human MSLN protein.

[0118] The above description contemplates preferred embodiments and is illustrative only, not limiting the combinations of features necessary to practice the invention. The provided headings are not meant to limit the various embodiments of the invention. Terms such as "including," "comprising," and "encompassing" are not meant to imply limitations. Furthermore, unless otherwise stated, plural forms are included in the absence of numerical modification, and the term "or" means "and / or." Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0119] All publications and patents mentioned in this application are incorporated herein by reference. Numerous modifications and variations of the methods and compositions described herein will be apparent to those skilled in the art. While the present invention has been described in terms of certain preferred embodiments, it should be understood that the invention is not limited to these embodiments. Indeed, many variations of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the appended patents.

Claims

1. Formula Ab-(LD) ｎ 1. An antibody drug conjugate having the formula: (a) Ab is a humanized antibody or functional fragment thereof that specifically binds to MSLN; (b) L is a linker or is absent; (c) D is a therapeutic agent; (d) n is 1, 2, 3, 4, 5, 6, 7, or 8; The antibody or functional fragment thereof that specifically binds to MSLN is (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7; and (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; Including, Antibody drug conjugates.

2. The antibody (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and (ii) a light chain comprising the amino acid sequence set forth in SEQ ID NO: 9; Including, The antibody-drug conjugate of claim 1.

3. The antibody-drug conjugate of claim 1 or 2, wherein the antibody is a monoclonal antibody.

4. The antibody drug conjugate of any one of claims 1 to 3, wherein the therapeutic agent is a dolastatin peptide.

5. The antibody drug conjugate of any one of claims 1 to 4, wherein the therapeutic agent is MMAD, MMAE, or MMAF.

6. The antibody drug conjugate of any one of claims 1 to 5, wherein the linker and the antibody are linked via a thiol group of the antibody.

7. The linker has the formula: 【Chemical 1】 or wherein the linker has the structure shown by the formula: 【Chemistry 2】 7. The antibody drug conjugate of claim 1, having the structure shown by:

8. 8. The antibody drug conjugate of any one of claims 1 to 7, having a structure represented by any one of the following formulas: 【Chemistry 3】

9. A humanized antibody or a functional fragment thereof capable of specifically binding to mesothelin, The antibody or functional fragment thereof is (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7; and (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; Including, An antibody or a functional fragment thereof.

10. The antibody comprising: (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10; and (ii) a light chain comprising the amino acid sequence set forth in SEQ ID NO: 9; Including, The antibody or functional fragment thereof according to claim 9.

11. The antibody or functional fragment thereof according to claim 9 or 10, wherein the antibody is a monoclonal antibody.

12. An isolated polynucleotide encoding the antibody or functional fragment thereof according to any one of claims 9 to 11.

13. 13. The polynucleotide of claim 12, the polynucleotides are operably linked to control sequences that allow expression of the polypeptides encoded thereby in a host cell or a cell-free expression system; Expression vector.

14. A composition comprising a polynucleotide encoding the heavy chain of the antibody or functional fragment thereof according to any one of claims 9 to 11 and a polynucleotide encoding the light chain of the antibody or functional fragment thereof according to any one of claims 9 to 11.

15. An expression vector comprising a polynucleotide encoding the heavy chain of the antibody or functional fragment thereof according to any one of claims 9 to 11, and An expression vector comprising a polynucleotide encoding the light chain of the antibody or functional fragment thereof according to any one of claims 9 to 11. Including, A composition in which the polynucleotides are operably linked to control sequences that allow for the expression of the polypeptides encoded thereby in a host cell or a cell-free expression system.

16. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 8 and / or the antibody or functional fragment thereof according to any one of claims 9 to 11, and a pharmaceutically acceptable carrier.

17. In the manufacture of a medicament for the treatment or prevention of mesothelin-positive cancer, A conjugate according to any one of claims 1 to 8. The polynucleotide of claim 12. The expression vector according to claim 13. A composition according to claim 14 or 15, or The pharmaceutical composition of claim 16 Use of.

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