Anti-GPC3 antibody drug conjugate

Abstract

Conventional cancer treatments are often ineffective, and therefore, there remains a need to develop more targeted and potent therapies to treat HCC. Antibody-drug conjugates (ADCs), which include antibodies that target GPC3 and are conjugated to cytotoxic drugs via a chemical linker, provide targeted therapies for treating patients with HCC tumors that express GPC3. [Solution] This disclosure provides GPC3 antibody-drug conjugates (ADCs), and includes compositions and methods for using such ADCs.

Description

[Technical Field]

[0001] 1. Sequence Listing This application includes a sequence listing, which is submitted electronically in XML format and is incorporated herein by reference in its entirety. A copy of the XML was prepared at [location].

[0002] 2. Technical Fields This application relates to an anti-GPC3 antibody drug conjugate (ADC). [Background technology]

[0003] 3. Background GPC3 is a hepatocellular carcinoma (HCC) tumor antigen that is highly expressed in the majority of tumors (Yamauchi et al., 2005, The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol 18, pp. 1591-1598). GPC3 expression is associated with low survival rates, is maintained in the post-treatment setting, and is independent of viral status or race / ethnicity (Guo et al., 2020, Glypican-3: A New Target for Diagnosis and Treatment of Hepatocellular Carcinoma. J Cancer. February 3, 2020; 11(8): pp. 2008-2021, doi:10.7150 / jca.39972). As a member of the glypican family of heparan sulfate proteoglycans, GPC3 is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor and translocates to the cell via antibody binding. GPC3 is expressed during development and functions to regulate WNT and Hedgehog signaling. However, the GPC3 protein is not expressed in most normal adult crude tissues (Guo et al., 2020, Glypican-3: A New Target for Diagnosis and Treatment of Hepatocellular Carcinoma. J Cancer. February 3, 2020; 11(8): 2008-2021, doi:10.7150 / jca.39972). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yamauchi et al., 2005, The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol 18, pp. 1591-1598

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] Conventional cancer therapeutic treatments are often not effective. Therefore, there is still a need to develop more targeted and powerful therapies for treating HCC. An antibody-drug conjugate (ADC) containing an antibody targeting GPC3 conjugated to a cytotoxic drug via a chemical linker provides a targeted therapy for treating patients with HCC tumors that express GPC3.

Means for Solving the Problems

[0006] 4. Abstract Formula (IV):

[0007]

Chemical Formula

[0008] In some embodiments, the anti-GPC3 antibody of the antibody-drug conjugate includes (a) a heavy chain variable region having the amino acid sequence shown as SEQ ID NO: 1, and (b) a light chain variable region having the amino acid sequence shown as SEQ ID NO: 2.

[0009] In some embodiments, the anti-GPC3 antibody in the antibody-drug conjugate is an IgG1 antibody.

[0010] In some embodiments, the anti-GPC3 antibody in the antibody-drug conjugate comprises (a) a heavy chain having the amino acid shown as SEQ ID NO: 9, and (b) a light chain having the amino acid sequence shown as SEQ ID NO: 10.

[0011] In some embodiments, the anti-GPC3 antibody drug conjugate is expressed by formula (IV):

[0012] [ka] [In the formula, n is an integer from 1 to 6, and Ab is an anti-GPC3 antibody comprising two heavy chains having the amino acid sequences shown as SEQ ID NO: 9 and two light chains having the amino acid sequences shown as SEQ ID NO: 10.] It has the structure of [the object].

[0013] In some embodiments, the anti-GPC3 antibody drug conjugate has n = 2.

[0014] In some embodiments, the anti-GPC3 antibody drug conjugate has n = 4.

[0015] In some embodiments, the anti-GPC3 antibody drug conjugate has n = 6.

[0016] In some embodiments, the anti-GPC3 antibody drug conjugate has n = 1.

[0017] A composition comprising a therapeutically effective amount of the anti-GPC3 antibody-drug conjugate disclosed herein is also provided herein, wherein the main species of the antibody-drug conjugate in the composition is n = 2.

[0018] Pharmaceutical compositions comprising a therapeutically effective amount of the anti-GPC3 antibody drug conjugate disclosed herein and pharmaceutically acceptable excipients, wherein the DAR is 2 or about 2, are also provided herein.

[0019] Also provided herein is a method for treating hepatocellular carcinoma (HCC), comprising administering a therapeutically effective amount of the anti-GPC3 antibody drug conjugate disclosed herein to a patient in need of treatment.

[0020] A method for treating HCC is also provided herein, comprising administering a therapeutically effective amount of a pharmaceutical composition disclosed herein, comprising a therapeutically effective amount of an anti-GPC3 antibody drug conjugate disclosed herein, to a patient in need of treatment.

[0021] Nucleic acids encoding antibodies containing the heavy chain sequence of SEQ ID NO: 9 or SEQ ID NO: 11 and the light chain sequence of SEQ ID NO: 10 are also provided. [Brief explanation of the drawing]

[0022] [Figure 1A] Comparative data between mAb1 and GC33 ADC are shown. Figure 1A shows the inhibition of proliferation of HepG2, Hep3B, and SK-HEP1-GPC3 cells in 2D culture after administration of mAb1 and GC33 with DAR=2 (top panel) and DAR=6 (bottom panel). [Figure 1B]Comparative data between mAb1 and GC33 ADC are shown. Figure 1B shows the inhibition of proliferation of HepG2, Hep3B, and SK-HEP1-GPC3 cells in 3D spheroids by ADC-1 and DAR2 GC33. [Figure 2] This shows the dose-response relationship between ADC-1 and DAR2 GC33 ADC treatment in a human HCC Hep3B CDX mouse model. [Figure 3] This shows the dose-response of ADC-1 treatment in a human HCC HepG2 CDX mouse model. [Figure 4] This shows the dose-response of ADC-1 treatment in a xenotransplant (PDX) mouse model derived from human HCC LI1068 patients. [Figure 5] This shows the dose-response to ADC-1 treatment in a human HCC LI6610 PDX mouse model. [Modes for carrying out the invention]

[0023] 6. Detailed explanation When used in this disclosure, the singular forms "a," "an," and "the" refer to multiple objects unless explicitly indicated by the context. The terms "a" (or "an"), as well as "one or more" and "at least one," may be used interchangeably herein unless explicitly indicated by the context.

[0024] When used in this disclosure, unless otherwise specified, the terms “about” and “approximately” generally refer to a range of numbers that a person skilled in the art would consider equivalent to (i.e., having the same function or result as) the listed values. In many cases, the terms “about” and “approximately” may include numbers rounded to the nearest significant figure. In certain embodiments, the terms “about” and “approximately” are interpreted as allowing for variations that would be considered normal by a person skilled in the art, such as variations of 20%, 10%, or 5% or less. In certain embodiments, the terms “about” and “approximately” encompass the exact listed values. Unless otherwise clearly indicated by the context, all numerical values ​​provided herein are modified by the term “about.”

[0025] In certain embodiments, anti-GPC3 ADCs containing TOP1 inhibitors and methods for using the ADCs are disclosed herein.

[0026] 6.1 Topoisomerase 1 inhibitors (TOP1i) Topoisomerase 1 (TOP1) removes supercoils formed during DNA replication. TOP1 inhibitors (TOP1i) can bind to and stabilize the TOP1-DNA complex, thereby inducing DNA strand breaks and apoptosis.

[0027] In certain embodiments, a topoisomerase I inhibitor drug ("TOP1i drug") with structural formula (I) is provided herein, which may be intended for targeted delivery to cells by conjugation with an anti-GPC3 antibody.

[0028] [ka]

[0029] In a particular embodiment, the TOP1i drug is (7S)-14-(3-aminobicyclo[1.1.1]pentan-1-yl)-7-ethyl-7-hydroxy-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-8,11(7H,13H)-dione.

[0030] In certain embodiments, the TOP1i drug intended herein may be conjugated to an antibody via a linker shown in structural formula (II).

[0031] [ka] During the ceremony

[0032] [ka] This represents the linker binding point to the TOP1i drug.

[0033] 6.1.1 Linker drug LD1 (structural formula (III)) In certain embodiments, the TOP1i linker drug (LD1) is a compound of formula (III). In certain embodiments, LD1 is (2S)-2-(2-bromoacetamide)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide.

[0034] [ka]

[0035] 6.2 Anti-GPC3 ADC In certain embodiments, the TOP1i drugs described herein (i.e., formula (I)) may be conjugated with an anti-GPC3 antibody to form an anti-GPC3 TOP1i antibody-drug conjugate. In certain embodiments, the disclosure provides an anti-GPC3 TOP1i ADC for therapeutic use in the treatment of GPC3-expressing HCC tumors. In certain embodiments, the anti-GPC3 antibody is mAb1, which was remarkably well tolerated as a TOP1i ADC in non-human primates in contrast to TOP1i ADCs using GC33 (codrituzumab) anti-GPC3 antibody (Zhu et al., 2013, First-in-man phase I study of GC33, a novel recombinant humanized antibody Against Glypican-3, in patients with advanced hepatocellular carcinoma. Clin. Cancer Res. DOI:10.1158 / 1078-0432. CCR-12-2616).

[0036] In certain embodiments, a TOP1i drug is conjugated to an anti-GPC3 antibody via a linker moiety. In certain embodiments, the linker connects the TOP1i drug to the anti-GPC3 antibody by forming a covalent linkage at one position on the TOP1i drug and another position on the antibody. In certain embodiments, the covalent linkage is formed by the reaction of a functional group of the linker with the functional groups of the TOP1i drug and the anti-GPC3 antibody. In certain embodiments, the anti-GPC3 ADC of the present disclosure comprises an anti-GPC3 antibody conjugated to a TOP1i linker of formula (III).

[0037] In certain embodiments, "Ab" refers to an anti-GPC3 antibody comprising a heavy chain having the amino acid sequence shown as SEQ ID NO: 9 and a light chain having the amino acid sequence shown as SEQ ID NO: 10. In certain embodiments, Ab is conjugated to a TOP1i linker drug of formula (III).

[0038] 6.2.1 Ab anti-GPC3 antibody In certain embodiments, Ab is a fully human anti-GPC3 IgG1 monoclonal antibody. In certain embodiments, Ab includes a variable region and a CDR (complementarity-determining region) identified by aligning its sequence with rules developed in the art and / or against a database of known variable regions.

[0039] Methods for verifying these areas are described in Kontermann and Dubel (eds.), Antibody Engineering, Springer, New York, NY, 2001, and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. For example, CDRs can be verified according to one of the schemes provided by Kabat et al. (1991), Sequences of Proteins of Immunological Interest (5th edition), USDept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242 (hereinafter referred to as "Kabat"), or according to AbM (Oxford Molecular / MSI Pharmacopia) (hereinafter referred to as "AbM"). AbM can be obtained from the Abysis database at www.bioinf.org.uk / abs (maintained by AC Martin at the Department of Biochemistry & Molecular Biology, University College London).

[0040] In a particular embodiment, Ab comprises a heavy chain having the amino acid sequence shown as SEQ ID NO: 9 (the variable region is in bold and has the amino acid sequence shown as SEQ ID NO: 1, the constant region is in italics, and the CDR is underlined and has the amino acid sequences shown as SEQ ID NOs: 3, 4, and 5, respectively).

[0041] [ka]

[0042] In a particular embodiment, Ab comprises a heavy chain having a C-terminal lysine cleavage, for example, the amino acid sequence shown as SEQ ID NO: 11 (the variable region is in bold and has the amino acid sequence shown as SEQ ID NO: 1, the constant region is in italics, and the CDR is underlined and has the amino acid sequences shown in order of appearance as SEQ ID NOs: 3, 4, and 5, respectively).

[0043] [ka]

[0044] In a particular embodiment, Ab includes a light chain having the amino acid sequence shown as SEQ ID NO: 10 (the variable region is in bold and has the amino acid sequence shown as SEQ ID NO: 2, the constant region is in italics, and the CDR is underlined and has the amino acid sequences shown as SEQ ID NOs: 6, 7, and 8, respectively, in order of appearance).

[0045] [ka]

[0046] In a particular embodiment, Ab comprises two heavy chains each having the amino acid sequence shown as SEQ ID NO: 9, and two light chains each having the amino acid sequence shown as SEQ ID NO: 10.

[0047] In a particular embodiment, Ab comprises two heavy chains each having the amino acid sequence shown as SEQ ID NO: 11, and two light chains each having the amino acid sequence shown as SEQ ID NO: 10.

[0048] In a particular embodiment, Ab includes CDR-H1 having the amino acid sequence shown as SEQ ID NO: 3, CDR-H2 having the amino acid sequence shown as SEQ ID NO: 4, CDR-H3 having the amino acid sequence shown as SEQ ID NO: 5, CDR-L1 having the amino acid sequence shown as SEQ ID NO: 6, CDR-L2 having the amino acid sequence shown as SEQ ID NO: 7, and CDR-L3 having the amino acid sequence shown as SEQ ID NO: 8. The amino acid sequence of CDR-H1 was verified using the AbM definition, and the remaining CDR amino acid sequences were verified using the Kabat definition.

[0049] In a particular embodiment, Ab includes a heavy chain variable domain having the amino acid sequence shown as SEQ ID NO: 1, and a light chain variable domain having the amino acid sequence shown as SEQ ID NO: 2.

[0050] In certain embodiments, one or more nucleic acids encoding an antibody comprising the heavy chain sequence of SEQ ID NO: 9 or SEQ ID NO: 11 and the light chain sequence of SEQ ID NO: 10 are provided herein. In other embodiments, the present invention comprises a vector comprising one or more of the above-described nucleic acids, or a host cell comprising the nucleic acids or vector. The nucleic acids of the present invention can be obtained using standard molecular biology techniques. In antibodies expressed by hybridomas (for example, hybridomas prepared as described in the examples below), the cDNA encoding the light and heavy chains of the antibody can be obtained by standard PCR amplification or cDNA cloning techniques.

[0051] In certain embodiments, the anti-GPC3 antibody Ab may be in a format such as an IgG1 or IgG4 format that is modified to confer desired properties, such as having a mutated Fc that reduces or "silences" effector function or extends half-life. In embodiments in which half-life extension and / or Fc silencing mutations are introduced, Ab may contain heavy chain sequences in the antibody combinations shown in SEQ ID NOs. 12-117 of the sequence listing in Table 12.

[0052] Embodiments of the anti-GPC3 antibody Ab may include an IgG1 heavy chain containing the amino acid sequence of SEQ ID NOs. 9, 11, and 12-45. In embodiments, Ab includes a wild-type IgG1 heavy chain containing the amino acid sequence of SEQ ID NO. 9 (having terminal lysine) and SEQ ID NO. 11 (not having terminal lysine). In embodiments, the IgG1 heavy chain includes an Fc silencing mutation selected from L234A, L235A(LALA) or L234S, L235T, G236R(STR). In embodiments, Ab includes an IgG1 heavy chain containing the L234A, L235A(LALA) mutations with or without terminal lysine. In embodiments, Ab includes an IgG1 heavy chain containing the L234S, L235T, G236R(STR) mutations with or without terminal lysine.

[0053] In embodiments, the anti-GPC3 antibody Ab may contain a heavy chain IgG1 containing half-life extension mutations selected from M252Y, S254T, T256E(YTE); Q311R, M428E, N434W(REW); M428L, N434S(LS); L309D, Q311H, N434S(DHS); or T250Q, M428L(QL). In embodiments, Ab contains an IgG1 heavy chain containing M252Y, S254T, T256E(YTE) with or without terminal lysine. In embodiments, Ab contains an IgG1 heavy chain containing Q311R, M428E, N434W(REW) with or without terminal lysine. In embodiments, Ab contains an IgG1 heavy chain containing M428L, N434S(LS) with or without terminal lysine. In the embodiment, Ab comprises an IgG1 heavy chain containing L309D, Q311H, and N434S(DHS) which may or may not have terminal lysine. In the embodiment, Ab comprises an IgG1 heavy chain containing T250Q and M428L(QL) which may or may not have terminal lysine.

[0054] In embodiments, the anti-GPC3 antibody Ab may include both Fc silencing mutations and half-life extension mutations. In embodiments, Ab includes an IgG1 heavy chain containing mutations with or without terminal lysine, L234A, L235A(LALA) and M252Y, S254T, T256E(YTE). In embodiments, Ab includes an IgG1 heavy chain containing mutations with or without terminal lysine, L234S, L235T, G236R(STR) and M252Y, S254T, T256E(YTE). In embodiments, Ab includes an IgG1 heavy chain containing mutations with or without terminal lysine, L234A, L235A(LALA) and Q311R, M428E, N434W(REW). In an embodiment, Ab comprises an IgG1 heavy chain containing mutations L234S, L235T, G236R(STR) and Q311R, M428E, N434W(REW) with or without terminal lysine. In an embodiment, Ab comprises an IgG1 heavy chain containing mutations L234A, L235A(LALA) and M428L, N434S(LS) with or without terminal lysine. In an embodiment, Ab comprises an IgG1 heavy chain containing mutations L234S, L235T, G236R(STR) and M428L, N434S(LS) with or without terminal lysine. In an embodiment, Ab comprises an IgG1 heavy chain containing mutations L234A, L235A(LALA) and L309D, Q311H, N434S(DHS) with or without terminal lysine. In one embodiment, Ab comprises an IgG1 heavy chain containing the mutants L234S, L235T, G236R(STR) and L309D, Q311H, N434S(DHS) with or without terminal lysine. In another embodiment, Ab comprises an IgG1 heavy chain containing the mutants L234A, L235A(LALA) and T250Q, M428L(QL) with or without terminal lysine. In yet another embodiment, Ab comprises an IgG1 heavy chain containing the mutants L234S, L235T, G236R(STR) and T250Q, M428L(QL) with or without terminal lysine.

[0055] Embodiments of the anti-GPC3 antibody Ab may include an IgG quadruplex containing the amino acid sequence of SEQ ID NOs. 46-117. In embodiments, Ab may include a wild-type IgG quadruplex containing the amino acid sequence of SEQ ID NOs. 82 (having terminal lysine) and SEQ ID NOs. 83 (not having terminal lysine). In embodiments, Ab may further include an IgG quadruplex containing the S228P mutation of SEQ ID NOs. 46 (having terminal lysine) and SEQ ID NOs. 47 (not having terminal lysine). In embodiments, the IgG quadruplex contains an Fc silencing mutation selected from F234A, L235A(FALA) or L234S, L235T, G236R(STR). In embodiments, Ab includes an IgG quadruplex containing mutations F234A and L235A(FALA) with or without terminal lysine, and with or without the S228P mutation. In the embodiment, Ab comprises an IgG quadruplex containing mutations L234S, L235T, and G236R(STR) that have or do not have terminal lysine, and that have or do not have the S228P mutation.

[0056] In embodiments, the anti-GPC3 antibody Ab may contain an IgG quadruplex containing a half-life extension mutation selected from M252Y, S254T, T256E(YTE); Q311R, M428E, N434W(REW); M428L, N434S(LS); L309D, Q311H, N434S(DHS); or T250Q, M428L(QL). In embodiments, Ab contains an IgG quadruplex containing M252Y, S254T, T256E(YTE) with or without terminal lysine, and with or without the S228P mutation. In embodiments, Ab contains an IgG quadruplex containing Q311R, M428E, N434W(REW) with or without terminal lysine, and with or without the S228P mutation. In embodiments, Ab comprises an IgG quadruplex containing M428L, N434S(LS) with or without terminal lysine, and with or without the S228P mutation. In embodiments, Ab comprises an IgG quadruplex containing L309D, Q311H, N434S(DHS) with or without terminal lysine, and with or without the S228P mutation. In embodiments, Ab comprises an IgG quadruplex containing T250Q, M428L(QL) with or without terminal lysine, and with or without the S228P mutation. In embodiments, Ab may contain both Fc silencing mutations and half-life extension mutations.

[0057] In one embodiment, the anti-GPC3 antibody Ab comprises an IgG quadruplex containing mutations F234A, L235A(FALA) and M252Y, S254T, T256E(YTE) that have or do not have terminal lysine, and S228P mutations. In another embodiment, Ab comprises an IgG quadruplex containing mutations L234S, L235T, G236R(STR) and M252Y, S254T, T256E(YTE) that have or do not have terminal lysine, and S228P mutations. In yet another embodiment, Ab comprises an IgG quadruplex containing mutations F234A, L235A(FALA) and Q311R, M428E, N434W(REW) that have or do not have terminal lysine, and S228P mutations. In one embodiment, Ab comprises an IgG quadruplex containing the mutations L234S, L235T, G236R(STR) and Q311R, M428E, and N434W(REW) which have or do not have the S228P mutation, and whether or not they have terminal lysine. In another embodiment, Ab comprises an IgG quadruplex containing the mutations F234A, L235A(FALA) and M428L, and N434S(LS) which have or do not have the S228P mutation. In yet another embodiment, Ab comprises an IgG quadruplex containing the mutations L234S, L235T, G236R(STR) and M428L, and N434S(LS) which have or do not have terminal lysine. In one embodiment, Ab comprises an IgG quadruplex containing mutations F234A, L235A(FALA) and L309D, Q311H, N434S(DHS) that have or do not have terminal lysine, and that have or do not have the S228P mutation. In another embodiment, Ab comprises an IgG quadruplex containing mutations L234S, L235T, G236R(STR) and L309D, Q311H, N434S(DHS) that have or do not have terminal lysine, and that have or do not have the S228P mutation. In yet another embodiment, Ab comprises an IgG quadruplex containing mutations F234A, L235A(FALA) and T250Q, M428L(QL) that have or do not have terminal lysine, and that have or do not have the S228P mutation.In the embodiment, Ab comprises an IgG quadruplex containing the mutations L234S, L235T, G236R(STR), T250Q, and M428L(QL), which may or may not have a terminal lysine mutation, and which may or may not have the S228P mutation.

[0058] The anti-GPC3 antibody disclosed herein is modified to facilitate efficient conjugation of TOP1i linker drugs. The cysteine ​​at position 214 of the light chain is mutated to alanine, thereby breaking the naturally formed disulfide crosslink by the cysteine ​​at position 220 of the heavy chain (Eu numbering by Kabat). This frees or unpairs the cysteine ​​at position 220 of the heavy chain, thus facilitating site-specific and controlled conjugation of TOP1i linker drugs.

[0059] 6.2.2 Number of linked drugs In certain embodiments, the ADCs disclosed herein include drug molecules conjugated to the antibody molar in various stoichiometric molar ratios, depending on the configuration of the antibody and, at least in part, on the method used to carry out the conjugation.

[0060] The term “drug load” or “drug loading” refers to the number of drug molecules per antibody in an individual ADC molecule. The number of TOP1i drugs linked to an anti-GPC3 ADC may vary and is limited by the number of available binding sites on the anti-GPC3 antibody. As intended for the anti-GPC3 ADC of the present invention, the linker links a single TOP1i drug to the antibody in the anti-GPC3 ADC. Anti-GPC3 ADCs having n up to 6 (i.e., formula (IV)) are intended, provided that the anti-GPC3 ADC does not exhibit unacceptable levels of aggregation under use and / or storage conditions. In certain embodiments, the anti-GPC3 ADC has n in the range of 1 to 6. In certain embodiments, the anti-GPC3 ADC has n selected from 1, 2, 3, 4, 5, or 6. In certain embodiments, n is 1, 2, 3, or 4. In certain embodiments, n is 2, or 4. In certain embodiments, n is 2. In a particular embodiment, the drug load may include one drug molecule, two drug molecules, three drug molecules, four drug molecules, five drug molecules, or six drug molecules.

[0061] A conjugation method for producing an anti-GPC3 ADC composition is provided herein, wherein the composition has n of 2 main species of ADCs and a drug-antibody ratio (DAR) of 2 or about 2. DAR is the average number of drugs conjugated to each antibody in the composition. Other conjugation methods are known in the art and can be used to produce ADCs having different numbers of conjugated drugs (e.g., n is 1, 2, 3, 4, 5, or 6) and compositions with different DARs (e.g., DAR is 1, 2, 3, 4, 5, or 6).

[0062] 6.2.3 Exemplary ADC In a particular embodiment, an anti-GPC3 antibody drug conjugate of formula (IV) is provided.

[0063] [ka]

[0064] In the formula, antibody Ab comprises two heavy chains, each having the amino acid sequence shown as SEQ ID NO: 9, and two light chains, each having the amino acid sequence shown as SEQ ID NO: 10. In certain embodiments, the conjugation of the linker drug to the antibody is via a linkage formed by sulfhydryl groups of cysteine ​​residues of the antibody. In certain embodiments, n has a value of 1, 2, 3, 4, 5, or 6.

[0065] In a particular embodiment, n has a value of 1, 2, 4, or 6. In a particular embodiment, n is 2. In a particular embodiment, the anti-GPC3 ADC of structural formula (IV) comprises an Ab having a heavy chain having the amino acid sequence shown as SEQ ID NO: 9 and a light chain having the amino acid sequence shown as SEQ ID NO: 10, and n has a value of 1, 2, 4, or 6. In a particular embodiment, the anti-GPC3 ADC of structural formula (IV) comprises an Ab having a heavy chain having the amino acid sequence shown as SEQ ID NO: 9 and a light chain having the amino acid sequence shown as SEQ ID NO: 10, and n has a value of 2.

[0066] In a particular embodiment, an anti-GPC3 antibody drug conjugate of formula (IV) is provided.

[0067] [ka]

[0068] In the formula, antibody Ab comprises two heavy chains, each having the amino acid sequence shown as SEQ ID NO: 11, and two light chains, each having the amino acid sequence shown as SEQ ID NO: 10. In certain embodiments, the conjugation of the linker drug to the antibody is via a linkage formed by sulfhydryl groups of cysteine ​​residues of the antibody. In certain embodiments, n has a value of 1, 2, 3, 4, 5, or 6.

[0069] In a particular embodiment, n has a value of 1, 2, 4, or 6. In a particular embodiment, n is 2. In a particular embodiment, the anti-GPC3 ADC of structural formula (IV) comprises an Ab having a heavy chain having the amino acid sequence shown as SEQ ID NO: 11 and a light chain having the amino acid sequence shown as SEQ ID NO: 10, and n has a value of 1, 2, 4, or 6. In a particular embodiment, the anti-GPC3 ADC of structural formula (IV) comprises an Ab having a heavy chain having the amino acid sequence shown as SEQ ID NO: 11 and a light chain having the amino acid sequence shown as SEQ ID NO: 10, and n has a value of 2.

[0070] As used herein, "ADC-1" is a composition comprising an ADC by structural formula (IV), wherein Ab has two heavy chains having the amino acid sequence shown as SEQ ID NO: 9 and two light chains having the amino acid sequence shown as SEQ ID NO: 10, with a drug-antibody ratio (DAR) of about 2 and a main species n of 2. The procedure used to prepare ADC-1 is described in the following examples. In one embodiment, ADC-1 is a composition comprising multiple ADCs by structural formula (IV), where n has two or more different values. In some embodiments, the ADC-1 composition has an average drug-antibody ratio (DAR) of about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, or about 2.3. In some embodiments, the ADC-1 composition has an average of two molecules of a topoisomerase 1 inhibitor (Top1i) conjugated for each anti-GPC3 antibody. In some embodiments, the main species of ADC in the ADC-1 composition has n=2. In some embodiments, the ADC-1 has an average DAR of approximately 2.

[0071] 6.3 How to use In a particular embodiment, a method for treating hepatocellular carcinoma (HCC) is provided, comprising administering a therapeutically effective dose of an ADC of formula (IV) to a patient in need of treatment.

[0072] The term "subject" as used herein refers to a human being. The terms "human being," "patient," and "subject" are interchangeable herein.

[0073] The terms “to treat,” “to treat,” and “treatment” as used herein refer to methods of reducing or suppressing a disease and / or its associated symptoms.

[0074] The term "therapeutic dose" refers to the amount of ADC (Acute Drug Concentrate) that, when administered for the treatment of a specific subject or population, is sufficient to prevent the onset of one or more symptoms of the condition or disorder being treated, or to alleviate one or more symptoms to some extent. [Examples]

[0075] 7. Examples The following examples, which highlight certain features and characteristics of exemplary embodiments of the ADCs, antibodies, and TOP1i described herein, are provided for illustrative purposes only.

[0076] [Example 1] Synthesis of linker drug [ka]

[0077] Example 1 (2S)-2-(2-bromoacetamide)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

[0078] [ka]

[0079] Example 1A tert-butyl(3-(methoxy(methyl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)carbamate

[0080] To a solution of 3-((tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentane-1-carboxylic acid (4.9 g), N,O-dimethylhydroxylamine hydrochloride (2.2 g), and N,N-diisopropylethylamine (11.30 mL) in dichloromethane (10 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate (8.61 g) was added in small amounts at 10 °C. The reaction mixture was stirred at 20 °C for 12 hours. Two additional reactions were set up as described and stirred at 20 °C for 12 hours. All three reaction products were combined. The reaction product was diluted with dichloromethane (200 mL) and added to a 1N aqueous solution of HCl (50 mL). The precipitate formed was filtered and the filtrate was separated. The organic layer was washed with saturated sodium bicarbonate aqueous solution (50 mL) and brine (50 mL), dehydrated with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography of silica gel eluted with 1-50% ethyl acetate in petroleum ether to obtain the labeled compound. 1 H NMR (400 MHz, CDCl3) δ ppm 4.97 (br s, 1H), 3.66 (s, 3H), 3.18 (s, 3H), 2.34 (s, 6H), 1.45 (s, 9H). MS (ESI+) m / z 271.2 (M+H) + .

[0081] [ka]

[0082] Example 1B tert-butyl(3-(benzo[d][1,3]dioxol-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)carbamate

[0083] To a solution of 5-bromobenzo[d][1,3]dioxol (8.83 g) in tetrahydrofuran (100 mL), n-butyllithium (17.57 mL, 2.5 M in hexane) was slowly added at -65°C under a nitrogen atmosphere. The mixture was stirred at -65°C for 30 minutes. A solution of Example 1A (4.75 g) in tetrahydrofuran (40 mL) was slowly added. The mixture was stirred at -65°C for 3 hours. Three additional reactions were set up and stirred at -65°C for 3 hours. All four reactants were combined. The mixture was quenched with saturated aqueous solution of ammonium chloride (500 mL) and extracted with ethyl acetate (3 × 500 mL). The combined organic layer was washed with brine (500 mL), dehydrated with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluted with 1-50% ethyl acetate in petroleum ether to obtain the labeled compound. 1 H NMR (400 MHz, CDCl3) δ ppm 7.63 (dd, 1H), 7.45 (d, 1H), 6.82 (d, 1H), 6.03 (s, 2H), 5.04 (br s, 1H), 2.50 (s, 6H), 1.46 (s, 9H). MS (ESI+) m / z 354.2 (M+Na) + .

[0084] [ka]

[0085] Example 1C N-(3-(benzo[d][1,3]dioxol-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)-2,2,2-trifluoroacetamide

[0086] Step 1: Trifluoroacetic acid (62 mL) was slowly added at 0°C to a solution of Example 1B (6.2 g) in dichloromethane (62 mL). The reaction mixture was stirred at 25°C for 4 hours. Two additional reactions were set up and stirred at 25°C for 4 hours. Each mixture was concentrated under reduced pressure. Each residue was used in the next step without further purification.

[0087] Step 2: To a solution of the crude product in dichloromethane (62 mL), N,N-diisopropylethylamine (16.34 mL) and trifluoroacetic anhydride (3.96 mL) were added dropwise at 0°C. The mixture was stirred at 25°C for 2 hours. Two additional reactions were set up as described and stirred at 25°C for 2 hours. All three reaction products were combined and poured into water (200 mL) and extracted with dichloromethane (2 × 200 mL). The organic layer was washed with brine (200 mL), dehydrated with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with 25% ethyl acetate in petroleum ether to obtain the labeled compound. 1 H NMR (501 MHz, CDCl3) δ ppm 7.61 (dd, 1H), 7.43 (d, 1H), 7.00 (s, 1H), 6.85 (d, 1H), 6.05 (s, 2H), 2.62 (s, 6H). MS (ESI+) m / z 328.2 (M+H) + .

[0088] [ka]

[0089] Example 1D 2,2,2-Trifluoro-N-(3-(6-nitrobenzo[d][1,3]dioxol-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)acetamide

[0090] To a solution of Example 1C (4.3 g) in acetic anhydride (25 mL) was added copper(II) nitrate trihydrate (4.76 g) portionwise at 0 °C. The mixture was stirred at 0 °C for 3 h. Three additional reactions were set up as described and stirred at 0 °C for 3 h. All four reactants were combined. The mixture was poured into water (50 mL) and extracted with ethyl acetate (5 × 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 75% ethyl acetate in petroleum ether to give the title compound. 1 H NMR (400 MHz, CDCl3) δ ppm 7.61 (s, 1H), 6.77 (br s, 1H), 6.64 (s, 1H), 6.21 (s, 2 H), 2.43 (s, 6 H). MS (APCI+) m / z 373.1 (M+H) + .

[0091]

Chem.

[0092] Example 1E N-(3-(6-Aminobenzo[d][1,3]dioxole-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)-2,2,2-trifluoroacetamide

[0093] To a solution of Example ID (4 g) in ethanol (40 mL) and water (8 mL) were added iron (5.4 g) and ammonium chloride (5.17 g) under nitrogen. The mixture was stirred at 100 °C for 3 h. Three additional reactions were set up as described and stirred at 100 °C for 3 h. After cooling to ambient temperature, all four reactants were combined. The mixture was poured into water (1 L) and extracted with ethyl acetate (5 × 500 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 10 - 75% ethyl acetate in petroleum ether to give the title compound. 1H NMR (400 MHz, CDCl3) δ ppm 7.22 (s, 1H), 6.70 (s, 1H), 6.50 (s, 2H), 6.14 (s, 1H), 5.92 (s, 2H), 2.63 (s, 6H). MS (ESI+) m / z 343.2 (M+H) + .

[0094] [ka]

[0095] Example 1F (S)-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-14-yl)bicyclo[1.1.1]pentan-1-yl)-2,2,2-trifluoroacetamide

[0096] To a suspension of Example 1E (3.5 g) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolidine-3,6,10(4H)-trione (2.69 g) in toluene (140 mL), para-toluenesulfonic acid monohydrate (1.945 g) was added. The mixture was stirred at 115 °C for 12 hours. Three additional reactions were set up as described and stirred at 115 °C for 12 hours. After cooling to ambient temperature, all four reactants were combined. The mixture was filtered, and the collected solid was pulverized with acetonitrile (200 mL) to obtain the labeled compound. 1 ¹H NMR (400 MHz, dimethyl sulfoxide -d6) δ ppm 10.28 (s, 1H), 7.64 (s, 1H), 7.52 (s, 1H), 7.24 (s, 1H), 6.47 (s, 1H), 6.30 (dd, 2H), 5.42 (s, 2H), 5.36 (s, 2H), 2.87 (s, 6H), 1.94 - 1.77 (m, 2H), 0.88 (t, 3H). MS (ESI+) m / z 570.3 (M+H) + .

[0097] [ka]

[0098] Example 1G (7S)-14-(3-aminobicyclo[1,1,1]pentan-1-yl)-7-ethyl-7-hydroxy-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-8,11(7H,13H)-dione

[0099] To a solution of Example 1F (3g) in methanol (30mL), HCl (60mL, 4M in methanol) was added. The mixture was stirred at 65°C for 4 hours. Four additional reactions were set up as described above and stirred at 65°C for 4 hours. After cooling to ambient temperature, all five reactants were combined. The mixture was concentrated under reduced pressure, and the residue was ground with methanol (200mL) to obtain the labeled compound. 1 ¹H NMR (400 MHz, dimethyl sulfoxide -d6) δ ppm 9.23 (s, 3H), 7.54 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.30 (d, 2H), 5.41 (s, 2H), 5.39 - 5.26 (m, 2H), 2.79 (s, 6H), 1.87 (hept, 2H), 0.88 (t, 3H). MS (ESI+) m / z 474.3 (M+H) + .

[0100] [ka]

[0101] Example 1H tert-butyl((S)-1-(((S)-1-((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-14-yl)bicyclo[1.1.1]pentan-1-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[0102] (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamide)propanoic acid (2.49 g), 2-hydroxypyridine 1-oxide (1.31 g), and N in acetonitrile (40 mL) 1 -((ethylimino)methylene)-N 3 ,N 3 To a suspension of dimethylpropane-1,3-diamine hydrochloride (2.26 g), 2,6-lutidine (2.74 mL) was added. The mixture was stirred at ambient temperature for 30 minutes. In a separate flask, Example 1G (4 g) and 2,6-lutidine (2.74 mL) were combined in N,N-dimethylformamide (40 mL), and the above solution was added. The mixture was stirred overnight at ambient temperature. The mixture was concentrated under reduced pressure, the residue was dissolved in dichloromethane (300 mL), washed with saturated aqueous solution of ammonium chloride (100 mL) and brine (100 mL), dehydrated with anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography of silica gel eluted with 0-10% methanol in dichloromethane to obtain the labeled compound. 1¹H NMR (400 MHz, dimethyl sulfoxide -d6) δ ppm 8.65 (s, 1H), 7.89 (d, 1H), 7.61 (s, 1H), 7.49 (s, 1H), 7.22 (s, 1H), 6.77 (d, 1H), 6.46 (s, 1H), 6.29 (d, 2H), 5.41 (s, 2H), 5.32 (s, 2H), 4.29 (q, 1H), 3.87 - 3.77 (m, 1H), 2.76 (s, 6H), 1.99 (q, 1H), 1.92 - 1.81 (m, 2H), 1.41 (s, 9H), 1.25 (d, 3H), 0.92 - 0.80 (m, 9H). MS (ESI+) m / z 744.4 (M+H) + .

[0103] [ka]

[0104] Example 1 (S)-2-amino-N-((S)-1-((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-14-yl)bicyclo[1.1.1]pentan-1-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[0105] Example 1H (5.5 g) was treated with trifluoroacetic acid (30 mL) at ambient temperature for 30 minutes. The mixture was concentrated under reduced pressure, and the residue was dissolved in 50% acetonitrile in water (200 mL). The solution was freeze-dried to obtain the labeled compound. 1¹H NMR (600 MHz, dimethyl sulfoxide-d6) δ ppm 8.80 (s, 1H), 8.59 (d, 1H), 8.10 (d, 3H), 7.60 (s, 1H), 7.48 (s, 1H), 7.23 (s, 1H), 6.33 - 6.26 (m, 2H), 5.41 (d, 2H), 5.35 - 5.23 (m, 2H), 4.35 (p, 1H), 3.66 - 3.63 (m, 1H), 2.77 (s, 6H), 2.17 - 2.06 (m, 1H), 1.91 - 1.83 (m, 2H), 1.31 (d, 3H), 1.01 - 0.96 (dd, 6H), 0.89 (t, 3H). MS (ESI+) m / z 644.4 (M+H) + .

[0106] [ka]

[0107] Example 1J (2S)-2-(2-bromoacetamide)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

[0108] To a solution of 2-bromoacetic acid (1.435 g) in N,N-dimethylformamide (26 mL), ethyl 2-ethoxyquinoline-1(2H)-carboxylate (2.55 g) was added. The mixture was stirred at ambient temperature for 10 minutes. In a separate flask, Example 1I (4.5 g) and 2,6-lutidine (3.61 mL) were combined in N,N-dimethylformamide (26 mL), and the above solution was added. The mixture was stirred at ambient temperature for 30 minutes. The mixture was acidified with trifluoroacetic acid (4 mL), and purified for 30 minutes by reverse-phase HPLC using a CombiFlash® Teledyne Isco system with a Luna® column (250 × 50 mm, 10 mm) and elution with 5-75% acetonitrile in water containing 0.1% trifluoroacetic acid. The compound was then lyophilized to obtain the marked compound. 1 H NMR (600 MHz, dimethyl sulfoxide-d6) δ ppm 8.57 (s, 1H), 8.32 (d, 1H), 8.16 (d, 1H), 7.67 (s, 1H), 7.52 (s, 1H), 7.24 (s, 1H), 6.30 (dd, 2H), 5.42 (s, 2H), 5.38 (d, 2H), 4.28 - 4.19 (m, 2H), 4.03 - 3.91 (m, 2H), 2.76 (s, 6H), 2.02 (h, 1H), 1.86 (ddp, 2H), 1.25 (d, 3H), 0.93 - 0.84 (m, 9H). MS (ESI+) m / z 764.46 (M+H) + .

[0109] [Example 2] Preparation and purification of antibodies mAb1, known as mAb hu GPC3(LC:C214A)[hu IgG1 / k]z,non-a, is a fully human IgG1 monoclonal antibody with a C214A mutation at the C-terminus of the light chain, enabling site-specific and controlled linker-drug conjugation of the heavy chain to cysteine ​​220. The discovery and modification of mAb1 are described below.

[0110] AMM-K C57BL / 6 mice (AlivaMab® Biologics, San Diego, CA, USA) possessing a human variable region were immunized with HEK-293 cells expressing cynomolgus monkey ("cyno") GPC3 (293H-cyno GPC3). After injection, plasma from the immunized mice was screened by ELISA for binding to human GPC3.

[0111] Splenocytes were collected from mice whose plasma tested positive for human GPC3 antibody, fused with NS0 cells to generate hybridomas, and the hybridoma supernatant was screened for binding to endogenously expressed GPC3 in HepG2 cells, HEK-293H cells expressing human GPC3, or HEK-293H cells expressing cynomolgus monkey GPC3. A total of 57 hits were provided for VH / VL sequence analysis from this screening. Following sequence analysis and refinement of the matrix assays performed, 27 antibodies were selected for recombinant production on a 35 mL scale and provided for MMAE conjugation in a secondary cell death assay against HepG2 cells.

[0112] Recombinant proteins were screened using cell-based binding assays, cell death assays, and Biacore® assays to determine which clones to scale up. After final clone selection, the GPC3 antibody was re-cloned with human IgG1 and designated as mAb1.

[0113] mAb1 was expressed in Chinese hamster ovary (CHO) cells, similar to IgG1 antibodies having the heavy chain and light chain sequences of SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0114] [Example 3] In vitro binding to GP3 The mAb1 binding affinity was characterized for both the recombinant extracellular domain (ECD) and cell surface GPC3. The binding affinity was compared with that of GC33 (codlituzumab) that binds to human GPC3 (Zhu et al., 2013, First-in-man phase I study of GC33, a novel recombinant humanized antibody Against Glypican-3, in patients with advanced hepatocellular carcinoma. Clin. Cancer Res. DOI:10.1158 / 1078-0432. CCR-12-2616).

[0115] ELISA showed that mAb1 and GC33 bind to recombinant human GPC3 extracellular domain (ECD) in the same manner, respectively. 50 GC33 has an affinity of 0.59 vs. 0.69 nM, similar to the affinity observed with cynoGPC3. In contrast, binding affinity measured by FACS and SPR (Biacore) showed a significant difference between mAb1 and GC33. According to FACS, GC33 bound to cell surface GPC3 of four different cell types with higher affinity than mAb1. A more dramatic difference was observed using SPR, showing that GC33 has a higher affinity than K D It had a molecular weight of 2.9 nM, compared to 121 nM for mAb1 (Table 1). In summary, these data indicate that GC33 has a higher affinity for GPC3 than for mAb1.

[0116] [Table 1]

[0117] method: Cell culture: All cells were cultured at 37°C in 5% CO2. HCC cell lines HepG2, Hep3B, and SK-Hep1 were obtained from the American Type Culture Collection (ATCC). HepG2 cells were maintained in MEM (Gibco, Cat. No. 11095-080) supplemented with 10% fetal bovine serum (FBS; Gibco, 10082-147). To produce SK-Hep1 cells that stably express GPC3 cells, full-length human GPC3 (NM_004484.4) encoding DNA was synthesized using GeneWiz and subcloned into a pLVX-IRES-Puro lentiviral vector (Clontech, Cat. No. 632183). Lentiviruses were generated by transfecting LentiX-293T cells (Clontech, Cat. No. 632180) with either a pLVX-IRES-Puro empty vector or pLVX-IRES-Puro containing human GPC3, using Lenti-X packaging single shots (Clontech, Cat. No. 631276). The virus-containing supernatant was collected and transduced into SK-Hep1 cells using a Lenti-X accelerator (Clontech, Cat. No. 631256). After transduction, the cells were treated with 1 μg / mL puromycin (Sigma, 540411) and sorted by FACS to generate stable empty vectors or GPC3-positive cells. SK-Hep1 empty vectors and SK-Hep1-GPC3 cells were maintained in MEM (Gibco, 10082-147) supplemented with 10% FAB and 1 μg / mL puromycin (Sigma, 540411-puromycin was removed during the purification assay).

[0118] HEK-293 cells were obtained from ATCC and maintained in RPMI basal medium supplemented with 10% FBS. To generate HEK-293 cells overexpressing human GPC3 or GPC3 from a potentially toxic species, full-length human (NM_004484.4), cynomolgus monkey (XM_005594608.4), mouse (NM_016697.3), or rat (NM_012774.2) GPC3-encoding DNA was synthesized and subcloned into pLVX-IRES-Puro lentiviral vectors using GeneWiz. Following the same protocol as for SK-Hep1 cells described above, including selection of GPC3-positive cells after sequential treatment with 1 μg / mL puromycin (Sigma, 540411), lentiviral generation and transduction of HEK-293 cells followed. All cells were cultured at 37°C in 5% CO2.

[0119] HCC cell lines were dissociated from storage flasks using 5 mL of non-enzymatic cell dissociation solution in PBS (Sigma, C5914-100ML). Before counting viable cells, the cells were filtered through a 20 μM pre-separation filter (Miltenyi Biotec, Cat. No. 130-101-812). 1000 cells were seeded into each well of a black 96-well plate (Corning, 3904) or into 300 cells / well of a white 384-well plate (Corning, 3765), and rotated briefly for 10 seconds at 800 rpm. In the three-dimensional amplification assay, 5000 cells were seeded into each well of a black / clear round-bottom ultra-low-adhesion surface spheroid 96-well microplate (Corning, 4520), and rotated briefly for 10 seconds at 800 rpm. Using a Tecan D300e Digital Dispenser, a 3x dose-response was applied to cells using a storage concentrate of ADC containing a surfactant (0.3% Tween20) or DMSO solution for the payload, and the cells were incubated (ADC and payload) for a further 10 days at 37°C in 5% CO2. The dose-response was initiated with 100 nM of ADC. Samples were run in duplicate. Cell viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Cat. No. G7573) according to the manufacturer's instructions, except that the plate was shaken for 30 minutes before reading. Luminescence was read using BioTek Synergy Neo2, and the data were analyzed using GraphPad Prism software. Inhibition percentages were calculated compared to positive control (staurosporine) and negative control (vehicle treatment). The calculations used were as follows: 100 * (Vehicle-ADC) / (Vehicle-Staurosporine). The maximum half-dose inhibitory value (IC50) was calculated using GraphPad Prism Software in n=3 independent biological replicates.

[0120] HEK-293 parent cells and stable GPC3 cells were washed with 5 mL of PBS-based non-enzymatic cell dissociation solution (Sigma, C5914-100 mL) at 37°C for 5 minutes. Medium was added to the dissociated cells and the cells were counted. 300 cells were seeded into each well of a white 384-well plate and rotated briefly at 800 × g for 10 seconds. All plates were treated in the same manner as described above for a total of 3 days.

[0121] Enzyme-linked immunosorbent assay (ELISA) binding: For mAb1 and ADC-1-binding ELISAs, 96-well Pierce® nickel-coated plates (ThermoFisher) were coated with 0.1 μg / ml of His-tagged human, cyno, mouse, or rat GPC3 extracellular domain (ECD) at 50 μl / well for 1 hour under ambient conditions. After incubation with GPC3 ECD, the plates were washed three times with phosphate-buffered saline (PBS-T) containing 0.05% Tween 20 and blocked at room temperature for 1 hour with 300 μl of 2% goat serum / phosphate-buffered saline (Jackson ImmunoResearch, West Grove, PA, USA). After 1 hour of blockage, the plates were washed three times again with PBS-T, and then a 1:3 dose titration of ADC / AB in blocking buffer starting at 66.67 nM was added to the wells in a double dose titration under ambient conditions for 1 hour. The plates were washed three times again with PBS-T and incubated with a barrier buffer (Jackson Immuno) containing 1:10,000 HRP goat anti-human IgG at room temperature for 30 minutes. After washing the plates three times with PBS-T, 50 ul / well of Ultra TMB substrate (ThermoFisher) was added, and the plates were incubated for 5-10 minutes under protection from light. Then, 50 ul / well of 1N NCl STOP solution was added, and the absorbance OD was measured. 450 Reads were measured using a Synergy Neo2 plate reader (Biotek Instruments, Winooski, VT, USA) with Gen5 3.10 software.

[0122] Surface Plasmon Resonance (SPR) Assay: The binding kinetics of GPC3 in humans, cynomolgus monkeys, mice, and rats were measured using Biacore® T200 SPR Instruments (Cytiva, Marlborough, MA, USA) as an analyte (Cytiva) conjugated to mAb1 and its ADC as a ligand. The assay format was based on the capture of a crystallizable antibody effector region (Fc) immobilized with anti-human Fc (Pierce ThermoFisher). Using a standard amine coupling protocol, the capture reagent was immobilized via a primary amine on the carboxymethyl dextran surface of the CM5 sensor tip (Cytiva), and the capture antibody was coupled at a level of approximately 2000 relative units (RU). For binding kinetics measurements, the assay buffer was HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20). During the assay, all measurements referenced only the capture surface. Each assay cycle consisted of the following steps: (1) capturing the ligand at approximately 80 RU; (2) injecting 240 μL of the analyte at 80 μL / min into both the reference and test surfaces, and then monitoring dissociation at 80 μL / min for 15 minutes; (3) rehydrating the capture surface with 10 mM glycine, pH 1.5. For kinetic determination, the analyte injections were 3-fold dilutions starting at 900 nM to arrive at a total of five concentrations, including an injection of buffer only for secondary reference. The data were processed and fitted to a 1:1 binding model using Biacore® T200 Evaluation Software to determine the binding kinetic rate constant k a (on-rate) and k d (Off-rate), and the equilibrium dissociation constant (affinity, K) D ) was decided.

[0123] Fluorescence-activated cell sorting (FACS) analysis: Cells used for FACS included HEK-293 stably transduced into human, cynomolgus monkey, rat, or mouse GPC3 cells. Cells were collected using 5 mL of enzyme-free cell dissociation buffer (MilliporeSigma) and counted using a ViCell XR cell viability analyzer (BeckmanCoulter, Indianapolis, IN, USA). Minimum viability was 2 × 10⁶. 6 Cells / mL were used. The cells were then centrifuged at 800×g for 5 minutes and resuspended in cold FACS buffer (5% FBS / PBS with 0.1% sodium azide). 50 μL of 2× concentration mAb / ADC (final concentration shown in figure) was added to a U-bottom 96-well plate. 50 μL of cells (100,000 cells) were added and the plate was incubated at 4°C for 1 hour. Next, each well was washed three times with 150 μL of cold FACS buffer by centrifugation at 320×g for 5 minutes. After the third wash, the cells were resuspended in 50 μL of FACS buffer containing 8 μg / mL of Alexa Fluor488 goat anti-human IgG (H+L) secondary (Invitrogen ThermoFisher) and 1:1000 live / dead exclusion dye (ThermoFisher). Cells were mixed and incubated in the dark at 4°C for 1 hour. The cells were then washed three times with FACS buffer and fixed in 100 μL of cold PBS with 1% formaldehyde (Polysciences, Niles, IL, USA). Cells were analyzed using a Becton Dickinson FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo® v10.10 flow cytometry software (Becton Dickinson).

[0124] [Example 4] Preparation of ADC-1 As described below, mAb1 was conjugated with a linker drug (Example 1J) to form the antibody-drug conjugate ADC-1.

[0125] The mAb1 antibody from Example 2 was partially reduced with an excess amount of reducing agent and then conjugated with an excess amount of drug linker. This yielded a final pharmaceutical product containing an ADC mainly comprising two drug molecules, i.e., an ADC of formula (IV) where n is 2 and DAR is 2 or approximately 2.

[0126] A 4°C solution of mAb1 antibody was treated with PBSE (dipotassium phosphate / sodium chloride / ethylenediaminetetraacetic acid buffer; 125 mM potassium phosphate, 150 mM NaCl, 6.3 mM EDTA, pH 7.2) to achieve a final concentration of 0.1 mM. Tris(2-carboxyethyl)phosphine (TCEP, 10 mM, 10 molar equivalents, Bond Breaker®, ThermoFisher) was added to the mAb1 antibody solution (approximately 10-20 mg / mL in 0.5 × PBSE) for 5 minutes while gently mixing the plate (225 rpm). After incubation at 4°C for 20 hours, the solution was brought to 22°C.

[0127] Pellicon(registered trademark)3 0.57m 2 Ultrafiltration / diafiltration (UFDF) was applied to the exchange of reduced Ab antibody buffer into PBSE buffer using a cassette (MilliporeSigma, Burlington, MA, USA). Ultrafiltration (UF) was performed until the mAb antibody concentration reached 40 mg / mL. Diafiltration (DF) was performed at 10 diavolumes (DV) of PBSE.

[0128] 6.6 molar equivalents of the linker drug (Example 1J) in a 10 mM solution of N,N-dimethylacetamide were incubated at 23°C for 16 hours with gentle plate mixing. After incubation, 8.0 equivalents of 100 mM N-acetyl-L-cysteine ​​(NAC, MilliporeSigma), prepared with sterile purified water, were added to the solution, gently mixed, and then incubated at 23°C for 2 hours.

[0129] Pellicon(registered trademark)3 0.57m 2 Ultrafiltration and diafiltration were performed using a cassette (MilliporeSigma, Burlington, MA, USA) to exchange the buffer of the obtained antibody-drug conjugate (ADC-1) with a mixture of dimethyl sulfoxide and 15 mM histidine buffer at pH 6, followed by 15 mM histidine buffer. The ADC-1 solution was collected and then further diluted with buffer. The final ADC-1 solution was sterile filtered through a 0.22 μM filter and stored at -80°C.

[0130] [Example 5] Comparison of the effectiveness of TOP1i drug and linker drug construct The antiproliferative activity of mAb1 and GC33 as antibody-drug conjugates was tested after conjugation to the linker drug of Example 1J using MSL-109ADC (anti-CMV antibody; Dobryski et al., 1991) as a non-targeting control. Human GPC3-expressing SK-Hep1, HepG2, and Hep3B cells in two-dimensional cell culture were treated with a 1:3 dilution of DAR=2 or DAR=6 type ADC.

[0131] As shown in Tables 2-4 and Figure 1A, GC33 ADCs (DAR2 and DAR6) exhibited greater antiproliferative activity than mAb1 ADCs against all three GPC-expressing cell lines when grown in a monolayer (two-dimensional) structure. As expected, MSL109 ADCs showed little to no activity.

[0132] [Table 2]

[0133] [Table 3]

[0134] [Table 4]

[0135] GC33 ADCs of type DAR=2 and mAb1 ADCs of type DAR=2 (ADC-1) were also tested in a spheroid three-dimensional cell culture system. Spheroid three-dimensional cultures are produced by culturing cancer cell lines on non-adherent biological plates, thereby forming spheroids from cell aggregates. Cancer cell line spheroids are hypothesized to better mimic the structural and physiological characteristics of tumors in terms of drug delivery and therapeutic response compared to two-dimensional conditions. (Kapalczynska et al., 2D and 3D cell cultures—a comparison of different types of cancer cell cultures. Arch Med Sci. June 2018; 14(4): pp. 910-919, doi: 10.5114 / aoms.2016.63743).

[0136] The antiproliferative activity of DAR2 GC33 ADC, ADC-1(DAR2), and DAR2 MSL ADC was tested in human GPC3-expressing SK-Hep1, HepG2, and Hep3B cells grown as spheroids in three-dimensional cell culture. Surprisingly, ADC-1 and GC33 ADC demonstrated similar antiproliferative activity against cells grown in three dimensions (Table 5, Figure 1B). This result was unexpected considering the excellent inhibition (Tables 2-4, Figure 1A) and cell binding (Table 1) of GC33 ADC in two-dimensional cell culture systems.

[0137] [Table 5]

[0138] Finally, the antitumor activity of ADC-1 (DAR2) and DAR2 GC33 ADC was compared in a human HCC Hep3B cell line-derived xenograft (CDX) mouse model. Three million cells were subcutaneously inoculated into the right flank of female CB17 SCID mice. The mouse tumors were size-matched 21 days after inoculation. The average tumor volume after size matching was approximately 215 mm². 3 The range is 159-271mm. 3 It was within the range.

[0139] ADC-1 or DAR2 GC33 were administered intraperitoneally as a single bolus at low or medium doses and compared to untargeted MSL ADC controls ("ADC-2"). Similar tumor growth inhibition and tumor growth delay were observed in mice treated with either ADC-1 or DAR2 GC33, as shown in Figure 1C (low dose) and Figure 1D (medium dose).

[0140] [Example 6] Testing of GC33 ADC, ADC-1 and ADC-1a in non-human primates To determine the toxicity and toxicological effects of ADC, a study was conducted in non-human primate (NHP) cynomolgus monkeys administered Q3W (two doses) over a four-week period.

[0141] Based on other ADCs using linker drugs as shown in Example 1J, ADCs with a DAR of 6 were expected to exhibit superior efficacy and safety at medium doses. However, treatment with medium-dose DAR6 GC33 ADCs resulted in severe adverse events, including gastrointestinal epithelial cytolysis, and death of test animals by day 9 (Table 6). Similar toxicity was observed with low-dose DAR2 GC33 ADCs, resulting in death of test animals by day 9.

[0142] In contrast, ADC-1 (DAR2 mAb1 ADC) was well tolerated at both low and moderate doses, and no significant clinical signs were observed.

[0143] Considering its excellent tolerability and comparable efficacy, ADC-1 was selected for further in vivo studies.

[0144] [Table 6]

[0145] [Example 7] Generation of tumor-carrying mice and determination of tumor volume of subcutaneous abdominal tumors To generate cell line-derived xenografts, tumor cells were subcutaneously inoculated into the right flank of female CB17 SCID mice (Charles River Laboratories, Wilmington, MA, USA) using NCI-H1581 cells (ATCC) or Hep3B cells, or into the right flank of female SCID-Beige mice (Charles River) using HepG2 cells. The injection volume was 0.1 mL and consisted of cells suspended in a 1:1 mixture of S-MEM (ThermoFisher) and Matrigel (Becton Dickenson, Franklin Lakes, NJ, USA). Animals with tumors were 158–335 mm before treatment. 3 The volume was randomized within the range of [specify range].

[0146] To generate LI1068 and LI6610 in vivo PDX models, tumors derived from the aforementioned lineage were excised from mice, and tumor fragments with a diameter of 2-3 mm were subcutaneously transplanted into the right flank of female Balb / c nude mice (GemPharmatech, Nanjing, CN). The tumor-bearing animals were 101-151 mm in diameter. 3 The volume was randomized within the range of [specify range].

[0147] Treatment was initiated within 24 hours of randomization of tumor-bearing animals to the required cohort. Tumor volume was estimated once or twice weekly. Tumor length (L) and width (W) measurements were obtained using an electronic caliper, and the volume was calculated according to the following equation: V = (L × W) 2 ) / 2.

[0148] In the following study, ADC-2 refers to a DAR2 ADC using MSL109 conjugated to a linker drug (Example J1), and is used as a non-targeted control antibody / negative control ADC.

[0149] Study 1. Inhibition of proliferation of xenotransplanted human HSCLC (NCI-H1581) by various doses of ADC-1. NCI-H1581 cells were grown in vitro for 3 passages. 5 million cells per mouse were subcutaneously inoculated into the right flank of female CB17 SCID mice. The mouse tumors were size-matched 13 days after inoculation. The average tumor volume after size matching was approximately 211 mm². 3 It is 158-335mm 3 It was within the range.

[0150] The efficacy of ADC-1 was determined by subcutaneous xenografts of NCI-H1581 cells derived from human NSCLC tumor samples. ADC-1 or ADC-2 was administered intraperitoneally as a single bolus to eight mice at low (1×), intermediate (2×), and high (3×) dose levels. Compared to vehicle controls, statistically significant (p<0.05) TGI (85%, 92%, and 95%, respectively, on day 11 post-administration) and TGD (100%, 218%, and 318%, respectively) were induced at all dose levels tested. Intermediate-dose ADC-1 induced an OR of 63% (63% PR), while high-dose ADC-1 induced a 100% OR (50% CR, 50% PR) (Table 7). Mice tolerated all treatments.

[0151] [Table 7]

[0152] Study 2. Inhibition of xenograft-transplanted human HCC (Hep3B) proliferation by various doses of ADC-1. Hep3B cells were grown in vitro for 3 passages. 3 million cells per mouse were subcutaneously inoculated into the right flank of female CB17 SCID mice. The mouse tumors were size-matched 21 days after inoculation. The average tumor volume after size matching was approximately 215 mm². 3 The range is 159-271mm. 3 It was within the range.

[0153] The efficacy of ADC-1 was determined by subcutaneous xenografts of Hep3B cells derived from human hepatocellular carcinoma. ADC-1 was administered intraperitoneally as a single bolus at low (1×), intermediate (2×), and high (3×) doses, inducing statistically significant (p<0.05) TGI (54%, 84%, and 98%) and TGD (52%, 295%, and >400%, respectively) at 24 days post-administration compared to vehicle controls at all dose levels tested. Intermediate dose levels of ADC-1 induced an OR of 75% (63% CR, 13% PR), while high dose levels induced a 100% OR (100% CR) (Table 8). Mice tolerated all treatments.

[0154] [Table 8]

[0155] Study 3. Inhibition of xenografted human HCC (HepG2) proliferation by various doses of ADC-1. HepG2 cells were grown in vitro for three passages. Five million cells per mouse were subcutaneously inoculated into the right flank of female SCID-Beige mice. The mouse tumors were size-matched 15 days after inoculation. The average tumor volume after size matching was approximately 226 mm². 3 It is 191-256mm 3 It was within the range.

[0156] The efficacy of ADC-1 was determined using subcutaneous xenografts of human HCC-derived HepG2 cells. ADC-1 was administered intraperitoneally as a single bolus at low (1×), intermediate (2×), and high (3×) doses, inducing statistically significant (p<0.05) TGI (58, 64, and 84%) and TGD (>50, 128, and 244%) at day 14 post-administration compared to vehicle controls at all dose levels tested. At high dose levels, ADC-1 treatment induced a 20% OR (20% PR) (Table 9 and Figure 3). Mice tolerated all treatments.

[0157] [Table 9]

[0158] Study 4. Inhibition of patient-derived xenograft (LI1068) proliferation by various doses of ADC-1. After proliferation in the right ventral region of stock mice, tumor fragments of LI1068, approximately 2–3 mm in diameter, were subcutaneously transplanted into the right ventral region of female Balb / c nude mice. The tumors were size-matched 37 days after inoculation. The average tumor volume after size matching was approximately 132 mm². 3 And, 101~151mm 3 It was within the range.

[0159] The efficacy of ADC-1 was determined in a subcutaneous PDX model of hepatocellular carcinoma. ADC-1 was administered intraperitoneally as a single bolus to 5 mice per group at either a low (1×) or high (3×) dose. Compared to vehicle controls, high-dose ADC-1 induced statistically significant (p<0.05) TGI (83% at 18 days post-administration) and TGD (382%), with a 40% complete response rate observed (Table 10 and Figure 4). Mice tolerated all treatments.

[0160] [Table 10]

[0161] Study 5. Inhibition of patient-derived xenograft (LI6610) growth by various doses of ADC-1. After proliferation in the right ventral region of stock mice, tumor fragments of LI6610, approximately 2–3 mm in diameter, were subcutaneously transplanted into the right ventral region of female Balb / c nude mice. The tumors were size-matched 20 days after inoculation. The average tumor volume after size matching was approximately 119 mm². 3 And, 103~135mm 3 It was within the range.

[0162] The efficacy of ADC-1 was determined in a subcutaneous PDX model of HCC LI6610. ADC-1 was administered intraperitoneally as a single bolus at low (1×) or high (3×) doses, inducing statistically significant (p<0.05) TGI (49% and 74%, respectively) and TGD (24% and 147%, respectively) at day 21 post-administration compared to vehicle controls (Table 11 and Figure 5). Mice tolerated all treatments.

[0163] [Table 11]

[0164] [Table 12]

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Claims

1. Formula (IV): 【Chemistry 1】 [In the formula, n is an integer from 1 to 6, and Ab is an anti-GPC3 antibody comprising a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3, and a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3.] CDR-H1 has the amino acid sequence shown as Sequence ID No. 3, CDR-H2 has the amino acid sequence shown as Sequence ID No. 4, CDR-H3 has the amino acid sequence shown as Sequence ID No. 5, CDR-L1 has the amino acid sequence shown as Sequence ID No. 6, CDR-L2 has the amino acid sequence shown as Sequence ID No. 7, CDR-L3 has the amino acid sequence shown as Sequence ID No.

8. Anti-GPC3 antibody drug conjugate.

2. The anti-GPC3 antibody drug conjugate according to claim 1, wherein the antibody is an IgG1 antibody.

3. The aforementioned antibody (a) A heavy chain variable region having the amino acid sequence shown as Sequence ID No. 1, and (b) Light chain variable region having the amino acid sequence shown as Sequence ID No. 2 The anti-GPC3 antibody drug conjugate according to claim 1, comprising:

4. The aforementioned antibody (a) Heavy chains having amino acids as shown in SEQ ID NO: 9 or SEQ ID NO: 11, and (b) Light chain having the amino acid sequence shown as Sequence ID No. 10 The anti-GPC3 antibody drug conjugate according to claim 1, comprising:

5. Formula (IV): 【Chemistry 2】 [In the formula, n is an integer from 1 to 6, and Ab is an anti-GPC3 antibody comprising two heavy chains having the amino acid sequences shown as SEQ ID NO: 9 and two light chains having the amino acid sequences shown as SEQ ID NO: 10.] Anti-GPC3 antibody drug conjugate.

6. An anti-GPC3 antibody drug conjugate according to any one of claims 1 to 5, wherein n is 2.

7. An anti-GPC3 antibody drug conjugate according to any one of claims 1 to 5, wherein n is 1.

8. An anti-GPC3 antibody drug conjugate according to any one of claims 1 to 5, wherein n is 4.

9. An anti-GPC3 antibody drug conjugate according to any one of claims 1 to 5, wherein n is 6.

10. A composition comprising a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 5, wherein the main species of the antibody-drug conjugate in the composition is n = 2.

11. A pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient, wherein the DAR is 2 or about 2.

12. A method for treating hepatocellular carcinoma, comprising administering a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 9 to a patient in need of treatment.

13. A method for treating hepatocellular carcinoma, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 11 to a patient in need of treatment.

14. One or more nucleic acids encoding an antibody comprising the heavy chain sequence of SEQ ID NO: 9 or SEQ ID NO: 11 and the light chain sequence of SEQ ID NO: 10.

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