Antibody-drug conjugates and anticancer agents

Abstract

The present invention provides a drug that can reduce the concentration of divalent cations within cancer cells and can specifically reach cancer cells. [Means] An antibody-drug conjugate comprising an antibody having properties to bind to cancer cells, a bisphosphonate preparation, and a crosslinking agent connecting them.

Description

Technical Field

[0001] The present invention relates to an antibody-drug conjugate and an anticancer agent.

Background Art

[0002] The homeostasis of intracellular calcium concentration is controlled by calcium channels and calcium pumps on the cell membrane and endoplasmic reticulum membrane. A decrease in the calcium concentration in the lumen of the endoplasmic reticulum causes endoplasmic reticulum stress and leads to disruption of cell function (Non-Patent Document 1). Therefore, if the intracellular calcium ion concentration can be decreased in cancer cells, the progression of cell division in cancer cells can be retarded.

[0003] One of the reasons why cancer cells are resistant to radiotherapy is chromatin aggregation, and the aggregated chromatin protects DNA. Since the phosphate groups contained in the structural units of DNA are negatively charged, when divalent cations (calcium ions, magnesium ions) are present in the cell, chromatin aggregation occurs, and DNA is protected from attacks by hydroxy radicals (active hydrogen, ·OH) generated by radiation and anticancer agents (Non-Patent Document 2).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] Based on the above-mentioned reports regarding the roles of calcium and magnesium ions within cells, the inventors hypothesized that if the concentration of divalent cations such as calcium and magnesium ions could be reduced within cancer cells, it would be possible to disrupt cancer cells or suppress chromatin aggregation, thereby proving effective in radiation therapy and chemotherapy.

[0006] However, it is difficult to specifically deliver drugs that reduce the concentration of divalent cations to cancer cells. In order for the drug to reach cancer cells sufficiently, it is necessary to administer an excessive amount of the drug to the body, which is a burden on the body.

[0007] Therefore, the object of the present invention is to provide a drug that can reduce the concentration of divalent cations in cancer cells and that can specifically reach cancer cells. In this specification, the term "cancer cells" includes both ordinary cancer cells and cancer stem cells. Furthermore, in this specification, the "divalent cation" is preferably a metal cation, specifically a calcium ion, a magnesium ion, or an iron ion, with calcium ions and magnesium ions being preferred, and calcium ions being more preferred. [Means for solving the problem]

[0008] As a result of diligent research into the above-mentioned problems, the inventors of this invention have obtained the following findings. • Bisphosphonate preparations, which are formulations that are taken up by osteoclasts and induce apoptosis in osteoclasts, thereby suppressing bone resorption by osteoclasts and increasing bone density, can be used as divalent cation supplements (ionic reaction or chelation reaction). A novel antibody-drug conjugate, prepared by linking an antibody having the property of binding to cancer cells with the above-mentioned bisphosphonate preparation via a predetermined crosslinking agent, specifically reaches cancer cells. The novel antibody-drug conjugate structure exhibits a divalent cation capture function within cancer cells without dissociating from the antibody (without cleavage of the crosslinking agent (linker) between the antibody and the drug).

[0009] Based on the above findings, the inventors have completed the following invention. [1] An antibody-drug conjugate comprising an antibody having properties to bind to cancer cells, a bisphosphonate preparation, and a crosslinking agent connecting them.

[0010] [2] The antibody-drug conjugate according to [1], wherein the bisphosphonate preparation is a compound having the following structure.

[0011] [ka]

[0012] [3] The antibody-drug conjugate according to [1] or [2], wherein the antibody having the property of binding to cancer cells is any of the following: anti-EGFR antibody, anti-VEGFR antibody, anti-HER2 antibody, anti-HER3 antibody, anti-FGFR antibody, anti-IGFR antibody, anti-HGFR antibody, anti-PDGFR antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD33 antibody, anti-CD52 antibody, anti-CEA antibody, anti-EpCAM antibody, anti-Nectin-4 antibody, anti-TROP-2 antibody, or anti-PD-L1 antibody.

[0013] [4] The antibody-drug conjugate according to any one of [1] to [3], wherein the crosslinking agent is any one of Sulfo-GMBS, Sulfo-EMCS, Sulfo-HMCS, Sulfo-KMUS, Sulfo-SMCC, Sulfo-AC5-SPDP, and DTSSP.

[0014] [5] An anticancer agent containing the antibody-drug conjugate according to any one of [1] to [4] as an active ingredient.

Advantages of the Invention

[0015] According to the antibody-drug conjugate of the present invention, the drug can be specifically delivered to cancer cells, and the drug can reduce the concentration of divalent cations in cancer cells. Thereby, cancer cells can be disrupted, or the effectiveness of radiotherapy and anticancer drug treatment can be improved by suppressing chromatin aggregation.

Brief Description of the Drawings

[0016] [Figure 1] Figure 1 is a conceptual diagram of the antibody-drug conjugate (ADC) of the present invention. [Figure 2] Figure 2 shows the results of SDS-PAGE analysis of each sample. [Figure 3] Figure 3 shows the results of absorbance photometric analysis of TNBSA and the reaction product of Vectibix and TNBSA. [Figure 4] Figure 4 shows the results of absorbance photometric analysis of DTNB and the reaction product of Vectibix and DTNB. [Figure 5] Figure 5 shows the DLS measurement results upon addition of calcium ions to Sample 22-008-2 (1 μM). (a) shows the particle size (nm) on the horizontal axis and the frequency (%) on the vertical axis, and (b) shows the concentration of calcium ions (μM) on the horizontal axis and the particle size (nm) of the main peak on the vertical axis. [Figure 6] Figure 6 shows the results of absorbance photometric analysis of the samples measured by DLS. [Modes for carrying out the invention]

[0017] <Antibody-drug conjugate> As shown in the conceptual diagram in Figure 1, the antibody-drug conjugate of the present invention comprises an antibody 10 having the property of binding to cancer cells, a bisphosphonate preparation 20, and a crosslinking agent 30 that connects them. In other words, the present invention is an antibody-drug conjugate (ADC) having a novel structure unlike anything seen before.

[0018] The antibody-drug conjugate of the present invention has the property that antibody 10 binds to an antibody-binding site present on cancer cells. Therefore, the antibody-drug conjugate of the present invention specifically reaches cancer cells, is taken up by cancer cells via endocytosis, and exerts a chelating effect on divalent cations present in the cells.

[0019] (Outline of intracellular calcium dynamics, centered on the endoplasmic reticulum) Intracellular calcium concentration homeostasis is regulated by calcium channels and calcium pumps on the cell membrane and endoplasmic reticulum membrane. The endoplasmic reticulum acts as a calcium reservoir in cells, storing calcium at a concentration approximately 10,000 times higher than that of the cytosol. Transient calcium release from the endoplasmic reticulum to the cytosol acts as a second messenger for various biological processes. However, excessive increases in cytosolic calcium concentration are harmful to cells and are rapidly removed into the extracellular space or endoplasmic reticulum lumen. The calcium pump SERCA2 is responsible for the uptake of calcium into the endoplasmic reticulum lumen on the endoplasmic reticulum membrane. SERCA2 dysfunction leads to increased calcium levels in the cytosol and decreased calcium concentration in the endoplasmic reticulum lumen. Many enzymes and molecular chaperones that function in the lumen of the endoplasmic reticulum require binding with calcium ions, and a decrease in calcium concentration in the lumen of the endoplasmic reticulum causes endoplasmic reticulum stress, leading to a breakdown of cellular function.

[0020] The antibody-drug conjugate of the present invention can be taken up by cancer cells and chelate divalent cations such as calcium ions, thereby causing a decrease in intracellular calcium concentration and, as described above, disrupting the cellular function of cancer cells.

[0021] (Inhibitory effect on chromatin aggregation) Chromatin is a fibrous substance formed when DNA is wrapped around nucleosomes. Because the phosphate groups in the structural units of DNA are negatively charged, chromatin condensation occurs when divalent cations (calcium ions, magnesium ions) are present in the cell. When chromatin condensation occurs, the DNA is protected. Therefore, when chromatin aggregates in cancer cells, they become resistant to radiation therapy and chemotherapy.

[0022] The antibody-drug conjugate of the present invention, upon being taken up by cancer cells, chelates divalent cations, calcium ions and magnesium ions, thereby causing a decrease in intracellular calcium ion concentration and / or magnesium ion concentration. This inhibits the aggregation of chromatin in cancer cells, thereby improving the effectiveness of radiation therapy and anticancer drug therapy.

[0023] Furthermore, the novel antibody-drug conjugate structure of the present invention exerts chelating activity against divalent cations within cancer cells without dissociating from the antibody (without cleavage of the crosslinking agent (linker) between the antibody and the drug). Therefore, cleavage of the crosslinking agent within the cell is not necessarily required.

[0024] (Antibodies that have the property of binding to cancer cells) Antibodies that have the property of binding to cancer cells are antibodies that have the property of binding to the antibody-binding site on cancer cells, and any antibody with such properties can be widely used.

[0025] Various antibodies can be used as such, including antibodies against cell surface markers, growth factor receptors, and angiogenesis-related receptors classified according to the type of cancer cell. Examples include anti-EGFR antibodies, anti-VEGFR antibodies, anti-HER2 antibodies, anti-HER3 antibodies, anti-FGFR antibodies, anti-IGFR antibodies, anti-HGFR antibodies, anti-PDGFR antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD33 antibodies, anti-CD52 antibodies, anti-CEA antibodies, anti-EpCAM antibodies, anti-Nectin-4 antibodies, anti-TROP-2 antibodies, and anti-PD-L1 antibodies.

[0026] Among these, anti-EGFR antibodies, anti-VEGFR antibodies, anti-HER2 antibodies, and anti-HER3 antibodies are preferred. Examples of anti-EGFR antibodies include panitumumab and cetuximab, examples of anti-HER2 antibodies include trastuzumab, and examples of anti-VEGFR antibodies include ramucirumab.

[0027] In addition, in this invention, if the antibody has the property of binding to cancer cells, it is also possible to use antibody fragments, which are antibody fragments obtained by fragmenting an antibody. Examples of antibody fragments include F(ab')2, Fab', Fab,Fv, and rlgG, but among these, Fab' and rlgG are preferred because they contain SH groups. Conventional methods utilizing specific enzymes can be applied to produce antibody fragments from antibodies.

[0028] (Bisphosphonate preparations) Generally, bisphosphonates are analogs of pyrophosphate, have a high affinity for hydroxyapatite, and adsorb to the bone surface once absorbed into the body. When bisphosphonates are specifically taken up by osteoclasts during bone resorption, they inhibit farnesyl pyrophosphate synthase activity and induce apoptosis in osteoclasts. Bisphosphonates used in the treatment of osteoporosis exert their effects by suppressing bone resorption by osteoclasts and increasing bone density.

[0029] In this invention, instead of utilizing the action of the general bisphosphonate preparations described above, we deliberately utilize the ionic or chelate reaction that bisphosphonate preparations undergo with divalent cations. In other words, we utilize an action different from the main action of bisphosphonate preparations.

[0030] Bisphosphonate preparations are compounds having two phosphonate groups, and examples include sodium alendronate hydrate, disodium etidronate, minodronate hydrate, sodium risedronate hydrate, zoledronic acid hydrate, and disodium pamidronate hydrate. Each of these can be used as an oral or injectable preparation that is commercially available. In particular, from the viewpoint of binding with the crosslinking agent described below, it is preferable to use alendronate sodium hydrate or pamidronate disodium hydrate, which have an amino group. Furthermore, other bisphosphonate formulations can also be used in the present invention by introducing reactive groups (for example, amino groups or thiol groups) that interact with the crosslinking agent, and by utilizing existing synthetic reactions.

[0031] (Crosslinking agent (linker)) Antibodies and drugs are bound together via crosslinking agents. Antibodies contain disulfide bonds and amino groups. Disulfide bonds can be reduced to thiol groups. It is also possible to introduce thiol groups into amino groups in antibodies by adding Trout's reagent. The crosslinking agent has a site at one end that can bond to a thiol group or an amino group. Examples of sites to which a thiol group or an amino group can be bonded include maleimide groups and N-hydroxysuccinimide active esters. The antibody is bound to at least one crosslinking agent, and may be bound to multiple crosslinking agents. For example, the antibody may be bound to 2 to 8 crosslinking agents.

[0032] Furthermore, the other end of the crosslinking agent has a site that can bind to a bisphosphonate formulation. One example of a site that can bind to bisphosphonate formulations is N-hydroxysuccinimide active ester.

[0033] Specific examples of crosslinking agents include: For example, GMBS(N-(4-Maleimidobutyryloxy)succinimide), EMCS (N-(6-Maleimidocaproyloxy)succinimide), HMCS(N-(8-Maleimidocapryloxy)succinimide), KMUS(N-(11-Maleimidoundecanoyloxy)succinimide), DSP (Dithiobis(succinimidyl propionate) Dithiobis(succinimidyl undecanoate), Dithiobis (succinimidyl octanoate), Dithiobis (succinimidyl hexanoate), Sulfo-GMBS(N-(4-Maleimidobutyryloxy)sulfosuccinimide, Sulfo-EMCS(N-(6-Maleimidocaproyloxy)sulfosuccinimide, Sulfo-HMCS(N-(8-Maleimidocapryloxy)sulfosuccinimide, Sulfo-KMUS(N-(11-Maleimidoundecanoyloxy)sulfosuccinimide, Sulfo-SMCC(N-[(4-Maleimidomethyl)cyclohexylcarbonyloxy]sulfosuccinimide, Sulfo-AC5-SPDP (N-{6-[3-(2-Pyridyldithio)propionamido]hexanoyloxy}sulfosuccinimide), Examples include, but are not limited to, DTSSP (Dithiobis(sulfosuccinimidyl propionate)).

[0034] Furthermore, preferred crosslinking agents include Sulfo-GMBS, Sulfo-EMCS, Sulfo-HMCS, Sulfo-KMUS, Sulfo-SMCC, Sulfo-AC5-SPDP, and DTSSP.

[0035] <Method for manufacturing antibody-drug conjugates> The antibody-drug conjugate of the present invention can be manufactured by mixing the above-mentioned "antibody," "bisphosphonate preparation," and "crosslinking agent."

[0036] More specifically, the method for producing an antibody-drug conjugate of the present invention comprises (1) introducing a reaction group with a crosslinking agent into an antibody, (2) reacting a bisphosphonate formulation with a crosslinking agent, and (3) mixing the products obtained in these steps.

[0037] More specifically, the product can be prepared by reacting a bisphosphonate preparation (alendronate) with a crosslinking agent (Sulfo-AC5-SPDP) in a pH 7.5 buffer, then reacting an anti-EGFR antibody prepared in a pH 6.8 buffer with Trout's reagent, adding it to a component into which thiol groups have been introduced, and then stirring and mixing for 60 minutes at a temperature of 20-30°C or reacting at 4°C for 72 hours.

[0038] <Anticancer drugs> The anticancer agent of the present invention contains the antibody-drug conjugate described above as an active ingredient. Because the anticancer agent of the present invention has the property of being specifically taken up by cancer cells, it does not need to be administered to the body in large quantities as in conventional methods, thereby reducing the burden on the body. By administering the anticancer agent of the present invention, the anticancer agent is specifically taken up by cancer cells, and within the cells, it captures divalent cations through chelation or ionic reactions, thereby causing the cancer cells to rupture or inhibiting chromatin aggregation in cancer cells. [Examples]

[0039] <Preparing the sample> I prepared the following sample. • Sample A: IgG + Trout reagent (reacted at room temperature for 15 minutes, then stored in a cool place at 4°C (not ultrafiltered)) Sample B: IgG + Trout reagent (reacted at room temperature for 15 minutes, then ultrafiltration) Sample 22-008-1: IgG + Trout reagent + Alendronate bound to Sulfo-AC5-SPDP (reacted at room temperature for 1 hour, then ultrafiltration) Sample 22-008-2: IgG + Trout reagent + Alendronate bound to Sulfo-AC5-SPDP (reacted in a cool place for 72 hours, then ultrafiltration) Sample 22-008-3: IgG + Trout reagent + Alendronate bound to Sulfo-AC5-SPDP (reacted in a cool place for 72 hours, without ultrafiltration) The "alendronate bound to Sulfo-AC5-SPDP" mentioned above was obtained by reacting alendronate with Sulfo-AC5-SPDP at room temperature for 2 hours.

[0040] All samples were replaced with 20 mM HEPES (pH 7.4) by ultrafiltration.

[0041] <SDS-PAGE analysis of each sample> Sample buffer 2ME+ (×2) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and each of the above samples, adjusted to 0.5 mg / mL, were mixed in equivalent volumes and heat-treated at 95°C for 5 minutes. Subsequently, each sample was loaded to a concentration of 5 μg / lane and SDS-PAGE analysis was performed (reducing conditions). Sample buffer 2ME-(×2) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was mixed with each sample adjusted to 0.5 mg / mL in equivalent volume, and the mixture was heat-treated at 70°C for 10 minutes. Subsequently, each sample was loaded to a concentration of 5 μg / lane, and SDS-PAGE analysis was performed (non-reducing conditions). The results are shown in Figure 2. As shown in Figure 2, it was found that almost no cleavage or decomposition occurred even as the reaction proceeded.

[0042] <Quantification of reactive primary amine groups in anti-EGFR antibody (Vectibix)> In 0.1 M sodium bicarbonate buffer (pH 8.5), the Vectibix stock solution or each of the above samples was mixed to a concentration of 1 μM, and 2,4,6-trinitrobenzenesulfonic acid (TNBSA) to a concentration of 95 μM, and the mixture was reacted at 37°C for 2 hours. Next, sodium dodecyl sulfate (SDS) was added to a concentration of 2.2% by mass, and HCl was added to a concentration of 110 mM to stop the reaction.

[0043] As shown in the formula below, the reaction product of a primary amine and TNBSA exhibits an absorption peak at 420 nm, as shown in Figure 3.

[0044] [ka]

[0045] For each of the obtained solutions, the absorbance at 420 nm was measured, and the concentration of the primary amine that reacted with TNBSA was calculated. ε420 = 19,200 M -1 cm -1 I used it. The results are shown in Table 1.

[0046] [Table 1]

[0047] Table 1 shows that in the stock Vectibix solution, the number of lysine molecules that reacted with TNBSA in one Vectibix molecule was 7.0, but in each sample it ranged from 5.7 to 6.1, indicating that one primary amine of lysine reacted with Trout's reagent to form a thiol group.

[0048] <Quantification of thiol groups in anti-EGFR antibodies (Vectibix)> In 20 mM sodium phosphate buffer (pH 7.4), the stock solution of Vectibix or each of the above samples was mixed to a concentration of 1 μM, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was mixed to a concentration of 220 μM (five times the amount of lysine), and the mixture was reacted at 37°C for 1 hour.

[0049] As shown in the formula below, the reaction product of the thiol group and DTNB exhibits an absorption peak at 412 nm, as shown in Figure 4.

[0050] [ka]

[0051] For each solution obtained, the absorbance at 412 nm was measured, and the TNB concentration (= thiol group concentration in each sample) was calculated. ε412 = 15,500 M -1 cm -1 I used it. The results are shown in Table 2.

[0052] [Table 2]

[0053] Table 2 shows that in sample B, one thiol group was added to each of two molecules of Vectibix in reaction with Trout's reagent. In samples 22-008-1 to 22-008-3, these thiol groups were removed due to a reaction with alendronate. From this, it was found that one molecule of alendronate is bound to every two molecules of Vectibix.

[0054] <Calcium ion capture> For sample 22-008-2, dynamic light scattering (DLS) measurements were used to confirm whether aggregation occurred upon addition of calcium ions. Specifically, DLS measurements were performed on solutions containing sample 22-008-2 (1 μM) in 20 mM HEPES buffer (pH 7.4) with calcium ions added at concentrations of 0.1 μM, 1 μM, and 100 μM, to confirm the aggregation state. The results are shown in Figure 5.

[0055] As shown in Figure 5, it was confirmed that calcium ions were captured and aggregated over a wide range of calcium ion concentrations, from 0.1 μM to 100 μM.

[0056] Furthermore, the samples measured for DLC as described above were also subjected to UV-vis absorption spectroscopy, but no differences were observed in any of them, indicating no change in the UV-vis absorption spectrum during calcium ion chelation. The results are shown in Figure 6. Figure 6 shows the superimposed UV-vis absorption spectra of all samples with calcium ion addition amounts of zero, 0.1 μM, 1 μM, and 100 μM.

Claims

1. An antibody-drug conjugate comprising an antibody having properties to bind to cancer cells, a bisphosphonate preparation, and a crosslinking agent connecting them.

2. The antibody-drug conjugate according to claim 1, wherein the bisphosphonate formulation is a compound having the following structure. 【Chemistry 1】

3. The antibody-drug conjugate according to claim 1, wherein the antibody having the property of binding to the cancer cells is any of the following: anti-EGFR antibody, anti-VEGFR antibody, anti-HER2 antibody, anti-HER3 antibody, anti-FFFR antibody, anti-IGFR antibody, anti-HGFR antibody, anti-PDGFR antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD33 antibody, anti-CD52 antibody, anti-CEA antibody, anti-EpCAM antibody, anti-Nectin-4 antibody, anti-TROP-2 antibody, or anti-PD-L1 antibody.

4. The antibody-drug conjugate according to claim 1, wherein the crosslinking agent is any of Sulfo-GMBS, Sulfo-EMCS, Sulfo-HMCS, Sulfo-KMUS, Sulfo-SMCC, Sulfo-AC5-SPDP, or DTSSP.

5. An anticancer agent comprising an antibody-drug conjugate as described in any one of claims 1 to 4 as an active ingredient.

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