KR20210085296A - Composition for thermotherapy of circulating tumor cells comprising magnetic nanoparticles bound with antibodies, preparation method thereof, and method for treating circulating tumor cells using the same - Google Patents

Abstract

The present invention relates to a composition for thermotherapy of circulating tumor cells comprising magnetic nanoparticles to which an antibody is bound, a method for manufacturing the same, and a method for treating circulating tumor cells using the same. The magnetic nanoparticles to which an anti-EpCAM antibody is bound according to the present invention recognize EpCAM expressed in circulating tumor cells or circulating tumor stem cells to selectively bind only to tumors, and thus can selectively remove only circulating tumor cells or circulating tumor stem cells without side effects such as apoptosis of normal cells or tissues, thereby having a useful effect of providing a composition, a kit, and a method for thermal treatment of circulating tumor cells or circulating tumor stem cells.

Description

Composition for thermotherapy of circulating tumor cells comprising magnetic nanoparticles bound with antibody, manufacturing method thereof, and treatment method of circulating tumor cells using the same {Composition for thermotherapy of circulating tumor cells comprising magnetic nanoparticles bound with antibodies, preparation method thereof; and method for treating circulating tumor cells using the same}

The present invention relates to a composition for thermotherapy of circulating tumor cells comprising magnetic nanoparticles to which an antibody is bound, a method for preparing the same, and a method for treating circulating tumor cells using the same.

Recently, with the rapid development of modern medicine, early diagnosis of cancer is possible, and at the same time, various cancer treatment methods such as surgery, radiation, and anticancer drug therapy have been developed, and the possibility of overcoming cancer is starting to appear. However, the cancer treatments developed so far simply extend the survival period of cancer patients rather than fundamentally cure the cancer. Therefore, there is an urgent need to develop an effective cancer treatment method with few side effects.

Treatments commonly performed in cancer treatment include surgery, chemotherapy, and radiation therapy. Surgery is the best treatment for early cancer, but it is difficult to expect a good therapeutic effect when the cancer has metastasized to other tissues.

Radiation therapy and anticancer drug therapy are known to not only have low cancer treatment effects, but also affect normal tissues, causing various side effects such as gastrointestinal disorders, decreased immune function, anorexia, general weakness, and hair loss. In order to supplement the limitations of these existing cancer treatment methods, various types of cancer treatment methods are currently being developed, and a representative one of them is heat treatment chemotherapy (Wust et al., The Lancet Oncology, 2002, 3:487-497). .

One of the unique characteristics of cancer cells is that their ability to adapt to heat is significantly lower than that of normal cells (Wust et al., The Lancet Oncology, 2002, 3:487-497). Heat treatment chemotherapy is an anticancer therapy that uses the characteristics of cancer cells that lack the ability to adapt to heat as described above to treat cancer by raising the temperature of the cancer tissue and its surroundings to 42°C or higher. Heat treatment If the temperature of cancer tissue is raised during chemotherapy, cancer cells cannot adapt to high heat and die because their ability to adapt to heat decreases. Heat treatment In the course of chemotherapy, several methods have been developed, such as a method using ultrasound to increase the temperature of cancer tissue, a method for heat transfer by contact, and a method using electromagnetic waves. However, the most common and effective method in current heat treatment anticancer therapy is "cancer heat treatment using electromagnetic waves", which generates heat in cancer tissues by irradiating electromagnetic waves (European Patent Publication No. 2174689, US Patent No. 4323056, international Patent Application No. 2002-172198, Korean Patent Application No. 1125200, International Patent Application No. 2010-043372, International Patent Application No. 2009-013630).

Electromagnetic waves are waves that occur when electric and magnetic fields change with time; Gamma rays, X-rays, ultraviolet rays, visible rays, infrared rays, microwaves, radio waves, etc. are all electromagnetic waves. When electromagnetic waves pass through polar substances, they stimulate molecular motion of polar substances to generate heat, so basically all electromagnetic waves can be used for heat treatment and anticancer therapy. However, in the current "cancer thermotherapy using electromagnetic waves," 13.56 MHz radio The high frequency of the frequency is most commonly used.

Electromagnetic waves used in "thermal cancer treatment using electromagnetic waves" generate heat through a process called dielectric heating. Water molecules that make up most of the human body have a dipole moment due to an asymmetric bond between oxygen and hydrogen atoms. Due to the dipole moment of this water molecule, during "thermal treatment of cancer using electromagnetic waves," water molecules exposed to electromagnetic waves rotate as many as the frequency of the electromagnetic waves, pushing, pulling, or collide with each other. As a result, electromagnetic waves are exposed heat is generated in the tissue. If only cancer cells can be exposed to electromagnetic waves, cancer cells can be efficiently killed due to the characteristics of cancer cells that lack the ability to adapt to heat. However, exposing electromagnetic waves only to cancer cells without exposing them to normal cells is almost impossible due to the structural/physical limitations of the body. Therefore, there is a limit to treating cancer by radiating a physical wavelength such as electromagnetic waves to cancer tissue.

The best way to increase the therapeutic efficacy of "thermal cancer treatment using electromagnetic waves" is to administer "thermal cancer treatment using electromagnetic waves" after administering a sensitizer for thermal treatment.

The sensitizers for thermotherapy that have been tried so far are nanoparticles based on metal components such as gold and iron oxide (International Patent Application No. 2009-091597, International Patent Application No. 2012-036978, International Patent Application No. 2012-177875, US Patent No. 6541039, Korean Patent No. 0802139). Metals react very well with electromagnetic waves to generate high heat. Therefore, the efficacy of cancer treatment can be maximized by emitting electromagnetic waves after the metal component targets only cancer cells, not normal cells. However, until now, a technology that can selectively deliver metal components only to cancer tissues has not been developed.

Metal nanoparticles such as gold and iron oxide do not have selectivity for cancer tissue and thus accumulate not only in cancer tissue but also in normal tissue. At this time, electromagnetic wave treatment causes damage to normal tissue by generating heat in all areas where nanoparticles are located. do.

To date, as a method for targeting cancer cells or tissues, there is, for example, a method of directly injecting MNP (magnetic nanoparticles) into a solid tumor (cancer), which enables direct heat transfer to the tumor (cancer) and thus the tumor It has been reported to be effective in (cancer) removal (J. Liu, N. Li, L. Li, D. Li, K. Liu, L. Zhao, J. Tang, and L. Li, Oncol Lett , 2013, 6 , 1550; K. Mahmoudi, and C. Hadjipanayis, Front Chem , 2014, 2 , 109), another method of administration is intravenous injection of MNP, in which case MNP is targeted to tumor (cancer) cells or tissues to improve It is to increase the effect of hyperthermia by selectively accumulating from the EPR effect.

However, all of the above methods are limitedly available only for use in solid tumors (cancer), or are only methods in which the problem of side effects of killing normal cells or tissues still exists.

Above all, there is still no such thing as cancer (oncology) in which a part of tumor cells, such as blood cancer or Circulating Tumor Cell (CTC), is separated from the primary cancer and flows into blood vessels or lymphatic vessels and moves to other tissues or organs. There is no treatment, and there have been no reports of cases where hyperthermia was applied.

Accordingly, the present inventors were trying to develop a tumor-selective hyperthermia treatment technology that can selectively exert thermal treatment effects on circulating tumor cells such as blood cancer and prevent side effects such as death of normal cells or tissues. , The present invention was confirmed by confirming that the EpCAM antibody-coupled magnetic nanoparticles of the present invention selectively bind to cancer cells in the bloodstream of a leukemia model mouse, and that it is possible to specifically remove leukemia cells without side effects such as death of normal cells or tissues. completed.

An object of the present invention is to selectively target circulating tumor cells (CTC) or circulating tumor stem cells (CTSC), such as blood cancer and leukemia, binding magnetic nanoparticles containing antibodies It is to provide a composition for thermotherapy.

Another object of the present invention is to provide a method for preparing the composition for thermotherapy.

Another object of the present invention is to provide a kit for thermotherapy comprising the composition for thermotherapy and a device for irradiating electromagnetic waves.

Another object of the present invention is to administer the composition for thermotherapy to a subject in need of treatment; and treating the subject with electromagnetic waves; to provide a method of treating circulating tumor cells or circulating tumor stem cells, including.

In order to achieve the above object,

EpCAM, CD34, CD38, TK1, CD200, CD56, CD7, CD3 and one or more selected from the group consisting of CD19 antibody-bound magnetic nanoparticles containing circulating tumor cells or circulating tumor stem cells warming A therapeutic composition is provided.

In addition, the present invention provides a composition for thermotherapy comprising: binding an antibody against one or more selected from the group consisting of EpCAM, CD34, CD38, TK1, CD200, CD56, CD7, CD3 and CD19 to magnetic nanoparticles; A method of manufacturing is provided.

Furthermore, the present invention provides a kit for thermotherapy of circulating tumor cells or circulating tumor stem cells, comprising the composition for thermotherapy and a device for irradiating electromagnetic waves.

In addition, the present invention provides the steps of administering the composition for thermotherapy to a subject in need of treatment; and treating the subject with electromagnetic waves; a method for treating circulating tumor cells or circulating tumor stem cells is provided.

Anti-EpCAM antibody-bound magnetic nanoparticles according to the present invention can recognize EpCAM expressed in circulating tumor cells or circulating tumor stem cells and selectively bind only to tumors, as well as side effects such as death of normal cells or tissues. Since it is possible to selectively remove only circulating tumor cells or circulating tumor stem cells, there is a useful effect that compositions, kits and treatment methods for thermal treatment of circulating tumor cells or circulating tumor stem cells can be provided.

1 is a photograph showing FE-TEM (A) and DLS (B) analysis of human EpCAM antibody-bound MNP (hEpCAM-MNP) and mouse EpCAM antibody-bound MNP (mEpCAM-MNP).
2 is a graph showing the magnetization properties of EpCAM-MNP evaluated using a SQUID magnetometer (abbreviations: hEpCAM-MNP (human EpCAM antibody-bound MNP); mEpCAM-MNP (mouse EpCAM antibody-bound MNP)) .
Figure 3 shows (A) a cell culture dish and device setup for magnetic hyperthermia, and (B) THP1 cell culture medium in the presence or absence of MNPs for 50 minutes in a magnetic field (316A, 1358W, 310kHz). Temperature change is shown, (C) Cell proliferation assay (CCK-8) after 50 min of autothermal treatment and an additional 16 h incubation, and relative proliferation rate (%) of (+)HT cells compared to (-)HT cells. is shown above each bar. For the (-)HT group, cells were incubated for 50 min at room temperature (23 °C to 25 °C). "Control" samples are MNP-free cells (abbreviations: hEpCAM-MNP (human EpCAM antibody bound MNP); and In-MNP (inactivated hEpCAM antibody-bound MNP). (D) AC/DC and heat transfer COMSOL Multiphysics analysis results for the in vitro experiment (B) using the module.
FIG. 4 is a Bio-TEM image of THP1 cells treated with In-MNP and hEpCAM-MNP and then treated with [(+)HT] or untreated [(-)HT] THP1 cells. An alternating magnetic field (AMF) was applied at a frequency of 310 kHz and a current of 316 A. (A) Treatment of In-hMNP without high heat treatment. (B) Treatment of hEpCAM-MNP without hyperthermia. (C) Treatment of In-hMNP subjected to high heat treatment. (D) Treatment of hEpCAM-MNP treated with hyperthermia. Arrows indicate the positions of MNPs.
5 shows in vivo hyperthermic treatment after intravenous injection of mEpCAM-MNP. An alternating magnetic field (AMF) was applied for 50 min for the (+)HT group at a frequency of 310 kHz and a current of 316 A. (A) shows the apparatus setup for in vivo hyperthermia, and (B) shows the temperature increase of the culture medium and mouse skin during hyperthermia with mEpCAM-MNP using the above setup (A).
6 shows the counting of the number of leukemia cells in the bloodstream of AKR mice after whole-body hyperthermia. (A) Shows Leishman staining for counting leukemia cells in mouse blood, magnification is ×400. (B) Shows the mean leukemia cell number determined by Leishman staining in each mouse group before and after autothermal treatment, control and (-)HT groups were not treated by hyperthermia, but their cell numbers were counted simultaneously with the hyperthermic group became *, p <0.05.
FIG. 7 . Leukemia cell numbers were counted by a commercial microfluidic chip (Celsee) by counting cells attached to antibodies to both EpCAM and CD45. (A) Leukemia cells captured in the holes of the microfluidic chip were counted, stained with 4',6-diamidino-2-phenylindole (DAPI) and fluorescently labeled with anti-CD45 and anti-EpCAM antibodies. (B) Ratio of the number of leukemia cells in mouse blood before and after hyperthermia treatment. The control group and the (-)HT group were not irradiated with a magnetic field, but their cell numbers were counted simultaneously with the (+)HT group. *, p <0.05; **, p < 0.01.
FIG. 8 shows pictures of Prussian blue staining of liver, lung, thymus and spleen tissues of the control group, In-mMNP (+)HT, mEpCAM-MNP (-)HT and mEpCAM-MNP (+)HT group. The blue spots in the picture indicate the localization of iron.
9 shows TUNEL for thymus tissue from (A) control, (B) mEpCAM-MNP (-)HT, (C) In-mMNP (+)HT and (D) mEpCAM-MNP (+)HT after magnetic hyperthermia. The analyzed picture is shown. Green spots in the picture indicate the distribution of dead cells.
10 is a photograph showing the Bio-TEM analysis of blood cells from (A) control group, (B) In-mMNP (+)HT and mEpCAM-MNP (+)HT groups. Blood samples were collected before and after hyperthermia. An alternating magnetic field (AMF) was applied for 50 min for the (+)HT group at a frequency of 310 kHz and a current of 316 A. The scale bar in (B) is 2.5 µm.
11 is a photograph illustrating a method of predicting temperature change by magnetic hyperthermia through in vivo heat transfer simulation using a constant temperature (37 °C) and removing leukemia cells from blood circulation. (A) An enlarged schematic of the uniformly dispersed particle model (mimicking EpCAM-MNP) and particles attached to leukemia cells, and (B) a uniformly dispersed particle model (mimicking In-mMNP) and particles isolated from the cells. shows an enlarged schematic diagram of (C) shows the FEM (Finite element method) result of the temperature distribution for the model (A), and (D) shows the FEM result of the temperature distribution for the model (B).
12 is a graph showing the nanoparticle concentration of hEpCAM-MNP by NanoSight LM10.
13 shows the results of in vitro hyperthermia (high heat treatment) for TK6 cells, (A) showing the temperature change of the TK6 cell culture medium according to the presence/absence of MNP for 50 minutes under a magnetic field (316A, 1358W, 310kHz) and (B) (C) cell viability assay (CCK-8) after autothermal therapy (hyperthermia) and an additional 16 h incubation, relative proliferation of (+)HT cells compared to (−)HT cells ( %) is shown above each bar. For the (-)HT group, cells were incubated at room temperature (23-25 °C) for 50 min.
14 shows AMF and heat transfer simulations using COMSOL multiphysics, (A) the modeling scheme of the in vitro experiment, and (B) multiphysics using 41 d (diameter) = 40 μm (dispersed particles) particles. FEM results of (AMF and heat transfer) simulations are shown, and (C) FEM results of particle-free multiphysics (AMF and heat transfer) simulations are shown.
15 shows a heat transfer simulation for agglomerated particles using a constant temperature (37° C.) to predict the effect of particle agglomeration on temperature change. (A) Schematic diagram of the modeling and particles of 6 aggregated particles (mimicking EpCAM-MNP) attached to leukemia cells, (B) Modeling and particle modeling of 6 aggregated particles (mimicking In-mMNPs) detached from cells. The schematic diagram of Fig. 1 is shown on an enlarged scale, (C) shows the FEM result of the temperature distribution for modeling (A), and (D) shows the FEM result of the temperature distribution for the modeling (B).
16 is a conceptual diagram of hyperthermia (high heat treatment) of circulating tumor cells (leukemia cells) in an in vivo experiment using magnetic nanoparticles to which EpCAM antibodies are bound to selectively attach to tumors in the circulation, provided by the present invention. to be.

Hereinafter, the present invention will be described in detail.

EpCAM, CD34, CD38, TK1, CD200, CD56, CD7, CD3 and one or more selected from the group consisting of CD19 antibody-bound magnetic nanoparticles containing circulating tumor cells or circulating tumor stem cells warming A therapeutic composition is provided.

Here, the anti-EpCAM antibody may be understood as a means for increasing the selectivity for tumor (cancer) by targeting EpCAM expressed in tumor (cancer) existing in the circulation of an individual, and the magnetic nanoparticles are thermotherapy It can be understood as performing a function of releasing heat by (high heat treatment) and transferring heat to the tumor (cancer) to which the particles are attached. From the composition of the present invention, EpCAM-expressing tumor (cancer) cells or tumor (cancer) stem cells present in the circulatory system of an individual can be killed or necrotic by thermotherapy.

On the other hand, as used herein, the term "antibody" includes reference to an immunoglobulin molecule reactive with a particular antigen and includes both polyclonal and monoclonal antibodies.

As used herein, the term “thermotherapy (hyperthermal treatment)” refers to treating a target cell or tissue at 40°C or higher, 41°C or higher, 42°C or higher, or 40°C-60°C, 40°C-50°C, 41 It means heating to a temperature of ℃-50 ℃, 40 ℃-48 ℃, 40 ℃-46 ℃ or 40 ℃-45 ℃.

In its most basic form, the invention described herein relates to a particle (antibody) bound to a ligand capable of binding a target for use in thermotherapy. Various embodiments of the present invention include a particle, at least one ligand (antibody), and optionally may further include a non-covalent or covalent molecule binding the particle and the ligand, for example, a conventional linker or It may be a crosslinking compound.

Meanwhile, in various embodiments, the particle is a magnetic particle, and in another embodiment, the particle refers to a magnetic particle capable of generating heat when placed in an alternating magnetic field (AMF) or other energy source. Such particles may be referred to as magnetic energy sensitive particles and may be useful in providing thermotherapy.

As used herein, the term “magnetic nanoparticles” refers to paramagnetic or ferromagnetic particles that are magnetized when a magnetic field is applied and can be attracted in a certain direction by magnetic force. The magnetic nanoparticles are specifically iron oxide, zinc ferrite, manganese (Mn), nickel (Ni), cobalt (Co), bismuth (Bi), platinum (Pt), gold (Au) , palladium (Pd), copper (Cu), alloys thereof, or magnetic nanoparticles selected from the group consisting of ferrites thereof, but is not limited thereto.

Referring specifically to the magnetic particles, heat generated by the magnetic particles when placed in the AMF is generally exhibited in response to the AMF. The amount of heat generated per period of the magnetic field and the mechanism resulting in energy loss depend on the specific properties of both the magnetic material of the magnetic particle and the applied magnetic field.

In some aspects, the magnetic particles are heated to a unique temperature called the Curie temperature when subjected to an AMF or other energy source. The Curie temperature is the temperature at which a reversible transition from ferromagnetism to paramagnetism of a magnetic material of a magnetic particle occurs. Below this temperature, the magnetic material generates heat in the applied AMF, but above the Curie temperature, the magnetic material is paramagnetic and non-reactive to the AMF. Thus, magnetic materials do not generate heat upon exposure to AMF above the Curie temperature. As the magnetic material cools to a temperature below the Curie temperature, the material recovers its magnetic properties and resumes heating upon application of AMF. This cycle can be repeated continuously during exposure to AMF. Accordingly, the magnetic material of the magnetic particles can self-regulate the heating temperature. In an embodiment of the present invention, the magnetic material may be selected to have a Curie temperature of about 40° C. to about 150° C., but is not limited thereto.

As used herein, the term "alternating magnetic field" or "AMF" means that the direction of its field vector is periodic, usually in a sine, triangular, square or similar pattern, from about 80 kHz to about 800 kHz. Represents a magnetic field that changes with a range of frequencies. AMF can also be added to the static magnetic field so that only the AMF component of the generated magnetic field vector changes direction. AMF may be accompanied by an alternating electric field and may be electromagnetic in nature.

The temperature to which the magnetic particles are heated depends, among other factors, on the magnetic properties of the magnetic particle material, the properties of the magnetic field, and the cooling ability of the target site. The choice of magnetic particle material and AMF properties can be tailored to optimize therapeutic efficacy for a particular target type. Many aspects of the magnetic particles, such as material composition, size and shape, can directly affect the heating properties, and these properties can be tailored to achieve the desired heating properties. For example, the size of magnetic particles used in thermotherapy may depend on the particular application for which the magnetic particles will be used (ie, the temperature to be achieved) and the material comprising the magnetic particles.

The size of the magnetic particle may also determine the overall size of a "magnetic particle conjugate" comprising, for example, a linker and a ligand. In various embodiments, the size of the magnetic particles may be from about 0.1 nm to about 250 nm, 1 nm-230 nm, 1 nm-200 nm, or 1 nm-100 nm. The size of the magnetic particle conjugate may also depend on the indication, the magnetic particle and the material comprising the magnetic particle conjugate, the route of administration, and the method of use. In some embodiments, magnetic particles or magnetic particle conjugates administered to a patient, eg, via intravenous injection, are administered to the reticuloendothelial system (RES) to achieve increased therapeutic composition concentrations and long residence times in the bloodstream. It may be desirable to avoid absorption by the liver and subsequent distribution to the liver, spleen, lung, kidney, heart and bone marrow. In order to successfully avoid absorption by RES, the diameter of the magnetic particle or the magnetic particle assembly may be less than about 30 nm, particularly in embodiments where the magnetic particle contains magnetite (Fe ₃ O ₄ ), the diameter of the magnetic particle assembly is about 8 nm to about 20 nm. The magnetic particles of this magnetic particle assembly retain a sufficient magnetic moment for heating in the applied AMF, while avoiding absorption by the RES of the therapeutic composition. In some embodiments, ferromagnetic particles greater than about 8 nm in diameter may be suitable for thermotherapy applications. In other embodiments, other elements, such as cobalt, are included in the magnetite magnetic particles. By including the secondary element, the size range of the magnetic particles and the magnetic particle assembly including the magnetic particles may be reduced. For example, cobalt has a magnetic moment that is smaller than that of magnetite but generally greater than that of magnetite, which can contribute to the overall magnetic moment of the cobalt-containing magnetite magnetic particles while reducing the size of the therapeutic composition.

Magnetic particles for use in embodiments of the present invention may include any number of materials that provide a suitable magnetic moment and size. The material composition of the magnetic particles can be selected based on a particular target. For example, in embodiments magnetic particles include, but are not limited to, _{iron oxide particles and FeCo/SiO 2 particles.} On the other hand, the iron content of the magnetic nanoparticles is not particularly limited as long as the tumor (cancer) removal effect by hyperthermia (high heat treatment) to be achieved by the present invention can be achieved, for example, 30% or more, 30% may be greater than, greater than 40%, or greater than 50%. Furthermore, because the self-limiting Curie temperature is directly related to the magnetic particle material that the total heat is transferred to the target, magnetic particle compositions can be designed for different target types. Such tuning may be necessary to achieve the desired heating of each target type, given the unique heating and cooling capabilities based on the location and composition of the target in the patient's body. For example, a tumor located in a relatively isolated area without a smooth blood supply to the patient may require a lower Curie temperature magnetic particle material than a tumor located near a major blood vessel for effective hyperthermia. A target placed in the bloodstream will also require a magnetic particle material with a specific Curie temperature. Accordingly, the magnetic particles of various embodiments may include, in addition to magnetite, for example, cobalt, iron, rare earth metals, and the like, and combinations thereof.

Electromagnetic Absorption Rate (SAR) may also be considered in the selection of magnetic particle materials. The SAR for a given material is commonly described as the rate at which the material absorbs radio frequency (RF) energy when exposed to an RF electromagnetic field. For example, series EMG 700 and EMG 1111 iron oxide particles of about 110 nm diameter available from Ferrotec Corp (Nashua, NH) have a SAR of about 310 Watts/gram of particle at 1,300 Oerstedt flow density and 150 kHz frequency. has other particles, such as Inframat Corp. _{FeCo/SiO 2} particles available from (Willington, Connecticut) have an SAR of about 400 Watts/gram of particle under the same magnetic field conditions.

In some embodiments, when the magnetic particle is formed from a magnetic material that is biocompatible, the surface of the magnetic particle itself can act as a biocompatible coating. In other embodiments, the coating material may help to make the magnetic particle biocompatible. In another embodiment, the coating material may facilitate transport of magnetic particles into cells, for example by transfection. Such coating materials, called transfection agents, can be, for example, vectors, such as plasmids, viruses, phages, virons or viral coats, prions, poly amino acids, cationic liposomes, amphiphiles, non-liposomal lipids, or any combination thereof. In other embodiments, the biocompatible coating material may be a composite of the transfection agent(s) and organic and/or inorganic materials. In such embodiments, the combination of coating material and/or transfection agent can be tailored for a specific type and specific location of a cell or diseased tissue within the patient's body.

In some embodiments of the invention, the linker is coupled to a magnetic particle or magnetic particle coating. A linker can, in some embodiments, be a bifunctional compound that contacts and binds to the external surface of the magnetic particle, while covalently binding to the ligand to attach the ligand to the external surface of the magnetic particle. In another embodiment, the linker binds to the outer surface of the magnetic particle and facilitates evasion of the magnetic particle from the reticuloendothelial system (RES). For example, functionalization of magnetic particles with a polyethylene glycol (PEG) linker, via a method known in the art as "pegylation", is effective to avoid detection and uptake by the RES, and increased permeation. and promotes the penetration of the altered vasculature of the tumor through the EPR effect, thereby preferentially accumulating magnetic particles in the tumor interstitium.

Depending on whether the linker is short or long, rigid or flexible, hydrophobic or hydrophilic, the linker can affect the properties of the final conjugate. Linkers of various embodiments include hydrophobic or hydrophilic organic or inorganic compounds, or mixtures of chemically different compounds. In some embodiments, the linker may comprise an alkyl, alkene, alkyne, haloalkyl, epoxide, vinyl or heterocumulline compound, and in other embodiments, the linker may comprise a multi-subunit compound. Linkers of such embodiments are not limited by the number and/or type of subunits in the multi-subunit compound, and include, for example, subunits such as epoxypropene, polyethylene glycol, polypropylene glycol, and combinations thereof. may include In another embodiment, the linker comprises one or more epichlorohydrins, diepoxides, or combinations thereof. Examples of linkers encompassed by embodiments of the present invention include poly(ethylene glycol), epoxyether, poly(ethylene glycol) diglycidyl ether, and epichlorohydrin and poly(ethylene glycol) diglycidyl ether. mixtures, but are not limited thereto.

Linkers of various embodiments also include one or more terminal reactive groups. The type of terminal reactive group will vary depending on the type of reaction chemistry required to couple a particular type of magnetic particle to any type of ligand, form a covalent bond with any ligand, or bind the magnetic particle to such a ligand. can In certain embodiments, the reactivity of an end group may be based on substitution or addition chemistry. Exemplary terminal reactive groups include, but are not limited to, carboxylic acids, amines, hydrazines, azides, thiols, disulfides, sulfonic acids, vinyls, 1,2-diacylethenes, and derivatives and combinations thereof, In certain embodiments, the terminal reactive group can be an amine, thiol, or carboxylic acid moiety. In some embodiments, the carboxylic acid end group can be a poly(ethylene glycol) etheric carboxylic acid, the azide end group can be 5-azido-2-nitrobenzamide, and the disulfide end group can be 3-(2-pyri) dilthio) propionamide, and the 1,2-diacylethene end group may be maleimide or 3-maleinidylpropionamide.

Ligands of embodiments of the invention may be selected to ensure that the magnetic particles selectively attach to or associate to a selected target. In some embodiments, the ligand enables targeting of EpCAM expressing tumor (cancer) cells or tumor (cancer) stem cells or tissues of the circulatory system. The anti-EpCAM antibody may be the whole or a fragment thereof.

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, the antibody refers to an antibody that specifically binds to a marker of the present invention or a protein constituting the marker, and includes polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

The "antibody" of the present invention includes anti-EpCAM (anti-Epithelial cell adhesion molecule). In addition, the antibody of the present invention may be an antibody mixture, and the antibody mixture of the present invention is anti-EpCAM (anti-Epithelial cell adhesion molecule) in addition to anti-EGFR (anti-Epidermal growth factor receptor), anti-N-cadherin, It may further include anti-TROP2 (anti-trophoblast cell-surface antigen) and anti-vimentin.

As described above, the generation of an antibody using a marker expressed on the surface of a cancer cell can be easily prepared using a technique well known in the art.

The polyclonal antibody can be produced by a method well known in the art for obtaining a serum containing the antibody by injecting the above-described marker protein antigen into an animal and collecting blood from the animal. Such polyclonal antibodies can be prepared from hosts of any animal species, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle and dogs.

The monoclonal antibody may be prepared by the hybridoma method well known in the art (see Kohler and Milstein, European Jounral of Immunology 6:511-519, 1976) or a phage antibody library (Clackson et al. Nature 352:624- 628, 1991; Marks et al. J. Mol. Biol., 222:58, 1-597, 1991).

The antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.

In the present invention, an antibody capable of specifically binding to the cancer cell is attached to the surface of the magnetic nanoparticles to form “antibody-nanoparticle” type particles. The antibody-nanoparticle can be easily prepared using techniques well known in the art. According to a specific embodiment of the present invention, the antibody-nanoparticle was prepared through an EDC/NHS coupling reaction (Anthony J. Di pasqua et al., Materials Letters 63 (2009), 1876-1879), but must be limited thereto it's not going to be

Preferably, the "antibody-nanoparticle" type particle of the present invention is a magnetic nanoparticle to which an anti-EpCAM antibody is bound, but is not limited thereto, and may be commercially available, for example, in the Examples below of the present invention , may be purchased from ltenyl Biotec (Bergisch Gladbach, Germany).

On the other hand, as used herein, the term "cancer cells in the blood" refers to cells found in the course of cancer in which a part of tumor cells are separated from the primary cancer and flow into blood vessels or lymphatic vessels and move to other tissues or organs.

The type of the term "cancer cell" used in the present invention is not limited, and the blood cancer cell of the present invention may be a circulating tumor cell (CTC) or a circulating tumor stem cell (CTSC). However, the present invention is not limited thereto.

In addition, the type of the term "cancer" used in the present invention is not limited, and liver cancer, colorectal cancer, rectal cancer, endometrial cancer, ovarian cancer, renal pelvis cancer, pancreatic cancer, small intestine cancer, hepato-pancreatic biliary tract cancer, stomach cancer, brain tumor and breast cancer, etc. It may include, and preferably may be a circulatory system cancer such as blood cancer or leukemia in the form of cancer existing in the circulatory system.

In addition, the present invention provides a composition for thermotherapy comprising: binding an antibody against one or more selected from the group consisting of EpCAM, CD34, CD38, TK1, CD200, CD56, CD7, CD3 and CD19 to magnetic nanoparticles; A method of manufacturing is provided.

A further embodiment of the present invention includes a method of making a ligand conjugated particle or "magnetic particle conjugate".

For example, preparing particle (I), amino-functionalizing the particle, reacting a linker as described above with an amino-functionalized particle, and reacting a second functional group on the linker with a ligand to form a ligand and forming a bonded particle or a magnetic particle bonded body.

Another embodiment of the present invention comprises the steps of functionalizing a particle forming a single magnetic domain with an amino or nitro group, contacting the functionalized particle with a linker, and coupling a ligand to the particle or linker to form a ligand conjugated particle. It may include a method for preparing a ligand conjugated particle comprising the step of:

For example, the preparation method of the present invention may be carried out as shown in Scheme 1 below.

[Scheme 1]

Furthermore, the present invention provides a kit for thermotherapy of circulating tumor cells or circulating tumor stem cells, comprising the composition for thermotherapy and a device for irradiating electromagnetic waves.

The electromagnetic waves are waves generated as electric and magnetic fields change with time, and may include gamma rays, X-rays, ultraviolet rays, visible rays, infrared rays, microwaves, radio waves, alternating magnetic fields (AMF), etc. And, in the present invention, a conventional electromagnetic wave irradiation device can be used.

In addition, the present invention provides the steps of administering the composition for thermotherapy to a subject in need of treatment; and treating the subject with electromagnetic waves; a method for treating circulating tumor cells or circulating tumor stem cells is provided.

In another aspect, the present invention comprises the steps of: targeting tumor (cancer) cells or tumor (cancer) stem cells present in the circulation by administering the composition for cancer thermotherapy to animals other than humans; And it relates to a treatment method of circulating tumor cells or circulating tumor stem cells comprising a; and the treatment of electromagnetic waves to kill or necrosis the targeted tumor (cancer) cells or tumor (cancer) stem cells.

As used herein, "administering" together with the therapeutic nanoparticle composition of the present invention means to have a therapeutic effect on cancer (tumor) cells or tissues existing in the target circulation by administering a therapeutic agent to a patient. However, this does not preclude administration targeting solid cancers other than the circulatory system. "Administering" a therapeutic agent may be accomplished by injection, infusion, or in combination with either method or other known techniques, to name a few.

As used herein, an “effective amount” or “therapeutically effective amount” of a composition is a predetermined amount calculated to achieve a desired effect.

As used herein, the subject may be a mammal or non-mammal, including a human, or a mammal other than a human, or a human.

Another embodiment includes a method of treating a disease using the magnetic particles and magnetic particle conjugates of the present invention. For example, in one embodiment, a magnetic particle conjugate in which a ligand (anti-EpCAM antibody) targets an EpCAM expressing (positive) cancer cell is administered to the patient, the magnetic particle conjugate becomes attached or bound to the cancer cell; , the patient is exposed to an alternating magnetic field (AMF). The heat generated by magnetic particles as a result of AMF destroys (ie, induces cell death) or inactivates cancer cells, either immediately or over time. In addition, heat generated by magnetic particles and/or cell death can stimulate the production and release of heat shock proteins, such as HSP 70, and the presence of heat shock proteins can stimulate an immune response against any residual cancer cells. Such a stimulated immune response may also help protect individuals from the future progression of cancer and other diseases.

In another embodiment of the present invention, the modified surface charge and particle size may contribute to the effectiveness of magnetic particles and magnetic particle conjugates in the treatment of diseases. For example, in one aspect of the invention, the magnetic particle surface charge is modified to reduce the clearance of the nanoparticles. In one embodiment of the invention, a substantial portion of the magnetic particle surface is functionalized to provide the desired surface charge and zeta potential. A magnetic particle or magnetic particle conjugate becomes a high-energy state, e.g., hot, as the AMF energy is absorbed, and the generated heat can pass to the target cell, e.g., by convection, conduction, radiation, or a combination of heat transfer mechanisms. have. Target cells exposed to heat are damaged, and in certain embodiments, such target cells may be irreversibly damaged. In certain embodiments, the target cell dies through necrosis, cell death, or other mechanisms when a sufficient amount of energy is transferred to the target cell.

The amount of magnetic particles or magnetic particle conjugate administered to a subject may vary and may depend on the disease to be treated and the location of the diseased tissue. Moreover, the dosage may vary depending on the mode of administration. For example, the dosage to be administered may also depend on a variety of factors, including the characteristics of the subject being treated (eg, age, weight, sex, health status, type of concurrent treatment (if any), and frequency of treatment). Given such information, one skilled in the art (eg, a clinician) can determine within its competence the amount of magnetic particles or magnetic particle conjugates that constitute a therapeutically effective amount. The choice of a particular route of administration and dosing regimen can be adjusted or titrated by such clinicians according to methods known to those skilled in the art to obtain an optimal clinical response.

Various routes of administration are contemplated in embodiments of the present invention, and magnetic particles and magnetic particle conjugates may be administered in a conventional manner by any route in which they are active. For example, administration may be by systemic, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, topical, transdermal, oral, buccal, or ophthalmic route, intravaginally, or by inhalation, by depot injection or implants, but are not limited thereto. Methods of administering the magnetic particles and magnetic particle conjugates of certain embodiments of the present invention may include, but are not limited to, intravascular injection, intravenous injection, intraperitoneal injection, subcutaneous injection, and intramuscular injection.

The method of any embodiment of the present invention may further comprise formulating the magnetic particle or magnetic particle conjugate together with a pharmaceutically acceptable excipient, vehicle or carrier into a pharmaceutical composition. For example, in one embodiment, the pharmaceutical composition disperses or separates the magnetic particles or magnetic particle conjugates, and comprises the magnetic particles or magnetic particle conjugates with a pharmaceutically acceptable excipient, vehicle or carrier, and any other ingredients. It is prepared by mixing to formulate a pharmaceutical composition.

With particular reference to parenteral administration routes, the magnetic particles and magnetic particle conjugates in any embodiment of the present invention may be formulated for parenteral administration by injection (eg, flash injection or continuous infusion). The magnetic particles and magnetic particle conjugates may be administered by continuous infusion subcutaneously over about 15 minutes to about 24 hours. Formulations for injection may be presented in single dosage form (eg, in ampoules or in multiple dosage containers) with an added preservative. The formulations may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

For example, the composition for thermal treatment is preferably dissolved in a solution such as water or physiological saline suitable for injection at a concentration of 0.01 to 100 mg/ml. Or preferably, the concentration of the anti-EpCAM antibody-bound magnetic nanoparticles of the composition of the present invention may be 10 ⁹ - 10 ¹³ , 10 ⁹ - 10 ¹² or 10 ⁹ - 10 ¹¹ particles/ml in the circulatory system of the subject.

For the effect of "thermal treatment using electromagnetic waves", the dosage of the composition for thermal treatment of the present invention can be considered in a complex manner, such as the subject to which it is applied, the distribution characteristics of the magnetic nanoparticles used, the exothermic characteristics, the applied electromagnetic field strength, etc. For example, a dose range of 1-50 mg/kg or 0.1-200 mg/kg may be applied to the subject.

For the desired effect of the "thermal treatment using electromagnetic waves", it is preferable to perform "thermal treatment using electromagnetic waves" within 1 to 48 hours after administration of the composition for heat treatment.

The "thermal therapy using electromagnetic waves" therapy may be easily determined and used by a known thermotherapy therapy. For example, a treatment irradiating for 30-60 minutes with a thermotherapy device that outputs 13.56 MHz high frequency can be performed more than twice a week for more than 4 weeks.

The treatment method according to the present invention can improve the therapeutic effect of cancer by using it in conjunction with or in parallel with the existing anticancer therapy. Existing anticancer therapies may include chemotherapy, radiation therapy, biological therapy, immunotherapy, and photodynamic therapy.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

<Example 1> Preparation of hEpCAM-MNP

Human EpCAM antibody-conjugated MNP (hEpCAM-MNP) was purchased from Miltenyl Biotec (Bergisch Gladbach, Germany).

Components: 2 mL CD326 (EpCAM) MicroBeads, human : MicroBeads conjugated to monoclonal antibody (isotype : mouse IgG1).

<Example 2> Preparation of mEpCAM-MNP

Mouse EpCAM antibody-conjugated MNP (mEpCAM-MNP) was purchased from Miltenyl Biotec (Bergisch Gladbach, Germany).

<Comparative Example 1> Preparation of In-hMNP

The inactivated form of hEpCAM-MNP (In-hMNP) was prepared by treating the hEPCAM-MNP of Example 1 at 100° C. for 30 minutes.

<Comparative Example 2> Preparation of In-mMNP

The inactivated form of mEpCAM-MNP (In-mMNP) was prepared by treating the mEPCAM-MNP of Example 2 at 100° C. for 30 minutes.

The above two types of MNP, hEpCAM-MNP (Example 1) and mEpCAM-MNP (Example 2) were used in vitro and in vivo, respectively. Since the THP1 cells used in this study are human-derived, hEpCAM-MNP (Example 1) was used for the in vitro test. In the in vivo test of mice, mEpCAM-MNP (Example 2) was used.

<Experimental Example 1> Analysis of MNP characteristics

In order to investigate the characteristics of each of the MNPs of Examples 1-2 and Comparative Examples 1-2, field emission transmission electron microscopy (FE-TEM) analysis using an HF-3300 microscope (Hitachi, Tokyo, Japan), ZetaSizer NanoZS (Malvern) Instruments, Malvern, UK, Malvern) were analyzed for dynamic light scattering (DLS) analysis, and a magnetometer using an MPMS SQUID VSM device (Quantum Design, San Diego, CA, USA) was analyzed.

The number of each MNP in the colloidal suspension was measured using a NanoSight LM10 instrument (Malvern Instruments).

Total iron (Fe) concentration was measured by inductively coupled plasma mass spectrometry (ICP-MS, NexION 300, PerkinElmer, Waltham, MA, USA).

Before ICP-MS analysis, MNPs were dispersed in 5% HCl solution and dissolved on a hot plate for 1 h. After cooling, samples were appropriately diluted with deionized water for ICP-MS.

The morphology and size of each MNP were analyzed by FE-TEM (Fig. 1A) and DLS (Fig. 1B). FE-TEM revealed that the size of the individual particles was approximately 5 nm. Some particles were associated with the formation of visible aggregates with a size of approximately 20-30 nm, and the hydrodynamic sizes of the aggregates measured by DLS were 79.8 nm and 88.2 nm for hEpCAM-MNP and mEpCAM-MNP, respectively.

The magnetization characteristics of two types of MNPs were also analyzed as shown in FIG. 2 . The MS of mEpCAM-MNP was slightly higher (approximately 1.2 fold) at both 5K and 300K temperatures compared to that of hEpCAM-MNP.

Particles Fe (mg/mL) hEpCAM-MNPs 365.50 mEpCAM-MNPs 1451.06

As shown in Figures 1A and 1B, although the morphology and size of the two particles were similar, the iron content was significantly different: mEpCAM-MNP had an almost 4-fold higher iron content than hEpCAM-MNP (Table 1).

Previous studies have reported that an increase in the polymer content of MNPs is associated with a decrease in MS due to a decrease in iron content. Therefore, the slightly increased MS for mEpCAM-MNP is due to the higher iron content compared to hEpCAM-MNP.

<Experimental Example 2> In vitro self-thermal therapy

<2-1> Preparation of THP1 and TK6 cells

THP1 and TK6 cells were purchased from the American Type Culture Collection (ATCC, Manassas, Va.). Streptomycin (Welgene, Gyeongsan, Korea) and 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO , USA) supplemented with 1% penicillin cells at 37 ℃ under a humid atmosphere consisting of 95% air and 5% CO ₂ It was cultured in RPMI-1640 medium (GE Healthcare, Pittsburgh, PA, USA).

<2-2> In vitro self-thermal therapy

For in vitro autothermal therapy (high heat treatment), 1 mL of a suspension of THP1 cells (approximately 105 cells/mL) was placed in a 35 × 10 mm tissue culture dish and 100 μl of hEpCAM-MNP (Example 1) or In-hMNP (Comparative Example) 1) was added to the cells. A control sample containing only THP1 cells was also prepared. Then, each cell culture dish was inserted into a coil set up for autothermal therapy (hyperthermia). The setup for magnetization here was purchased from Ambrell (Rochester, NY, USA) and used with an infrared thermal imaging camera (Testo, Lenzkirch, Germany). The system consisted of a generator (EASYHEAT; 1.2 kW with RF power from 150 to 400 kHz), a three-turn coil and a closed cooling water system. An alternating magnetic field (AMF) was applied with a frequency of 310 kHz and a current of 316 A.

The hyperthermic group [(+)HT] was treated with autothermal therapy (hyperthermic treatment) for 50 min, and the non-hyperthermic group [(-)HT] was incubated at room temperature (23-25 °C) for 50 min. Cell viability of all samples was measured using Cell Counting Kit-8 (CCK-8; Rockville, Dojindo Molecular Technologies, Inc., MD, USA) according to the manufacturer's instructions. The cell suspension (100 μL) used for hyperthermia was added to the wells of a 96-well cell culture plate. Cells were grown for 16 h at 37 °C in a 5% CO _{2 atmosphere under humidified conditions.} After incubation, 10 μl of CCK-8 solution was added to each well and incubated at 37° C. for 2 hours, and the absorbance of the supernatant from each well was measured at 450 nm. Interactions between THP1 cells and each MNP, and cell morphology analysis after high heat treatment were observed by bio-TEM analysis using a Tecnai G2 F20 TWIN TMP device (FEI, Hillsboro, OR, USA).

It was confirmed that the temperature of the MNP-treated THP1 cells was increased to almost 42 °C, while the temperature of the control cells without MNP was almost 37 °C (Fig. 3B). In previous reports, a temperature rise without MNP was observed, but this is due to the thermal radiation of the induction coil.

For a time period of 24-41 min, the temperature of hEpCAM-MNP was significantly higher than that of In-hMNP. The average temperature difference at this time was 1.9 °C. The slightly reduced temperature by In-hMNPs could be attributed to the change in surface properties that occurred during the heat-inactivation process of In-hMNPs, because the thermogenesis efficiency by AMF was reduced by partial aggregation of MNPs. .

Although there was a slight difference between the heating capacities of hEpCAM-MNP and In-hMNP, the two MNPs almost had similar thermogenic capacity from 40°C to 42°C, which is effective in eliminating cancer cells.

As shown in Fig. 3A, the heating coil of the instrument was exposed to room temperature (23 °C to 25 °C), and the temperature of the (+)HT group at the beginning of the high heat treatment was 23 °C to 26 °C (Fig. 3B). Therefore, the cells of the (-)HT group were cultured at room temperature for the same time as that of the (+)HT group, making the experimental conditions comparable between the (-)HT and (+)HT groups as much as possible. After high heat treatment, an aliquot of (+)HT and (-)HT group cells was transferred to the wells of a 96-well plate and further incubated at 37°C for 16 hours.

As shown in Fig. 3C, the cell viability of all (-)HT groups was very similar to each other. However, the viability of In-hMNP and hEpCAM-MNP-treated cells in the (+)HT group was reduced to 66.5% and 40.8% in the (-)HT group, respectively.

The results showed that hEpCAM-MNP was more effective in eliminating THP1 cells compared to In-hMNP. hEpCAM-MNPs can attach specifically to THP1 cells, and thus efficient heat transfer from hEpCAM-MNPs to THP1 cells leads to more effective tumor cell death. On the other hand, since the In-hMNP antibody has a structure inactivated by heat treatment, it has no or low binding ability to the THP1 cell surface, so it is expected that In-hMNP will be dispersed in the cell suspension. However, the temperature of the medium was increased to almost 40 °C in the In-hMNPs (+)HT group, and thus cell growth was delayed to 66.5% of the (-)HT group ( FIG. 3C ).

Specific binding of hEpCAM-MNP to THP1 cells can be confirmed by treating TK6 cells (human lymphoblasts) with hEpCAM-MNP and then irradiating the same magnetic field as in THP1 cells. The temperature of In-hMNP and hEpCAM-MNP-treated TK6 cells was increased to 39.1 °C and 42.0 °C during 50 min of hyperthermia, a pattern similar to that in THP1 cells. However, the cell viability of In-MNP and hEpCAM-MNP-treated TK6 cells in the (+)HT group was similarly reduced to 69.2 and 69.9% of the (−)HT group, respectively. These proportions were higher than those of THP1 cells (66.5% and 40.8%, respectively, in Fig. 3C). This result is due to the presence of EpCAM in TK6 and THP1 cells, respectively. The higher cell viability for TK6 cells than for THP1 cells by irradiating magnetic fields with hEpCAM-MNPs suggested that hEpCAM-MNPs could specifically bind to THP1 cells but not TK6 cells. Thus, for the (+)HT group of TK6 cells treated with In-hMNP and hEpCAM-MNP, heat induction appeared to normally arise from free MNPs in the culture medium and not from bound MNPs on the cell surface.

Collectively, the induced cell death rate was achieved by magnetic hyperthermia, the mechanism of which can be explained by the specific binding of hEpCAM-MNP to THP1 cells and the increase in the temperature of the cell medium during magnetization of MNPs.

A slight decrease in temperature for 17 min caused by In-hMNP could affect the reduced apoptosis rate of THP1 cells in Figure 3C, but specific binding of hEpCAM-MNP to THP1 cells induces apoptosis. more effective

<2-3> In vitro heat transfer simulation

To support the experimental results, the temperature of AMF-induced MNPs in cells and surrounding was analyzed using Multiphysics® 5.4 software (COSMOL, Inc., Stockholm, Sweden). To analyze the calorific value of MNPs in AMF, the AC/DC module of the software was used. For the analysis of temperature changes in cells and their surroundings by heated MNPs, the heat transfer module of the software was used simultaneously.

First, the equation of the _{calorific value Q EM} (W/m ³ ) per unit volume generated from electromagnetic heating of MNPs by AMF is calculated as follows.

(1)

(One)

where J, E, B, ω and H are current density (A/m ² ), electric field (V/m), magnetic flux density (T), frequency (s ^-1 ) applied to AMF, and magnetic field strength (A/m), respectively m) is shown.

Q _EM consists of an electric heating term and a self heating term. In electrical heating terms, the current density (J) is defined as:

(2)

(2)

where σ and D denote electrical conductivity (S/m) and electromagnetic (C/m ² ), respectively.

Here, electromagnetism refers to the degree of charging of the MNP by an electric field and is expressed as follows:

(3)

(3)

where ε denotes the permittivity, which is divided into a real part (ε') and an imaginary part (ε"). Therefore, from equations (2) and (3), the electric heating term in equation (1) can be written as can:

(4)

(4)

In the magnetic heating term of equation (1), the magnetic flux density (B) and magnetic field strength (H) have the following relationship:

(5)

(5)

where μ is the transmittance, which is divided into a real part (μ′) and an imaginary part (μ″), and the transmittance can be obtained from the magnetization characteristic curve as shown in Fig. 2. Therefore, based on Equation (5), Equation (1) ) can be rewritten as:

(6)

(6)

Therefore, Q _EM can be explained by combining the electric heating in equation (4) and self heating in equation (6) as follows:

(7)

(7)

Next, the temperature change due to heat transfer generated from MNP by AMF can be analyzed with the following equation:

(8)

(8)

where ρ, c _p , k and T represent the density, specific heat, thermal conductivity and temperature of the material, respectively. Q denotes the term that considers water cooling to reduce the heat of the coil in AMF and consists of uρ _w c _w (T _in - T), where u, ρ _w , c _w and T _in are the circulation number, density, and specific heat, respectively. and the temperature of the feed water.

Concentrations of MNPs for simulation were obtained by a NanoSight LM10 instrument (Malvern Instruments). For simplicity of multiphysics analysis, it was assumed that MNPs were aggregated in a symmetric three-dimensional form around the z-axis. Using the in vitro experimental conditions and parameters, the Q _EM of MNPs produced by AMF was measured. The power and frequency of the AMF used for multiphysics analysis were 1358 W and 310 kHz, respectively. The material properties used in equations (7) and (8) are summarized in Table 2.

Parameters Values

5800

1050

1000

1035

1.2

5000

1.0

3.8

0.58

0.60

0.56

To support the cytotoxic effect of magnetic hyperthermia with hEpCAM-MNP caused by the specific adhesion of MNPs to the cell surface, the present inventors conducted simulations to predict thermogenesis capacity from MNPs based on in vitro experimental conditions. The model was built. The concentration of MNP for the simulation ^{was set to 3.6 × 10 10} particles/mL, which was measured by NanoSight measurement.

The heat generating capacity by MNP was calculated using COMSOL Multiphysics software, and the result was calculated that the Q _EM generated by AMF was approximately 3.57 × 10 ¹⁷ W/m ³ .

Under these conditions, the temperature of the cell suspension containing MNP increased to about 42 °C, and the solution without MNP increased to about 37 °C. A time course simulation of the temperature change for a 50 min magnetic annealing is shown in Figure 3D. The calculated results are very similar to the experimental data in Fig. 3B. In real experiments, slight differences between hEpCAM-MNP and In-hMNP-treated samples cannot be distinguished using simulations. Differences may be due to material properties shown in actual experiments, such as particle aggregation in the culture medium. Although the microscopic changes that have occurred cannot be reflected, the simulation results are consistent with the in vitro high heat treatment experimental results.

<2-4> Morphological analysis of THP1 cells after hyperthermia (high heat treatment)

4 shows bio-TEM images of THP1 cells before and after autothermal therapy (hyperthermal treatment). Cells treated with In-hMNP (Fig. 4A) and hEpCAM-MNP (Fig. 4B) that were not subjected to high heat treatment showed spherical and intact cell morphology. Tens to hundreds of hEpCAM-MNPs were observed on the surface of THP1 cells, indicating that specific binding of hEpCAM-MNPs to THP1 cells occurred (Fig. 4B). In the (+)HT group, some cells treated with In-hMNP (Fig. 4C) had intact morphologies similar to those observed in the (-)HT group (Fig. 4C, two left panels). However, some other cells appeared to be destroyed and small debris was observed (Fig. 4C, two right panels). In contrast, hEpCAM-MNP-treated cells were morphologically disrupted and MNPs adhered to the cell surface (Fig. 4D).

Collectively, these results indicated that magnetic hyperthermia with hEpCAM-MNP had cytotoxicity due to the specific binding of MNPs to THP1 cells, and that heat was transferred to the adhered cells. On the other hand, In-hMNPs not attached to THP1 cells were not confirmed as cytotoxic as hEpCAM-MNPs, and sufficient heat transfer could not be achieved.

<Experimental Example 3> In vivo self-thermal therapy

<3-1> Preparation of the mouse

6-week-old male AKR mice were purchased from the Central Animal Research Institute (Seoul, Korea). Mice were housed in the central animal facility of Daegu Gyeongbuk Institute of Science and Technology (DGIST) for acclimatization to experimental conditions. An optimal supply of food and water was ensured for the duration of the experiment. The animal testing protocol was approved by the DGIST Institutional Animal Care and Use Committee. Experiments were performed according to institutional protocols for animal welfare. All studies involving animals were reported according to the ARRIVE guidelines for experiments involving animals.

AKR mice were grown to 8 months of age for leukemia development and were randomly divided into four groups: control, In-mMNP (+)HT, mEpCAM-MNP (-)HT, and mEpCAM-MNP (+)HT.

<3-2> In vivo self-thermal therapy

For autothermal therapy (hyperthermal treatment) of the mouse whole body, the mice were first anesthetized with 30 µl of xylazine and ketamine by intraperitoneal injection. Each group was administered 100 μL phosphate-buffered saline (PBS, pH 7.4), In-mMNP or mEpCAM-MNP via tail vein injection. Thirty minutes after injection, mice in the (+)HT group were placed in a magnetized (Ambrell) coil and subjected to hyperthermia (hyperthermia) for 30 minutes with body (skin) temperature recording using a thermal camera (Testo). performed. Except for the diameter of the coil, the energy of the magnetizing device and equipment was the same as that of the in vitro test (Experimental Example 2).

Blood samples were collected before hyperthermia (hyperthermia) and 24 hours after treatment. To observe the interaction of MNPs with blood cells, bio-TEM analysis was performed using a Tecnai G2 F20 TWIN TMP instrument (FEI). After 24 h of hyperthermia (hyperthermia), all mice were sacrificed and tissues were collected and paraffin-blocked for subsequent analysis.

<3-3> Quantification of leukemia cells

Leukemia cancer cells were counted in two ways. First, Leishman staining was performed on whole blood cells. A small amount of blood was kept on a slide glass, and a thin smear was prepared and then air dried. The air-dried smears were stained with Leishman staining solution (Sigma-Aldrich, Co., St. Louis, MO, USA) for 2 min, double distilled water was added, and incubated for 10 min. Samples were rinsed with distilled water and air dried before mounting the cover glass. Abnormal lymphocytes were visualized and then quantified with an optical microscope (Leica Microsystems, Wetzlar, Germany) at X 400 magnification.

For enumeration of leukemia cells based on the expression of molecular markers, a PREP100 instrument (Celsee, Inc. Ann Arbor, MI, USA) was used for detection of cancer cells in 100 μl of whole blood according to the manufacturer's instructions. Prior to instrument use, cells were incubated with rat anti-EpCAM antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-CD45 antibody (1:100, Santa Cruz Biotechnology). Alex Fluor 488-labeled anti-rat IgG and Alex Fluor 568-labeled anti-rabbit IgG were used as secondary antibodies for EpCAM and CD45 detection, respectively. For nuclear staining, a 4',6-diamidino-2-phenylindole (DAPI) solution (Sigma-Aldrich) was used. Confocal microscopy (FV1200, Olympus, Tokyo, Japan) was used for observation of cell distribution in microfluids by fluorescence labeling of cells.

The tool setup for in vivo testing is shown in Figure 5A. 30 min high heat treatment increased the temperature of the culture medium without MNP to 34 °C, and the temperature of the medium containing mEpCAM-MNP increased to 42 °C, similar to that observed with hEpCAM-MNP (Fig. 5B). . However, after intravenous administration of 100 μl of mEpCAM-MNP to mice, there was no apparent temperature change in the skin during 30 min hyperthermia (Fig. 5B). The skin temperature measured by the thermal imaging camera is 27 ° C to 28 ° C, which is lower than the normal body temperature. This is due to the thermal emissivity of mouse hair, and the emissivity coefficient is reported to be 0.86 ± 0.01. Since the emissivity is less than 1, the actual temperature is higher than the temperature measured by the thermal imaging camera.

After 30 minutes of high heat treatment, mice in each of 4 groups were sacrificed 24 hours later. The number of abnormal cells in the blood was counted by Leishman staining and compared with the number of blood samples collected before hyperthermia. Figure 6A shows images of abnormal cells stained by Leishman solution. Cells stained purple and exhibiting an irregularly shaped membrane were classified as leukemia cells. As a result, magnetic hyperthermia to the mEpCAM-MNP(+)HT group of mice significantly reduced the number of abnormal cells to 44% of the number before hyperthermia, whereas control, In-mMNP(+)HT and mEpCAM-MNP ( -) There was no significant change in the HT group (Fig. 6B).

Leukemia cells were also counted using a commercial microfluidic chip-based instrument (Celsee) according to their specific fluorescent labeling with anti-CD45 and anti-EpCAM antibodies (lymphocytic leukemia cells expressing both CD45 and EpCAM markers on their surface). . Before using whole blood cells via microfluidic chip, cells were incubated with two primary antibodies, followed by secondary antibodies labeled with different fluorescent dyes. At the bottom of each microfluidic chip there is a hole through which normal blood cells can pass, but larger cancer cells cannot. Thus, leukemia cells with fluorescent labels of both CD45 and EpCAM could be maintained inside each well and counted (Fig. 7A). The ratio of the number of leukemia cells after hyperthermia compared to the number measured before hyperthermia was significantly decreased compared to the control, In-mMNP (+)HT and mEpCAM-MNP (-)HT groups, respectively (Fig. 7B). The reduced ratio of leukemia cells in the mEpCAM-MNP (+)HT group to the mEpCAM-MNP (-)HT group was 61%. These results show that the number of leukemia cells in the bloodstream can be directly reduced by systemic-magnetic hyperthermia after intravenous administration of mEpCAM-MNP having specific binding ability to leukemia cells in mice.

Removal efficiencies were determined significantly differently in the mEpCAM-MNP(+)HT group by Leishman staining and Celsee instruments, 56% and 39%, respectively. The difference is due to a completely different type of analysis method: counting by Leishman staining was performed based on cell morphology, whereas the Celsee instrumentation used immunological detection. The Celsee instrument method involved filtering the cells through the wells of the microfluidic chip, which may affect the results. However, the patterns of cell numbers in each group before and after heat treatment were very similar: only the mEpCAM-MNP-treated group showed a significant decrease in the number of leukemia cells after heat treatment.

<3-4> Prussian Blue Staining for MNP Localization in Tissue

Tissue sections from each group were de-paraffinized and rehydrated in deionized water. The sections were then stained with an iron staining kit (Sigma-Aldrich) for 15 min at room temperature. After the nuclei were stained with pararosaniline for 1 min, the slides were washed with distilled water, then briefly washed with an ascending series of alcohols, and then washed with xylene. The samples were then covered with coverslips. The bluish color of iron by Prussian blue staining was observed with an optical microscope (Leica Microsystems) at about 100 times magnification.

Since the thymus is the origin of T-cell lymphoblastic leukemia, mEpCAM-MNP can also be distributed in the thymic tissue. 8 shows representative results of Prussian blue staining of liver, lung, thymus and spleen tissues to analyze the distribution of MNPs. In-mMNPs and mEpCAM-MNPs were distributed in the thymus tissue, but no iron distribution was found in the lung and liver tissues in any mouse group. Because the spleen removes old red blood cells, retains blood and recycles iron, the iron distribution in the spleen is derived from the hemosiderin present in the spleen. Therefore, iron was observed in spleen tissues of all groups regardless of injection of mEpCAM-MNP.

Taken together, mEpCAM-MNPs can be distributed in the thymus of AKR mice, but these MNPs were not found in other tissues within 24 h after injection of the particles. Although the difference in thymic iron distribution between In-mMNP and mEpCAM-MNP could not be quantitatively measured in this study, Prussian blue staining revealed a relatively stronger signal in the thymus of the mEpCAM-MNP treated group.

<3-5> Detection of apoptosis in the thymus

Mouse thymus sections were stained using the DeadEnd Fluorescent-Based Terminal Transferase dUTP Nick End Labeling (TUNEL) Assay Kit (Promega, Madison, WI) to visualize dead cells after hyperthermia (high heat treatment).

Briefly, paraffin-embedded tissue sections (5 μm) were dewaxed in xylene and rehydrated in PBS. The sections were then coated with a solution of proteinase K in PBS (20 μg/mL) and incubated for 15 minutes at room temperature. The proteinase K solution was removed by immersing the slides in PBS. Then, reaction buffer containing rTdT enzyme, nucleotide mixture and equilibration buffer was added and incubated in a humidified chamber at 37° C. for 1 hour in the dark. The reaction was then terminated by immersing the slides in 2X saline citrate solution for 15 min at room temperature and washed thoroughly with PBS to remove unbound rTdT from the tissues. Finally, propidium iodide was used for counterstaining and mounted with extended gold antifade (Thermo Fisher Scientific, MA, USA, MA). Dead cells were visualized and images were captured at ×400 magnification with a confocal microscope (FV 1200, Olympus).

The discovery that mEpCAM-MNP is distributed in thymic tissue indicates that leukemia cells in thymic tissue may be affected by autothermal treatment. To support the specific clearance of leukemia cells by adhesion of mEpCAM-MNPs and subsequent magnetic hyperthermia, TUNEL analysis was performed on the thymus tissue. The results showed that apoptosis was highest in the mEpCAM-MNP (+)HT group (Fig. 9), indicating that the specific adhesion of mEpCAM-MNP to leukemia cells occurred in the thymus and circulatory system.

<3-6> Morphological change of blood cells by hyperthermia (high heat treatment)

The morphology of blood cells before and after autothermal therapy was analyzed by bio-TEM. Blood cells collected from control mice were intact and had a normal shape and appearance (Fig. 10A). As shown in Figure 10B, it is clear that some cells adhered to mEpCAM-MNP but not to In-mMNP. After hyperthermia, some blood cells in the mEpCAM-MNP (+)HT group were destroyed in their cell membranes, but membrane damage was not evident in the In-mMNP (+)HT group mice.

The data support that, due to the specific adhesion of mEpCAM-MNP to leukemia cells, magnetic hyperthermia can induce selective clearance of leukemia cells from circulation in AKR mice. The side effects of high heat treatment can be reduced in the case of normal tissue. This is because the binding specificity of MNP to the leukemia cell surface may limit the localization of mEpCAM-MNP in the bloodstream and leukemia cells in the thymic tissue for 24 h after injection.

<3-7> Heat transfer simulation during in vivo autothermal therapy (high heat treatment)

The temperature change of the leukemia cell surface was predicted by heat transfer simulations from mEpCAM-MNPs. The temperature of blood flow could not be confirmed during high heat treatment. However, the skin temperature of mice was measured with a thermal imaging camera (Fig. 5B). Although the body temperature was maintained below 30 °C during hyperthermia, the number of abnormal cells in whole blood was significantly reduced (Figs. 6 and 7). To confirm the apoptosis mechanism in the bloodstream, the temperature change of the leukemia cell surface was predicted by simulation of heat transfer from MNPs under experimental conditions.

In Figure 3D, temperature changes were predicted by applying AMF to THP1 cells, and the results showed that the model could simulate heat transfer from MNPs fairly well (Figures 3B and 3D). Based on these results, the temperature of leukemia cells with attached mEpCAM-MNPs was also predicted to support the reduced cell number of leukemia cells in mouse blood by whole-body magnetic hyperthermia.

It is a model constructed for in vivo prediction as shown in FIG. 11 using COMSOL Multiphysics software. To simplify modeling, the system volume ^{was defined as 10 × 10 × 10 μm 3} , and the initial temperature surrounding the leukemia cells was set to 37 °C to reflect body temperature. In addition, the concentration of MNP ^{was set to 3.6 × 10 10} particles/mL, 3.6 μl X 10 ¹⁰ particles of mEpCAM-MNP were injected into mice, and a total volume of mouse blood was assumed ^{to be approximately 1 mL (10 −6} m ^{3 ).} Because. Therefore, the number of MNPs in the system volume (10 × 10 × 10 μm ³ = 10 ⁻¹⁵ m ³ ) was calculated to be approximately 36. MNPs were assumed to be attached to the cell surface, and the diameters of MNPs and cells were set to 25 nm (Fig. 1A) and 5 μm (Fig. 10B), respectively. Based on the in vitro simulation results (Fig. 3D), the Q _EM of each MNP was considered to be 3.57 × 10 ¹⁷ W/m ^{3 .}

11A shows simulation conditions and parameters for predicting thermogenesis from mEpCAM-MNPs attached to leukemia cells. 11B shows the status of In-mMNPs floating in the bloodstream. The temperature distribution of cells and the surrounding environment was predicted for mEpCAM-MNP and In-mMNP in FIGS. 11C and 11D, respectively. The temperature of hEpCAM-MNPs attached to the cell surface significantly increased to 82 °C and the internal temperature of the cells increased to 41 °C (Fig. 11C). The temperature of the isolated In-mMNPs increased to 72 °C, and the internal temperature of free cells induced by In-mMNPs in the bloodstream was about 38 °C (Fig. 11D). It was predicted that the temperature of free cells and blood flow was not significantly increased by floating MNPs. The results support that when In-mMNPs are not attached to cells and are uniformly dispersed in the bloodstream, normal cells may not be damaged by the temperature rise of MNPs.

Blood flow was not taken into account in this simulation, but it was assumed that the temperature condition was kept constant at 37 °C. We also modeled a simplified distribution of particles in the simulation. In the case of in vitro modeling, modeling is performed assuming that the particles are distributed with some degree of dispersion according to the temperature distribution over time obtained from the actual experimental results. For in vivo modeling (Fig. 11), it was assumed that the particles were evenly distributed on the cell surface. The simplified heating model of MNP by AMF may have the following characteristics.

When MNP particles agglomerate, the dissipation of heat generated by AMF generally decreases, so the temperature of the particle itself becomes higher and the ambient temperature can rise faster than that of uniformly dispersed MNPs. However, it is known that when the number of MNPs per unit volume is the same, the final ambient temperature of the MNPs has a nearly similar temperature distribution regardless of the degree of dispersion or agglomeration of the particles. Since the number of MNPs per unit volume is the same, the total Q _EM is expected to be the same. Compared with uniformly distributed MNPs, simulations of aggregated MNPs (the same number of particles per unit volume) were performed. As a result, when MNPs aggregated, the temperature of the particles themselves increased faster compared to uniformly dispersed MNPs, but the final temperature distribution inside the cells was very similar regardless of the distribution of MNPs. Therefore, if the total Q _EM is the same, it can be confirmed that the temperature distribution around the particles is similar regardless of the distribution of the particles. Consequently, it was predicted that mEpCAM-MNPs attached to leukemia cells could specifically induce cell mortality by heat transfer generated under AMF.

Consequently, it was predicted that mEpCAM-MNPs attached to leukemia cells could specifically induce cell mortality by heat transfer generated under AMF. It has been reported that cells undergo mainly necrosis at temperatures exceeding the threshold of 45 °C, whereas they are prone to apoptosis below this threshold during self-hyperthermal treatment. The morphology of THP1 cells was different between In-hMNP and hEpCAM-MNP-treated cells by bio-TEM analysis (Fig. 4C and D): Fig. 4C appears to be a form of apoptosis, Fig. 4D is a form of necrotic cell death. showed In addition, according to the simulation results, the surface temperature of cells bound to MNPs could be as high as 82 °C under magnetic hyperthermia, but the surface temperature of cells without bound MNPs was predicted to be 38 °C (Figs. 11C and D). This result suggests that the mechanism of leukemia cell death due to magnetic hyperthermia may be necrotic, which is also supported by the bio-TEM image of blood cells in FIG. 10B .

A magnetic field was used to achieve the temperature of the ambient buffer solution up to 42 °C when the MNPs were dispersed (Fig. 5B). This is the minimum temperature for cancer cell removal. Under these conditions, the removal efficiency of leukemia cells in two different detection methods was determined to be 39-56%. In order to increase the removal efficiency, the size and concentration of MNPs and the duration and energy of AMF may be changed accordingly in consideration of the predicted results of heat generation simulation by magnetic hyperthermal treatment.

On the other hand, the concentration in FIG. 12 was measured while being diluted, and the average concentration derived from the equipment analysis result was used as an input of the model in consideration of the dilution factor. According to the present invention, the preferred concentration of the anti-EpCAM-coupled magnetic nanoparticles of the present invention to achieve a desired therapeutic effect was determined to be ^{10 9} - 10 ^{11 particles/ml.}

In the present invention, magnetic hyperthermia (hyperthermal treatment) was applied to selectively remove leukemia cells from the bloodstream, and specific clearance was achieved by targeted adhesion of MNPs to cancer cells. According to the detection method, the removal efficiency was measured to be 39 to 56%, and from this, it can be seen that hyperthermia (high heat treatment) is a very effective method for cancer (tumor) removal from the circulatory system through tumor selective targeting. It can be seen that in addition to (tumor), it is possible to treat harmful microorganisms such as viruses and bacteria through hyperthermia (high heat treatment) targeting them.

Claims (15)

EpCAM, CD34, CD38, TK1, CD200, CD56, CD7, CD3 and a composition for thermal treatment of circulating tumor cells or circulating tumor stem cells comprising magnetic nanoparticles bound to one or more antibodies selected from the group consisting of CD19 .
According to claim 1,
The composition, characterized in that the antibody is an anti-EpCAM antibody.
According to claim 1,
The magnetic nanoparticles include iron oxide, zinc ferrite, manganese (Mn), nickel (Ni), cobalt (Co), bismuth (Bi), platinum (Pt), gold (Au), palladium (Pd), copper (Cu), an alloy thereof, characterized in that at least one selected from the group consisting of ferrite (ferrite), the composition.
According to claim 1,
The magnetic nanoparticles are characterized in that the diameter is 1-100 nm, the composition.
According to claim 1,
wherein the magnetic nanoparticles have an iron content of greater than 30% (w/w).
3. The method of claim 2,
The EpCAM is a human or mouse-derived EpCAM, characterized in that the composition.
According to claim 1,
The binding of the antibody to the magnetic nanoparticles is a non-covalent bond or a covalent bond, the composition.
EpCAM, CD34, CD38, TK1, CD200, CD56, CD7, CD3, and binding to magnetic nanoparticles at least one antibody selected from the group consisting of CD19 Method for producing a composition for thermotherapy of claim 1 comprising a; .
A kit for thermotherapy of circulating tumor cells or circulating tumor stem cells, comprising the composition for thermotherapy of claim 1 and a device irradiating electromagnetic waves.
administering the composition for thermotherapy of claim 1 to an individual in need of treatment; and treating the subject with electromagnetic waves; and a method of treating circulating tumor cells or circulating tumor stem cells.
11. The method of claim 10,
The concentration of the magnetic nanoparticles to which the anti-EpCAM antibody of the composition is bound is 10 ⁹ - 10 ¹¹ particles/ml in the circulatory system of the individual, the treatment method.
11. The method of claim 10,
The electromagnetic wave treatment is to heat the tumor cells targeted to the circulatory system to 40 ℃ or more, the treatment method.
11. The method of claim 10,
The treatment method is to kill (apotosis) or necrosis (necrosis) tumor cells that target the circulatory system.
11. The method of claim 10,
The electromagnetic wave treatment is to apply that the electromagnetic wave is selected from the group consisting of alternating magnetic field (AMF), gamma rays, X-rays, ultraviolet rays, visible rays, infrared rays, microwaves and radio waves.
11. The method of claim 10,
characterized in that one or more treatment methods selected from the group consisting of chemotherapy, radiation therapy, biological therapy, immunotherapy, and photodynamic therapy are combined cancer treatment method.

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