KR101884987B1 - Complex comprising radionuclide-embedded gold nanoparticles, imaging agent and pharmaceutical composition for cancer immunotherapy comprising the same - Google Patents

Abstract

The present invention relates to radionuclide-embedded gold nanoparticles (RIe-AuNP) -based complexes, a molecular imaging contrast agent comprising the same, and a pharmaceutical composition for use in the anticancer immunotherapy using the same. More specifically, the present invention relates to a gold nanoparticle-radioactive isotope complex containing a radioactive isotope, a contrast agent containing the radioactive isotope, and a pharmaceutical composition for anticancer immunotherapy.
The complex according to the present invention has about 2000 or more radioactive isotopes bonded to each nanoparticle, and can provide stable nuclear and optical images in vivo with very strong signal intensity. In addition, when the Au envelope is additionally formed, very high in vivo stability can be achieved, and even if it is absorbed into the body by dendritic cells, it exhibits strong and enhanced antitumor immunity without inducing side effects on the biological function of body cells .

Description

FIELD OF THE INVENTION The present invention relates to a complex comprising radionuclide-embedded gold nanoparticles, a contrast agent containing the same, and a pharmaceutical composition for anti-cancer immunotherapy,

The present invention relates to radionuclide-embedded gold nanoparticles (RIe-AuNP) -based complexes, a molecular imaging contrast agent containing the same, and a pharmaceutical composition for anticancer immunotherapy using the same.

Because gold nanoparticle (AuNP) based imaging agents exhibit excellent biocompatibility and inherent optical properties and have simple surface chemistry for targeting and surface stabilization, they are widely studied as molecular imaging agents (Non-Patent Document 1). The use of a contrast agent with a high signal sensitivity can significantly reduce the dosage required for biomolecule imaging and thus reduce the potential for adverse effects from the use of contrast agents. In addition, in order to maintain the sensitivity of the contrast agent for a long time in the body, The protection of bound radioisotopes is very important. For this reason, easy and rapid synthesis methods capable of producing a contrast agent with high signal sensitivity and capable of maintaining such signal sensitivity while maintaining effective signal sensitivity have been intensively studied (Non-Patent Document 2).

In this regard, when ¹²⁵ I is conjugated to gold nanoparticles by noncovalent bonding to the surfaces of gold nanoparticles, the radioisotope per nanoparticle is loaded as low as about six. In addition, despite the presence of a protective layer composed of a polymer such as PEG, significant amounts of radioactive isotopes are released from the surface of the gold nanoparticles, and therefore, it is necessary to inject a large amount (10 mg / kg) of contrast agent 3).

Although signal sensitivity and stability are critical issues in all molecular formulations, in the field of cancer-based immunotherapy based on dendritic cells, in order to accurately evaluate the results of such immunotherapy, bone marrow- And it is particularly important to precisely track whether it has been well transferred to the lymphatic system (Non-Patent Document 4).

In this context, nuclear imaging (i.e., positron emission tomography (PET), single photon emission computed tomography (SPECT)), magnetic resonance and optical imaging (i.e., quantum dots, organic fluorescent sources and upconverted light emitting nanoparticles) (Non-Patent Documents 5 and 6), it is very difficult to track dendritic cells with high sensitivity for a long time due to sensitivity and stability problems of the contrast agent in vivo have.

PET and SPECT are routinely used in clinical nuclear medicine because of their high sensitivity (SPECT: 10 ^-10 M, PET: 10 ^-11 -10 ^-12 M) and no depth limitation (Non-Patent Document 7) In view of the development of a low-cost, high-sensitivity optical imaging technique, there is a need to develop an optical-based imaging technique comparable to nuclear imaging in terms of sensitivity and penetration depth (Non-Patent Document 8). In view of this, cheren Corp. luminescence imaging method (Cerenkov luminescence imaging, CLI) are promising optical-based technologies bar, which the charged particles from the light emitting radionuclides if the (mostly beta ⁺⁾ passes through the water at a higher speed than the speed of light (Non-patent documents 8 and 9). Many attempts have been made to use PET radionuclides such as ⁶⁴ Cu, ⁶⁸ Ga and ¹²⁴ I in CLI-based optical imaging techniques (Non-Patent Document 10), but the quantitative relationship between PET and CLI imaging methods has not yet been established Bar, establishing this relationship is required when applying CLI-based imaging methods to clinical applications.

Non-Patent Document 1: Boisselier, E. & Astruc, D. Gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity. Chem. Soc. Rev. 38, 17591782 (2009). Non-Patent Document 2: Lim, DK, Jeon, K.-S., Hwang, J.-H., Kim, H., Kwon, S., Suh, YD & Nam, JMHighly uniform and reproducible surface- scattering from DNA-tailorable nanoparticles with 1-nm interior gap. Nat. Nanotechnol. 6, 452460 (2011). Non-Patent Document 3: Harris, J. M. & Chess, R. B. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discov. 2, 214221 (2003). Non-Patent Document 4: Palucka, K. & Banchereau, J. Cancer immunotherapy via dendritic cells. Nat. Rev. Cancer 12, 265277 (2012). Non-Patent Document 5: Wu, C., Xu, Y., Yang, L., Wu, J., Zhu, W., Li, D., Cheng, Z., Xia, Gong, Q.Negatively charged magnetite nanoparticle clusters as efficient MRI probes for dendritic cell labeling and in vivo tracking. Adv. Funct. Mater. 25, 35813591 (2015). Non-Patent Document 6: Xiang, J., Xu, L., Gong, H., Zhu, W., Wang, C., Xu, J., Feng, L., Cheng, L., Liu, Z. Antigen-loaded upconversion nanoparticles for dendritic cell stimulation, tracking, and vaccination in dendritic cell-based immunotherapy. ACS Nano 9, 64016411 (2015). Non-Patent Document 7: Adonai, N., Nguyen, KN, Walsh, J., Iyer, M., Toyokuni, T., Phelps, ME, McCarthy, T., McCarthy, DW & Gambhir, SS Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis (N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positronemission tomography. Proc. Natl Acad. Sci. USA 99, 30303035 (2002). Non-Patent Document 8: Thorek, D. L. J., Robertson, R., Bacchus, W. A., Hahn, J., Rothberg, J., Beattie, B. J. & Grimm, J. Cerenkov imagingA new modality for molecular imaging. Am. J. Nucl. Med. Mol. Imaging 2, 163 (2012). Non-Patent Document 9: Robertson, R., Germanos, M. S., Li, C., Mitchell, G. S., Cherry, S. R. & Silva, M. D. Optical imaging of Cerenkov light generation from positron-emitting radiotracers. Phys. Med. Biol. 54, N355N365 (2009). Non-Patent Document 10: Xu, Y., Liu, H. & Cheng, Z. Harnessing the power of radionuclides for optical imaging: Cerenkov luminescence imaging. J. Nucl. Med. 52, 20092018 (2011).

Accordingly, in order to solve the problems of the conventional art described above, the present invention has been made to solve the above-mentioned problems of the prior art, and it is an object of the present invention to provide a nanoparticle composition that exhibits very strong signal intensity by bonding a very large number of radioactive isotopes per nanoparticle, The present invention also provides a complex comprising radioisotope-embedded gold nanoparticles capable of exhibiting strong anti-tumor immunity without inducing side effects, and a pharmaceutical composition for contrast agent immunotherapy comprising the same.

In order to solve the above problems, the present invention provides a gold nanoparticle-radioisotope complex in which a radioactive isotope is contained.

According to one embodiment of the present invention, the complex may be a conjugate of a radioisotope to gold nanoparticles to which an oligonucleotide having an adenine base is bonded.

According to another embodiment of the present invention, the complex may be one in which a radioisotope is conjugated to gold nanoparticles to which a peptide sequence including a tyrosine amino acid is bound.

According to another embodiment of the present invention, the conjugation is performed such that the primary amine group in the adenine base of the oligonucleotide forms an amide bond with the 4-hydroxyphenyl group of sulfosuccininmyl-3- (4-hydroxylphenyl) propionate (sulfo-SHPP) And the 2,5-position of the 4-hydroxyphenyl ring is substituted with a radioactive isotope.

According to another embodiment of the present invention, the conjugation may be formed by replacing the 2,5-position of the 4-hydroxyphenyl ring in the tyrosine of the peptide sequence with a radioactive isotope.

According to another embodiment of the present invention, the radioisotope may be ¹²⁴ I, ¹²⁵ I, ⁶⁴ Cu, ⁶⁸ Ga, ¹²⁴ I, ¹²⁵ I or a mixture thereof.

According to another embodiment of the present invention, the complex may comprise an additional gold envelope.

According to another embodiment of the present invention, the composite may have an average particle size of 1 nm to 1000 nm.

The present invention also provides a contrast agent comprising the gold nanoparticle-radioisotope complex.

According to an embodiment of the present invention, the contrast agent can be applied to nuclear medicine imaging or optical imaging.

According to another embodiment of the present invention, the nuclear medicine imaging may be positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

According to another embodiment of the present invention, the optical imaging may be CLI (Cerenov luminescence imaging).

The present invention also provides a pharmaceutical composition for immunotherapy wherein the gold nanoparticle-radioactive isotope complex labeled dendritic cells.

According to one embodiment of the invention, the composition can track the migration of dendritic cells in vivo during immunotherapy.

According to another embodiment of the present invention, the dendritic cell may be a bone marrow derived cell.

According to another embodiment of the present invention, the dendritic cell may be stimulated with a lysate of a tumor to be treated, a tumor-specific peptide, a tumor RNA, a DNA encoding a tumor-specific antigen, or a mixture thereof.

According to another embodiment of the present invention, the dendritic cell may be stimulated with a lysate of the tumor to be treated.

The complex according to the present invention has about 2000 or more radioactive isotopes bonded to each nanoparticle, and can provide stable nuclear and optical images in vivo with very strong signal intensity. In addition, when the Au envelope is additionally formed, very high in vivo stability can be achieved, and even if it is absorbed into the body by dendritic cells, it exhibits strong and enhanced antitumor immunity without inducing side effects on the biological function of body cells .

FIG. 1 is a view schematically showing a process of labeling dendritic cells using the complex according to the present invention and performing immunotherapy, lymph node imaging and biodistribution monitoring using labeled dendritic cells.
FIG. 2 is a schematic diagram showing the reaction scheme (a) for synthesizing RI-AuNP, the change in the chemical structure of adenine in the reaction (b), and the time course of TLC chromatography (c to e) Fig.
FIG. 3 is a schematic diagram of a reaction scheme (a) for synthesizing RIe-AuNP, UVs for A10-AuNPs, SHPP-A10-AuNPs, RI- AuNPs (red line) and radioactive nuclide- embedded AuNPs (RIe- AuNPs, blue line) - EDX-based element mapping results for the visible spectrum (b), XPS data for RIe-AuNP (c), transmission electron microscopy images for RIe-AuNPdp and Au (blue), iodine (red) Fig.
Figure 4 is a graph showing the radioactivity of RIe-AuNP ( ¹²⁵ I) as a function of core particle size (5 nm (black), 10 nm (red) and 20 nm (blue) (B) showing the sensitivity of RIe-AuNP ( ¹²⁵ I) as a function of the concentration (1.0 pM to 0.5 nM), the photograph of the RIe-AuNP ( ¹²⁴ I) solution and the PET / CT image (c) (d) showing the sensitivity of RIe-AuNP ( ¹²⁴ I) as a function of the concentration of RIe-AuNP ( ¹²⁴ I) (1) and RIe-AuNP ( ¹²⁴ I) (The blue value indicates the relative area% of the peak).
Figure 5 is a graph showing the relationship between nanoparticle size and radioactivity.
FIG. 6 shows the effect of absorption efficiency of RIe-AuNP in DC and biological function and anti-cancer immunity of DC. After incubation with three different concentrations of RIe-AuNP (1.04.0 nM), DC PET / CT images (b) of tube phantoms containing bright-field images (a), unlabeled DCs (1) or labeled DCs (2), time- (D) showing the DC survival as a function of RIe-AuNP concentration (1.04.0 nM) and incubation time (24 hours (black bars) or 48 hours (empty bars)), phenotypic markers (E), unlabeled and nonpulsed DCs (1), DCs pulsed with unlabeled and LLC lysates (2), and markers for MHC-I, MHC-II, CD86, CD54 and CCR- And PET / CT images (f) on tumor bearing mice and incised tumors after treatment with non-pulsed DC (3), label and DC pulsed with LLC lysate (4) Of cytotoxicity of splenocytes by bioluminescence Monitored plots (LLC / effluc cells were used as target cells). Data were expressed as mean ± standard deviation (7 mice) (* P < 0.05).
FIG. 7 is a graph showing the effect of the dose on the cell uptake efficiency and the cell uptake efficiency (b) over time of RIe-AuNP (2.0 nM).
FIG. 8 is a graph showing the radioactivity of DC treated with RI-AuNP at 0 hour and the change of radioactivity in DC and cell medium after 3 hours.
FIG. 9 is a graph comparing cell death levels after incubation of unlabeled DCs and labeled DCs for 24 hours. Cells were stained with iodidated propidium (PI) and Annexin-V and analyzed by flow cytometry
FIG. 10 is an analysis of phenotypic markers of unlabeled DCs and RIe-AuNP labeled DCs, showing FITC-conjugated anti-CD11c, anti-MHC I, anti-MHC II, anti-CD86, anti- (Black graph shows isotype-matched cells; level indicates relative increase (%) in marker expression level).
Figure 11 shows the level (a) of cytokines (TNF- alpha and IL-6), the results of transwell migration analysis (labeled or unlabeled DCs were applied to the upper chamber, followed by the lower chamber containing CCL- (B), antigen uptake ability (unlabeled and labeled DCs were incubated with Texas Red-OVA at 4 or 37 ° C for 1 hour, cells were washed with PBS , And absorption of nanoparticles is analyzed using a flow cytometer) (c).
Figure 12 is a schematic outline of the process for in vivo treatment. C57BL / 6 mice were immunized once a week for 2 weeks with DC by the following procedure: (1) unlabeled and nonpulsed DC, (2) (3) labeled and non-pulsed DC, (4) labeled and LLC pulsed DC pulses, respectively. For pulsatile DC, total LLC solubles were prepared by repeated freezing and thawing and then LLC cell lysates (5-10 ug / ml) were treated with DC overnight. Mice were injected intramuscularly once a week for 2 weeks with the appropriate DC formulation. One week after the final vaccination, LLC cells were injected into mice by subcutaneous injection, and tumor growth was monitored by palpation and ¹⁸ F-furopoxyglucose ( ¹⁸ F-FDG) PET / CT at designated times . In-vivo experiments were performed 3 times (7 mice per group).
Figure 13 shows the results after treatment with unlabeled and nonpulsed DC (black), unlabeled and pulsed DC (red), labeled and nonpulsed DC (blue) or labeled and pulsed DC (green) Fig. 3 is a graph showing the tumor volume measured by a caliper. Fig.
FIG. 14 is a process schematic diagram for an animal experiment in which DC was generated from bone marrow cells by treatment with GM-CSF for 7 days. Immature DCs were then activated overnight with LPS and LLC tumor lysates. Mature DCs were treated with 2.0 nM RIe-AuNP for 3 hours and injected subcutaneously through the sole of the foot. The mobility of the moved DCs was monitored by PET / CT. In-vivo experiments were performed 3 times (5 mice per group).
FIG. 15 is a graph showing the time-dependent 3D-PET / CT images (top) and axial PET / CT images (bottom) (yellow circles represent multiple dendritic lymph nodes (C, d) for analysis of DPLN and non-DPLN in PET / CT and CLI imaging (a), (b) Data were expressed as mean ± SD and n = 5), the distribution of labeled DCs in all organs after injection through the soles (e) (the red box was enlarged to clearly show changes in radioactivity during DPLN (1), CD79a-specific antibody staining for B-cell region (2), T-cell (2) (3) and S100-specific antibody staining for DC (4) (black circles and arrows indicate RIe-AuNPs and data are mean ± standard deviation, and n = 5) (f).
FIG. 16 shows the time-dependent PET / CT, cherenkov luminescence image and biodistribution of the intravenously injected labeled DCs, and the 3D PET / CT image (top) and axial image (bottom) (A), and (c) cherenkov luminescence images were obtained using an optical imaging system (IVIS Lumina III) (b) and the distribution of DCs labeled in the mouse DC) were taken intravenously, and obtained after 0.5 hours (black), 4 hours (red), 1 day (blue), 2 days (green), 3 days (orange) and 4 days (purple). Data are mean ± standard deviation (N = 5).

Hereinafter, the present invention will be described in detail.

The present invention provides a gold-based complex and a contrast agent containing the same that can be applied as a dual bioimaging mode by combining nuclear imaging and Cherenkov luminescence imaging methods. In addition, a simple and simple synthesis method present.

In the present invention, multivalent adenine-rich oligonucleotide sequences present on gold nanoparticles enable conjugation of large amounts of radioactive isotopes per gold nanoparticle. In addition, the additional Au envelope creates a stable protective layer that can completely defend the enzyme's attack in the biological system.

Because the complex according to the present invention exhibits high radiation sensitivity of radioisotope embedded-AuNP (RIe-AuNP), only a small amount of Au (0.3 mg / kg) Can be obtained.

Furthermore, as can be seen from the following examples, the RIe-AuNP according to the present invention had no adverse effect on the biological function of the dendritic cells and showed a strong anti-cancer immune response against lung cancer.

In addition, due to the strong radioactivity of RIe-AuNP, it is possible to monitor the migration of dendritic cells to the lymph nodes with high sensitivity using PET and CLI simultaneously. In connection with this, an in-vivo study was conducted using intravenous injection as described in the following examples. As a result, due to the strong Cerenkow emission intensity of RIe-AuNP, CLI-based images are comparable to corresponding PET images I showed the video.

Accordingly, the present invention provides a complex comprising radioisotope embedded-gold nanoparticles (RI-AuNP). In the present invention, the RI-AuNP may be prepared by conjugating a radioisotope to gold nanoparticles bound with an oligonucleotide containing an adenine base or with a peptide sequence comprising a tyrosine amino acid The conjugation means that the primary amine group in the adenine nucleotide of the oligonucleotide forms an amide bond with the 4-hydroxyphenyl group of sulfosuccininmyl-3- (4-hydroxylphenyl) propionate (sulfo-SHPP), and the 4-hydroxyphenyl Position may be formed by substituting the radioisotope for the 2,5-position of the ring, or by substituting the radioisotope for the 2,5-position of the 4-hydroxyphenyl ring in the tyrosine of the peptide sequence.

Further, as will be described later in the following examples, it is also possible to achieve further excellent stability in the body by forming an additional gold skin (RIe-AuNP) on the surface of the composite (RI-AuNP) according to the present invention.

Radioisotopes embedded in the complex according to the present invention include, but are not limited to, ¹²⁴ I, ¹²⁵ I, ⁶⁴ Cu, ⁶⁸ Ga or mixtures thereof, preferably ¹²⁴ I, ¹²⁵ I or And mixtures thereof.

In addition, the composite according to the present invention may have an average particle diameter of 1 nm to 1000 nm.

Meanwhile, the present invention provides a contrast agent comprising the gold nanoparticle-radioactive isotope complex, and the contrast agent according to the present invention can be effectively used for nuclear medicine imaging or optical imaging, The present invention can be effectively applied to a dual-body image capturing method in which image capturing is combined. The nuclear magnetic resonance imaging (MRI) includes, but is not limited to, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) For example.

In addition, when various ligands such as a hydrophilic functional group, a target binding ligand and the like are bound to the surface of the contrast agent according to the present invention and used as a molecular imaging contrast agent, it is possible to diagnose various diseases using the ligand.

Further, the present invention also provides a pharmaceutical composition for immunotherapy comprising the dendritic cells to which the complex is labeled, and as described in detail in the following examples, the pharmaceutical composition for immunotherapy according to the present invention comprises The movement of dendritic cells in vivo can be tracked very accurately.

The dendritic cell may be, but is not limited to, a bone marrow-derived cell and may be a lysate of a tumor to be treated, a tumor-specific peptide, a tumor RNA, a DNA encoding a tumor-specific antigen, Or may be stimulated with the mixture.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are intended to assist the understanding of the present invention and are not intended to limit the scope of the present invention.

Experimental course

Materials and reagents

All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Citrate-stabilized gold nanoparticles (20 nm) were purchased from Ted Pella Inc. (Redding, CA, USA). Sulfosuccimidyl-3- [4-hydroxyphenyl] propionate (sulfo-SHPP) was purchased from Thermo Scientific (Rockford, IL, USA). Radioactive sodium iodide (Na ¹²⁵ I) was purchased from Perskin Elmer (Waltham, MA, USA) and sodium iodide (Na ¹²⁴ I) was purchased from Duchem Bio (Daegu, Korea) and KIRAMS (Seoul, Korea). Bone marrow-derived dendritic cells produced by differentiation of bone marrow cells were used for all experiments. Lewis lung carcinoma (LLC) cells and cervical cancer cells were treated with 10% heat inactivated fetal bovine serum (HyClone), 50 μg / ml gentamicin (Gibco, Grand Island, NY, USA) and 1.0% penicillin- Were cultured in complete Dulbecco modified Eagle medium supplemented with Gibco (HyClone, Logan, UT, USA). For the experiments, SLC Inc. Sterile, 6-week immunocompetent C57BL / 6 rats obtained from Shizuoka, Japan were used. All animal testing procedures were carried out in strict accordance with the appropriate institutional guidelines for animal studies. The protocol was approved by the Animal Experimentation Ethics Committee of Kyungpook National University (Approval No.: KNU 2012-43).

Synthesis of RIe-AuNP (oligonucleotide-linked AuNP-based synthesis)

A reduced thiolated adenine-rich oligonucleotide (3'-HS- (CH ₂ ) ₃ -A ₁₀ -5 ') was added to citrate-Au NP (20 nm) via standard salt aging procedures . Subsequently, the amine group (A ₁₀ -AuNP) of the adenine-rich oligonucleotide on AuNP was modified with sulfo-SHPP to conjugate with a radioactive isotope such as sodium iodide (Na ¹²⁵ I or NA ¹²⁴ I) (Fig. 2) . To prepare 20.0 mL of SHPP-modified AuNP (1.0 nM), the immediately prepared A ₁₀ -AuNP solution (20 mL) was reacted with sulfo-SHPP (1.0 mg) for 12 hours at room temperature and then centrifuged (12,000 rpm / 15 min). The supernatant was removed and the precipitate redispersed in distilled water (20 mL). Particle concentration was determined using a UV-visible spectrophotometer. For radioisotopic labeling, SHPP-AuNP (1.0 nM, 1 mL) was incubated at room temperature for 2 hours in the presence of 90 μL of chloramine-T (3 mg / mL) and 100 μL of 1% dodecyl sulfate , 150 μL Na ¹²⁵ I (129.5 MBq) or NA ¹²⁴ I (81.4 MBI), followed by purification by centrifugation (12,000 rpm / 15 min). The supernatant was removed and the precipitate redispersed in distilled water. The decay-corrected radiochemical yield was 90-95% (NA ¹²⁵ I (116.5-118.4 MBq), Na ¹²⁴ I (73.2-74 MBq)). 1 mL of radioisotope-AuNP (RI-AuNP) (1.0 nM) was added to 500 μL of 1.0% (w / v) poly ( N- vinyl-2-pyrrolidone) _w 40 kDa), radioisotope embedded-gold nanoparticles (RIe-AuNP) containing ¹²⁵ I or ¹²⁴ I were added by adding 100 μL of 100 mM phosphate buffer (pH 7.4) and 165 μL of 2.0 M NaCl, . Finally, the solution was reacted with 434 μL of hydroxylamine hydrochloride (10 mM) and 434 μL of HAuCl ₄ (5 mM). The reaction mixture was stirred and mixed at room temperature for 30 minutes and purified by centrifugation (6,000 rpm / 15 minutes). The supernatant was removed and the precipitate redispersed in distilled water (1 mL) (FIGS. 2 and 3).

Synthesis of RIe-AuNP (tyrosine-coupled AuNP-based synthesis)

To 20 ml of citric acid-bound AuNP (20 nm) was added 1 mg of cysteine-linked amino acid at the end of the amino acid chain in which 10 tyrosine amino acids were connected in series, and the mixture was reacted for 12 hours. The supernatant is removed by centrifugation (12,000 rpm / 15 minutes), and 20 ml of distilled water is added to prepare 20 ml of tyrosine-bound gold nanoparticles. For radioisotope labeling, tyrosine-bound gold nanoparticles (1.0 nM, 1 mL) were incubated with 90 μL of chloramine-T (3 mg / mL) and 100 μL of 1% dodecylsulfate in the presence, sikyeotgo reacted with Na ¹²⁵ I (129.5 MBq) or NA ¹²⁴ I (81.4 MB1) of 150 μL, was then purified by centrifugation (12,000 rpm / 15 minutes). The supernatant was removed and the precipitate redispersed in distilled water. The decay-corrected radiochemical yield was 90-95% (NA ¹²⁵ I (116.5-118.4 MBq), Na ¹²⁴ I (73.2-74 MBq)). 1 mL of radioisotope-AuNP (RI-AuNP) (1.0 nM) was added to 500 μL of 1.0% (w / v) poly ( N- vinyl-2-pyrrolidone) _w 40 kDa), radioisotope embedded-gold nanoparticles (RIe-AuNP) containing ¹²⁵ I or ¹²⁴ I were added by adding 100 μL of 100 mM phosphate buffer (pH 7.4) and 165 μL of 2.0 M NaCl, . Finally, the solution was reacted with 434 μL of hydroxylamine hydrochloride (10 mM) and 434 μL of HAuCl ₄ (5 mM). The reaction mixture was stirred and mixed at room temperature for 30 minutes and purified by centrifugation (6,000 rpm / 15 minutes). The supernatant was removed and the precipitate redispersed in distilled water (1 mL).

Analysis of RIe-AuNP

The dispersibility, chemical composition and structure analysis of RIe-AuNP prepared as described above were performed through UV-Vis spectrometer, X-ray photoelectron spectroscopy, energy dispersive X-ray based element mapping and transmission electron microscopy (Fig. 3).

Radioactivity sensitivity and in vitro ( In vitro ) stability

The signal intensities of SPECT and PET were examined with various concentrations of RIe-AuNP (1.0 pM-0.5 nM) using a gamma counter (Gamma-10; Sinjin Medics, Korea) (FIGS. For in vitro stability experiments, RI-AuNP (100 μL, 1.0 nM) and RIe-AuNP (100 μL, 1.0 nM) were incubated with human serum (900 μL) at 37 ° C. The released radioisotope was then quantified using thin layer chromatography (eluent: acetone) and the results were recorded using a scanner (AR-2000; Bioscan, USA) (FIG.

Absorption efficiency and cytotoxicity of RIe-AuNP in dendritic cells

RIe-AuNP was diluted in cell culture medium at various concentrations of 0.5-4.0 nM and incubated with dendritic cells (DC) for 3 hours at 37 ° C. After incubation, DC was washed with phosphate buffer and free RIe-AuNP was removed from the supernatant after two centrifugations (1,300 rpm / 5 min). The absorption efficiency of RIe-AuNP in DC was measured with a gamma counter (Gamma-10; Sinjin Medics, Korea). The time-dependent absorption efficiency was investigated using RIe-AuNP at a concentration of 2.0 nM (Fig. 7). By measuring the radioactivity of the supernatant over time, changes in radioactivity over time in DC were determined (FIGS. 4 and 8).

Meanwhile, cytotoxicity of RIe-AuNP in DC was evaluated by performing cell proliferation assay using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (Dojindo Laboratories, Tokyo, Japan) Respectively. Apoptosis analysis was performed using FITC-conjugated annexin V and propidium iodide (BD Biosciences) using a flow cytometer (Accuri C6, BD Biosciences) (FIG. 9).

Biological function of DC

Phenotype marker analysis : DCs labeled with unlabeled DC or RIe-AuNP (2.0 nM) were incubated for 30 min at 4 ° C with the picoerythrin-conjugated CD11c, H-2Kb (MHC-I) or (BD Bioscience, San Jose, Calif., USA). The cells were stained with anti-IL-1 / IA (MHC-II), CD86 and CD54 and allophycocyanin-Cy7-conjugated CD197 (CCR-7) The washed cells were analyzed using flow cytometry. As a control, isotype-matched monoclonal antibodies were used. Data were analyzed using FlowJo analysis software (FlowJo, USA) (Figure 10).

Were stimulated with while the immature DC 12 sigan lipopolysaccharide (1.0 μg / mL): cytokine analysis (cytokine analysis). Culture plates were inoculated with mature DC or unlabeled mature DCs labeled with RIe-AuNP. After 24 hours, the contents of TNF-α and interleukin-6 in the supernatant were quantified by a multiplexed bead-based immunoassay (BDTM Cytometric Bead Array) system and flow cytometry (BD Biosciences) (FIG.

Transwell (Transwell) migration assay: cultured DC with for 24 hours, 1.0 μg / mL prostaglandin E2 (Sigma), and added to the culture medium containing CCL 19 (Pepro Tech) in the lower chamber and then, 5 μm pore 2x10 &lt; ⁵ &gt; cells were added to the upper chamber containing the polycarbonate membrane (BD Falcon). After incubation at 37 ° C for 2 hours, cells migrating to the lower chamber were collected and counted by flow cytometry. The events occurring during the fixed time (60 seconds) were analyzed using FlowJo analysis software.

Antigen Absorption Efficiency : DCs labeled with unlabeled DC or RIe-AuNP were incubated with 100 μg / mL Texas Red albumin (Life Technologies, Grand Island, NY, USA) at 37 ° C for 1 hour. After labeling, the cells were washed with phosphate buffered saline and analyzed using a BD Accuri C6 flow cytometer (BD Biosciences).

DC-based cancer immunotherapy in the body

As described in more detail in Figure 12, rats were grouped into four groups according to treatment regime: 1) unlabeled nonpulsed DC, 2) DC pulsed with unlabeled LLC lysate, 3) labeled nonpulsed DC, and 4) Pulse DC as a lid for cover LLC. For the pulses of DC, the entire LLC lysate was prepared by repeated freezing and thawing, and then the LLC cell lysate (5-10 [mu] g / mL) prepared was treated overnight in DC. The mice were injected intramuscularly with corresponding DCs for 2 weeks at intervals once a week. One week after the last inoculation, LLC cells were injected subcutaneously into mice and tumor growth was monitored with palpation and ¹⁸ F-furopoxyglucose ( ¹⁸ F-FDG) PET / CT at designated times. The size of the tumor (mm ³ ) was calculated as (A x B ² ) / 2, where A is the longitudinal direction diameter and B is the unidirectional diameter (Figure 13).

For cytotoxic T lymphocyte analysis, spleen cells collected from tumor-bearing mice were re-stimulated with total LLC lysate and IL-2 for 4 days. LLC / effluc cells were used as target cells. Target cells were inoculated into black clear bottom 96-well plates (1 x 10 ⁴ cells). After overnight incubation, splenocytes were added to the wells containing 10: 1 ratio of the effector and target cells as effectors. After 11 hours, luciferase activity was measured with an IVIS Lumina III instrument (Perkin Elmer).

In-vivo PET imaging and Cerenkov luminescence imaging

For lymph node imaging, 30 ng TNF-alpha was injected subcutaneously into the hindpaw of the rats, and then on day 1, RIe-AuNP-labeled DCs (50 μL of cells per mouse x 10 ⁶ cells, 6.44 μg (Au), 0.2 mg / kg) were injected and PET / CT images were taken at the designated time (FIG. 14). The body experiment was performed three times.

For quantitative comparison of PET and CLI, 50 μL of cells (3 × 10 ⁶ cells, 6.44 μg (Au), 0.2 mg / kg) were injected intraperitoneally via RIe-AuNP labeled DCs PET / CT images were taken at the specified time. For PET / CT imaging, a 20-minute scan (DC imaging) was performed with a Triumph II PET-CT system (LabPET8; Gamma Medica-Ideas, Waukesha, WI, USA). CT scans were performed with an X-ray detector immediately after acquiring PET images. PET images were reconstructed by 3D-OSEM repetitive image reconstruction, and CT images were reconstructed by filtration back projection. All rats were anesthetized with 1-2% isoflurane gas during imaging. PET images were registered with anatomical CT images using 3D visualization and analysis software (VIVID: Gamma Medica-Ideas, Northridge, CA, USA). To determine the rate of absorption (% ID / cc) for the volume of interest, we manually subdivided the volume of interest in each image from the combined CT images using both VIVID and PMOD software (PMOD Technologies, Zurich, Switzerland) Absorbance in the region of interest was measured using PMOD 3.5 software.

Cerenkov luminescence imaging was performed using the IVIS Lumina III imaging system (Perkin Elmer). Black and white photographic images and bioluminescence color images were superimposed using LIVINGMAGE (version 2.12, Perkin Elmer) and IGOR image analysis FX software (WaveMetrics, Lake Oswego, OR, USA). CLI signals are expressed as units of photons per cm ² per second per steradian ^{(P / cm 2 / s /} sr).

Biological distribution studies and in vitro imaging

Rats were sacrificed at designated times after administration of labeled DC through the plantar or tail vein. Interested organs were separated, weighed, and analyzed using a gamma counter. Results are expressed as percentage of injected dose per gram of tissue (% ID / g). After obtaining the images, mice were sacrificed and the draining popliteal lymph node (DPLN) was cut out. Each cut - off lymph node was placed in a tube and the PET / CT images were taken for 5 minutes. In vitro experiments were performed three times.

Histological data and immunohistochemistry

Histologic data were obtained from draining and non-draining lymph nodes (formalin fixed, paraffin-embedded) and analyzed by microscopic examination of hematoxylin and eosin (H & E) staining slides. Immunohistochemical staining was performed according to the manufacturer's guidelines using a Benchmark XT slide dyer (Ventana Medical Systems, Inc., Tucson, AZ, USA). Anti-CD3 (1: 200 dilution; Clone SP7; Thermo Fisher Scientific), anti-CD79a (pre-dilution; clone SP18; Ventana Medical Systems, Inc., Tucson, AZ, USA) -100 (1: 400 dilution; clone B-8; Dako, Glostup, Denmark).

Results and review

Synthesis of RIe-AuNP

Because oligonucleotide-modified AuNP has a high loading capacity per nanoparticle, it has been extensively studied as a sensitive biosensor or gene delivery platform. The present invention focuses on the chemical function of the oligonucleotide base on AuNP which can be used as a reaction site for radioactive labeling. The primary amine group in the adenine base can be functionalized with sulfosuccininmyl-3- (4-hydroxylphenyl) propionate (sulfo-SHPP), thereby forming an amide bond between the primary amine of the 4-hydroxyphenyl group and adenine (FIG. 2) . The 2,5-positions of the 4-hydroxyphenyl ring are likely to be readily iodinated to Na ¹²⁴ I or Na ¹²⁵ I. A plurality of adenine bases at a single oligonucleotide per AuNP ²² and multiple oligonucleotide strands provide many reactive sites for such iodination. For example, when raising thiolated consisting of AuNP of 20 nm to dozens of adenine base (A ₁₀₎ modified by a nucleotide, A ₁₀ - oligonucleotide there is a oligonucleotide strands of 100 A ₁₀ per area, and AuNP 20 of reaction per strand Therefore, 2,000 iodine molecules can be added to each gold nanoparticle. Therefore, adenine-rich DNA-modified AuNP (A ₁₀ -AuNP) can be a promising strategy to improve iodine loading into AuNP. In addition, the additional gold envelope significantly improves the body's stability of radiolabeled DNA on AuNPs by preventing the possibility of degradation by enzymes or other factors present in living cells by embedding radioisotopes in the gold envelope can do.

The iodination reaction was carried out by preparing a radionuclide-Au NP (FIG. 3 a) by introducing Na ¹²⁴ I or Na ¹²⁵ I into a SHPP-A ₁₀ -AuNP solution. As monitored using a radio-TLC (eluent: acetone) scanner, the reaction was completed within 120 minutes (Figure 2). After the reaction was completed, the solution was centrifuged to remove unreacted radioactive NaI. The radioactive chemical yield of the isotope labeled reaction was found to be 99% (FIG. 2). In addition, an additional Au shell formation reaction was performed to produce RIe-AuNP. In the present invention, ¹²⁵ I was used to characterize the stability of RIe-AuNP, and ¹²⁴ I was selected for imaging with PET and CLI combined. The UV-visible spectrum of SHPP-A ₁₀ -AuPN and RI-AuNP was the same as the spectrum of A ₁₀ -AuNP, as shown in Figure 3b, indicating that no significant particle aggregation occurred during the radionuclide labeling reaction . After the Au envelope was formed, the RIe-AuNP showed a characteristic absorption band at 540 nm, which means spherical shape (FIG. 3 b). The hydrodynamic radius of A ₁₀ -AuNP, RI-AuNP and RIe-AuNP were 29.3 ± 0.8 nm, 41.2 ± 1.1 nm, and 65.7 ± 1.2 nm, respectively, as a result of the modification city). Two characteristic peaks corresponding to Au (4f, 4p ¹ ) and iodine (3d ⁵ ) were observed in X-ray photoelectron spectroscopy (FIG. As can be seen from the transmission electron microscope image, the RIe-AuNP had a spherical shape, and the energy dispersive X-ray mapping clearly showed the dispersion of iodine around the AuNP (FIG. 3 d).

SPECT and PET sensitivity and in-vitro stability of RIe-AuNP

The radioactivity sensitivities of RI-AuNP and RIe-AuNP were investigated in depth. In the case of detection of ¹²⁵ I by SPECT and gamma counters (FIGS. 4A and 4B), RI-AuNP (center diameters of 5 and 10 nm) showed low activity due to the less loading of thiolated DNA into AuNP. RI-AuNP (center diameter 20 nm) showed the strongest radioactivity signal. There was a clear linear relationship between AuNP size and radioactivity (Fig. 5).

On the other hand, RIe-AuNP (42 nm in diameter) prepared by forming the Au sheath on the RI-AuNP (5, 10, 20 nm core) showed a slight decrease but a strong radioactivity (FIG. The gamma count response of RIe-AuNP (diameter 42 nm) as a function of NP concentration (1.0 pM-0.5 nM) was linear (Figure 4b). The RIe-AuNP was also detected at 1.0 pM and the count was 1.0 x 10 ⁵ (insert drawing of b in Fig. 4). The number density of radioactive isotopes per AuNP was calculated as 2,681 iodine molecules per AuNP. In the case of ¹²⁴ I detection by PET / CT (c and d in FIG. 4), the solution was not observed for the RIe-AuNP concentration of 1.6 pM, but the PET / CT images showed strong radioactivity signals (FIG. 4c). According to the PET / CT images of the tube phantom, the measurement of radioactivity using a gamma counter proved a good correlation between the concentration of RIe-AuNP (1.6-200 pM) and radioactivity (FIG. 4 d). The stability of radiolabeled RI-AuNP (or RIe-AuNP) in human serum was investigated because the stability of the radionuclide on the particle in vivo is very important for obtaining a biologic image sensitively and stably over a long period of time. The separated radioactive nuclear species appeared rapidly within 30 minutes in RI-AuNP (e (1) in FIG. 4). In contrast, RIe-AuNP showed only minute changes in radioactivity TLC after 24 hours at 37 ° C, indicating that the radioactive labeling substances in RIe-AuNP exhibit excellent stability due to the Au envelope e (2)).

Effect of RIe-AuNP labeling on the biological function of DC

DCs were labeled with RIe-AuNP and used to visualize their migration in the lymphatic system in real time. In order to achieve this goal, we analyzed the effect of RIe-AuNP-induced cell markers on cell viability as well as DC biological functions, which are very important factors for efficient cancer immunotherapy and imaging applications to be. The cell uptake efficiency of RIe-AuNP on DC depends on the concentration of RIe-AuNP, as can be seen in Fig. 6a. As the concentration of RIe-AuNP increases (1.0-4.0 nM) DC exhibits a darker color. A strong PET signal was observed in RIe-AuNP-labeled DC (Fig. 6b (2)). The optimum concentration and incubation time of RIe-AuNP were 2.0 nM and 3 hours, respectively (Fig. 7).

More importantly, for quantitative long-term tracking of DC, the RIe-AuNP must remain inside the DC. As a result of radioactivity analysis of RIe-AuNP over time in DC, the radioactivity decreased gradually. However, RIe-AuNP retained more than 60% of the initial radioactivity in the cells after 3 days, indicating that the radioactive labeling of RIe-AuNP in DC showed excellent stability due to effective protection by the Au envelope (C in Fig. 6). On the other hand, RI-AuNP in DC showed a drastic decrease in radioactivity in DC even after a short time (3 hours), since there is no protective layer (FIG. 8).

Cell viability experiments showed that at all concentrations of RIe-AuNP (1.0-4.0 nM) over 48 hours, several differences were observed between labeled and unlabeled DCs. Furthermore, RIe-AuNP did not induce apoptosis in DC (Fig. 9). Expression of phenotypic markers related to adaptive T-cell immune responses such as CD11c, MHC I, MHC II, CD86, CD54 and CCR7 is shown in Figure 6, e and Figure 10, I did not get it. Various cytokine concentrations, cell migration and antigen uptake capacity were not altered by absorption of RIe-AuNP in DC (FIG. 11).

Antitumor immune response of RIe-AuNP-labeled DC

More importantly, the present invention investigated antitumor immune responses caused by RIe-AuNP-labeled DCs in a mouse model to rodent LLC. To generate a DC-based tumor vaccine capable of inducing anti-tumor immunity associated with the LLC, DC was pulsed with a total lysate of LLC cells, as shown in FIG. Non-labeled and non-pulsed DC, unlabeled and LLC pulse pulsed DC, labeled and non-pulsed DC, and labeled and LLC pulse pulsed DC were compared (Fig. 6, fg). Antitumor effects were confirmed by caliper measurements and ¹⁸ F-fu- cleoxyglucose ( ¹⁸ F-FDG) PET / CT images. ¹⁸ F-FDG can be used to monitor cancer glucose metabolism and is a replacement for cell proliferation. As a result, tumor growth was effectively suppressed in rats immunized with DC pulsed with LLC lysate (labeled f (2), (4) in FIG. 6) with or without labeling, This was not the case in rats immunized with seaweed non-pulsed DCs (f (1), (3) in FIG. 6). Interestingly, rats immunized with pulsed and labeled DCs with LLC lysates showed the smallest tumor size, indicating strong antitumor activity of DCs labeled with RIe-AuNP (Fig. 6 (f) ). To further examine the relationship between antitumor immunity and cytotoxic T lymphocyte killing kinetics, enhanced firefly luciferase-expression LLC &lt; RTI ID = 0.0 &gt; Cytotoxicity assays using cells (LLC / effluc as target) were performed. Consistent with the results in vivo, luciferase activity in spleen cells of mice immunized with pulsed DC (g (2), (4) in Fig. 6) (G (1), (3) in Fig. 6). In addition, there was a slight increase in the killing activity of cytotoxic T lymphocytes immunized with pulsed and labeled DCs from LLC lysates (Fig. 6, g (4)), indicating the smallest tumor size in mice (4)). Taken together, this result implies that labeling DC with RIe-AuNP does not negatively affect the antitumor immunity of DCs. Indeed, although no precise mechanism to explain this enhanced antitumor immunity is known, increased antitumor activity has been observed. This enhanced antitumor immunity is thought to be due to the stimulatory immune response of DC regulated by RIe-AuNP.

Movement tracking of DC to drainage lymph nodes by PET and CLI

The efficacy of RIe-AuNP as an internal contrast agent for DC tracking was evaluated using a combination of PET / CT and Cerenkov luminescence (CLI) imaging and using biodegradation studies (Fig. 14). DCs labeled with RIe-AuNP were injected subcutaneously through the soles of the rats to track the movement of DCs to the draining nodes and lymph nodes (DPLN). From the PET / CT images, DPLN-labeled DCs migrated very rapidly, 6 hours after the injection, and the migration peaked on the third day, and the radial signal decreased on the fourth day (Fig. 15 a). Most of the labeled DC remained in the implanted soles, but due to the inherent nature of DC, the labeled DC was found primarily in the DPLN. In contrast to the present invention, conventional imaging methods for monitoring DC movement into draining lymph nodes using NIR fluorescent nanomaterials or MR imaging require longer accumulation times to clearly visualize the DPLN At least 12 or 24 hours). Although the CLI has relatively lower optical image sensitivity compared to PET, it can not be used to visualize the movement of labeled DC into DPLN 6 hours after delivery, but CLI-based imaging is consistent with PET / CT imaging , Clearly showing the movement of DC to the DPLN (Fig. 15 (b)). The strong correlation between the change in PET signal over time and the intensity of CLI signal indicates the superiority of RIe-AuNP as a new optical contrast agent (Fig. 15c, d). The biodistribution study results were also in good agreement with the combined PET / CT images and CLI data (Fig. 15e). Most of the DC remained in the injection site (55-60%) and 2-3.5% of the DC moved to the DPLN. Histopathological evaluation revealed the location of the labeled DCs in the DPLN in more detail (f (1), (4) in FIG. 15). As a result of the histological analysis of DPLN, the B-cell region (f (2) in FIG. 15) stained with anti-CD79a-specific antibody and the T-cell region stained with anti-CD3- 3)) could be confirmed clearly. Notably, from the representative immunohistochemical evaluation of DPLN, the presence of S-100 (DC-specific marker) -positive DCs labeled with RIe-AuNP, but mainly in the T- , And such DCs contained dark brown particles (f (4) insertion drawing in Fig. 15). These results demonstrate that DC is actively migrating toward the T-cell region of DPLN.

Quantitative comparison between PET and CLI images

The systemic biodistribution of labeled DCs after intravenous infusion was investigated to assess the overall likelihood of RIe-AuNP on combined PET / CT and CLI images (FIG. 16). DCs labeled with RIe-AuNP showed definite localization in the lungs, liver, and spleen within 0.5 hours after transfection, and labeled DCs were clearly detectable at day 4 (Fig. 16a). Although there have been reports of the diffusion of DC into the lungs, spleen, and liver using a variety of imaging modalities, it is not possible to detect localized DCs in the spleen within 0.5 hours of transfusion due to the low sensitivity of existing contrast agents did. The accumulation of radioactive iodine in the thyroid was not observed due to the excellent stability of the radioactive labeling system utilizing the technique of embedding in the Au envelope. Intra-body CLI imaging also showed a strong optical signal in the lung, liver and spleen at 0.5 hour post-transplantation, where the quality of the imaging was the same as the PET / CT image quality (Fig. Typically, due to depth limitations, it is difficult to detect cells moving in deep tissues such as the lungs, spleen, and liver using optical imaging. However, the in-body CLI using RIe-AuNP clearly revealed the location of DC in deep tissue for a short period of time (ie, 0.5 hour and 4 hours), which is a dual (nuclear and optical) Proves the strong possibility of. Considering that CLI has high spatial resolution, low cost and high speed imaging capability compared to PET, CLI is a promising imaging method. In addition, consistent with PET / CT images in the body, biodistribution data clearly showed DC in the lungs 0.5 hour after metastasis, followed by rapid localization in the liver and spleen (Fig. 16c and d).

Claims (17)

As gold nanoparticle-radioisotope complexes,
The radioisotope is contained inside,
Wherein said complex is conjugated with a radioisotope to a gold nanoparticle to which a peptide sequence comprising a tyrosine amino acid is bound,
The conjugation is characterized in that the 2,5-position of the 4-hydroxyphenyl ring in the tyrosine of the peptide sequence is formed by substituting the radioisotope by an iodination reaction,
Wherein the radioisotope is ¹²⁴ I, ¹²⁵ I, or a mixture thereof. delete delete delete delete The method according to claim 1,
Wherein said complex comprises an additional gold envelope. &Lt; RTI ID = 0.0 &gt; 15. &lt; / RTI &gt; delete The method according to claim 1,
Wherein said complex has an average particle size of 1 nm to 1000 nm. A contrast agent comprising the gold nanoparticle-radioisotope complex according to claim 1. 10. The method of claim 9,
Wherein the contrast agent is applied to nuclear medicine imaging or optical imaging. 11. The method of claim 10,
Wherein the nuclear medicine imaging is positron emission tomography (PET) or single-photon emission computed tomography (SPECT). 11. The method of claim 10,
Wherein the optical imaging is CLI (Cerenov luminescence imaging). A pharmaceutical composition for immunotherapy, comprising a dendritic cell labeled with a gold nanoparticle-radioisotope complex according to claim 1. 14. The method of claim 13,
Wherein said composition is capable of tracking the migration of dendritic cells in vivo during immunotherapy. 15. The method of claim 14,
Wherein said dendritic cells are bone marrow-derived cells. 15. The method of claim 14,
Wherein the dendritic cells are stimulated with a lysate of a tumor to be treated, a tumor-specific peptide, a tumor RNA, a DNA encoding a tumor-specific antigen, or a mixture thereof. 15. The method of claim 14,
Wherein the dendritic cells are stimulated with a lysate of the tumor to be treated.

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