Method for evaluating therapeutic efficacy of near-infrared photoimmunotherapy

Abstract

A method for evaluating the therapeutic efficacy of a NIR-PIT in real time has been developed. A discharge coefficient Ke value of an ultrasonic imaging contrast agent or the attenuation of contrast luminance can be used as a measure for the evaluation of the therapeutic effect of a NIR-PIT. According to this method, SUPR can be evaluated by measuring an ultrasonic signal at a high frame rate for several minutes. Thus, the method, as a method for rapidly predicting the therapeutic efficacy of the NIR-PIT, makes it possible to evaluate the therapeutic efficacy in real time and to provide a personalized treatment. Conventionally, it has been believed that the EPR effect is observed with particles having sizes of 10 to 200 nm, and it has also been believed that particles of 10 to 200 nm are retained also with respect to the enhancement of the EPR effect (SUPR effect) by a NIR-PIT. It has newly been found that microsized particles of up to about 5 μm are retained. This can be a useful finding that can be utilized in other treatment methods.

Description

Method for evaluating therapeutic effect of near-infrared photoimmunotherapy

[0001] The present invention relates to a method for evaluating in real time or immediately after treatment whether the amount of near-infrared light irradiated onto a tumor site is appropriate in near-infrared photoimmunotherapy (NIR-PIT, hereinafter referred to as NIR-PIT or photoimmunotherapy).

[0002] NIR-PIT is a treatment approved in Japan in September 2020, ahead of the rest of the world, for unresectable, locally advanced, or locally recurrent head and neck cancer with high EGFR expression. NIR-PIT involves creating a complex by binding a molecule called IR700DX, which is excited by near-infrared light, to an antibody or other substance that binds to a target molecule specifically expressed in target cells. The complex is then bound to the target molecule and irradiated with near-infrared light, inducing necrotic cell death in the target cells through a photochemical reaction. By binding the antibody to the desired tumor cells and then irradiating the target tumor tissue with light to induce a reaction, it is possible to locally remove only the target cells without damaging surrounding cells, allowing treatment with almost no side effects.

[0003] Currently, the application of NIR-PIT is limited to head and neck cancers where EGFR is highly expressed, but EGFR expression has also been observed in various solid cancers such as esophageal cancer, lung cancer, colon cancer, and pancreatic cancer, and it is expected that the range of application will expand. In addition, since it is possible to prepare a conjugate by binding IR700 to a molecule such as an antibody that specifically binds to a target molecule expressed on the cell surface of the target cell, it is possible to target so-called cancer antigens that are specifically expressed in tumors, such as HER2, PSMA, and CEA, and this is a treatment that can be widely applied to various tumors (Patent Document 1, Non-Patent Document 1).

[0004] However, means for predicting therapeutic efficacy are limited. Currently, NIR-PIT irradiates tumor sites with a dose of near-infrared light considered sufficient. However, a characteristic of phototherapy is that a fixed dose of light is irradiated, but uniform irradiation of the tumor is difficult due to factors such as attenuation over distance, scattering within tissue, and the variability in tumor size and shape and vascular distribution among patients. Therefore, tumors may not completely disappear even with the standard dose of light. Currently, therapeutic efficacy is confirmed several days after irradiation. For example, the therapeutic efficacy of NIR-PIT for head and neck cancer is evaluated by blood sampling after irradiation, image evaluation several days later (RECIST (Response Evaluation Criteria in Solid Tumors) using CT), and discoloration of the treatment site. However, efficacy is not confirmed in real time during treatment. Drugs are excreted over time, but the rate of excretion depends on factors such as gender, age, and renal function. Therefore, it is very difficult to predict the amount of drug remaining in each patient's body after a certain period of time has passed. Because NIR-PIT is a treatment that uses near-infrared light, if it is found that the treatment is not effective immediately after treatment, it is possible to re-irradiate the light and aim to eliminate the tumor. Therefore, there is a need to develop a method for predicting and evaluating the therapeutic effects of NIR-PIT in real time.

[0005] Patent Document 2 discloses a composite for use in NIR-PIT that can also be used for imaging. Specifically, the composite includes nanoparticles with an average particle size of 100 nm or less, antibody molecules, and a photosensitive moiety. The core of the nanoparticles is made of magnetic particles containing MRI contrast agents such as iron oxide or gadolinium compounds, or fluorescent particles such as semiconductor nanoparticles or CdSe quantum dots with amino groups introduced onto the particle surface. Therefore, the characteristics of the core particles can be utilized to perform MRI and fluorescent imaging. However, while this method has good sensitivity, both require large-scale measuring equipment, necessitating the patient to be moved to a room equipped with the measuring equipment for measurement. This does not allow for real-time confirmation during or on-site after treatment.

[0006] The mechanism of cell death induced by photosensitizers was thought to be primarily oxidative stress caused by free radicals, but the detailed mechanism of tumor cell death by NIR-PIT remained unclear. It has recently been elucidated that the mechanism of tumor therapeutic efficacy by NIR-PIT is that the photosensitizer in an antibody conjugate bound to the tumor cell surface becomes hydrophobic through a photochemical reaction, causing aggregation of the antibody conjugate, damaging the tumor cell membrane, resulting in an osmotic pressure difference between the inside and outside of the cell, inducing cell death (Non-Patent Document 2). As is clear from this mechanism of cell death induction, tumor changes induced by NIR-PIT are induced immediately after light irradiation. Therefore, if changes in tumor size and shape due to treatment can be measured in real time, it is possible to evaluate whether light irradiation was sufficient, i.e., whether an appropriate amount of light was irradiated to the appropriate area. If changes can be measured in real time, additional light irradiation can be performed immediately if the initial light irradiation does not have the desired effect on the tumor. This reduces the burden on the patient and eliminates the need to consider the loss of antibody-photosensitizer conjugates due to metabolism or excretion in the body. However, no method has yet been developed to measure the size and shape of tumors in real time.

[0007] JP 2018-528268 A JP 2022-047517 A

[0008] Kobayashi, H. & Choyke, PL, 2019, Acc. Chem. Res., Vol.52, pp.2332-2339. Sato, K., "Elucidation of the mechanism of near-infrared photoimmunotherapy," Journal of the Japanese Society for Laser Surgery and Medicine, Vol. 41, No. 2, 2020, pp.104-109. Sano, K. et al., ACS Nano., 2013 Jan22;7(1):717-24., Publication Date: December 7, 2012, doi:10.1021 / nn305011p. Tainaka, K., et al., Cell Rep. 2018, 24(8):2196-2210.e9. doi: 10.1016 / j.celrep.2018.07.056. Wang, AD, et al. al., 2015, PLOS ONE, https: / / doi.org / 10.1371 / journal.pone.0123461

[0009] Until now, there has been no cancer treatment like NIR-PIT in which the amount of light exposure and technique directly reflect the therapeutic effect. Therefore, there is no established method for evaluating it. As mentioned above, if the amount of light is insufficient and the tumor cannot be widely irradiated with light, additional light exposure is necessary. If it is determined that the light exposure is insufficient, additional light exposure is required. However, currently, the effectiveness is confirmed several days after light exposure. If the light exposure is insufficient, it is necessary to consider factors such as the reduction in the administered drug due to metabolism and excretion. Therefore, the current practice is to administer the drug again and perform light exposure, which places a significant burden on the patient.

[0010] The present invention aims to provide a method for evaluating whether the amount of near-infrared light irradiated to a tumor site is appropriate in real time or on-site after treatment, and for confirming and predicting the therapeutic effect during or immediately after irradiation treatment. This evaluation method makes it possible to immediately and flexibly add more light irradiation if the amount of near-infrared light during the initial treatment is insufficient, enabling flexible, customized treatment tailored to the tumor and patient.

[0011] The present invention relates to the following examination methods: (1) A method for predicting the therapeutic effect of near-infrared photoimmunotherapy (NIR-PIT), the method comprising using an ultrasound echo method. (2) The method for supporting therapeutic effect prediction according to (1), wherein the ultrasound echo method is a harmonic echo method. (3) The method for supporting therapeutic effect prediction according to (2), wherein measurement is performed using an ultrasound contrast agent. (4) The method for supporting therapeutic effect prediction according to (3), wherein the treatment target is a tumor, and the SUPR effect is evaluated from images acquired by ultrasound echo method before and after irradiation with near-infrared light, thereby predicting the therapeutic effect. (5) The method for supporting therapeutic effect prediction according to (4), wherein the retention of an ultrasound contrast agent within a tumor is evaluated. (6) The method for supporting therapeutic effect prediction according to (5), wherein an excretion coefficient Ke value from a tumor of the ultrasound contrast agent is calculated, and the therapeutic effect is predicted from the Ke value. (7) A method for supporting prediction of therapeutic effect according to (5), in which the attenuation rate of the ultrasound contrast agent is measured and the therapeutic effect is predicted from the ratio of the attenuation rates. (8) A method for supporting prediction of therapeutic effect according to (3), in which the ultrasound contrast agent is perfluorobutane microbubbles. (9) Near-infrared photoimmunotherapy, in which a therapeutic agent comprising a compound photosensitive to near-infrared light and an antibody molecule that binds to target cells is combined, near-infrared light is irradiated, and the therapeutic effect is evaluated by the method according to any one of (1) to (8), to determine the need for additional near-infrared light irradiation. (10) Near-infrared photoimmunotherapy according to (9), in which additional near-infrared light irradiation is immediately performed if the therapeutic effect is determined to be insufficient.

[0012] Schematic diagram of an experimental scheme for evaluating the SUPR effect using QD800 nanoparticles. Diagram showing the timing of the SUPR evaluation experiment. Diagram showing the results of analyzing the tumor shrinkage effect after NIR-PIT using luciferase activity. Diagram showing the results of quantitatively analyzing the tumor shrinkage effect after NIR-PIT using luciferase activity. In vivo IR700 fluorescence image after NIR-PIT. Diagram showing the correlation between IR700 fluorescence and therapeutic effect. Diagram showing the SUPR effect using QD800. Image showing the time course of QD800 fluorescence in vivo after NIR-PIT. Graph quantifying QD800 fluorescent signal. Diagram showing the correlation between QD800 fluorescent signal and therapeutic effect at each time point. Diagram showing the results of analysis of excised tissue after clearing. Representative images before and after tumor tissue clearing are shown. Stained images with CD31 (PECAM-1) and QD800 are shown. A diagram showing a 3D image obtained by light sheet microscopy. The bar indicates 300 μm. A diagram showing the results of measuring the diameter of microvessels. A diagram showing the results of quantifying the overlap of microvessels and QD800 fluorescence. A representative microscopic image of 2 μm Poly(lactic-co-glycolic acid) (PLGA) particles is shown. The bar indicates the timing of 50 μm SUPR assessment. A diagram showing luciferase activity before and after NIR-PIT. A diagram showing the results of SUPR assessment after NIR-PIT using 2 μm PLGA. Fluorescence images of consecutive PLGA particles after in vivo NIR-PIT are shown. A diagram showing the results of quantifying PLGA fluorescence signals after NIR-PIT. A diagram showing the results of observations using an excised tumor. Fluorescence image of PLGA using frozen sections. Figure showing the results of quantifying PLGA fluorescence. Image of a tumor observed by fluorescence microscope. Figure showing the results of quantifying PLGA fluorescence signals along intratumoral blood vessels. Representative microscopic images of 5 μm PLGA particles. Bar represents 50 μm. Figure showing the results of SUPR evaluation after NIR-PIT using 5 μm PLGA particles. Sequential PLGA fluorescence images after in vivo NIR-PIT. Figure showing the results of quantification of PLGA fluorescence signals after NIR-PIT. Figure showing the results of ex vivo quantification of PLGA fluorescence signals after tumor excision. Representative microscopic photographs of the ultrasound contrast agent used, Sonazoid (registered trademark).1 is a diagram showing the evaluation scheme for SUPR after NIR-PIT using an ultrasound contrast agent. FIG. 2 is a diagram showing the timing of imaging by echo. FIG. 3 is a diagram quantitatively showing luciferase activity one day after NIR-PIT. FIG. 4 is a diagram showing a representative harmonic echo image of a tumor after near-infrared irradiation. FIG. 5 is a diagram showing an analysis method using a two-compartment model. FIG. 6 is a diagram showing an example of data used to evaluate SUPR after NIR-PIT using an ultrasound contrast agent. FIG. 7 is a diagram showing the change over time in brightness values ​​(Contrast Mean Power) before and after NIR-PIT. FIG. 8 is a diagram showing the change in Ke values ​​before and after treatment. FIG. 9 is a diagram showing the results of a correlation analysis between Sonazoid Ke values ​​and therapeutic effect (luciferase activity). FIG. 10 is a diagram showing the timing of imaging by echo. FIG. 11 is a diagram showing an example of data used to evaluate SUPR after NIR-PIT using an ultrasound contrast agent. This figure shows the results of an analysis of the correlation between the ratio of early to late luminance values ​​after NIR-PIT and the therapeutic effect (luciferase activity).

[0013] In NIR-PIT technology, which measures tumor changes and antitumor effects in real time or immediately after treatment, ease of application to patients is key. Specifically, it allows for on-site measurements using portable equipment, rather than large equipment such as MRI or CT, which require patient movement during treatment. The inventors developed a method for evaluation using existing ultrasound echo equipment found in any hospital and an approved microbubble ultrasound contrast agent (galactose-palmitic acid mixture). The use of existing equipment and an approved ultrasound contrast agent facilitates clinical application and allows for easy bedside evaluation. Furthermore, even when targeting gastrointestinal tumors, evaluation can be performed using endoscopic ultrasound (EUS) and endobronchial ultrasound (EBUS). Therefore, this evaluation method can be applied even when the indications are expanded beyond head and neck cancer, for which it has already been approved, to include esophageal cancer, gastric cancer, and other cancers.

[0014] It has been discovered that the use of harmonic echo imaging (also called contrast echo imaging) as an ultrasound diagnostic method allows for highly sensitive detection. Unlike B-mode imaging, which displays an image using a reflected signal with the same frequency component as the transmitted signal, harmonic echo imaging is an imaging method that utilizes harmonics. Harmonic echo imaging selectively displays harmonic signals generated by nonlinear effects of ultrasound incident on an ultrasound contrast agent (generally microbubbles). The use of an ultrasound contrast agent generates a larger amount of harmonics than tissue harmonic imaging, which is another imaging method that utilizes harmonic signals, and therefore aims to improve the sensitivity of the target area and enhance the strength of the harmonic signals. In addition, although perfluorobutane microbubbles (Sonazoid, registered trademark) were used in the following examples, any contrast agent may be used, and ultrasound contrast agents other than Sonazoid, such as Albunex, Levovist (registered trademark), Optison (registered trademark), Definity, Imagment, SonoVue (registered trademark), Echogen, PB127, and AI-700, may also be used.

[0015] Nanoparticles with particle sizes controlled to 100 nm or less are known to have an enhanced permeation and retention (EPR) effect, which allows them to reach and remain in cancer tissue from tumor blood vessels without leaking into normal tissue. Neovascularization around tumors is incomplete, leaving gaps between vascular endothelial cells. Therefore, tumor tissue exhibits significantly increased vascular permeability compared to normal tissue, allowing macromolecules and microparticles to easily penetrate the vascular wall and penetrate into the tissue. Furthermore, tumors lack a well-developed lymphatic system, preventing the excretion of foreign substances from the tissue. As a result, substances that reach tumor tissue accumulate. This characteristic is known as the EPR effect, and it is said that particles with a particle size of 10 to 200 nm tend to accumulate and remain in tumor tissue. The EPR effect is an important factor in passive targeting of tumor tissue, and some anticancer drugs have already been developed with the intention of utilizing the EPR effect.

[0016] Furthermore, after NIR-PIT, tumors near blood vessels with high antibody concentrations instantly necrotize, creating spaces that result in nanoparticle retention that exceeds the typical EPR effect described above. Quantitative analysis has shown that retention increases 10 to 20 times compared to the EPR effect (Non-Patent Document 3). This retention effect is called the Super Enhanced EPR effect (SUPR effect). The present inventors conceived and investigated the use of the SUPR effect to predict real-time therapeutic efficacy in NIR-PIT. As described above, the EPR effect is said to cause accumulation and retention of particles 10 to 200 nm in tumor tissue. Since ultrasound contrast agents are larger in size (micrometers), there was concern about signal enhancement due to ultrasound contrast agents, and this investigation was therefore undertaken. As a result, it was revealed that not only the nano-sized SUPR effect described above was observed, but also that micro-sized particles (2 μm to 5 μm) were retained. This effect was named the micro-sized SUPR effect and was demonstrated as a proof of concept. This is the first time in the world that an improvement in the retention of micro-sized particles has been demonstrated.

[0017] The present invention will be described below with reference to examples. [Example 1] Verification using nanoparticles QD800 First, we analyzed whether there was a correlation between the SUPR effect and the therapeutic effect of NIR-PIT. A431-lucGFP cells, in which the luciferase gene and the GFP gene were introduced into human epidermoid carcinoma-derived cells A431, and 5 × 10 ６ The cells were subcutaneously transplanted into both dorsal and rump sides of 8- to 10-week-old nude mice (BALB / cSlc-nu / nu). Six days later, 100 μg of panitumumab-IR700 was injected into the tail vein, and one day later, near-infrared light was applied at 30-100 J / cm. ２ The tumor was irradiated at an intensity of 100 μg / cm. After treatment, nanoparticles QD800 (Thermo Fisher) were injected into the tail vein, and their retention was evaluated. One day after NIR-PIT, the therapeutic effect was evaluated by an in vivo luciferase assay (Figure 1A). The right tumor was irradiated with near-infrared light, and 1 hour later, 32 pmol of QD800 was injected into the tail vein. Analysis was performed using a Pearl Trilogy Imaging System (LI-COR) from before QD800 injection until 120 minutes after near-infrared light irradiation (Figure 1B).

[0018] One day after treatment with near-infrared light irradiation, the therapeutic effect was evaluated by measuring luciferase activity. Luciferase activity was measured using an IVIS-SPECTRUM-CT imaging system after intraperitoneal administration of D-luciferin (7.5 mg / ml, 200 μl). Luciferase activity was reduced only in the tumor on the side irradiated with near-infrared light. Luciferase activity on the side not irradiated with near-infrared light increased as the tumor progressed (Figure 1C). Figure 1D shows the results of quantifying luciferase activity. The luciferase activity of the tumor one day after treatment is shown as a ratio (%) to the luciferase activity of the same tumor in the same mouse before treatment. A significant decrease in luciferase activity was observed in the tumor on the side irradiated with near-infrared light (n=6, ＊ p=0.000980195<0.05, Student's t-test).

[0019] Furthermore, in vivo IR700 fluorescence was measured (Figure 1E). The upper panel shows a superposition of reflected light and fluorescence, and the lower panel shows the fluorescence observation results. It was demonstrated that IR700 fluorescence rapidly disappeared in the tumor on the side irradiated with near-infrared light. We analyzed the correlation between IR700 fluorescence intensity (the ratio of fluorescence intensity after light irradiation to that before light irradiation) and the change in luciferase luminescence intensity before and after light irradiation (the ratio of luciferase activity one day after treatment to that before treatment in the same tumor in the same mouse) (Figure 1F). A positive correlation was observed between the ratio of IR700 fluorescence intensity and the ratio of luciferase luminescence intensity, i.e., the therapeutic effect (P = 0.023584, r = 0.56161).

[0020] Near-infrared light at 100 J / cm ２The tumors were irradiated with near-infrared light, and 60 minutes later, 32 pmol of QD800 was injected into the tail vein (see Figure 1B). The time-dependent changes in QD800 were observed by fluorescence (Figure 2A). The upper panel shows a superposition of reflected light and fluorescence, and the lower panel shows the results of fluorescence observation. In tumors irradiated with near-infrared light (indicated by arrows in the figure), fluorescence due to QD800 was observed up to 60 minutes, indicating that QD800 remained for a long time. When the tumors were removed after 60 minutes and observed by fluorescence, strong fluorescence due to QD800 was observed in the tumors irradiated with near-infrared light (Figure 2A, ex vivo). Quantification of the QD800 fluorescence signal showed that the signal remained significantly higher in tumors that had undergone NIR-PIT compared to the control (side not irradiated with light) (Figure 2B, n=6, ＊ p = 30 min: 0.012807878, 60 min: 0.006226658 < 0.05, Student's t-test).

[0021] Since the SUPR effect of NIR-PIT was confirmed in this experimental system, we analyzed the correlation between the fluorescence signal from QD800 and the therapeutic effect one day later (Figure 2C). Plotting the ratio of luciferase activity one day after NIR irradiation to that before irradiation and the QD800 influx ratio (fluorescence intensity with pre-irradiation set to 1) in the tumor 10, 30, and 60 minutes after QD800 intravenous injection revealed a significant negative correlation (10 minutes: r = -0.51077, p = 0.015136 < 0.05; 30 minutes: r = -0.63794, p = 0.001402 < 0.005; 60 minutes: r = -0.65118, p = 0.001191 < 0.005). These results indicate a correlation between the decrease in luciferase activity in the tumor, i.e., the therapeutic effect, and the SUPR effect.

[0022] The location of the retention effect of QD800 in the tumor tissue was analyzed (Figure 3). ２After irradiation with near-infrared light, the tumor was excised and cleared using the CUBIC-R method (Non-Patent Document 4, Tokyo Chemical Industry Co., Ltd.). Tumor tissue before and after clearing is shown (Figure 3A). After clearing, the tumor was stained with an anti-CD34 antibody (Proteintech) to detect CD34 present in the capillary endothelium, and 3D observation of the tumor was performed using a light sheet microscope. The fluorescence of QD800 injected after near-infrared light irradiation was also detected (Figure 3B). On the near-infrared light-irradiated side, QD800 fluorescence was observed in the area where microvessels were present. Furthermore, on the near-infrared light-irradiated side, CD34 was strongly stained. This is thought to be due to the fact that near-infrared light irradiation disrupts cells near blood vessels where antibodies are highly bound, destroying the structure of vascular endothelial cells and promoting the diffusion and regeneration of CD34 molecules.

[0023] Quantification of microvessel diameter revealed that the diameter of microvessels in the tumors exposed to near-infrared light was approximately 4-6 times larger (Figure 3C, n = 5, *p = 0.0000000061 < 0.05, vs. untreated tumor, Student's t-test). Furthermore, quantification of the CD34-containing area, i.e., the overlap between microvessels and QD800 fluorescence, revealed a 4-fold increase in QD800 fluorescence intensity, consistent with the presence of microvessels (Figure 3D, n = 5, *p = 0.011954 < 0.05, vs. untreated tumor, Student's t-test).

[0024] Example 2: Verification of the SUPR effect using 2 μm PLGA particles As mentioned above, the EPR effect is said to be an effect in which particles of 10 to 200 nm accumulate and remain in tumor tissue. Since the SUPR effect after NIR-PIT is also an EPR effect, it was thought that the particles that accumulate and remain in tumor tissue were 10 to 200 nm in size. The QD800 used in Example 1 is also a nanoparticle with a particle size of approximately 20 nm. However, since the diameter of ultrasound contrast agents is larger, there were concerns about whether the SUPR effect would occur. Therefore, we investigated whether retention in tumor tissue would occur using NIR-PLGA, which is a NIR-labeled version of Poly(lactic-co-glycolic acid:PLGA) particles, which are larger particles.

[0025] First, we investigated NIR-IR fluorescent PLGA nanoparticles (Sigma-Aldrich, hereafter referred to as 2 μm PLGA particles) with a diameter of approximately 2 μm. Figure 4A shows, from the left, an image taken by a differential interference microscope, an image taken by a fluorescence microscope (detected in the Cy7 channel, which can detect near-infrared light), and a superposition of these images. As in Example 1, A431-lucGFP cells, 5 × 10 ６ The cells were subcutaneously transplanted into both dorsal buttocks of 8- to 10-week-old nude mice, and 6 days later, 100 μg of panitumumab-IR700 was injected into the tail vein. One day later, near-infrared light was applied at 100 J / cm. ２ The tumors were irradiated at an intensity of 1 μm. Sixty minutes after treatment, 2 μm PLGA particles were injected into the tail vein, and retention was evaluated using a Pearl Trilogy imaging system (Figure 4B). The therapeutic effect was also analyzed by luciferase activity. Luciferase activity was measured before and one day after near-infrared light irradiation (Figure 4C). One day later, luciferase activity was reduced only in tumors irradiated with near-infrared light. In vivo, PLGA fluorescence was continuously photographed after NIR-PIT (Figure 4D). Stronger PLGA fluorescence was observed in tumors irradiated with near-infrared light compared to tumors not irradiated with near-infrared light, and PLGA fluorescence was confirmed to persist for up to five minutes. Tumors subjected to in vivo fluorescence observation were then excised and subjected to fluorescence observation (Figure 4D, ex vivo). Reflected light images (top) and fluorescence images (bottom) are shown. Significantly higher PLGA fluorescence was observed in the tumors on the near-infrared light-irradiated side (n = 3, *p = 0.004542163 < 0.05, vs. untreated tumor, Student's t-test). Quantification of PLGA fluorescence signals after near-infrared light irradiation revealed that the near-infrared light-irradiated side (PIT) maintained higher fluorescence intensity than the control side (n = 4, *p = 4 min: 0.034319917, 5 min: 0.017826908 < 0.05, vs. untreated tumor, Student's t-test, Figure 4E).

[0026] Frozen sections of the excised tumors were prepared and PLGA fluorescence was observed (Figure 5A). Significantly enhanced PLGA fluorescence signals were observed in tumors irradiated with near-infrared light. Quantification of the observed PLGA signal revealed a significant increase in PLGA fluorescence signals in the near-infrared light-irradiated group (Figure 5B, n = 4, *p = 0.002937117 < 0.05, vs. untreated tumors, Student's t-test). Blood vessels were stained with anti-PECAM-1 antibody (Cell Signaling Technology), and tumor regions along the blood vessels were observed (Figure 5C). In tumors irradiated with near-infrared light, PLGA fluorescence was observed in the blood vessel regions detected by anti-PECAM-1 antibody. Quantification of PLGA fluorescence signals along intratumoral blood vessels revealed that PLGA fluorescence signals around blood vessels were significantly increased on the near-infrared light-irradiated side (Figure 5D, n = 3, *p = 0.036197743 < 0.05, vs. untreated tumor, Student's t-test).

[0027] Example 3: Verification of the SUPR effect using 5 μm PLGA particles The SUPR effect by NIR-PIT was verified using NIR-IR fluorescent PLGA particles with a diameter of approximately 5 μm, in the same manner as in Example 2. Figure 6A shows a representative image of the 5 μm PLGA particles used. From the left, a differential interference microscope image, a fluorescence microscope image, and a superposition of these images are shown. As in Example 1, A431-lucGFP cells were subcutaneously transplanted into both dorsal buttocks of nude mice, and 6 days later, 100 μg of panitumumab-IR700 was injected via the tail vein. One day later, near-infrared light at 100 J / cm was applied. ２The tumors were irradiated with near-infrared light at an intensity of 1000 kJ / s. Sixty minutes after treatment, 5 μm PLGA particles were injected into the tail vein and their retention was evaluated. The PLGA fluorescence was continuously imaged after NIR-PIT in vivo (Figure 6B). No fluorescent signal from the 5 μm PLGA particles was detected in either tumor. This was likely due to the majority of the 5 μm PLGA particles being trapped in the pulmonary capillaries, resulting in weak PLGA fluorescence that could not be observed through the skin. Therefore, when the excised tumors were subjected to fluorescence observation, PLGA fluorescence was observed on the side irradiated with near-infrared light (Figure 6B, ex vivo bottom panel). The results of quantitative analysis of the PLGA fluorescence signal in vivo after near-infrared light irradiation are shown in Figure 6C. No significant difference in the fluorescence signal of 5 μm PLGA was observed between the NIR-irradiated and unirradiated sides (n = 3, p = 0.746189325, vs. untreated tumor, Student's t-test). However, quantification of PLGA fluorescence signals in excised tumors showed significantly higher fluorescence intensity in the NIR-irradiated side (Fig. 6D, n = 6, *p = 0.0409931, vs. untreated tumor, Student's t-test).

[0028] Example 4: Verification using an ultrasound contrast agent Example 1 demonstrated a correlation between therapeutic efficacy and the SUPR effect, and Examples 2 and 3 demonstrated that micro-sized particles of 2 to 5 μm remained around tumor blood vessels. Therefore, we investigated whether the SUPR effect could be measured using ultrasound to assess therapeutic efficacy. As mentioned above, ultrasound echo equipment is available in every hospital, and because the equipment is compact, measurements can be performed at the bedside. Therefore, clinical application is readily possible. If the retention of ultrasound contrast agents due to the SUPR effect can be detected, the SUPR effect can be used to predict and assess therapeutic efficacy through analyses such as correlation analysis, prediction formulas, and comparative analysis.

[0029] The ultrasound contrast agent Sonazoid is a microbubble ultrasound contrast agent with a diameter of approximately 2-3 μm (Figure 7A). The PLGA results in Examples 2 and 3 demonstrated that micro-sized particles were retained, leading to the name "micro-sized SUPUR." The size of the ultrasound microbubbles was also approximately 2 μm, and retention was assessed using ultrasound, assuming retention.

[0030] In the same manner as in Example 1, whether the SUPR effect can be detected by ultrasound imaging was examined (FIG. 7B). A431-lucGFP cells, in which the luciferase gene and the GFP gene were introduced into A431 cells, were used. ６ The cells were subcutaneously transplanted into both dorsal and rump sides of 8- to 10-week-old nude mice (BALB / cSlc-nu / nu), and 6 days later, 100 μg of panitumumab-IR700 was injected into the tail vein. One day later, the mice were exposed to near-infrared light at 30-100 J / cm. ２ The tumors were irradiated at an intensity of 1000 nm. Before and after treatment, Sonazoid (GE Healthcare Pharma) was injected into the tail vein, and the retention of the ultrasound contrast agent was analyzed using harmonic echo analysis to evaluate the effect of NIR-PIT. Specifically, Sonazoid was administered intravenously before near-infrared light irradiation and detected using harmonic echo analysis (control). Sonazoid was then intravenously injected again immediately after NIR-PIT, and the echo signal from the tumor area was measured and evaluated (Figure 7C).

[0031] 30 J / cm ２ , 60 J / cm ２ The cells were irradiated with near-infrared light at 60 J / cm 2 and luciferase activity was quantified one day after irradiation. ２ It was confirmed that luciferase activity was significantly reduced in tumors irradiated with light (Fig. 7D, n = 5, ＊ p=0.011181862<0.05, Student's t-test). Harmonic echo images of the tumor over time on the near-infrared light-irradiated and non-irradiated sides are shown (Figure 7E). Retention of Sonazoid was observed on the near-infrared light-irradiated side.

[0032] Method for Evaluating the Effect of NIR-PIT Therapy (1) Analysis Using a Two-Compartment Model The concentration of a drug in the blood and tissues from the time it is administered until it is excreted can be explained using pharmacokinetic theory. Following rapid intravenous administration, a drug is instantly and uniformly distributed in the blood compartment, gradually distributed in the tissue compartment, then returned to the blood compartment, and gradually excreted from the body. Using this method to analyze the EPR effect using a two-compartment model (Non-Patent Document 5), an analysis was conducted to determine whether the Sonazoid concentration in tumors could be explained. Since Sonazoid is distributed not only in blood vessels but also in muscle and tumor tissue, a regression analysis was performed taking this into account (Figure 8A). C １ (t) and C ２ (t) represents the drug concentration in the blood (peripheral compartment) and tumor tissue (tumor tissue compartment), respectively. The first-order rate constant Ke represents the first-order rate constant for the drug excretion from all excretion routes, including excretion from the tumor tissue compartment. In other words, the smaller Ke is, the more the drug remains in the tissue. The first-order rate constant K １２ and K. ２１ is the first-order rate constant describing the exchange of Sonazoid between the two compartments, blood and tumor tissue.

[0033] A regression analysis of signal intensity after administration of Sonazoid was performed using a two-compartment model. Tumors treated with NIR-PIT showed a signal intensity curve that was clearly different from the control (not irradiated with near-infrared light) or the tumor before near-infrared light irradiation (Figure 8B). The vertical axis represents the brightness value (contrast mean power), and the horizontal axis represents the time elapsed since treatment. The control represents the tumor not treated with NIR-PIT. Calculation of the Ke value (excretion coefficient) before and after NIR-PIT revealed a decrease dependent on the light dose due to treatment, demonstrating a SUPR effect on ultrasound contrast agents (Figure 8C). Near-infrared light irradiation reduced the Ke value of Sonazoid, demonstrating a delay in the excretion of Sonazoid. (control: n=5, 30J: n=6, 60J: n=16, ＊ p = 0.023022825 < 0.05, Student's t-test).

[0034] The correlation between the excretion coefficient Ke value of Sonazoid and therapeutic efficacy was analyzed (Figure 8D). A significant correlation was observed when the ratio of luciferase activity before and after near-infrared light irradiation and the Ke value in the tumor were plotted (r = 0.74292, p = 1.37998E-05 < 0.005). Therefore, the Ke value can be used as a predictive indicator for rapid clinical evaluation of the therapeutic efficacy of NIR-PIT. This method, which uses the clinically used ultrasound contrast agent Sonazoid and measures ultrasound signals at a high frame rate for several minutes, allows for rapid evaluation of SUPR. This method can be used to predict and evaluate therapeutic efficacy in real time as an NIR-PIT efficacy prediction method, and can be utilized for personalized cancer treatment.

[0035] (2) Evaluation by Attenuation Rate This section describes a method for evaluating the therapeutic effect by evaluating the change in attenuation rate within the tumor after treatment. This method is simpler because it involves intravenously injecting microbubbles immediately after NIR-PIT and measuring the contrast mean power of the tumor (Figure 9A). Here, the results obtained in Example 4 are used for analysis (Figure 9B).

[0036] Because Sonazoid accumulates and remains at the treatment site, the area where NIR-PIT was performed maintains a high intensity value for a longer period of time compared to the control (untreated) area (Figure 9B). After treatment, the decay rate is clearly different between the early (e.g., in the example of Figure 9B, frame number time (ft) 10-500, 0-20 seconds) and late (frame number time 1000-5000, 40-200 seconds) periods. Therefore, the ratio of the intensity value (contrast mean power) at either the early or late period can be calculated to calculate the decay delay and evaluate the therapeutic effect. Specifically, the higher the therapeutic effect, the more the contrast agent remains in the tumor, indicating a lower decay rate.

[0037] The correlation between the therapeutic effect and the decay rate was analyzed by the ratio of luciferase activity before NIR-PIT to that one day after treatment (Figure 9C). The decay rate ratio is expressed as the ratio of ft100 to ft2000, and luciferase activity is expressed as the ratio (%) of the luciferase activity of the tumor one day after treatment to the luciferase activity of the same tumor in the same mouse before treatment, as in Example 1. Therefore, the lower the luciferase activity ratio, the more the tumor shrinks. It was shown that there is a positive correlation between the luciferase activity ratio and the ratio of the early and late decay rates (R ２ = 0.8725). Therefore, the ratio of the early and late attenuation rates can be used to evaluate treatment. This evaluation method is very simple, as it only requires measuring the signal intensity with an echo after NIR-PIT and calculating the ratio of the early and late attenuation rates.

[0038] This method, which uses ultrasound echo to measure drug retention due to the SUPR effect, allows for real-time evaluation of the therapeutic efficacy of NIR-PIT at the bedside. Therefore, if the therapeutic effect is deemed insufficient, additional light irradiation can be immediately administered, ensuring sufficient treatment. This not only reduces the need for additional treatment on a different day, but also reduces the burden on the patient by eliminating the disadvantage of insufficient treatment. Furthermore, as mentioned above, the EPR effect has been observed with particles ranging from 10 to 200 nm. Previously reported enhancement of the EPR effect (SUPR effect) by NIR-PIT has also been thought to involve retention of nano-sized particles (nano-sized SUPR). In this study, evaluations using PLGA particles (2 μm, 5 μm) and Sonazoid (2-3 μm) revealed new findings, demonstrating that particles up to approximately 5 μm in size retain their properties. This effect has been named the micro-sized SUPR effect. The micro-sized SUPR effect, which utilizes the retention of substances with particle sizes larger than previously reported, may also be useful for other treatments. For example, by using particles with a size of 10 nm to 5 μm, drugs, nucleic acids, gases, liquids, etc. can be encapsulated and used in treatments by utilizing their retention properties.

Claims

1. A method for predicting the therapeutic effect of near-infrared photoimmunotherapy (NIR-PIT), Using ultrasound echotherapy, The SUPR effect is evaluated from the acquired images. A method for supporting the prediction of treatment effectiveness.

2. The method for supporting the prediction of therapeutic effect according to claim 1, characterized in that the ultrasound echo method is a harmonic echo method.

3. The method for supporting the prediction of treatment effect according to claim 2, characterized in that it is measured using an ultrasound contrast agent.

4. The method for predicting therapeutic effect according to claim 3, characterized by evaluating the retention of an ultrasound contrast agent within a tumor.

5. The Ke value, which is the excretion coefficient of the ultrasound contrast agent from the tumor, is determined. A method for supporting the prediction of therapeutic effect according to claim 4, which predicts therapeutic effect from Ke value.

6. The attenuation rate of the aforementioned ultrasound contrast agent is measured, A method for supporting the prediction of therapeutic effect according to claim 4, which predicts the therapeutic effect from the ratio of decay rates.

7. The method for supporting the prediction of therapeutic effect according to claim 3, characterized in that the ultrasound contrast agent is perflubutane microbubbles.