Enhancement of treatments using immunotherapy agents

Abstract

The present invention relates to ultrasound-mediated enhancement of immunotherapeutic agents and regimens, particularly for the immunotherapeutic treatment of cancer and autoimmune diseases. In particular, the present invention provides cluster compositions and pharmaceutical compositions for use in the delivery of immunotherapeutic agents to target areas of interest within the body and in enhancing activated immune cell infiltration into target pathological conditions, as well as for the treatment of solid tumors and autoimmune pathologies.

Description

[Technical Field]

[0001] Field of the present invention The present invention relates to ultrasound-mediated targeted delivery of immunotherapeutic agents to pathological sites, particularly to drug treatment using such therapeutic agents. Accordingly, the present invention provides cluster compositions and pharmaceutical compositions for use in the delivery and preparation of immunotherapeutic agents for the administration of pathological conditions, such as cancer and autoimmune / inflammatory diseases. [Background technology]

[0002] Background of the present invention Immunotherapy is the treatment of disease by activating or suppressing the immune system. The use of immunotherapeutic agents (ITAs) is a rapidly developing new class of drugs, and new uses for existing drugs are continuously being developed. Different classes of immunotherapeutic agents include: monoclonal antibodies (mAbs), fusion proteins, soluble cytokine receptors, recombinant cytokines, small molecule mimetic drugs, cell therapies, cancer vaccines, and oncolytic viruses. Of these, mAbs are by far the most important class, and include a wide range of approved drugs for the treatment of various pathological conditions, including cancer and autoimmune diseases.

[0003] Monoclonal antibody therapy is a form of immunotherapy that uses mAbs (metabolic antibodies) that specifically bind to certain cells or proteins. The goal is for the treatment to stimulate the patient's immune system to attack those cells, or to use mAbs to bind to molecules involved in T cell regulation, thereby eliminating inhibitory pathways that block the T cell response. This is known as immune checkpoint therapy. Currently, more than 80 different mAbs have been approved by the FDA for the treatment of a wide range of diseases, particularly within the fields of oncology and autoimmune diseases. The therapeutic use of mAbs is a rapidly growing segment: in 2019, seven of the ten best-selling drugs in the United States were mAbs, led by Humira (AbbVie, autoimmune diseases), Opdivo (BMS, oncology), and Keytruda (Merck, oncology).

[0004] Immuno-oncology (IO) is the artificial stimulation of the immune system to treat cancer, enhancing the immune system's natural ability to fight disease [Carter and Thurson, Immune-oncology agents for cancer therapy, The Pharmaceutical Journal, May 7, 2020]. While normal antibodies of the immune system bind to external pathogens, modified immunotherapy antibodies bind to tumor antigens, marking and identifying cancer cells for the immune system to inhibit or kill. IO agents explore a variety of mechanisms of action (MoA) that differ from conventional chemotherapeutic agents; for example, checkpoint inhibitors enhance the body's own cytotoxic T cells that recognize and attack tumor cells. Monoclonal antibodies represent the most important class of IO agents in current clinical practice. However, the IO segment also includes oncolytic viruses (e.g., tarimodine-raherparepbec), cytokines (e.g., interferons and interleukins), and a broad range of cancer vaccines.

[0005] Immunotherapy is also widely used to suppress autoimmunity and to treat allergies or reduce transplant organ rejection. For example, tumor necrosis factor alpha (TNF-α) inhibitors are conventionally used to reduce inflammation in diseases such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis.

[0006] A prerequisite for successful drug therapy is that the drug reaches the stromal tissue outside the vascular compartment. This applies both to direct effects on pathology (e.g., when using chemotherapeutic agents) and interactions with immune cells. However, for the vast majority of therapeutic agents, only a small portion of the administered drug reaches the target pathological disease area (e.g., tumor) in the body, while most is taken up by healthy tissue and broken down or excreted before reaching its target. For example, for many chemotherapy regimens, less than 0.01% of the administered dose accumulates in the target cancerous tissue [Kurdziel et al.: "Human Dosimetry and Preliminary Tumour Distribution of 18F-Fluoropaclitaxel in Healthy Volunteers and Newly Diagnosed Breast Cancer Patients Using PET / CT", J Nucl Med. 2011 September; 52(9): pp. 1339-1345]. Following systemic administration, drug spillage from vascular compartments into the interstitial space can occur via three basic processes: passive diffusion, convective transport, and transcytosis through vascular epithelial cells. For the vast majority of small molecule drugs, passive diffusion is by far the most important pathway for spillage and distribution. However, due to the physiological and chemical properties and large size of most ITAs, passive diffusion does not play a significant role in the spillage process. Reliance on large molecular structures or nanoscale constructs is common to the majority of ITAs; for example, mAbs have a molecular weight of approximately 150,000 daltons and a diameter of 10–15 nm, while oncolytic viruses are typically large constructs with a diameter of 50–150 nm. This is in contrast to the molecular weight of the vast majority of chemotherapeutic agents, which have molecular weights of less than 1000 daltons and exhibit dimensions of less than 1000 nm. These sizes effectively prevent drug spillage from vascular compartments, thus significantly reducing the potential efficacy of the treatment [Ryman and Meibohm, CPT Pharmacometrics Syst Pharmacol. September 2017; 6(9): pp. 576-588].Therefore, the vascular wall presents a significant barrier to the effective use of ITAs, and methods to increase drug spillage could lead to a significant increase in the therapeutic effect of these drugs.

[0007] Furthermore, within the realm of immuno-oncology, it is not ITA itself that produces the therapeutic effect, but rather inflammatory cytokines (e.g., IL-1, IL-12, IL-18, TNFα, and INFγ) and activated immune cells (e.g., activated T cells, e.g., CD3, CD4, and CD8-positive cells). In this case, ITA blocks receptors and allows immune cells to initiate the necessary cytotoxic processes against the pathological cells involved. Therefore, for the therapy to function, activated immune cells must be activated and made tumor immunologically "hot" rather than immunologically "cold" before being able to infiltrate the pathology. Here, the vascular barrier also presents significant interference, often limiting immune cell infiltration, and methods to enhance it could potentially lead to a significant increase in the therapeutic effect of these drugs.

[0008] For example, despite improvements in toxicity profiles compared to chemotherapeutic agents, immunotherapy remains hampered by unwanted systemic effects and dose-limiting toxicity. Due to their mechanisms of action, ITAs have an inherent but variable toxicity spectrum. The most serious concern is the potential hyperphysiological stimulation of the immune system, which can lead to the production of uncontrolled, rapid inflammatory cytokines that may be life-threatening, but ITAs may also exhibit a range of dermatological, endocrine, hepatic, and gastrointestinal toxicity. Increasing doses to overcome limited overflow is therefore not an option. However, if overflow efficacy can be improved as well, it may be possible to explore relatively low doses of the drug that still maintain therapeutic efficacy while simultaneously reducing cost and systemic toxicity.

[0009] Numerous immunotherapy regimens are effective in only a small proportion of the treated patient population; some respond well to therapy, while others do not respond at all. There are several possible reasons for these differences in clinical response, including the presence of different gene mutations and varying degrees of activity of specific signaling pathways in individual patients. However, drug overflow and / or activated cell infiltration differ from patient to patient, and variability in response may also be due in part to insufficient concentration of the drug in the stromal tissue of a given subject, or insufficient infiltration of activated cells into the pathology.

[0010] There are significant cost implications associated with immunotherapy-based therapies. For example, the annual worldwide cost of treating non-small cell lung cancer with selected mAbs has been estimated to exceed US$80 billion. The estimated annual cost per patient for various ITAs exceeds £100,000, which puts considerable pressure on healthcare systems. The cost of carrying out these relatively new targeted therapies has risen dramatically, and the duration of treatment is also increasing as many diseases are increasingly treated as chronic conditions. In the UK, the National Institute for Health and Care Excellence (NICE) is responsible for determining whether new treatments are cost-effective for the NHS. The cost of new therapies is evaluated in relation to their clinical effectiveness using a standardized metric known as quality-adjusted life years (QALYs). To be considered cost-effective for the NHS, the cost of a therapy must be £20,000–£30,000 per QALY obtained, or £50,000 or less for end-of-life care. New ITAs are increasingly exceeding these thresholds, leading to NICE rejection and reduced patient access. Identifying methods to increase spillage from vascular compartments and enhance drug distribution in pathological tissues could lead to dose reductions, while maintaining therapeutic efficacy and ensuring a significant reduction in treatment costs.

[0011] Therapeutic options currently under investigation include nanoparticles, molecular targeting, and ultrasound-mediated microbubble therapy, i.e., sonoporation, which specifically focus on targeted drug delivery in the treatment of localized pathologies, such as solid tumors. Interest in ultrasound-mediated drug delivery has grown over the past 20 years. For a recent review, see Castle et al. [Castle et al., Am J Physiol Heart Circ Physiol 2013 / 2 / 1 304:H350~H357]. Numerous approaches are based on the use of microbubbles similar to those used as ultrasound contrast agents for medical imaging applications, for the release of incorporated or bound drugs, and / or for enhanced uptake of systemically (co)administered drugs. Sonoporation is a methodology in which gaseous microbubbles are injected into the vascular system and stimulated by ultrasound (US) to induce a biomechanical effect that increases the permeability of the vascular barrier and the spillage of drugs at specific locations (e.g., within solid tumors). Microbubbles are stabilized bubbles (2-3 μm in diameter) that are injected into blood vessels and remain stable for up to 1-2 minutes without known in vivo side effects. Upon application of ultrasound, these microbubbles vibrate and interact with adjacent endothelial / vascular wall cells to form fenestrations through various biomechanical effects. These interactions can increase drug overflow from vascular compartments, intracellular drug uptake, and allow therapeutic agents to penetrate deeper into tissues as well as within the vascular wall. While this technique is promising, the true potential of sonoporation is limited by the use of commercially available microbubbles (contrast-enhanced microbubbles) designed and optimized for ultrasound imaging rather than therapeutic enhancement. As a result, substantial research has focused on developing “next-generation” microbubbles optimized for ultrasound-mediated targeted enhancement therapy. The main limitation is the size of the microbubbles. When they are small, the level of biomechanical effect they can exert within vascular compartments is limited. Furthermore, physical contact with the endothelial wall is limited, and the resulting biomechanical effect typically decreases exponentially with distance from the blood vessel wall, thus limiting the effectiveness of inducing fenestration.As a result of these limitations, sonoporation using conventional contrast-enhanced microbubbles requires the use of relatively high-energy US fields (mechanical index, MI). Although microbubble-mediated delivery mechanisms have been clearly demonstrated in vivo, there are associated biological effects that raise safety concerns for the approach. These likely include microbubble cavitation mechanisms, particularly microbleeding and irreversible vascular damage. These processes can also lead to vascular occlusion, i.e., vascular collapse (temporary or permanent), effectively halting blood flow perfusion and therefore drug uptake.

[0012] International Publication No. 2015 / 047103 proposes the concept of ultrasound-mediated targeted delivery, suggesting that administering microdroplet cluster compositions together with a therapeutic agent and irradiating the target pathology with ultrasound may result in increased therapeutic efficacy compared to using the therapeutic agent alone. This concept, known as acoustic cluster therapy (ACT sonoporation or ACT), has since been investigated in a series of preclinical proof-of-principle and proof-of-concept studies treating various cancerous conditions with various chemotherapy agents. For example, van Wamel et al. [Acoustic Cluster Therapy enhances the therapeutic efficacy of paclitaxel and Abraxane® for treatment of human prostate adenocarcinoma in mice, J Control Release, 236 (2016) pp. 15-21] demonstrates the potential of ACT in combination with standard small molecule chemotherapy regimens. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] International Publication No. 2015 / 047103 [Patent Document 2] International Publication No. A-9416379 [Patent Document 3] International Publication No. 9953963 [Non-licensed literature]

[0014] [Non-licensed Document 1] Carter and Thurson, Immune-oncology agents for cancer therapy, The Pharmaceutical Journal, May 7, 2020 [Non-licensed Document 2] Kurdzielら: "Human Dosimetry and Preliminary Tumour Distribution of 18F-Fluoropaclitaxel in Healthy Volunteers and Newly Diagnosed Breast Cancer Patients Using PET / CT", J Nucl Med. 2011 September; 52(9): 1339~1345 pages] [Non-licensed Document 3] Ryman and Meibohm, CPT Pharmacometrics Syst Pharmacol. Sep 2017;6(9):576-588. [Non-licensed Document 4] Castleら、Am J Physiol Heart Circ Physiol February 1, 2013 304:H350~H357 [Non-licensed Document 5] van Wamel, Acoustic Cluster Therapy enhances the therapeutic efficacy of paclitaxel and Abraxane (registered trademark) for treatment of human prostate adenocarcinoma in mice, J Control Release, 236 (2016) pages 15~21 [Non-licensed Document 6] American Institute of Ultrasound in Medicine., "Acoustic Output Measurement Standard for Diagnostic Ultrasound Equipment", 1st Edition, 2nd Edition, Laurel, MD: American Institute of Ultrasound in Medicine; 1998, 2003

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0015] Based on the above, there is a clear need for novel and alternative compositions and methods for treatment using immunotherapeutic agents that can increase uptake into interstitial tissue and / or increase activated immune cell infiltration into target pathologies. Such compositions and methods may also be explored as having the potential to improve therapeutic outcomes, reduce systemic dose-limiting toxicity, and significantly reduce the cost of treatment. [Means for solving the problem]

[0016] Brief Overview of the Invention The object of the present invention is to provide compositions and methods for use in drug treatment and immunotherapy methods using immunotherapeutic agents (ITAs), particularly for the treatment of cancer and autoimmune diseases. The present invention relates to ultrasound-mediated targeted delivery of ITAs to pathological sites and drug treatment using such therapeutic agents.

[0017] The inventors have discovered that Acoustic Cluster Therapy (ACT®) can be used to enhance the delivery of ITAs and the infiltration of activated immune cells. Presented in International Publication No. 2015 / 047103, ACT is a concept of ultrasound-mediated targeted delivery in which a microbubble / microdroplet cluster composition is administered together with a therapeutic agent, and ultrasound irradiation of the target pathology can result in increased therapeutic efficacy compared to the use of the therapeutic agent alone. While International Publication No. 2015 / 047103 provides a comprehensive description of the characteristics of the ultrasound field applied during the ACT procedure, the inventors have now surprisingly found that specific use of the ultrasound irradiation frequency and pressure of this method fully utilizes the potential of ACT, resulting in increased permeability of the vascular barrier, increased overflow of co-administered therapeutic agents and / or inflammatory cytokines, and / or infiltration of activated immune cells, as well as avoidance of adverse effects.

[0018] Based on the studies conducted and planned, the applicant has now surprisingly found that the specific use of ACT techniques, which involves careful selection of the ultrasound irradiation frequency and mechanical index of the present method, is useful in treatments using ITA, and in particular, results in improved therapeutic effects of co-administered ITA compared to treatment of subjects using ITA alone.

[0019] A cluster composition and procedure for this use in a method of enhancing the therapeutic effect of ITA has now been confirmed. This utilizes ACT technology to generate large phase-shift bubbles in vivo from an administered composition containing microbubble / microdroplet clusters. Combined with local ultrasound irradiation, this promotes enhanced overflow and uptake of separately, pre-, and / or simultaneously and / or post-administered therapeutic agents and / or inflammatory cytokines, as well as enhanced infiltration of activated immune cells into the target pathology, resulting in a significant increase in therapeutic effect compared to the use of the therapeutic agent alone.

[0020] Furthermore, the inventors have confirmed that by combining ACT technology with clinically relevant drugs, the compositions and methods of the present invention can deliver larger amounts of ITA to target pathological sites and improve the overflow of ITA from vascular compartments into the target tissue interstitial space.

[0021] Accordingly, the present invention provides microbubble / microdroplet cluster compositions for use in methods for enhancing the delivery of ITA to target pathological tissue. Similarly, the present invention provides methods for enhancing the delivery of ITA to target tissue stroma, including administration of a microbubble / microdroplet cluster composition to a subject. This disclosure demonstrates that a two-component microbubble / microdroplet cluster composition, in which microbubbles as a first component are physically bound to microdroplets as a second component in the cluster, can be used in methods to promote enhanced uptake of ITA administered separately, prior to, and / or simultaneously to and / or subsequently. The methods offer a significant increase in potential therapeutic effect compared to the use of the therapeutic agent alone.

[0022] In one embodiment, the present invention relates to a microbubble / microdroplet cluster composition for use in a method for treating pathological conditions in mammals, wherein the method is: (i) A step of administering at least one immunotherapy agent (ITA) to the subject, (ii) A step of administering a microbubble / microdroplet cluster composition to a target, wherein at least one ITA is administered separately from the cluster composition, beforehand, and / or simultaneously, and / or afterward. (iii) A step of activating the phase shift of the diffusive component of the microdroplets of the cluster composition from step (i) by ultrasonic irradiation of a region of interest within the subject at a first frequency of 1 to 10 MHz and a first mechanical index of 0.1 to 0.4. (iv) Further irradiation with ultrasound at a second frequency of 0.4 to 0.6 MHz and a second mechanical index of 0.1 to 0.3. The present invention provides a microbubble / microdroplet cluster composition containing the following:

[0023] Similarly, the present invention relates to a method for treating a pathological condition in a mammal using at least one immunotherapy agent (ITA), (i) A step of administering at least one immunotherapy agent (ITA) to the subject, (ii) A step of administering a microbubble / microdroplet cluster composition to a target, wherein at least one ITA is administered separately from the cluster composition, beforehand, and / or simultaneously, and / or afterward. (iii) A step of activating the phase shift of the diffusive component of the microdroplets of the cluster composition from step (i) by ultrasonic irradiation of a region of interest within the subject at a first frequency of 1 to 10 MHz and a first mechanical index of 0.1 to 0.4. (iv) Further irradiation with ultrasound at a second frequency of 0.4 to 0.6 MHz and a second mechanical index of 0.1 to 0.3. This provides a method that includes [something].

[0024] Further irradiation in step (iv) promotes the exudation of ITA administered in step (i) and the infiltration of activated immune cells into the target pathology. Brief explanation of the drawing [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 visualizes cluster size versus in vivo product efficacy, with the Y-axis showing the calculated correlation coefficient for grayscale enhancement from US imaging (i.e., amount of bubbles deposited after activation), and the X-axis showing cluster diameter in μm. [Figure 2] Figure 2 shows the attenuation spectrum of the large bubble cluster after ultrasonic activation of the cluster composition. The Y-axis represents the attenuation in dB / cm, and the X-axis represents the frequency in MHz. [Figure 3] Figure 3 shows the results from modeling the response of activated bubbles to low-frequency enhanced irradiation fields with various incident US fields and mechanical index (MI). The stationary bubble radius was 20 μm, and the incident field consisted of 8 cycles including the frequencies and MIs listed in each panel. For each panel, the Y-axis represents the radius of the activated bubble in μm, and the X-axis represents the time in μs. [Figure 4] Figure 4 shows the results regarding tumor-specific uptake of a fluorescent dye (Evans Blue) during ACT treatment using enhanced step irradiation fields at 500 kHz and mechanical indices (MI) of 0, 0.1, 0.2, 0.3, and 0.4 (lower panel). The Y-axis shows tumor-specific uptake in mg Evans Blue / mg tumor tissue units. The X-axis shows the mechanical index. The four upper panels show the modeling results of the response of activated bubbles to the incident US field at various MIs examined. The Y-axis shows the radius of the activated bubbles in μm. The X-axis shows time in μseconds. [Figure 5]Figure 5 shows the results regarding the therapeutic efficacy of nab-paclitaxel (nab-PTX) ± ACT for the treatment of prostate cancer in mice. The Y-axis shows overall survival in % for all treated animals. The X-axis shows the time since the start of the study in days. Groups: saline control (gray dotted line), nab-PTX alone (gray solid line), nab-PTX + ACT with a 500 kHz enhancement field (MI 0.2) (black solid line), and nab-PTX + ACT with a 900 kHz enhancement field (MI 0.2) (black dotted line). [Figure 6] Figure 6 shows the results of the therapeutic efficacy of nab-paclitaxel ± ACT for the treatment of mammary cancer in mice. The Y-axis represents the normalized tumor diameter (mm). The X-axis represents the time (days) after the start of the study. Groups: saline control (black, open rectangle), nab-PTX alone (black, open circle), nab-PTX + ACT with enhancement field MI0.1 (500kHz) (gray, filled circle), and nab-PTX + ACT with enhancement field MI0.2 (500kHz) (black, filled circle). [Figure 7] Figure 7 provides a photograph of the A) setup and a schematic diagram of the B) setup of the apparatus used in the study of Example 3 for the application of an ACT sonoporation procedure including ultrasonic activation and enhancement. In Figure 7B, the numbers indicate the following: 1 is a dual-frequency ultrasonic transducer (2.7 MHz and 500 kHz output), 2 is an ultrasonic waveguide, 3 is a water bath, 4 is an ultrasonic gel, 5 is an ultrasonic absorption pad, 6 is an injection syringe containing a cluster composition, 7 is a VeVo imaging stage, 8 is a catheter, and 9 is a tumor. [Figure 8] Figure 8 shows the results from the study in Example 3: the therapeutic effect of reovirus combined with ACT on the treatment of hepatocellular carcinoma. The Y-axis shows tumor volume as a function of time for the treatment of hepatocellular carcinoma (HCC) in mice using oncolytic reovirus (filled triangle), saline control (filled square), and oncolytic reovirus combined with ACT (filled circle). The X-axis shows the time from the start of treatment. The gray triangle below the X-axis indicates the treatment day. [Figure 9]Figure 9 shows a schematic diagram of the apparatus setup used in the Example 4 test for the application of the ACT sonoporation procedure, including ultrasonic activation and enhancement. The numbers indicate: 1- Amplifier, 2- Signal generator, 3- Switchbox between 0.5 and 2.7 MHz, 4- Dual frequency transducer, 5- Water-filled cone, 6- Water-filled bag, 7- Ultrasonic gel, 8- Mouse in prone position, 9- Ear bar, 10- Acoustic absorption pad. [Figure 10] Figure 10 shows a box plot of results from Example 4: Test of ACT-induced delivery of nanoparticles across the blood-brain barrier. Top panel: Representative images from near-infrared fluorescence (NIRF) imaging of CCPM nanoparticle uptake into brain tissue. Control brain and ACT-treated brain 1 hour and 24 hours after ACT treatment. Bottom left panel: The Y-axis shows uptake as measured by NIRF, as a percentage of the injected dose per gram of brain tissue in the control and ACT groups 1 hour and 24 hours after ACT treatment. Black filled circles represent individual observations. Lines and asterisks indicate statistical significance between groups (***p<0.001) obtained from t-tests. Bottom right panel: The Y-axis shows uptake as measured by confocal microscopy, as a percentage of the brain area containing nanoparticles for the control and ACT groups 1 hour after ACT treatment. Black filled circles represent individual observations. Lines and asterisks indicate statistical significance between groups (*p<0.05) obtained from the Mann-Whitney rank-sum test. [Figure 11]Figure 11 provides a graph of possible ACT procedures performed during treatment with a combination regimen including a 30-minute infusion of nivolumab followed by a 90-minute infusion of ipilimumab. The ACT procedure is applied three times during administration, as indicated by the gray ACT® sonoporation bars. Panel A: The ACT procedure includes a.: injection of the cluster composition, b.: activation of the cluster with, for example, 60 seconds of standard medical imaging ultrasound irradiation, and c.: enhancement step with, for example, 5 minutes of ultrasound irradiation at 400–600 kHz with an MI of 0.1–0.3. Panel B: The y-axis shows the plasma concentration of the administered therapeutic agent as a percentage of the peak, and the x-axis shows the time in minutes. In this example, three ACT procedures are performed at approximately 30, 80, and 120 minutes to show treatment of the entire area of ​​interest, including both drugs. [Figure 12] Figure 12 shows a graph of possible ACT procedures performed during treatment with a standard combination immunotherapy + chemotherapy regimen for metastatic squamous cell non-small cell lung cancer, i.e., a combination regimen including pembrolizumab, followed by paclitaxel and carboplatin. Panel A: ACT procedures as detailed in Figure 11. Panel B: The Y-axis shows the plasma concentration of the administered therapeutic agent in percentage of peaks, and the X-axis shows the time in minutes. In this example, three ACT procedures are performed at approximately 160, 200, and 240 minutes to include all drugs and represent treatment of the entire area of ​​interest. [Figure 13] Figure 13 provides a volcano plot from the Example 6 study showing the effect of ACT on the immunogenicity status of tumors as expression of immunogenes alone when ACT is combined with an isotype antibody (IgG), compared to treatment with IgG alone. [Figure 14]Figure 14 provides two graphs from the Example 6 study showing the genetic effects of ACT as shown by the downregulated (A) or upregulated (B) pathways when ACT is combined with an isotype antibody (IgG), compared to treatment with IgG alone. The x-axis represents -log10 (p-value). Panel A: Downregulated pathway with IgG + ACT. Panel B: Upregulated pathway with IgG + ACT. The letters indicate the following: GO:0001666 - Response to hypoxia, WP4206 - Hereditary leiomyomatosis and renal cell carcinoma pathway, GO:0001525 - Angiogenesis, GO:0051235 - Position maintenance, GO:0061061 - Muscle structure development, GO:0006936 - Muscle contraction, GO:0097435 - Supramolecular fiber composition, GO:0043462 - Regulation of ATPase activity, GO:0030199 - Collagen fibril composition, GO:0030239 - Myofibrils assembly, GO:0044057 - Regulation of system processes. [Figure 15] Figure 15 provides a plot of preliminary immunohistochemical findings from automated analysis of whole-stained tumor sections; see Example 6. The percentage of cells positively stained for CD8 T cells is shown by treatment group, group mean (bar), and standard deviation (error bar) for individual animals (black dots). The x-axis represents A: ACT + PD1 / CTL4, B: PD1 / CTL4, C: ACT + ISO, D: ISO, and E: normal saline. [Modes for carrying out the invention]

[0026] Detailed description of the present invention Definition: Unless otherwise defined, all technical terms, notations, and other scientific terms or vocabulary used herein are intended to have meanings that are generally understood by those skilled in the art to which the invention pertains. In some cases, terms that have generally understood meanings are defined herein for clarity and / or for ease of reference, but the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from those generally understood in the art.

[0027] As used herein, acoustic cluster therapy (ACT) comprises the administration of a cluster composition (see definition below) in combination with at least one therapeutic agent, as further defined below, and subsequent application of ultrasound to a target area of ​​interest within the object (e.g., a tumor, stromal tissue, or lymph node). The terms “ACT treatment” or “ACT procedure” are used to describe the administration and irradiation of the cluster, and therefore steps (ii), (iii), and (iv) of the method.

[0028] As used herein, “Subject” means any human or non-human animal individual selected for treatment or therapy, and may include, but may not be limited to, patients, particularly subjects with cancer or autoimmune disease.

[0029] As used herein, the term "therapeutic dose" means the amount of therapeutic agent that is effective in producing the desired therapeutic effect in a subject, based on a reasonable benefit-to-risk ratio applicable to any treatment.

[0030] The terms “microbubbles” or “conventional contrast-enhanced microbubbles” are used in this document to describe microbubbles having a diameter in the range of 0.2 to 10 microns, typically with an average diameter of 2 to 3 μm. “Conventional contrast-enhanced microbubbles” include commercially available formulations such as Sonazoid® (GE Healthcare), Optison® (GE Healthcare), Sonovue® (Bracco Spa.), Definity® (Lantheus Medical Imagin), Micromarker® (VisualSonics Inc.), and Polyson L® (Miltenyi Biotec GmbH).

[0031] The term HEPS / PFB microbubbles is used in this document to describe microbubbles formed by reconstituting the first component (as provided in Example 1) using 2 ml of water.

[0032] The terms "phase-shifted bubble," "large phase-shifted bubble," "large activated bubble," and "activated bubble" are used in this document to describe large (>10 μm) bubbles that form after ultrasound (US) induces activation of a cluster composition.

[0033] In this book, the term "microdroplet" is used to describe emulsion microdroplets having a diameter in the range of 0.2 to 10 microns.

[0034] "Irradiation" or "US irradiation" are terms used to describe exposure to ultrasound or procedures using ultrasound.

[0035] The term “conventional medical imaging ultrasound” is used to describe ultrasound from shelf US scanners and probes intended for medical imaging, i.e., ultrasound with frequencies between 1 and 10 MHz and an MI of less than 1.9, preferably less than 0.7, and more preferably less than 0.4.

[0036] In this book, the term "sediment tracer" is used in relation to activated phase-shift bubbles, meaning that the transient mechanical capture of large bubbles in the microcirculation indicates that the local deposition of phase-shift bubbles in tissue reflects the amount of blood flowing through the tissue's microcirculation at the time of activated bubble deposition. Therefore, the number of captured "deposited" phase-shift bubbles depends linearly on tissue perfusion at the time of deposition.

[0037] In this book, the term "phase shift (process)" is used to describe the phase transition of a substance from a liquid state to a gaseous state. Specifically, it refers to the transition (process) in which the state of the oil component of microdroplets of a cluster composition changes from liquid to gaseous upon US irradiation.

[0038] In this book, the terms "therapeutic delivery / therapeutic agent" and "drug delivery / drug" are understood to include the delivery of drug molecules, nanoparticles, nanoparticle delivery systems, and liposome delivery systems containing at least one therapeutic agent.

[0039] The term "first component" (or component 1, or C1) is used in this document to describe the dispersed gas (microbubble) component. The term "second component" (or component 2, or C2) is used in this document to describe the dispersed oil phase (microdroplet) component, which includes a diffusible component.

[0040] The term "cluster composition" is used herein to describe a composition resulting from a combination, for example, a mixture, of a first (microbubble) component and a second (microdroplet) component. Therefore, cluster compositions having the features further described herein refer to formulated compositions ready for administration to a subject and for use in acoustic cluster therapy.

[0041] In this book, the term "diffusible component" is used to describe the chemical composition of the oil phase of a second component that can diffuse in vivo into the microbubbles of the first component and transiently increase their size.

[0042] As used in this book, the term "pharmaceutical composition" has its usual meaning and is in a form particularly suitable for administration to mammals. A composition preferably comprises two distinct compositions, namely a cluster composition (a) and a therapeutic agent (b), both suitable for administration to mammals via the same or different routes of administration, for example, parenteral injection, intraperitoneal injection, or intramuscular injection. The phrase "form suitable for administration to mammals" means a composition that is sterilized, free of pyrogens, free of compounds that cause excessive toxicity or adverse effects, and formulated at a biocompatible pH (approximately pH 4.0 to 10.5). Such a composition does not precipitate upon contact with biological fluids (e.g., blood), contains only biocompatible excipients, and is preferably formulated to be isotonic.

[0043] In this book, the term "sonometry (system)" refers to an in vitro measurement system that uses acoustic technology to dynamically determine and count activated phase-shifted bubbles.

[0044] In this document, the term "reactivity" is used to describe the ability of microbubbles of the first component and microdroplets of the second component to form microbubble / microdroplet clusters upon mixing. The Coulter count is suitable for quantifying the concentration and size distribution of microbubbles and microdroplets in C1 and C2, and for characterizing particles in cluster compositions (pharmaceuticals, DPs). The reactivity (R) of a cluster composition is defined as follows: R = (C C1 + C C2 - C DP )·100 / (C C1 + C C2 ) In the formula, C C1 , C C2 and C DP These are the number concentrations observed in C1, C2, and DP, respectively. Therefore, reactivity is a measure of the number of individual microbubbles and microdroplets of C1 and C2 contained in the cluster morphology of DP. Reactivity also correlates with the size of these clusters (i.e., the number of individual microbubbles and microdroplets constituting a single cluster). Reactivity can be easily calculated from the Coulter analysis of C1, C2, and DP.

[0045] In this book, the terms "microbubble / microdroplet cluster" or "cluster" refer to a group of microbubbles and microdroplets that are permanently held together by electrostatic attraction within an aggregated entity of a single particle. In this book, the term "clustering" refers to the process by which microbubbles (a first component) and microdroplets (a second component) form a cluster.

[0046] In medical ultrasound, acoustic power is typically described by the "mechanical index" (MI). This parameter is defined as the peak negative pressure (PNP) in the ultrasound field, divided by the square root of the center frequency of the ultrasound field in MHz (Fc) units [American Institute of Ultrasound in Medicine, "Acoustic Output Measurement Standard for Diagnostic Ultrasound Equipment," 1st and 2nd editions, Laurel, MD: American Institute of Ultrasound in Medicine; 1998, 2003].

[0047]

number

[0048] A standard requirement during medical ultrasound imaging is the use of microbubble contrast (MI) of less than 1.9. During ultrasound imaging with microbubble contrast agents, MI of less than 0.7 is recommended, and MI of less than 0.4 is considered "best practice," to avoid adverse biological effects such as microbleeding and irreversible vascular damage. When MI is referred to in this document, please understand that it refers to in situ MI, that is, MI applied to the targeted area of ​​interest.

[0049] In the context of the ACT procedure described in this book, the term "activation" or "activation process" refers to the induction of a phase shift in microbubble / microdroplet clusters by ultrasonic (US) irradiation, i.e., the generation of large activated bubbles.

[0050] The term frequency is defined as the number of (ultrasonic) cycles per second (Hz). As used herein, this term refers to the center frequency of the applied sound field.

[0051] The term "enhancement" or "enhancement process" refers to the induction of volumetric oscillations of large activated bubbles and the associated biomechanical effects by US irradiation in the context of the ACT procedure in this book. The term "resonance frequency" or "microbubble resonance frequency", when used in this book, means to describe the acoustic resonance frequency of a single bubble in an infinite matrix domain (ignoring the effects of surface tension and viscous attenuation). The resonance frequency is given as follows:

Number

[0052] The term "immunotherapeutic agent" relates to a treatment intended to treat a disease or condition by inducing, enhancing or suppressing an immune response. This term also relates to the manipulation of an immune response in which an inappropriate immune response is adjusted to a more appropriate one in the context of a particular disease.

[0053] The term "molecular target" should be understood as a molecule or group of molecules in human cells that is essentially related to the course of a particular disease, e.g., etiology, progression, and / or drug resistance. To be termed a target, there must be evidence that directing a small molecule, biological product, or other intervention at the target results in a desired therapeutic effect and a change in the course of the disease. In this book, molecular targets are named according to the Human Cell Differentiation Molecule (HCDM) nomenclature committee, as agreed upon during the ongoing series of workshops on human leukocyte differentiation antigens (HLDA) officially approved by the International Union of Immunological Societies (IUIS) and endorsed by the World Health Organization (WHO). Most often, molecular targets are named by their cluster of differentiation antigen (CD) numbers as published by HCDM in this book. However, other chemically acceptable terms that detail the nature of the target are also used, e.g., CTLA-4 denotes cytotoxic lymphocyte antigen 4, PD-l denotes programmed cell death protein 1, etc.

[0054] This invention provides cluster compositions for use in treatment methods using immunotherapy agents (ITAs), particularly for the treatment of cancer and autoimmune diseases. Using ACT technology, the invention generates large phase-shifted bubbles in vivo from a drug composition containing microbubble / microdroplet clusters, thereby facilitating the delivery and uptake of separately, pre-administered, and / or simultaneously and / or post-administered therapeutic agents. The therapeutic effect of the therapeutic agent is significantly increased compared to administration of the agent alone, due to the biomechanical mechanisms of the microvascular system, as further described below. This disclosure demonstrates that specific use of ACT technology, including two-step ultrasound irradiation at different frequencies and mechanical indices, enables increased delivery of separately administered ITAs and enhanced activated immune cell infiltration. Activated phase-shifted bubbles are approximately 1000 times larger in volume (10 times larger in diameter) than typical normal contrast-enhanced microbubbles.

[0055] The present invention provides a cluster composition for enhancing activated immune cell infiltration for use in a method of delivering at least one ITA to the target tissue stroma, the method comprising a phase-shift technique to facilitate the delivery and uptake of separately administered ITAs to generate large phase-shift bubbles in vivo from the administered cluster composition. The compositions and methods for use of the present invention enhance the therapeutic effect of separately administered co-administered therapeutic agents and provide improved therapeutic outcomes compared to treatments that do not use the compositions of the present invention.

[0056] Therefore, in a first aspect, the present invention relates to a microbubble / microdroplet cluster composition for use in a method for treating pathological conditions in mammals, wherein the method is (i) A step of administering at least one immunotherapy agent (ITA) to the subject, (ii) A step of administering a microbubble / microdroplet cluster composition to a target, wherein at least one ITA is administered separately from the cluster composition, beforehand, and / or simultaneously, and / or afterward. (iii) A step of activating the phase shift of the diffusive component of the microdroplets of the cluster composition from step (i) by ultrasonic irradiation of a region of interest within the subject at a first frequency of 1 to 10 MHz and a first mechanical index of 0.1 to 0.4. (iv) Further irradiation with ultrasound at a second frequency of 0.4 to 0.6 MHz and a second mechanical index of 0.1 to 0.3. The present invention provides a microbubble / microdroplet cluster composition containing the following:

[0057] The method steps, particularly step (iv), promote the exudation of ITA administered in step (i) and the infiltration of activated immune cells into the target pathology.

[0058] In one embodiment, a microbubble / microdroplet cluster composition and at least one ITA can be considered a pharmaceutical composition comprising these two, preferably as separate compositions. Accordingly, the present invention provides a pharmaceutical composition comprising a microbubble / microdroplet cluster composition for use in a method of treating a pathological condition in a mammalian subject with at least one ITA, wherein the at least one ITA is administered separately from the cluster composition, beforehand, and / or simultaneously, and / or afterward.

[0059] In addition to the required steps (i) to (iv), the method may optionally include a step (iib) of imaging the cluster using ultrasound imaging to identify a region of interest for treatment within the subject. Such a step should be performed after step (ii) and before step (iii).

[0060] The Acoustic Cluster Therapy (ACT®) technology used in this invention is an ultrasound-mediated targeted drug delivery platform that utilizes microbubble / microdroplet clusters activated by the application of ultrasound to create local openings or fenestrations in the vascular system of target tissue, thereby resulting in a transient increase in vascular permeability, and thereby allowing drugs and activated immune cells to penetrate the pathological stroma more effectively. Accordingly, the compositions and methods of this invention can create fenestrations in the endothelial barrier, improve the overflow and intratumoral distribution of activated immune cells (e.g., T cells), improve intratumoral uptake and distribution of antibodies, and stimulate the release of tumor antigens. The van Wamel paper demonstrated the potential of ACT to enhance treatment regimens using conventional small molecule chemotherapeutic agents. However, this paper does not show the potential of combining ACT with larger drug constructs, e.g., ITAs, and it has now been shown that this delivery concept is particularly useful. Furthermore, this paper does not describe the potential of applying ACT to the treatment of autoimmune diseases. Finally, ACT is consistently described in the current state of technology as a concept for enhancing the local delivery of drug molecules to target tissues. The inventors have found that, in addition to improving the stimulation of antibody uptake and distribution within tumors and the release of tumor antigens because ACT creates a window in the endothelial barrier, this technique can also be applied to improve the overflow and distribution within tumors of activated immune cells (e.g., T cells). Essentially, the ACT concept solves most of the limiting attributes associated with the use of conventional contrast-enhanced microbubbles for sonoporation; 1) It generates large activated bubbles in vivo that are approximately three orders of magnitude larger than normal contrast-enhancing microbubbles, resulting in a much greater biomechanical effect. 2) These bubbles are in close contact with the endothelium and are therefore far more effective, 3) It is functional with a stable cavitation mode using a low MI US field, avoiding safety concerns and vascular damage associated with inertial cavitation.

[0061] This invention is based in part on findings from several preclinical studies. In Example 3, the applicant investigated the ability of ACT to enhance the therapeutic effect of ITA in the form of an oncolytic virus for the treatment of hepatocellular carcinoma. As can be observed from the results shown in Table 3 and visualized in Figure 8, treatment with reovirus alone did not show significant inhibition of tumor growth at the investigated doses compared to the saline control group. However, when the same dose of virus was combined with ACT treatment, a remarkable and significant tumor inhibition was observed, with a reduction of over 95% in tumor volume at day 25 compared to virus alone. This study demonstrates the ability of the ACT concept to enhance the therapeutic efficacy of large construct ITA for the treatment of local pathological conditions.

[0062] In Example 4, the applicant investigated the ability of ACT to deliver cytotoxic nanoparticles across the blood-brain barrier (BBB). The BBB is a highly selective, semipermeable endothelial cell boundary that prevents solutes in circulating blood from non-selectively crossing into the extracellular fluid of the central nervous system where neurons reside. The blood-brain barrier is formed by endothelial cells of the capillary wall, stellate terminal foot covering the capillaries, and pericelles embedded in the capillary basement membrane. This system allows for the penetration of several selected small molecules by passive diffusion, as well as the selective and active transport of various nutrients, ions, organic anions, glucose, water, and amino acids that are important for neuronal function. The blood-brain barrier restricts the penetration of pathogens into the cerebrospinal fluid and the diffusion of solutes and large or hydrophilic molecules in the blood, while allowing the diffusion of small hydrophobic and small polar molecules. The blood-brain barrier also restricts the penetration of peripheral immune factors such as signaling molecules, antibodies, and immune cells into the CNS, thus protecting the brain from damage by peripheral immune events.

[0063] Therefore, the blood-brain barrier (BBB) ​​represents the tightest vascular barrier in the body. In the absence of damage, the BBB is completely closed to therapeutic agents larger than approximately 4-500 Daltons. Example 4 demonstrates the ability of ACT to enable the uptake of large constructs such as nanoparticles into brain tissue, indirectly demonstrating the usefulness of the ACT concept for local delivery of large ITAs, such as mAbs, across any vascular barrier in the body. A 280-290% increase in uptake into brain tissue was observed when nanoparticles were administered in combination with ACT compared to when nanoparticles were administered alone.

[0064] In Example 2, the applicant investigated the attributes of the US field applied during the second irradiation step (enhancement step) and its effect on the function of the applied procedure. Surprisingly, contrary to the teachings of International Publication 2015 / 047103, which discloses a preferred frequency range of 0.2–1 MHz and proposes an MI of less than 0.4, the applicant found that the functionality of the concept is highly sensitive to these parameters. Based on these studies, the applicant found that the preferred frequency range is 0.4–0.6 MHz and that the applied MI should be maintained above 0.1 but below 0.3. Using lower frequencies and higher MI during step (iv), which is the enhancement step, the applicant surprisingly found that the induced activated bubble oscillations were too strong, resulting in a significant loss of effectiveness and vascular damage. On the other hand, as demonstrated by the comparative example investigating 0.5 MHz versus 0.9 MHz (see Figure 3), at higher frequencies and lower MIs, the induced bubble oscillations are too small, resulting in insufficient biomechanical effects and, consequently, a significant decrease in therapeutic efficacy.

[0065] The applicant has found that the present invention's method, utilizing the ACT concept, is an effective method for overcoming biological barriers to improve the uptake of therapeutic agents and activated immune cells. This has been found to be particularly beneficial for treatments using immunotherapeutic agents, due to the generally low rates of drug overflow exhibited by this class of agents. The present invention's method, and in particular the enhancement step (iv), promotes the overflow of the ITA administered in step (i) and the infiltration of activated immune cells into the target pathology.

[0066] Therefore, the administered microdroplet-microbubble clusters are induced by a local irradiation method including ultrasound irradiation at different frequencies. When the clusters are irradiated (activated) with ultrasound, the vibrating bubbles initiate immediate vaporization (phase shift) of the attached microdroplets. The resulting expanding bubbles have been shown to form in vivo within capillary-sized vessels and, when further stimulated by low-frequency ultrasound, induce a biomechanical effect that promotes overflow and increases drug and / or cell penetration within the irradiated tissue. It has been confirmed that the treatment method should include two irradiation steps, one activation step and one enhancement step. Different ultrasound frequencies are used in these irradiation steps. During the activation step (step (iii) of the method), the microbubbles of the cluster vibrate, transferring energy to the microdroplets to induce droplet evaporation and form larger ACT bubbles designed to transiently remain in the microvascular system. Therefore, the clusters are activated to generate large bubbles by applying external ultrasound energy under imaging control, for example, after administration from a clinical ultrasound imaging system, and further irradiation at lower frequencies induces increased biomechanical effects, overflow, and drug penetration.

[0067] The steps of the method of the present invention are further described below: Administration steps i) and ii): As further described herein in the section on "Route of Administration," the cluster composition is administered parenterally, preferably intravenously, to the mammalian subject, and the therapeutic agent is administered separately from the cluster composition, as a separate composition, before, and / or simultaneously with, and / or after.

[0068] Optional imaging, step iib): Since clusters are not activated at low MI (below the cluster activation threshold of approximately 0.1), standard medical ultrasound contrast imaging can be performed, for example, to identify tumor microvascular pathology without cluster activation. Therefore, in one embodiment, the method includes an imaging step using a low MI contrast imaging mode (MI<0.1) to image the microbubble component, i.e., dispersed gas, without cluster activation and to identify the pathological location for treatment. Thus, since clusters are not activated at low MI (below the activation threshold), standard medical ultrasound contrast imaging can be performed before the activation step, for example, to identify tumor microvascular pathology.

[0069] Activation, step iii): The acoustic resonance frequencies of the microbubble components of the cluster are within the diagnostic frequency range (1-10 MHz). When the cluster composition is administered to a subject, activation of the cluster can be easily achieved by irradiating the region of interest with a low to medium range mechanical index, i.e., MI less than 0.4 but greater than 0.1, using standard diagnostic ultrasound imaging pulses used in conventional abdominal and cardiac applications of medical ultrasound.

[0070] Phase shift activation is performed by ultrasonic irradiation of the region of interest within the target at a first frequency of 1–10 MHz, for example, particularly 2–3 MHz. Cluster activation for phase shift to generate larger (diameter 10 μm or larger) activated bubbles can be achieved using imaging pulses with clinical imaging systems up to a spatial resolution of less than a millimeter. Upon activation, the oil in the microdroplets vaporizes, and the resulting large activated bubbles temporarily accumulate in the microvascular system.

[0071] In one embodiment, activation, i.e., US irradiation at a first frequency, is initiated immediately after each administration of the cluster composition, for example, within 20 seconds, and lasts for, for example, 60 to 120 seconds. In one embodiment, if imaging is performed (step iib), this is done before and during the injection of the cluster composition, and then the activation clock is started when the inflow of contrast agent is observed.

[0072] Activation under medical ultrasound imaging control using imaging pulses enables spatially targeted activation of clusters within a tissue region exposed to the ultrasound field. After activation, the generated large phase-shifted bubbles are transiently trapped in the microvascular system of the target region of interest due to their size. The resulting large phase-shifted bubbles are approximately 1000 times the volume of the vaporized emulsion microdroplets (from 2 μm diameter oil microdroplets to 20 μm diameter bubbles). The scattering cross-sections of these large phase-shifted bubbles are several orders of magnitude larger than those of the micron-sized microbubbles contained in the cluster before activation. As a result, the large phase-shifted bubbles generate a large amount of backscattered signal, which can be easily imaged in basic imaging modes using a diagnostic imaging system. Furthermore, the resonance frequency of the large phase-shifted bubbles is an order of magnitude lower than that of the microbubbles contained in the cluster before activation (see Example 2).

[0073] Enhancement, step iv): This step uses a second frequency lower than that used during the activation step to induce controlled volume vibrations of ACT bubbles, thereby exerting biomechanical forces on the capillary walls and enhancing local drug delivery. This further application of low-frequency ultrasound after activation and deposition promotes enhancement of delivery mechanisms and increases the efficiency of drug delivery to spatial target tissues by effectively overcoming biological barriers. These mechanisms may include sonoporation processes, i.e., the process by which irradiation of microbubbles within vascular compartments and the resulting volume vibrations increase the permeability of the vascular barrier. In other words, the ACT procedure increases the permeability of the endothelial wall and therefore enhances the overflow, distribution, and cellular uptake of therapeutic or activated immune cells. Other mechanisms, such as the generation of cellular signaling pathways to enhance therapeutic effects and the mechanical degradation of interstitial structures to enhance drug permeability, may also be induced.

[0074] With regard to compositions and methods for use of the present invention, it will be understood that this further irradiation of large activated bubbles by the application of low-frequency ultrasound further enhances the uptake of therapeutic agents or cells. Thus, it has been found that by using the application of low-frequency ultrasound close to the resonant frequency of large activated bubbles, a mechanical bioeffect mechanism can be generated to enhance vascular permeability and / or sonoporation and / or interstitial distribution and / or endocytosis, and therefore enhance therapeutic outcomes.

[0075] Applying sound fields corresponding to the resonant frequencies of bubbles with larger phase shifts generates relatively large radiated oscillations in MI within the medical diagnostic range. Therefore, applying low-frequency ultrasound in the range of 0.2–1 MHz, most preferably 0.4–0.6 MHz, can generate a bioeffect mechanism that enhances the uptake of administered drugs and thus promotes their overflow. Surprisingly, as demonstrated in Example 2, greater therapeutic benefits were found when irradiating activated bubbles with MI of 0.1–0.3, e.g., 0.2, and inducing enhanced uptake by applying ultrasound in the range of 0.4–0.6 MHz, e.g., 500 Hz, as used in the example. After activation in vivo, the volume-weighted average diameter of the activated bubbles was found to be approximately 20 μm. The resonant frequency of free microbubbles of this size is approximately 0.33 MHz. However, the resonant frequency of such bubbles is expected to be somewhat higher when trapped in microvessels. Therefore, the most preferred frequency range for the low-frequency enhancement process is 0.4–0.6 MHz. The utilization of the resonance effect of activated bubbles allows for better control over the initiation of these biological effects at lower acoustic intensities and frequencies than is possible with other techniques. The fact that large phase-shifted bubbles are activated under imaging control and deposit in the tissue microvascular system (enabling spatial targeting of large activated bubbles within tissue), coupled with their long residence time, enables more efficient and controlled implementation of drug delivery mechanisms.

[0076] Therefore, in one embodiment, this method includes an enhancement step (step iv) in which irradiation is performed at a second frequency in the range of 0.4 to 0.6 MHz. The MI for this enhancement step is preferably less than 0.3, greater than 0.1, and preferably greater than 0.15. If the MI applied during the enhancement step is less than 0.1, the resulting biomechanical effect is expected to be insufficient, and therefore the therapeutic benefit is expected to be significantly reduced. If the MI applied during the enhancement step is greater than 0.3, the resulting biomechanical effect is expected to be too strong, inducing undesirable effects, such as vascular damage or occlusion, and the therapeutic benefit is expected to be significantly reduced.

[0077] Irradiation with low-frequency ultrasound typically lasts for 3 to 10 minutes, for example, about 5 minutes, after the activation step. It is preferable to start step (iii) immediately after step (iv).

[0078] In the method of the present invention, when the phase shift of the diffusive component of the second component of the cluster composition is activated by ultrasonic irradiation of the region of interest within the subject, the microbubbles of the cluster are expanded by the diffusive component, resulting in expanded bubbles localized in the region of interest due to transient capture in microcirculation in the region of interest. Further ultrasonic irradiation at a low frequency (step iv) promotes the overflow of the administered therapeutic agent and the infiltration of activated immune cells into the target pathology. Thus, the method promotes enhanced overflow and, separately, pre-administered and / or simultaneously and / or post-administered therapeutic agent uptake and / or enhanced infiltration of activated immune cells into the target pathology. Thus, increased permeability of the vascular barrier, increased overflow of co-administered therapeutic agents and / or inflammatory cytokines and / or infiltration of activated immune cells are provided by the present invention.

[0079] Therefore, the present invention further relates to a microbubble / microdroplet cluster composition for use in a method for the overflow and uptake of at least one separately administered ITA and / or inflammatory cytokine and / or enhanced infiltration of activated immune cells into a target pathology of a mammalian subject, the method being: (i) A step of administering at least one immunotherapy agent (ITA) to the subject, (ii) A step of administering a microbubble / microdroplet cluster composition to a target, wherein at least one ITA is administered separately from the cluster composition, beforehand, and / or simultaneously, and / or afterward. (iii) A step of activating the phase shift of the diffusive component of the microdroplets of the cluster composition from step (i) by ultrasonic irradiation of a region of interest within the subject at a first frequency of 1 to 10 MHz and a first mechanical index of 0.1 to 0.4. (iv) Further irradiation with ultrasound at a second frequency of 0.4 to 0.600 MHz and a second mechanical index of 0.1 to 0.3. The present invention provides a microbubble / microdroplet cluster composition containing the following:

[0080] Similarly, the present invention provides a method comprising the steps described above for the exudation and uptake of at least one separately, pre- and / or simultaneously and / or post-administered ITA and / or for enhanced infiltration of activated immune cells into a target pathology in a mammalian subject. The steps and embodiments of the above method for improving or enhancing the exudation and uptake of at least one ITA or the infiltration of activated immune cells into a target pathology are disclosed with respect to a first embodiment relating to the treatment.

[0081] In some embodiments of these methods, it is not ITA itself that produces the therapeutic effect, but rather inflammatory cytokines (e.g., IL-1, IL-12, IL-18, TNFα, and INFγ) and / or activated immune cells (e.g., activated T cells, e.g., CD3, CD4, and CD8-positive cells), in which case ITA blocks receptors and allows immune cells to initiate the necessary cytotoxic processes against the pathological cells involved.

[0082] Cluster composition: In the method of the present invention, in addition to the separate administration of one or more immunotherapy agents, a pre-mixed cluster composition is administered to the subject. The administered clusters can be activated by ultrasound. The cluster composition is a pre-mixture of microbubbles (first component) and microdroplets (second component), resulting in small microbubble-microdroplet clusters held together by electrostatic force. The microdroplets typically contain an oil component having a boiling point below 50°C and low blood solubility. The cluster composition, i.e., the combination of the first and second components, contains clusters of gas microbubbles and oil microdroplets, i.e., a suspension or dispersion of individual microbubbles and microdroplets in the form of stable microbubble / microdroplet clusters. Analytical methods for the quantitative detection and characterization of the clusters are described in Example 1. In this document, the term "cluster" refers to a group of microbubbles and microdroplets permanently held together by electrostatic attraction in a single particle, an aggregated entity. The cluster content and size in the cluster composition are intrinsically stable for a certain period of time (e.g., >3 hours) after the first and second components are combined in vitro; that is, the clusters do not spontaneously disintegrate, form larger aggregates, or spontaneously activate (phase shift), and remain intrinsically stable for a certain period of time after dilution, even with continuous stirring. Therefore, it is possible to detect and characterize the clusters in the cluster composition using various analytical techniques that require dilution and / or stirring. Furthermore, the stability of the cluster composition allows for necessary clinical procedures (e.g., reconstitution, dose dispensing, and administration). The first and second components, as well as the cluster composition, are prepared in accordance with Good Manufacturing Practices (GMP).

[0083] In one embodiment, the cluster composition comprises a suspension of clusters in an aqueous biocompatible medium, wherein the clusters have an average diameter in the range of 1 to 10 μm and a circularity of less than 0.9. (i) A first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles, (ii) A second component comprising a microdroplet containing an oil phase and a second stabilizer for stabilizing the microdroplet, wherein the oil contains a diffusible component that can diffuse into the gas microbubbles to increase their size at least temporarily, The microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via electrostatic interaction attraction.

[0084] For example, by reconstituting a microbubble component, such as a freeze-dried microbubble component, with a microdroplet component in the form of an emulsion, the first and second components are combined (in vitro). After this combination, the cluster composition prepared according to the present invention exhibits stability in use suitable for its intended use and maintains stable properties over a suitable time frame for administration, for example, more than one hour, or preferably more than three hours, after the components have been combined. The cluster composition should be administered to the subject within this time frame.

[0085] Each cluster in the cluster composition contains at least one microbubble and one microdroplet, typically 2 to 20 individual microbubbles / microdroplets, and the clusters typically have an average diameter in the range of 1 to 10 μm and can flow freely within vascular compartments. They are further characterized by a circularity parameter, which separates them from the individual microbubbles and microdroplets. The circularity of a two-dimensional morphology (e.g., a projection of a microbubble, microdroplet, or microbubble / microdroplet cluster) is the ratio of the circumference of a circle having the same area as the morphology to the actual circumference of the morphology. Thus, a perfect circle (i.e., a two-dimensional projection of a spherical microbubble or microdroplet) has a theoretical circularity value of 1, while any other arbitrary geometric shape (e.g., a projection of a cluster) has a circularity of less than 1. The clusters of the present invention have a circularity of less than 0.9. The definition of the circularity parameter is further provided in International Publication No. 2015 / 047103.

[0086] According to the present invention, as shown in the examples, compositions comprising clusters defined as having an average size in the range of 1 to 10 μm, particularly 3 to 10 μm, and a circularity of less than 0.9 are considered particularly useful. In one embodiment, the average cluster diameter is in the range of 3 to 10 μm, preferably 4 to 9 μm, and more preferably 5 to 7 μm. Clusters of this size range flow freely within the vascular system before activation, are easily activated by US irradiation, and generate activated bubbles of sufficient size to temporarily accumulate and remain in the microvascular system, such as cancerous or inflammatory tissue. The microbubbles within the clusters enable efficient energy transfer of ultrasonic energy in the diagnostic frequency range (1 to 10 MHz), i.e., the ultrasonic energy at activation, and enable vaporization (phase shift) of emulsion microdroplets at low MI (preferably less than 0.4 but greater than 0.1), diffusion of the vaporized liquid into the microbubbles, and / or fusion between the vapor bubbles and the microbubbles. Subsequently, the activated bubbles expand further due to inward diffusion of the matrix gas (e.g., blood gas) to reach a volume-weighted median diameter of over 10 μm but less than 40 μm.

[0087] The formation of these clusters, i.e., the preparation of cluster compositions from the first and second components before administration, is a prerequisite for efficient phase-shift events, and their number and size characteristics have been found to be strongly related to the efficacy of the composition, i.e., its ability to form large activated (i.e., phase-shift) bubbles in vivo, and a prerequisite for its intended functionality in vivo. The number and size characteristics can be controlled through various formulation parameters, for example, but not limited to, the strength of the attractive force between the microbubbles of the first component and the microdroplets of the second component (e.g., the difference in surface charge between microbubbles and microdroplets); the size distribution of microbubbles and microdroplets; the ratio between microbubbles and microdroplets; and the composition of the aqueous matrix (e.g., pH, buffer concentration, ionic composition, and strength). When the cluster composition is prepared and administered, the average equivalent circle diameter of the clusters formed is preferably greater than 3 μm, more preferably between 5 and 7 μm, but less than 10 μm.

[0088] The cluster concentration in the combined preparation (cluster composition) should be greater than 3 million / mL, preferably greater than 10 million / mL, and more preferably greater than 20 million / mL. As shown in Example 1, the cluster composition for use according to the present invention had a cluster concentration of 400,000 to 44 million / mL with an average diameter of 5.8 to 6.2 μm, measured 0 to 3 hours after mixing the first and second components.

[0089] Figure 11 of the applicant's International Publication No. 2015047103 shows a correlation between in vivo enhancement of the ultrasound signal at injection (measured as an increase in grayscale units) and activation of the cluster composition with a variable concentration of 5-10 μm clusters. The observed level of GS enhancement is a direct measure of the amount of large activated bubbles generated and retained in the target tissue, and represents a direct measure of the potential for increased overflow and therapeutic benefit (i.e., product efficacy). In one embodiment, the composition for administration should contain at least 3 million clusters / mL with a diameter of 5-10 μm. According to Figure 11 of the applicant's International Publication No. 2015047103, the minimum value ensures an enhancement of >150 GS units, as well as a certain minimum level of product efficacy and therapeutic benefit. In another embodiment, the concentration of clusters in the size range of 1-10 μm should be at least about 10 million / mL, e.g., at least about 25 million / mL.

[0090] Accordingly, drug delivery to targeted overflow tissue and treatment using immunotherapeutic agents according to the present invention are achieved by the use of a two-component microbubble / microdroplet formulation system (i.e., cluster composition), in which microbubbles in the first component are physically bound to micron-sized emulsion microdroplets in the second component via electrostatic attraction before administration. The composition for use in the treatment method according to the present invention provides improved uptake of at least one ITA, resulting in beneficial treatment including, for example, reduction of tumor volume or complete remission. Accordingly, the present invention further provides a two-component formulation system for the preparation of a composition of microbubble / microdroplet clusters dispersed in an aqueous biocompatible medium, comprising the first and second components as disclosed above, for use in the method of the present invention.

[0091] The direct mechanism of action, i.e., the resulting mechanical and / or thermal bioeffects, increases the delivery and enhances the distribution of the therapeutic agent. However, it should be understood that the nature of these biomechanical effects is a direct result of the chemical attributes of the cluster composition, i.e., the chemical composition and properties of the clusters. For example, the lifetime of bubbles in an aqueous matrix is ​​inversely proportional to the solubility and diffusion coefficient of the gas in the matrix and proportional to the gas density. Therefore, bubbles made from heavy gases with low solubility and diffusivity are larger and have a longer lifetime than bubbles made from light gases with high solubility and diffusivity. As an example, a 5 μm perfluorobutane bubble lasts 500 times longer in water than a 5 μm air bubble. Therefore, the chemical composition of the microdroplet components governs the lifetime of the activated bubbles in vivo, and thus the level of biomechanical force that can be induced, and the level of therapeutic effect that can be achieved in the ACT procedure. Therefore, perfluoro oils are particularly useful for use in microdroplets of the second component because the gases from such droplets have very low water solubility and diffusivity, and high density.

[0092] When comparing the compositions and methods of the present invention with methods using freely flowing conventional contrast microbubbles, the large phase-shifted microbubbles generated in vivo from the administration clusters of the present invention are trapped in a region of a blood vessel, and the activated bubble surface is in close contact with the endothelium. In addition, the volume of the activated bubbles is typically 1000 times that of conventional microbubbles. At equal mechanical indices (MI) irradiated at frequencies close to the resonance of both bubble types (approximately 0.5 MHz for phase-shifted microbubbles and approximately 3 MHz for conventional contrast agent microbubbles), the absolute volume displacement during oscillation (i.e., the biomechanical force exerted) was shown to be three orders of magnitude greater for phase-shifted bubbles than for conventional contrast microbubbles. Therefore, as demonstrated in [Ng et al., Abstract A099: Acoustic Cluster Therapy enhances the efficacy of chemotherapeutic regimens in patient-derived xenograft mouse models for pancreatic ductal adenocarcinoma, AACR Conference on Molecular Targets and Cancer Therapeutics, Boston, October 2019], irradiation with phase-shifted bubbles produces a completely different level of biomechanical effects with significantly larger effect size and penetration depth than irradiation with conventional contrast-enhanced microbubbles. The biomechanical effects observed with freely flowing conventional contrast-enhanced microbubbles are likely dependent on the cavitation mechanism, which raises safety concerns such as microbleeding and irreversible vascular damage. However, larger phase-shifted bubbles from clusters can oscillate in a more flexible way (lower MI, e.g., <0.3) while avoiding the cavitation mechanism, but can still generate sufficient mechanical force to enhance drug uptake from the vascular system to target tissue. Capture of large phase-shifted bubbles also functions as a deposit tracer. This further enables the quantification of the number of activated clusters and tissue perfusion, allows contrast imaging of the tissue vascular system, and identifies the spatial extent of the tissue being treated.

[0093] The chemical composition of the administered clusters and the processes that occur during cluster activation are important for the cluster's effect. For example, the chemical properties of the encapsulated oil droplets affect the amount of activated bubbles deposited upon US irradiation, as well as their lifetime in vivo. The physicochemical properties of the oil, such as vapor pressure, boiling point, and water solubility, all correlate with the amount of activated bubbles deposited and the duration of their deposition. In C4-C6 homologous perfluorohydrocarbon chains, as water solubility and vapor pressure decrease and the boiling point increases, the amount and lifetime of activated bubbles increase with chain length. Furthermore, it should be noted that the large bubbles of activated clusters mechanically act on vascular cells, potentially generating biochemical signals and leading to increased uptake of therapeutic agents.

[0094] The cluster composition, and its first and second components, are designed to cluster and phase-shift in a controlled manner. The size of the activated bubbles (in vivo) can be designed by changing the different formulation parameters of the first and second components and the size characteristics of the prepared clusters (see Example 1).

[0095] When the target tissue is exposed to ultrasound, for example, at standard medical imaging frequencies and intensities, the microbubbles of the administered cluster composition transfer acoustic energy to attached oil microdroplets, which then act as seeds for evaporation or merge with the microbubbles, causing the oil to undergo a phase transition (vaporization) from liquid to gas. The resulting bubbles undergo an initial rapid expansion due to the vaporization of oil, followed by a slower expansion due to the inward diffusion of blood gases, temporarily blocking the microcirculation (meta-arterioles and capillary networks) for about 1 minute or more, preferably 2-3 minutes or more, most preferably 3-6 minutes or more. In the method of the present invention, or for pharmaceutical compositions for use, a therapeutic agent is further administered to the subject, for example, co-administered with the cluster composition, or pre-administered or post-administered. The clusters are activated to generate larger bubbles by applying external ultrasound energy, which are captured in the microvascular system of the target tissue (e.g., tumor or inflammatory site). Further application of low-frequency ultrasound after capture facilitates the overflow of the therapeutic agent or activated immune cells into the target tissue. Therefore, the main limitations of existing technologies in the limited exudation and uptake of immunotherapeutic agents and the infiltration of activated immune cells can be overcome by the technology of the present invention, as it has been found that the accessibility of therapeutic agents or activated cells to target tissue is significantly increased. Large activated bubbles temporarily remain in the microvascular system of the irradiated tissue, and by further application of low-power, low-frequency ultrasound, drug or cell uptake into the target tissue is promoted. Activated phase-shifted bubbles have a diameter about 10 times that of typical microbubbles, resulting in the following: - Transient deposition / trapping of activated bubbles in the microvascular system of targeted (i.e., irradiated) tissue; - Close contact between activated bubbles and the endothelium; - Compared to conventional contrast-enhanced microbubbles, the inertial cavitation mechanism is avoided, resulting in significantly larger biomechanical effects during post-activation ultrasound treatment.

[0096] The above attributes result in a significant enhancement of drug and immune cell overflow, distribution, and uptake.

[0097] The first component of the cluster composition: The first component comprises gas microbubbles and a first stabilizer for stabilizing the microbubbles. Thus, the first component is an aqueous medium for injection containing a dispersed gas and a material for stabilizing the gas. The microbubbles may be similar to conventional ultrasound contrast agents that are commercially available and approved for use in preclinical applications, e.g., Sonazoid®, Optison®, Definity®, or Sonovue®, or similar agents used for preclinical applications, e.g., Micromarker® and Polyson L®. Any biocompatible gas may be present in the gas dispersion. As used herein, the term “gas” includes any substance (including mixtures) in at least partially, for example, substantially or completely gaseous (including vapor) form at 37°C, the normal body temperature. Therefore, the gas may include, for example: air; nitrogen; oxygen; carbon dioxide; hydrogen; inert gases, e.g., helium, argon, xenon, or krypton; sulfur fluorides, e.g., sulfur hexafluoride, disulfur decafluoride, or trifluoromethylsulfur pentafluoride; selenium hexafluoride; optionally halogenated silanes, e.g., methylsilane or dimethylsilane; low molecular weight hydrocarbons (e.g., those containing up to 7 carbon atoms), e.g., alkanes, e.g., methane, ethane, propane, butane, or pentane; cycloalkanes, e.g., cyclopropane, cyclobutane, or cyclopentane; alkenes, e.g., ethylene, propene, propadiene, or butene; or alkynes, e.g., acetylene or propyne; ethers, e.g., dimethyl ether; ketones; esters; halogenated low molecular weight hydrocarbons (e.g., those containing up to 7 carbon atoms); or any mixture of the above. Preferably, the gas is a halogenated gas, more preferably a perfluorinated gas. Advantageously, at least some of the halogen atoms in the halogenated gas are fluorine atoms.Therefore, biocompatible halogenated hydrocarbon gases can be selected from, for example, bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethyl fluoride, 1,1-difluoroethane, and perfluorocarbons. Typical perfluorocarbons include perfluoroalkanes, such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (e.g., perfluoro-n-butane, optionally a mixture with other isomers such as perfluoro-iso-butane), perfluoropentane, perfluorohexane or perfluoroheptane; perfluoroalkenes; perfluoroalkynes; and perfluorocycloalkanes.

[0098] The use of perfluoroemission gases, such as sulfur hexafluoride, and perfluorocarbons, such as perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane, is particularly advantageous given the recognized high stability of microbubbles containing such gases in the bloodstream. In one embodiment, the gas of the first component is selected from the group of sulfur fluoride and halogenated low molecular weight hydrocarbons (e.g., those containing up to 7 carbon atoms). Other gases having physicochemical properties that allow for the formation of very stable microbubbles in the bloodstream may also be useful. Most preferably, the dispersion gas includes sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane (i.e., C3-6 perfluorocarbons), nitrogen, air, or a mixture thereof. More preferably, the dispersion gas includes sulfur hexafluoride, perfluoropropane, or perfluorobutane, or a mixture thereof. Even more preferably, the dispersion gas is perfluorobutane.

[0099] The dispersed gas may be in any convenient form, for example, any suitable gas-containing ultrasound contrast agent formulation, such as Sonazoid®, Optison®, Sonovue®, or Definity®, or a preclinical agent, such as Micromarker® or PolySon L®, may be used as the gas-containing component. The first component also includes a material referred to herein as the “first stabilizer” for stabilizing the microbubble dispersion. Typical examples of such formulations include a first stabilizer, such as microbubbles of gas stabilized (e.g., at least partially encapsulated) by a bonding-resistant surface film (e.g., gelatin), a membrane-forming protein (e.g., albumin such as human serum albumin), a polymer material (e.g., synthetic biodegradable polymer, elastic interface synthetic polymer film, microparticle biodegradable polyaldehyde, microparticle N-dicarboxylic acid derivatives of polyamino acid-polycyclic imide), a nonpolymer and nonpolymerizable wall-forming material, or a surfactant (e.g., polyoxyethylene-polyoxypropylene block copolymer surfactant, e.g., Pluronic, polymer surfactant, or film-forming surfactant, e.g., phospholipid). Preferably, the dispersed gas is in the form of phospholipid, protein, or polymer-stabilized gas microbubbles. Therefore, in one embodiment, the first stabilizer is selected from the group consisting of phospholipids, proteins, and polymers. Particularly useful first stabilizers are selected from the group of surfactants containing molecules with a net overall negative charge, such as naturally occurring (e.g., derived from soy or egg yolk), semi-synthetic (e.g., partially or fully hydrogenated), and synthetic phospholipids including phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, and / or cardiolipin. Alternatively, the phospholipid applied for stabilization may have an overall neutral charge and may be compounded with a negative surfactant, such as a fatty acid, for example, palmitic acid-added phosphatidylcholine, or a mixture of phospholipids with different charges, such as phosphatidylethanolamine and / or phosphatidylcholine and / or phosphatidic acid.Regarding the first stabilizer, that is, the stabilizer that stabilizes the microbubbles of the first component, various examples are shown in Example 5 of International Publication No. 2015 / 047103, and in Tables 9 and 10, and various microbubble formulations with different excipients have been tested. The results demonstrate that the ACT concept used in the present invention is applicable to a wide variety of microbubble formulations, even in terms of the composition of the stabilized membrane.

[0100] The size of the microbubbles of the dispersed gas component should preferably be less than 7 μm, more preferably less than 5 μm, and most preferably less than 3 μm, even within microbubble / microdroplet clusters, in order to facilitate their passage through the lung system without obstruction.

[0101] The second component of the cluster composition: The second component comprises microdroplets containing an oil phase and a second stabilizer for stabilizing the microdroplets, the oil containing a diffusible component. This diffusible component can diffuse into the gas microbubbles of the first component to increase their size, at least temporarily. With respect to the second component, the “diffusible component” is preferably a gas / vapor, a volatile liquid, a volatile solid, or a precursor thereof that can generate gas, for example upon administration, and the main requirement is that the component has or can generate sufficient gas or vapor pressure (e.g., at least 50 torr, preferably more than 100 torr) in vivo so that it can facilitate the inward diffusion of gas or vapor molecules into the dispersed gas. The “diffusible component” is preferably formulated as an emulsion (i.e., a stabilized suspension) of microdroplets in a suitable aqueous medium, because in such a system the vapor pressure of the diffusible component in the aqueous phase is substantially equal to the vapor pressure of the pure component material in the aqueous phase, even in a highly diluted emulsion.

[0102] The diffusible components in such microdroplets are advantageously liquid at processing and storage temperatures, which may be as low as -10°C, for example, when the aqueous phase contains a suitable antifreeze material, while at body temperature they are gaseous or exhibit substantial vapor pressure. Suitable compounds may be selected from a variety of emulsifiable low-boiling liquids given, for example, in International Publication A-9416379 or 2015 / 047103 of the patent application, the contents of which are incorporated herein by reference. Specific examples of emulsifiable diffusible components include aliphatic ethers, e.g., diethyl ether; polycyclic oils or alcohols, e.g., menthol, camphor, or eucalyptol; heterocyclic compounds, e.g., furan or dioxane; saturated or unsaturated, and potentially linear or branched aliphatic hydrocarbons; alicyclic hydrocarbons, e.g., cyclobutane, cyclobutene, methylcyclopropane, or cyclopentane; and halogenated low molecular weight hydrocarbons, e.g., halogenated low molecular weight hydrocarbons containing up to seven carbon atoms. Representative halogenated hydrocarbons include dichloromethane, methyl bromide, 1,2-dichloroethylene, 1,1-dichloroethane, 1-bromoethylene, 1-chloroethylene, ethyl bromide, ethyl chloride, 1-chloropropene, 3-chloropropene, 1-chloropropane, 2-chloropropane, and t-butyl chloride.Advantageously, at least a portion of the halogen atoms are fluorine atoms, for example, dichlorofluoromethane, trichlorofluoromethane, 1,2-dichloro-1,2-difluoroethane, 1,2-dichloro-1,1,2,2-tetrafluoroethane, 1,1,2-trichloro-1,2,2-trifluoroethane, 2-bromo-2-chloro-1,1,1-trifluoroethane, 2-chloro-1,1,2-trifluoroethyldifluoromethyl ether, 1-chloro-2,2,2-trifluoroethyldifluoromethyl ether, partially fluorinated alkanes (e.g., pentafluoropropane, such as 1H,1H,3H-pentafluoropropane) These are ropane, hexafluorobutane, nonafluorobutane, e.g., 2H-nonafluoro-t-butane, and decafluoropentane, e.g., 2H,3H-decafluoropentane, partially fluorinated alkenes (e.g., heptafluoropentene, e.g., 1H,1H,2H-heptafluoropenta-1-ene, and nonafluorohexene, e.g., 1H,1H,2H-nonafluorohexa-1-ene), and fluorinated ethers (e.g., 2,2,3,3,3-pentafluoropropyl methyl ether or 2,2,3,3,3-pentafluoropropyl difluoromethyl ether), preferably perfluorocarbons.Examples of perfluorocarbons include perfluoroalkanes, such as perfluorobutane, perfluoropentane, perfluorohexane (e.g., perfluoro-2-methylpentane), perfluoroheptane, perfluorooctane, perfluorononane, and perfluorodecane; perfluorocycloalkanes, such as perfluorocyclobutane, perfluorodimethylcyclobutane, perfluorocyclopentane, and perfluoromethylcyclopentane; perfluoroalkenes, such as perfluorobutene (e.g., perfluorobuta-2-ene or perfluorobuta-1,3-diene), perfluoropentene (e.g., perfluoropenta-1-ene), and perfluorohexene (e.g., perfluoro-2-methylpenta-2-ene or perfluoro-4-methylpenta-2-ene); perfluorocycloalkenes, such as perfluorocyclopentene or perfluorocyclopentadiene; and perfluorinated alcohols, such as perfluoro-t-butanol. Therefore, the second component oil (diffusible component) can be selected from the group consisting of aliphatic ethers, heterocyclic compounds, aliphatic hydrocarbons, halogenated low molecular weight hydrocarbons, and perfluorocarbons. In one embodiment, the oil phase of the second component contains a perfluorocarbon.

[0103] Particularly useful in this invention are 1.10 -4 Less than M, comfort level 1.10 -5 These are diffusible components with water solubility of less than M. However, it should be noted that when using mixtures of diffusible components and / or cosolvents, substantial fractions of the mixture may contain compounds with higher water solubility. Based on water solubility, examples of suitable oils (diffusible components) are perfluorodimethylcyclobutane, perfluoromethylsilopentane, 2-(trifluoromethyl)perfluoropentane, and perfluorohexane.

[0104] It will be understood that a mixture of two or more diffusible components may be used in accordance with the present invention, if necessary. References to “diffusible components” herein should be interpreted as including such mixtures.

[0105] The second component also includes a material referred to in this document as the “second stabilizer” to stabilize the microdroplet dispersion. The second stabilizer may be the same as or different from any material used to stabilize the gas dispersion, such as surfactants, phospholipids, polymers, or proteins. The properties of any such material can significantly affect factors such as the growth rate of the dispersed gas phase. Generally, a wide range of surfactants can be useful as stabilizers, and typical examples of useful surfactants include fatty acids (e.g., linear saturated or unsaturated fatty acids, e.g., those containing 10 to 20 carbon atoms) and their carbohydrates and triglyceride esters, phospholipids (e.g., lecithin), fluorine-containing phospholipids, proteins (e.g., albumin, e.g., human serum albumin), polyethylene glycol, and polymers, such as block copolymer surfactants (e.g., polyoxyethylene-polyoxypropylene block copolymer, e.g., Pluronics), elongated polymers, e.g., acyloxyacyl polyethylene glycol, e.g., polyethylene glycol methyl Examples include ether 16-hexadecanoyloxy-hexadecanoate, for example, those with a polyethylene glycol portion having a molecular weight of 2300, 5000, or 10000, and fluorine-containing surfactants (e.g., those commercially available under the trade names Zonil and Fluorad). Particularly useful surfactants include phospholipids, especially phospholipids containing molecules with an overall neutral charge, such as distearoyl-sn-glycerol-phosphocholine (DSPC). For the second component, various different stabilizers may be used to stabilize the microdroplets. Furthermore, a wide range of ionic, preferably cationic, substances may be used to facilitate a suitable surface charge.

[0106] It will be understood that, in order to facilitate electrostatic mutual attraction for achieving clustering between microbubbles of the first component and emulsion microdroplets of the second component, they should have opposite surface charges. Therefore, if the microbubbles of the first component are negatively charged, the microdroplets of the second component should be positively charged, and vice versa. In a preferred embodiment, the surface charge of the microbubbles of the first component is negative, and the surface charge of the microdroplets of the second component is positive. To facilitate appropriate surface charges for oil microdroplets, a cationic surfactant may be added to the stabilizing structure. A wide range of cationic substances, such as compounds having a basic nitrogen atom that are at least somewhat hydrophobic and / or substantially water-insoluble, e.g., primary, secondary, or tertiary amines and alkaloids, may be used. A particularly useful cationic surfactant is stearylamine. In one embodiment, the second stabilizer is a neutral phospholipid to which a cationic surfactant has been added, e.g., a DSPC film having stearylamine.

[0107] In one embodiment, the first stabilizer and the second stabilizer each independently include a phospholipid, a protein, a polymer, polyethylene glycol, a fatty acid, a positively charged surfactant, a negatively charged surfactant, or a mixture thereof. In particular, the first stabilizer includes a phospholipid, a protein, or a polymer to which a negatively charged surfactant is optionally added, and the second stabilizer includes a phospholipid, a protein, or a polymer to which a positively charged surfactant is optionally added.

[0108] In one embodiment, the first component comprises a dispersed gas selected from the group consisting of sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, nitrogen, and air or mixtures thereof, and is stabilized by a first stabilizer selected from the group consisting of phospholipids, proteins, and polymers. The second component comprises a diffusible component selected from the group consisting of perfluorocarbons, such as perfluorocycloalkanes, stabilized by a second stabilizer selected from the group consisting of surfactants, which include, for example, phospholipids, polymers, and proteins. More specifically, one of the stabilizers is selected from phospholipids.

[0109] The first and second components of a two-component formulation system are combined immediately before intended use to prepare a composition of microbubbles / microdroplets and to use within a suitable timeframe by the method of the present invention. It will also be understood that mixing of the first and second components can be achieved in various ways depending on the form of the components, for example, by mixing two fluid components, reconstituting one component in dry powder form with one component in fluid form, or by mixing two components in dry form and then reconstituting them with a fluid (e.g., water for injection or buffer). Accordingly, in one embodiment of the present invention, the method includes a step of preparing a microbubble / microdroplet cluster composition prior to the administration step (step ii). In a preferred embodiment, the microbubble / microdroplet cluster composition is prepared by reconstituting a first component (microbubbles) in dry powder form with a second component (microdroplets) in fluid form. More specifically, a first vial containing the first component is reconstituted with the second component, taken from a second vial, using a sterile, disposable syringe and needle. The contents of the syringe are added through the stopper of the first vial, and the resulting cluster composition is homogenized, for example, by manual mixing.

[0110] It will also be understood that other components may affect the ability of microbubbles and microdroplets to form clusters upon mixing, such as, but not limited to, the level of surface charge of microbubbles / microdroplets, the concentration of microbubbles / microdroplets in the two components, the size of microbubbles / microdroplets, the composition and concentration of ions in the liquid matrix, pH, and the composition and concentration of excipients (e.g., buffering or tensioning components) (see International Publication No. 2015 / 047103, Example 1). The characteristics of such components and compositions may also affect the size and stability of the resulting clusters (both in vitro and in vivo) and may be important factors influencing the biological attributes (e.g., efficacy and safety profile). It will also be understood that not all microbubbles / microdroplets in a cluster composition may exist in cluster form, and substantial portions of microbubbles and / or microdroplets may exist in a free (non-clustered) form together with the collection of microbubble / microdroplet clusters. In addition, the method by which the two components are mixed can affect these aspects, for example, the shear stress applied during homogenization (e.g., gentle manual homogenization or strong mechanical homogenization) and the time range for homogenization. The cluster composition should be administered to the subject within a time frame in which the properties of the cluster do not substantially change, for example, within 5 hours from the combination of the two components, for example, within 3 hours. The applicant's use stability tests have shown that the cluster exhibits stable properties for at least 3 hours. See Example 1.

[0111] The droplet size of the dispersed diffusible components in an emulsion intended for intravenous injection should preferably be less than 7 μm, more preferably less than 5 μm, most preferably less than 4 μm, and greater than 0.5 μm, more preferably greater than 1 μm, and most preferably greater than 2 μm, in order to facilitate unimpeded passage through the pulmonary system, while still maintaining sufficient volume for retaining activated bubbles in the microvascular system.

[0112] The growth of a dispersed gas phase in vivo may involve, for example, the expansion of any encapsulating material (if it is sufficiently flexible) and / or the extraction of excess surfactant from the administered material to the growing gas-liquid interface. However, the extension of the encapsulating material and / or the interaction of the material with ultrasound may substantially increase its porosity. While such fracture of the encapsulating material has been found to often result in a rapid decrease in echo intensity through outward diffusion and dissolution of the exposed gas, the inventors have found that when the composition is used according to the present invention, the exposed gas exhibits substantial stability. Without wishing to dwell on theoretical calculations, the inventors believe that, for example, the exposed gas in the form of free microbubbles may be stabilized against the collapse of microbubbles by a supersaturated environment generated by the diffusive component, which provides an inward pressure gradient to counteract the outward diffusion tendency of the microbubble gas. The substantial absence of encapsulating material allows the exposed gas surface to exhibit exceptionally favorable acoustic characteristics for activated bubbles, as evidenced by high backscattering and low energy absorption (e.g., high backscattering as expressed by the attenuation ratio) at typical diagnostic imaging frequencies: this echogenic effect can persist for considerable time, even while ultrasonic irradiation continues.

[0113] The ACT concept for the delivery of ITA and / or activated immune cells to target tissues, i.e., the compositions and methods for use of the present invention, are assumed to be applicable to a wide range of combinations of components (first and second components) and also to a wide range of immunotherapeutic agents in combination with chemotherapeutic agents of choice. Accordingly, any of the components listed with respect to the first component, including the gas and the first stabilizer, can be combined with the components listed with respect to the second component, including the diffusible component and the second stabilizer.

[0114] In summary, in one embodiment, a two-component formulation system for preparing microbubble / microdroplet cluster compositions for use by the method of the present invention is (i) A first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles, wherein the gas of the gas microbubbles is selected from the group of halogenated gases, preferably a perfluorinated gas, more preferably perfluorobutane; and the first stabilizer is selected from the group of phospholipids, proteins, and polymers, more preferably a phospholipid, and most preferably sodium hydrogenated egg phosphatidylserine (HEPS-Na), to which a negatively charged surfactant is optionally added. (ii) A second component comprising a microdroplet containing an oil phase and a second stabilizer for stabilizing the microdroplet, wherein the oil comprises a diffusible component that can diffuse into the gas microbubbles to at least temporarily increase their size, the oil being selected from the group of aliphatic ethers, heterocyclic compounds, aliphatic hydrocarbons, halogenated low molecular weight hydrocarbons and perfluorocarbons, preferably a perfluorocarbon, most preferably perfluoromethylcyclopentane (pFMCP); the second stabilizer being selected from the group of phospholipids, polymers and proteins, more preferably a phospholipid with a positively charged surfactant added, most preferably 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) with stearylamine (SA) added, and The microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via electrostatic interaction attraction.

[0115] Treatment drugs: The therapeutic agent or group of therapeutic agents, also called “drugs” or “drug group” for use in the present invention, is selected from the group of immunotherapeutic agents (ITAs), which are optionally combined with drugs from the group of chemotherapeutic agents. These or these are administered as a composition separate from the cluster composition. The therapeutic agent is administered according to standard treatment, i.e., according to the respective product description (SmPC).

[0116] Classes of ITAs include, but are not limited to, monoclonal antibodies (mAbs), fusion proteins, soluble cytokine receptors, recombinant cytokines, small molecule mimetic drugs, cell therapies, cancer vaccines, and oncolytic viruses. The term “immuno-oncological agents (IOs)” tends to be used as a general term to describe the group of ITAs collectively, and therefore, the classes of ITAs useful in this invention also include (IOs) as further described below.

[0117] Within the field of oncology, ITAs are used to enhance the immune system and attack cancer cells, while within the field of treating autoimmune diseases, their function is to suppress the autoimmune response that attacks healthy cells in the body.

[0118] Within the realm of cancer therapy, immunotherapy antibodies bind to tumor antigens (molecular targets), marking and identifying cancer cells for the immune system to inhibit or kill. Various classes of cancer immunotherapies target different tumor antigens. Below, the molecular target is indicated in parentheses after the drug name.

[0119] Immuno-oncology is a rapidly developing field, with drugs targeting previously unexplored antigens continuously entering preclinical and clinical development. Embodiments of the present invention involve the use of novel IO agents targeting all or any of the antigens named and identified (CD1-CD371) according to the Human Cell Differentiation Molecules (HCDM) Council nomenclature and to be identified and agreed upon during future Human Leukocyte Differentiation Antigen (HLDA) workshops.

[0120] Monoclonal antibodies (mAb): Immune checkpoint inhibitors (ICIs) are monoclonal antibody drugs that block proteins called checkpoints, which are produced by certain types of immune system cells, such as T cells, and some cancer cells. These checkpoints help prevent an excessive immune response and, at times, can prevent T cells from killing cancer cells. When these checkpoints are blocked, T cells can kill cancer cells more effectively. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 (antibody) / PD-L1 / PD-L2 (antigen) and CTLA-4 (antibody) / CD80 / CD86 (antigen). ICIs are used to treat a variety of cancerous diseases, including melanoma, lung cancer, kidney cancer, lymphoma, and urothelial carcinoma. ICIs, such as anti-PD-1 / PD-L1 drugs, interfere with the interaction between PD-L1 on tumor cells and PD-1 on T cells, allowing the immune system to initiate an anti-tumor response. Within the scope of the present invention, delivery of ICIs, particularly anti-PD1 agents or anti-PD-L1 / L2 agents, represents a preferred embodiment. Preferred agents within the scope of ICIs include, but are not limited to, ipilimumab (CTLA-4), nivolumab (PD-1), pembrolizumab (PD-1), atezolizumab (PD-L1), avelumab (PD-L1), durvalumab (PD-L1), and semiprimab (PD-1). In one embodiment, at least one ITA is an immune checkpoint inhibitor selected from the group consisting of nivolumab (PD-1), pembrolizumab (PD-1), atezolizumab (PD-L1), avelumab (PD-L1), durvalumab (PD-L1), and semiprimab (PD-1).

[0121] mAbs directed at other targets: Other IO agents targeting various internal and external targets (antigens) that represent preferred embodiments of the present invention include, but are not limited to, alemtuzumab (CD52), rituximab (CD20), tositumomab (CD20), obinotuzumab (CD20), ofatumumab (CD20), ibritumomab (CD20), dinutuximab (GD2), blinatumumab (CD19 / CD3), daratumumab (CD38), isatuximab (CD38), elotuzumab (SLAMF7), cetuximab (EGFR), panitumumab (EGFR), necitumumab (EGFR), and catumakisomab (Ep Examples include CAM, trastuzumab (HER2), pertuzumab (HER2), olaratumab (PDGF-Ra), bevacizumab (VEGF), ramucirumab (VEGF R2), imiquimod (TLR7), and tocilizumab (IL-R6).

[0122] Drug-conjugated mAbs: Another class of IO agents is represented by drug-antibody conjugates. These include, but are not limited to, preferred embodiments of the present invention, moxetumumab-pasdotox-tdfk (CD22), brentuximab-vedotin (CD30), trastuzumab-emtansine (HER2), inotuzumab-ozogamicin (CD22), gemtuzumab-ozogamicin (CD33), tagraxofusp-erzs (CD123), polatuzumab-vedotin-piiq (CD79B), elfortumab-vedotin-ejfv (nectin 4), tratuzumab-deruxtocan (HER2), and sacituzumab-govitecan-hziy (Trop 2).

[0123] Cytokine therapy: Cytokines are proteins produced by numerous types of cells present in tumors, often utilizing them to proliferate and reduce immune responses. These immunomodulatory effects make them suitable for use as drugs to induce immune responses.

[0124] Interleukin-2 and interferon-α are cytokines, which are proteins that regulate and coordinate the behavior of the immune system, and represent embodiments of the present invention. They have the ability to enhance antitumor activity and can therefore be used as passive cancer treatments. Interferon-α is used in the treatment of hairy cell leukemia, AIDS-associated Kaposi's sarcoma, follicular lymphoma, chronic myeloid leukemia, and malignant melanoma. Interleukin-2 is an approved treatment for malignant melanoma and renal cell carcinoma.

[0125] Cell therapy: The rationale for CAR-T and other cell-based immunotherapies is to modulate immune cells to recognize cancer cells in order to more effectively target and destroy them. For example, T cells are collected from a patient, genetically modified to add chimeric antigen receptors (CARs) that specifically recognize cancer cells, and then injected back into the patient to attack the tumor. Agents in this classification that represent preferred embodiments of the present invention include, but are not limited to, ciproisel-T (Provenge), tisagenlecleucel (Kymriah), and axicapbutazine-silolucel (Yescarta).

[0126] Oncolytic viruses: Oncolytic viruses are viruses that preferentially infect and replicate in cancer cells. Infected cancer cells are destroyed by oncolysis, releasing new infectious viral particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought to not only cause direct destruction of tumor cells but also stimulate a host anti-tumor immune response to long-term immunotherapy. Oncolytic viruses can also be used as vectors for transgene delivery to cancer cells, enabling localized expression of transgene products. Such encoding products include antibody fragments, bispecific antibodies, T-cell mating ligands, secreted immunomodulators, or other immunoisotopes.

[0127] Numerous viruses, including adenoviruses, reoviruses, measles viruses, herpes simplex viruses, Newcastle disease viruses, and vaccinia viruses, have been clinically tested as oncolytic agents, and several are in clinical development. T-Vec (tarimodine-rherparepvec) is currently the only FDA-approved oncolytic virus (for the treatment of melanoma). However, many other oncolytic viruses, including, but not limited to, Ad2 / 5 dl1520 (Onyx-015), GLV-1h68 (GL-ONC1), and CV706, are in Phase II-III development. The use of oncolytic viruses represents a preferred embodiment of the present invention.

[0128] Cancer vaccines: Therapeutic cancer vaccines are vaccines that treat existing cancers. Antigens found on the surface of cells are substances that the body considers harmful. The immune system attacks antigens and, in most cases, eliminates them. This leaves a "memory" in the immune system, which helps it fight those antigens in the future. Cancer treatment vaccines enhance the immune system's ability to find and destroy antigens. In many cases, cancer cells have specific molecules called cancer-specific antigens on their surface that healthy cells do not have. When a vaccine gives a person these molecules, the molecules function as antigens. The molecules instruct the immune system to find and destroy cancer cells that have these molecules on their surface.

[0129] Two therapeutic cancer vaccines are currently used in clinical practice: Bacillus Calmette-Guélain (BCG) for the treatment of early bladder cancer and Ciproisel-T for the treatment of prostate cancer. Furthermore, Oncophage® is approved in Russia for the treatment of bladder cancer. Various other vaccines are currently in clinical development. The use of cancer vaccines represents preferred embodiments of the present invention.

[0130] New IO appearances: Anti-CD47 therapy: Many tumor cells overexpress CD47, evading the immune surveillance of the host immune system. CD47 binds to its receptor, signal regulatory protein alpha (SIRPa), downregulating the phagocytosis of tumor cells. Therefore, anti-CD47 therapy aims to restore tumor cell clearance. In addition, increasing evidence supports the use of tumor antigen-specific T cell responses in response to anti-CD47 therapy. Numerous therapeutic agents are under development, including anti-CD47 antibodies, genetically modified decoy receptors, anti-SIRPa antibodies, and bispecific agents. The use of anti-CD47 therapeutic agents represents a preferred embodiment of the present invention.

[0131] Anti-GD2 Antibodies: Carbohydrate antigens on the surface of cells can be used as targets for immunotherapy. GD2 is a ganglioside found on the surface of numerous types of cancer cells, including neuroblastoma, retinoblastoma, melanoma, small cell lung cancer, brain tumors, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, liposarcoma, fibrosarcoma, leiomyosarcoma, and other soft tissue sarcomas. GD2 is not normally expressed on the surface of normal tissues, which makes GD2 a good target for immunotherapy. The use of anti-GD2 agents represents a preferred embodiment of the present invention.

[0132] Anti-TIM3: Recent studies have highlighted the crucial role TIM3 plays in T cell depletion and its correlation with anti-PD-1 therapy outcomes. Targeting TIM3 may be a promising approach to cancer immunotherapy. The use of anti-TIM3 agents represents a preferred embodiment of the present invention.

[0133] Anti-LAG3: LAG3 is an ICI with multiple biological effects on T cell function. LAG3 is highly expressed in various types of tumor-infiltrating lymphocytes and participates in tumor immune evasion mechanisms. For this reason, LAG3 is currently being clinically explored as an indicator of tumor prognosis and as a targeted tumor therapy. The use of this LAG3 agent represents a preferred embodiment of the present invention.

[0134] Very often, an IO regimen includes a combination therapy in which one or more chemotherapeutic agents are administered in combination with an IO agent. A preferred embodiment of the present invention is the application of such a combination regimen. A list of preferred chemotherapeutic agents is enumerated below.

[0135] Alkylating agent: Nitrogen mustards: Mechloretamine hydrochloride Nitrosoureas: carmustine, streptozocin, lomustine Tetrazines: Dacarbazine, Temozolomide Aziridines: Thiotepa, mitomycin, aziridinylbenzoquinone Cisplatins: cisplatin, carboplatin, oxaliplatin antimetabolites Antifolic acid drugs: methotrexate, pemetrexed Fluoropyrimidines: Fluorouracil, capecitabine Deoxyribonucleotide analogs: cytarabine, decitabine, azacitidine, gemcitabine, fludarabine, nelarabine, pentostatin Thiopurines: Thioguanine, mercaptopurine Antimicrotubule agents: Vinca alkaloids: vinorelbine, vinicristine, vindesine, vinflunin Taxanes: Paclitaxel or nab-paclitaxel, cabazitaxel, docetaxel Podophyllotoxin; etoposide, teniposide Topoisomerase inhibitors Topoisomerase I: Irinotecan or liposomal irinotecan, topotecan Topoisomerase II: Doxorubicin or liposomal doxorubicin, mitoxantrone, teniposide, nobiosin, melbaron, acralubicin cytotoxic antibiotics Anthracyclines: Doxorubicin, daunorubicin, epirubicin, idarubicin, bleomycin, mitomycin

[0136] In addition to the use of ITA according to the present invention in oncology, other major areas of use are, but are not limited to, for the treatment of autoimmune diseases, including: psoriasis, lupus, rheumatoid arthritis, Crohn's disease, multiple sclerosis, and alopecia areata. This class of agents is also frequently used to avoid organ rejection after transplantation. The pathological conditions treated according to the methods of the present invention are preferably cancer or autoimmune diseases.

[0137] Immunotherapy for the treatment of autoimmune diseases is a rapidly developing field, with drugs targeting previously unexplored antigens continuously entering preclinical and clinical development. Embodiments of the present invention involve the use of novel ITAs targeting all or any of the antigens named and identified (CD1-CD371) according to the Human Cell Differentiation Molecules (HCDM) Council nomenclature and to be identified and agreed upon during future Human Leukocyte Differentiation Antigen (HLDA) workshops. Accordingly, in one embodiment, at least one ITA for use in the method of the present invention is selected from ITAs having the ability to target any of the antigens named CD1-CD371.

[0138] Preferred ITAs for the treatment of autoimmune diseases under the present invention include, but are not limited to, the following: Small molecule inhibitors, such as prednisone, budesonide, prednisolone, tofacitinib, cyclosporine, tacrolimus, sirolimus, everolimus, azathioprine, leflunomide, and mycophenolic acid.

[0139] Monoclonal antibodies and other biologics, such as abatacept (CD80 and CD86), adalimumab (TNFα), anakinra (IL-1), certolizumab (TNFα), etanercept (TNFα), golimumab (TNFα), infliximab (TNF), ixekizumab (IL17A), natalizumab (α-4 integrin), rituximab (CD20), secukinumab (IL17A), tocilizumab (IL-6), ustekinumab (IL-12 and IL-23), vedolizumab (integrin a4b7), basiliximab (CD25), and daclizumab (CD25). These monoclonal antibodies and biologics represent preferred embodiments of the present invention.

[0140] Therefore, in one embodiment, the immunotherapy agent is selected from the group consisting of IOs, monoclonal antibodies (mAbs), fusion proteins, soluble cytokine receptors, recombinant cytokines, small molecule mimetic drugs, cell therapies, cancer vaccines, and oncolytic viruses.

[0141] In another embodiment, one or more ITAs are for use in combination therapy, in which one or more chemotherapeutic agents are given in combination with one or more ITAs. Thus, a treatment using one or more immunotherapeutic agents is combined with a treatment using one or more chemotherapeutic agents, for example, for the treatment of cancer, and the chemotherapeutic agents are selected from an unspecified group including alkylating agents, antimetabolites, antimicrotubule agents, topoisomerase inhibitors, anthracyclines, and cytotoxic antibiotics.

[0142] In another embodiment, the immunotherapy agent has a molecular weight greater than 15,000 daltons, preferably greater than 30,000 daltons, more preferably greater than 50,000 daltons, and most preferably greater than 100,000 daltons. In some embodiments, the immunotherapy agent has a molecular weight of up to 1,500 daltons.

[0143] The compositions and methods of use of the present invention may be useful for autoimmune diseases or cancer, or at sites of inflammation (e.g., joints). In one embodiment, the compositions and methods of use / treatment are for the treatment of any of the following cancers: local pathological lesions, such as solid tumors, or metastatic cancers, such as melanoma, sarcoma, prostate cancer, colon cancer, anal cancer, esophageal cancer, gastric cancer, rectal cancer, small intestine cancer, liver cancer, pancreatic cancer, lung cancer, kidney cancer, breast cancer, brain cancer, bile duct cancer, head and neck cancer, lymphoma, urothelial carcinoma, adrenocortical carcinoma, Merkel cell carcinoma, parathyroid cancer, paraganglioma, pheochromocytoma, pituitary tumor, thyroid cancer, bladder cancer, penile cancer, testicular cancer, cervical cancer, endometrial cancer, fallopian tube cancer, gestational trophoblastic tumor, ovarian cancer, peritoneal cancer, vaginal cancer, and vulvar cancer.

[0144] In one embodiment, the composition and method of use / treatment are for the treatment of any of the following autoimmune diseases: psoriasis, lupus, rheumatoid arthritis, Crohn's disease, multiple sclerosis, and alopecia areata.

[0145] In one embodiment, pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC), is abandoned from the pathological condition treated by the method of the present invention.

[0146] In another embodiment, the composition and method of use / treatment are for post-organ transplantation treatment.

[0147] In one embodiment, any of the following monoclonal antibodies are discarded from the use and methods of the present invention: bevacizumab, cetuximab, ipilimumab, ofatumumab, ocrelizumab, panitumab, and rituximab.

[0148] In one embodiment, the therapeutic agent is formulated in a vehicle and, for example, in the form of liposomes, conjugates, nanoparticles, or microspheres used as a vehicle for the therapeutic agent. Thus, the therapeutic agent may be part of a larger drug construct such as a nanodrug, for example, in a liposome formulation or particle formulation, or as part of a monoclonal antibody. Thus, in one embodiment, the therapeutic agent is formulated in a closed lipid sphere, for example, containing the therapeutic agent in a liposome formulation, or formulated in a polymer micelle.

[0149] As demonstrated by Examples 3 and 4, the ACT concept may be particularly useful for combinations with larger drug molecules or constructs.

[0150] Furthermore, a combination regimen between one or more chemotherapeutic agents and one or more immunotherapeutic agents is preferred. Moreover, a newer generation of liposomes containing two types of anticancer drugs, each containing a single liposome, and immunoliposomes containing antibodies conjugated to liposomes are also included in the present invention.

[0151] In a second preferred embodiment, the therapeutic agent is an immunotherapeutic agent selected from the group of oncolytic viruses, including, but not limited to, adenoviruses, reoviruses, measles viruses, herpes simplex viruses, Newcastle disease viruses, and vaccinia viruses. The virus can cause cancer cells to "burst," killing them and releasing cancer antigens. These antigens can then stimulate an immune response that can locate and eliminate any remaining tumor cells nearby and potentially elsewhere in the body.

[0152] In another embodiment, several therapeutic agents, including at least one ITA, are administered as a combination regimen. Examples of suitable combination regimens, but are not limited to, include the following: 1) A combination regimen including pembrolizumab followed by paclitaxel and carboplatin, and 2) Combination regimens including nivolumab, followed by ipilimumab.

[0153] Therefore, in one embodiment of the method, the ITA is selected from the group of monoclonal antibodies anti-PD1, anti-PDL1, or CTLA4 and used in combination with a chemotherapeutic agent, for example, pembrolizumab in combination with cisplatin + oxaliplatin or capecitabine.

[0154] Disclaimer: In one embodiment, the chemotherapeutic agent is not paclitaxel, i.e., in its free, albumin-unbound form. In one embodiment, the therapeutic agent is not a combination of gemcitabine and nab-paclitaxel.

[0155] Disclaimer: In one embodiment, the disease being treated is not pancreatic cancer.

[0156] Disclaimer: In one embodiment, the immunotherapy agent does not target CTLA-4, CD-20, VEGF, or EGF.

[0157] Route of administration: The cluster composition is administered parenterally, preferably intravenously, to the mammalian subject. The route of administration may also be selected from intra-arterial, intramuscular, intraperitoneal, intratumoral, or subcutaneous. For administration to the subject, the therapeutic agent is administered separately from the cluster composition, as a separate composition, before, and / or simultaneously, and / or afterward. The therapeutic agent is administered according to the approved product description. Typically, the route is selected from the group including, but is not limited to, intravenous, intraperitoneal, intratumoral, and intramuscular administration. Thus, the two compositions, namely the cluster composition (a) and the therapeutic agent composition (b), may be administered via the same route of administration or via different routes of administration.

[0158] Treatment schedule: It will be understood that the compositions, treatment methods, and / or methods for drug delivery for use of the present invention may be used, for example, as part of a multi-drug treatment regimen. In one embodiment of the present invention, this involves the use of two or more therapeutic agents. Such a chemotherapy combination regimen may include, for example, paclitaxel and cisplatin in addition to at least one ITA, for example, pembrolizumab.

[0159] The applicant has surprisingly found that perfusion patterns in target tissue (e.g., tumors) can change on short timescales. US imaging studies have demonstrated that the deposition patterns of activated bubbles change significantly with each injection within the same target area and region of interest, likely due to temporal variations in perfusion patterns. This change translates to therapeutic benefits when the target tissue region is treated multiple times to enhance overflow within the overall tissue volume. Furthermore, treatment regimens often involve the administration of several different therapeutic agents at different time points within the regimen. This, too, translates to the benefit of performing ACT treatments several times to provide enhanced overflow of all agents.

[0160] Therefore, in one embodiment, several ACT treatments may be performed during the period of therapeutic agent administration, for example, as illustrated in Figures 11 and 12. In one embodiment, the treatment method comprises 1 to 5 ACT treatments, for example, 2 to 4 ACT treatments. An "ACT treatment" or "ACT procedure" includes at least the administration of a cluster composition, activation of the cluster by conventional medical imaging US irradiation, and subsequent low-frequency US irradiation to induce enhanced uptake, i.e., as described in method steps ii), iii) and iv). Figures 11 and 12 provide examples of treatment schedules in which nivolumab and ipilimumab, and pembrolizumab combinations, followed by paclitaxel and carboplatin, are used. Other ITAs that may be combined with chemotherapeutic agents as disclosed herein may also be used with one or more ACT treatments.

[0161] Figure 11 provides a graph of possible ACT procedures for use according to the present invention, performed during treatment with a combination regimen including a 30-minute infusion of nivolumab followed by a 90-minute infusion of ipilimumab. The ACT procedure is applied three times during administration, as indicated by the gray ACT® sonoporation bars.

[0162] Panel A in Figure 11: The ACT procedure is, a.: Injection of cluster composition, b.: Activation of clusters accompanied by 60 seconds of standard medical imaging ultrasound irradiation, and • c.: Enhancement process with 5 minutes of 400-600 kHz ultrasound irradiation at MI of 0.1-0.3 It consists of.

[0163] In panel B of Figure 11, the y-axis shows the plasma concentration of the administered therapeutic agent as a percentage of the peak, and the x-axis shows the time in minutes. In this example, three ACT procedures are performed at approximately 30 minutes, 80 minutes, and 120 minutes to show treatment of the entire area of ​​interest, including both drugs.

[0164] Figure 12 provides a graph of possible ACT treatments during treatment with a combination regimen including standard therapy-combined immunotherapy + chemotherapy regimen; pembrolizumab followed by paclitaxel and carboplatin for the treatment of metastatic squamous cell non-small cell lung cancer.

[0165] Panel A of Figure 12: The ACT procedure as detailed in Figure 11. Panel B of Figure 12: The y-axis shows the plasma concentration of the administered therapeutic agent as a percentage of the peak, and the x-axis shows the time in minutes. In this example, three ACT procedures are performed at approximately 160 minutes, 200 minutes, and 240 minutes to show treatment of the entire area of ​​interest, including all three drugs.

[0166] The inventors found that repeating the ACT procedure multiple times is beneficial even when a single therapeutic agent is administered. Using US imaging during ACT activation, the inventors observed a noteworthy effect on the deposition of ACT bubbles in the tumor. Strong variability in the deposition pattern from injection to injection was observed in the same animals, with the density of deposited ACT bubbles differing across different segments of the tumor, and this pattern changing between injections. Although not fully elucidated, these effects are hypothesized to be due to temporal variations in perfusion across different tumor segments. Based on these observations, in the examples, the inventors applied the ACT procedure three times consecutively to reach as much tumor volume as possible. This also points to the advantages of applying several ACT procedures during clinical use, as described above for the regimens visualized in Figures 11 and 12.

[0167] Therefore, in one embodiment, more than one therapeutic agent, for example, 1 to 5 therapeutic agents, are administered simultaneously or sequentially over a specific time span, for example, up to 3 hours, while at least one, for example, 1 to 5 ACT treatments (ACT procedures) are performed during the same period.

[0168] In one embodiment, the ACT procedure is provided comprising the following steps: administration of a cluster composition, e.g., intravenous administration, followed by local ultrasound irradiation of the tissue of interest region using conventional medical imaging (US) (activation), then low-frequency ultrasound irradiation to induce enhanced exudation of the drug and activated immune cells. These steps are performed 2 to 5 times consecutively, for example, 3 times consecutively. Thus, steps (i) to (iv) are repeated 1 to 4 times. These steps are performed in conjunction with the administration of one or more therapeutic agents, including at least one ITA. Activation, i.e., the initial US irradiation, should be initiated before or immediately after each administration of the cluster composition, for example, within 20 seconds, and should last for, for example, 30 to 120 seconds. Irradiation with low-frequency ultrasound follows the activation step and typically lasts for 3 to 10 minutes, for example, about 5 minutes. Preferably, step (iv) is initiated immediately after step (iii). Dual-frequency transducers may be usefully used in the procedure for both the activation and enhancement steps. Such use allows for a seamless transition from activation irradiation in step (iii) to enhancement irradiation in step (iv). Applying the enhancement field immediately after activation may be important for the therapeutic benefits obtained. In this regard, it would be beneficial to apply both activation and enhancement irradiation using a broadband or dual-frequency US transducer. That is, a transducer capable of delivering sufficient US pressure (i.e., MI) across all frequencies required within the indicated preferred range. For example, a transducer capable of delivering up to 0.4 MI at both 1–10 MHz and 0.1–1 MHz, more preferably at 0.4–0.6 MHz.

[0169] Furthermore, it may be beneficial to apply the activation and enhancement fields simultaneously or intermittently using transducers capable of emitting both fields simultaneously or in short time sequences (e.g., an activation field for 1 second, followed by an enhancement field for 1 second, followed by another activation field for 1 second, etc.).

[0170] In another embodiment, the multi-drug regimen includes an anti-PD1, anti-PDL1, or CTLA4 monoclonal antibody and a chemotherapeutic agent, such as pembrolizumab + cisplatin + oxaliplatin or capecitabine. Therefore, several therapeutic agents can be used in the treatment regimen, and several ACT procedures can be applied. In a preferred embodiment, the ACT procedure is performed when the active therapeutic molecule reaches or near-maximum concentration in the blood after administration. Therefore, the timing of the ACT procedure may vary depending on the pharmacokinetics of the therapeutic agent.

[0171] In one embodiment, the pharmaceutical composition of the present invention is intended for use in delivering a therapeutic agent to a target tissue, particularly to a subject diagnosed with cancer or autoimmune disease. The composition for use provides site-specific delivery of the therapeutic agent to achieve an effective local concentration of the therapeutic agent using ACT technology, and further provides improved uptake of the therapeutic agent and activated immune cells in the region of interest.

[0172] Therefore, the present invention also provides a microbubble / microdroplet cluster composition for use in a method of local delivery of at least one ITA to a target, the method being: (i) A step of administering at least one ITA to the subject, (ii) A step of administering a cluster composition to a subject, wherein at least one therapeutic agent is administered to the cluster composition before, and / or simultaneously with, and / or after. (iii) A step of activating the phase shift of the diffusive component of the microdroplets of the cluster composition from step (i) by ultrasonic irradiation of a region of interest within the subject at a first frequency of 1 to 10 MHz and a first mechanical index of 0.1 to 0.4. (iv) Further irradiation with ultrasound at a second frequency of 0.4 to 0.6 MHz and a second mechanical index of 0.1 to 0.3. The present invention provides a microbubble / microdroplet cluster composition containing the following:

[0173] Similarly, the present invention is (i) A step of administering at least one ITA to the subject, (ii) A step of administering a microbubble / microdroplet cluster composition to a target, wherein at least one ITA is administered to the cluster composition before, and / or simultaneously with, and / or after. (iii) A step of activating the phase shift of the diffusive component of the microdroplets of the cluster composition from step (i) by ultrasonic irradiation of a region of interest within the subject at a first frequency of 1 to 10 MHz and a first mechanical index of 0.1 to 0.4. (iv) Further irradiation with ultrasound at a second frequency of 0.4~0.6 Hz and a second mechanical index of 0.1~0.3. This provides a method for delivering at least one ITA to a mammalian target, including [specific example].

[0174] With regard to this composition for use and a method for delivering at least one ITA, the steps of the method may optionally include a step (iib) of imaging the cluster using ultrasound imaging to identify a region of interest for treatment within the subject.

[0175] The use and method promotes the enhanced overflow and uptake of ITA administered separately beforehand, and / or simultaneously, and / or afterward, and / or the enhanced infiltration of activated immune cells into the target pathology. In the method, the therapeutic agent is administered to the cluster composition beforehand, and / or simultaneously, and / or afterward, and before steps ii) to iv) or after any of steps ii) to iv).

[0176] In some embodiments, only a cluster composition without a therapeutic agent is administered to the subject for the purpose of preparing the subject for later administration of the therapeutic agent. In such embodiments, the administration of the cluster composition is not a treatment, but rather preparation for a treatment, for example, preparation for a treatment by ITA.

[0177] The methods and compositions for use of the present invention, as disclosed herein, may involve diagnostic imaging, such as ultrasound imaging, including the use of cluster microbubbles as a contrast agent, but can typically be combined with other types of imaging studies to diagnose and / or evaluate the outcome of a procedure. Such imaging may include, for example, abdominal computed tomography (CT) or abdominal magnetic resonance imaging (MRI), abdominal ultrasonography (US), and endoscopic ultrasound (EUS) as initial tests to identify target pathologies.

[0178] The present invention is not limited to the embodiments and examples shown. While various embodiments of the disclosure are described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Several modifications and changes, as well as variations and substitutions, to the embodiments described herein will be apparent to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments described herein can be used when carrying out the disclosure.

[0179] It should be understood that all embodiments disclosed in one aspect are equally and fully applicable to other aspects. Therefore, for example, features disclosed with respect to compositions for therapeutic use are also applicable to methods of local delivery as well as methods for increasing the uptake and enhanced infiltration of activated immune cells.

[0180] Each embodiment of this disclosure may, at its discretion, be combined with one or more of the other embodiments described herein.

[0181] It should be understood that each component, compound, or parameter disclosed herein is disclosed for use alone or in combination with one or more other components, compounds, or parameters disclosed herein. Furthermore, each quantity / value or range of quantity / value of each component, compound, or parameter disclosed herein is also disclosed in combination with any other quantity / value or range of quantity / value disclosed herein. Therefore, it should be understood that any combination of quantities / values ​​or ranges of quantity / value disclosed for two or more components, compounds, or parameters disclosed herein is also disclosed in combination with each other for the purposes of this description. Any and all features described herein, and any combination of such features, are included within the scope of the invention, provided that the features are not contradictory to each other.

[0182] It should be understood that each lower limit of each range disclosed herein should be interpreted as being disclosed in combination with each upper limit of each range disclosed herein for the same component, compound, or parameter. Accordingly, a disclosure of two ranges should be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. A disclosure of three ranges should be interpreted as a disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, and so on. Furthermore, any specific amount / value of a component, compound, or parameter disclosed herein or in the examples should be interpreted as a disclosure of either a lower or upper limit of a range, and thus can be combined with any other lower or upper limit, range, or specific amount / value of the same component, compound, or parameter disclosed elsewhere in this application to form a range for that component, compound, or parameter.

[0183] In the context of one embodiment, embodiments and features described, for example, with respect to an embodiment relating to a composition for use in therapy, also apply to other embodiments of the present invention relating to use in delivery or in a method for treatment or delivery.

[0184] The following embodiments are provided to illustrate the present invention in accordance with the principles of the present invention, but should not be construed as limiting in any way. [Examples]

[0185] (Example 1) Cluster preparation, analytical tools, and basic characteristics International Publication No. 2015 / 047103, the applicant's application, in particular Examples 1 and 2, which are summarized herein by reference, provide a description of analytical methods for the properties of cluster compositions arising from the use of clusters, etc.

[0186] Hereinafter, the first component is denoted as C1, the second component as C2, and the cluster composition, i.e., the composition resulting from the combination of the first and second components, is denoted as DP (pharmaceutical). The microbubble / microdroplet clusters formed when C1 and C2 are combined, i.e., present in the DP, are important quality attributes of the composition, namely its functionality for drug delivery. Therefore, analytical methodologies for characterizing and controlling the formed clusters with respect to concentration and size are essential tools for evaluating the present invention and for pharmaceutical quality control (QC). Three different analytical tools applicable to this purpose have been identified: Coulter counting, flow particle imaging (FPIA), and microscopy / image analysis.

[0187] In addition to these techniques applied to the characterization of clusters in cluster compositions, analytical methodologies have been developed to investigate the activation of clusters in vitro, i.e., the generation of large activated bubbles upon ultrasonic irradiation. This methodology, "sonometry," is described in detail in International Publication No. 2015 / 047103, E1-6. The primary report response from sonometric analysis includes decay spectra, as well as the number and volume of activated bubbles and their size distribution, both against time after activation. The activation response may also be explored by microscopy / image analysis, as detailed in International Publication No. 2015 / 047103, E1-5.

[0188] Ingredients and composition: The first component (C1) in the compositions investigated in the included examples consisted of perfluorobutane (PFB) microbubbles embedded in lyophilized sucrose, stabilized by a hydrogenated egg phosphatidylserine sodium (HEPS-Na) membrane. HEPS-Na possesses negatively charged head groups that ensure a negative surface charge for the microbubbles. Each vial of C1 contains approximately 16 μL or 2-10 9 It contains microbubbles, with an average diameter of approximately 2.0 μm. The freeze-dried formulation exhibits a long shelf life, particularly 3 years, when stored at room temperature.

[0189] The second component (C2) in the composition investigated in this example consisted of perfluoromethylcyclopentane (pFMCP) microdroplets stabilized by a 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) membrane with 3% mol / mol of stearylamine (SA) added to provide a positive surface charge. The microdroplets in C2 were dispersed in 5 mM Tris buffer. The standard formulation of C2 investigated in this test was approximately 4 μL or 0.8-10 μL per 1 ml. 9 It contains microdroplets with an average diameter of approximately 1.8 μm. The second component exhibits a long shelf life under refrigeration, particularly 18 months or more.

[0190] In some cases, various formulation variables, such as SA content, microdroplet size, microdroplet concentration, TRIS concentration, and pH, were varied in a controlled manner to elucidate their effects on cluster characteristics. These modifications, when using such samples, are described in this document.

[0191] The cluster composition (DP) was aseptically prepared by reconstituting the C1 vial with 2 mL of C2, followed by manual homogenization for 30 seconds. 2 mL was withdrawn from the C2 vial using a sterile, disposable syringe and needle. The contents of the syringe were added through the stopper of the C1 vial, and the resulting DP was homogenized to prepare the administration composition. The prepared cluster composition is also referred to herein as PS101.

[0192] As shown in International Publication No. 2015 / 047103, the first and second components, namely the microbubble formulation and the microdroplet formulation, can be varied. For example, as shown in Tables 9 and 10 of International Publication No. 2015 / 047103, both the gas and the stabilizing membrane of the first component can be varied to prepare clusters having suitable properties that are expected to be useful for treatment according to the present invention.

[0193] Cluster stability in the cluster composition under analysis: Clusters within DP are formed and held together by electrostatic attraction between microbubbles and microdroplets. This attraction is finite, and clusters can decompose after formation through various pathways / influences such as mechanical stress and thermal (Brownian) motion. For precise and accurate characterization, it is important that the clusters remain stable during analysis. This stability was investigated using all the methodologies described above. To evaluate stability, a single DP sample was analyzed 3 to 5 times over time spans exceeding 5 minutes. No significant changes were observed in either concentration or size over these repetitions, demonstrating that microbubbles, microdroplets, and clusters remain stable for more than 5 minutes under the described analytical conditions, i.e., after dilution in PBS or water, under continuous homogenization (stirring).

[0194] Form of the formulation: Several different formulations may be explored to control the cluster content and size within the DP and target optimal properties. Parameters that can be used to manipulate the cluster content and size distribution include, but are not limited to, the difference in surface charge between microbubbles and microdroplets, e.g., SA%, the size of the C2 microdroplets, pH, the TRIS concentration in C2, and the concentrations of microbubbles and microdroplets. In addition, chemical degradation of components, such as chemical degradation during long-term storage at high temperatures, can affect the ability of C1 and C2 to form clusters during the preparation of the DP.

[0195] As reported in International Publication No. 2015 / 047103, several important correlations can be extracted from the in vitro characterization of 30 different compositions to elucidate the properties and characteristics of the system. The inventors found that the size of the clusters formed is strongly related to the reactivity of the system. At relatively low levels of reactivity (e.g., <20%), only small clusters (i.e., 1-5 μm) and medium-sized clusters (i.e., 5-10 μm) are formed. As reactivity increases, larger clusters begin to form. At R > approximately 20%, clusters of 10-20 μm begin to form, and at R > approximately 50%, clusters of 20-40 μm begin to form. When larger clusters are formed, small and medium-sized clusters are sacrificed. The inventors found clear optimal conditions for content vs. reactivity for cluster concentrations of 1-5 μm and 5-10 μm. The inventors have found that the formation of larger clusters (i.e., larger than 10 μm or larger than 20 μm) is detrimental to the effectiveness of the composition, and therefore the clustering potential needs to be balanced.

[0196] Based on the applicant's experiments and the results shown in Tables 5 and 6 of International Publication No. 2015 / 047103, the effectiveness of the cluster composition (linear enhancement in grayscale units (GS)) correlates with the average cluster size and cluster concentration (millions / ml). Grayscale enhancement is the increase in brightness (contrast) observed by US imaging after administration and activation of the cluster composition in vivo, and is a measure of the amount of activated bubbles in the imaged tissue. The reported results are from multivariate principal component analysis (PCA) of the contribution of clusters of various size classes to the linear enhancement (in grayscale units) of the ultrasound signal from canine myocardium upon IV administration and activation of the cluster composition in the left ventricle. See Example 2 of International Publication No. 2015 / 047103. PCA was performed on data from 30 samples detailed in Tables 5 and 6. The results demonstrate that small to medium-sized clusters (<10 μm) significantly contribute to the effectiveness of the cluster composition, while larger clusters (>10 μm) do not contribute significantly. These results and conclusions also apply to the present invention. The results of the PCA analysis are shown in Table 1 below. Figure 1 shows a visualization of cluster size versus product effectiveness based on the data in Table 1, demonstrating that clusters with an average diameter in the range of 3–10 μm have optimal effectiveness. Therefore, Figure 1 shows product effectiveness vs. cluster diameter. The Y-axis shows the correlation coefficient for grayscale enhancement from US imaging of canine myocardium after cluster injection and activation in the left ventricle, reflecting the amount of accumulated activated bubbles. The X-axis shows the cluster diameter in μm. The gray boxes represent the different cluster sizes evaluated: 1–5 μm, 5–10 μm, 10–20 μm, and 20–40 μm. The solid line represents a continuous function of effectiveness vs. cluster diameter. Error bars represent standard errors. Figure 1 is an alternative visualization of Figure 12 (left) from International Publication No. 2015 / 047103. As can be observed, the average cluster diameter should be in the range of 3–10 μm, preferably 4–9 μm, and more preferably 5–7 μm.

[0197] [Table 1]

[0198] The cluster concentration and average diameter of the cluster composition prepared according to Example 1 were analyzed, and it was found that over several hours, it had a cluster concentration of approximately 40 to 44 million clusters / ml and an average cluster diameter of approximately 5.8 to 6.2 μm. The results are shown in Table 2 below. The results are consistent with the results in Table 6 of International Publication No. 2015 / 047103. The data in Table 2 indicate that the prepared cluster composition has acceptable stability and can achieve optimal cluster size and concentration.

[0199] [Table 2]

[0200] Applying the concept of the present invention, namely by preparing a cluster composition from C1 and C2 before administration and forming microbubble / microdroplet clusters, enables an increase in efficacy of more than 10 times, in contrast to the simultaneous injection of the two components taught in International Publication No. 99 / 53963. Forming microbubbles / microdroplets upon combination of the first and second components and administering these pre-formed clusters is a prerequisite for its intended functionality in vivo. The cluster composition should be administered to the subject within a time frame in which the properties of the clusters do not substantially change, for example, within 3 hours of combining the two components.

[0201] (Example 2) Frequency and mechanical index of the enhancement process As mentioned earlier, the application of further ultrasound irradiation after activation of large ACT bubbles, i.e., the enhancement step, results in increased drug spillage from vascular compartments into the target tissue interstitial space and increased infiltration of activated immune cells.

[0202] However, with respect to frequency and MI, the attributes of this enhanced field can strongly influence the effectiveness of the procedure. A certain minimum level of biomechanical effect is necessary to induce increased permeability, but if it is too strong, it can induce inertial cavitation mechanisms, potentially leading to vascular damage and reduced effectiveness.

[0203] The level of bubble oscillation depends on several parameters, most importantly the US frequency and pressure, the latter of which is defined by the mechanical index (MI). Five tests were conducted to investigate the effects of frequency and MI on the nature of the induced bubble oscillation and the effectiveness of the ACT procedure:

[0204] • The decay spectrum of the activated bubble population was measured.

[0205] The radiative oscillations of typical activated bubbles with a resting diameter of 20 μm, induced during the enhancement process, were modeled for a range of frequencies and MIs using a modified Rayleigh-Plesset model [Postema and Schmitz, "Ultrasonic bubbles in medicine: influence of the shell," Ultrason Sonochem, 2007. 14(4): pp. 438-44].

[0206] The tissue uptake of a drug-mimicking chromophore (Evans blue) was investigated as a function of MI with an enhancement process US frequency of 0.5 MHz. In this study, bubble oscillations were modeled using a modified Rayleigh-Plesset model.

[0207] In mice, the therapeutic efficacy of treating prostate cancer with nab-paclitaxel ±ACT was investigated at both 0.5 MHz and 0.9 MHz with a 0.2 augmentation MI.

[0208] In mice, the therapeutic efficacy of treating breast cancer with nab-paclitaxel ±ACT was investigated at MI values ​​of 0.1 and 0.2 with an enhancement frequency of 0.5 MHz.

[0209] Materials and methods: The cluster compositions investigated were as detailed in Example 1.

[0210] The decay spectrum of the activated bubble population was measured by sonometry, as detailed in International Publication No. 2015 / 047103, E1-6.

[0211] Modeling of bubble oscillations as a function of frequency and MI was performed by solving the partially differential modified Rayley-Plesset equations in MATLAB 2020b (MathWorks, Natick, MA, USA). Specifically, a 20 μm diameter bubble was modeled in blood using negligible shell stiffness and C4F10 gas properties. Linear ultrasonic pulses were simulated using a sine wave with a 3-cycle start and end Gaussian ramp.

[0212] To investigate the effect of US-enhanced field MI dispersion, tumor-specific uptake of Evans Blue (EB, fluorescent dye) was examined in a mouse subcutaneous prostate cancer model (PC3). Five groups with enhanced irradiation MI of 0, 0.1, 0.2, 0.3, and 0.4 were investigated (N=3 mice per group). Immediately after intravenous injection of EB, a single dose of cluster composition (2 mL / kg, (iv)) was administered, followed by 45 seconds of activated US (2.25 MHz, MI 0.4) focused on the tumor volume and 5 minutes of enhanced US (0.5 MHz, variable MI). Thirty minutes after treatment, the tumor was resected, and the amount of EB was measured by spectrophotometric analysis at 620 nm.

[0213] The therapeutic effect of ACT (Acute Compression Therapy) using a cluster dispersion of nab-paclitaxel (Abraxane®, ABR) ± 2 mL / kg was investigated in a subcutaneous prostate cancer model (PC3) for the treatment of human prostate cancer in mice. ABR (12 mg / kg, iv) was administered weekly for 4 weeks, and the ACT procedure was performed three times consecutively immediately after each administration. N=9-12 mice per group. In addition, a saline control group (N=4) was also subjected to the procedure. Activated ultrasound consisted of 45 seconds of irradiation at 2.5 MHz, MI=0.4, and enhanced ultrasound consisted of 5 minutes of irradiation at 0.5 or 0.9 MHz, both with an MI of 0.2. The endpoint was overall survival. The tumor had a maximum volume of 1000 mm³. 3 When the situation reached a certain point, the animals were sorted and disposed of.

[0214] The therapeutic effect of ACT (Acute Compression Therapy) using a cluster dispersion of nab-paclitaxel (Abraxane®, ABR) ± 2 mL / kg was investigated in a subcutaneous breast cancer model (Ca-MDA-MB231) for the treatment of human breast cancer in mice. ABR (12 mg / kg, iv) was administered weekly for 4 weeks, and immediately after each administration, the ACT procedure was performed three times consecutively with an MI of 0.1 or 0.2. In addition, a group receiving the drug alone was also investigated. N=9-12 mice per group. Activated ultrasound consisted of 45 seconds of irradiation at 8 MHz, MI=0.33, and enhanced ultrasound consisted of 5 minutes of irradiation at 0.5 MHz, MI of 0.1 or 0.2. The endpoint was tumor volume (daily) measured by caliper, and the measured tumor volume was normalized by the tumor volume on day 1 of treatment. The tumor had a maximum volume of 1000 mm³. 3 When the condition was reached, the animals were sorted and disposed of.

[0215] result: Attenuation spectrum of activated bubbles The decay spectrum of a typical population of activated ACT bubbles was measured, and the results are visualized in Figure 2. As can be noted, the resonant frequency was determined to be approximately 0.3 MHz. Essentially, the decay spectrum explains the interdependence between the bubble population and the incident US field. At or near the resonant frequency, the bubbles are most effective in decay and therefore most effective in converting the incident US energy into volume oscillations and biomechanical effects. Thus, the spectrum demonstrates that the bubble response is very limited for frequencies outside the range of approximately 0.15 MHz to 0.6 MHz. However, this is the case when the bubbles are dispersed in a substantially infinite matrix, and not when the bubbles are retained in microvessels. In this case, contact with the vessel wall suppresses the bubble response and shifts the decay spectrum upward, depending on the diameter and elasticity of the vessel. While it is difficult to provide an accurate model of such decay effect, based on empirical evidence, a shift of the resonant frequency to approximately 0.5 MHz is a reasonable estimate. Therefore, the optimal coupling between ACT bubbles, which ensures the optimal generation of biomechanical effects, is expected to occur between approximately 0.4 and 0.6 MHz. This represents a preferred frequency range for the enhancement process under the present invention.

[0216] Modeling of bubble oscillations as a function of frequency and MI Figure 3 visualizes the modeling of bubble oscillations at MIs of 0.1, 0.20.3, and 0.4 at 300, 400, 600, and 900 kHz. Strong oscillations are evidence of the induction of increased biomechanical effects. However, sharp, sawtooth responses indicate the onset of nonlinear and / or inertial cavitation behavior. As can be noted, at 900 kHz, radiated oscillations are small for all MIs. As predicted from the decay spectra above, they are likely too limited to induce the necessary biomechanical effects required to produce a significant therapeutic benefit. On the other hand, at 300 kHz, even at low MIs, radiated oscillations are very strong and nonlinear, likely causing some degree of inertial cavitation and potentially leading to vascular damage. However, for frequencies between 400 kHz and 600 kHz, radiated oscillations appear to induce sufficient biomechanical effects to induce therapeutic effects while simultaneously avoiding excessive nonlinear behavior and vascular damage.

[0217] Evans Blue's tissue uptake and bubble vibration as a function of MI The results are visualized in Figure 4. As can be noted, tissue uptake increases from no ultrasound (MI=0) to MI=0.1, further increases at MI=0.2, then decreases again at MI=0.3, and further decreases at MI=0.4. At MI=0.2, an increase of approximately 60% in tumor-specific uptake is observed compared to MI=0 (no ultrasound). Simultaneously, the maximum radiated vibration from the embedded bubble vibration panel increases from approximately 3 μm at MI=0.1 to approximately 6 μm at MI=0.2, approximately 10 μm at MI=0.3, and over 20 μm at MI=0.4. Importantly, the subsequent onset of the decrease in tissue uptake (from MI=0.2 to MI=0.3) coincides with the onset of significant nonlinear behavior in which inertial cavitation begins to occur.

[0218] Therapeutic efficacy of nab-paclitaxel ± ACT for the treatment of prostate cancer - Effect of US frequency during the enhancement process Figure 5 visualizes the results showing overall survival versus time. As can be noted, in the ACT (0.5 MHz group), 100% of animals survived to the end of the study, and 80% were in stable complete remission (i.e., cancer-free). The ACT (0.9 MHz) group also showed a therapeutic benefit compared to the drug alone, but its effect was significantly inferior to that of ACT (0.5 MHz). In the 0.9 MHz group, most tumors began to regrow, and only 25% of animals were in stable complete remission at the end of the study, with a survival rate of 57%. These results suggest that a specific minimum radiated oscillation is required to induce the optimal therapeutic effect.

[0219] As a pilot study for this trial, a 0.40 MI was also tested at 0.9 MHz to evaluate whether a higher MI was applicable. However, clear evidence of superficial bleeding was observed in this MI.

[0220] Therapeutic efficacy of nab-paclitaxel ± ACT for breast cancer treatment - Effect of MI during the enhancement process Figure 6 visualizes the results showing tumor growth rate vs. time. As can be noted, the ACT(MI 0.1) group showed a slight but non-significant reduction in tumor growth rate, with tumor growth inhibition at day 31 being only 8% compared to drug alone. However, the ACT(MI 0.2) group showed a strong and significant reduction in tumor growth rate, with tumor growth inhibition at day 31 being 52% compared to drug alone. Again, these results demonstrate that specific minimum radiative oscillations are necessary to induce therapeutic benefits.

[0221] Conclusion: Based on the results obtained in these examples, the functionality of the ACT concept is demonstrated to be highly sensitive to variations in the frequency and MI applied during the augmentation process. Based on these tests, it is concluded that the preferred frequency range is 0.4–0.6 MHz combined with MI applied at 0.1–0.3 MHz. Surprisingly, it was demonstrated that applying lower frequencies and higher MI during the augmentation process resulted in excessively strong induced activated bubble oscillations, leading to significant loss of efficacy and vascular damage. On the other hand, with higher frequencies and lower MI, the induced bubble oscillations were too weak, resulting in insufficient biomechanical effect and therefore significant loss of therapeutic efficacy.

[0222] (Example 3) Therapeutic efficacy of reovirus ± acoustic cluster therapy in a subcutaneous mouse model of hepatocellular carcinoma (HCC) - Cancer immunotherapy This example reports the efficacy of ACT in combination with oncolytic viruses for the treatment of HCC.

[0223] Oncolytic viruses are a form of cancer immunotherapy that uses viruses to infect and destroy cancer cells. Viruses are particles that infect or invade our cells, then use the cell's genetic mechanisms to produce copies of themselves, and subsequently spread to surrounding uninfected cells. In recent years, viruses have been used to target and attack already formed tumors. These viruses are known as oncolytic viruses and represent a promising approach to cancer treatment. Often, cancer cells have damaged antiviral defenses, which makes them susceptible to infection. After infection, these oncolytic viruses can cause cancer cells to "burst," killing them and releasing cancer antigens. These antigens can then stimulate an immune response that can seek out and eliminate any remaining tumor cells nearby and potentially elsewhere in the body.

[0224] Viruses derived from the family Reoviridae (reovirus) have been extensively studied for cancer treatment with clear evidence or therapeutic benefits (NCT01280058, NCT02620423, NCT03723915). However, a factor that can reduce the effectiveness of reovirus is limited uptake by cancer cells. In this regard, combining ACT with virus therapy is hypothesized to strongly increase virus uptake and thus lead to an increase in treatment effectiveness.

[0225] Materials and Methods: Acoustic cluster therapy (ACT) and the composition for use according to the present invention (referred to as ACT) were investigated for treatment efficacy levels in combination with oncolytic reovirus.

[0226] The investigated cluster composition was as detailed in Example 1. The cluster composition was intravenously administered at 2 mL / kg, followed by local ultrasound (US) irradiation of the tumor using a 45-second US activation field (2.7 MHz, MI 0.3), and then irradiation using a 5-minute US enhancement field (500 kHz, MI 0.2). The procedure was performed three times consecutively immediately after the administration of reovirus on each treatment day.

[0227] HCC tumor xenografts were transplanted by subcutaneous injection in balb / C mice. 1×10 7 cancer cells were injected into the flank and allowed to grow freely for approximately 21 days until enrollment in the study. Mice (N = 5 per group) were treated with reovirus alone or in combination with ACT. In addition, a phosphate-buffered saline (PBS) control group was investigated. The virus was administered six times on days 0, 5, 7, 10, 14, and 16. A dose of 1×10 7 plaque-forming units (PFU) was intravenously administered immediately before the ACT treatment. Animals were monitored twice weekly for tumor size (volume) via body weight and caliper measurements over the duration of the study (25 days).

[0228] Results: The mean tumor volume and mean standard error (SEM) results are shown in Table 3 below and visualized in Figure 8.

[0229] [Table 3]

[0230] Figure 8 shows the therapeutic efficacy of ACT combined with oncolytic reovirus for the treatment of hepatocellular carcinoma. The Y-axis represents tumor volume in mm². 3 The data is shown in units. The X-axis represents the time since the start of the study in days. The gray triangle below the X-axis indicates the treatment day. The treatment groups are: saline control (inverted triangle), oncolytic reovirus saline (filled square), and oncolytic reovirus with ACT (filled circle).

[0231] As can be observed from the results shown in Table 3 and visualized in Figure 8, treatment with reovirus alone did not show significant inhibition of tumor growth at the investigated doses compared to the PBS control group. However, when the same dose of virus was combined with ACT treatment, a remarkable and significant tumor inhibition was observed, resulting in a reduction of over 95% of tumor volume at day 25 compared to virus alone. At day 25, the p-value was calculated as p=0.037 using a two-sided ANOVA test with a 95% confidence interval (non-parametric Kruskal-Wallis test with Dunn's multiple comparison correction).

[0232] Conclusion: Therefore, this study confirms the potent synergistic effect of immunotherapy treatments, such as the combination of reovirus with ACT, according to the present invention. Similar synergistic effects are expected with regard to the treatment of other diseases (e.g., cancer and autoimmune diseases) using other types of immunotherapeutic agents.

[0233] (Example 4) Delivery of nanodrugs across the blood-brain barrier (BBB) This embodiment investigates the ability of ACT to deliver large nanoconstructions across BBBs by applying the US field according to the present invention.

[0234] Materials and methods: The ACT cluster compositions investigated were as detailed in Example 1. The nanoparticles investigated were core-crosslinked polymer micelles (CCPMs) from Cristal Therapeutics (Maastricht, Netherlands). These CCPMs had a diameter of 70 nm and were labeled with rhodamine B Cy7 for imaging purposes. The formulations contained 44 mg / ml of polymer and 40 nmol / ml of Cy7.

[0235] CCPM overflow in healthy mouse brains was measured using near-infrared fluorescence (NIRF) imaging, and the minute distribution of CCPM in brain sections was imaged using a confocal laser scanning microscope (CLSM).

[0236] Thirteen female albino BL6 mice (Janvier Labs, France), purchased at 6-8 weeks of age, were housed in groups of five in individually ventilated cages under conditions free from specific pathogens. The cages were well-equipped with housing, resting materials, and chewing wood, and maintained a controlled environment (20-23°C, 50-60% humidity) with a 12-hour day-night cycle. The animals had free access to food and sterile water. All experimental procedures were approved by the Norwegian Food Safety Authority.

[0237] An illustration of the ultrasonic setup is shown in Figure 9. A custom-made dual-frequency transducer (center frequencies of 0.5 MHz and 2.7 MHz) [Andersen et al., "A Harmonic Dual-Frequency Transducer for Acoustic Cluster Therapy," Ultrasound Med Biol, September 2019; 45(9): 2381-2390] was mounted on the apex of a custom-made cone filled with degassed water. The transducer had a diameter of 42 mm, and the -3 dB widths of the 0.5 MHz and 2.7 MHz beam profiles were 16 and 6 mm at a distance of 220 mm from the transducer surface. The signal was generated by a signal generator (33500B, Agilent Technologies, USA) and amplified by a 50 dB RF amplifier (2100L, E&I, USA). The amplifier was connected to a switchbox, which could switch the US field from activation to enhancement. A bag was formed by covering the base of a cone with optically and acoustically transparent plastic foil (Jula Norge AS, Norway). The animal was placed face down on an acoustically absorbing material (Aptflex F28, Precision Acoustics, UK), and an ultrasonic gel was used to connect the acoustically absorbing material, the animal's head, and the acoustically transparent foil.

[0238] The ACT procedure used consisted of an activation step and an enhancement step. Attenuation through the mouse skull was measured to be approximately 21±17% and 42±21% at frequencies of 0.5 MHz and 2.7 MHz, respectively. These values ​​were used to calculate the in situ sound pressure / MI. The following ultrasound parameters were used in each step. • Activation: Center frequency 2.7MHz, average in situ sound pressure of 0.18 corresponding to the mechanical index (MI), pulse length of 8 cycles, pulse repetition frequency of 1kHz, and irradiation time of 60 seconds. • Enhancement: Center frequency 0.5MHz, average in situ sound pressure of 0.15 corresponding to MI, pulse length of 4 cycles, pulse repetition frequency of 1kHz, and irradiation time of 300 seconds.

[0239] One round of ACT consisted of a bolus intravenous injection of 2 mL / kg of the cluster composition followed by 360 seconds of irradiation. Each animal received three rounds of ACT, resulting in a total of 75 μl of ACT preparation and 18 minutes of ultrasound. CCPM was administered intravenously immediately before the first ACT procedure.

[0240] The animals were anesthetized using 2% isoflurane (78%) and oxygen (20%) in medical air (Baxter, USA), and then a cannula was inserted into their lateral tail vein. Body hair was removed using a hair trimmer and depilatory cream (Veet, Canada). During the ACT procedure, the animals were anesthetized using 1.5–2% isoflurane in medical air. Respiratory rate was monitored using a pressure-sensitive probe (SA Instruments, USA), and body temperature was maintained by external heating. Each animal received three rounds of ACT immediately after injection of CCPM. Control animals were treated in the same manner as the animals that received ACT, but instead of the cluster composition, they were given three injections of 50 μl of saline at 6-minute intervals.

[0241] The animals were investigated at two time points: 1 hour and 24 hours after the completion of ACT treatment. At these time points, the animals were euthanized by intraperitoneal injection of pentobarbital (200 μl) and maintained under anesthesia until respiration stopped. Subsequently, the animals were perfused transcardiacally with 30 mL of PBS, and then the brain was resected and imaged using a NIRF imaging device. The control / 1 hour group consisted of N=3, control / 24 hour group N=3, ACT / 1 hour group N=5, and ACT / 24 hour group N=2.

[0242] The excised brain was placed in a NIRF imaging device (Pearl Impulse Imager, LI-COR Biosciences Ltd., USA) to evaluate the accumulation of CCPM (registered trademark) in the brain. The brain was stimulated at 785 nm and fluorescence emission was detected at 820 nm. The images were analyzed using ImageJ (ImageJ 1.51j, USA). Regions of interest (ROIs) were drawn around the brain to obtain the total fluorescence intensity of the brain, which was normalized against the wet mass of the brain. Using a standard curve, the total fluorescence intensity was converted to the percentage of the injected dose per gram of brain tissue (% ID / g). The results were plotted for each time point and treatment group.

[0243] For confocal microscopy, the excised brain was laterally mounted on a cork piece using Optimum Cutting Temperature Tissue Tek (Sakura, Netherlands), and then the sample was slowly immersed in liquid nitrogen. After removing the first 500 μm from the top of the frozen brain, 5×10 μm thick sections and 5×25 μm thick sections were cut laterally. This was repeated every 800 μm throughout the brain.

[0244] Results: To examine whether increased permeability promotes the spillover of CCPM, the excised brain was imaged with a NIRF imaging device. Representative NIRF images of the control and animals injected with nanoparticles are shown in Figure 10 (upper panel). As can be noted, clear accumulation can be observed in the brains that received ACT, contrary to the control brains.

[0245] Quantitative analysis of the NIRF images revealed a statistically significant increase in accumulation (% ID / g) between ACT and control animals at both time points (Figure 10, lower left panel). Compared to the control, in ACT, the median % ID / g increased from 0.9% ID / g to 2.6% ID / g at 1 hour after ACT and from 0.8% ID / g to 2.2% ID / g at 24 hours after ACT. Increases of 290 and 280% were observed in % ID / g units, respectively.

[0246] Brain sections were imaged by CLSM to examine the increased accumulation of CCPM in brain tissue after ACT treatment and to investigate the location of CCPM relative to blood vessels. Tile scans of ACT-treated brains showed several fluorescence "clouds" that were not observed in the brains of control animals. 24 hours after ACT, tile scans of ACT-treated brains showed a similar cloud pattern to the 1-hour treatment group. The number of pixels representing CCPM was extracted from threshold tile scans of both control and ACT-treated animals and normalized by the size of the ROI used to outline the hemispheres. As can be seen in Figure 10 (bottom right panel), a clear and statistically significant 4.7-fold increase can be observed in sections 1 hour after ACT treatment, contrary to the control brain.

[0247] High-magnification CLSM images were acquired at various locations in both control and ACT-treated brains to investigate the position of the cytoplasmic sperm artery (CCPM) relative to blood vessels. In ACT-treated brains, the CCPM was clearly overflowing, while in control brains, it was observed mainly intravascular or slightly displaced from blood vessels.

[0248] Conclusion: Applying the two-stage irradiation approach of the present invention, ACT significantly increased the blood-brain barrier (BBB) ​​penetration of large nanoparticle constructs, such as the 70 nm CCPM compounds investigated. ACT resulted in improved accumulation, overflow, and penetration of CCPMs into the brain parenchyma. Thus, we demonstrate ACT's ability to deliver large drug molecules or constructs, such as ITAs, across any vascular barrier in the body.

[0249] (Example 5 (Predictive)) Pilot study of acoustic cluster therapy (ACT) using PD-1 antibodies in a syngeneic mouse model of melanoma Until recently, metastatic melanoma was considered refractory to systemic therapy. A better understanding of the interaction between tumors and the mechanisms of immune system and T-cell regulation has led to the development of immune checkpoint inhibitors. While this class of immunotherapies has improved patient clinical outcomes, the response rate remains in the 30-40% range, and therefore there is a clear medical need to improve this therapy regimen.

[0250] By using an ACT approach to increase vascular permeability, it may be possible to increase activated T cell infiltration and enhance the delivery of immunotherapeutic agents, thereby improving T cell activation. Therefore, this could have a significant impact on the treatment outcomes of immunotherapy in patients with melanoma.

[0251] The purpose of this study is to evaluate the antitumor activity of ACT combined with PD-1 antibody in a syngeneic mouse model of melanoma. The applicable cluster composition and ACT procedure will be as described in Examples 1 and 3.

[0252] Materials and methods: B16-F1 cells are subcutaneously transplanted into immune C57BL / 6J Black 6 mice, and disease progression is monitored weekly by measuring primary tumor calipers. Tumor volume is 30-40 mm. 3 Once the target is reached, mice (n=12 per group) will be randomized into six groups: control (saline), US+PS101, anti-mouse PD-1 (CD279), isotype antibody control, anti-mouse PD-1+US+PS101, and isotype antibody control+US+PS101.

[0253] The research period continued until the tumor volume reached 150 mm. 3The treatment continues until the target is reached. Four animals from each group are euthanized on day 7 for gene expression RNA sequencing analysis (see 3.b). Anti-mouse PD-1 or isotyped antibodies are administered intravenously twice weekly at 200 μg / mouse, followed by PS101 administration for the duration of the study. Endpoints include primary tumor volume, body weight curve, and overall survival.

[0254] Four animals from each group are euthanized on day 7, and tumors are excised for whole transcriptome mRNA analysis (NanoString analysis). Based on the results of this analysis, immunohistochemical (IHC) analysis for selected immune pathways, such as F4 / 80, CD3, CD4, CD8, and Foxp3 antibodies, is performed on the excised tumors at the end of the study. The endpoints are mRNA analysis at day 7 and IHC analysis at the end of the study.

[0255] Results (predictive): Results will likely indicate that the combination of ACT with anti-PD1 treatment, compared to anti-PD1 treatment alone, will lead to the fulfillment of one or more of the following therapeutic efficacy endpoints: a significant increase in tumor growth inhibition, a significant increase in median overall survival, and a significant increase in tumor infiltration by immune cells.

[0256] Therefore, this study will confirm the potent synergistic effect when an immunotherapy agent, such as an anti-PD1 monoclonal antibody, is combined with ACT in accordance with the present invention.

[0257] (Example 6) Preliminary investigation of synergistic effects between acoustic cluster therapy (ACT) and immuno-oncology (IO) The objective of the study was to test the efficacy of immune checkpoint inhibitors in combination with ACT in a subcutaneous melanoma model (B16-F1, ATCC). The pilot study had two objectives: a) Based on the idea that ACT enhances the delivery of chemotherapeutic agents, and therefore ACT should enhance the delivery of checkpoint inhibitors to tumors, we will test whether ACT enhances the efficacy of immune checkpoint inhibitors in a subcutaneous melanoma model, and b) Whether preliminary evidence can be obtained that ACT stimulates an immunogenic response in tumors (i.e., turns an immunologically "cold" tumor into an immunologically "hot" tumor).

[0258] method: The study was conducted in two parts. First, an inhibitor selection study was performed to establish the optimal checkpoint inhibitor in the proposed model (B16-F1, ATCC in immune-capable C57BL / 6J Black 6 mice) using either anti-PD-1, anti-CTLA4, or a combination of anti-PD-1 and anti-CTLA4. Based on the premise that the selected inhibitor regimen alone showed only moderate efficacy, data were analyzed to establish the regimen most likely to enable demonstration of improved efficacy when combined with ACT. For brevity, the results of this part of the study are not reported below. The results led to the decision to use the combination of anti-PD-1 and anti-CTLA4 as the checkpoint inhibitor in the second part of the study, which was conducted with the addition of ACT.

[0259] The cluster composition was as described in Example 1. "US+PS101" means the use of ultrasound and the cluster composition in the ACT procedure as disclosed herein.

[0260] Tumor volume, mouse body weight, and survival time were monitored for the following treatment groups (n=12): i) Control (physiological saline), ii) Checkpoint inhibitors, iii) Isotype antibody control (e.g., InVivoMab rat IgG2a), iv) Checkpoint inhibitors (anti-PD-1 and anti-CTLA4) + US + PS101 (ACT), v) Isotype antibody control (IgG2a) + US + PS101 (ACT).

[0261] Data were analyzed to determine whether ACT® enhances the effectiveness of checkpoint inhibitors compared to checkpoint inhibitors alone.

[0262] To test for any stimulated changes in the immunogenicity of the tumors, four animals from each group were euthanized on day 7 for transcriptome mRNA (TempO-Seq(c)) analysis of 20,000 genes and immunohistochemistry (IHC) for analysis of selected immune pathway biomarkers. Tumors excised at the end of the study were fixed and preserved for potential retrospective RNA sequencing analysis and IHC.

[0263] Each subsequent cannula insertion presented several challenges in the model, including difficulty in inserting the cannula into the tail vein in enlarged black mice, extremely soft, highly cellular tumors that were difficult to measure by palpation and caliper, and very different rapid growth rates from tumor to tumor. This resulted in significant inter-tumor variability, which limited the ability to interpret the results. Significant inter-tumor variability in growth rate and possibly introductory tumor volume led to substantial heterogeneity in the growth curves. Nevertheless, differences in mean growth rates across all mice were not statistically significant for different treatment groups, although a tendency toward careful interpretation was observed. Treatment with the isotype antibody IgG2a alone appeared to have a similar therapeutic effect compared to saline control with treatment with the checkpoint inhibitor combination of anti-PD-11 and anti-CTLA-4 alone. The addition of ACT appeared to slightly improve the mean therapeutic effects of both the isotype and checkpoint inhibitors. Further analysis of these results could have included calculating tumor doubling times to determine if any significant differences occurred. However, examination of individual tumor growth curves suggests that differences between treatment groups may be more attributable to differences in the spread of growth rates among treated mice than to differences between means. There was even a tendency toward an increase in the number of outliers showing improved responses, or even a suggestion of the generation of a bimodal distribution with the addition of ACT. In this model, studies using more mice would be needed to confirm whether this is a genuine finding.

[0264] The findings regarding the survival curve, namely that isotype antibodies appeared to be effective and that the non-significant trend suggested that ACT could extend survival, were similar to those regarding the mean proliferation ratio.

[0265] Growth curves, tumor genetics analysis, and IHC analysis of a subset of mice selected for disposal suggest a similar story, with a tendency for isotypes to induce a response and ACT to improve the response. Herein lies the fact that further analysis, such as calculation of tumor doubling time, may also be meaningful to determine whether any significant differences occur between the groups.

[0266] Immunohistochemical (IHC) analysis of selected immune pathway biomarkers (genes): Heatmaps, normalized gene expression graphs, and other IHC graphs were created for each treatment group. Since the maps and graphs include color coding, they are not suitable for inclusion; the findings are summarized below.

[0267] - One analysis provided a heatmap showing the intensity of immune pathway gene expression for each treatment group compared to the average expression across all animals. The selected genes were: BCR signaling, M1 activation, MHC class I antigen presentation, T cell checkpoint signaling, IFNg gene, CTLA4 signaling, MHC class II antigen presentation, PP1 signaling, and TLR signaling. The heatmap clearly showed that for treatment group iv) treated with checkpoint inhibitors (anti-PD-1 and anti-CTLA4) and ACT, all biomarkers were activated and appeared red in the heatmap. The other treatment groups appeared yellow to blue for all genes, indicating relatively little or no activation.

[0268] - Another analysis provided a heatmap showing the intensity of gene expression associated with specific immune types in the following cells: T cells, CD8 T cells, monocytes, fibroblasts, NK cells, Masaat cells, lymphatic vessels, B-derived endothelial cells, and blood vessels, for different treatment groups. The heatmap clearly showed that, for treatment groups iv) treated with checkpoint inhibitors (anti-PD-1 and anti-CTLA4) and ACT, T cells, CD8 T cells, and monocytes in particular were activated and appeared red in the heatmap.

[0269] Since there were no treatment groups treated with ACT alone, the potential of ACT to alter the immunogenicity of tumors was evaluated as differential (log2 change ratio) gene expression between treatment with isotype antibody alone and treatment with isotype antibody + ACT. Figure 13 provides a "volcano plot" showing the effect of ACT on the immunogenicity of tumors as expression of immunogenes only when ACT is combined with an isotype antibody (IgG) compared with treatment with IgG alone. The median is shown. Genes that did not show significant differential expression are not labeled and are plotted as points only in (B), while genes that were significant are plotted as points and labeled. A relatively small number of immunogenes showed differential sequences that reached statistical significance, which highlights the need for studies using more animals, less heterogeneous models, and groups treated with ACT alone.

[0270] Further differential expression analysis of biochemical pathways suggested that ACT combined with isotype antibodies tended to significantly downregulate several pathways, including the response to hypoxia (-log10(P)>1.3), and upregulate others, compared to isotype antibodies alone. Before considering the significance of these findings, larger-scale studies and studies using ACT alone are recommended. Therefore, there is potential for future correlational imaging (e.g., using in vivo photoacoustic imaging or quantitative morphohistology) to confirm the relationship between gene expression and phenotypic characteristics.

[0271] Figure 14 shows the genetic effects of ACT as indicated by the downregulated (A) or upregulated (B) pathways when ACT is combined with an isotype antibody (IgG), compared to treatment with IgG alone, with the x-axis showing -log10 (p-value).

[0272] Panel A: Pathways downregulated by IgG+ACT.

[0273] Panel B: Pathways upregulated by IgG+ACT.

[0274] The characters represent the following: GO:0001666 - Response to hypoxia, WP4206 - Hereditary leiomyomatosis and renal cell carcinoma pathway, GO:0001525-Angiogenesis, GO:0051235 - Maintain position, GO:0061061 - Muscle structure development, GO:0006936 - Muscle contraction, GO:0097435 - Supramolecular fiber structure, GO:0043462 - Regulation of ATPase activity, GO:0030199 - Collagen fibril composition, GO:0030239 - Myofibrils Assembly, GO:0044057 - System process adjustment.

[0275] Figure 15 provides immunohistochemical findings from automated analysis of whole-stained tumor sections. The percentage of cells positively stained for CD8 T cells is shown for each treatment group for individual animals (black dots), along with the group mean (bar) and standard deviation (error bars). The x-axis represents the treatment group, with A: ACT + PD1 / CTL4, B: PD1 / CTL4, C: ACT + ISO, D: ISO, and E: normal saline.

[0276] The mean normalized intensity of immune pathway gene expression appears to increase with ACT, but this is attributable to increases in single tumors, and genetic differences can be observed between mice within a large group, which are reproducible for other genes. Such genetic heterogeneity is characteristic of cancer and is seen in patient-derived tumor samples. When analyzing patient-derived tumor samples, previous studies have drawn useful conclusions from studying gene expression in extreme responders and non-responders as defined by the RECIST (tumor reduction) criteria (reference). A similar approach here, or even comparing the intensity of expression in specific groups of responsive genes measured from growth curves in individual animals, may be beneficial in future studies, but would require more animals than the four used for genetic analysis in this pilot study. The use of tumor models that are easier to handle and produce growth curves with less heterogeneity may also allow for greater significance in the differences between groups with respect to the mean.

[0277] A similar pattern emerged with respect to tissue inflammation signature (TIS) genes and those related to the immune type of cells. TIS is being developed to guide potential treatments.

[0278] In this pilot study, IHC analysis has so far been limited to staining for CD8 T cells, but sections remain available for further staining (e.g., for CD4, etc.). CD8 stains a subpopulation of T cells important for mediating adaptive immunity, including cytotoxic (so-called "killer") T cells. It is interesting to note that, although caution must be exercised regarding the small number of animals and the corresponding lack of significance, the addition of ACT to treatment with checkpoint inhibitors or isotypes appeared to increase the number of tumors with a high percentage of CD8-positive T cells.

[0279] Despite the lack of significant significance among many of the individual results, the combined findings appear to indicate a pattern in which ACT can improve the therapeutic efficacy of checkpoint inhibitors and induce an enhanced immunogenic response in tumors.

[0280] (Example 7 (Predictive)) Production of cluster compositions using various microbubble and microdroplet compositions To demonstrate that the present invention is applicable to various chemical compositions of the first and second components (C1 and C2), several formulations can be manufactured or commercially procured, and the in vitro properties of the resulting cluster compositions can be explored.

[0281] Example of C1: Commercially available microbubble US imaging agents, Sonovue® (Bracco Spa, Italy) and Micromarker® (VisualSonics, USA), can be procured and used as component C1. Sonovue is a sulfur hexafluoride microbubble stabilized with distearoylphosphatidylcholine, dipalmitoylphosphatidylglycerol sodium, palmitic acid, and a PEG4000 membrane, and is presented in a lyophilized form for reconstitution with a 5 mL aqueous matrix. Micromarker is a perfluorobutane / nitrogen microbubble stabilized with phospholipids, polyethylene glycol, and fatty acids, and is presented in a lyophilized form for reconstitution with a 0.7 mL aqueous matrix.

[0282] Example of C2: Microdroplet (C2) component containing diffusible components; a) Perfluorodimethylcyclobutane, b) 2-(trifluoromethyl)perfluoropentane, and c) perfluorohexane can be prepared as follows: Measure 790 mg of distearoylphosphatidylcholine (DSPC) and 8.1 mg of stearylamine (SA) into a 250 ml round-bottom flask and add 50 ml of chloroform. Heat the sample under hot tap water until a clear solution is obtained. Remove the chloroform by evaporation to dryness using a rotary evaporator at 350 mmHg and 40°C, and further dry in a desiccator at 50 mmHg overnight. Then add 160 ml of water, place the flask back on the rotary evaporator, and rehydrate the lipids for 25 minutes at the maximum rotation speed and a water bath temperature of 80°C. Transfer the resulting lipid dispersion to a suitable vial and store in the refrigerator until use.

[0283] Emulsions are prepared by transferring 1 ml aliquots of the chilled lipid dispersion to 2 ml chromatography vials. 100 μl of fluorocarbon oil is added to each of the six vials as described above. The chromatography vials are shaken on a CapMix (Espe) for 75 seconds. The resulting emulsions are washed three times by centrifugation, removal of the supernatant (infranatant), and then addition of an equivalent volume of 5 mM aqueous TRIS buffer. The vials are immediately cooled on ice, pooled, and kept cool until use.

[0284] Coulter counting analysis was performed to determine the volume concentration and diameter of the microdroplets, and then the emulsion was diluted with 5 mM TRIS buffer to a dispersed phase concentration of 4 μl microdroplets / ml.

[0285] The cluster composition is prepared by reconstituting Sonovue or Micromarker using 5 mL or 0.7 mL of the above C2 component, respectively.

[0286] Results (predictive) When components C1 and C2 are mixed, all six possible combinations are expected to contain more than 10 million clusters per milliliter, with an average diameter of 3 to 10 μm. [Explanation of Symbols]

[0287] Example 3 1. Dual-frequency ultrasonic transducer (2.7MHz and 500kHz output) 2. Ultrasonic waveguide 3 Bathing 4. Ultrasonic gel 5. Ultrasonic absorption pads 6. Injection syringe containing cluster composition 7 VeVo Imaging Table 8 Catheters 9. Tumors Example 4 1 Amplifier 2. Signal Generator 3. Frequency switch box (between 0.5MHz and 2.7MHz) 4. Dual-frequency transducer 5. A cone filled with water 6. Bag filled with water 7. Ultrasonic gel 8. A mouse in a prone position. 9 Ear Bars 10 Acoustic absorbing pads

Claims

1. A pharmaceutical combination comprising a microbubble-microdroplet cluster composition and at least one immunotherapy agent (ITA), wherein the microbubble-microdroplet cluster composition has a cluster concentration of at least 25 million clusters / ml of clusters in the size range of 1 to 10 μm, and the microbubble-microdroplet cluster composition comprises a group of negatively charged microbubbles and positively charged microdroplets that are permanently held together by their opposing electrostatic attraction to form a single aggregated entity, and the pharmaceutical combination comprises, Autoimmune diseases in humans; or Cancers in humans (the aforementioned cancers do not include pancreatic cancer) It is intended for use in methods of treating, The method described above is (i) A step of administering the at least one immunotherapy agent (ITA) to the human subject, (ii) A step of administering the microbubble-microdroplet cluster composition to the human subject, The process involves administering at least one ITA separately from the cluster composition, beforehand, and / or simultaneously, and / or afterward. (iii) A step of activating the phase shift of the diffusible component of the microdroplets of the cluster composition from step (ii) by ultrasonic irradiation of a region of interest within the human subject at a first frequency of 1 to 10 MHz and a first mechanical index of 0.1 to 0.

4. (iv) Further irradiation with ultrasound at a second frequency of 0.4–0.6 MHz and a second mechanical index of 0.1–0.

3. A combination of pharmaceuticals, including [specific compound / product name].

2. The pharmaceutical combination according to claim 1, wherein steps (ii) to (iv) are repeated 1 to 4 times.

3. The pharmaceutical combination according to claim 1, wherein the irradiation in step (iii) is started immediately after step (ii), and the irradiation in step (iv) is followed immediately afterward.

4. The pharmaceutical combination according to claim 1, wherein the irradiation in step (iii) lasts for 30 to 120 seconds, followed by the irradiation in step (iv) lasting for 3 to 10 minutes.

5. A pharmaceutical combination according to claim 1, used as part of a polypharmacy treatment.

6. The pharmaceutical combination according to claim 1, wherein 1 to 5 therapeutic agents, including at least one ITA, are administered simultaneously or sequentially over a certain period of time, and at least one ACT treatment including steps (ii) to (iv) is performed during the same period.

7. The method described above is Enhanced overflow and uptake of at least one ITA (the at least one ITA including an inflammatory cytokine) administered separately, prior to, and / or simultaneously, and / or afterward, and / or Enhanced infiltration of activated immune cells into target pathologies A pharmaceutical combination according to claim 1, which promotes the following:

8. The pharmaceutical combination according to claim 1, wherein a broadband or dual-frequency US transducer is used in both the activation irradiation of step (iii) and the further irradiation of step (iv).

9. The pharmaceutical combination according to claim 1, wherein the cluster has an average diameter in the range of 3 to 10 μm.

10. The pharmaceutical combination according to claim 1, wherein the cluster has an average diameter in the range of 4 to 9 μm.

11. The pharmaceutical combination according to claim 1, wherein the gas of the microbubbles in the microbubble-microdroplet cluster comprises sulfur hexafluoride or C3-6 perfluorocarbon or a mixture thereof.

12. The pharmaceutical combination according to claim 1, wherein the oil phase of the microdroplets in the microbubble-microdroplet cluster comprises a partially or completely halogenated hydrocarbon or a mixture thereof.

13. The pharmaceutical combination according to claim 1, wherein the microbubbles contain a first stabilizer comprising a phospholipid, protein, or polymer, and the microdroplets contain a second stabilizer comprising a phospholipid, protein, or polymer.

14. The pharmaceutical combination according to claim 1, wherein the immunotherapy agent is formulated in a vehicle.

15. The pharmaceutical combination according to claim 14, wherein the vehicle on which the immunotherapy agent is formulated comprises liposomes, micelles, conjugates, nanoparticles, core-crosslinked polymer micelles (CCPM), or microspheres.

16. The pharmaceutical combination according to claim 1, wherein the at least one ITA is selected from the group consisting of immuno-oncology agents, monoclonal antibodies (mAbs), fusion proteins, soluble cytokine receptors, recombinant cytokines, small molecule mimetic drugs, cell therapies, cancer vaccines, and oncolytic viruses.

17. The pharmaceutical combination according to claim 1, wherein the immunotherapy agent is selected from the group of monoclonal antibodies.

18. The pharmaceutical combination according to claim 1, wherein the treatment using at least one ITA is combined with a treatment using one or more chemotherapeutic agents.

19. The pharmaceutical combination according to claim 1, wherein one or more ITAs are selected from ITAs having the ability to target any of the antigens named CD1 to CD371.

20. The pharmaceutical combination according to claim 1, wherein the ITA is selected from the group of monoclonal antibodies anti-PD1, anti-PDL1, and CTLA4, and is used in combination with a chemotherapeutic agent.

21. The pharmaceutical combination according to claim 1, wherein the one or more ITAs are selected from the group of immune checkpoint inhibitors.

22. The pharmaceutical combination according to claim 1, wherein the ITA, or a formulation of the ITA, has a molecular weight exceeding 15,000 daltons.

23. The pharmaceutical combination according to claim 1, wherein the cluster composition is administered within a 3-hour time frame by combining microbubbles of a first component and microdroplets of a second component to prepare the microbubble-microdroplet cluster composition.

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