Device and method for pre-treating solid tumors with pressure pulses to enhance anti-cancer therapy

Abstract

The present disclosure relates to devices and methods for pre-treating a tumor microenvironment by pressure pulses to enhance the efficacy of an anti-cancer therapeutic drug, suitable for subjects in need of such treatment. In addition, the present disclosure also discloses enhanced response of solid tumors locally exposed to stress waves to systemically administered therapeutic drugs. An apparatus according to the present disclosure comprises a pulsed laser system (1), a light guide (2) for guiding laser pulses to one or more optical pressure transducers (3) that absorb laser pulses from the pulsed laser system and generate pressure pulses, a tumor positioning support structure (4) configured to couple the one or more optical pressure transducers with a solid tumor (5), and a control system (6) for limiting exposure of the solid tumor to the pressure pulses. The anti-cancer therapeutic drug may be administered before, after, or during the pressure pulse pretreatment of the solid tumor.

Description

Technical Field

[0001] The present disclosure generally relates to the field of remodeling (or preconditioning) the tumor microenvironment (TME) to enhance the efficacy of anti-cancer therapeutic agents. More specifically, the present disclosure relates to a pressure pulse tumor preconditioning device and methods of using the same, as well as methods of preconditioning the tumor microenvironment using pressure pulses to enhance the efficacy of anti-cancer therapeutic agents. Background Art

[0002] The study of triggering anti-tumor immune responses capable of controlling tumor growth and spread by actively stimulating the host immune system has been ongoing for a century and has recently become a first-line treatment for certain cancer indications [1-3]. A notable example is immune checkpoint blockade therapy (ICB), which uses monoclonal antibodies (mAbs) to block inhibitory immune receptors (checkpoints), thereby releasing the anti-tumor function of immune cells present in the immunosuppressive tumor microenvironment (TME). To promote a therapeutic response, CD8 + T cells strongly activated by tumor antigens must be unrestricted by negative regulators. These negative regulators are referred to as "checkpoints" because they can detect, resist, and reverse over-reactions. Although checkpoint inhibitors are crucial for maintaining self-tolerance and preventing autoimmune diseases, they can also be hijacked by tumor cells to evade immune surveillance. Inhibitory immune receptors include, but are not limited to, CTLA4 (cytotoxic T lymphocyte-associated protein 4), PD1 (programmed cell death protein 1), LAG3 (lymphocyte activation gene-3), TIM3 (T cell immunoglobulin and mucin domain-3), TIGIT (T cell immunoreceptor with Ig and ITIM domains), ICOS (inducible T cell co-stimulatory receptor), BTLA (B and T lymphocyte attenuator), and VISTA (V-domain Ig-containing T cell activation inhibitor). CTLA4 and PD1 are currently the most well-known and effective T cell immune checkpoint molecules.

[0003] The sequence of clinical applications of ICBs approved by the US Food and Drug Administration (FDA) is very impressive [4]: ipilimumab (2011), pembrolizumab (2014), nivolumab (2014), atezolizumab (2016), durvalumab (2017), avelumab (2017), cemiplimab (2018), dostarlimab (2021), tislelizumab (2021), relatlimab (2022). In China, other ICBs have also been approved for clinical use, such as toripalimab (2018), camrelizumab (2019), and sintilimab (2019). In addition, some of these ICBs have also been approved for the treatment of different types of cancer. This remarkable progress in immunotherapy explains why, in 2019, it was estimated that approximately 44% of cancer patients in the United States were eligible for ICB treatment. However, the proportion of cancer patients who do not respond to ICBs exceeds 87% [5]. There is an urgent need to improve the efficacy of immunotherapy.

[0004] CTLA4 molecules are contained in intracellular vesicles of naive T cells and are located in CD4 + CD25 + Regulatory T(T (reg) ) cells. When the T cell receptor binds to the cognate antigen presented by the antigen presenting cell (APC) in the presence of a co-stimulatory signal, the naive T cell is activated. This co-stimulatory signal is the binding between CD28 expressed on the surface of the T cell and the B7 molecule (B7.1, also known as CD80 or B7.2, also known as CD86) on the APC. APCs are immune cells that process and present antigens for T cell recognition, including B lymphocytes, dendritic cells, macrophages, and other immune cells. CD28 and CTLA4 competitively bind to B7-1 and B7-2 on APCs, but CTLA4 binds more tightly to B7-1 and B7-2 and transmits negative rather than cost-stimulatory signals to T cells. CTLA4 can counteract multiple internal signaling nodes to hinder the activation and proliferation of T cells [6]. T cells that constitutively express CTLA4 (reg) Cells can suppress T cell responses. CTLA4 is a negative regulator of T cell activity, suggesting that blocking the action of CTLA4 could rescue T cell responses against cancer cells. In fact, neutralizing anti-CTLA4 mAbs can enhance anti-tumor immunity. In addition to enhancing CD8+ effector T cell responses, anti-CTLA4 therapy can also deplete local intratumoral T cells through antibody-dependent cell-mediated cytotoxicity. (reg)cells and shift the balance of the TME away from the immunosuppressive state [7].

[0005] Human PD1 is expressed on T cells after T cell receptor stimulation and binds to the B7 homologs PDL1 and PDL2, which are constitutively expressed on APCs and can be induced in non-hematopoietic tissues. PDL1 (programmed death ligand 1, also known as B7-H1) is a transmembrane protein that can downregulate immune responses by binding to its two inhibitory receptors, PD1 and B7.1. It is present on a variety of cell types, including T cells, tumor cells, epithelial cells, and endothelial cells, and is more common than PDL2 (programmed death ligand 2, also known as B7-DC). PD1 mainly restricts immune responses through inhibitory signaling in CD8+ effector T cells and T (reg) cells [7]. When PD1 binds to its ligand, it can induce T cell dysfunction, i.e., T cell exhaustion. Tumor cells can upregulate PD1 ligands. PDL1 expressed by cells in the TME binds to PD1 on T cells, subsequently triggering inhibitory signaling, blocking effector function, and reducing the killing ability of T cells. Therefore, tumor cells can induce T cell exhaustion and create a TME that is favorable for tumor growth and invasion. Since the PD1 / PDL1 pathway can protect cells from T cell attack, anti-PD1 and anti-PDL1 antibodies can enhance the functional properties of CD8 + effector T cells at the tumor site.

[0006] Activating T cells enables T cell lymphocytes to recognize antigens on specific target cells. Activated CD8 + T cells gradually transform into effector T cells (or cytotoxic T lymphocytes, CTLs), which recognize target cells and kill them through different pathways. In one pathway, Fas ligand (or CD95L) expressed on the surface of CTLs binds to the Fas receptor (or CD95) on target cells and triggers apoptosis through the caspase cascade. In another pathway, CTLs release granzyme B, perforin, granulin C, and / or granzyme into the intercellular space between CTLs and target cells, and these substances are highly cytotoxic to target cells. Although the mechanisms of these immunotherapies are complex, interconnected, and still under investigation, these pathways suggest that the anti-tumor activity of CTLs requires them to penetrate solid tumors. This requirement for TME penetration also applies to other immunotherapies, such as adoptive T cell therapy or cancer vaccines.

[0007] Immunotherapy fails to treat the vast majority of patients, while achieving remarkable success in a small number of patients, which has attracted intense attention. There are many reasons for the poor treatment effect, including the abnormal tumor microenvironment (TME), which is characterized by dysfunctional blood vessels that impede the delivery of immunotherapy drugs and lead to immunosuppression. In fact, the insufficient tumor blood perfusion in space and time can result in hypoxia, low pH, and insufficient drug delivery, thereby affecting the efficacy of cancer therapies including immunotherapy. Chemotherapy [8] has recognized that it is difficult for anti-cancer drugs to reach all cells in the TME, and considering the size of the corresponding therapeutic drugs, this is expected to become a greater limiting factor for biologics and immunotherapy. The rapid proliferation of cancer cells will force the blood vessels in solid tumors to separate, reduce the vascular density, compress the blood and lymphatic vessels, limit the delivery of oxygen and nutrients, accumulate metabolites, and lower the pH value. Since mAbs are very large macromolecules with a molecular weight of about 150 kDa or a combination of macromolecules, it is more difficult to penetrate solid tumors when using mAbs. The physicochemical properties and large volume of mAbs impede their passive diffusion in vascular epithelial cells. The main mechanism for mAbs to be distributed from the body to tissues is convective transport, that is, the blood-tissue hydrostatic pressure gradient. The affinity of mAbs for target antigens in the interstitial space or on the cell surface and their convection to the lymph determine the retention of mAbs in tissues. The convective efficiency into the interstitial space is much lower than the convective efficiency out of the interstitial space. Therefore, the clinical central compartment volume of most mAbs is between 2 and 3 liters, similar to the vascular water, while the total distribution volume at steady state is 8 to 20 liters [9]. The limited distribution volume of mAbs, especially ICB, indicates that they are mainly confined to the vascular compartment.

[10]

[0008] The increased vascular leakage in solid tumors and the inability of intratumoral lymphatics to effectively drain these fluids have been exploited by nanoparticle drug delivery systems to accumulate therapeutic drugs in tumors through the enhanced permeability and retention effect

[11] . Compared with free drugs, nanoparticle drug carriers have been shown to mediate more tumor drug deposition, and the delivered drug concentration can be sustained for several days, exceeding the peak concentration reached by free drugs in tumors. However, nano-drugs tend to accumulate in reticuloendothelial system tissues (spleen, liver, lung) and tend to stay near the pores of tumor blood vessels

[12] . The probability of nano-drugs reaching most target cells in tumors is low.

[0009] The mechanical microenvironment of solid tumors is characterized by elevated interstitial fluid pressure (IFP) and solid stress. The difference between the contributions of these two stresses in the TME is that IFP is an isotropic stress generated by increased leakage and poor drainage of fluid within the tumor, while solid stress is generated by non-fluid components. IFP originates from the combination of high vascular permeability in the TME and mechanical compression of downstream blood vessels and draining lymphatics. Solid stress is associated with the excessive proliferation of cancer cells, which exert forces on nearby tumor and normal tissue structural elements, and according to the law of action-reaction, these elements exert equal but opposite forces. Vascular compression has two consequences for treatment: (i) vascular collapse impedes the entry of drugs and immune cells, and (ii) loss of lymphatic function reduces drainage and increases IFP, and the transport of large therapeutic agents such as antibodies that block inhibitory checkpoint molecules or nanodrugs becomes diffusive and reduced due to their large size. These consequences are even more severe in immunotherapies that rely on the infiltration of tumor antigen-specific cytotoxic T lymphocytes into solid tumors, including adoptive T cell therapy and cancer vaccines.

[0010] Abnormal and disordered tumor blood vessels have become targets for various therapies, namely anti-angiogenic drugs such as anti-vascular endothelial growth factor (anti-VEGF). Low-dose anti-angiogenic therapy can normalize tumor blood vessels and improve tumor perfusion and drug delivery

[13] . Therapies that overcome the IFP and high growth-induced solid stress in solid tumors are rare. In normal tissues, IFP is in the range of 0 - 3 mmHg. However, the leakage of fluid in solid tumor blood vessels and the inability of intratumoral lymphatics to effectively drain this fluid result in IFP values reaching the range of 5 - 40 mmHg, and even up to 75 - 130 mmHg in sclerosing pancreatic tumors

[14] , although it drops suddenly at the tumor margin. Anti-solid stress strategies are different from vascular normalization strategies, which use anti-angiogenic drugs to prune immature blood vessels and strengthen the remaining blood vessels. Anti-solid stress aims to decompress blood vessels to increase perfusion

[15] . If it can also successfully reduce venous resistance and reconstruct lymphatic drainage, it will reduce IFP. IFP and solid stress limit the delivery of anticancer drugs to tumor cells within solid tumors. The limitation on the delivery of immune cells to solid tumors is even more stringent. This has a huge impact on the efficacy of immunotherapy. In particular, CD8 + T cells are excluded or trapped by the dense fibrotic extracellular matrix produced by cancer-associated fibroblasts

[16] . The infiltration of CTLs in tumors is related to the efficacy of ICBs.

[0011] In pharmacological approaches to reducing solid stress, tumor-targeted angiotensin receptor blockers have been shown to reduce the activity of cancer-associated fibroblasts and exhibit enhanced efficacy when used in combination with ICB

[17] . For example, combining an angiotensin receptor blocker with an ICB mixture of aCTLA-4 plus aPD-1 extended the median survival of mice with orthotopic 4T1 tumors from 17 days in the control group to 24 days (the median survival of mice treated with aCTLA-4 plus aPD-1 was 20 days)

[17] . Although such pharmacological approaches can reduce solid stress in solid tumors and enhance the efficacy of immunotherapy, they have systemic effects.

[0012] Given the relatively high diffusion coefficients of small molecule drugs, the effects of elevated IFP and solid stress may be considered less important when using small molecule drugs for chemotherapy. However, this consideration overlooks the fact that most of the above small molecule drugs are sparingly soluble or insoluble in aqueous media and bind extensively to plasma proteins (such as albumin, low density and high density lipoproteins) shortly after administration. For example, human serum albumin (HSA) has a molecular weight of approximately 67 kDa, almost half that of an mAb, and small molecule drugs bound to HAS may face the same problems as macromolecular drugs, nanodrugs, biologics, or CTLs when penetrating solid tumors.

[0013] Given that the consequences of vascular normalization and solid stress alleviation are interrelated and both contribute to enhanced drug delivery to solid tumors, "tumor pretreatment" has been proposed to refer to these two strategies

[14] . Photodynamic tumor pretreatment can also enhance the penetration of drugs into solid tumors, in which sub-lethal concentrations of photosensitizers are used to enhance the permeability of tumors to chemotherapy and biologics

[18] . This approach uses spatiotemporal control of the permeability of solid tumors with low toxicity photosensitizers, but remains a pharmacological approach that requires the use of additional drugs. Acoustic tumor pretreatment using focused ultrasound has also been described

[19] . Applying focused ultrasound to biological tissues produces thermal and cavitation effects, leading to changes in the physiology of target cells.

[19]

[0014] Acoustic pretreatment therapy uses a piezoelectric transducer configured to produce a spatial-peak-temporal-average intensity (I 2 ) of 10 to 1000 W / cm SPTA and ultrasound with a frequency between 0.01 and 10 MHz, and the ultrasound acts continuously on any specific volume of the treatment area within a time range of 0.5 to 5 seconds

[19] . The I SPTA value in acoustic pretreatment therapy greatly exceeds the current FDA limit for ultrasound diagnostic output: I (SPTA) <0.72 W / cm 2 .

[0015] Current pre-treatment drug therapies for solid tumors have achieved some success, but there are also off-target effects. Radiological solid tumor pre-treatment methods increase the body's exposure to harmful radiation. Acoustic tumor pre-treatment requires ultrasonic waves with a spatial-peak-temporal-average intensity that greatly exceeds the output safety limits set by the FDA for diagnostic ultrasound. This disclosure for the first time involves using pressure pulses with a spatial peak temporal average intensity within the output safety limits set by the FDA for diagnostic ultrasound to achieve solid tumor pre-treatment and enhance the tumor's response to drug therapy.

[0016] Optical pressure Summary of the Invention

[0017] This disclosure discloses the use of a Pressure Pulse Tumor Pre-Treatment Therapy (PPTPT). In one embodiment, the PPTPT utilizes laser light pulses and piezoelectric (opto-pressure) transducers to generate high-pressure and broadband photoacoustic waves that pass through solid tumors and enhance the penetration of chemical, biological, immunological agents, or any combination thereof. In another embodiment, the PPTPT uses laser light pulses to ablate the surface of a material and generate shock waves that pass through the solid tumor, promoting the penetration of chemical, biological, immunological agents, or any combination thereof in the tumor. The PPTPT combines solid tumors exposed to pressure pulses with chemotherapy, biological, or immunotherapy. The main role of the pressure pulse is to pre-treat the solid tumor, enabling chemical / biological / immunological agents to penetrate deep into the solid tumor, thereby enhancing the tumor's response to chemical, biological, immunological agents, or any combination thereof.

[0018] According to one aspect of the present disclosure, there is provided a device for tumor pre-treatment by pressure pulses, the device comprising: a pulsed laser system with a pulse repetition rate between 0.1 Hz and 100 Hz; an optical waveguide configured to direct laser pulses to one or more opto-pressure transducers; one or more opto-pressure transducers configured to absorb laser pulses from the pulsed laser system and generate pressure pulses, wherein the peak compression pressure of the pressure pulse is between 0.1 MPa and 100 MPa, and the 90% duration of each pressure pulse is between 0.1 ns and 500 ns; a tumor positioning support structure configured to couple one or more opto-pressure transducers to a selected area of the solid tumor, not more than 3 cm away from the area; and a control system configured to limit the exposure time of the solid tumor to the pressure pulse between 1 second and 60 minutes.

[0019] In a further embodiment, the optical waveguide comprises one or more optical fibers or light pipes.

[0020] In a further embodiment, the optical waveguide comprises mirrors, lenses, prisms, diffusers, polarizers, or any combination thereof.

[0021] In a further embodiment, the optopneumatic transducer includes a laser light absorption system and a material having a Grüneisen parameter higher than 0.5, wherein each pressure pulse is a wavefront of a photoacoustic wave.

[0022] In a further embodiment, the optopneumatic transducer includes a laser light absorption system and a material having an ablation threshold lower than 200 mJ / cm 2 and wherein each pressure pulse is a wavefront of a shock wave.

[0023] In a further embodiment, the tumor localization support structure is configured to fix one or more optopneumatic transducers together with an acoustic coupling element disposed between the transducer and the surface of the solid tumor.

[0024] In a further embodiment, the tumor localization support structure is an endoscope and the light guide is one or more optical fibers configured to transmit laser light from a light source through the endoscope and to one or more optopneumatic transducers at the distal end of the optical fiber.

[0025] In a further embodiment, the endoscope is configured to be inserted into a hollow organ through a natural body opening or through a body incision less than 2 cm in length.

[0026] In a further embodiment, the tumor localization support structure is a catheter and the light guide is one or more optical fibers configured to transmit laser light from a light source through the catheter to one or more optopneumatic transducers at the distal end of the optical fiber.

[0027] In a further embodiment, the catheter is configured to be inserted into a body cavity, duct, blood vessel, brain, skin, or adipose tissue.

[0028] In a further embodiment, the tumor localization support structure includes a sharp tip configured to enable one or more optopneumatic transducers to be inserted into the solid tumor.

[0029] According to another aspect of the present disclosure, there is provided a method for treating a solid tumor in a cancer subject, the method comprising: exposing the solid tumor to one or more pressure pulses and performing pressure pulse tumor pretreatment, wherein the peak compression pressure of the pressure pulse is between 0.1 MPa and 100 MPa and the 90% duration of each pressure pulse is between 1 ns and 500 ns; and administering to the subject one or more anti-cancer therapeutic agents, thereby treating the solid tumor in the cancer subject. In a further embodiment, the pressure pulse tumor pretreatment of the solid tumor is performed using the device according to the present disclosure.

[0030] In a further embodiment, the method further comprises the step of repeating at least once the pressure pulse tumor pretreatment, administering one or more anti-cancer therapeutic agents, or both, at a dose that improves the response of the solid tumor to the treatment.

[0031] In a further embodiment, the anti-cancer therapeutic agent is selected from the group consisting of inhibitors of inhibitory checkpoint molecules, activators of stimulatory checkpoint molecules, antibodies, cytokines, interferons, interleukins, vaccines, oncolytic viruses, chimeric antigen receptor T cells, and any combination thereof.

[0032] In a further embodiment, the therapeutic agent is a biotherapeutic agent.

[0033] In a further embodiment, the therapeutic agent is a monoclonal antibody (mAb) for treating cancer, or any combination of mAbs for treating cancer.

[0034] In a further embodiment, the therapeutic agent is selected from the group consisting of ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab, sintilimab, or any combination thereof.

[0035] In a further embodiment, the therapeutic agent is a cytostatic or cytotoxic drug that binds to plasma proteins.

[0036] In a further embodiment, the therapeutic agent is a macromolecule.

[0037] In a further embodiment, the therapeutic agent is a nanodrug.

[0038] According to another aspect of the present disclosure, there is provided an apparatus including means for generating pressure pulses having a peak compression pressure of 0.1 MPa to 100 MPa, and a 90% duration of each pressure pulse being between 1 ns and 500 ns, for treating solid tumors in a cancer subject.

[0039] In a further embodiment, the apparatus is the apparatus according to the present disclosure.

[0040] According to another aspect of the present disclosure, there is provided a system including at least one apparatus according to the present disclosure.

[0041] In a further embodiment, the anti-cancer therapeutic agent is selected from the group consisting of inhibitors of inhibitory checkpoint molecules, activators of stimulatory checkpoint molecules, antibodies, cytokines, interferons, interleukins, vaccines, oncolytic viruses, chimeric antigen receptor T cells, and any combination thereof.

[0042] In a further embodiment, the therapeutic agent is a biotherapeutic preparation.

[0043] In a further embodiment, the therapeutic agent is a monoclonal antibody (mAb) for treating cancer, or any combination of monoclonal antibodies for treating cancer.

[0044] In a further embodiment, the therapeutic agent is selected from the group consisting of ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab, sintilimab, or any combination thereof.

[0045] In a further embodiment, the therapeutic agent is a cytostatic or cytotoxic drug that binds to plasma proteins.

[0046] In a further embodiment, the therapeutic agent is a macromolecule.

[0047] In a further embodiment, the therapeutic agent is a nanodrug.

[0048] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present disclosure, exemplary methods and / or materials are described below. In case of conflict, the patent specification shall prevail, including the definitions therein. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting necessarily.

[0049] Further embodiments and the full scope of applicability of the present disclosure will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Some embodiments of the present disclosure are described only by way of example in the drawings. It is to be emphasized that the details shown in the drawings are by way of example only and for the purpose of illustrative discussion of embodiments of the present disclosure. In this regard, the description in conjunction with the drawings enables those skilled in the art to clearly understand how embodiments of the present disclosure will be practiced. The dimensions of the components and features in the drawings are chosen for convenience and clarity and are not necessarily shown to scale.

[0051] Figure 1A -B presents a piezoelectric material made of carbon nanoparticles and PDMS at a laser flux of ( Figure 1A) Approximately 60 mJ / cm 2 and Figure 1B ) approximately 126 mJ / cm 2 The absolute pressure pulses generated under excitation with a duration of 8 ns were detected using a hydrophone calibrated in the range of 1 to 30 MHz;

[0052] Figure 2 The Fourier transform of the stress wave generated by the piezophotonic material composed of carbon nanoparticles and polymers under laser fluence of approximately 60 mJ / cm 2 and excitation with a duration of 8 ns was measured using a 225 MHz contact transducer;

[0053] Figure 3 The in vitro viability of immortalized monkey fibroblasts (COS-7) in control (CTR) cell culture plates and plates exposed to photoacoustic waves for 5 minutes (5 min) or 10 minutes (10 min) was presented. The laser repetition rate was 6 Hz or 20 Hz. Exposure to up to 12,000 pressure pulses did not impair cell viability;

[0054] Figure 4 The magnetic resonance imaging (MRI) of the neck tissue of a Sprague Dawley rat was presented. In photoacoustic waves with a pulse repetition frequency of 20 Hz and a peak compressive pressure of approximately 3 MPa, the left side was exposed 5 times a week for 5 minutes each time for 4 weeks. No differences were observed on the left side compared to the right side not exposed to photoacoustic waves. No adverse effects were observed in the area where photoacoustic waves were applied;

[0055] Figure 5 The hematoxylin-eosin staining of the tissue in the left carotid artery region of a Sprague Dawley rat exposed to photoacoustic waves with a peak compressive pressure of approximately 3 MPa and a pulse repetition frequency of 20 Hz, 5 times a week for 5 minutes each time for 4 weeks was presented. No abnormalities were found in the carotid artery and surrounding tissues. No adverse effects were observed in the area where photoacoustic waves were applied;

[0056] Figure 6 is a cross-sectional schematic diagram of a tumor pretreatment device according to some embodiments of the present disclosure, not to scale. The device includes a pulsed laser system (1) with a control system (6) to limit the number of generated laser pulses, an optical waveguide (2) for guiding the laser pulses to the photoacoustic converter (3). The tumor positioning support structure (4) places the photoacoustic conversion transducer (3) near a selected area of a solid tumor (5) growing in the middle of healthy tissue (7). The photoacoustic converter absorbs each pressure pulse generated by a laser pulse, passes through a small section (less than 3 cm) of tissue or acoustic coupling medium, and then passes through at least part of the tumor mass;

[0057] Figure 7A cross-sectional schematic view of a tumor pretreatment device according to some embodiments of the present disclosure, not to scale, for generating pressure pulses near a solid tumor using an endoscope, including a pulsed laser system (1) with a control system (6) to limit the number of generated laser pulses, an optical waveguide (2) for guiding the laser pulses to an optoacoustic transducer (3). The tumor positioning support structure (4) is an endoscope that can place the optoacoustic transducer (3) within 3 cm of a selected area of a solid tumor (5) growing in the abdominal healthy tissue. Each pressure pulse is guided to the optoacoustic transducer, where a pressure pulse is generated that passes through the intestinal wall and reaches the tumor;

[0058] Figure 8 A cross-sectional schematic view of a tumor pretreatment device according to some embodiments of the present disclosure, not to scale, for generating pressure pulses near a solid tumor using a catheter, including a pulsed laser system (1) with a control system (6) to limit the number of generated laser pulses, an optical waveguide (2) that may have a lens at the distal end for guiding the laser pulses to an optoacoustic transducer (3). The tumor positioning support structure (4) is a urinary catheter with an inflatable balloon (8) that can place the optoacoustic transducer (3) within 3 cm of a selected area of a solid tumor (5) growing in the bladder. Each pressure pulse is guided to the optoacoustic transducer, where a pressure pulse is generated that passes through the bladder and reaches the tumor; and

[0059] Figure 9 A Kaplan-Meier plot of BALB / c mice with orthotopic 4T1 tumors. Control group (dashed line, no pretreatment and no treatment), group with tumors exposed to photoacoustic waves (dotted line, with pretreatment but no treatment), group receiving intraperitoneal injection of aCTLA4 for treatment (solid line, no pretreatment but with treatment), and group with tumors exposed to light waves and intraperitoneal injection of aCTLA4 for treatment (dash-dotted line, with pretreatment and treatment). Day 0 is the date when the procedure started, and tumors with a longest diameter of at least 3 mm were selected. Mice were sacrificed when the longest diameter of the tumor reached 12 mm. Detailed implementation manners

[0060] Definitions

[0061] The following definitions apply to the present disclosure:

[0062] The term "therapeutic agent" refers to small molecule drugs, biopharmaceuticals or immune cells that can be used to treat tumors, including chemotherapeutic drugs, radiosensitizers, photosensitizers, nanoparticles, senescent cell scavengers, biologics, immunomodulators, immune molecules or CD8 that are activated by tumor antigens and not restricted by negative regulators +T cells. The therapeutic agent can be a drug that has been approved by a regulatory agency for treating tumors or cancers, a drug that is undergoing clinical trials awaiting regulatory approval, or a drug that is being studied for treating tumors or cancers.

[0063] The term "small molecule drug" refers to an organic compound having a molecular weight equal to or less than 1 kDa. This term includes drugs having the desired pharmacological properties, including compounds that are orally administrable or injectable. Small molecule drugs include cytostatic or cytotoxic agents for cancer chemotherapy.

[0064] The term "photosensitizer" refers to a dye that may bind to a target molecule and has no detectable therapeutic effect in the electronic ground state but can trigger a process that ultimately leads to cell death, such as the generation of reactive oxygen species in cancer photodynamic therapy and photoimmunotherapy when electronically excited.

[0065] The term "macromolecule" refers to an organic molecule or bioorganic molecule having a molecular weight greater than 1 kDa, which can be a protein, bioconjugate, RNA molecule, or DNA molecule, or a fragment of the above molecules.

[0066] The term "biological agent" or "biotherapeutic agent" refers to a diverse group of drugs, including vaccines, growth factors, immunomodulators, monoclonal antibodies, and products derived from human blood and plasma. This definition specifically includes proteins purified from living culture systems or blood

[0067] The term "immunomodulator" refers to checkpoint inhibitors, co-stimulators of immune pathways, antibodies targeting immune cell antigens and / or cancer antigens, and cell therapy methods (such as adoptive cell transfer with genetically modified receptors, such as chimeric antigen receptor therapy), specifically including immunomodulators such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab, and sintilimab.

[0068] The term "nanodrug" refers to a nanoparticle drug delivery system in which more than 50% of the particles, by number particle size distribution, range from 1 nm to 500 nm in one or more external dimensions and contain small molecule drugs, photosensitizers, or macromolecules.

[0069] The term "tumor preconditioning" refers to the normalization of blood vessels and the relief of solid stress in solid tumors, including the subsequent or simultaneous reduction of the interstitial fluid pressure within the solid tumor to facilitate the infiltration of therapeutic drugs into the solid tumor.

[0070] The term "photoacoustic transducer" or "photo-pressure converter" refers to a material that can absorb a large amount of pulsed laser light and convert the absorbed light energy into a pressure pulse.

[0071] The term "pressure pulse" refers to a perturbation that carries a transient density change within its propagation medium. The definition of "pressure pulse" clearly encompasses shock waves and photoacoustic waves, which are also collectively referred to as "stress waves".

[0072] The term "pressure-pulse tumor induction" refers to tumor preconditioning by stress waves (which can be photoacoustic waves and / or shock waves).

[0073] The terms "comprising", "including", "having" and their conjugations mean "including but not limited to".

[0074] The term "example" is used herein to mean "as an example, instance or illustration". Any embodiment described as an "example" is not necessarily to be understood as more preferred or advantageous than other embodiments, and / or does not exclude incorporating features from other embodiments.

[0075] Throughout this application, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, the description of a range should be considered to have expressly disclosed all possible sub-ranges as well as individual numerical values within that range. For example, the description of a range such as from 1 to 6 should be considered to have expressly disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numerical values within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0076] Any numerical range recited herein is meant to include any cited numeral (fractional or integral) within the recited range. The phrases "between a first recited numeral and a second recited numeral" and "from a first recited numeral to a second recited numeral" may be used interchangeably herein and are meant to include the first and second recited numerals and all the decimal and integral numerals therebetween.

[0077] As used herein, the term "treating" or "curing" a disease, disorder or condition includes alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder or condition is completely cured. To be an effective treatment, the active ingredient in the present disclosure only needs to reduce the severity of the disease, disorder or condition, reduce the severity of the related symptoms, or improve the quality of life of the patient or subject.

[0078] As used herein, the term "subject" refers to an animal, more specifically a non-human mammal, a human, and a human organism. Non-human animal subjects may also include prenatal forms of animals, such as embryos or fetuses. Non-limiting examples of non-human animals include: horses, cows, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, pigs. In one embodiment, the subject is a human.

[0079] It should be understood that certain features of the present disclosure described in the context of separate embodiments for clarity can also be provided in a single embodiment in combination. Conversely, the various features of the present disclosure described in the context of a single embodiment for brevity can also be provided separately or in any suitable sub-combination, or appropriately provided in any other described embodiment of the present disclosure. Certain features described in the context of various embodiments should not be considered as essential features of these embodiments unless the embodiment cannot operate without these elements.

[0080] Description

[0081] According to some embodiments, the present disclosure provides devices and methods for pretreating solid tumors by absorbing pressure pulses generated by laser pulses with piezoelectric photon materials, thereby improving the therapeutic effect of administering therapeutic drugs.

[0082] Optical pressure, optical pressure, optical pressure. Prior to the present disclosure, it was completely unexpected that pressure pulses with a spatial peak temporal average intensity lower than the output safety limit set by the FDA for diagnostic ultrasound, although very well tolerated by normal tissues, could alter the TME. Example 1 shows a typical stress wave generated by the absorption of a laser pulse in an optical pressure (piezoelectric photon) transducer. Examples 2 and 3 show that such stress waves are safe for in vitro cells and in vivo tissues. The pressure pulses of these stress waves do not produce any detectable effect on normal tissues, which is consistent with the fact that their spatial peak temporal average intensity is lower than the output safety limit set by the FDA for diagnostic ultrasound. The pressure pulses are completely safe and do not alter normal tissues. However, as shown in Example 4, exposing solid tumors to the pressure pulses results in pretreatment of the solid tumors and enhanced response to anti-cancer therapeutic drugs. Those skilled in the art could not anticipate that pressure pulses well tolerated by normal tissues are very effective in the pretreatment of solid tumors.

[0083] According to some embodiments, the present disclosure provides an apparatus for tumor pretreatment by pressure pulses, comprising: a pulsed laser system; an optical waveguide configured to direct laser pulses to one or more photoacoustic transducers; one or more photoacoustic transducers configured to absorb laser pulses from the pulsed laser system and generate pressure pulses; a tumor positioning support structure configured to couple one or more photoacoustic transducers to a selected region of a solid tumor; and a control system configured to limit the exposure of the solid tumor to the pressure pulses.

[0084] In some embodiments, the pulse repetition rate of the pulsed laser system is between 0.1 Hz and 100 Hz.

[0085] In some embodiments, the apparatus includes a control system configured to limit the exposure time of the solid tumor to the pressure pulses to 1 second (s) to 60 minutes (min).

[0086] In some embodiments, the optical waveguide configured to direct laser pulses to one or more photoacoustic transducers includes one or more optical fibers or light pipes.

[0087] In some embodiments, the optical waveguide includes mirrors, lenses, prisms, diffusers, or polarizers, or any combination thereof.

[0088] In some embodiments, the tumor positioning support structure is configured to couple one or more photoacoustic transducers to an acoustic coupling element placed between the transducer and the surface of the solid tumor.

[0089] In some embodiments, the pressure pulse is a photoacoustic wave. In some embodiments, it is a shock wave. The pretreatment of a solid tumor by pressure pulses involves exposing the solid tumor to one or more pressure pulses.

[0090] The photoacoustic wave is a high-pressure ultrasonic pulse that can reach a peak compression pressure p max = 15 MPa and a frequency exceeding 100 MHz. The instantaneous peak intensity,

[0091] I = p max 2 / (ρv),

[0092] where is the density of the medium (water: ρ = 997 kg / m 3 ), and v is the speed of sound in the same medium (water: v = 1480 m / s). The instantaneous peak intensity of such a photoacoustic wave is very high: I = 15 kW / cm 2However, since the pulse duration of the photoacoustic wave is similar to that of the laser pulse, and the laser repetition rate of a laser pulse with mJ energy is usually a few hertz, the time to reach the peak intensity is very short. For example, the duty cycle of a laser pulse with a duration of 10 nanoseconds and a frequency of 10 hertz is 10 -7 . This means that even if the instantaneous peak intensity is 15 kW / cm 2 , the spatial-peak-temporal-average intensity is only ISPTA = 1.5 mW / cm 2 . Such photoacoustic waves are safe, below the ultrasonic diagnostic output limit of 0.72 W / cm 2 specified by the FD. They do not cause cavitation, and their effects are mainly mechanical. A pulse duration of nearly 500 ns and a pulse repetition rate of 100 Hz are required to achieve a duty cycle of 5x10 -5 and approach the safety limit of the FDA for ultrasonic diagnosis.

[0093] What shock waves and photoacoustic waves have in common is that they temporarily change the density inside the material during propagation. The difference between them is that the propagation speed of the shock wave is higher than the speed of sound inside the material. Here, photoacoustic waves and shock waves are collectively referred to as "pressure pulses". Those skilled in the art know that shock waves can be generated by various processes, including explosions, objects hitting the surface, objects moving at supersonic speeds, or intense pulsed laser ablation of a target. In the context of the present disclosure, shock waves generated by intense laser pulses are of particular interest. The laser flux rate (in W / m^2^) required to generate a plasma and thus a shock wave is higher than the flux rate required for thermoelastic expansion and thus the generation of photoacoustic waves. This means that, when using the same material, the shock wave generated by a pulsed laser usually has a higher peak pressure than the photoacoustic wave. However, the peak intensities of both shock waves and photoacoustic waves are reached in a very short time. Therefore, a low pulse repetition rate (≤100 Hz) can result in a very low duty cycle, and when the pressure pulse is generated by a laser pulse with a nanosecond duration, a pressure pulse of up to 100 MPa can be used without causing significant damage to the tissue.

[0094] According to some embodiments of the present disclosure, a superficial solid tumor can be exposed to pressure pulses by directly placing a material that absorbs laser pulses above the tumor or above the skin layer covering the tumor, and achieving good acoustic coupling with the skin and the tumor. The laser pulse is directed onto the material, and the energy of the laser pulse is absorbed by the material, generating photoacoustic waves or shock waves on the material, passing through the material and reaching the skin and the tumor. Good acoustic coupling can be achieved by appropriately matching the acoustic impedance of the material with that of human tissue, and an acoustic coupling gel layer can be used to improve it. From the thermoelastic expansion that generates photoacoustic waves to ablation that generates shock waves, it mainly depends on the ablation limit of the material, the energy of the laser pulse, and the size of the irradiation area. Please refer to Example 1 in the example section of the present disclosure, which describes the method of generating photoacoustic waves and its characteristics.

[0095] In many clinical situations, solid tumors are not superficial, that is, located more than 3 centimeters below the body surface. In this case, the stress waves generated on the body surface may be strongly attenuated by healthy tissues before reaching the tumor mass, thus losing the guiding effect on the tumor. According to FDA regulations, the attenuation of ultrasonic waves in tissues can be calculated using a derating factor of 0.3 dB / (cm MHz). This means that at a distance of 3 centimeters from a 3.3 MHz transducer, the attenuation time-intensity ratio of ultrasonic waves is 3 decibels lower (i.e., half) than the value measured in water. However, the higher the frequency, the greater the impact on tumor induction. If the same derating factor is applied to a 33 MHz frequency at 3 centimeters, the derated time-averaged intensity is 30 decibels (i.e., 0.001) of the value measured in water. This indicates that the photoacoustic transducer must be placed within 3 centimeters of the surface of the solid tumor for the solid tumor to be significantly affected by the stress waves. Part of the present disclosure is based on a surprising discovery that normal cells and healthy tissues are not affected by stress waves. Please refer to Examples 2 and 3 in the embodiment section of the present disclosure, which show that normal cells and healthy tissues are not affected by stress waves.

[0096] In some embodiments, even if the solid tumor is not superficial, an endoscopic examination method can be used to approach the tumor through a natural body cavity. Minimally invasive surgery can be used to generate stress waves near solid tumors in the gastrointestinal tract, respiratory tract, urinary tract, or female reproductive system. Since the endoscope has a channel for inserting optical fibers, stress waves can be generated near solid tumors in these locations. Additionally, in surgeries such as laparoscopy or thoracoscopy, optical fibers can also enter the normally closed body cavity through small incisions less than 2 centimeters in length. Furthermore, optical fibers can be inserted into catheters to reach many desired locations in the human body.

[0097] According to some embodiments of the present disclosure, a laser can be delivered near a solid tumor using an optical fiber. Non-limiting examples of solid tumors that can be reached using an optical fiber include gastric cancer, intestinal cancer, lung cancer, breast cancer, uterine cancer, esophageal cancer, ovarian cancer, pancreatic cancer, pharyngeal cancer, sarcoma, liver cancer, bladder cancer, upper jaw cancer, head and neck cancer, gastric cancer, pancreatic cancer, bladder cancer, liver cancer, lung cancer, breast cancer, uterine cancer, esophageal cancer, ovarian cancer, pancreatic cancer, pharyngeal cancer, sarcoma, liver cancer, bladder cancer, upper jaw cancer, bile duct cancer, head and neck cancer, tongue cancer, brain tumor, skin cancer, malignant goiter, prostate cancer, colorectal cancer, parotid gland cancer, and kidney cancer.

[0098] In certain embodiments, when delivering a laser to a solid tumor using an optical fiber, the laser is directed to the proximal end of the optical fiber, and a piezophotonic converter can be coupled to its distal end near the solid tumor. The piezophotonic converter absorbs most of the intensity of the laser pulse and generates a stress wave. A stress wave is generated whenever a laser pulse is absorbed by the piezophotonic converter. Under thermal confinement and stress confinement conditions, the stress wave can be generated by the thermoelastic expansion of the piezophotonic converter, in which case the stress wave is a photoacoustic wave. The stress wave may be generated by ablation of the piezophotonic converter, which means that some material of the converter is removed or damaged, in which case the stress wave is a shock wave. In either case, the stress wave propagates through the piezophotonic converter, from the side that absorbs the laser pulse to the other side, and then is transmitted to the tissue near the solid tumor or directly to the solid tumor.

[0099] According to the present disclosure, a piezophotonic converter can be made of a variety of dyes or pigments, but to generate a pressure pulse with a high peak compression amplitude, the dye or pigment must have a high absorption coefficient (μ) at the laser pulse wavelength and be able to quickly and efficiently convert the absorbed light energy into heat energy. In addition, when generating a photoacoustic stress wave, the dye or pigment is preferably incorporated into a material with a high Grüneisen parameter (G > 0.5), because the peak pressure of the photoacoustic wave is

[0100] p0 = ΓμF,

[0101] where F is the local light flux. When generating a stress wave of a shock wave, the dye or pigment is preferably combined with a material having a low ablation threshold. For example, polyethylene terephthalate, polyimide, and triazine polymers are such cases. These polymers can be used to produce piezophotonic materials that generate shock waves during ablation at a laser fluence below 200 mJ / cm 2 of the laser flux.

[0102] According to the present disclosure, non-limiting examples of dyes or pigments that can be used to fabricate piezophotonic materials include: ortho-hydroxybenzophenone and similar molecules that undergo ultrafast photoinduced intramolecular proton or hydrogen atom transfer and quickly return to the original ground state; manganese of tetraphenylporphyrin IIIComplexes and other paramagnetic complexes that have ultrafast metal-ligand and / or ligand-metal charge transfer relaxation processes, complexes with charge transfer bands that can return to the ground state via ultrafast charge recombination; β-carotene and other systems that can rapidly decay to the ground state via conical intersections; graphite or carbon nanoparticles or carbon nanotubes or carbon soot and other materials that can ultrafast transfer electronic energy to phonon modes and then cool on a sub-nanosecond timescale; semiconductor materials with short-lived transients, or other materials or material mixtures with ultrafast non-radiative relaxation processes. When ablation occurs, in addition to converting light energy into heat energy, the structural volume of dyes and pigments may also change, thereby increasing the intensity of the stress wave.

[0103] In some embodiments, the photoacoustic transducer comprises a laser light absorption system and a material having a Grüneisen parameter greater than 0.5, wherein each pressure pulse is the wavefront of a photoacoustic wave.

[0104] Some non-limiting examples of materials having a high Grüneisen parameter (G > 0.5) include polymers (polydimethylsiloxane, polystyrene, polyamide, polyvinyl chloride, polyethylene, polyacrylonitrile, polyethylene terephthalate, polychloroprene, p-xylene), metal films, glass, and layered materials containing these materials. These materials can absorb the light of the laser pulse, or are designed to incorporate dyes or pigments that absorb the light of the laser pulse, and the optical path is very short, which is very convenient for manufacturing piezoelectric photon materials for endoscopes and catheters.

[0105] In some embodiments, the photoacoustic transducer comprises a laser light absorption system and a material having an ablation threshold below 200 mJ / cm2, wherein each pressure pulse is the wavefront of a shock wave.

[0106] In certain embodiments, the piezoelectric transducer made of a dye or pigment and a material having a high Grüneisen parameter or a low ablation threshold can have various forms and shapes. Considering that the dye or pigment must have a high absorption coefficient, the piezoelectric photon converter can absorb most of the laser pulse in an optical path shorter than 200 microns, or preferably shorter than 100 microns, or most preferably shorter than 50 microns. Given the very small thickness of the piezoelectric photon converter, they can be used to cover the distal end of the optical fiber.

[0107] In certain embodiments, the peak compressive pressure of the stress wave is between 0.1 MPa and 100 MPa.

[0108] In certain embodiments, 90% of the duration of each pressure pulse is between 0.1 ns and 500 ns.

[0109] Please refer to Figure 6 、 Figure 7 and Figure 8, illustrating various embodiments of the device according to the present disclosure, wherein the piezoelectric transducer is coupled to the distal end of the optical fiber.

[0110] In certain embodiments, the optical fiber is fixed by a tumor localization support structure that can direct laser pulses and the photoacoustic transducer to a solid tumor less than 3 centimeters from the body surface ( Figure 6 ). In another embodiment, the optical fiber is inserted into an endoscope, serving as a tumor localization support structure, and its distal end is optically connected to an optical diffuser that is at least partially coated with a piezoelectric photon converter located within 3 centimeters of the solid tumor ( Figure 7 ). In certain embodiments, the optical fiber is inserted into a catheter and, with the assistance of an airbag, serves as a tumor localization support structure with a lens at its distal end that can direct laser pulses to a piezoelectric photon converter within 3 centimeters of the solid tumor ( Figure 8 ). In another embodiment, the optical fiber is connected to an optical diffuser coated with a piezoelectric photon converter and inserted into the solid tumor. The system for puncturing the tumor and inserting the photoacoustic converter is the tumor localization support structure, which has a tip.

[0111] In some embodiments, the tumor localization support structure is an endoscope and the light guide is one or more optical fibers configured to transmit laser light from a light source through the endoscope to one or more photoacoustic transducers at the distal end of the optical fiber.

[0112] In certain embodiments, the endoscope is configured to be inserted into a hollow organ through a natural body opening or an incision in the body less than 2 centimeters in length.

[0113] In some embodiments, the tumor localization support structure is a catheter and the light guide is one or more optical fibers configured to transmit laser light from a light source through the catheter to one or more photoacoustic transducers at the distal end of the optical fiber.

[0114] In certain embodiments, the catheter is configured to be inserted into a body cavity, duct, blood vessel, brain, skin, or adipose tissue.

[0115] In some embodiments, the tumor localization support structure includes a sharp tip configured to enable one or more photoacoustic converters to be inserted into a solid tumor.

[0116] Tumor pretreatment using pressure pulses includes placing a piezoelectric material within 3 cm of the solid tumor, where the path between the piezoelectric material and the solid tumor is filled with a medium capable of transmitting pressure pulses, exposing the piezoelectric material to a laser pulse that generates a peak compressive pressure of 0.1 to 100 MPa in the piezoelectric material, and directing such a pressure pulse to at least a portion of the solid tumor for a time ranging from 1 second to 1 hour. The duration (full width at half maximum) of the laser pulse can be femtosecond, picosecond, or nanosecond. The duration of the laser pulse is preferably less than 500 nanoseconds because it is easier to meet the thermal confinement conditions under these conditions, and 90% of the duration of the pressure pulse is less than 500 nanoseconds.

[0117] In some embodiments, the time for which the solid tumor is exposed to the pressure pulse can be a short time (e.g., 1 second), a long time (e.g., 1 hour), or an intermediate time. The exposure of the solid tumor to the pressure pulse can be performed before, during, or after the administration of a therapeutic agent and can be timed according to the plasma lifetime of the therapeutic agent.

[0118] This disclosure is in part based on the discovery that in the case of repeatedly administered therapeutic agents and / or therapeutic agents with a long plasma half-life, the exposure of the solid tumor to the pressure pulse can be performed multiple times, which can be several times a day, several times a week, several times a month, or several times a year.

[0119] This disclosure is in part based on the discovery that the pressure pulse solid tumor pretreatment described herein promotes the penetration of therapeutic agents into solid tumors and enhances the response to treatment. Pretreatment of solid tumors by exposure to pressure pulses enhances the penetration of various therapeutic agents into solid tumors without affecting the delivery of therapeutic agents to healthy host tissues and without enhancing host toxicity. It is particularly valuable for delivering small molecule drugs and macromolecular drugs that are extensively bound to plasma proteins, especially biopharmaceuticals. This disclosure is in part based on the discovery that pressure pulse pretreatment of solid tumors improves the efficacy of immunotherapy, especially when the therapeutic agent is an mAb used for ICB therapy. Pressure pulse solid tumor pretreatment promotes the penetration of tumor antigen-specific T lymphocytes into tumors and their integration with the tumor microenvironment (TME), contributing to enhancing the response of tumors to immunotherapy. This disclosure is in part based on this unexpected discovery that pressure pulses well tolerated by normal tissues are effective in solid tumor pretreatment.

[0120] According to some embodiments, the present disclosure provides a method of treating a tumor in a subject. In some embodiments, the method comprises administering to the subject (i) a quantity of high-intensity photoacoustic waves and (ii) a quantity of a therapeutic agent, wherein the quantities of (i) and (ii) together are sufficient to treat a solid tumor, and the order of administration can be selected as (i) before (ii), (i) simultaneous with (ii), or (i) after (ii). In some embodiments, a method of sensitizing a tumor in a subject to a quantity of anti-cancer therapy is disclosed, the method comprising administering to the subject a quantity of pressure pulses before or during the anti-cancer therapy, the pressure pulses being effective to improve the response of the tumor in the subject to the anti-cancer therapy administered to the subject. In some embodiments, the subject is a cancer patient.

[0121] According to some embodiments, the present disclosure provides a method of treating a solid tumor in a cancer patient, the method comprising: subjecting the solid tumor to pressure pulse tumor pretreatment by exposing the solid tumor to one or more pressure pulses, wherein the peak compression pressure of the pressure pulses is between 0.1 MPa and 100 MPa, and the 90% duration of each pressure pulse is between 1 ns and 500 ns; and administering to the subject one or more anti-cancer therapeutic agents so as to treat the solid tumor of the cancer subject. In some embodiments, the subject suffering from cancer is a cancer patient.

[0122] In certain embodiments, the step of subjecting the solid tumor to pressure pulse tumor pretreatment by exposing the solid tumor to one or more pressure pulses is carried out before, during, or after administering to the subject one or more anti-cancer therapeutic agents.

[0123] In some embodiments, the method further comprises repeating the steps of (i) pressure pulse tumor pretreatment, (ii) administering one or more anti-cancer therapeutic agents, or both. In some embodiments, the repetition is at least once, at least twice, at least three times, at least five times, or the number of times required to relieve the symptoms of the cancer patient, and the dose is sufficient to improve the response of the solid tumor to the treatment.

[0124] In certain embodiments, the anti-cancer therapeutic agent is selected from the group consisting of inhibitors of inhibitory checkpoint molecules, activators of stimulatory checkpoint molecules, antibodies, cytokines, interferons, interleukins, vaccines, oncolytic viruses, chimeric antigen receptor T cells, and any combination thereof.

[0125] In certain embodiments, the therapeutic agent is a biotherapeutic agent.

[0126] In certain embodiments, the therapeutic agent is a monoclonal antibody (mAb) for treating cancer, or any combination of mAbs for treating cancer.

[0127] In certain embodiments, the therapeutic agent is selected from the group consisting of ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab, sintilimab, and any combination thereof.

[0128] In certain embodiments, the therapeutic agent is a cytostatic or cytotoxic agent that binds to plasma proteins.

[0129] In certain embodiments, the therapeutic agent is a macromolecule.

[0130] In certain embodiments, the therapeutic agent is a nanomedicine.

[0131] According to some embodiments, the present disclosure provides a kit comprising the device as described above and the anti-cancer therapeutic agent as described above.

[0132] According to some embodiments, the present disclosure provides a system comprising at least one device as described above.

[0133] According to some embodiments, the present disclosure provides a device comprising means for generating pressure pulses having a peak compression pressure of from 0.1 MPa to 100 MPa, and a 90% duration of each pressure pulse between 1 ns and 500 ns, for treating solid tumors in a cancer patient. In some embodiments, the device is the device as described above.

[0134] Pressure Pulse Tumor Pretreatment Therapy (PPTPT) combines exposing a solid tumor to pressure pulses (as described above) with administering a therapeutic agent having anti-cancer effects. Refer to Example 4 and Figure 9, which illustrates PPTPT in mice with orthotopic 4T1 breast cancer. 4T1 cells were inoculated into the mammary fat pads of BALB / c mice and allowed to grow to a maximum diameter of 3 mm before intervention began. The pressure pulse tumor preconditioning in this example included exposing the orthotopic tumor to photoacoustic waves for 5 minutes on days 0 and 2. The treatment in this example included intraperitoneal administration of anti-mouse CTLA-4 monoclonal antibody (aCTLA4) on days 0, 2, 6, and 10. The control group showed that there was no statistical difference in the survival rate of mice in the group that received only tumor preconditioning (with preconditioning, without treatment) compared to the control group (without preconditioning, without treatment). In the group that received four administrations of aCTLA4 (without preconditioning, with treatment), only one animal responded to the treatment. In contrast, all animals in the group that received PPTPT (with preconditioning, with treatment) responded to the treatment.

[0135] By comparing with tumor preconditioning using angiotensin receptor blockers

[17] , the remarkable achievement of photoacoustic wave tumor preconditioning can be recognized. The median survival time of mice with orthotopic 4T1 tumors increased from 20 days for the aCTLA4 plus aPD1 combination to 24 days when this combination was combined with angiotensin receptor blocker tumor preconditioning

[17] . In the same animal model, namely mice with orthotopic 4T1 tumors, Example 4 shows that when photoacoustic preconditioning was combined with aCTLA4 treatment, the median survival time increased from 21 days for aCTLA4 treatment to 36 days. Those skilled in the art could not have anticipated that exposing the orthotopic 4T1 tumor to photoacoustic waves in two independent 5-minute sessions could increase the median survival time of mice with orthotopic 4T1 tumors by 15 days, especially considering that the preconditioning with angiotensin receptor blockers only increased the median survival time by 4 days. The orthotopic 4T1 tumor is widely considered to be very difficult to treat, and it is completely unexpected that all mice's response to immunotherapy can be increased by simply exposing the tumor to harmless photoacoustic waves in two 5-minute local sessions.

[0136] This disclosure is partly based on the finding that PPTPT is a novel and surprisingly effective method for enhancing the response of solid tumors to therapeutic drugs. High-intensity broadband stress waves exert mechanical forces at the microscopic level and can reshape the TME. As shown in Example 1, a pressure wave with a peak pressure of about 7 MPa and a related frequency of about 20 MHz corresponds to a change of 50 bar in 10 nanoseconds, or considering the speed of sound propagation in tissue, a change of 50 bar in 15 micrometers. A huge pressure change is generated at the scale of cell size. This disclosure is partly based on the finding that cells can withstand these high pressures, as shown in Example 2, and normal tissues do not show any adverse effects, as shown in Example 3. However, the mechanical forces exerted by these pressure pulses in the TME can achieve microscopic mechanical preconditioning of solid tumors, as shown in Example 4.

[0137] The various embodiments and aspects of the present disclosure, as described above and in the claims section below, have received experimental support in the following examples.

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[0158] Example

[0159] Now, please refer to the following examples, together with the above description, to illustrate some embodiments of the present disclosure in a non-limiting manner.

[0160] Example 1

[0161] Generate photoacoustic waves with a peak pressure of 10 megapascals

[0162] Carbon nanoparticles are a very convenient light absorption system as they strongly absorb light over a large wavelength range from ultraviolet to visible to infrared. Carbon nanoparticles are difficult to disperse in solution. Therefore, 160 mg of carbon nanoparticles produced from candle soot were added to 5 mL of toluene and sonicated for 5 minutes at 60 MHz using a tip sonicator. Immediately after mechanical sonication, 2 mg of polystyrene was added to the suspension and heated to 60 °C in a water bath. Polystyrene has a relatively high Grüneisen parameter (G≈0.7), which is very convenient for fabricating thin piezoelectric photon converters. A polystyrene film with dispersed carbon nanoparticles was made using a mechanical applicator (Elcometer) and dried overnight to allow the residual solvent to evaporate.

[0163] Alternatively, depositing carbon nanoparticles produced from the combustion of paraffin lamps on a borosilicate glass window can also produce a piezoelectric photon converter. The glass window was directly exposed to the flame for 2 minutes to collect soot. Then, a thin layer of soot deposited on the glass window was covered with 0.1 mL of polydimethylsiloxane (PDMS) and treated in a vacuum for 10 minutes to remove air bubbles. Next, a 100 g weight was placed on the system (glass + soot + PDMS) to form a thin layer of PDMS and then exposed to vacuum for another 10 minutes to remove the excess air remaining in the system. Finally, the entire assembled system was placed in an oven at 50 °C and heated overnight to obtain complete curing of the PDMS.

[0164] The piezoelectric photon transducers made of carbon nanoparticles and polystyrene or PDMS as described above were studied under pulsed laser excitation at 1064 nm to determine the characteristics of the pressure pulses they can generate. Laser excitation used a Nd:YAG laser (Monfort M-NANO) and nanosecond pulses to generate photoacoustic waves. Two types of ultrasonic measurements were carried out. An absolute pressure was measured using a 0.2 mm needle hydrophone (Precision Acoustics, model NH0200) with a calibration frequency range from 1 to 30 MHz. The ultrasonic frequency distribution was studied using a 225 MHz contact sensor (Panametrics / Olympus, model V2113). Figure 1A -B shows the absolute pressure pulses measured using the hydrophone at laser energies of ~60 mJ / cm 2 and ~250 mJ / cm (2) . Figure 2 shows the ultrasonic frequency distribution at a laser fluence of ~60 mJ / cm 2 measured using the contact sensor.

[0165] Example 2

[0166] Photoacoustic waves with a peak pressure of 10 MPa are not toxic to fibroblasts in vitro

[0167] The immortalized monkey fibroblast cell line (COS-7) was cultured as a monolayer in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 1% penicillin, and streptomycin (Invitrogen), and cultured at 37 °C in a humidified environment containing 5% CO2. First, COS-7 cells were seeded at a density of 30,000 cells / well in a 12-well culture dish with 2 mL of medium; the medium was changed after 24 hours to allow the cells to grow rapidly; after 48 hours of seeding, the medium was removed and 300 μL of fresh medium was added. Then, the piezoelectric material of Example 1 was immersed in the medium and placed within 3 mm of the surface of the COS-7 cell monolayer. Then, using a Nd:YAG laser (Monfort M-NANO), with a laser repetition rate of 6 Hz or 20 Hz and a laser fluence of ~60 mJ / cm (2) , the cells were exposed to photoacoustic waves for 5 minutes (5 min) or 10 minutes (10 min). Twenty-four hours after the cells were exposed to the photoacoustic waves, cell viability was measured using the Alamar assay.

[0168] Example 3

[0169] Exposing the healthy tissues of rats to photoacoustic waves for 5 minutes per day, 5 days per week for 4 weeks had no adverse effects

[0170] The Portuguese Animal Health Authority approved the animal experiment (DGAV authorization 0420 / 000 / 000 / 2011). Male Sprague Dawley rats (Charles River Laboratories, Barcelona, Spain) were used in this study. The hair on the necks of the rats was removed, and a circle was drawn in the area that would be subjected to the stress wave. Stress wave exposure was performed 5 days a week for 4 weeks. In each exposure, a piezoelectric sensor made of carbon nanoparticles and PDMS and a Monfort M-NANO Nd:YAG laser were used to generate stress waves at a frequency of 20 Hz for 5 minutes. Under the conditions employed, the peak compressive pressure of each pulse was approximately 3 MPa. The acoustic coupling between the piezoelectric transducer and the rat neck was optimized using an ultrasonic gel (EcoSupergel). The carotid artery was imaged using magnetic resonance imaging ( Figure 4 ). After the experiment, histological examinations were performed on neck sections ( Figure 5 ).

[0171] Example 4

[0172] Pressure pulse tumor pretreatment therapy

[0173] The Portuguese Animal Health Authority approved the animal experiment (DGAV authorization 0420 / 000 / 000 / 2011). 4T1 cells (ATCC CRL-2539) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich, Saint-Louis, MO, USA) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (GIBCO TM , Life Technologies, Bleiswijk, The Netherlands), 100 U / mL penicillin, and 100 ng / mL streptomycin (Invitrogen TM , Thermo Fisher Scientific, Grand Island, NY, USA). Tumors were established by orthotopically injecting 20,000 4T1 cells into the right mammary gland of female BALB / c mice, which were approximately 8 to 12 weeks old (20 g). 20,000 4T1 cells were orthotopically injected into the right mammary gland of female BALB / c mice.

[0174] Before tumor inoculation, the abdomen of the mice, i.e., the mammary gland area where the tumor was to be inoculated, was depilated. The piezoelectric transducer used was prepared with carbon nanoparticles and polydimethylsiloxane. The piezoelectric transducer was placed above the tumor, and a layer of acoustic coupling gel was applied between the tumor and the piezoelectric transducer to improve acoustic coupling. Photoacoustic waves were generated by the Monfort M-NANO Nd:YAG laser emitting laser pulses at a laser repetition rate of 20 Hz towards the piezoelectric transducer. Under the conditions used, the peak compressive pressure of each pulse was ~6.5 MPa.

[0175] This protocol used four study groups with 4 or 5 animals per group: (i) a control group with an in situ tumor without tumor pretreatment and treatment; (ii) a pretreatment control group with an in situ tumor pretreated with pressure pulses without treatment; (iii) an anti-mouse CTLA4 treatment group with an in situ tumor without tumor priming and treated with InVivo mAb anti-mouse CTLA4 (CD152); (iv) a tumor priming treatment group with an in situ tumor primed with pressure pulses and treated with InVivo mAb anti-mouse CTLA4 (CD152). Day 0 (zero) was defined as the day of the first treatment, and the tumor diameter in all groups was approximately 3 mm. Day 0 corresponded to 8 days after orthotopic tumor inoculation.

[0176] Group (i) did not undergo tumor pretreatment or treatment, and the tumors grew naturally. Group (ii) had the tumors pretreated by exposing them to photoacoustic waves for 5 minutes on days 0 and 2. Group (iii) received InVivo mAb anti-mouse CTLA4 (CD152) treatment on days 0, 2, 6, and 10 by intraperitoneal injection of InVivoMab anti-mouse CTLA4 (CD152) (Bio Cell, Lebanon, NH, USA). Group (iv) had the same treatment protocol as group (iii), but, like group (ii), had the tumors pretreated 10 minutes after antibody injection on days 0 and 2. The tumors were measured twice a week with calipers, and the animals were euthanized when the tumor diameter reached 12 mm. Figure 9 The survival of the mice to this endpoint is shown.

[0177] Although the disclosure has been described in connection with specific embodiments, it will be apparent to those skilled in the art that various alternatives, modifications, and variations are possible. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0178] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein. In addition, the citation or identification of any reference in this application shall not be construed as an admission that such reference is prior art to the present disclosure. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

1. An apparatus for tumor pretreatment by pressure pulses, comprising: - A pulsed laser system with a pulse repetition frequency between 0.1 Hz and 100 Hz; - An optical waveguide for directing laser pulses to one or more photoacoustic transducers; - One or more photoacoustic transducers configured to absorb laser pulses from the pulsed laser system and generate pressure pulses, wherein the peak compression pressure of the pressure pulses is between 0.1 MPa and 100 MPa, and the 90% duration of each pressure pulse is between 0.1 nanoseconds and 500 nanoseconds; - A tumor localization support structure for coupling one or more photo - acoustic transducers to a selected area of a solid tumor, with a distance to the area less than 3 cm; and - A control system for limiting the exposure time of the solid tumor to the pressure pulses between 1 second and 60 minutes.

2. The apparatus according to claim 1, wherein the optical waveguide comprises one or more optical fibers or light pipes.

3. The apparatus according to claim 1, wherein the optical waveguide comprises mirrors, lenses, prisms, diffusers or polarizers, or any combination thereof.

4. The apparatus according to claim 1, wherein the photoacoustic transducer comprises a laser light absorption system and a material with a Grüneisen parameter higher than 0.5, and each pressure pulse is the wavefront of a photoacoustic wave.

5. The apparatus according to claim 1, wherein the photoacoustic transducer comprises a laser light absorption system and a material with an ablation threshold lower than 200 mJ / cm2, and each pressure pulse is the wavefront of a shock wave.

6. The apparatus according to claim 1, wherein the tumor localization support structure is configured to accommodate one or more photoacoustic sensors, and at the same time accommodate an acoustic coupling element disposed between the sensor and the surface of the solid tumor.

7. The apparatus according to claim 1, wherein the tumor localization support structure is an endoscope, and the optical waveguide is one or more optical fibers configured to transmit laser light from a light source through the endoscope and to one or more photoacoustic transducers located at the distal end of the optical fiber.

8. The apparatus according to claim 7, wherein the endoscope is configured to be inserted into a hollow organ through a natural body opening or through a body incision less than 2 cm in length.

9. The apparatus according to claim 1, wherein the tumor localization support structure is a catheter, and the optical waveguide is one or more optical fibers configured to transmit laser light from a light source through the catheter and to one or more photoacoustic sensors at the distal end of the optical fiber.

10. The apparatus according to claim 9, wherein the catheter is configured to be inserted into a body cavity, duct, blood vessel, brain, skin or adipose tissue.

11. The apparatus according to claim 1, wherein the tumor localization support structure includes a tip configured to enable one or more photoacoustic transducers to be inserted into the solid tumor.

12. A method for treating a solid tumor in a cancer patient, the method comprising – subjecting the solid tumor to pressure-pulse tumor pretreatment by exposing the solid tumor to one or more pressure pulses, wherein the peak compression pressure of the pressure pulse is between 0.1 megapascal and 100 megapascals, and 90% of the duration of each pressure pulse is between 1 nanosecond and 500 nanoseconds; and – administering to the subject one or more anti-cancer therapeutic agents, thereby treating the solid tumor of a cancer patient.

13. The method according to claim 12, wherein the pressure-pulse tumor pretreatment of the solid tumor is performed using the device according to claim 1.

14. The method according to claim 12, further comprising repeating at least once the steps of the pressure-pulse tumor pretreatment, administering one or more anti-cancer therapeutic agents, or both, in a dose that improves the response of the solid tumor to the treatment.

15. The method according to claim 12, wherein the anti-cancer therapeutic agent is selected from the group consisting of inhibitors of inhibitory checkpoint molecules, activators of stimulatory checkpoint molecules, antibodies, cytokines, interferons, interleukins, vaccines, oncolytic viruses, chimeric antigen receptor T cells, and any combination thereof.

16. The method according to claim 12, wherein the therapeutic agent is a biotherapeutic agent.

17. The method according to claim 12, wherein the therapeutic agent is a monoclonal antibody (mAb) for treating cancer, or any combination of mAbs for treating cancer.

18. The method according to claim 12, wherein the therapeutic agent is selected from the group consisting of ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab, sintilimab, or any combination thereof.

19. The method according to claim 12, wherein the therapeutic agent is a cytostatic or cytotoxic agent that binds to plasma proteins.

20. The method according to claim 12, wherein the therapeutic agent is a macromolecule.

21. The method according to claim 12, wherein the therapeutic agent is a nanomedicine.

22. An apparatus comprising means for generating a pressure pulse having a peak compression pressure in the range of 0.1 megapascal to 100 megapascals and a 90% duration of each pressure pulse of 1 nanosecond to 500 nanoseconds for treating a solid tumor in a subject suffering from cancer.

23. The apparatus according to claim 22, wherein the apparatus is the apparatus according to claim 1.

24. A system comprising at least one apparatus according to claim 1.

25. A kit comprising any of the apparatuses according to claims 1 to 11 and an anti-cancer therapeutic agent.

26. The kit according to claim 25, wherein the anti-cancer therapeutic agent is selected from one or more of the following categories: inhibitory checkpoint molecule inhibitors, stimulatory checkpoint molecule agonists, antibodies, cytokines, interferons, interleukins, vaccines, oncolytic viruses, chimeric antigen receptor T cells (CAR-T), and any combination thereof.

27. The kit according to claim 25, wherein the therapeutic agent is a biotherapeutic agent.

28. The kit according to claim 25, wherein the therapeutic agent is a monoclonal antibody (mAb) for treating cancer, or a combination of multiple monoclonal antibodies for treating cancer.

29. The kit according to claim 25, wherein the therapeutic agent is selected from one or more of the following drugs: ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab, sintilimab, or any combination thereof.

30. The kit according to claim 25, wherein the therapeutic agent is a cell inhibitor or cytotoxic drug that binds to plasma proteins.

31. The kit according to claim 25, wherein the therapeutic agent is a macromolecule.

32. The kit according to claim 25, wherein the therapeutic agent is a nanodrug.

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