WO2024042447A1 - Dispositifs et procédés d'amorçage de tumeurs solides avec des impulsions de pression pour renforcer des thérapies anticancéreuses - Google Patents

Dispositifs et procédés d'amorçage de tumeurs solides avec des impulsions de pression pour renforcer des thérapies anticancéreuses Download PDF

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WO2024042447A1
WO2024042447A1 PCT/IB2023/058316 IB2023058316W WO2024042447A1 WO 2024042447 A1 WO2024042447 A1 WO 2024042447A1 IB 2023058316 W IB2023058316 W IB 2023058316W WO 2024042447 A1 WO2024042447 A1 WO 2024042447A1
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tumor
pressure
light
therapeutic agent
solid tumor
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PCT/IB2023/058316
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Luís Guilherme DA SILVA ARNAUT MOREIRA
Maria Inês PIMENTEL MENDES
Diogo António DA CRUZ FIGUEIREDO PEREIRA
Carlos Alberto LOURENÇO DE SERPA SOARES
Celso Manuel DE FIGUEIREDO PAIVA JOÃO
João Pedro FREIRE BARRACA CARDOSO SANTOS
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Laserleap Technologies, S.A.
Universidade De Coimbra
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Priority claimed from US17/892,355 external-priority patent/US20240058061A1/en
Application filed by Laserleap Technologies, S.A., Universidade De Coimbra filed Critical Laserleap Technologies, S.A.
Publication of WO2024042447A1 publication Critical patent/WO2024042447A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/18—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves
    • A61B18/20—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser
    • A61B18/22—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser the beam being directed along or through a flexible conduit, e.g. an optical fibre; Couplings or hand-pieces therefor
    • A61B18/26—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser the beam being directed along or through a flexible conduit, e.g. an optical fibre; Couplings or hand-pieces therefor for producing a shock wave, e.g. laser lithotripsy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/067—Radiation therapy using light using laser light
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/18—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves
    • A61B18/20—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser
    • A61B18/22—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser the beam being directed along or through a flexible conduit, e.g. an optical fibre; Couplings or hand-pieces therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/18—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves
    • A61B18/20—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser
    • A61B18/22—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser the beam being directed along or through a flexible conduit, e.g. an optical fibre; Couplings or hand-pieces therefor
    • A61B18/24—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser the beam being directed along or through a flexible conduit, e.g. an optical fibre; Couplings or hand-pieces therefor with a catheter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0092—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin using ultrasonic, sonic or infrasonic vibrations, e.g. phonophoresis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0601—Apparatus for use inside the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152

Definitions

  • the present disclosure relates generally to the field of remodeling (or priming) of a tumor microenvironment (TME) to enhance the efficacy of anticancer therapeutic agents. More specifically, the disclosure relates to a pressure-pulse tumor-priming device and method of using same, and methods of priming a tumor microenvironment with pressure pulses, thereby enhancing the efficacy of anticancer therapeutic agents.
  • TME tumor microenvironment
  • IBD immune checkpoint blockade
  • mAb monoclonal antibodies
  • TEE immunosuppressive tumor microenvironment
  • CD8 + T cells that are strongly activated by tumor antigens must be unrestrained by negative regulators. These negative regulators have been called “checkpoints” since they detect, resist and reverse overreaction.
  • Inhibitory immunoreceptors 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 Suppressor of T cell Activation).
  • CTLA4 and PD1 are the most potent examples of T cell immune checkpoint molecules known today.
  • CTLA4 molecules are contained within intracellular vesicles in naive T cells and constitutively expressed on the surface of CD4 + CD25 + regulatory T (T reg ) cells.
  • Naive T cells are activated when their T cell receptors bind to their cognate antigen presented by antigen-presenting cells (APCs) in the presence of a co-stimulatory signal.
  • APCs antigen-presenting cells
  • This costimulatory signal is the binding between CD28 expressed on the surface of the T cell with B7 molecules (B7.1, also named CD80, or B7.2, also named CD86) on APCs.
  • APCs are immune cells that process and present antigens for recognition by T cells, and include B lymphocytes, dendritic cells, macrophages and other immune cells.
  • CTLA4 counteracts several internal signaling nodes to impede activation and proliferation of T cells [6], Treg cells, which constitutively express CTLA4, can arrest T cell responses.
  • Treg cells which constitutively express CTLA4
  • CTLA4 is a negative regulator of T cell activity suggested that blocking its actions could rescue T cells response to cancer cells. Indeed, neutralizing anti-CTLA4 mAbs enhance antitumoral immunity.
  • anti-CTLA4 therapy depletes local intra-tumoral T reg cells through antibodydependent cell-mediated cytotoxicity and shifts the balance of the TME away from immunosuppression [7],
  • Human PD1 is expressed on T cells after T cell receptor stimulation, and binds to the B7 homologues PDL1 and PDL2, which are constitutively expressed on APCs and can be induced in non-hematopoietic tissues.
  • PDL1 programmed cell death ligand 1, also known as B7-H1
  • B7-H1 is a transmembrane protein that down-regulates immune responses through binding to its two inhibitory receptors PD1 and B7.1, and is present on many cell types, including T cells, tumor cells, epithelial cells and endothelial cells, much more frequently than PDL2 (programmed cell death ligand 2, also known as B7-DC).
  • PD1 restrains immune responses primarily through inhibitory signaling in CD8 + effector T cells and in T reg cells [7], When PD1 engages its ligands, it can induce a state of T cell dysfunction called T cell exhaustion.
  • Tumor cells can upregulate PD1 ligands.
  • PDL1 expressed by cells in the TME, engages PD1 on T cells and subsequently triggers inhibitory signaling, blocking effector functions and reducing T-cell killing capacity.
  • tumor cells can induce T cell exhaustion and generate a TME that facilitates tumor growth and invasion.
  • anti- PD1 and anti-PDLl antibodies can enhance the functional properties of CD8 + effector T cells at the tumor site.
  • Activation of T cells allows T cell lymphocytes to recognize an antigen on a specific target cell.
  • Activated CD8 + T cells gradually transform into effector T cells (or cytotoxic T lymphocytes, CTLs), which recognize target cells and kill them by distinct pathways.
  • the Fas ligand (or CD95L) expressed on the surface of CTL binds to the Fas receptor (or CD95) on the target cell and triggers apoptosis through the caspase cascade.
  • the CTL releases granulysin, perforins, cathepsin C and/or granzymes into the intercellular space between the CTL and the target cell, which are highly cytotoxic to the target cell.
  • Nanoparticle delivery systems to accumulate therapeutic agents in the tumor using the enhanced permeation retention effect [11]
  • Nanoparticulate drug carriers have been shown to mediate greater tumor diug deposition compared with free diugs, and the delivered drug can persist for days at concentrations that exceed peak tumor concentrations achieved with free diug.
  • nanomedicines tend to accumulate in tissues of the reticuloendothelial system (spleen, liver, lungs) and tend to stay in the vicinity of the tumor vascular fenestrae [12], There is a low probability that nanomedicines reach a majority of target cells within the tumor.
  • the mechanical microenvironment of solid tumors is characterized by elevated interstitial fluid pressure (IFP) and by solid stress.
  • IFP interstitial fluid pressure
  • solid stress is exerted by nonfluid components.
  • IFP is originated by the high vascular permeability of the TME coupled with mechanical compression of downstream blood vessels and draining lymphatic vessels.
  • the solid stress is associated with the hyperproliferation of cancer cells, which exert a force against nearby structural elements of tumor and normal tissue that according to the law of action-reaction, exert a force with equal magnitude but opposite direction.
  • Vessel compression has two consequences for therapeutics: (i) the collapse of blood vessels hinders the access of drugs and immune cells, (ii) the lack of lymphatic vessel function reduces drainage, increases IFP and the transport of large therapeutics (e.g., antibodies that block inhibitory checkpoint molecules) or nanomedicines becomes diffusional and is reduced because of their large size. These consequences are aggravated in immunotherapies that depend on the infiltration of tumor antigen-specific cytotoxic T lymphocytes into solid tumors, including in adoptive T cell therapies and in cancer vaccines.
  • anti-VEGF anti-vascular endothelial growth factor
  • Anti-solid stress strategy is distinct from the vessel normalization strategy, which employs anti -angiogenic agents to prune immature vessels and fortify the remaining vessels. Anti-solid stress aims at decompressing vessels to increase perfusion [15], If it succeeds also in lowering the venous resistance and re-establishing lymphatic drainage, it will also reduce IFP. IFP and solid stress contribute to limit anticancer drugs to reach tumors cells inside a solid tumor.
  • CD8 + T cells can be excluded from, or trapped within, tumors by the dense, fibrotic extracellular matrix produced by cancer-associated fibroblasts [16], The infiltration of CTLs in tumors correlates with the therapeutic efficacy of ICBs.
  • tumor-targeted angiotensin receptor blockers were shown to reduce the activity of cancer-associated fibroblasts, and when combined with ICBs displayed enhanced efficacy [17],
  • angiotensin receptor blockers with an ICB mixture of aCTLA-4 plus aPD- 1 increased the median survival of mice with orthotopic 4T1 tumors from 17 days in controls to 24 days in the combination (the median survival of mice treated with aCTLA- 4 plus aPD-1 was 20 days) [17]
  • pharmacological approaches can reduce solid stress in solid tumors and increase the efficacy of immunotherapies, they have systemic effects.
  • HSA human serum albumin
  • tumor priming was suggested to designate both these strategies [14]
  • Drug penetration into solid tumors was also enhanced using photodynamic tumor priming, where a photosensitizer is employed in sub-tumoricidal concentrations to enhance tumor permeability to chemo and biological agents [18]
  • This offers spaciotemporal control of solid tumor permeability with low-toxicity photosensitizers, but remains a pharmacological approach that requires the use of an additional medicine.
  • Acoustic priming of solid tumors using focused ultrasound has also been described [19]
  • the application of focused ultrasound to biological tissues is associated with the generation of thermal and cavitation effects that cause changes in target cell physiology [19].
  • Acoustic priming therapy uses piezoelectric transducers configured to produce spatial -peak temporal-average intensities (ISPTA) between 10 and 1000 W/cm 2 in the treatment zone and ultrasound with frequencies between 0.01 and 10 MHz, and the ultrasound is applied continuously from a time in the range of 0.5 to 5 seconds for any particular volume of the treatment zone [19],
  • ISPTA spatial -peak temporal-average intensities
  • the values of ISPTA in acoustic priming therapy largely exceed the current FDA output limits for diagnostic ultrasound: ISPTA ⁇ 0.72 W/cm 2 .
  • PPTPT tumor-priming therapy
  • PPTPT uses laser light pulses and piezophotonic (light-to- pressure) transducers to generate high-pressure and broadband photoacoustic waves that traverse a solid tumor and enhance the infiltration of chemo/biological/immunological agents.
  • PPTPT uses laser light pulses to ablate the surface of a material and generate shock waves that pass through a solid tumor and facilitate the infiltration of chemo, biological, immunological agents, or any combination thereof, in the tumor.
  • PPTPTPT combines the exposure of the solid tumor to pressure pulses with the administration of chemotherapy, biologicals or immunotherapies.
  • the pressure pulses work mainly by priming the solid tumor so that the chemo/biological/immunological agents infiltrate deeply into the solid tumor and, consequently, the response of the tumor to the chemo, biological, immunological agents, or any combination thereof, is stronger.
  • a device for tumor priming by pressure pulses comprising: a pulsed laser system with a pulse repetition rate from 0.1 Hz to 100 Hz; a light guide configured to direct laser pulses to one or more light- to-pressure transducers; one or more light-to-pressure transducers configured to absorb laser pulses from the pulsed laser system and generate pressure pulses, where the pressure pulses have peak compressional pressures from 0.1 MPa to 100 MPa, and 90% of each pressure pulse lasts from 0.1 ns to 500 ns; a tumor-positioning support structure configured to couple one or more light-to-pressure transducers with a selected area from a solid tumor, at a distance shorter than 3 cm from the area; and a control system configured to limit a exposure of the solid tumor to the pressure pulses for a period of time from 1 second to 60 minutes.
  • the light guide comprises one or more optical fibers or light pipes.
  • the light guide comprises mirrors, lenses, prisms, diffusers, polarizers, or any combination thereof.
  • the light-to-pressure transducer comprises a laser light absorbing system and a material with a Griineisen parameter higher than 0.5, and wherein each pressure pulse is a wavefront of a photoacoustic wave.
  • the light-to-pressure transducer comprises a laser light absorbing system and a material with an ablation threshold below 200 mJ/cm 2 , and wherein each pressure pulse is a wavefront of a shock wave.
  • the tumor-positioning support structure is configured to hold one or more light-to-pressure transducers together with an acoustic coupling element disposed between the transducers and the surface of a solid tumor.
  • the tumor-positioning support structure is an endoscope and the light guide is one or more optical fibers configured to carry a laser light from a light source through the endoscope, to one or more light-to-pressure transducers at the distal ends of the optical fibers.
  • the endoscope is configured for insertion in a hollow organ through a natural body opening or through an incision in the body with less 2 cm in length.
  • the tumor-positioning support structure is a catheter and the light guide is one or more optical fibers configured to carry a laser light from a light source through the catheter, to one or more light-to-pressure transducers at the distal ends of the optical fibers.
  • the catheter is configured for insertion into a body cavity, duct, vessel, brain, skin or adipose tissue.
  • the tumor-positioning support structure comprises a sharp end configured to enable the insertion of one or more light-to-pressure transducers into the solid tumor.
  • a method for treating a solid tumor in a subject afflicted with cancer comprising: pressure-pulse tumor priming of the solid tumor by exposure of the solid tumor to one or more pressure pulses, wherein the pressure pulses have peak compressional pressures from 0.1 MPa to 100 MPa, and 90% of each pressure pulse lasts from 1 ns to 500 ns; and administering of one or more anticancer therapeutic agents to the subject, thereby treating the solid tumor in a subject afflicted with cancer.
  • the pressure-pulse tumor priming of the solid tumor is performed with the device according to the present disclosure.
  • the method further comprises a step of repeating the pressure-pulse tumor priming, the administering of one or more anticancer therapeutic agents, or both, at least one time with doses that improve a response of the solid tumor to the treatment.
  • the anticancer therapeutic agent is selected from the group consisting of an inhibitor to an inhibitory checkpoint molecule, an activator of a stimulatory checkpoint molecule, an antibody, a cytokine, an interferon, an interleukin, a vaccine, an oncolytic virus, chimeric antigen receptor T cells, and any combination thereof.
  • the therapeutic agent is a biological therapeutic.
  • the therapeutic agent is a monoclonal antibody (mAb) used to treat cancer, or any combination of mAbs used to treat cancer.
  • mAb monoclonal antibody
  • the therapeutic agent is selected from ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab, sintilimab, or any combination thereof.
  • the therapeutic agent is a cytostatic or a cytotoxic drug bound to a plasma protein.
  • the therapeutic agent is a macromolecule.
  • the therapeutic agent is a nanomedicine.
  • a device comprising means for generating pressure pulses having peak compressional pressures from 0.1 MPa to 100 MPa, and 90% of each pressure pulse lasts from 1 ns to 500 ns, for treating a solid tumor in a subject afflicted with cancer.
  • the device is the device according to the present disclosure.
  • kits comprising a device according to the present disclosure, and an anticancer therapeutic agent.
  • the anticancer therapeutic agent is selected from the group consisting of an inhibitor to an inhibitory checkpoint molecule, an activator of a stimulatory checkpoint molecule, an antibody, a cytokine, an interferon, an interleukin, a vaccine, an oncolytic virus, chimeric antigen receptor T cells, and any combination thereof.
  • the therapeutic agent is a biological therapeutic.
  • the therapeutic agent is a monoclonal antibody (mAb) used to treat cancer, or any combination of mAbs used to treat cancer.
  • mAb monoclonal antibody
  • the therapeutic agent is selected from ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab, sintilimab, or any combination thereof.
  • the therapeutic agent is a cytostatic or a cytotoxic drug bound to a plasma protein.
  • the therapeutic agent is a macromolecule.
  • the therapeutic agent is a nanomedicine.
  • Figures 1A-B present absolute pressure pulses generated by piezophotonic materials made of carbon nanoparticles and PDMS when excited with laser fluences of (Figure 1 A) ⁇ 60 mJ/cm 2 and ( Figure IB) -126 mJ/cm 2 and 8 ns duration, detected with a hydrophone calibrated to the 1 to 30 MHz range;
  • Figure 2 presents Fourier transform of a stress wave generated by a piezophotonic material made of carbon nanoparticles and polymer when excited with a laser fluences -60 mJ/cm 2 and 8 ns duration, measured with a 225 MHz contact transducer;
  • Figure 3 presents in vitro viability of immortalized monkey fibroblast (COS-7) cells in control (CTR) cell culture plates and in plates exposed to photoacoustic waves for 5 minutes (5 mins) or for 10 minutes (10 mins), at the laser repetition rates of 6 Hz or 20 Hz. The viability of the cells is not compromised by exposure of up to 12000 pressure pulses;
  • Figure 4 presents magnetic resonance imaging (MRI) of tissues in the region of the neck of a Sprague Dawley rat. The left side was exposed for 5 minutes, 5 times a week for 4 weeks to photoacoustic waves with peak compressional pressures of ⁇ 3 MPa at a pulse repetition rate of 20 Hz. No differences were observed in the left side relative to the right side, which was not exposed to photoacoustic waves. No adverse effects were observed in the area where photoacoustic waves were applied;
  • Figure 5 presents hematoxylin and eosin stain of tissues in the region of the left carotid of a Sprague Dawley rat exposed for 5 minutes, 5 times a week for 4 weeks, to photoacoustic waves with peak compressional pressures of ⁇ 3 MPa at a pulse repetition rate of 20 Hz. No anomalies were detected in carotids and surrounding tissues. No adverse effects were observed in the area where photoacoustic waves were applied;
  • FIG. 6 is a schematic cross-sectional view, not to the scale, of a tumor-priming device, according to some embodiments of the present disclosure, comprising a pulsed laser system (1) with a control system (6) to limit the number of laser pulses generated, a light guide (2) to direct the laser pulses to a light-to-pressure transducer (3).
  • the tumorpositioning support structure (4) places the light-to-pressure transducer (3) in the proximity of a selected area of a solid tumor (5) growing in the middle of healthy tissue (7).
  • Each pressure pulse generated by the absorption of one laser pulse by the light-to- pressure transducer crosses a small (less than 3 cm) path of tissue or acoustic coupling medium and then traverses at least part of the tumor mass;
  • Figure 7 is a schematic cross-sectional view, not to the scale, of a tumor-priming device, according to some embodiments of the present disclosure, to generate pressure pulses in the vicinity of a solid tumor using endoscopy, comprising a pulsed laser system
  • the tumor-positioning support structure (4) is an endoscope that places the light-to-pressure transducer (3) at less than 3 cm of a selected area of a solid tumor (5) growing in the middle of healthy tissues in the abdomen.
  • FIG. 8 is a schematic cross-sectional view, not to the scale, of a tumor-priming device, according to some embodiments of the present disclosure, to generate pressure pulses in the vicinity of a solid tumor using a catheter, comprising a pulsed laser system (1) with a control system (6) to limit the number of laser pulses generated, a light guide
  • the tumor-positioning support structure (4) is a urinary catheter with a balloon (8) that places the light-to-pressure transducer (3) at less than 3 cm of a selected area of a solid tumor (5) growing in the bladder. Each pressure pulse is directed to the light-to-pressure transducer where it generates a pressure pulse that crosses the bladder and reaches the tumor; and
  • Figure 9 is Kaplan-Meier plot of BALB/c mice with orthotopic 4T1 tumors.
  • Control group (dashed-dotted line, no priming and no therapy), group with tumors exposed to photoacoustic waves (dashed line, priming but no therapy), group treated with intraperitoneal administration of aCTLA4 (full line, no priming but therapy) and group with tumors exposed to photoacoustic waves and treated with intraperitoneal administration of aCTLA4 (dotted line, priming and therapy).
  • Day 0 is the day when the procedures started, chosen that the longest diameter of the tumor was at least 3 mm. The mice were sacrificed when the longest diameter of the tumor reached 12 mm.
  • the term “therapeutic agent” refers to a small molecule drug or a biologic drug or an immune cell, that can be used to treat a tumor, and include a chemotherapeutic drug, a radiosensitizer, a photosensitizer, a nanoparticle, a senolytic agent, a biological, an immunomodulatory agent, an immune molecule, or CD8+ T cells activated by tumor antigens and unrestrained by negative regulators.
  • the therapeutic agent can be an agent approved by a regulatory agency for treating tumors or cancer, undergoing clinical trials prior to regulatory approval, or that is under investigation for treating tumors or cancer.
  • small molecule drug refers to an organic compound having a molecular weight equal or less than 1 kDa.
  • the term includes drugs having desired pharmacological properties and includes compounds that can be administered orally or by injection.
  • Small molecule drugs include cytostatic or cytotoxic drugs used in chemotherapy of cancer.
  • photosensitizer refers to a dye, possibly bound to a targeting moiety, that has no detectable therapeutic effect in the electronic ground state but when electronically excited can trigger processes that eventually lead to cell death, as illustrated by the photogeneration of reactive oxygen species in photodynamic therapy of cancer and by photoimmunotherapy.
  • micromolecule refers to an organic, or bioorganic, molecule with a molecular weight higher than 1 kDa, which can be a protein, a bioconjugate, a RNA molecule or a DNA molecule, or a fragment of the molecules.
  • biologicals or “biological therapeutic” refer to a diverse group of medicines which includes vaccines, growth factors, immune modulators, monoclonal antibodies, as well as products derived from human blood and plasma. This definition specifically includes proteins purified from living culture systems or from blood.
  • immunomodulatory agent refers to a checkpoint inhibitor, a costimulator of immune pathways, an antibody targeting immune cell antigens and/or cancer antigens, and cell therapy approaches (e.g., adoptive cell transfers with genetically modified receptors such as chimeric antigen receptor therapies), and specifically includes immunomodulatory agents such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab and sintilimab.
  • immunomodulatory agents such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalim
  • nanomedicine refers to a nanoparticulate drug carrier system with a size in the range of 1 nm to 500 nm in one or more external dimensions, for more than 50% of the particles, according to the number size distribution, incorporating a small molecule drug, a photosensitizer or a macromolecule.
  • tumor priming refers to vascular normalization and solid-state alleviation in solid tumors, including subsequent or concomitant reduction of interstitial fluid pressure inside the solid tumor, to facilitate the infiltration of therapeutic agents into a solid tumor.
  • piezophotonic transducer or “light-to-pressure transducer” refer to a material that substantially absorbs the light of a laser pulse and transforms the optical energy absorbed into a pressure pulse.
  • pressure pulse refers to a perturbation that carries temporary density changes inside the medium where it propagates. This definition of “pressure pulse” covers explicitly both shock waves and photoacoustic waves, which are also designated collectively as “stress waves”.
  • pressure-pulse tumor priming refers to tumor priming by stress waves, which can be photoacoustic and/or shock waves.
  • treatment encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured.
  • a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.
  • subject refers to an animal, more particularly to nonhuman mammals, human, and human organism. Non-human animal subjects may also include prenatal forms of animals, such as, e.g., embryos or fetuses.
  • Non-limiting examples of non-human animals include: horse, cow, camel, goat, sheep, dog, cat, non- human primate, mouse, rat, rabbit, hamster, guinea pig, pig.
  • the subject is a human.
  • the present disclosure provides devices and methods for priming solid tumors with pressure pulses generated by piezophotonic materials when the materials absorb laser pulses, thereby improving the therapeutic outcome of administration of a therapeutic agent.
  • EXAMPLE 1 shows typical stress waves generated with the absorption of laser pulses in light-to-pressure (piezophotonic) transducers.
  • EXAMPLES 2 and 3 show that such stress waves are safe for cells in vitro and for tissues in vivo. The fact that the pressure pulses of these stress waves do not produce any detectable effect in normal tissues is consistent with the fact that their spatial-peak temporal-average intensities are below the output safety limits set by FDA for diagnostic ultrasound. The pressure pulses are completely safe and do not change normal tissues.
  • the present disclosure provides a device for tumor priming by pressure pulses comprising: a pulsed laser system; a light guide configured to direct laser pulses to one or more light-to-pressure transducers; one or more light-to-pressure 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 light-to-pressure transducers with a selected area from a solid tumor; and a control system configured to limit the exposure of the solid tumor to the pressure pulses.
  • the pulsed laser system has a pulse repetition rate between 0.1 Hz and 100 Hz.
  • the device comprises a control system configured to limit the exposure of the solid tumor to the pressure pulses for a period of time from 1 second (s) to 60 minutes (min).
  • the light guide configured to direct laser pulses to one or more light-to-pressure transducers comprises one or more optical fibers or light pipes.
  • the light guide comprises mirrors, lenses, prisms, diffusers or polarizers, or any combination thereof.
  • the tumor-positioning support structure is configured to couple one or more light-to-pressure transducers to an acoustic coupling element disposed between the transducers and the surface of a solid tumor.
  • the pressure pulses are photoacoustic waves. In some embodiments, the pressure pulses are shock waves. Solid tumor priming with pressure pulses involves exposing the solid tumor to one or more pressure pulses.
  • the instantaneous peak intensity, [089] I pmax 2 /(p v),
  • shock waves have in common with photoacoustic waves the fact that they carry temporary density changes inside the materials where they propagate. They differ because shock waves propagate with a velocity that is higher than the local speed of sound in the material.
  • photoacoustic waves and shock waves are collectively designated as “pressure pulses”.
  • shock waves can be generated by a variety of process, including detonation, a projectile hitting a surface, an object travelling at supersonic speed, or an intense pulsed laser producing ablation of a target. In the context of the present disclosure, the generation of shock waves by intense laser pulses is of particular interest.
  • the laser fluence rates (in W/m 2 ) required to generate plasma in a given target, and consequently to generate shock waves, are higher than those required for thermoelastic expansion of the target, and consequently to generate photoacoustic waves.
  • pulsed laser generation of shock waves typically gives higher peak pressures than photoacoustic waves using the same material. Nevertheless, peak intensities of both shock waves and photoacoustic waves are reached for a very small fraction of the time. Therefore, low pulse repetition rates ( ⁇ 100 Hz) lead to very low duty cycles and pressure pulses up to 100 MPa can be used without significant damage to tissues when the pressure pulses are generated by laser pulses with nanosecond duration.
  • a superficial solid tumor can be exposed to pressure pulses by placing the material absorbing the laser pulse directly over the tumor, or over the skin layer covering the tumor, with good acoustic coupling with the skin and with the tumor.
  • the laser pulse is directed to the material, the energy of the laser pulse is absorbed by the material and either a photoacoustic wave or a shock wave is generated on the material, crosses the material and is transmitted to the skin and to the tumor.
  • Good acoustic coupling can be achieved with proper matching of the acoustic impedances of the material and of human tissues, and can be improved using a layer of acoustic coupling gel.
  • thermoelastic expansion generating photoacoustic waves to ablation generating shock waves depends mostly on the ablation limit of the material, on the energy of the laser pulse and on the size of the area illuminated.
  • EXAMPLE 1 in the example section of the present disclosure, that describes a method to generate a photoacoustic wave and its characterization.
  • the solid tumor is not superficial, i.e., it is more than 3 cm beneath the surface of the body.
  • the stress waves generated at the surface of the body may be strongly attenuated by healthy tissues before reaching the tumor mass and may lose efficiency in tumor priming.
  • the attenuation of ultrasound in tissues can be calculated with the derating factor 0.3 dB/(cm MHz). This means that 3 cm from a 3.3 MHz transducer, the derated temporal -average intensity of ultrasound is 3 dB (i.e., half of) the value measured in water.
  • the derated temporal -average intensity becomes 30 dB (i.e., 0.001 of) the value measured in water.
  • the present disclosure is based, in part, on the surprising finding that normal cells and healthy tissues are not affected by stress waves. Reference is made to EXAMPLES 2 and 3 in the example section of the present disclosure, that show that normal cells and healthy tissues are not affected by stress waves.
  • the solid tumor even when the solid tumor is not superficial, it may nevertheless be possible to approach the tumor mass through natural body orifices using methods of endoscopy.
  • Minimally invasive procedures can be used to generate stress waves in the vicinity of solid tumors in the gastrointestinal tract, respiratory tract, urinary tract or female reproductive system. Stress waves can be generated close to a solid tumor in these locations because endoscopes have channels that allow for the insertion of optical fibers.
  • small incisions with a length smaller than 2 cm, can be made to give access of optical fibers to normally closed body cavities in procedures such as laparoscopy or thoracoscopy.
  • optical fibers can be inserted in catheters and reach many desired locations inside the human body.
  • optical fibers allows for the delivery of laser light in the vicinity of solid tumors.
  • solid tumors that can be reached with optical fibers include gastric cancer, enteric cancer, lung cancer, breast cancer, uterine cancer, esophageal cancer, ovarian cancer, pancreatic cancer, pharyngeal cancer, sarcomas, hepatic cancer, cancer of the urinary bladder, cancer of the upper jaw, cancer of the bile duct, head and neck cancer, cancer of the tongue, cerebral tumor, skin cancer, malignant goiter, prostatic cancer, colorectal cancer, cancer of the parotid gland, and renal cancer.
  • the laser light is directed to proximal end of the optical fiber and a piezophotonic transducer can be coupled to its distal end, which is in the vicinity of the solid tumor.
  • the piezophotonic transducer absorbs most of the intensity of the laser pulse and generates a stress wave.
  • a stress wave is generated each time that a laser pulse is absorbed by the piezophotonic transducer.
  • the stress wave may be generated by thermoelastic expansion of the piezophotonic transducer, under the conditions of thermal confinement and stress confinement, and in this case the stress wave is a photoacoustic wave.
  • the stress wave may be generated by ablation of the piezophotonic transducer, which implies the removal or destruction of some material of the transducer, and in this case the stress wave is a shock wave.
  • the stress wave propagates in the piezophotonic transducer, from the side where the laser pulse was absorbed to the opposite side, and then is transferred to tissues in the vicinity of the solid tumor, or directly to the solid tumor.
  • F is the local light fluence.
  • the dyes or pigments must preferably be incorporated in a material with a low ablation threshold. This is the case, for example, of poly(ethylene terephthalate), polyimide and triazene polymers. These polymers can be used to produce piezophotonic materials that undergo ablation with the production of shock waves at laser fluences below 200 mJ/cm 2 .
  • Non-limiting examples of dyes or pigments that can be used to make piezophotonic materials according to the present disclosure are ortho- hydroxybenzophenone and similar molecules undergoing ultrafast photoinduced intramolecular proton or hydrogen-atom transfers that return rapidly to the original ground state, Mn 111 complexes of me o-tetraphenyl porphyrin and other paramagnetic complexes with ultrafast metal-to-ligand and/or ligand-to-metal charge-transfer relaxation processes, complexes with charge-transfer bands that return to the ground state by ultrafast charge recombination, P-carotene and other systems that rapidly decay to the ground state through conical intersections, graphite or carbon nanoparticles or carbon nanotubes or carbon soot and other materials capable of ultrafast transfer of their electronic energy to phonon modes followed by cooling in the sub-nanosecond time scale, semiconductor materials with short-lived transient states, or other materials, or mixtures of materials, with ultrafast radiationless relaxation processes.
  • the light-to-pressure transducer comprises a laser light absorbing system and a material with a Griineisen parameter higher than 0.5, and wherein each pressure pulse is a wavefront of a photoacoustic wave.
  • Some non-limiting examples of materials with high Griineisen parameters are polymers (poly dimethylsiloxane, polystyrene, polyamide, poly(vinyl chloride), polyethylene, polyacrylonitrile, poly(ethylene terephthalate), polychloroprene, parylene), metallic films, glasses and layered materials containing them.
  • Such materials can absorb light of a laser pulse, or be designed to incorporate dyes or pigments that absorb light of a laser pulse, in a very short optical path, which is very convenient to fabricate piezophotonic materials to work with endoscopes or catheters.
  • the light-to-pressure transducer comprises a laser light absorbing system and a material with an ablation threshold below 200 mJ/cm 2 , and wherein each pressure pulse is a wavefront of a shock wave.
  • piezophotonic transducers made of dyes or pigments and of a material with a high Griineisen parameter or a low ablation threshold can take various forms and shapes. Considering that the dyes or pigments must have high absorption coefficients, the piezophotonic transducers may absorb most of the laser pulse in an optical path shorter than 200 pm, or preferably shorter than 100 pm, or most preferably shorter than 50 pm. In view of the very small thickness of piezophotonic transducers, they can be used to cover the distal end of an optical fiber.
  • the peak compressional pressures of the stress waves are from 0.1 MPa to 100 MPa.
  • each pressure pulse lasts from 0.1 ns to 500 ns.
  • the optical fiber is held by a tumor-positioning support structure that orients the laser pulse and a light-to-pressure transducer to a solid tumor less than 3 cm from the surface of the body ( Figure 6).
  • the optical fiber is inserted in an endoscope, that works as a tumor-positioning support structure, and at the distal end the optical fiber is optically connected to an optical diffuser and the optical diffusor is at least partially coated with a piezophotonic transducer placed within 3 cm from a solid tumor ( Figure 7).
  • the optical fiber is inserted in a catheter, that works as a tumor-positioning support structure with the assistance of a balloon, and at the distal end the optical fiber has a lens that directs the laser pulse to a piezophotonic transducer placed within 3 cm from a solid tumor ( Figure 8).
  • the optical fiber is connected to an optical diffuser, the optical diffusor is coated with a piezophotonic transducer and inserted in a solid tumor, and the system of perforating the tumor and inserting the light-to-pressure transducer is the tumorpositioning support structure and this structure has a sharp end.
  • the tumor-positioning support structure is an endoscope and the light guide is one or more optical fibers configured to carry the laser light from the light source through the endoscope, to one or more light-to-pressure transducers at the distal ends of the optical fibers.
  • the endoscope is configured for insertion in a hollow organ through a natural body opening or through an incision in the body with less 2 cm in length.
  • the tumor-positioning support structure is a catheter and the light guide is one or more optical fibers configured to carry the laser light from the light source through the catheter, to one or more light-to-pressure transducers at the distal ends of the optical fibers.
  • the catheter is configured for insertion into a body cavity, duct, vessel, brain, skin or adipose tissue.
  • the tumor-positioning support structure comprises a sharp end configured to enable the insertion of one or more light-to-pressure transducers into the solid tumor.
  • Tumor priming using pressure pulses consists in bringing a piezophotonic material within 3 cm of the solid tumor, where the path between the piezophotonic material and the solid tumor is filled by a medium capable of transmitting pressure pulses, exposing the piezophotonic material to laser pulses that generate peak compressional pressures from 0.1 to 100 MPa in the piezophotonic material, and directing such pressure pulses to at least part of the solid tumor for a time in the range of 1 second to 1 hour.
  • the laser pulses may have durations (full-width at half height) of femtoseconds, picoseconds or nanoseconds.
  • the laser pulse durations should be less than 500 nanoseconds because under these conditions the thermal confinement condition is more easily met and 90% of the pressure pulse lasts less 500 ns.
  • the exposure of the solid tumors to the pressure pulses can be performed for a short period of time (e.g., 1 sec), for a long period of time (e.g., 1 hour) or for an intermediate period of time.
  • the exposure of a solid tumor to the pressure pulses can be performed before, during or after, the administration of the therapeutic agent, and can be timed according to the plasma lifetime of the therapeutic agent.
  • the present disclosure is based, in part, on the finding that, in repeated administrations of the therapeutic agent, and/or for therapeutic agents with long plasma half-lives, the exposure of solid tumors to the pressure pulses can be performed several times, which can be several times a day, several times a week, several times a month, or several times a year.
  • the present disclosure is based, in part, on the finding that, solid tumor priming with pressure pulses as described herein, facilitates the infiltration of a therapeutic agent in a solid tumor and increases the response to therapy.
  • Solid tumor priming by exposure to pressure pulses enhances the infiltration of a variety of therapeutic agents in a solid tumor without affecting the delivery of the therapeutic agents to healthy tissues of the host or enhancing host toxicity. It is particularly valuable for the delivery of small molecule drugs extensively bound to plasma proteins and of macromolecules, especially when they are biological pharmaceuticals.
  • the present disclosure is based, in part, on the finding that, solid tumor priming by pressure pulses improves the therapeutic efficacy of immunotherapies, notably when the therapeutic agents are mAb employed in ICB therapy.
  • Solid tumor priming by pressure pulses facilitates the infiltration of tumor antigen-specific T lymphocytes into tumors and their integration into the tumor microenvironment (TME), contributing to enhance tumor responses to immunotherapies.
  • TAE tumor microenvironment
  • the present disclosure is based, in part, on the unexpected finding that, pressure pulses that are well tolerated by normal tissues can be effective in solid tumor priming.
  • the present disclosure provides a method of treating a tumor in a subject.
  • the method comprises administering to the subject (i) an amount of high-intensity photoacoustic waves and (ii) an amount of a therapeutic agent, wherein the amounts of (i) and (ii) together are sufficient to treat a solid tumor, and the order of administration can be selected from (i) before (ii), (i) simultaneously with (ii) or (i) after (ii).
  • a method of sensitizing a tumor in a subject to an amount of an anti-cancer therapy comprising of administering to the subject, prior to or during the course of the anti-cancer therapy, an amount of pressure pulses effective to improve the response of a tumor in a subject to an amount of an anti -cancer therapy administered to the subject.
  • a subject is a cancer patient.
  • the present disclosure provides a method for treating a solid tumor in a subject afflicted with cancer, the method comprising: pressurepulse tumor priming of the solid tumor by exposure of the solid tumor to one or more pressure pulses, wherein the pressure pulses have peak compressional pressures from 0.1 MPa to 100 MPa, and 90% of each pressure pulse lasts from 1 ns to 500 ns; and administering of one or more anticancer therapeutic agents to the subject, thereby treating the solid tumor in a subject afflicted with cancer.
  • a subject afflicted with cancer is a cancer patient.
  • the step of pressure-pulse tumor priming of the solid tumor by exposure of the solid tumor to one or more pressure pulses is performed before administering of one or more anticancer therapeutic agents to the subject, during administering of one or more anticancer therapeutic agents to the subject, or after administering of one or more anticancer therapeutic agents to the subject.
  • the method further comprises a step of repeating (i) the pressure-pulse tumor priming, (ii) the administering of one or more anticancer therapeutic agents, or both.
  • repeating is at least one time, at least 2 times, at least 3 times, at least 5 times or the number of times required to alleviate the symptoms of the subject afflicted with cancer, with doses that improve a response of the solid tumor to the treatment.
  • the anticancer therapeutic agent is selected from the group consisting of an inhibitor to an inhibitory checkpoint molecule, an activator of a stimulatory checkpoint molecule, an antibody, a cytokine, an interferon, an interleukin, a vaccine, an oncolytic virus, chimeric antigen receptor T cells, and any combination thereof.
  • the therapeutic agent is a biological therapeutic.
  • the therapeutic agent is a monoclonal antibody (mAb) used to treat cancer, or any combination of mAbs used to treat cancer.
  • mAb monoclonal antibody
  • the therapeutic agent is selected from ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiplimab, dostarlimab, tislelizumab, relatlimab, toripalimab, camrelizumab, sintilimab, or any combination thereof.
  • the therapeutic agent is a cytostatic or a cytotoxic drug bound to a plasma protein.
  • the therapeutic agent is a macromolecule.
  • the therapeutic agent is a nanomedicine.
  • the present disclosure provides a kit comprising a device as described hereinabove, and an anticancer therapeutic agent as described hereinabove.
  • the present disclosure provides a system comprising at least one device as described hereinabove.
  • the present disclosure provides a device comprising means for generating pressure pulses having peak compressional pressures from 0.1 MPa to 100 MPa, and 90% of each pressure pulse lasts from 1 ns to 500 ns, for treating a solid tumor in a cancer patient.
  • the device is the device as described hereinabove.
  • Pressure-pulse tumor-priming therapy combines the exposure of solid tumors to pressure pulses, as described hereinabove, with the administration of a therapeutic agent with an anti-cancer effect.
  • PPTPT Pressure-pulse tumor-priming therapy
  • aCTLA4 anti-CTLA-4 mAbs
  • mice bearing orthotopic 4T1 tumors increases from 20 days in the combination of aCTLA4 plus aPDl, to 24 days when this combination is associated with tumor priming with angiotensin receptor blockers [17].
  • EXAMPLE 4 shows that the median survival time increases from 21 days when the animals are treated with aCTLA4 to 36 days when photoacoustic priming is associated with the aCTLA4 treatment.
  • orthotopic 4T1 tumors are widely recognized as very difficult to treat and increasing the response to immunotherapy in all the mice with just two sessions of 5 minutes local exposure of the tumors to harmless photoacoustic waves is totally unexpected.
  • the present disclosure is based, in part, on the finding that PPTPT is a novel and surprising effective approach to increase solid tumor response to therapeutic agents.
  • High-intensity broadband stress waves exert mechanical forces at the microscopic level that can remodel the TME.
  • peak pressures of ⁇ 7 MPa of pressure waves with relevant frequencies of about 20 MHz correspond to changes of 50 bar in 10 nsec or, considering the speed of sound propagation in tissues, a change of 50 bar in 15 pm. Dramatic pressure changes are produced on the scale of the size of a cell.
  • the present disclosure is based, in part, on the finding that cells survive these high pressures, as shown in EXAMPLE 2, and normal tissues do not exhibit any adverse effects, as shown in EXAMPLE 3.
  • the mechanical forces exerted by such pressure pulses in the TME enable micro-mechanic priming of solid tumors, as shown in EXAMPLE 4.
  • Carbon nanoparticles are very convenient light-absorbing systems because they strongly absorb light over a large range of ultraviolet-visible-infrared wavelengths. Carbon nanoparticles are difficult to disperse in solution, hence 160 mg of the carbon nanoparticles, produced as candle soot, were added to 5 mL of toluene and sonicated, using a tip sonicator, for 5 minutes at 60 MHz. Immediately after mechanical sonication, 2 mg of polystyrene were added to the suspension and heated in a water bath to 60 °C. Polystyrene has a high Griineisen parameter (G ⁇ 0.7) and is very convenient to make thin piezophotonic transducers. Polystyrene films with dispersed carbon nanoparticles were fabricated using a mechanical applicator (Elcometer) and dried overnight to allow the remaining solvent evaporate.
  • piezophotonic transducer were produced depositing carbon nanoparticles from the combustion of a paraffin lamp on a borosilicate glass window. This glass window was directly exposed to the flame for 2 min to collect carbon soot. Then, the thin layer of carbon soot deposited on the window was covered with 0.1 mL of poly dimethylsiloxane (PDMS) and subject to vacuum for 10 min to remove any air bubbles. Next, a weight of 100 g was placed over the system (glass + soot + PDMS) to make a thin layer of PDMS and exposed for 10 additional minutes to vacuum to remove the excess of air trapped in the system. Finally, the complete assembly system was heated in an oven at 50°C overnight to obtain a full cure of PDMS.
  • PDMS poly dimethylsiloxane
  • Laser excitation employed a Nd: YAG laser (Monfort M-NANO) with nanoseconds pulses to generate photoacoustic waves.
  • Nd: YAG laser Monitoring M-NANO
  • Two types of ultrasound measurements were made. Absolute pressures were measured using a 0.2 mm needle hydrophone (Precision Acoustics, model NH0200), calibrated to the 1 to 30 MHz range.
  • Ultrasonic frequency distributions were investigated with a 225 MHz contact transducer (Panametrics/Olympus, model V2113).
  • Figures 1A-B show the absolute pressure pulses obtained with laser fluences of -60 mJ/cm 2 and -250 mJ/cm 2 , measured with the hydrophone.
  • Figure 2 shows the ultrasonic frequency distribution using a laser fluence of -60 mJ/cm 2 measured with the contact transducer.
  • Photoacoustic waves with peak pressures of 10 MPa are not toxic to fibroblasts in vitro
  • COS-7 Monolayers of immortalized monkey fibroblast cell line (COS-7) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat- inactivated fetal bovine serum (Gibco) and 1% penicillin and streptomycin (Invitrogen), in humidified atmosphere with 5% CO2 at 37 °C.
  • DMEM Dulbecco
  • Gibco heat- inactivated fetal bovine serum
  • Invitrogen penicillin and streptomycin
  • the piezophotonic material of EXAMPLE 1 was immersed in the culture medium and placed within 3 mm from the surface of the COS-7 cells monolayer. Then, the cells were exposed to photoacoustic waves for 5 minutes (5 mins) or for 10 minutes (10 mins), at the laser repetition rates of 6 Hz or 20 Hz and laser fluence ⁇ 60 mJ/cm 2 , using a Nd: YAG laser (Monfort M-NANO). Cell viability was measured 24 h after exposure to photoacoustic waves using the Alamar Blue® assay.
  • the Portuguese Animal Health Authority approved the animal experiments (DGAV authorization 0420/000/000/2011).
  • This study employed male Sprague Dawley rats (Charles River Laboratories, Barcelona, Spain). The rats were depilated around the neck and a circle was drawn in the area to be subject to stress waves. The exposure to stress waves was performed 5 days a week, for 4 weeks. In each exposure, stress waves were generated for 5 min at 20 Hz with piezophotonic transducers made of carbon nanoparticles and PDMS and using a Monfort M-NANO Nd:YAG laser. Under the conditions employed, the peak compressional pressure of each pulse was ⁇ 3 MPa.
  • 4T1 cells (ATCC CRL-2539) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Sigma- Aldrich, Saint-Louis, MO, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (GIBCOTM, Life Technologies, Bleiswijk, The Netherlands), 100 U/mL penicillin, and 100 ng/mL streptomycin (InvitrogenTM, Thermo Fisher Scientific, Grand Island, NY, USA). Tumors were established by orthotopic injection of 20,000 4T1 cells in the right mammary gland of female BALB/c mice ca. 8 to 12 weeks old (20 g).
  • mice Prior to tumor priming, the mice were depilated in the abdominal area, namely the mammary gland where the tumor was inoculated.
  • the piezophotonic transducer employed was prepared with carbon nanoparticles and polydimethylsiloxane.
  • the piezophotonic transducer was positioned over the tumor, and acoustic coupling was improved with a layer of acoustic coupling gel between the tumor and the piezophotonic transducer.
  • Photoacoustic waves were generated directing laser pulses from the Monfort M-NANO Nd:YAG laser, with a laser repetition rate of 20 Hz, to the piezophotonic transducer. Under the conditions employed, the peak compressional pressure of each pulse was ⁇ 6.5 MPa.
  • Group (i) was not subject to tumor priming or treatment and the orthotopic tumors grew naturally.
  • Group (ii) was subject to tumor priming by exposing the tumors to photoacoustic waves for 5 min in days 0 and 2.
  • Group (iii) was treated with InVivo mAb anti-mouse CTLA4 (CD 152) on days 0, 2, 6 and 10, by intraperitoneal injection of //d wiMab anti-mouse CTLA4 (CD152) (Bio Cell, Riverside, NH, USA).
  • Group (iv) was subject to the same treatment protocol as group (iii) but additionally tumor priming was performed 10 minutes after antibody administration on days 0 and 2, as done in group (ii).

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Abstract

La présente divulgation concerne des dispositifs et un procédé d'amorçage d'un micro-environnement tumoral avec des impulsions de pression pour améliorer l'efficacité d'agents thérapeutiques anticancéreux, chez un sujet en ayant besoin. En outre, la divulgation concerne une réponse accrue de tumeurs solides localement exposées à des ondes de stress à des agents thérapeutiques administrés de manière systémique. Un dispositif selon la présente divulgation comprend : un système laser pulsé (1), un guide de lumière (2) pour diriger des impulsions laser vers un ou plusieurs transducteurs lumière-pression (3), le ou les transducteurs lumière-pression absorbant les impulsions laser provenant du système laser pulsé et générant des impulsions de pression, une structure de support de positionnement de tumeur (4) conçue pour coupler un ou plusieurs transducteurs lumière-pression avec une tumeur solide (5), et un système de commande (6) pour limiter l'exposition de la tumeur solide aux impulsions de pression. Des agents thérapeutiques anticancéreux peuvent être administrés avant, après ou pendant l'amorçage des tumeurs solides avec des impulsions de pression.
PCT/IB2023/058316 2022-08-22 2023-08-21 Dispositifs et procédés d'amorçage de tumeurs solides avec des impulsions de pression pour renforcer des thérapies anticancéreuses WO2024042447A1 (fr)

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