Combination of bubble-ultrasound mediated histotripsy and chimeric antibody receptor t-cells (car-t) for cancer therapy

Abstract

Disclosed herein are systems, compositions and methods for combined tumor therapy using CAR-T cells and administered bubbles accompanied by application of low frequency ultrasound (US).

Description

[0001] COMBINATION OF BUBBLE-ULTRASOUND MEDIATED HISTOTRIPSY AND CHIMERIC ANTIBODY RECEPTOR T-CELLS (CAR-T) FOR CANCER THERAPY

[0002] FIELD OF THE INVENTION

[0003] The present disclosure is directed to systems, compositions and methods for combined tumor therapy using CAR-T cells and administration of bubbles (microbubbles and / or nanobubbles) accompanied by application of low frequency ultrasound (US).

[0004] BACKGROUND

[0005] CAR T cells are engineered T cells expressing a chimeric antigen receptor (CAR) that recognizes a specific tumor-associated antigen (TAA) which may distinguish cancer cells from healthy ones. Upon recognition of the TAA, the CAR T cell is stimulated and can efficiently kill its target cells. CAR T cells-based therapy is used in the treatment of hematological cancers, showing exceptional efficacy in eradicating cancer cells in blood malignancies. Such therapy involves the genetic engineering of T cells to express specific receptors that can recognize and bind to specific antigens, thereby initiating cytotoxic responses that kill cancer cells. Despite its success in hematological applications, CAR T therapy has faced substantial obstacles when applied to solid tumors. Administering CAR T cells directly into tumors is both invasive and ineffective in transforming the tumor microenvironment, as it only targets a small fraction of the tumor. Thus, treating solid tumors with CAR T cells remains a great challenge.

[0006] Focused ultrasound (US) is a noninvasive clinical therapy that induces mechanical or thermal effects through the local delivery of powerful acoustic energy. Such technique has been used to treat solid tumors deep within the body. Mechanical US surgery via histotripsy or mechanical ablations utilizes short and high intensity US pulses to mechanically destroy deep tissues, fractioning the targeted soft tissue into subcellular debris while leaving the surrounding organs and tissues unaffected. These mechanical effects result from the production of inertial cavitating or boiling bubbles. Inertial cavitation is a strong physical effect, where gas bubbles are formed, expand and violently collapse, excreting powerful mechanical effects on the surrounding tissue.

[0007] Ultrasound bubbles include micron bubbles (MBs) and sub-micron bubbles (nanobubbles, NBs), having different shells and formulations, impacting their resulting diameter that typically varies between 50-2000 nm. It has been shown that upon application of low-frequency ultrasound, bubbles that accumulate in the tumor can act as mechanical therapeutic agents that locally fractionate the tumor and lead to mechanical ablation of solid tumors at low pressure.

[0008] Due to the substantial obstacles faced by the CAR T therapy when applied to tumors, there is an unmet need for improved effective methods to enhance the utilization of CAR T therapy, to treat such tumors and to modify the tumor microenvironment, which can increase immunogenicity of the tumor.

[0009] SUMMARY

[0010] According to some embodiments, there are provided herein advantageous systems, methods and compositions for the treatment of cancer, including solid tumors, which utilize a combination therapy, combining the use of CAR-T cells and administration of bubbles (NBs and / or MBs) and corresponding application of low energy ultrasound (US), to facilitate a synergistic effect of the applied low energy US effect on bubbles’ oscillations in target tissues and of the CAR-T cells, to thereby provide enhanced therapeutic effect on the target cells and tissues.

[0011] According to some embodiments, without wishing to be bound by any theory or mechanism, it is surprisingly demonstrated herein that exciting the MBs and / or NBs disclosed herein, with low ultrasound (US) frequencies (for example, on the order of 10s- 1000s kilohertz), bubbles oscillations are triggered in the target tissue, which enhances the effect of administered CAR-T cells on the cancer cells, for example, by enhancing tumor infiltration by the CAR-T cells.

[0012] According to some embodiments, the present invention is based, at least in part, on unexpected results showing that a combinatory administration of CAR T cells (for example, anti-HER2 CAR-T cells), and administered bubbles (for example, nanobubbles (NBs)) accompanied by application of low frequency ultrasound (US) provide an enhanced and synergistic anticancer effect, in cells and tissues, both on in-vitro and in-vivo.

[0013] According to some embodiments, advantageously, combining bubbles (such as, MBs or NBs) with low frequency US and CAR-T treatment synergistically reduces off target toxicity (for example, diminishing or reducing the need to perform lymphodepletion due to the combined treatment), and increases on-target effect, in particular in solid tumors, which are otherwise inaccessible to the CAR-T cells. In some embodiments, the combination therapy of low frequency US with MBs and / or NBs, together with CAR-T cells is safe, cost effective and clinically available, while facilitating tumor targeting, partially due to the ability of the bubbles to be visualized by US imaging. Moreover, the bubbles can be specifically targeted to the tumor cells and increase effectiveness of the treatment by increasing the effective concentration of the bubbles into the targeted tumor and by improving tumor imaging and alignment.

[0014] In some embodiments, advantageously, the compositions and methods disclosed herein, can successfully aid in treating deep-seated solid tumors and facilitate the treatment of larger tumor volumes simultaneously.

[0015] According to some embodiments, the methods and compositions disclosed herein are suitable for treating various types of cancers and tumors, including primary tumors, relapse tumors, and metastases, in particular, solid tumors (including, for example, breast, lung, prostate, colon, melanoma, brain, ovarian, bladder, kidney, sarcomas, carcinomas, and lymphomas). In some embodiments, the term “solid tumor” also encompasses solid-like tumors (such as, for example, some types of lymphoma (e.g., Hodgkin and non -Hodgkin lymphoma) that can form solid masses in lymph nodes or other tissues). In some embodiments, the cancer is breast cancer tumor.

[0016] According to some embodiments, the NBs and compositions including the same, can be administered systemically or localized, together with, prior to, or after of administration of CAR-T cells compositions. In some embodiments, advantageously, the CAR-T cells (or compositions including the same) are administered systemically, or in some cases, may be administered locally (for example, intratumorally). In some embodiments, one or more additional therapeutic agents may be administered or co-expressed, for example, as a pretreatment to the CAR-T administration. Such agents may include, for example, but not limited to: checkpoint inhibitors and cytokines. In some embodiments, the CAR-T administration may be facilitated after at least part of the tumor has been surgically (or otherwise) removed.

[0017] According to some embodiments, combining bubbles-mediated low energy ultrasound mechanical ablation with Chimeric Antigen Receptor T cells can enhance Chimeric Antigen Receptor T cell solid tumor infiltration, as well as other immune cells. To this aim, a non- invasive ultrasound system that utilizes micro or nanoscale bubble and low-frequency ultrasound to remotely damage cancer cells is used. For example, the nanobubbles can accumulate in target tissue (such as solid tumors), following a systemic and administration (for example, by intravascular injection). Then, using low frequency ultrasound, the nanobubbles can be locally detonated, causing tumor fractionation that increases immune cells and Chimeric Antigen Receptor T cells infiltration and overall synergistically and significantly enhance the anti-cancer treatment.

[0018] According to some embodiments, the systems, compositions and methods disclosed herein, which integrate bubble-mediated low-energy histotripsy with CAR-T cell therapy, offer a distinctive synergy capable of diminishing tumor burden, augmenting CAR-T cell infiltration into solid tumors, modifying the tumor microenvironment, and ultimately enhancing therapeutic efficacy. In some embodiments, the therapeutic efficacy may include eradication or at least partial reduction in tumor growth / size and / or reduction or prevention of relapse. .

[0019] According to some embodiments, there is provided herein a method for treating a tumor in a target tissue in subject in need thereof, the method includes: administering bubbles (MBs and / or NBs) to the subject; applying low frequency ultrasound (US) to the target tissue; and administering CAR-T cells to the subject.

[0020] According to some embodiments, the method of treating the tumor includes at least partially inducing mechanical damage to the tissue (e.g., tumor fractionation) by the bubbles and the applied low frequency US, and allowing the CAR-T cells and optionally immune cells present in the blood stream to induce a target cytotoxic effect on the cancer cells in the tumor.

[0021] According to some embodiments, the CAR-T are administered concomitantly with the NBs. In some embodiments, the CAR-T cells are administered prior to administration of the NBs. In some embodiments, the CAR-T cells are administered after administration of the NBs. In some embodiments, the CAR-T cells may be administered before or after a selected period of time after administration of the NBs.

[0022] In some embodiments, the NBs and / or the CAR-T cells are administered systemically or locally. In some embodiments, the NBs and / or the CAR-T cells are administered intratum orally.

[0023] In some embodiments, the CAR-T cells are administered in suitable pharmaceutical compositions. In some embodiments, the CAR-T cells and the NBs are administered in separate compositions. In some embodiments, the CAR-T cells and the NBs are administered in a single composition (formulation). In some embodiments, each of the disclosed compositions may be administered a plurality of times. According to some embodiments, the bubbles are nanobubbles, that may have an average diameter in the range of about 50-800nm. According to some embodiments, the nanobubbles may have an average diameter in the range of about 110-300nm. According to some embodiments, the bubbles are microbubbles and may have an average diameter in the range of about 800-3000nm.

[0024] According to some embodiments, the bubbles may be essentially spherical. According to some embodiments, the bubbles may be essentially rod-shaped.

[0025] According to some embodiments, the bubbles may include one or more lipids. According to some embodiments, the bubbles may include one or more proteins. According to some embodiments, the bubbles may include one or more polymers. According to some embodiments, the bubbles may include a combination of lipids, proteins or polymers. According to some embodiments, the bubbles may further include a targeting moiety on a shell thereof. According to some embodiments, the targeting moiety may be a cell type-specific antibody conjugated to the shell. According to some embodiments, the targeting moiety may be a protein or a peptide conjugated to the shell. According to some embodiments, the targeting moiety may be an aptamer conjugated to the shell. According to some embodiments, the targeting moiety may be a small molecules, or others (for example, vitamins or carbohydrates) conjugated to the shell.

[0026] According to some embodiments, the ultrasound is a low-energy US. In some embodiments, the US is in the frequency of less than about 500, less than about 250, less than about 200kHz. In some embodiments, the US is in the frequency of less than about 100kHz. In some embodiments, the US is in the frequency of less than about 1 MHz.

[0027] According to some embodiments, the US may be characterized as having a peak negative pressure (PNP) of about 200-2000 kPa, for example 200-1000kPa. In some embodiments, the US may be characterized as having a peak negative pressure (PNP) of about 800kPa or less, 500kPa or less. In some embodiments, the US may be characterized as having a peak negative pressure (PNP) of about 350kPa.

[0028] According to some embodiments, the low-energy US may have a center frequency of about 1MHz or less, about 850kHz or less, about 500kHz or less, about 250kHz or less, about 110 kHz or less, 105kHz or less, 100 kHz or less, 90 kHz or less, 80 kHz or less, or about 80 kHz. In some embodiments, the US may have a peak negative pressure (PNP) of about 1000 kPa or less, 800 kPa or less, 650 kPa or less, 550 kPa or less, 500 kPa or less, 450 kPa or less, 400 kPa or less, 350 kPa or less, 300 kPa or less, 280 kPa or less, 250 kPa or less, or about 250 kPa. In some embodiments, The US may be characterized by having a mechanical index (MI) that is less than about 1.9, less than 1.85, less than about 1.8, less than 1.5, or less than 1, wherein the MI is calculated as equaling the PNP of the low frequency US divided by the square root of the center frequency of the US. In some exemplary embodiments, the US may have a center frequency of 80 kHz or less and a PNP of 350 kPa or less.

[0029] According to some embodiments, the US may be applied after a time interval from the administration of the bubbles. In some embodiments, the time interval is at least 10 minutes.

[0030] According to some embodiments, the CAR T cells (or composition including the same) may be administered after a time interval from the application of US preceded by administration of the bubbles. In some embodiments, the time interval may be about 1-60 minutes, about 1-24 hours; about 1-30 days, or any subranges thereof. In some embodiments, the time interval may be about 2-7 days.

[0031] According to some embodiments, the CAR T cells (or composition(s) including the same) may be administered prior to the application of US preceded by administration of the nanobubbles. In some embodiments, the time interval may be about 1-60 minutes, about 1-24 hours; about 1-14 days, or any subranges thereof. In some embodiments, the time interval may be about 2-7 days.

[0032] In some embodiments, the damage to the cancer tissue by the bubbles may include ablation, debulking and / or lesion of the tissue. In some embodiments, the damage to the cancer tissue by the bubbles may enhance immune cells (CAR-T cells, lymphocytes, macrophages, etc.) infiltration to the tissue.

[0033] According to some embodiments, the CAR-T cells may include a CAR which is, for example, but not limited to: anti-HER2, anti-CD19, anti EGFR, anti CD276, anti-CD38, anti- CD24-anti CD 138; or any other tumor associated antigen(s). Each possibility is a separate embodiment.

[0034] In some embodiments, the CAR-T cells may include one or more CARs which are directed against one or more antigens, including, for example, but not limited to: ErbB2 (HER2), CD 19, BCMA, CD38, CD 138, EGFR, CD276, CD24, GD2, EGF, Mesothelin (MSLN), MUC1, FAP, PSCA, EPCAM, CEA, PSMA, GPC3, LMP1, CD133, cMET, ROR1, CD70, MUC16, RAO, RA9, or any combinations thereof. Each possibility is a separate embodiment. According to some embodiments, the CAR-T cells may include a dual CAR construct. In some embodiments, the dual CAR construct may include a first CAR specific to CD24 and a second CAR specific to CD 138 (CD24 / CD138).

[0035] According to some embodiments, there is provided a use of a composition which includes bubbles (such as NBs and / or MBs) and a composition which includes CAR-T cells, for treating a solid tumor in a subject in need thereof.

[0036] In some embodiments, the bubbles composition and the CAR-T composition are administered systemically. In some embodiments, the CAR-T composition may be administered after a time period following administration of the bubbles and application of low frequency ultrasound.

[0037] In some embodiments, the administration of CAR-T composition may be facilitated by systemic (including, injection, inhalation, and the like), or localized routes. In some embodiments, CAR-T compositions may include one or more types of CAR-T cells (i.e., CAR- T cells expressing different CARs, directed against a different or similar antigen). In some embodiments, CAR-T compositions may be administered a plurality of times, wherein the compositions may be identical, similar or different with respect of ingredients (e.g., amount of cells, excipients, etc.) and / or type of CAR-T cells.

[0038] According to some embodiments, there is provided a system for inducing damage to a target tumor tissue of a subject, the system includes a low frequency focused ultrasound transmitter configured to emit low frequency ultrasound (US), towards the target tissue, wherein the subject has been administered with a composition which includes bubbles (MBs and / or NBs) and a composition which includes CAR-T cells; wherein said low frequency ultrasound causes the nanobubbles to induce at least partial damage to the target tissue, thereby allowing infiltration of the CAR-T cells to environment of the target tissue to thereby induce a cytotoxic effect and / or immune effect on the target tissue.

[0039] According to some embodiments, there is provided a formulation for use in treating a solid tumor in a subject in need thereof, the formulation includes a composition including bubbles and / or a pharmaceutical composition including CAR-T cells, said formulation is administered systemically and wherein low frequency ultrasound is applied at least after administration of the bubbles composition.

[0040] In some embodiments, the composition including the bubbles and the pharmaceutical composition including the CAR-T cells are included in a single (same) composition. In some embodiments, the composition including the bubbles and the pharmaceutical composition including the CAR-T cells are included in separate compositions.

[0041] Other objects, features and advantages of the present invention will become clear from the following description, examples and drawings.

[0042] Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more other technical advantages may be readily apparent to those skilled in the art from the figures, descriptions, and claims included herein. Moreover, while specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Some embodiments of the disclosure are described herein with reference to the accompanying figures. The description, together with the figures, makes apparent to a person having ordinary skill in the art how some embodiments may be practiced. The figures are for the purpose of illustrative description and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the disclosure. For the sake of clarity, some objects depicted in the figures are not to scale.

[0045] In the Figures:

[0046] Fig. 1 - FACS analysis of transduction efficiency of T-cells (lymphocytes) with a CAR T-GFP construct;

[0047] Figs. 2A-C- Bar graphs showing treatment effect in-vitro, in various types of cell lines. Combination therapy (NB+US+CAR-T) was compared to each treatment alone (NB+US or CAR-T), or control (NTC). Significant reduction in cell viability to 2% or lower was observed in the cell lines, demonstrating the synergistic effect of the combination treatment. Fig. 2A- 4T1-HER2 cells (breast cancer cells); Fig. 2B- A549 cells (lung cancer cells); Fig. 2C- OVCAR8 cells (Ovarian cancer cells);

[0048] Fig. 3A- Shows a schematic illustration of combination treatment of immunocompetent mice subcutaneously injected spontaneous tumors; Fig. 3B- shows images of control or treated mice (US+NB+CAR-T), comparing tumor established the mice. The arrow mark the tumor in the control mouse, whereas, in the treated mouse, no tumor growth is detected.

[0049] Fig. 3C- shows line graphs of tumor growth rate, monitored for 17 days, in the various treatment groups (NTC- control; CAR-T- treatment with CAR-T cells (targeting HER2); NB+US- treatment with nanobubbles and low frequency ultrasound (250 kHz frequency, 500 kPa pressure, 0.5 millisecond pulse length, pulse repetition frequency 30ms); NB+US+CAR- T- treatment with NB, ultrasound followed by CAR T treatment). The results demonstrate a significant reduction in tumor volume in the combination group when compared to the individual treatments;

[0050] Fig. 3D- shows graphs of survival experiment showing significant increase in survival rate in the mice treated with combination treatment. * indicates rechallenge to the mice at day 162. As shown, the tumors were not reestablished after rechallenge, and the survival rate was unaffected, in the combination therapy group; and

[0051] Figs. 4A-B Show images of immunohistochemistry staining of treated mice, showing a significant increase in f4 / 80 (Fig. 4A) and CD3 cells (Fig. 4B), in the combination treated group compared to the individual treatments; and

[0052] Fig. 4C shows flow cytometry analysis of expression of CD45, CD8 and CD4 lymphocytes in the tested mice. The results demonstrate a significantly higher expression of the CD45, CD8 and CD4 lymphocytes in the group treated with the combination therapy (US+NB+CAR-T).

[0053] DETAILED DESCRIPTION

[0054] The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description and figures. Upon perusal of the description and figures present herein, one skilled in the art will be able to implement the teachings herein without undue effort or experimentation. In the figures, same reference numerals refer to same parts throughout. In the figures, same reference numerals refer to same parts throughout.

[0055] According to some embodiments provided herein are systems, compositions and methods for treating a solid tumor in a subject in need thereof, by synergically combining advantageous nanobubbles (compositions including the same) and application of low frequency US, together with CART therapy (combination therapy).

[0056] According to some embodiments, there are provided herein advantageous therapeutic methods which combine bubbles (microbubbles and / or nanobubbles) mediated low energy histotripsy with CAR-T cells, to enhance CAR-T cell solid tumor infiltration, modify the tumor microenvironment and improve therapeutic outcomes.

[0057] According to some embodiments, the methods disclosed herein can be used for treatment of various types of cancers and tissues,

[0058] The term “bubbles” is directed to substantially spherical or rod-like bodies having a shell (for example, a phospholipid shell) and a gaseous core, capable of serving as low energy cavitation nuclei for US mechanotherapy of target region (such as, tumor region). In some embodiments, the bubbles are microbubbles (MBs) (having a diameter of over about 800nm (for example, in the range of about 800-3 OOOnm). In some embodiments, the bubbles are nanobubbles (NBs) (having a diameter of less than about 800 nm). In some embodiments, the cavitation nuclei used are nanodroplets with a liquid core that can be vaporized and become a microbubble or a nanobubble.

[0059] The terms “CART cells”, “CAR-T cells” and “CAR T cells” are used interchangeably and refer to chimeric antigen receptor (CAR)-engineered T lymphocytes. Such CAR-T cells include a chimeric antigen receptor (CAR) that includes an extracellular antigen-binding domain, for example a single chain variable fragment (ScFv), capable of specifically recognizing and binding to an antigen on the surface of target cells such as cancer cells. A typical CAR includes, in operable order, an extracellular antigen recognition domain (ScFv), an optional hinge region, a transmembrane (TM) domain (for example derived from CD28, CD8, or an analog thereof), one or more intracellular costimulatory domains (for example selected from CD28, 4-1BB, 0X40, iCOS, CD27, CD80, or CD70, or analogs thereof), and an intracellular signaling / activation domain (for example CD3-(^ or FcRy, or analogs thereof, including one or more immunoreceptor tyrosine-based activation motifs (IT AMs)). The CAR may include any hinge, costimulatory, or activation domains known in the art, including but not limited to 4- IBB, CD28, or CD40 co-stimulation and zeta or gamma chain IT AMs. The CAR-T cells may be of any generation (first, second, third, or fourth generation) and may include dual CAR-T cells, tandem CAR-T cells, or universal / allogeneic CAR-T cells. The CAR and / or the CAR-T cells may be produced by any suitable genetic engineering method known in the art, including but not limited to viral vector transduction (e.g., retroviral or lentiviral), mRNA transfection, transposon-based delivery, CRISPR / Cas genome editing, or in vivo / in situ expression systems. In general, CAR-T cell therapy involves modifying T cells to express a CAR that recognizes and induces cytotoxic and / or immune-stimulatory effects (by recruiting other immune cells) specifically against cells expressing the targeted antigen. In some embodiments, the term CAR-T cells also encompasses CAR-expressing T cell subtypes, including CD4+helper T cells, CD8+cytotoxic T cells, tumor-infiltrating lymphocytes (TILs), and mixed CD4+ / CD8+populations, as well as other immune effector cells genetically engineered to express CARs, including but not limited to CAR-NK cells, CAR-macrophages (CAR-M), and CAR-invariant NKT cells.

[0060] According to some embodiments, the T cell may be any type of thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes, a T helper (Th) cell, CD4+ T cell, a cytotoxic T cell, a tumor infiltrating cytotoxic T cell (TIL), CD8+ T cell, CD4+CD8+ T cell, CD4-CD8-T cell, CD45+, NK-CAR cell, NK-T cell, lymphocytes, and the like, or any combinations thereof. In some embodiments, the T-cell may be autologous, allogeneic, syngeneic or xenogeneic to the subject. In some embodiments, the CAR-T cells may be autologous or allogeneic, including universal CAR-T cells lacking endogenous TCR / MHC, or may be CAR-NK cells or CAR- macrophages.

[0061] According to some embodiments, the CAR may bind specifically to a tumor-associated antigen, including, for example, but not limited to: ErbB2 (HER2), CD 19, BCMA, CD38, CD 138, EGFR, CD276, CD24, GD2, EGF, Mesothelin (MSLN), MUC1, FAP, PSCA, EPC AM, CEA, PSMA, GPC3, LMP1, CD133, cMET, ROR1, CD70, MUC16, RAO, RA9, and the like, or any combinations thereof. Each possibility is a separate embodiment. In some embodiments, a CAR may recognize / bind / be directed against more than one antigen.

[0062] In some embodiments, the CAR-T cells are engineered to express dual CAR constructs. In some embodiments, the dual CAR construct include two distinct chimeric antigen receptors on the same T cell, each with a different antigen specificity. In some embodiments, the CAR- T cells are engineered to include logic-gated CAR constructs, designed to require the presence of two or more distinct antigens on the same target cell to trigger full activation. For example, in certain embodiments, a dual CAR is used that is designed to recognize CD24 and CD138 antigens, such that activation occurs only when both antigens are present on the same target cell. In some embodiments, this “gated” configuration allows selective activation of the CAR- T cells in the presence of tumor cells co-expressing both CD24 and CD 138, thereby reducing off-target toxicity against cells expressing only one of the antigens.

[0063] In some embodiments, such gating may be achieved by incorporating two different CARs on the same T cell, each specific to a different antigen, wherein one CAR delivers a primary activation signal (for example, comprising a CD3-(^ intracellular domain) and the other CAR delivers a costimulatory signal (for example, comprising a 4- IBB or CD28 intracellular domain). In some embodiments, the two CARs each contain full activation domains, such that co-engagement of both antigens produces synergistic activation.

[0064] In certain embodiments, the gating antigens may be selected from tumor-associated antigens, such as CD24 and CD 138, CD38 and CD 138, or any other pair or combination of antigens expressed on target pathological cells. In some embodiments, a dual CAR may be designed to recognize CD24 and gate with CD138 antigens of a target cells.

[0065] In some embodiments, such gating can enhance tumor specificity and safety by reducing off-target cytotoxicity against normal tissues that express only one of the target antigens.

[0066] According to some embodiments, the compositions and methods disclosed herein may be used for the treatment of cancer.

[0067] In some embodiments, the cancer may be selected from breast cancer, ovarian cancer, lung adenocarcinoma, stomach, mammary carcinomas, melanoma, skin neoplasms, lymphoma, leukemia, gastrointestinal tumors, including colon carcinomas, stomach carcinomas, pancreas carcinomas, colon cancer, small intestine cancer, ovarian carcinomas, cervical carcinomas, lung cancer, prostate cancer, kidney cell carcinomas and / or liver metastases. In another embodiment, the cancer is a solid tumor (e.g. sarcoma, bladder cancer, bone cancer, brain cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, liver cancer, lung cancer, glioblastoma multiforme, glioma, melanoma, castration-resistant prostate cancer, triple negative breast cancer, squamous cell carcinoma, or colorectal cancer). Each possibility is a separate embodiment.

[0068] According to some embodiments, the cancer may be selected from breast cancer, ovarian cancer, lung adenocarcinoma, stomach, liver, pancreatic and brain cancers and hematology malignancies.

[0069] In some embodiments, the disclosed systems, compositions and methods are used for treating relapsed or refractory disease, including relapsed tumors or recurrent lesions following prior therapy. In some embodiments, the treatment is used for preventing or reducing the likelihood of relapse after achieving remission, for example by eliminating residual tumor cells that may lead to disease recurrence. In some embodiments, the methods are used for eradicating minimal residual disease (MRD) to prolong remission duration.

[0070] According to some embodiments, the compositions and methods disclosed herein may be administered as an initial therapy, as a salvage or rescue therapy for relapsed or refractory disease, or as a consolidation or maintenance therapy to prevent relapse. For example, in certain embodiments, bubble-mediated ultrasound disruption is applied to residual tumor sites or tissue niches following primary therapy to facilitate infiltration of subsequently administered CAR- T cells, thereby eliminating residual malignant or pathogenic cells that could cause relapse.

[0071] According to some embodiments, although much of the present disclosure describes treatment of solid tumors, the methods and systems described herein may also be used for treating other conditions where pathological tissues impede immune cell infiltration. Such conditions include hematological malignancies (such as leukemias, lymphomas, and myelomas), chronic inflammatory and autoimmune disorders (such as rheumatoid arthritis, inflammatory bowel disease, or systemic lupus erythematosus), viral reservoir diseases (such as HIV or hepatitis B), benign proliferative diseases (such as adenomas, fibromas), and fibrotic disorders (such as liver, lung, or cardiac fibrosis). In such cases, bubble-mediated ultrasound may locally disrupt the diseased tissue or stromal barrier, facilitating penetration of therapeutic engineered cells and enhancing therapeutic efficacy.

[0072] According to some embodiments, following bubbles administration (for example, systemic or localized, by injection), and coupled with low-frequency tumor insonation, bubbles (such as NBs and / or MBs) that are located in the tumor (or in vicinity thereof), are used as mechanical therapeutic warheads, creating large lesions in the tumor, synergistically facilitating the effect of administered CAR-T cells on the tumor.

[0073] According to some embodiments, the MBs may have a diameter in the range of about 800-3000nm. In some embodiments, the MBs may have an average diameter in the range of about 900-2500nm. In some embodiments, the MBs may have an average diameter in the range of about 1000-2000nm. In some embodiments, the MBs may have an average diameter in the range of about 1200-1800nm. In some embodiments, the MBs may have an average diameter in the range of about 1300-1500nm. In some embodiments, the MBs may have an average diameter in the range of about 750-1200nm. In some embodiments, the MBs may have an average diameter of about 750nm. In some embodiments, the MBs may have an average diameter of over about 800nm. Each possibility is a separate embodiment.

[0074] According to some embodiments, the NBs may have a diameter in the range of about 30-800nm. In some embodiments, the NBs may have an average diameter in the range of about 75-600nm. In some embodiments, the NBs may have an average diameter in the range of about 80-400. In some embodiments, the NBs may have an average diameter in the range of about 90-300nm. In some embodiments, the NBs may have an average diameter in the range of about 100-250nm. In some embodiments, the NBs may have an average diameter of about 110-230 nm. In some embodiments, the NBs may have an average diameter in the range of about 150- 200nm. In some embodiments, the NBs may have an average diameter of about 170nm. In some embodiments, the NBs may have an average diameter of less than about 250nm, less than about 200nm, each possibility is a separate embodiment.

[0075] According to some embodiments, the low-energy US (LE-US) may have a center frequency of 1000kHz or less, 800kHz or less, 500kHz or less, 250kHz or less, 200 kHz or less, 150kHz or less, 100 kHz or less, 90 kHz or less, 80 kHz or less, or about 80 kHz. In some embodiments, the US may have a peak negative pressure (PNP) of 550 kPa or less, 500 kPa or less, 450 kPa or less, 400 kPa or less, 350 kPa or less, 300 kPa or less, 280 kPa or less, 250 kPa or less, or about 250 kPa. In some embodiments, the US may be characterized by having a mechanical index (MI) that is less than about 1.9, less than about 1.5, less than about 1. In some embodiments, the MI may be calculated as equaling the PNP of the US divided by the square root of the center frequency of the US.

[0076] According to some exemplary embodiments, 80 kHz US may be applied, using an MI of 1.1-1.5 (for example, 1.3), burst length of about 1-5 ms (milliseconds), a PRF of about 10- 80Hz (for example, about 30 Hz), and a total US application duration of about 1-60 minutes (for example, 2 minutes).

[0077] According to some embodiments, the US methods disclosed herein utilize a mechanical index (MI) within the safety limits. The MI parameter is defined as the peak negative pressure (PNP) divided by the square root of frequency, is a parameter that determines the likelihood of creating mechanical damage as a result of US application.

[0078] According to some embodiments, the bubbles are lipid nanobubbles or microbubbles. In some embodiments, the bubbles have an external lipid shell, that may include 1,2- dibehenoyl-sn-glycero-3 -phosphocholine (C22), l,2-dipalmitoyl-sn-glycero-3 -phosphate (DPPA), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-mPEG 2000). In some embodiments, the lipids may be in a molar ratio of 18.8:4.2:8.1 : 1 with a final lipid concentration of 10 mg / mL. In some embodiments, the lipid mixture may be sonicated. In some embodiments, the shell may include such components as, but not limited to: disteroylphosphatidylcholine (DSPC), 2-dibehenoyl-sn-glycero-3 -phosphocholine (C22), 1 ,2-dipalmitoyl-sn-glycero-3 -phosphate (DPPA), 1 ,2-dipalmitoyl-sn-glycero-3 - phosphoethanolamine (DPPE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(poly ethylene glycol)-2000] (ammonium salt) (DSPE-PEG2K) and 1,2- distearoylsnglycero-3-phosphoethanolamine-N-[biotinyl(poly ethylene glycol) 2000] (DSPE- PEG2000-Biotin).

[0079] In some embodiments, the bubbles include a fluid core. In some embodiments, the fluid is gas. In some embodiments, the gas is selected from: perfluorobutane (C4F10), octafluoropropane C3F8, perfluorocarbons, sulfur hexafluoride, air and nitrogen.

[0080] According to some embodiments, the size distribution and concentration of freshly prepared bubbles may be measured using a particle sizing system. In some embodiments, the concentration may be 3.3 x 1012particles / ml. According to some embodiments morphology of the bubbles may be characterized using transmission election microscopy (TEM).

[0081] According to some embodiments, the bubbles may exhibit an essentially spherical morphology and / or rod-shape morphology.

[0082] According to some embodiments, the bubbles may further include a targeting moiety on an external region thereof. In some embodiments the targeting moiety may be on the shell of the bubbles. In some embodiments, the targeting moiety may be a cell-type specific targeting moiety. In some embodiments, the targeting moiety may be a cell-type specific antibody. In some embodiments, the targeted to diseased tissue by conjugated ligands (e.g., anti-HER2 antibodies, anti-EGFR antibodies, folate, RGD peptides).

[0083] According to some embodiments, the amount / concentration / number of the nanobubbles may be determined according to the target tissue, size of tissue, type of tumor, size of tumor, location of the tumor, and the like. In some embodiments, the amount of NBs administered may be about 1x1011NBs. In some embodiments, the amount of NBs administered may be about 5xl0nNBs. In some embodiments, the amount of NBs administered may be about 6.6xlOnNBs. In some embodiments, the amount of NBs administered may be at least about 1x106NBs. In some embodiments, the amount of NBs administered may be at least about 1x108NBs. In some embodiments, the amount of NBs administered may be at least about 1x108NBs. In some embodiments, the amount of NBs administered may be at least about 1x1011NBs. In some embodiments, the concentration of NBs administered may be about 1x1011NBs / 200pl.

[0084] According to some embodiments, the amount / concentration / number of the microbubbles may be determined according to the target tissue, size of tissue, type of tumor, size of tumor, location of the tumor, and the like. In some embodiments, the amount of MBs administered may be about 1x105MBs. In some embodiments, the amount of MBs administered may be about 1x106MBs. In some embodiments, the amount of MBs administered may be about 5xl06MBs. In some embodiments, the amount of MBs administered may be about 6.6xl06MBs. In some embodiments, the amount of MBs administered may be at least about 1x107MBs. In some embodiments, the amount of MBs administered may be at least about 1x108MBs. In some embodiments, the amount of MBs administered may be at least about 1X109MBS. In some embodiments, the amount of MBs administered may be at least about 1x1010MBs. In some embodiments, the concentration of

[0085] MBs administered may be about 1x1011MBs / 20 l.

[0086] According to some embodiments, the bubbles may be administered for a plurality of times, according to a treatment regime. In some embodiments, when a plurality of administration events are completed, each administration event may be identical, similar or different from the previous and / or following administration event (for example, with respect of: type of bubbles, amount of bubbles, administration route, properties of applied ultrasound, timing of US application, and the like, or any combinations thereof).

[0087] According to some embodiments, the amount / concentration / number of the CAR-T cells may be determined according to the target tissue, size of tissue, type of tumor, size of tumor, location of the tumor, and the like. According to some embodiments, CAR-T cells may be administered at concentration of about 0.2xl06to 10xl06CAR-T cells per kilogram of body weight of the subject, e.g., about 0.5x l06-2>< 106, about 1 X 106-5X 106, about 2x l06-8x l06, about 3x l06-6x l06CAR-T cells / kg, and the like.

[0088] In some embodiments, the CAR-T cells may be administered for a plurality of times, according to a treatment regime. In some embodiments, when a plurality of administration events are completed, each administration event may be identical, similar or different from the previous and / or following administration event (for example, with respect of: type of cells (i.e., type of CAR), amount of cells, administration route, excipients, timing with respect of bubbles and / or US application, and the like, or any combinations thereof).

[0089] In some embodiments, the CAR-T disclosed herein may be administered following a preconditioning regimen (i.e., lym phodepletion) to enhance CAR-T engraftment and expansion.

[0090] In some embodiments, the subject may be administered a preconditioning regimen before CAR-T cell administration. Suitable preconditioning regimens include, for example, lymphodepl eting chemotherapy agents such as cyclophosphamide and fludarabine, bendamustine, or other known lymphodepleting regimens, as well as low-dose total body irradiation. In some embodiments, the ultrasound and bubble-mediated mechanical disruption is performed before, during, or after the preconditioning regimen.

[0091] In some embodiments, the preconditioning regimen may be administered about 1- 14 days before CAR-T administration, for example about 1-5 or about 2-7 days before CAR-T administration. In some embodiments, the preconditioning regimen may include one or more doses of cyclophosphamide, fludarabine, bendamustine, or low-dose total body irradiation, administered over a period of 1 -5 days, followed by a rest period of about 1-3 days before CAR-T cell infusion.

[0092] In some embodiments, the bubble administration and ultrasound application may be performed after completion of the preconditioning regimen but before CAR-T administration, for example within about 1-72 hours after preconditioning and about 1-72 hours before CAR- T infusion. In some embodiments, the bubbles and ultrasound may be administered concurrently with or shortly after the CAR-T infusion even if preconditioning occurred several days earlier.

[0093] In some embodiments, the bubble-mediated ultrasound treatment may reduce or eliminate the need for preconditioning by mechanically disrupting the target tissue to enhance CAR-T cell infiltration without systemic lymphodepletion.

[0094] In some embodiments, administration of the CAR-T cells further encompasses the administration of a composition including viral carriers, capable of targeting T-cells and inducing the expression of desired CARs in the T-cells, wherein the CARs are at least partially encoded by the viral carriers.

[0095] According to some embodiments, exemplary CAR-T cells may include a targeting region directed against HER-2. According to some embodiments, exemplary CAR-T cells may include a targeting region directed against Carcinoembryonic antigen (CEA). According to some embodiments, exemplary CAR-T cells may may include a targeting region directed against CA-125. According to some embodiments, exemplary CAR-T cells may include a targeting region directed against MUC-1. According to some embodiments, exemplary CAR- T cells may include a targeting region directed against EGFR. According to some embodiments, exemplary CAR-T cells may include a targeting region directed against CD276. According to some embodiments, exemplary CAR-T cells may include a targeting region directed against CD24. According to some embodiments, exemplary CAR-T cells may include a targeting region directed against CD38. According to some embodiments, exemplary CAR- T cells may include a targeting region directed against CD138. According to some embodiments, exemplary CAR-T cells may include a targeting region directed against dual CAR of any combination, such as, but not limited to, CD138-CD24, CD38-CD138, and the like.

[0096] In some embodiments, CAR-T cells compositions may include a pharmaceutically acceptable carrier, excipients, saline, buffers, etc.

[0097] In some embodiments, immune checkpoint inhibitors (e.g., anti-PD-1, anti-PD-Ll, anti- CTLA-4) or cytokines (e.g., IL-2, IL-12, IL-15, IL-18, GM-CSF) may be co-administered or co-expressed (e.g., by being constructed with the CAR expressing cassette (for example, CAR- IL18 secreting construct), to enhance CAR-T persistence and function.

[0098] According to some embodiments, systemic administration may include parenteral administration, including, for example: intravenously, intra-arterially, intramuscularly, intraperitoneally, intradermally, intravitreally, by inhalation, or subcutaneously administration. In some embodiments, the systemic administration is by injection.

[0099] According to some embodiments, compositions formulated for injection may be in the form of solutions, suspensions, dispersions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0100] According to some embodiments, the bubbles and / or CAR-T cells compositions may be administered intravenously, and may thus be formulated in a form suitable for intravenous administration. According to another embodiment, the compositions may be administered intra-arterially, and are thus formulated in a form suitable for intra-arterial administration.

[0101] In some embodiments, the administration may be localized, for example, intratumorally (i.e., in the tumor). According to some embodiments, the application of US may be performed at a time period after administration of the bubbles. In some embodiments, the time period may be at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 60 minutes. Each possibility is a separate embodiment.

[0102] According to some embodiments, the CAR-T cells (or compositions including the same) and the bubbles may be administered in a time interval before or after the ultrasound treatment. In some embodiments, the time interval may be in the range of 1-60 minutes, 1-24 hours, 1-7 days, or any time period therebetween.

[0103] In some embodiments, the CAR-T cells and the bubbes are included in the same composition. In some embodiments, the CAR-T cells and the bubbles are included in separate compositions. In some embodiments, the CAR-T may be administered together with the bubbles (in a single or separate compositions). In some embodiments, the CAR-T may be administered after the administration of the bubbles. In some embodiments, the CAR-T may be administered prior to the administration of the bubbles. In some embodiments, the administration of the CAR-T composition(s) and / or the bubbles composition(s) may be repeated for any number of rounds.

[0104] According to some embodiments, as exemplified hereinbelow, in tumor cells, the combination of low-frequency insonation at a peak negative pressure of 0.35 MPa and nanobubbles resulted in a decrease in viability to 17.3 ± 1.7% compared to control groups. In- vivo, using a breast cancer mouse model, confirmation of nanobubble tumor accumulation was achieved through fluorescence microscopy and ultrasound imaging. Nanobubble-mediated histotripsy effectively fragmented tumors, as evidenced by histological examination. Moreover, the combined method of nanobubble-mediated histotripsy and CAR-T cells was shown to significantly reduce tumor growth rate and led to increased infiltration of immune cells (F4 / 80 macrophages and CD3 T-cells) into the tumor, as detected by FACS and immunohi stochemi stry .

[0105] According to some embodiments, the pharmaceutical compositions disclosed herein (including the CAR-T cells and / or bubbles), are for use in treating cancer. According to some embodiments, the cancer expresses a tumor antigen associated, to which the CAR specifically binds. According to some embodiments, the cancer is a solid cancer.

[0106] According to some embodiments, there is provided a composition comprising bubbles selected from microbubbles (MBs) and nanobubbles (NBs), and chimeric antigen receptor (CAR)-T cells, for use in treating a pathological condition in a subject, wherein the treatment comprises applying low-frequency ultrasound to a target tissue after administration of the bubbles and / or CAR-T cells, thereby inducing bubble-mediated disruption of the tissue to enhance infiltration and activity of the CAR-T cells.

[0107] According to some embodiments, the condition is a cancer selected from solid tumors, leukemias, lymphomas, and myelomas, primary tumors, relapsing tumors, and the like. Each possibility is a separate embodiment.

[0108] According to some embodiments, the bubbles have an average diameter of 50-800 nm or 800-2000 nm.

[0109] According to some embodiments, the CAR-T cells are allogeneic CAR-T cells, universal CAR-T cells, CAR-NK cells, or CAR-macrophages.

[0110] According to some embodiments, the treatment may further include administration of an immune checkpoint inhibitor or cytokine.

[0111] According to some embodiments, the treatment may further include imaging the bubbles prior to insonation.

[0112] According to some embodiments, there is provided a system which includes an ultrasound transducer, configured to deliver ultrasound at a center frequency of about 800 kHz or less, and PNP of less than about 800 kPa; and a control module configured to apply the ultrasound after administration of bubbles, wherein the subject is further administered with CAR-T cells, wherein the ultrasound induces bubble-mediated disruption of tissue to enhance infiltration and activity of the CAR-T cells.

[0113] In some embodiments, the system may include a low-frequency focused ultrasound transmitter, an ultrasound imaging module to visualize bubble accumulation, a feedback controller to adjust parameters based on cavitation or tissue perfusion, an infusion module to coordinate timed delivery of bubble and CAR-T compositions, or any combinations thereof.In some embodiments, the system may include real-time ultrasound imaging and an automated feedback controller to adjust ultrasound parameters based on bubble cavitation or tissue perfusion.

[0114] According to some embodiments, there is provided a composition comprising bubbles and CAR-T cells, for use in combination with low-frequency ultrasound to treat a pathological condition in a subject. According to some embodiments, there is provided a combination which includes a composition including lipid CAR-T cells, and a composition including bubbles.

[0115] As used herein the term "about" refers to the designated value ± 10%.

[0116] In the description and claims of the application, the words “include” and “have”, and forms thereof, are not limited to members in a list with which the words may be associated.

[0117] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0118] EXAMPLES

[0119] Materials and Methods

[0120] Bubble preparation and characterization

[0121] NB synthesis was performed as follows: l,2-dibehenoyl-sn-glycero-3 -phosphocholine (C22), l,2-dipalmitoyl-snglycero-3 -phosphate (DPP A), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-mPEG 2000) (Sigma-Aldrich) were dissolved in propylene glycol by heating to 80 °C and sonication. Glycerol was mixed with phosphate buffer solution (PBS) and the mixture was preheated to 80 °C before adding it to the lipid solution. The lipids were mixed at a molar ratio of 18.8 : 4.2 : 8.1 : 1 and a final lipid concentration of 10 mg mL-1. The resulting mixture was then sonicated at room temperature for 10 min. One mL of the resulting lipid mixture was transferred to a 3 mL headspace vial. The vial was saturated with octafluoropropane (C3F8) gas, then capped with a rubber septum and sealed with an aluminum seal. The vial was stored at 4 °C until usage. At a later time, a vial was activated by mechanical shaking for 45 s in a Vialmix shaker (Bristol- Myers Squibb Medical Imaging Inc., N. Billerica, MA). The vial was placed inverted into a centrifuge (581 OR centrifuge, Eppendorf AG, Hamburg, Germany) and then centrifuged at 50 ref for 5 min. 200 μL of the NB solution was taken out of the inverted vial with a 21 G needle at a distance of 5 mm from the bottom of the vial. A particle sizing system (AccuSizer FX-Nano, Particle Sizing Systems, Entegris, MA, USA) was used to measure the size and concentration of the purified NBs. The size distribution and concentration varied by less than 10% between measurements.

[0122] TEM experiments were conducted to visualize the NB morphology used a TEM (JEM- 1400Plus, JEOL, Tokyo, Japan) that was operated at 120 kV. Briefly, 5 μL of the NB sample were pipetted onto glow-discharged carbon grids. After 30 s of incubation, the sample was washed with buffer. 5 μL of 1% uranyl acetate were added for 30 s, then removed, and left to air-dry. The grids were then imaged.

[0123] Low frequency ultrasound setup

[0124] The experimental therapeutic configuration included a 64-mm-diameter spherically focused single-element transducer (Hl 15, Sonic Concepts, Bothell, WA, USA) placed on its bottom of a degassed and deionized water tank facing upwards. The transducer was focused at a distance of 45 mm. The transducer transmits a sinusoid at the desired frequency, in this case 250kHz. The waveform was generated using a transducer power output unit combining an arbitrary waveform generator together with a radiofrequency amplifier (TPO-200, Sonic Concepts). Calibration measurements of the transmitted pressure were previously described. Beam pattern measurements using a calibrated hydrophone (NH0500) reveal a focal width of 7 mm and a focal length of 50 mm for the for the 250 kHz center frequency configuration. For each experiment, the desired target was placed at the focal spot of the transducer. For the in vitro experiments, a 0.5 mL Eppendorf tube containing a mixture of cancer cells and NBs was used. For the in vivo experiments, a mouse was positioned such that the breast cancer tumor was located precisely at the focal spot of the transducer.

[0125] Cell lines

[0126] The 4T1-HER2 cell line derived from 4T1 cells, a metastatic triple negative murine breast carcinoma cell line, was used for in vitro studies. To generate the HER2-expressing variant, 4T1 cells were transfected with the construct, resulting in stable expression of the human HER2 protein. The 4T1-HER2 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% v / v fetal bovine serum (FBS), 1% v / v penicillinstreptomycin and 0.11 g L'1sodium pyruvate.

[0127] The human ovarian cancer (OC) cell line OVCAR8 was provided by Prof. Dan Peer from Tel-Aviv University. OVCAR8 mCherry was created following transduction of the mCherry gene. OVCAR8 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% v / v penicillin-streptomycin and 0.11 g L'1sodium pyruvate.

[0128] Cells were incubated in a humidified 37 °C incubator with 5% CO2. The cells were frozen at low passage, and the number of passages after thawing was recorded.

[0129] Packaging cell lines GP+E-86 (ATCC CRL-9642) murine packaging cell line was cultured in DMEM and maintained in a humidified 37°C incubator with 7.5% CO2.

[0130] CAR T preparation

[0131] Antibodies and reagents- Anti-mCD3 (BioLegend) and anti m-CD28 (SouthernBiotech) were used.

[0132] Mouse HER2-specific CAR-T cells were generated. The HER2 specific CAR construct includes a GFP marker to monitor transduction efficiency precentage. Briefly, Retroviral transduction of T cells was performed. Briefly, CD3+CD28 activated splenocytes were transduced using “spin-infection” on RetroNectin (Takara) coated-plates in the presence of a vector-containing supernatant and IL-2. After transduction, cells were cultured for 2 days in the presence of 350 p / ml human IL-2 (Proleukin, Novartis).

[0133] Construction of CAR T- Variable fragment light (VL) chain was linked to a variable fragment heavy (VH) chain through a flexible linker. The N29 monoclonal antibody against human ErbB2 was used as a source of the single-chain fragment variable (scFv) for construction of the ErbB2-directed CAR (Deshet-Unger, N., et.al, Biomedicines 2022, 10(9), 2216). The ErbB2 scFv was ligated with the cytoplasmic domain of the costimulatory domain of the CD28 gene, followed by the activating domain of the human FcyR / CD3 molecule at the 3’ end.

[0134] Another CAR dual construct was used, which was designed to recognize CD24 and gate with CD138 antigens displayed on the OVCAR8 cell line.

[0135] Preparation of packaging cells.

[0136] Transduction- Retroviral transduction of T cells was performed as described previously (Globerson-Levin, A., et al. Molecular Therapy, Volume 22, Issue 5, 1029 - 1038). Briefly, CD3+CD28 activated murine T cells were transduced using “spin-infection” on RetroNectin (Takara) coated-plates in the presence of vector-containing supernatant and IL-2. Shortly after transduction the T cells were used for in vitro and in vivo experiments. Transduction efficiency of ErbB2CAR was estimated based on green fluorescence protein (GFP) expression, analyzed by a BD CANTO flow cytometer. Data analysis was carried out with FCS Express software.

[0137] In Vitro IFNy Secretion Assay

[0138] Before the cell viability was assessed, cell culture supernatants were harvested, frozen, and later assayed for interferon (ZFN)-y using the mouse or human fFN-y ELISA Kit (R&D Systems), according to the manufacturer’s instructions.

[0139] In vitro NB-mediated low frequency insonation

[0140] About 85% cell confluency was reached on the day of each experiment. Cell collection was performed using TrypLE Express dissociation reagent (Gibco Corp, 12604-013, Grand Island, NY, USA). The cells were then suspended at a concentration of 3.3 x io6cells per mL in degassed PBS containing calcium and magnesium (PBS+ / +). A mixture of 2 x 105cells and NBs at a concentration of 12.5 x 108NBs per μL was then transferred into 0.5 mL Eppendorf tubes and degassed PBS+ / + was added to a final volume of 0.48 mL. Each Eppendorf tube was positioned at the focal spot of the low frequency US setup, and 250 kHz US with an MI of 1.3 (PNP of 650 kPa), a PRF of 30 Hz and a 0.5 ms burst length was applied to the tube for 30 seconds. After each US treatment, 30 μL of the cell suspension was taken and Cell drop (DeNovix Inc., Wilmington, USA) and 0.4% trypan blue (Sigma- Aldrich) at a 1 : 1 ratio to the cell suspension were used for live cell counting. Next, the cells were seeded in six-well tissue culture dishes containing complete medium supplemented with 2.5% v / v penicillinstreptomycin. After, T lymphocytes transduced with the N29-derived anti-HER2 CAR or the dual-CAR anti-CD24 / CD138 were added at differing effector to target ratio (2: 1, 1 :1, 0.5: 1 or 4: 1,2: 1,0.5: 1 respectively) relative to the live cell count. 72 hours post-US treatment (the cells were maintained at 37°C in a humidified incubator with 5% CO2), cell viability was assessed. The cells were collected in 500 μL of TrypLE Express. Cell drop (DeNovix Inc., Wilmington, USA) and 0.4% trypan blue at a 1 : 1 ratio to the cell suspension and were used for live cell counting. All treatments were analyzed in triplicate. The control groups were composed of the sham group, and groups treated with US only, NB only, US+NB only, untransduced (UT) lymphocytes and CAR-T only.

[0141] Breast cancer animal model

[0142] In vivo experiments were conducted using a genetically modified FVB spontaneous breast cancer model. Briefly, Female FVB / NHan®Hsd mice (8 to 12 weeks old, 20-25 g) were used as breast cancer animal models for evaluation. Her2NG transgenic male mice (Jackson) were routinely mated with wild-type FVB / N strain female mice to obtain Fl heterozygotic Her2NG offspring. The transgene positive females developed mammary tumors. Those tumors were harvested, minced in a sterile dish, filtered, washed, and counted. The cells collected are injected subcutaneously into 8 to 12 weeks old female (20-25 gram) FVB / NHanHsd mice (Envigo, Jerusalem, Israel), 1 x 106cells for each mouse. The cells were subcutaneously injected into the #4 inguinal mammary fat pad. The tumor size was then monitored until it reached approximately 6 mm in diameter, which occurred approximately 21 days post injection, before US treatment. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Tel Aviv University and were carried out in compliance with institutional guidelines for care and use of animal models.

[0143] In vivo experiments

[0144] In the in vivo experiments, tumor bearing mice were anesthetized with 2% isoflurane using a low flow vaporizer system (SomnoFlo, Kent Scientific). The tumor area was completely shaved, additional fur was removed using a depilatory cream to enhance coupling, and US gel was applied. The mice were positioned on their side, on top of an agarose pad, such that the tumor was located at the focal spot of the transducer. To prepare the agarose spacer, agarose powder (Alfa Aesar, MA) was mixed with deionized water to form a 1.5% solution at ambient temperature. The mixture was heated until all powder dissolved, poured into a custom mold, and allowed 19 to cool at ambient temperature. The Hl 15 spherically-focused singleelement transducer was positioned at the bottom of a degassed water tank, facing upwards and aligned to focus the agar spacer. The agar spacer, created in a custom mold, was designed to place the tumor at the transducer's focal depth (z = 45 mm). 6.6 x 1011Bs in 200 pl were systemically injected. Ten minutes post-injection, 250 kHz US was applied to the tumor using an MI of 1.3 (a PNP of 650 kPa), a burst length of 0.5 ms, a PRF of 30 Hz, and a total treatment duration of 2 minutes. The parameters were chosen while ensuring that the mechanical index remained below the required 1.9 by US guidelines. 3 days post US+NB treatment the mice were injected intravenously with U 107CAR T cells. The control groups were composed of a sham group, a group treated only with US+NB, and a group only injected with CAR T cells.

[0145] In vivo analysis of NB-mediated low frequency insonation of tumors

[0146] The mice were sacrificed 24 hours after treatment for tumor extraction and histology analysis. The tumors were harvested, covered in Leica OCT cryocompound ‘tissue freezing medium’ (Leica Microsystems, Bensheim, Germany) and flash-frozen in 2-methylbutane (Sigma- Aldrich) using liquid nitrogen. The tumors were then transferred to a -80° refrigerator until sectioning. A -20° Leica CM1950 cryostat (Leica Biosystems Inc.) was used to cut the tumors into 12 gm slices that were mounted on microscope slides and placed in dark slide boxes for air-drying at room temperature. Frozen tumors were cryo-sectioned to 12 pmthick slices and stained with hematoxylin (Leica 3801542) and eosin (Leica 3801602) (H&E) according to the standard procedure. The slides were then scanned with an Aperio Versa 200 slide scanner (Leica Biosystems, Buffalo Grove, IL) at 20* optical magnification.

[0147] Immune cell infiltration experiments

[0148] For this set of experiments the mice were sacrificed 1 week after US+NB+CAR T treatment for tumor extraction and immune cell infiltration analysis. The tumors were then frozen and at a later time were used for histology. Frozen tumors were cryo-sectioned to 12 pmthick slices. Immunohistochemistry was conducted manually. For staining of macrophages, the rabbit anti-mouse F4 / 80 primary antibody (1 : 1000; Abeam; ab300421) was used. Initially, the slices of frozen tumors were fixed in cold acetone for 15 minutes and then air-dried for 10 minutes. This was followed by a wash in PBS. For blocking, a solution containing 5% Normal Goat Serum (NGS) and 5% Bovine Serum Albumin (BSA) in PBS was used, and the slides were incubated in this solution for 30 minutes followed by a was in PBS. The primary and secondary antibodies were diluted in a solution with 1% NGS and 1% BSA in PBS. The slides were first incubated with the primary antibody at room temperature for 90 minutes, followed by an overnight incubation at -4°C. Post-primary antibody incubation, the slides were washed twice with 0.5% NGS in PBS for 10 minutes each. This was followed by incubation with the secondary antibody for 1 hour at room temperature and subsequent similar washes. For visualization, the slides were incubated with DAB (Diaminobenzidine) for 15 minutes and then washed in distilled water. Hematoxylin staining was applied for nuclear visualization. Throughout the process, a mouse spleen sample was included as a positive control.

[0149] Separately but with the same timetable for tumor extraction, flow cytometry analysis was conducted. Single-cell suspensions were prepared from tumors and spleens by mechanical dissociation. Cells were filtered, washed, and incubated with the appropriate fluorescent antibodies (anti-CD45, anti-CD8, anti CD4 (Biogen)) in staining buffer (5% fetal calf serum, 0.05% sodium azide in phosphate-buffered saline, 2 mM EDTA) for 45 minutes on ice. Cells were spun down, washed, resuspended in Buffer, and analyzed by a BD CANTO flow cytometer. Data analysis was carried out with FCS Express software. Lymphocytes (0.5-1 x 106cells) were incubated with the appropriate fluorescent antibodies in staining buffer (5% fetal calf serum, 0.05% sodium azide in phosphate-buffered saline, 2 mM EDTA) for 45 minutes on ice. Cells were spun down, washed, resuspended in Buffer, and analyzed by a BD CANTO flow cytometer. Data analysis was carried out with FCS Express software.

[0150] In vivo growth rate and survival experiments

[0151] The anti-tumor response was evaluated by monitoring Tumor growth rate. Tumor growth was monitored by measuring the length (L), width (W), and depth (D) of each tumor using a digital caliper two to three times per week. Tumor volume was calculated using the formula:

[0152] V(mm3) = 0.5 x πT X L X W X D

[0153] Further, the response was also monitored via survival. Mice were euthanized at the experimental endpoint or when tumor volume reached 1,500 mm3, in accordance with institutional animal care and use guidelines.

[0154] Statistics

[0155] Statistical analyses were performed using Prism9 software (GraphPad Software Inc.). Results are presented as mean ± SD. Statistical tests are reported in the relevant captions. P values less than 0.05 were considered significant and were adjusted for multiple comparisons as indicated in the captions.

[0156] RESULTS

[0157] A particle sizing system was employed to assess both the size distribution and particle concentration of freshly synthesized lipid-shelled C3F8 gas core NBs. The analysis showed that the NBs had an average diameter of 170 ± 60 nm, with a concentration measured at 3.3 x 1012particles per milliliter. The same instrument was utilized for both sizing and concentration measurements.

[0158] Initially, the transduction efficiency of lymphocytes with the CAR-T GFP construct was confirmed. The results are presented in Fig. 1. In vitro nanobubble-mediated low frequency insonation of breast cancer cells combined with CAR T cell therapy

[0159] In vitro experiments were conducted to assess the effect of combining NB low- frequency insonation (250 kHz) and CAR T cell therapy on the viability of 4T1 breast cancer cells. Eppendorf tubes containing a mixture of NBs and tumor cells were subjected to US at a mechanical index (MI) of 1.3. Shortly after, CAR T cells were added to the mixture at differing ratios of effector to target cells. The impact of treatment duration was evaluated using a fixed NB concentration of 12.5 x 108NBs per pL. Following treatment, the cell viability was measured to determine the effect of the combined US+NB and CAR T cell therapy.

[0160] As shown in Figs. 2A-C, there was a statistically significant reduction in 4T1, A549 and OVCAR8 cell viability following the combined US+NB and CAR T cell treatment compared to either US+NB treatment alone or CAR T cell treatment alone. The combined therapy resulting in a enhanced decrease in cell viability than that observed with either single treatment demonstrates the enhanced efficacy of the combination approach relative to either modality alone.

[0161] Supernatants collected from the in vitro experiments were analyzed to quantify IFN-y secretion as a measure of CAR T cell activation. Using a cytokine release assay, it was shown that IFN-y levels were significantly higher in the group receiving the combined US+NB and CAR T cell treatment compared to either US+NB or CAR T cell treatment alone. This indicates that the combined therapy not only enhanced cytotoxicity but also promoted greater CAR T cell activation as reflected by elevated IFN-y production, as further exemplified below.

[0162] In vivo nanobubble-mediated low frequency insonation of breast cancer tumors

[0163] When the induced tumors reached a designated size (for example, approximately 7 mm in the mice), a NB treatment (systemic administration) combined with US (250 kHz frequency, 500 kPa pressure, 0.5 millisecond pulse length and a total treatment of 2 min) was performed. In vivo, the tumors were subjected to low-frequency US exposure in the presence of NBs. Ten minutes after systemic injection of NBs, breast cancer tumors were treated with 250 kHz US at an MI of 1.3. Control groups included a sham group and a US-only group. Tumors were collected 24 hours post-treatment for histological evaluation. Histology of tumors treated with US alone showed no damage, resembling the sham group. In contrast, tumors receiving the combined NBs + 250 kHz US treatment exhibited extensive regions of cellular loss and debris.

[0164] Combination treatment with CAR T cell therapy Three days after the initial US+NB treatment, mice bearing breast cancer tumors received an intravenous injection of CAR T cells. Fig. 3A shows a schematic illustration of steps in combination treatment of immunocompetent mice subcutaneously injected with spontaneous tumors.

[0165] This timing of the steps was selected to allow for the maximal effect of US+NB- mediated tumor disruption and to enhance CAR-T cell and immune cell infiltration and activity within the tumor microenvironment. The control groups included a sham group and two groups that received either US+NB treatment or CAR-T cell therapy only.

[0166] The results presented in Fig. 3B demonstrate that tumor established in a control mouse was not affected in control mice, whereas in mouse treated with the combination treatment, no tumor growth was observed.

[0167] Further, following CAR-T cell administration, tumor volumes were measured at regular intervals to assess treatment efficacy. As shown in Fig. 3C, mice treated with the combination of therapy of US+NB and CAR-T cells showed a marked and statistically significant reduction in tumor volume compared to the control groups, including those receiving CAR-T cells alone, US+NB alone, or sham treatment. The decrease in tumor size in the combination group was evident as early as several days post-infusion and persisted throughout the whole observation period. In contrast, while the US+NB initially showed a modest response, ultimately all of the control groups showed significant tumor growth. These findings indicate that pre-treatment with US+NB not only disrupts the integrity of the tumor tissue but also creates a more permissive environment for CAR-T cell activity, resulting in enhanced antitumor efficacy. Overall, the data demonstrate that the sequential administration of US+NB followed by CAR- T cell therapy leads to superior tumor control compared to either modality alone.

[0168] Additionally, as shown in Fig. 3D, combined treatment (US+NB and CAR-T cells) resulted in a marked extension of survival in tumor-bearing mice. In contrast, none of the mice in the control groups survived beyond 21 days. Moreover, the combined therapy was further able to prevent relapse. As shown in Fig. 3D, when the treated mice were challenged at day 162, the tumors were not reestablished and the survival rate was unaffected. These results demonstrate that the US+NB+CAR-T cell combination not only significantly prolongs survival compared to either single treatment or sham controls, but can also prevent, reduce or diminish underscoring the superior and synergistic efficacy of this combined therapy.

[0169] Enhanced infiltration of immune cells combined US+NB+CAR-T Cell To assess the impact of treatment on tumor immune microenvironment, the infiltration of F4 / 80+macrophages and CD3+T cells within the tumor microenvironment one week after US+NB administration (and four days after CAR-T cell injection) were performed. As shown in Fig. 4A Fig. 4B, both F4 / 80+and CD3+were evaluated by immunohistochemistry staining of tumor sections, with quantitative analysis performed using ImageJ software. The staining revealed a significant increase in the number of both F4 / 80+macrophages and CD3+T cells in tumors from mice treated with the combined US+NB+CAR-T cell regimen compared to all control groups. The enhanced infiltration of these immune cells was not observed in tumors from mice receiving either treatment alone, or the sham control. This indicates that the combination therapy promotes extensive remodeling of the tumor innate and adaptive immune compartments, contributing to the observed antitumor effects.

[0170] In addition, the infiltration of lymphoid immune cell populations was tested at the same post-treatment timepoint. Flow cytometry analysis demonstrated a robust accumulation of CD45+leukocytes within the tumor, as shown in Fig. 4C. The percentages of CD4+and CD8+T cells were determined as proportions within the CD45+immune cell population. Both CD4+helper and CD8+cytotoxic T cell were significantly increased in tumors from the combination therapy group. Quantitative analysis confirmed a substantial elevation in the percentage and absolute numbers of CD4+and CD8+T cells when compared to all control groups.

[0171] Collectively, these findings indicate that the combined administration of US+NB and CAR-T cells synergistically promotes the infiltration of both macrophage and T cell populations into the tumor, supporting an enhanced intertumoral immune response and therapeutic efficacy.

[0172] Thus, the results clearly demonstrate a synergistic effect of NB mediated US histotripsy with CAR-T treatment of target tissues.

[0173] While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0174] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the patent specification, including definitions, governs. As used herein, the indefinite articles “a” and “an” mean “at least one” or “one or more” unless the context clearly dictates otherwise.

Claims

CLAIMS1. A method for treating a tumor in a target tissue in a subject in need thereof, the method comprising: administering bubbles or a composition comprising the same to the subject; applying low frequency ultrasound (US) to the target tissue; and administering CAR-T cells to the subject.

2. The method according to claim 1, wherein the CAR-T cells are administered concomitantly with the bubbles.

3. The method according to claim 1, wherein the CAR-T cells are administered prior to administration of the bubbles.

4. The method according to claim 1, wherein the CAR-T cells are administered after the bubbles and the ultrasound application.

5. The method according to any one of claims 1-4, wherein the CAR-T cells are administered in a suitable pharmaceutical compositions.

6. The method according to any one of claims 1-5, wherein the bubbles and / or the CAR- T cells are administered systemically or locally.

7. The method according to any one of claims 1-6, wherein the bubbles comprise microbubbles (MBs) and / or nanobubbles (NBs).

8. The method according to any one of claims 1-7, wherein the bubbles comprise nanobubbles having an average size of about 50-800nm and / or microbubbles, having an average size of about 800-3000nm.

9. The method according to any one of claims 1-8, wherein the bubbles comprise one or more lipids.

10. The method according to any one of claims 1-9, wherein the bubbles comprise a targeting moiety on a shell thereof.

11. The method according to claim 10, wherein the targeting moiety comprises a cell typespecific antibody conjugated to the shell.

12. The method according to any one of claims 1-11, wherein the US is in the frequency of less than about 850kHz.

13. The method according to any one of claims 1-11, wherein the US is in the frequency of less than about 500kHz.

14. The method according to any one of claims 1-13, wherein the US is characterized as having a peak negative pressure (PNP) of less than about 800kPa.

15. The method according to any one of claims 1-14, wherein the mechanical index of US is 1.9 or less.

16. The method according to any one of claims 1-15, wherein the US is applied after a time interval from the administration of the bubbles.

17. The method according to claim 16, wherein the time interval is at least 10 minutes.

18. The method according to any one of claims 1-17, further comprising imaging the bubbles prior to, during and / or after insonation.

19. The method according to any one of claims 1-18, wherein bubbles induced tissue damage comprises: ablation, debulking and / or lesion of the tissue.

20. The method according to any one of claims 1-19, wherein the tumor is selected from: breast cancer, lung cancer, prostate cancer, colon cancer, melanoma, brain cancer, ovarian cancer, bladder cancer, kidney cancer, sarcomas, carcinomas, and / or lymphomas.

21. The method according to any one of claims 1-20, wherein the tumor is breast cancer.

22. The method according to any one of claims 1-20, wherein the CAR is directed against an antigen comprising ErbB2 (HER2), CD 19, BCMA, CD38, CD 138, EGFR, CD276, CD24, GD2, EGF, Mesothelin (MSLN), MUC1, FAP, PSCA, EPCAM, CEA, PSMA, GPC3, LMP1, CD133, cMET, ROR1, CD70, MUC16, RAO, RA9, or any combinations thereof.

23. The method according to any one of claims 1-22, wherein the CAR is directed against an HER2 antigen and / or against CD24 / CD138 antigens.

24. A formulation for use in treating a tumor in a subject in need thereof, the formulation comprises a composition comprising bubbles and / or a pharmaceutical composition comprising CAR-T cells, said formulation is administered systemically and wherein low frequency ultrasound is applied at least after administration of the bubbles composition.

25. The formulation for use according to claim 24, wherein the composition comprising the bubbles and the pharmaceutical composition comprising the CAR-T cells are comprised in a single composition.

26. The formulation for use according to claim 24, wherein the composition comprising the bubbles and the pharmaceutical composition comprising the CAR-T cells are comprised in separate compositions.

27. The formulation for use according to any one of claims 24-26, wherein the bubbles comprise nanobubbles having an average size of about 50-800nm and / or microbubbles, having an average size of about 800-3000nm.

28. The formulation for use according to any one of claims 24-27, wherein the US is in the frequency of about 850kHz or less.

29. The formulation for use according any one of claims 24-28, US is characterized as having a peak negative pressure (PNP) of less than about 800kPa.

30. The formulation for use according to any one of claims 24-29, wherein the US is applied after a time interval from the administration of the bubbles.

31. The formulation for use according to any one of claims 24-30, wherein the tumor is selected from: breast cancer, lung cancer, prostate cancer, colon cancer, melanoma, brain cancer, ovarian cancer, bladder cancer, kidney cancer, sarcomas, carcinomas, and / or lymphomas.

32. The formulation for use according to any one of claims 24-31, wherein the CAR is directed against an antigen comprising ErbB2 (HER2), CD 19, BCMA, CD38, CD 138, EGFR, CD276, CD24, GD2, EGF, Mesothelin (MSLN), MUC1, FAP, PSCA, EPCAM, CEA, PSMA, GPC3, LMP1, CD133, cMET, ROR1, CD70, MUC16, RAO, RA9, or any combinations thereof.

33. A system for facilitating CAR-T infiltration to a target tissue, the system comprising: a low frequency focused ultrasound transmitter configured to emit low frequency ultrasound (US) towards the target tissue, wherein the subject has been administered with a composition comprising bubbles and a composition which comprises CAR-T cells; wherein said low frequency ultrasound causes the bubbles to induce at least partial damage to the target tissue, thereby allowing infiltration of the CAR-T cells toenvironment of the target tissue to thereby induce a cytotoxic and / or immune effect on the target tissue.

34. The system according to claim 33, wherein the US transmitter is configured to deliver ultrasound at a center frequency of about 500 kHz or less, and PNP of about 800 kPa o less.

35. The system according to any one of claims 33-34, wherein the system further comprises a control module configured to apply the ultrasound after administration of the bubbles.

36. The system according to any one of claims 33-35, wherein the CAR-T cells are administered before, concomitantly with, or after administration of the bubbles.

37. The system according to any one of claims 33-36, wherein the CAR-T cells are administered or after ultrasound application.

38. The system according to any one of claims 33-37, wherein the CAR-T cells and the bubbles are formulated in a single or separate compositions.

39. The system according to any one of claims 33-38, wherein the CAR-T cells and / or the bubbles are administered systemically.

40. The system according to any one of claims 33-39, wherein the target tissue is or comprises a tumor.

41. The system according to any one of claims 33-40, for use in treating cancer.

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