Controlled Lesion and Immune Responses to Pulsed Electric Field Therapy

Abstract

Treatment of damaged, diseased, abnormal, obstructed, cancerous or unwanted tissue (such as tumors, benign tumors, malignant tumors, cysts, or zones of diseased tissue) is accomplished by delivering specialized pulsed electric field (PEF) energy to a target tissue area at specific doses to achieve superior outcomes. The PEF energy and delivery is optimized to provide advanced treatment of the target tissue area, including destruction of the unwanted tissue and generation of improved inflammatory and immune responses. These various types of treatments are controlled by a variety of factors, including the electrode geometry, the dose of PEF energy delivered, the time the energy is delivered, and the waveform of the PEF energy itself. The PEF energy is delivered in the form of doses that are considered to be one application of the specialized energy. Each dose creates a lesion in the target tissue area.

Description

[Background technology]

[0001] background This application claims the benefit of U.S. Provisional Patent Application No. 63 / 246,239, filed September 20, 2021, U.S. Provisional Patent Application No. 63 / 290,529, filed December 16, 2021, U.S. Provisional Patent Application No. 63 / 322,319, filed March 22, 2022, and U.S. Provisional Patent Application No. 63 / 351,562, filed June 13, 2022, the contents of which are incorporated by reference in their entireties.

[0002] Abnormal tissue can take a variety of different forms, such as damaged tissue, diseased tissue, obstructed tissue, cancerous tissue, or unwanted tissue. In some cases, the abnormal tissue is a tumor, such as a benign or malignant tumor, a cyst, or an area of ​​diseased tissue. One of the most troublesome types of abnormal tissue is associated with cancer.

[0003] Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. If the spread is uncontrolled, it can lead to death. Although the causes of cancer are not fully understood, many factors are known to increase the incidence of the disease, including many modifiable factors (e.g., tobacco use and excess weight) and others that are not modifiable (e.g., inherited genetic mutations). These risk factors may act simultaneously or sequentially to initiate and / or promote cancer growth. Cancer is the second most common cause of death in the United States, surpassed only by heart disease.

[0004] Lung, liver, and pancreatic cancers have some of the lowest survival rates. Lung cancer is the leading cause of cancer death, surpassing colorectal, breast, and prostate cancers combined. Overall changes in 5-year survival rates for all stages combined have shown only slight improvements over time: 1970s (approximately 13%), 2010s (approximately 17.2%), and 2019 (approximately 21.7%). While liver cancer incidence has more than tripled since 1980, mortality rates have more than doubled during this time. While some progress has been made in liver cancer patient survival, 5-year survival rates remain low, even for patients diagnosed at limited stages. Pancreatic cancer is projected to be the second leading cause of cancer-related death in 2020. The 5-year survival rate for all stages is 9%, a rate that has not substantially improved over 40 years. These outcomes persist despite advances in conventional therapy.

[0005] Many types of cancer are not successfully cured or recur at a later time. Recurrence typically occurs because the original treatment did not successfully eliminate all cancer cells, causing the remaining cancer cells to proliferate. In some instances, cancer cells spread to other parts of the body in undetectable amounts, known as micrometastases. If these micrometastases are not overcome by the body, they grow to detectable levels, requiring further treatment. Ultimately, many patients lose the battle against cancer.

[0006] As a result, improved therapies that more successfully treat cancers and reduce or prevent their recurrence, as well as improved therapies for all types of abnormal tissue, are needed. At least some of these objectives are met by the present invention. Summary of the Invention [Means for solving the problem]

[0007] Abstract DETAILED DESCRIPTION OF THE INVENTION Described herein are embodiments of devices, systems and methods for treating target tissue, particularly cardiac tissue. The present invention also relates to the following numbered clauses:

[0008] 1. A system for treating tissue within a patient's body, comprising: an instrument having at least one energy delivery body configured to be positioned to direct pulsed electric field energy to tissue producing a lesion; and A system comprising: a generator in electrical communication with at least one energy delivery entity, the generator comprising at least one energy delivery algorithm configured to provide pulsed electric field energy in a manner that produces multiple zones within a lesion that induce an increased adaptive immune response in a patient.

[0009] 2. The system of clause 1, wherein the plurality of zones have a bull's-eye configuration.

[0010] 3. The system of any of the preceding clauses, wherein the plurality of zones includes at least a pulsed electric field zone.

[0011] 4. The system of any of the preceding clauses, wherein the plurality of zones comprises an immune response zone.

[0012] 5. The system of any of the preceding clauses, wherein the plurality of zones includes a thermal zone.

[0013] 6. The system of any of the preceding clauses, wherein the plurality of zones includes an inflammatory zone.

[0014] 7. The system of clause 1, wherein the plurality of zones includes at least a pulsed electric field zone and a thermal zone.

[0015] 8. The system of clause 7, wherein the pulsed electric field zone and the thermal zone together cover an area within the lesion, and the thermal zone does not exceed 50% of the area.

[0016] 9. Systems as described in clause 8 where the thermal zone does not exceed 25% of the area.

[0017] 10. A system as described in clause 9, in which the thermal zone is 10-20% of the area.

[0018] 11. A system described in any one of clauses 7 to 10, wherein at least one energy delivery algorithm includes an inter-cycle delay in the waveform of the pulsed electric field energy, and the inter-cycle delay is configured to control the size of the thermal zone.

[0019] 12. The system of clause 11, wherein the cycle-to-cycle delay is at least 10 microseconds.

[0020] 13. The system of clause 12, wherein the cycle-to-cycle delay is between 10 microseconds and 1000 microseconds.

[0021] 14. A system as described in any one of clauses 11 to 13, wherein the inter-cycle delay is configured to minimize or eliminate cavitation zones.

[0022] 15. A system as described in any one of clauses 11 to 14, wherein the cycle-to-cycle delay is 1000 microseconds.

[0023] 16. The system of clause 11, wherein at least one energy delivery algorithm includes an inter-packet delay in the waveform of the pulsed electric field energy, the inter-packet delay configured to control the size of the thermal zone.

[0024] 17. The system of clause 16, wherein the inter-packet delay is between 3 and 5 seconds.

[0025] 18. A system according to any of the preceding clauses, wherein the lesion is produced by a single dose within 5-6 minutes.

[0026] 19. The system of any of the preceding clauses, wherein the adaptive immune response comprises an increase in the ratio of CD8+ / CD4+ T cells.

[0027] 20. The system of any of the preceding clauses, wherein the adaptive immune response comprises an increase in PDL1 expression.

[0028] 21. The system of any of the preceding clauses, wherein the adaptive immune response includes upregulation of lymphatic pathways.

[0029] 22. The system of any of the preceding clauses, wherein the adaptive immune response includes the generation of tumor antigen release.

[0030] 23. The system of any of the preceding clauses, wherein the adaptive immune response includes downregulation of regulatory T cells.

[0031] 24. The system according to any of the preceding clauses, wherein the adaptive immune response includes the production or increased production of tertiary lymphoid structures in the tissue.

[0032] 25. The system of any of the preceding clauses, wherein at least one energy delivery algorithm produces a synergistic effect when pulsed electric field energy is combined with an immune checkpoint inhibitor.

[0033] 26. The system of clause 25, wherein the immune checkpoint inhibitor comprises anti-PD1 therapy.

[0034] 27. A system according to any one of clauses 25-26, wherein the synergistic effect increases the abscopal effect.

[0035] 28. The system of any one of clauses 25 to 27, wherein the synergistic effect increases PDL-1 expression.

[0036] 29. The system of any one of clauses 25 to 28, wherein the tissue comprises a tumor that is non-responsive or has reduced responsiveness to anti-PD1 therapy, such that the tumor is responsive to anti-PD1 therapy.

[0037] 30. A system according to any of the preceding clauses, wherein at least one energy delivery algorithm generates a pulsed electric field energy waveform having a voltage of 3000V.

[0038] 31. A system described in any of the preceding clauses, wherein at least one energy delivery algorithm generates a pulsed electric field energy waveform having a fundamental frequency of 400 kHz.

[0039] 32. The system of any of the preceding clauses, wherein at least one energy delivery algorithm generates a waveform of pulsed electric field energy having packets of biphasic pulse cycles.

[0040] 33. The system of clause 32, wherein each packet has 40 biphasic pulse cycles.

[0041] 34. The system of any one of clauses 32-33, wherein an inter-cycle delay of 1000 microseconds is placed between each biphasic pulse cycle within a packet.

[0042] 35. A system as described in any one of clauses 32 to 34, wherein the waveform includes 100 packets in a single line charge.

[0043] 36. A system according to any one of clauses 32 to 35, wherein an inter-packet delay of 3 seconds is placed between each packet of dose.

[0044] 37. A system according to any of the preceding clauses, wherein at least one energy delivery algorithm generates a waveform of pulsed electric field energy having packets, each packet having an active time of 100 microseconds.

[0045] 38. The system of clause 37, wherein each packet has 40 cycles.

[0046] 39. A system as described in any one of clauses 37-38, wherein the waveform has a voltage of 3000V.

[0047] 40. A system for treating target tissue within a patient's body, comprising: an instrument having at least one energy delivery body configured to be positioned to direct pulsed electric field energy to a target tissue; and 1. A system comprising: a generator in electrical communication with at least one energy delivery entity, the generator comprising at least one energy delivery algorithm configured to provide pulsed electric field energy to create a lesion in a target tissue, the at least one energy delivery algorithm generating a waveform of the pulsed electric field energy having a combination of parameters configured to cause a synergistic effect when combined with an immune checkpoint inhibitor.

[0048] 41. The system of clause 40, wherein the synergistic effect increases the abscopal effect.

[0049] 42. The system of clause 40, wherein the immune checkpoint inhibitor comprises anti-PD1.

[0050] 43. The system according to clause 42, wherein the synergistic effect increases PDL-1 expression.

[0051] 44. The system of any one of clauses 40-43, wherein the tissue comprises a tumor that is non-responsive or has reduced responsiveness to anti-PD1 therapy, such that the tumor is responsive to anti-PD1 therapy.

[0052] 45. A system described in any one of clauses 40-44, wherein at least one energy delivery algorithm generates a pulsed electric field energy waveform having a voltage of 3000V.

[0053] 46. ​​A system described in any one of clauses 40-45, wherein at least one energy delivery algorithm generates a pulsed electric field energy waveform having a fundamental frequency of 400 kHz.

[0054] 47. A system described in any one of clauses 40-46, wherein at least one energy delivery algorithm generates a waveform of pulsed electric field energy having packets of biphasic pulse cycles.

[0055] 48. The system of clause 47, wherein each packet has 40 biphasic pulse cycles.

[0056] 49. The system of any one of clauses 47-48, wherein an inter-cycle delay of 1000 microseconds is placed between each biphasic pulse cycle within a packet.

[0057] 50. A system as described in any one of clauses 47 to 49, wherein the waveform includes 100 packets in a single line charge.

[0058] 51. A system described in any one of clauses 47 to 50, wherein an inter-packet delay of 3 seconds is placed between each packet of dose.

[0059] 52. A system described in any one of clauses 47-51, wherein at least one energy delivery algorithm generates a waveform of pulsed electric field energy having packets, each packet having an active time of 100 microseconds.

[0060] 53. A system for treating a tumor in a patient that is non-responsive or has a reduced response to therapy, comprising: an instrument having at least one energy delivery body configured to be positioned to direct pulsed electric field energy at a tumor; and A system comprising: a generator in electrical communication with at least one energy delivery body, the generator comprising at least one energy delivery algorithm that generates a waveform of pulsed electric field energy having a combination of parameters configured to make the tumor responsive to therapy.

[0061] 54. The system of clause 53, wherein the therapy comprises immune checkpoint inhibitor therapy.

[0062] 55. The system of clause 54, wherein the immune checkpoint inhibitor therapy comprises anti-PD1 therapy.

[0063] 56. A system for treating tissue within a patient's body, comprising: an instrument including a shaft having a proximal end and a distal end, and at least one energy delivery entity disposed near the distal end of the shaft, the distal end of the shaft configured to deliver pulsed electric field energy to tissue producing a lesion, the pulsed electric field energy maintaining an extracellular matrix within at least a portion of the lesion; and A system comprising: a generator in electrical communication with at least one energy delivery entity, the generator comprising at least one energy delivery algorithm configured to provide pulsed electric field energy in a manner that reduces or eliminates a cavitation zone within a lesion.

[0064] 57. The system described in clause 56, wherein at least one energy delivery algorithm includes an inter-cycle delay in the waveform of the pulsed electric field energy, the inter-cycle delay configured to reduce or eliminate cavitation zones.

[0065] 58. The system of clause 57, wherein the cycle-to-cycle delay is at least 10 microseconds.

[0066] 59. The system of clause 58, wherein the cycle-to-cycle delay is between 10 microseconds and 1000 microseconds.

[0067] 60. The system of clause 59, wherein the cycle-to-cycle delay is 1000 microseconds.

[0068] 61. A system for treating tissue within a patient's body, comprising: an instrument including a shaft having a proximal end and a distal end, and at least one energy delivery entity disposed near the distal end of the shaft, the distal end of the shaft configured to deliver pulsed electric field energy to tissue producing a lesion, the pulsed electric field energy maintaining an extracellular matrix within at least a portion of the lesion; and A system comprising: a generator in electrical communication with at least one energy delivery entity, the generator comprising at least one energy delivery algorithm configured to provide pulsed electric field energy in a manner that produces at least a thermal zone and a pulsed electric field zone within a lesion.

[0069] 62. A system for treating target tissue within a patient's body, comprising: an instrument having at least one energy delivery body configured to be positioned to direct pulsed electric field energy to a target tissue; and 1. A system comprising: a generator in electrical communication with at least one energy delivery entity, the generator comprising at least one energy delivery algorithm configured to provide pulsed electric field energy to create a lesion in a target tissue, the at least one energy delivery algorithm generating a waveform of pulsed electric field energy having a combination of parameters configured to cause the generation or increased generation of tertiary lymphoid structures in the target tissue.

[0070] 63. The system of clause 62, including at least one energy delivery algorithm configured to provide pulsed electric field energy in a manner that produces multiple zones within the lesion that induce an increase in the patient's adaptive immune response.

[0071] 64. The system of clause 63, wherein the plurality of zones includes at least a pulsed electric field zone.

[0072] 65. A system according to any of clauses 63-64, wherein the plurality of zones comprises an immune response zone.

[0073] 66. A system according to any of clauses 63 to 65, wherein the plurality of zones includes a thermal zone.

[0074] 67. A system described in any of clauses 63 to 66, wherein multiple zones are devoid of a cavitation zone.

[0075] 68. A system described in any of clauses 63 to 67, wherein the plurality of zones includes an inflammatory zone.

[0076] 69. A system for treating an unwanted tissue cell mass in a patient's body, comprising: an instrument including a shaft having a proximal end and a distal end and at least one energy delivery entity disposed near the distal end of the shaft, the distal end of the shaft configured to deliver non-thermal energy to the mass of unwanted tissue cells; and A system comprising: a generator in electrical communication with at least one energy delivery body, the generator comprising at least one energy delivery algorithm configured to provide an electrical signal of non-thermal energy deliverable to an undesirable tissue mass to destroy at least a portion of the undesirable tissue mass.

[0077] These and other embodiments are described in further detail in the following description taken in conjunction with the accompanying drawings. Incorporation by Reference

[0078] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0080] [Figure 1] 1A-1B provide a schematic diagram of an exemplary therapeutic system for use in the delivery of specialized PEF energy.

[0081] [Figure 2] 2A to 2C show an example of a treatment method.

[0082] [Figure 3] Figures 3A-3B show tissue lesions resulting from a study focusing on the effect of adding inter-cycle and inter-packet delays within an energy waveform on local tissue properties.

[0083] [Figure 4] FIG. 4 illustrates one embodiment of a waveform of a signal defined by an energy delivery algorithm.

[0084] [Figure 5] FIG. 5 is a table showing various exemplary effects of parameter changes.

[0085] [Figure 6] 6A-6D show tissue samples with lesions produced by waveforms with no inter-cycle delay or different inter-cycle delays.

[0086] [Figure 7] 7A-7B show in vivo tissue samples from preclinical studies in porcine lung tissue.

[0087] [Figure 8] FIG. 8 shows how integrating inter-packet delays can help mitigate the thermal effects of PEF energy delivery.

[0088] [Figure 9] FIG. 9 shows in vivo data captured from preclinical studies in porcine liver tissue.

[0089] [Figure 10] 10A-10B show additional in vivo data captured from preclinical studies in porcine liver tissue.

[0090] [Figure 11] 11A-11B show in vivo samples captured from preclinical trials in porcine liver tissue.

[0091] [Figure 12] 12A-12B show the results of flow cytometry.

[0092] [Figure 13] 13A-13B provide CT images of a chest with a 3.4 cm lesion before PEF delivery (FIG. 13A) and the same lesion before surgery with a longest diameter of 2.8 cm (FIG. 13B).

[0093] Figures 13C-13D provide macroscopic sections of resected tumors 20 days after PEF delivery (Figure 13C) and corresponding hematoxylin & eosin (H&E) (Figure 13D).

[0094] [Figure 14] Figure 14 shows the evaluation schedule.

[0095] [Figure 15] FIG. 15 provides a table of the H&E and immunohistochemistry (IHC) performed.

[0096] [Figure 16] Figure 16 shows a Luminex Multiplex 71plex panel.

[0097] [Figure 17] Figure 17 shows the baseline characteristics and treatment details.

[0098] [Figure 18] FIG. 18 shows various tissue samples with tertiary lymphoid structures.

[0099] [Figure 19A] Figures 19-19B show the results of preliminary serum cytokine analysis. [Figure 19B]Figures 19-19B show the results of preliminary serum cytokine analysis.

[0100] [Figure 20] FIG. 20 shows a tumor treated with PEF energy, which kills cells in a way that is meaningful to the immune system.

[0101] [Figure 21] Figure 21 shows PEF energy delivered to a target treatment area, such as one containing a tumor, in a manner that promotes upregulation of PD-L1 by tumor cells.

[0102] [Figure 22] FIG. 22 provides a Kaplan-Myer plot showing survival outcomes in an EMT6 tumor model study comparing various treatment protocols.

[0103] [Figure 23] FIG. 23 provides control group outcome data for the EMT6 tumor model study.

[0104] [Figure 24] FIG. 24 shows the results of three groups (PEF+αPD-1, PEF energy alone, and αPD-1 alone) in an EMT6 tumor model re-challenge study.

[0105] [Figure 25] Figures 25A-25D show example results of cytokine analysis for a 4T-1 tumor model study.

[0106] [Figure 26] FIG. 26 shows one embodiment of a delivery system.

[0107] [Figure 27] FIG. 27A shows the direct injection of a drug into a target tissue through a needle.

[0108] FIG. 27B shows energy delivery from an energy delivery device inserted in place in the needle of FIG. 27A.

[0109] [Figure 28] FIG. 28A shows the direct injection of a drug into a target tissue via an energy delivery device.

[0110] FIG. 28B shows the delivery of energy from the energy delivery body of FIG. 28A.

[0111] [Figure 29] FIG. 29 shows a drug being delivered locally while energy is being delivered locally, and optionally the drug is also delivered locally.

[0112] [Figure 30] FIG. 30 shows an energy delivery device comprising a shaft having an energy delivery body near its distal end, the energy delivery body comprising a plurality of tines.

[0113] [Figure 31] FIG. 31 provides a heat map showing cytokine expression from contralateral whole tumor lysates after 18 days of treatment.

[0114] [Figure 32] Figures 32A-32D refer to the presence of cytokines in secondary (untreated) tumors.

[0115] [Figure 33] FIG. 33A provides a heat map showing cytokine expression from contralateral whole tumor lysates after 18 days of treatment.

[0116] FIG. 33B shows an analysis of how cytokine expression from lung tissue affects pathways associated with cancer, as determined by Ingenuity Pathway Analysis (IPA) software.

[0117] [Figure 34A]Figures 34A-34B provide ELISA quantification of Ki-67 from whole lung tissue lysates after 18 days of treatment, and Figure 34C provides ELISA quantification of CD8 from whole lung tissue lysates after 18 days of treatment.

[0118] [Fig. 34B-C] Figures 34A-34B provide ELISA quantification of Ki-67 from whole lung tissue lysates after 18 days of treatment, and Figure 34C provides ELISA quantification of CD8 from whole lung tissue lysates after 18 days of treatment.

[0119] [Figure 35] FIG. 35 shows primary tumor growth curves after 14 days of PEF in the EMT-6 mouse tumor model.

[0120] [Figure 36] FIG. 36 shows sequential tumor growth curves after 14 days of PEF in the EMT-6 mouse tumor model.

[0121] [Figure 37] Figure 37 shows the survival plot over 24 days.

[0122] [Figure 38] Figures 38A-38B show the primary tumor growth curve (Figure 38A) and the secondary tumor growth curve (Figure 38B).

[0123] [Figure 39] Figure 39A shows the frequent anti-PD-1 dosing scheme initiated at the time of primary tumor inoculation.

[0124] Figure 39B shows a low-frequency, late anti-PD-1 dosing scheme in which CPB was initiated during PEF treatment.

[0125] [Figure 40] Figure 40 is a Kaplan-Meier curve showing survival 60 days after treatment.

[0126] [Figure 41]Figures 41A-41B show EMT6 tumor growth in naive control mice not previously challenged with EMT6 tumor cell injections, and in animals from the combination of PEF and CPB that previously achieved a complete response.

[0127] [Figure 42] FIG. 42 is the dosing schedule for Balb / c mice orthotopically challenged with 200,000 cells (4T-1) in the fifth mammary fat pad (bilateral, bilateral tumor model).

[0128] [Figure 43] FIG. 43 is a growth plot of primary or directly treated 4T-1 tumors.

[0129] [Figure 44] Figure 44 shows a comparison of contralateral or untreated 4T-1 tumor volumes between biological groups at 18 days after PEF or 13 days after inoculation. DETAILED DESCRIPTION OF THE INVENTION

[0130] Detailed Description of the Invention Specific embodiments of the disclosed devices, systems, and methods will now be described with reference to the drawings. Nothing in this detailed description is intended to imply that any particular component, feature, or step is essential to the invention. I. Overview

[0131] Devices, systems, and methods are provided for treating damaged, diseased, abnormal, obstructed, cancerous, or unwanted tissue (e.g., tumors, benign tumors, malignant tumors, cysts, or zones of diseased tissue) by delivering specialized pulsed electric field (PEF) energy to a target tissue area at specific doses to achieve superior outcomes. The PEF energy and delivery are optimized to provide advanced treatment of the target tissue area, including destruction of the unwanted tissue and generation of improved inflammatory and immune responses. These various types of treatment are controlled by various factors, including electrode geometry, the dose of PEF energy delivered, the time the energy is delivered, and the waveform of the PEF energy itself. The PEF energy is delivered in doses that are considered to be one application of the specialized energy. Each dose creates a lesion in the target tissue area. In some embodiments, the desired outcome is achieved by delivering a single dose. This reduces treatment time and any complications that may arise from providing additional doses, such as those associated with repositioning the energy delivery device or re-treating the same tissue area. The particular parameter values ​​defining the energy waveform allow the waveform to be configured to deliver a desired effect in a single dose, and it will be understood that if desired, additional doses may be delivered to the target tissue, or energy may be delivered to additional target tissues, which may or may not include the original target tissue.

[0132] Conventional PEF energy delivery systems have focused solely on tissue destruction. When treating cancer with PEF energy, complete destruction of the tumor is desirable. Consequently, it is desirable to create a lesion that covers the tumor's size and destroys the tumor's cells. This technology remains relatively unrefined and therefore has yet to truly fulfill its promise. Specifically, although the energy itself is conceptualized as "non-thermal," several studies have shown that thermal sequelae are plagued by a variety of different factors. These studies have shown that while higher delivered electrical energies can be associated with larger treatment zones, they also cause increased cavitation, pale tissue intracellular changes, and white-to-tan tissue extracellular coagulation due to Joule heating effects. Conventional PEF can also cause cavitation through partial discharge (i.e., arcing). Arcing can occur due to several factors, one of which is higher delivered electrical energy. These effects limit any potential benefits to the immune system while increasing the likelihood of complications as a result of thermal damage. Thus, there is a lack of formalized understanding of how conventional PEF energy can be deployed to maximize field effects while managing potential thermal and cavity effects that may be associated with the procedure.

[0133] In contrast, the devices, systems, and methods described herein utilize specific doses of specialized PEF energy to create lesions with various zones, and manipulation of the zones provides benefits that result in more comprehensive and therefore successful treatment of the patient, particularly when treating cancer and other diseases. Typically, the zones are manipulated to eliminate or reduce cavitation zones, eliminate or reduce thermal zones, and maximize PEF zones along with potential inflammatory and immune response zones. The size or quantity of the zones is specifically engineered so that their combination elicits an increased adaptive immune response in the patient, such as by promoting a cascade of increased recruitment of Tregs, CD4, CD8, and other adaptive immune cell types. These lesion classifications (i.e., zones) can be measured macroscopically but can also be monitored in real time based on tissue characteristics (i.e., tissue density, impedance changes, etc.) and, perhaps most importantly, via imaging modalities such as CT. II. Example Delivery Systems

[0134] Specialized PEF energy is delivered using systems and devices advantageously designed for superior access to target tissues throughout the body, particularly in locations previously considered inaccessible via percutaneous approaches. While such access is typically minimally invasive and relies on an endoluminal approach, it can be appreciated that other approaches, such as percutaneous, laparoscopic, or open surgical approaches, may be used in some situations, if desired. Figure 1 provides a schematic diagram of an exemplary treatment system 100 for use in delivering specialized PEF energy. In this embodiment, the system 100 includes an elongated instrument 102 including a shaft 106 having a distal end 103 and a proximal end 107. The instrument 102 includes an energy delivery body 108 near the distal end 103 of the shaft 106. It will be appreciated that the energy delivery body 108 can take a variety of forms. The energy delivery body 108 may be externally attached to the shaft 106 or may be integral therewith, such that it is visible from the outside. Alternatively, the energy delivery element 108 may be housed internally within the shaft 106 and exposed by advancing or retracting the shaft 106 itself. Similarly, multiple energy delivery elements 108 may be present, and they may be external, internal, or both. In some embodiments, the shaft 106 is comprised of a polymer, such as an extruded polymer. It will be appreciated that in some embodiments, the shaft 106 is constructed from multiple layers of material having different durometers to control flexibility and / or stiffness. In some embodiments, the shaft 106 is reinforced with various elements, such as individual wires or wire braids. In either case, such wires may be rectangular or round. The wire braids have a braid pattern, and in some embodiments, the braid pattern is tailored to the desired flexibility and / or stiffness. In other embodiments, the wire braid reinforcing the shaft 106 can be advantageously combined with multiple layers of material having different durometers to provide additional control of flexibility and / or stiffness along the length of the shaft.

[0135] In either case, each energy delivery body 108 includes at least one electrode for delivering PEF energy. Typically, the energy delivery body 108 includes a single delivery electrode and operates in a monopolar configuration, achieved by supplying energy between the energy delivery body 108 positioned near the distal end 103 of the instrument 102 and a return electrode 140 positioned on the patient's skin. However, it will be understood that bipolar energy delivery and other configurations may alternatively be used. When using bipolar energy delivery, the instrument 102 may include multiple energy delivery bodies 108 configured to function in a bipolar manner, or may include a single energy delivery body 108 with multiple electrodes configured to function in a bipolar manner. The instrument 102 typically includes a handle 110 positioned near the proximal end 107. The handle 110 is used to operate the instrument 102 and typically includes an actuator 732 for operating the energy delivery body 108. In some embodiments, the energy delivery entity 108 transitions from a closed or retracted position (during access) to an open or exposed position (for energy delivery) controlled by an actuator 732. As such, the actuator 732 typically has the form of a knob, button, lever, slide, or other mechanism. It will be appreciated that in some embodiments, the handle 110 includes a port for introducing a liquid, agent, substance, tool, or other device for delivery through the instrument 102. Examples of liquids include suspensions, mixtures, chemicals, fluids, chemotherapeutics, immunotherapeutics, micelles, liposomes, embolic agents, nanoparticles, drug-eluting particles, genes, plasmids, and proteins, to name a few.

[0136] The instrument 102 is in electrical communication with a generator 104 configured to generate PEF energy. In this embodiment, the generator 104 includes a user interface 150, one or more energy delivery algorithms 152, a processor 154, a data storage / retrieval unit 156 (e.g., memory and / or database), and an energy storage subsystem 158 that generates and stores the delivered energy. In some embodiments, the user interface 150 of the generator 104 is used to select the desired treatment algorithm 152. In other embodiments, the algorithm 152 is automatically selected by the generator 104 based on information obtained by one or more sensors. A variety of energy delivery algorithms may be used. In some embodiments, one or more capacitors are used for energy storage / delivery, although any other suitable energy storage elements may be used. Additionally, one or more communication ports are typically included.

[0137] The distal end 103 of the instrument 102 is typically advanceable through a delivery device, such as an endoscope. The endoscope typically includes a control body attached to an elongated insertion tube having a distal tip. The endoscope has an internal lumen accessible by a port through which the distal end 103 of the instrument 102 passes. The shaft 106 of the instrument 102 is advanceable through the internal lumen and exits the distal tip of the endoscope. Imaging is achieved through the endoscope using a light guide tube with an endoscope connector that connects to a light source and an energy source. The distal tip of the endoscope may be equipped with visualization technologies, including, but not limited to, video, ultrasound, laser scanning, and the like. These visualization technologies collect signals consistent with their design and transmit the signals via wires or wirelessly down the length of the shaft to a video processing unit. The video processing unit then processes the video signal and displays the output on a screen. It will be appreciated that endoscopes are typically specific to the anatomical location in which they are being used, such as a gastroscope (upper GI endoscopy including the stomach, esophagus, and small intestine (duodenum)), colonoscope (large intestine), bronchoscope (lungs), laryngoscope (larynx), cystoscope (urinary tract), duodenoscope (small intestine), enteroscope (digestive system), ureteroscope (ureter), hysteroscope (cervix), etc. It will be appreciated that in other embodiments, instrument 102 can be delivered via a catheter, sheath, introducer, needle, or other delivery system.

[0138] Intraluminal access allows treatment of target tissue from within various lumens within the body. Lumens are spaces inside tubular or hollow structures within the body, including passageways, canals, ducts, and cavities, to name a few. Examples of luminal structures include blood vessels, the esophagus, stomach, small and large intestines, colon, bladder, urethra, urinary tract, uterus, vagina, fallopian tubes, ureters, kidneys, renal tubules, spinal canal, spinal cord, and other structures throughout the body, as well as structures within and containing organs such as the lungs, heart, and kidneys, to name a few. In some embodiments, the target tissue is accessed through a nearby luminal structure. In some cases, the treatment instrument 102 is advanced through various luminal structures or branches of the luminal system to reach the target tissue location. For example, when accessing a target tissue site via a blood vessel, the treatment instrument 102 may be inserted remotely and advanced through various branches of the vasculature to reach the target site. Similarly, if the luminal structure originates from a natural orifice, such as the nose, mouth, urethra, or rectum, it can be entered through the natural orifice, and the treatment instrument 102 can then be advanced through a bifurcation of the luminal system to reach the target tissue location. Alternatively, the luminal structure can be entered near the target tissue via an incision or other method. This may be the case when accessing a luminal structure that is not part of a larger system or is otherwise difficult to access.

[0139] Once the target tissue area is close to the lumen, energy can be delivered to the target tissue in a variety of ways. In one arrangement, the energy delivery body 108 is positioned within the body cavity, and energy is delivered to the target tissue entering the body cavity through and / or at least partially surrounding the lumen wall to target tissue within the lumen wall, or through the lumen wall to target tissue outside and adjacent the lumen wall. In another arrangement, the energy delivery body 108 is advanced through the lumen wall and inserted into or near the target tissue outside the lumen wall. It will be appreciated that such an arrangement may include at least two energy delivery bodies 108, one positioned within the body cavity and one extending through the wall of the body cavity. In some embodiments, each energy delivery body 108 functions in a monopolar manner (e.g., utilizing a return electrode positioned at a distance). In other embodiments, at least some of the energy delivery bodies 108 function in a bipolar manner. These delivery options are possible because the lumen itself is preserved throughout the procedure, allowing treatment of tissue within, on, or near the lumen itself. Delivery of such therapies allows access to previously inaccessible tissue, such as tumors or diseased tissue that infiltrate the lumen wall or at least partially wrap around the body cavity, making them too close to be surgically removed or treatable with conventional focal therapy. Many conventional focal therapies, such as treatments with thermal energy, damage or destroy the structure of the lumen wall through thermal protein coagulation. In particular, bowel damage caused by radiofrequency ablation is one of the most feared complications and is associated with death from sepsis and abscess formation. As a result, most physicians will postpone radiofrequency ablation in tumors adjacent to the intestine. Other conventional focal therapies are ineffective near certain body cavities. For example, cryotherapy relies on sufficient cooling of tissue that is compromised by flow through the body cavity, such as blood flowing through the vasculature, which reduces the cooling effect. Such intraluminal access is also less invasive than other types of procedures, such as percutaneous delivery of energy, which involves placing multiple needle probes through the skin deep into tissues and organs.Because natural openings within the body are utilized, wound healing is reduced along with fewer potential points of infection. Similarly, locations deep within the body can be accessed, along with locations that are otherwise difficult to access externally, such as behind other organs or near major blood vessels. It will be understood that a variety of anatomical locations can be treated with the systems and methods described herein. Examples include the luminal structures themselves, soft tissue throughout the body located near luminal structures, and solid organs accessible from luminal structures (including, but not limited to, the liver, pancreas, gallbladder, kidneys, prostate, ovaries, lymph nodes and lymphatic drainage ducts, underlying muscle tissue, bone tissue, brain, eye, thyroid, etc.). It will also be understood that a variety of tissue locations can be accessed percutaneously.

[0140] The endoscopic approach also lends itself to monopolar energy delivery. As discussed above, monopolar delivery involves passing electrical current from the energy delivery body 108 (near the distal end of the instrument 102) to the target tissue and through the patient to a return pad 140 positioned against the patient's skin to complete the current circuit. Thus, in some embodiments, the instrument 102 includes only one energy delivery body 108 or electrode. This allows the instrument 102 to have a low profile so that it can be placed in smaller body cavities. This also allows for deep penetration of the tissue surrounding the energy delivery body 108. Similarly, when penetrating a lumen wall with such a device, the use of only one energy delivery body 108 requires only one penetration per procedure. While additional penetrations may occur due to various device designs or treatment protocols, it will be appreciated that in some embodiments, a monopolar delivery design reduces the invasiveness of the procedure, simplifies device and treatment design, and provides a superior treatment zone at the target tissue.

[0141] The devices, systems, and methods described herein may be used alone or in combination with other treatments. Such combination treatments may be particularly applicable to cancer treatments. For example, the PEF treatments described herein may be used in combination with neoadjuvant and adjuvant therapies, such as various non-surgical therapies, radiation therapy, chemotherapy, targeted therapy / immunotherapy, focal therapy, gene therapy, and plasmid therapy, to name a few. Examples of focal therapies include microwave ablation, radiofrequency ablation, cryoablation, high-intensity focused ultrasound (HIFU), and other pulsed electric field ablation therapies. Such combinations may condition tissue for improved responsiveness and, in some cases, a synergistic response greater than that of either therapy alone. Furthermore, the PEF treatments described herein may produce an abscopal effect due to the nature of the therapy.

[0142] 1A-1B illustrate a treatment system 100 including an energy delivery catheter 102 connectable to a generator 104. As shown, the catheter 102 includes a shaft 106 having a distal end 103, a proximal end 107, and at least one lumen 105 extending at least partially therethrough. Similarly, the catheter 102 also includes at least one energy delivery body 108. In this embodiment, the energy delivery body 108 takes the form of a probe 700 disposed within the lumen 105 of the shaft 106. The probe 700 is advanceable through the lumen 105 and has a probe tip 702 extendable from the distal end 103 of the shaft 106 (enlarged in FIG. 1A to show detail). In this embodiment, the tip 702 has a pointed shape configured to penetrate tissue, similar to a needle. Thus, in this embodiment, the probe tip 702 is utilized to penetrate the lumen wall W and surrounding tissue so that the probe can be inserted into target tissue outside the body cavity. Thus, the probe 700 is flexible enough to be delivered intraluminally, yet has sufficient column strength to penetrate the lumen wall W and target tissue. In some embodiments, the catheter 102 has markings that indicate to the user the distance the probe tip 702 has been advanced to ensure desired placement.

[0143] In some embodiments, the probe extends approximately less than 0.5 cm, 0.5 cm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, or more than 8 cm from the distal end 103 of the shaft 106. In some embodiments, the probe extends 1-3 cm or 2-3 cm from the distal end of the shaft 106. In some embodiments, the probe is 18 gauge, 19 gauge, 20 gauge, 21 gauge, 22 gauge, 23 gauge, 24 gauge, or 25 gauge. In some embodiments, the probe 700 is made of a conductive material to function as an electrode. Thus, the electrode has the size of the exposed probe. Examples of materials include stainless steel, nitinol, cobalt-chromium alloy, copper, and gold. In some embodiments, the exposed probe conductive material is coated with a different material, examples of which include platinum-iridium, gold, platinum black, palladium, or other materials. The conductive material or conductive material coating can be designed to, among other purposes, reduce tissue biological interaction, reduce the generation of electrochemical effects from the PEF process, or more efficiently distribute PEF energy to the tissue. The material may be smooth, electropolished, sandblasted with various grits, or treated with other mechanical or chemical preparations to alter the surface roughness, which may be done to, among other purposes, reduce tissue biological interaction, facilitate easier electrode deployment and retraction, reduce the generation of electrochemical effects from the PEF process, or more efficiently distribute PEF energy to the tissue. Thus, in these embodiments, PEF energy can be transmitted through the probe 700 to the probe tip 702. Consequently, the shaft 106 is constructed of an insulating material or is covered by an insulating sheath. Examples of insulating materials include polyimide, silicone, polytetrafluoroethylene, and polyether block amide. The insulating material may be consistent or vary along the length of the shaft 106 or sheath. Similarly, in either case, the insulating material typically includes complete electrical insulation. However, in some embodiments, the insulating material allows some leakage current to penetrate.

[0144] When the probe 700 is energized, the insulated shaft 106 protects the surrounding tissue from the treatment energy and directs the energy to the probe tip 702 (and any exposed portions of the probe 700), which can deliver the treatment energy to the surrounding tissue. The tip 702 thus acts as a delivery electrode, the size of which can be selected based on the amount of exposure of the probe 700. A larger amount of exposure of the probe 700 can create a larger electrode, and a smaller amount of exposure can create a smaller electrode. In some embodiments, the exposed tip 702 (measured from its distal end to the distal edge of the insulated shaft) during energy delivery has a length of 0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1 cm, 2 cm, 3 cm, greater than 3 cm, up to 8 cm, 0.1 cm or less, 0.3 cm or less, 0.5 cm or less, 1 cm or less, 0.2-0.3 cm, 0.1-0.5 cm, 0.1-1 cm, and all ranges and subranges therebetween. In addition to varying electrode size, the tip 702 is retractable within the shaft 106 to enable atraumatic endoscopic delivery and then advanceable as desired to reach the target tissue. In this embodiment, advancement and retraction are controlled by an actuator 732 (e.g., a knob, button, lever, slide, or other mechanism) on the handle 110 attached to the proximal end 107 of the shaft 106. It will be appreciated that the shaft 106 itself can be advanced toward the target tissue with or without a probe being advanced from the distal end 103 of the shaft 106. In some embodiments, the distal end of the shaft 106 is advanced up to 20 cm into the tissue, such as from the exterior surface of a luminal structure or from the exterior surface of a patient's body.

[0145] The handle 110 connects to the generator 104 using a specialized energy plug 510. The energy plug 510 has a first end 512 that connects to the handle 110 and a second end 514 that connects to the generator 104. The connection of the first end 512 to the handle 110 is shown in expanded detail in FIG. 1B . In this embodiment, the first end 712 has an adapter 716 that includes a connecting wire 718 extending therefrom. The connecting wire 718 is insertable into the proximal end of the probe 700 within the handle 110. This allows energy to be transferred from the generator 104 to the probe 700 via the connecting wire 718. Thus, the probe 700 can be energized throughout its entire length, but only the exposed tip 702 delivers energy to tissue due to the presence of the insulated shaft 106.

[0146] In this embodiment, the generator 104 includes a user interface 150, one or more energy delivery algorithms 152, a processor 154, a data storage / retrieval unit 156 (such as a memory and / or database), and an energy storage subsystem 158 that generates and stores the energy to be delivered. In some embodiments, one or more capacitors are used for energy storage / delivery, although any other suitable energy storage elements may be used. Additionally, one or more communication ports are included.

[0147] In some embodiments, the generator 104 includes three subsystems: 1) a high-energy storage system, 2) a high-voltage, mid-frequency switching amplifier, and 3) a system controller, firmware, and user interface. The system controller includes a cardiac synchronization trigger monitor that allows the pulse energy output to be synchronized to the patient's cardiac rhythm. The generator incorporates an alternating current (AC) power source to power multiple direct current (DC) power sources. The generator controller can cause the DC power source to charge a high-energy capacitor storage bank before energy delivery begins. At the start of therapeutic energy delivery, the generator controller, high-energy storage bank, and biphasic pulse amplifier can operate simultaneously to produce a high-voltage, mid-frequency output.

[0148] It will be appreciated that numerous generator electrical architectures can be used to implement energy delivery algorithms. In particular, some embodiments employ advanced switching systems that can separately direct pulsed electric field circuits to energy delivery electrodes from the same energy storage and high-voltage delivery system. Furthermore, generators used for advanced energy delivery algorithms that employ rapidly changing pulse parameters (e.g., voltage, frequency, etc.) or multiple energy delivery electrodes may utilize modular energy storage and / or high-voltage systems to facilitate highly customizable waveforms and geographic pulse delivery paradigms. It will be further appreciated that the electrical architectures described herein above are for illustrative purposes only, and that systems that deliver pulsed electric fields may or may not include additional switching amplifier components.

[0149] The user interface 150 may include a touch screen and / or more traditional buttons that allow an operator to enter patient data, select a treatment algorithm (e.g., energy delivery algorithm 152), initiate energy delivery, view records stored in the storage / retrieval unit 156, and / or otherwise communicate with the generator 104. The user interface 150 may include a voice activation mechanism for entering patient data, or may be capable of communicating with additional equipment in the suite such that control of the generator 104 is through a secondary, separate user interface.

[0150] In some embodiments, the user interface 150 is configured to receive operator-defined input. The operator-defined input can include the duration of energy delivery, one or more other timing aspects of the energy delivery pulse, power, and / or operational mode, or a combination thereof. Exemplary operational modes can include, but are not limited to, system startup and self-test, operator input, algorithm selection, pre-treatment system status and feedback, energy delivery, post-energy delivery display or feedback, treatment data review and / or download, software update, or any combination or subcombination thereof.

[0151] In some embodiments, the system 100 also includes a mechanism for acquiring an electrocardiogram (ECG), such as an external cardiac monitor 170. An exemplary cardiac monitor is available from AccuSync Medical Research Corporation. In some embodiments, the external cardiac monitor 170 is operably connected to the generator 104. The cardiac monitor 170 can be used to continuously acquire ECG signals. External electrodes 172 may be applied to the patient P to acquire the ECG. The generator 104 analyzes one or more cardiac cycles to identify the beginning of a period in which it is safe to apply energy to the patient P, thus providing the ability to synchronize energy delivery with the cardiac cycle. In some embodiments, this period is within milliseconds of the R wave (of the ECG QRS complex) to avoid induction of arrhythmias, which can occur if the energy pulse is delivered at the T wave. It will be appreciated that such cardiac synchronization is typically utilized when using unipolar energy delivery, but may be utilized as part of other energy delivery methods.

[0152] In some embodiments, processor 154, among other activities, changes and / or switches between energy delivery algorithms, monitors energy delivery and any sensor data, and reacts to the monitored data via a feedback loop. In some embodiments, processor 154 is configured to execute one or more algorithms for implementing a feedback control loop based on one or more measured system parameters (e.g., current), one or more measured tissue parameters (e.g., impedance), and / or combinations thereof.

[0153] The data storage / retrieval unit 156 stores data related to delivered treatments, etc., which can be downloaded, as needed, by connecting a device (e.g., a laptop or thumb drive) to the communications port. In some embodiments, the device has local software that is stored in the data storage / retrieval unit 156 and used to direct the download of information, such as instructions executable by the processor 154. In some embodiments, the user interface 150 allows an operator to select to download data to a device and / or system, such as, but not limited to, a computing device, tablet, mobile device, server, workstation, cloud computing appliance / system, etc. The communications port, which can allow for wired and / or wireless connections, can enable data downloads as described above, but can also enable data uploads, such as uploading custom algorithms or providing software updates.

[0154] The data storage / retrieval unit 156 may be, for example, a random access memory (RAM), a memory buffer, a hard drive, a database, an erasable programmable read-only memory (EPROM), an electrically erasable read-only memory (EEPROM), a read-only memory (ROM), a flash memory, etc. The data storage / retrieval unit 156 may store instructions that cause the processor 154 to execute modules, processes, and / or functions associated with the system 100.

[0155] In some embodiments, the data storage / retrieval unit 156 includes a computer storage product having a non-transitory computer-readable medium (also called a non-transitory processor-readable medium) having instructions or computer code for performing various computer-implemented operations. The computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not include a transient propagating signal itself (e.g., a propagating electromagnetic wave carrying information over a transmission medium such as space or a cable). The medium and computer code (also called code) may be designed and constructed for a specific purpose or purposes. Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tape; optical storage media such as compact disks / digital video disks (CDs / DVDs), compact disk read-only memories (CD-ROMs), and holographic devices; magneto-optical storage media such as optical disks; carrier wave signal processing modules; and hardware devices specially configured to store and execute program code, such as ASICs, programmable logic devices (PLDs), read-only memories (ROMs), and random access memory (RAM) devices. Other embodiments described herein relate to computer program products that may include, for example, the instructions and / or computer code described herein.

[0156] Examples of computer code include, but are not limited to, microcode or microinstructions, machine instructions such as those produced by a compiler, code used to generate a web service, and files containing higher-level instructions that are executed by a computer using an interpreter. For example, embodiments may be implemented using an imperative programming language (e.g., C, Fortran, etc.), a functional programming language (Haskell, Erlang, etc.), a logic programming language (e.g., Prolog), an object-oriented programming language (e.g., Java, C++, etc.), or other suitable programming language and / or development tool. Further examples of computer code include, but are not limited to, control signals, encryption code, and compression code.

[0157] In some embodiments, system 100 may be communicatively coupled to a network, which may be any type of network, such as, for example, a local area network (LAN), a wide area network (WAN), a virtual network, a telecommunications network, a data network, and / or the Internet, implemented as a wired and / or wireless network. In some embodiments, any suitable type and / or method of secure communications (e.g., Secure Sockets Layer (SSL)) and / or encryption may be used to protect any or all communications. In other embodiments, any or all communications may not be secure.

[0158] As described herein, the various energy delivery algorithms 152 may be programmable or pre-programmed into the generator 104, such as stored in a memory or data storage / retrieval unit 156. Alternatively, the energy delivery algorithms may be added to the data storage / retrieval unit and executed by the processor 154. The processor 154 may be, for example, a general-purpose processor, a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), and / or a digital signal processor (DSP), etc. The processor 154 may be configured to implement and / or execute application processes and / or other modules, processes and / or functions associated with the system 100 and / or networks associated with the system 100. As used herein, the term “module” refers to any assembly and / or set of operably coupled electrical components that may include, for example, memory, a processor, electrical traces, optical connectors, software (implemented in hardware), etc. For example, a module executed by a processor may be any combination of hardware-based modules (e.g., FPGA, ASIC, DSP) and / or software-based modules (e.g., modules of computer code stored in memory and / or executed by a processor) that can perform one or more specific functions associated with that module.

[0159] Each of these algorithms 152 may be executed by the processor 154. In some embodiments, the instrument 102 includes one or more sensors 160 that can be used to determine temperature, impedance, resistance, capacitance, conductivity, pH, optical properties (coherence, echogenicity, fluorescence), electrical or optical permittivity, and / or conductance, to name a few. In some embodiments, one or more electrodes function as the one or more sensors. In other embodiments, the one or more sensors are separate from the electrodes. It will be appreciated that the one or more sensors 160 can be positioned in a variety of locations, depending particularly on the parameter being sensed. For example, sensor placement may be along the energy delivery body 108, along the interior of the instrument, along the shaft 106, along elements protruding from the instrument 120, etc. Multiple sensors 160 may be present to sense the same parameter at multiple locations, to sense different parameters at different locations, or to sample a parameter at different locations to compile a single metric value measurement (e.g., average temperature, average voltage exposure, average conductivity, etc.). Alternatively, or in addition, one or more sensors 160 may be located on a separate device. Sensor data can be used to plan therapy, monitor therapy, and / or provide direct feedback via processor 154, which can then modify the energy delivery algorithm 152. For example, impedance measurements can be used to determine the initial dose to apply, as well as whether further treatment is required.

[0160] It will be appreciated that the system 100 may include automatic treatment delivery algorithms that can dynamically respond, adjust, and / or terminate treatment depending on inputs such as temperature, impedance at various voltages or AC frequencies, treatment duration or other timing aspects of the energy delivery pulse, treatment power, and / or system state.

[0161] In some embodiments, imaging is accomplished using a commercially available system, such as an endoscope connected to a separate imaging screen. It will be appreciated that the imaging modality can be incorporated into the instrument 102 or can be used together or in conjunction with the instrument 102. The imaging modality can be mechanically, operatively, and / or communicatively coupled to the instrument 102 using any suitable mechanism. III. Intraluminal Placement and Energy Delivery

[0162] As mentioned above, in one arrangement, the energy delivery body 108 is placed within a body cavity and energy is delivered to or through the lumen wall to target tissue either within the lumen, within the lumen wall, at least partially surrounding the lumen wall, or outside the lumen wall. Thus, the target tissue can be treated from the energy delivery body 108 placed within the body cavity.

[0163] In some embodiments, treatment devices and systems are configured to access the lumen and deliver therapeutic energy toward the lumen wall to treat nearby target tissue. The therapeutic energy is generally characterized by high-voltage pulses that allow removal of the target tissue with little or no disruption of critical anatomical structures, such as tissue-level structural proteins in the extracellular matrix. This prevents dangerous collateral effects, such as stenosis, thrombus formation, or fistula formation, to name a few, and also allows for the regeneration of healthy new luminal tissue within days of the procedure. Examples of systems providing this type of therapeutic treatment include International Patent Application No. PCT / US2017 / 039527, entitled "GENERATOR AND A CATHETER WITH AN ELECTRODE AND A METHOD FOR TREATING A LUNG PASSAGEWAY," which claims priority to U.S. Provisional Patent Application Nos. 62 / 355,164 and 62 / 489,753, and International Patent Application No. PCT / US2017 / 039527, entitled "METHODS, APPARATUSES, AND SYSTEMS FOR THE TREATMENT OF LUNG PASSAGEWAYS," which claims priority to U.S. Provisional Patent Application Nos. 62 / 610,430. and International Patent Application No. PCT / US2018 / 067501 entitled "OPTIMIZATION OF ENERGY DELIVERY FOR VARIOUS APPLICATIONS," which claims priority to U.S. Provisional Patent Application No. 62 / 610,430, filed December 26, 2017, and U.S. Provisional Patent Application No. 62 / 693,622, filed July 3, 2018, all of which are incorporated herein by reference for all purposes.

[0164] As previously mentioned, one or more energy delivery algorithms 152 can be programmable or pre-programmed into the generator 104 for delivery to the patient. The one or more energy delivery algorithms 152 specify electrical signals that provide energy delivered to the luminal wall that is non-thermal (e.g., below the threshold for thermal ablation; below the threshold for inducing coagulation thermal damage), reduces or avoids inflammation, and / or prevents denaturation of interstitial proteins in the luminal structure. Generally, the algorithms 152 are tailored to affect tissue to a predetermined depth and / or to target specific types of cellular responses to the delivered energy. It will be understood that depth and / or targeting can be influenced by parameters of the energy signal defined by the one or more energy delivery algorithms 152, the design of the instrument 102 (particularly the one or more energy deliverers 108), and / or the selection of monopolar or bipolar energy delivery. Typically, the depth is up to 0.01 cm, up to 0.02 cm, 0.01-0.02 cm, up to 0.03 cm, 0.03-0.05 cm, up to 0.05 cm, up to 0.08 cm, up to 0.09 cm, up to 0.1 cm, up to 0.2 cm, up to 0.5 cm, up to 0.7 cm, up to 1.0 cm, up to 1.5 cm, up to 2.0 cm, up to 2.5 cm, up to 3.0 cm, up to 3.5 cm, up to 4.0 cm, up to 4.5 cm, or up to 5.0 cm, to name a few. These depths may be greater relative to the circumferential focal target or may exist throughout the entire circumferential depth through the lumen and parenchyma.

[0165] Thus, the treatment is minimally invasive, quick and easy to perform, and relatively insensitive to electrode placement (e.g., due to monopolar placement), thus allowing technicians of various skill levels to achieve a high level of consistency and successful outcomes. In some embodiments, monopolar placement is possible without the need for muscle paralysis due to the waveform characteristics of the energy used. This allows muscle contraction from depolarization of motor neurons and skeletal muscles to be relaxed to tolerable levels, regardless of whether neuromuscular paralysis occurs. Therefore, monopolar directional treatment delivery through the lumen to a distant pad can be performed, creating a more predictable and desirable treatment zone. It will be appreciated that paralysis may be used as needed, depending on the type of energy and depth of penetration desired. IV. Extraluminal Placement and Energy Delivery

[0166] 2A-2C illustrate an example of a treatment method. FIG. 2A shows abnormal or diseased tissue D, e.g., a tumor, near a luminal structure LS. In this example, the diseased tissue D is near the luminal structure LS but is spaced apart from the luminal wall W. This luminal structure LS is used to access and extraluminally treat the diseased tissue D near the luminal structure LS. In this embodiment, the elongated insertion tube 14 of the endoscope 10 is advanced into the luminal structure LS, and its distal tip 16 is maneuvered toward the luminal wall W, beyond which the diseased tissue D resides. Once desirably positioned, the treatment catheter 102 is advanced through the lumen within the insertion tube 14 so that the distal end 103 of the shaft 106 extends beyond the tip 16 of the endoscope 10, as shown in FIG. 2B. In this embodiment, the probe tip 702 aids in penetrating the wall W, and the shaft 106 is advanced across the wall W until the probe tip 702 is desirably positioned within the diseased tissue D. 2C, in this embodiment, the probe tip 702 is then advanced from the shaft 106 to create the desired delivery electrode size. Energy is then delivered through the probe 700 to the diseased tissue D according to one or more energy delivery algorithms 152, as shown in FIG. 2C by the wavy arrows extending radially outward from the probe tip 702. It can be understood that the distance into the diseased tissue can vary based on parameter values, treatment time, and tissue type, to name a few. It can also be understood that treatment depths greater or less than those shown herein can be achieved. The delivered energy appropriately treats the diseased tissue D. In the case of cancer, cancerous cells are destroyed, eliminated, killed, removed, etc. Examples include immunogenic cell death (e.g., necroptosis, pyroptosis, etc.) and programmed cell death (apoptosis), to name a few. In the PEF zone, such cell death occurs while preserving non-cancerous non-cellular elements such as collagen, elastin, and matrix proteins. These non-cellular elements maintain the tissue architecture that enables and promotes normal cell regeneration. Similarly, any energy reaching the wall W of the nearby luminal structure LS maintains the integrity and mechanical properties of the luminal structure LS. In some instances, the energy can be understood to directly kill cells of the diseased tissue D, such as through accumulated systemic cellular injury and irreversible disruption of cellular homeostasis. In other instances, cells are killed by the action of the immune system or other biological processes. In some instances, remaining diseased tissue is surgically or otherwise removed. A. Alternative Probe Designs

[0167] It will be appreciated that the probe 500 can have a variety of forms and configurations. In some embodiments, the probe 500 is hollow, e.g., having a tubular shape. In such embodiments, the probe 500 may be formed from a hypotube or metal tube. Such tubes can be optimized for desired pushability and torque capabilities, kink performance, compression resistance, and flexibility to ensure consistent and reliable maneuverability to the target treatment site. Similarly, such tubes can include custom-designed transitions, such as laser cut and skive features, along with optional coatings to optimize manufacturability. In some embodiments, the tube has a sharp point with multiple cutting edges to form the probe tip 502. In other embodiments, the tube has a blunt, atraumatic tip. In some embodiments, the probe 500 is solid, e.g., has a rod shape. These probes can also be optimized and customized similarly to hypotubes. In some embodiments, the solid probe 500 has a sharp point with symmetrical or asymmetrical cuts to form the probe tip 502. In other embodiments, the solid probe 502 has a blunt, atraumatic tip.

[0168] It will be appreciated that the probe 500 may include lumens for delivering fluids or agents. Such lumens may be internal or external to the probe. Similarly, fluids or agents may be delivered directly from the shaft 106, such as through an internal lumen or a port disposed along the shaft 106.

[0169] In some embodiments, the probe 500 is comprised of multiple probe elements, each having similar features and functionality to the individual probes 500 described above. Thus, in some embodiments, they may be considered separate probes, but for simplicity, they are described as probe elements comprising a single probe 500 because they pass through the same shaft 106 of the instrument 102. It will be understood that there may be any number of probe elements, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. Similarly, the probe elements may extend the same or different distances from the shaft 106 and may have the same or different curvatures. In another embodiment, the probe elements do not have any curvature and exit the shaft 106 in a straight line. Typically, the probe elements are pre-curved such that advancement of the probe tip from the shaft 106 allows the probe element to assume its pre-curved shape. Thus, in some embodiments, various curvatures can be utilized by advancing the probe tip different amounts from the shaft 106.

[0170] It will be appreciated that the size of the probe tip 502 capable of transmitting energy can be further adjusted using an insulating sheath 552 that extends at least partially over the probe. As previously mentioned, the size of the active portion of the probe tip 502 can be adjusted based on its extension from the shaft 106. However, this can be further refined using an insulating sheath 552 that covers a portion of an individual probe element, particularly when multiple probe elements are present.

[0171] It will be understood that any of the probe elements described herein can have the same structure and characteristics as any of the probes described herein. For example, the probe elements may be composed of the same materials, have the same functions, and have sharp or atraumatic tips. Similarly, it will be understood that any of the probe elements can be independently or simultaneously deployed and independently or simultaneously energized. The delivered energy may be provided by the same energy delivery algorithm 152 or different energy delivery algorithms 152, thus delivering the same or different energies. Any of the probe elements can function in a monopolar or bipolar fashion between pairs of probe elements. It will also be understood that the probe elements can function in a combination of monopolar and bipolar fashions.

[0172] As mentioned above, in many of these extraluminal delivery embodiments, the energy delivery body 108 takes the form of a probe 500 disposed within the lumen 105 of the shaft 106. In some embodiments, the probe 500 includes multiple wires or ribbons 120 to form a basket 555 that functions as an electrode. Alternatively, it will be appreciated that the basket 555 can be laser cut from a tube. It will be appreciated that a variety of other designs may be used. Typically, the basket 555 is delivered to the target area in a collapsed configuration and then expanded for use. V. Imaging

[0173] Imaging-related methods that may be useful include (a) detecting diseased target tissue, (b) identifying the area to be treated, (c) evaluating the treated area to determine how effective the energy delivery was, (d) evaluating the target area to determine if the area was missed or insufficiently treated, (e) using pre- or intra-procedure imaging to measure the target treatment depth and using that depth to select a specific energy delivery algorithm to achieve a tissue effect at that depth, (f) using pre- or intra-procedure imaging to identify a target cell type or cell interface and using that location or depth to select a specific energy delivery algorithm to achieve a tissue effect at that target cell type or cell interface, and / or (g) using pre-, intra-, or post-procedure imaging to identify the presence or absence of pathogens with or without inflamed tissue.

[0174] In some embodiments, confocal laser endomicroscopy (CLE), optical coherence tomography (OCT), ultrasound, static or dynamic CT imaging, X-ray, magnetic resonance imaging (MRI), and / or other imaging modalities can be used as separate devices / systems or can be incorporated / integrated (functionally and / or structurally) into the treatment system 100 by being incorporated into the instrument 102 or a separate device. The imaging modality (or multiple modalities) can be used to locate and / or access various sections of the target tissue. In some embodiments, it can be used to measure the target depth of the treatment and select a treatment algorithm 152 sufficient to treat to the target depth. At least one energy delivery entity can then be deployed at the target tissue site, and energy can be delivered to affect the target tissue. The imaging modality (or multiple modalities) can be used before, during, between, and / or after treatment to determine where treatment was delivered, not delivered, or whether the energy adequately affected the airway wall. If it is determined that an area was missed or not adequately affected, the energy delivery and subsequent imaging modality (or modalities) can be repeated until adequate treatment is achieved. Additionally, imaging information can be utilized to determine whether a particular cell type and / or desired therapy depth was applied. This can allow for customization of energy delivery algorithms to treat a wide variety of patient anatomies.

[0175] In some embodiments, access through a body cavity is visualized with one or more instruments inserted into the body. Similarly, in some embodiments, one or more of a variety of imaging modalities (e.g., CLE, OCT) are used in conjunction with or in place of direct visualization. As an example, a bronchoscope may be delivered through the mouth to allow direct visualization and delivery of the instrument 102, while an alternative imaging modality may be delivered adjacent to the bronchoscope through a separate working channel of the bronchoscope, through the nose, or through the mouth. In some embodiments, the imaging modality (e.g., direct visualization, CLE, and / or OCT) is incorporated into the instrument 102 with appropriate mechanisms for connecting the imaging modality to either the system generator 104 or a commercially available console. VI. Conditioning

[0176] It will be appreciated that the PEF ablation treatment provided by system 100 may be used as conditioning for other treatments, but the target tissue cells may alternatively be conditioned prior to the PEF ablation treatment provided by system 100.

[0177] In some embodiments, cells targeted for treatment are conditioned to modify cellular behavior in response to delivery of an energy signal. Such conditioning can occur before, during, or after delivery of the energy signal. In some embodiments, conditioning before energy delivery is considered preconditioning, and conditioning after energy delivery is considered postconditioning. This distinction is based solely on timing, not how the conditioning treatment affects the cells. In other embodiments, preconditioning relates to affecting what happens to cells during energy delivery, such as how the cells take up energy, while postconditioning relates to affecting what happens to cells after energy delivery, such as how the cells behave after receiving energy. In some examples, conditioning can occur before energy delivery, but such a distinction may not be as relevant to timing, as it only affects cellular responses after energy delivery. Therefore, it will be understood that unless otherwise specified, "conditioning" may be considered to apply to each of these situations.

[0178] Typically, conditioning is achieved by delivering a conditioning solution. In the case of intraluminal therapy, the conditioning solution may be delivered via a luminal structure. Alternatively or additionally, the conditioning solution may be delivered via direct fluid injection of the conditioning solution into the target area, either intraluminally or via other approaches. In some embodiments, the conditioning solution selectively alters the electrical properties of target cells, such as by affecting the way pulsed energy delivery is distributed. In other embodiments, the conditioning solution affects the activity of target cells. For example, in the lung, such a conditioning solution may promote differentiation of basal cells into ciliated cells and / or downregulate goblet cells and submucosal gland cells. In other embodiments, the conditioning solution increases the likelihood that target cells will die after pulsed energy delivery. In still other embodiments, the conditioning solution alters the response of non-target cells to pulsed electric fields. In alternative embodiments, conditioning is achieved by non-solution-based exposure of tissue, including radiation therapy, radiotherapy, proton therapy, and the like. In some embodiments, conditioning affects enzymes and energy-producing components of the cellular infrastructure.

[0179] The conditioning solution may be comprised of a variety of agents, such as drugs, genetic material, bioactive compounds, and antimicrobial agents, to name a few. For embodiments in which the conditioning solution increases the likelihood that target cells will die after pulsed energy delivery, the conditioning solution may include chemotherapeutic agents (e.g., cisplatin, doxorubicin, paclitaxel, bleomycin, carboplatin, etc.), calcium, antibiotics, or toxins, to name a few. For embodiments in which the conditioning solution alters the response of non-target cells to the pulsed electric field, the conditioning solution may include cytokines (e.g., immunostimulants such as interleukins), genes, VEGF (e.g., to promote more blood vessel growth into the area), cell differentiation factors (e.g., molecules that promote the conversion of goblet cells to ciliated cells), and / or other small molecules that interact with cells, such as agonists and antagonists of receptors such as programmed death (PD-1) or programmed death ligand (PD-L1). The conditioning solution may also be delivered to the target site to interact directly at the site of PEF energy delivery. They can also be delivered systemically (e.g., intravenously, intraperitoneally) or locally (e.g., intravenously to the arterial supply to the target area, higher supply airway generation, intraparenchyma to tissues surrounding tissues directly affected by PEF treatment).

[0180] In some embodiments, the conditioning solution includes cells, such as stem cells, autologous cells, allogeneic cells, or other cell types. In these embodiments, the cells may be used to alter tissue response to pulsed electric fields. In other embodiments, the cells may be used to repopulate the affected area with healthy or desirable cells. For example, if the target cells are weakened or killed by the delivered pulsed energy treatment, the cells from the conditioning solution may migrate into pores, such as a decellularized extracellular matrix. In some embodiments, the area is flushed with, for example, a mild detergent, surfactant, or other solution to remove dead cells prior to delivery of the conditioning solution containing new cells. In other embodiments, mechanical stimulation, such as aspiration, debriding, or ultrasonic hydrodissection, is used to physically remove dead cells prior to delivery of the conditioning solution containing new cells.

[0181] In some embodiments, the provided conditioning can induce a targeted immune response. The immune response can result in several factors that alter the outcome of the treatment. This can result in increased upregulation of systemic immunity using specific markers associated with certain target tissues, such as tumors or bacteria or viruses associated with infection. It can also result in upregulation of innate immunity, which broadly affects immune system function to detect common abnormal cells, bacteria, or other infectious organisms present in the body, which can occur locally, regionally, or systemically.

[0182] In some embodiments, the conditioning solution is heated or cooled to alter how target cells respond. Generally, heated solutions promote increased treatment effects (e.g., increased susceptibility to cell death), while cooled solutions reduce the magnitude of treatment effects or increase cell survival after exposure to reversibly designed protocols. In some embodiments, cooled conditioning solutions composed of genes and / or drugs are used to precondition cells to withstand energy delivery treatments and increase the number of cells that survive the treatment. In some embodiments, the effects of the heated / cooled conditioning solution are combined with the general effects caused by other agents in the solution (e.g., heated calcium solution, cooled gene-containing solution). In other embodiments, the heated / cooled conditioning solution does not produce any effect other than temperature change. In such embodiments, the conditioning solution typically consists of isotonic saline, phosphate buffer solution, or other benign solutions.

[0183] It will be appreciated that such heating or cooling may alternatively be achieved by other methods that do not involve the delivery of a conditioning solution. For example, the target tissue may be heated or cooled by contacting the tissue with a heated / cooled device, intentionally heating / cooling a pulsed electric field delivery catheter, delivering mild cryotherapy, or delivering mild radiofrequency or microwave energy. As previously mentioned, this may facilitate an enhanced lethal or permeabilizing effect on the tissue, or may provide a protective aspect to the cells that allows them to survive the procedure and exhibit the desired targeted changes as a result of the therapy.

[0184] In some embodiments, the conditioning solution is delivered systemically, such as by intravenous injection, ingestion, or other systemic method. In other embodiments, the conditioning solution is delivered locally to the area of ​​the target cells, such as via a delivery device or instrument 102 itself. VII. Energy Delivery Algorithms and Lesion Optimization

[0185] The specific energy is provided by one or more energy delivery algorithms 152. In some embodiments, the algorithm 152 defines a signal having a waveform including a series of energy packets, each including a series of high-voltage pulses. In such embodiments, the algorithm 152 specifies parameters of the signal, such as the number of packets, the number of pulses within a packet, the fundamental frequency of the pulse sequence, the duration of individual pulses, or the energy amplitude (e.g., voltage) and duration of the applied energy, consisting of the sequence of pulses comprising the packets (which may vary within the packets themselves), to name a few. Additional parameters may include a switch time between polarities of biphasic pulses, a dead time or inter-cycle delay between biphasic cycles, and a pause time or inter-packet delay between packets, which are described in more detail in later sections. In some embodiments, there is a fixed inter-packet delay between packets. In some embodiments, the packets are gated to the cardiac cycle and therefore variable depending on the patient's heart rate, or fixed to and synchronized with the cardiac cycle. In some embodiments, there may be an intentional, variable inter-packet delay algorithm, or no pause period may be applied between packets. Packet delivery may also be coordinated with multiple factors, such as a minimum inter-packet delay, where a subsequent trigger signal (e.g., a cardiac synchronization signal) is used to coordinate the timing of subsequent packet delivery. It will be appreciated that feedback loops based on sensor information, automatic shut-off specifications, etc. may also be included.

[0186] The specialized energy delivered to the target tissue by the probe 702 or other energy delivery device creates a lesion with multiple zones. The zones emanate radially outward from the probe 702, e.g., in a ring shape. Each zone has a different cellular effect and, therefore, a different effect on the overall outcome of the treatment. In some examples, the zones include a cavitation zone, a thermal zone, a PEF zone, an inflammatory zone, and an immune response zone.

[0187] Figure 3A shows actual tissue lesions in tissue T produced by delivering specific energy to a porcine liver maintained viably using a machine-perfused organ preservation model. Figure 3B shows the lesions defined by zones for clarity. The energy waveform produced a very small cavitation zone 200 (in this example, it is likely not an actual cavity, but a needle tube formed by placing an energy delivery device (needle) within it; however, the cavitation zone 200 would typically be located there), a thermal zone 202, and a PEF zone 204. It can be seen that inflammatory and / or immune response zones are located around the PEF zone and thus follow its edge. The presence and size of the zones can be manipulated by the waveform, particularly the parameter values. It can be seen that in these examples, the lesions are produced by a single dose or application of energy. The energy on-time during this dose and its application depend on the waveform, more specifically, the waveform parameters.

[0188] Energy delivery can be activated by various mechanisms, such as the use of an actuator 132 on the instrument 102 or a footswitch operably connected to the generator 104. Such activation typically provides a single energy dose. The energy dose is defined by the number of packets delivered and the voltage of the packets. Each energy dose delivered to the target tissue maintains the temperature of the target tissue or temperatures within the target tissue to create a desired zone within the lesion. A zone, such as the PEF zone, is maintained below the threshold for thermal ablation, particularly thermal ablation or denaturation of interstitial proteins. Furthermore, the dose can be titrated or relaxed over time to further reduce or eliminate heat accumulation during the treatment procedure. Instead of inducing thermal damage in the PEF zone (defined as protein coagulation at sites at risk for therapy), the energy dose provides a level of energy that induces treatment of conditions such as cancer without damaging sensitive tissue.

[0189] FIG. 4 illustrates one embodiment of a waveform 400 of a signal defined by the energy delivery algorithm 152. Two packets are shown: a first packet 402 and a second packet 404, separated by a rest period or inter-packet delay 406. In this embodiment, each packet 402, 404 consists of a first biphasic cycle (including a first positive pulse peak 408 and a first negative pulse peak 410) and a second biphasic cycle (including a second positive pulse peak 408' and a second negative pulse peak 410'). The first and second biphasic pulses are separated by a dead time or inter-cycle delay 412 (i.e., a pause) between each pulse. In this embodiment, the biphasic pulses are symmetrical such that the set voltage 416 is the same at the positive and negative peaks. Here, the biphasic symmetrical wave is also a square wave, such that the magnitude and duration of the positive voltage wave are approximately equal to the magnitude and duration of the negative voltage wave.

[0190] It will be appreciated that manipulation of various parameters (e.g., voltage, fundamental frequency, number of pulses per packet, number of packets, and various delays) will have different effects on the resulting lesion and the body itself. In some cases, parameter changes can balance each other, and the effects of changing one or more parameters can be balanced by changing one or more different parameter values ​​that produce the same or similar results. In other examples, parameter values ​​can be adjusted to create or produce different effects, such as different lesion characteristics (e.g., the presence, size, and / or characteristics of different zones) and / or different effects on the body. These effects on the body may be immediate, such as muscle stimulation, or delayed, such as the generation of a specific immune response.

[0191] Figure 5 is a table showing various exemplary effects of parameter changes. Typically, the electrode style is monopolar rather than bipolar. In a monopolar configuration, one or more delivery electrodes are placed near the target tissue site and at least one remote return electrode is placed against the patient's skin. Utilizing a monopolar electrode configuration increases muscle contraction strength. To counteract this effect, a biphasic waveform or waveforms are generally used, consisting of sufficiently short individual pulse durations with packets having appropriate delays between pulses to offset the degree of muscle contraction. While this waveform variation results in a reduction in treatment effect, the reduction is more subtle than a reduction in muscle contraction, thus still resulting in effective PEF therapy application. A monopolar configuration also reduces the risk of electrical arcing compared to bipolar or multipolar configurations, as all affecting electrodes are positioned within a similar area that may allow electrical arcing between them. Typically, the waveform utilizes biphasic pulses as opposed to monophasic pulses. The use of biphasic pulses reduces treatment size, reduces muscle contraction, and also reduces the risk of arcing. Therefore, the use of biphasic pulses counters the increased muscle contraction caused by unipolar electrode styles. Because both unipolar electrode styles and biphasic pulses reduce treatment size, treatment size can be increased by altering other variables. For example, increasing voltage, packet duration, and packet number increases treatment size. However, increasing these parameters has various other effects. For example, increasing voltage and packet duration increases the risk of muscle contraction, temperature rise, and electrical arcing, respectively. Increasing the fundamental frequency can reduce muscle contraction but also reduces treatment size. Similarly, increasing the number of packets increases treatment size but also increases temperature rise and treatment delivery time. Treatment delivery time can be reduced by increasing the packet delivery rate, but this increases temperature rise. Therefore, managing the effects of various parameter changes is a complex endeavor, which is further complicated by the increments at which these changes are made. Given the number of parameters related to the PEF waveform and the dose itself and the increments at which they can be altered, the set of combinations becomes enormous.This is further exacerbated by the effects of electrode geometry, such as when larger electrodes delivering a particular voltage have different characteristics than smaller electrodes, or when using monopolar versus bipolar and multipolar configurations (and the separation distance between electrodes in these configurations). These characteristics also include temperature rise, treatment effect size, delivered current, muscle contraction, risk of electrical arcing, and the time required to achieve coverage of the target treatment effect size. Furthermore, certain conditioning solutions provided to patients can be used specifically to target and reduce induced muscle contractions. For example, neuromuscular blockade with pancuronium bromide, vecuronium, succinylcholine, and other occlusions may be used. This reduction in muscle contractions can be used to facilitate treatment doses at lower frequencies, longer packet durations, or higher voltages to achieve greater treatment effects while maintaining acceptable and safe muscle contractions.

[0192] Thus, determining appropriate parameter values ​​for a particular outcome involves a high level of skill and dexterity. Furthermore, determining which particular outcome is desired also involves a high level of skill. For example, the existence, type, and size of various zones in the resulting lesion that lead to the desired clinical outcome are previously unknown. The methods, systems, and devices described herein provide both the specific parameter values ​​and the characteristics of the desired resulting lesion that will lead to the desired clinical outcome. Thus, the specific energy typically delivered to cancerous or otherwise undesirable target tissue is generated from an algorithm 152 in the generator 104 that generates waveforms according to these parameter values.

[0193] In one embodiment, the treatment dose is provided by a waveform generated from a combination of parameter values ​​including: voltage = 3000 V, fundamental frequency = 400 kHz, number of biphasic pulses (i.e., cycles) per packet = 40 cycles, inter-cycle delay = 1000 microseconds, number of packets = 100 packets, and inter-packet delay = 3 seconds. With this combination, the dose delivery time is approximately 5-6 minutes. This treatment time is substantially shorter than treatment times using conventional energies such as microwave (10 minutes), RF (20 minutes), or cryoablation (30-40 minutes). In some cases, this dose can be delivered in an area of ​​approximately 1x1x1 cm in any direction. 3 + The lesions measured 0.3 cm in size. The characteristics of the lesions obtained are described below. Cavitation Effect

[0194] In some instances, when delivering conventional PEF energy, cavitation occurs in the area closest to the probe 702, considered the cavitation zone. However, the specialized energies described herein are configured to minimize or eliminate the occurrence of cavitation. Here, cavitation is minimized or eliminated through manipulation of parameter values, particularly by including and manipulating specific inter-cycle delays.

[0195] Referring to Figures 6A-6D, tissue samples T with lesions created by energy having waveforms with different inter-cycle delays are shown. In a perfusion organ preservation model, each lesion was created by placing an energy delivery device 108 (e.g., a needle / probe 702, not shown) in a portion of liver tissue. Figure 5A shows a lesion created from energy with a specific waveform without an inter-cycle delay. Here, energy delivery is prone to electrical arcing, which results in irregularly shaped cavity effects (i.e., cavitation zones 200) within the lesion. Although these cavity effects are irregular, the cavities are well contained within the core of the treated lesion. Minimal secondary thermal effects are observed due to tissue voids (i.e., cavities) filling with perfusate resulting from cavity formation. As shown in Figures 6B-6D, adding inter-cycle delays such as 10 μs, 100 μs, and 1000 μs, respectively, completely eliminated the cavity effect. The small hole in the center of the lesion is due to the insertion of the probe 702, not the formation of a cavity. Thus, the amount of cavitation, including its complete elimination, can be controlled and manipulated by aspects of the waveform itself, particularly by including a specific inter-cycle delay. Thus, for example, if the PEF waveform has a fundamental frequency of 400 kHz, 40 cycles per packet is provided, and 100 packets per dose, an inter-cycle delay of at least 10 μs in the PEF waveform is desirable to eliminate cavitation. It will be appreciated that as the length of the inter-cycle delay increases, arcing and discharge, thereby reducing cavitation, will decrease. However, such reduction will begin to plateau, such as at an inter-cycle delay near 1000 μs. Therefore, in some embodiments, a 1000 μs delay is preferred to most effectively minimize or eliminate cavitation without excessively extending the total treatment time. It will be appreciated that such a reduction in cavitation will deliver more of the intended energy to the target tissue to form the lesion. Thus, in this example, the thermal zone 202 increases, indicating that more energy than desired was delivered to create the PEF zone.

[0196] Similarly, Figures 7A-7B show an in vivo tissue sample T from a preclinical study in porcine lung tissue. Figure 7A shows macroscopic lung tissue with a lesion 210 formed by delivering energy having a particular waveform with an inter-cycle delay. As shown, tissue cavitation was prevented within the lung parenchyma. It will be appreciated that in some embodiments, a similar effect can be achieved by including an inter-packet delay instead of or in addition to an inter-cycle delay. Figure 7B shows lung tissue with a lesion formed from delivering energy having a waveform without an inter-cycle delay. As shown, the core treatment area is mostly composed of a large hemorrhagic (blood-filled) region 212 well contained within the anatomical interlobular septum. Treatment without an inter-cycle delay resulted in the largest cavitation area. Thermal effect

[0197] It will be appreciated that in some instances, PEF energy, when used with certain waveforms and methodologies, to name a few factors, can produce lesions with areas of thermal sequelae. This occurs most frequently when attempting to create larger treatment zones. While using higher levels of energy delivery can achieve larger treatment zones, this can also result in increased white tissue coagulation due to Joule heating effects. This typically occurs in areas closer to the delivery electrode. As a result, specialized energies reduce, eliminate, or otherwise control the presence of thermal effects in the created lesions. It will be appreciated that in some cases, a small amount of thermal effect can be beneficial for eliciting a specific immune response in the patient. Thus, in some cases, a thermal effect may be desired. However, controlling the amount of thermal effect maintains a balance between benefits and undesirable effects. Thus, in some embodiments, the ratio or combination of effects (e.g., thermal effect vs. PEF effect) is optimized to maximize and / or customize the patient's immune response. Thus, in some cases, there is a "golden ratio" of effects or waveform parameter combinations that tailor local tissue properties to elicit specific outcomes from the body. Thus, in some embodiments, the resulting improved waveforms and energy delivery algorithms ultimately maximize and / or beneficially control the patient's immune response.

[0198] In some embodiments, thermal effects are minimized or controlled by manipulating parameter values, particularly by including and manipulating specific inter-cycle delays. For example, in some embodiments, adding inter-cycle and inter-packet delays enhances the ability to maximize the PEF-influenced volume of the generated lesion while minimizing thermal and cavity zones. In particular, in some embodiments, including specific inter-packet delays minimizes thermal zones.

[0199] Figure 8 illustrates how integrating an inter-packet delay into an energy waveform can help mitigate the thermal effects of PEF energy delivery. Two curves are shown. The first curve 220 shows the temperature rise over time when the waveform includes a 3-second inter-packet delay 406. The second curve 222 shows the temperature rise over time when the waveform includes a 5-second inter-packet delay 406. As shown, the 5-second inter-packet delay 406 results in a lower temperature rise over time. This is achieved simply by manipulating the waveform, rather than by adding a coolant or other additive. In some cases, the thermal effect occurs at temperatures above 65°C. Thus, in some cases, a waveform with a 5-second inter-packet delay 406 may eliminate the thermal effect, while a 3-second inter-packet delay 406 may result in a small thermal effect. While the inter-packet delay 406 reduces the temperature rise over time, it also increases the overall treatment time. Because a small thermal effect is tolerable and potentially beneficial, a 3-second inter-packet delay 406 was included in the specific energy to balance the factors. However, it will be understood that an inter-packet delay of 3 to 5 seconds may be acceptable, including 3 seconds, 3.5 seconds, 4 seconds, 4.5 seconds, and 5 seconds, and all subranges therebetween. Similarly, if a longer procedure time is acceptable, an inter-packet delay 406 of greater than 5 seconds may be used.

[0200] It will be appreciated that when synchronized with the heartbeat, the 3-second delay may be slightly longer than 3 seconds. For example, in some cases, the energy delivery algorithm has a 3-second inter-packet delay and waits until the next heartbeat triggers energy delivery. Thus, the actual inter-packet delay may be 3.1 seconds, 3.2 seconds, 3.3 seconds, etc. In some cases, the delay may be as long as 4.49 seconds (e.g., for a 40 bpm patient, a 3-second inter-packet delay results in maximum misalignment). It will be appreciated that the overall lesion size is not significantly affected by the introduction of an inter-cycle delay.

[0201] In the PEF zone, energy is delivered to treat tissue nonthermally (i.e., below the threshold for causing thermal ablation). As a result, if extracellular matrix is ​​present, it is preserved, and the target tissue maintains its structural architecture, including blood and lymphatic vessels. Therefore, sensitive structures, such as biological lumens, blood vessels, and nerves, important for maintaining tissue integrity and functionality, can be preserved. This offers several advantages. First, it enables the treatment of tissues often considered untreatable by conventional methods. Target tissues close to sensitive structures are typically untreatable by surgical methods because the tissue cannot be completely and effectively surgically separated from the sensitive structures. Similarly, many conventional nonsurgical therapies are contraindicated due to the potential for damage to sensitive structures by the therapy or because the therapy is deemed ineffective due to the proximity of sensitive structures. Furthermore, the ability to treat tissues adjacent to sensitive structures also provides a more comprehensive treatment in that no malignant margins remain near the sensitive structures. Once the tissue is treated, the survival of the structural architecture also allows for the natural influx of biological elements, such as components of the immune system, or the introduction of various agents for further therapeutic treatment.

[0202] Fine-tuning and adjusting the ratio of tissue zones (cavitation zone 200, thermal zone 202, PEF zone 204) within a particular treatment site promotes a cascade of increased recruitment of immunological elements specific to the energy delivered and the resulting lesion zone ratio, such as regulatory T cells (Tregs), CD4 (T helper), CD8 (T cytotoxic), CD3 (T cells), CD20 (B cells), FoxP3 (Tregs), Pan-CK (tumor), PD-L1, M1F & M2F, DCs, NKs, MDSCs, NFs, and other adaptive immune cell types, as described in more detail herein below.

[0203] Lesion classifications such as cavitation zone 200, thermal zone 202, and PEF zone 204 can be measured macroscopically, but can also be monitored in real time based on tissue properties (i.e., tissue density, impedance changes, etc.) and imaging modalities such as computed tomography. Additionally, in some embodiments, these ratios are centered around measurements of other clinically relevant and quantifiable tools such as local temperature, tissue impedance, and tissue density, to name a few.

[0204] In addition to manipulating parameter values ​​to produce the desired lesion zone, parameter values ​​were further manipulated to maximize lesion size while ensuring patient safety, desirable procedure time, reduced risk of electrical arcing, reduced muscle contraction, and reduced temperature rise, to name a few. Extensive research was conducted to determine the desired dose parameters. Research included evaluating combinations of the following parameters:

[0205] [Table 1]

[0206] Treatments were delivered to the liver parenchyma because this tissue provides excellent tissue for accurately measuring lesion size. One study utilized two pigs. Both animals were euthanized on postoperative day 3. Needle electrodes were applied to deliver treatments to the liver parenchyma of each 3-day surviving animal. A laparotomy was used to improve visibility and ensure the desired positioning of the needle electrodes within the intended target. Following laparotomy, 24 treatments were delivered to the liver parenchyma of each 3-day surviving animal. All treatment sites were excised and sectioned for macroscopic ablation analysis.

[0207] As shown in Table 1, all treatments were delivered in 100 packets at a fundamental frequency of 400 kHz. Treatments were delivered using R-trigger cardiac synchronization, with a 3-second rest period between subsequent packets (rhythmically approximately 20 packets per minute (pkts / min)). Intercycle delays of 500 μs or 1000 μs were used to confirm no change in lesion size from these values. In this study, no statistical difference was observed in overall treatment zone size or treatment characteristics between treatments using 500 μs and 1000 μs cycle delays, as shown in Figure 9. Figure 9 shows in vivo data captured from this preclinical study in porcine liver tissue. The column plots demonstrate the lack of effect of intercycle delay on overall treatment size across five varying energy protocols (varying voltage amplitude and on-time per packet).

[0208] Treatment volume was calculated based on the short axis measurement and can be found in Equation 1, where r is the short axis radial dimension. V=4 / 3*π*(r)^2*(r+applicator exposed length) [Equation 1]

[0209] Figures 10A-10B show volumetric treatment zone measurements after fixation for both animals. Because these calculated volumes were based on short-axis measurements, the same trends as the short-access measurement analysis emerged, with an increase in treatment volume with increasing voltage and packet on-time, except for the 3300 V / 60-cycle protocol. In this case, this may be due to partial discharge at the treatment site. The column plots in Figures 10A-10B show the effect of voltage amplitude and on-time per packet on the ratio of total affected tissue to tissue undergoing thermal effects. Figure 10A shows the short-axis treatment volume calculations for varying voltage amplitudes and cycle counts, while Figure 10B shows the percentage of thermal necrosis and PEF within the treatment volume. Overall, interpretation of the maximum total PEF zone volume (and therefore maximum cellular debris and antigen bioavailability to the immune system) coupled with an acceptable percentage of thermally apparent treatment effects determined that parameter values ​​of 3000 V and 40 cycles (with a 100 μs packet active time) provided the desired outcomes, as later confirmed by human studies.

[0210] Liver samples were histologically reviewed in a blinded fashion without knowledge of the treatment delivered to any given liver site. Based on the histology on the slides, an overall assessment of the size of the treated liver parenchyma was provided (e.g., 0.7 x 0.7 cm to 1.5 x 1.2 cm). A qualitative assessment of the lesions was also provided. Overall, the lesions were determined to be very similar with reproducible patterns of injury. Histological analysis of the tissue lesions revealed additional zones within each lesion. These zones are inflammatory zones, an effect of PEF energy delivery. Histological analysis performed on the tissue samples demonstrates a peripheral inflammatory response 3 days postoperatively. Figures 11A-11B show in vivo samples captured from a preclinical study in porcine liver tissue. Figure 11A is a photograph of the lesion in tissue sample T. Figure 11B is an overlaid "bullseye" image showing the zones seen in the lesion in Figure 11A. From inside to outside, the zones include a thermal zone 202, a PEF zone 204, and an inflammatory zone 206. Tissue samples show the ratio of total affected tissue to tissue subjected to the thermal effect, as well as the effect of frequency on the peripheral inflammatory band or zone 206. In some embodiments, lower frequencies produce a larger peripheral inflammatory response or zone 206 and a larger area of ​​the PEF energy effect or zone 204. One variable that appeared to differ somewhat between treatments was the width of the peripheral inflammatory zone 206 / partially devitalized hepatocytes compared to the size of the central, completely ablated liver parenchyma. There is a reproducible pattern of damage in terms of the size of the ablated liver parenchyma, as well as the "bull's-eye" nature of the central tissue ablation, the partially viable / inflammatory zone, and the rapid loss of completely normal / uninvolved adjacent parenchyma.

[0211] Thus, the refined dose of specialized energy described herein produces treatment lesions (e.g., 10 mm in diameter) with minimal thermal volume, particularly compared to lesions produced by conventional pulsed electric field technologies. In some embodiments, treatment duration is 5 minutes. The refined dose provided by the specialized waveform maximizes the PEF treatment effect zone while minimizing thermal effects. At this specially tailored dose, the PEF energy triggers tumor antigen release and stimulates the body's innate and adaptive immune responses through enhanced neoantigen release, stimulated immunogenic cell death (e.g., necroptosis, pyroptosis, etc.), and enhanced susceptibility to antigens due to limited encapsulation and scarring of the treatment area. Furthermore, in some embodiments, the specialized energy disrupts the immunosuppressive tumor immune microenvironment (TME). Thus, embodiments of the treatment system maximize antigen release and increase immune responses. VIII. Immune Response

[0212] It has been determined that refined doses of specialized energy produce treated lesions that induce a superior immune response compared to conventional PEF treatment. When energy is used to treat cancerous tumors, this immune response utilizes the body to further eliminate cancerous cells, such as residual cancer cells, metastatic cancer cells, or cancer cells that subsequently develop. This has been studied in multiple mouse models of cancer, where specialized PEF treatment induces tumor-specific immune responses. For example, data were generated from a syngeneic orthotopic model of triple-negative breast cancer (EMT6 tumors) and a metastatic model of melanoma (B16-F10 / B16-OVA tumors). To understand the impact of this specialized PEF treatment on tumor suppression, survival studies were performed in EMT6 tumor-bearing mice. Next, the antitumor mechanisms mediated by PEF were identified to better explain the tumor suppression observed in the EMT6 model. Furthermore, studies were conducted to determine whether tumor antigen release induced by PEF is sufficient to drive tumor-specific T cell responses.

[0213] Furthermore, to evaluate the safety and initial feasibility of the refined dose of the specialized PEF energy described herein for treating solid tumors, we conducted a prospective, two-arm, randomized, concurrently controlled, multicenter, open-label treatment and ablation study. We analyzed pre- to post-treatment changes in immune cell populations in the blood of patients whose tumors were treated with PEF. [Example]

[0214] the study Example 1: Local treatment with specific pulsed electric fields produces tumor-specific responses

[0215] Objective: PEF is an emerging technology being evaluated for the cytoreductive treatment of tumors. PEF energy does not thermally denature proteins, allowing the release of intact tumor-associated antigens to antigen-presenting cells to drive tumor-specific responses. This may promote improved local responses and abscopal effects in treated tumors. Refined doses of specialized PEF energy can optimize the PEF zone and induce additional zones that promote these responses, including enhanced effects associated with antigen-specific adaptive immune responses. The purpose of this study was to evaluate the presence of antigen-specific cytotoxic T cells in preclinical tumors treated with specialized PEF therapy.

[0216] Methods: C57BL / 6J mice were inoculated into the right flank with either the B16-F10 tumor cell line (B16-OVA) genetically modified to express chicken ovalbumin protein or a negative control tumor (B16-F10) (subcutaneous injection, 1 x 10 cells per 50 µL of Matrigel: PBS- / -). Nine days after inoculation, mice were randomized into treatment groups containing PEF and a no-treatment control (NTC). PEF energy was delivered using a single needle with a grounded electrical pad configuration. For PEF-treated mice, a modified 25 g electrode was used to deliver the specialized PEF treatment at a reduced clinical dose. Ten days after treatment, tumors were harvested and processed for flow cytometry (Table 2). [Table 2]

[0217] Results: Flow cytometry demonstrated that the frequency of CD8+ T cells was significantly increased in tumors directly treated with PEF compared with NTC (Figure 12A). To quantify the proportion of tumor-specific T cells within the immune infiltrate, we used SIINFEKL, a tetrameric complex containing H2-Kb MHC class I molecules bound to OVA peptide. B16-OVA tumors directly treated with PEF showed a 125% (p=0.03) increase in OVA-specific CD8+ T cells compared with B16-OVA tumors in the NTC group (Figure 12B). The percentage of all CD45+ cells positive for the CD8+ tetramer was increased compared with PEF-treated and NTC B16-F10 negative controls (p=0.05).

[0218] Conclusions: Local treatment with specialized PEF generates tumor antigen release in an immunocompetent manner that allows for clonal expansion of tumor-specific T cells. Furthermore, specialized PEF treatment increases CD8+ T cell infiltration, enhancing the immunogenicity of cold tumors. Taken together, local treatment with specialized PEF provides tumor-specific immune responses that result in a robust abscopal effect, providing systemic outcomes from focal therapy. Example 2: Lesion Treatment of Early Stage NSCLC with Specialized Pulsed Electric Fields: Safety and 30-Day Results from the INCITE ES Study

[0219] Objective: To evaluate the safety and initial feasibility of specialized pulsed electric field (PEF) treatment of non-small cell lung cancer (NSCLC) tumors before surgical resection using a PEF system to deliver energy to soft tissue.

[0220] Overview: Early-stage NSCLC is generally not very sensitive to chemotherapy and is treated by surgical resection with curative intent when possible. An additional treatment is specialized non-thermal focal ablation modalities that use high-frequency electrical pulses to destabilize cell membranes and promote cell death. Compared to traditional ablation modalities used in soft tissues (e.g., radiofrequency, microwave, cryotherapy), PEF ablation has several potential advantages, including an improved safety profile and the ability to treat lesions near critical structures due to the preservation of surrounding architecture, including blood vessels, lymphatic vessels, and extracellular matrix. Furthermore, cell death induced by specialized PEF delivery results in enhanced efficacy through stimulation of the body's innate immune response. In contrast to thermal ablation mechanisms, non-thermal cell death induced by specialized PEF releases a larger pool of antigens from the tumor that are accessible to cells of the immune system. Furthermore, limited encapsulation and scarring of the treatment area allows immune cells better access to these antigens and the tumor itself.

[0221] Methods: In this treatment and resection study, specialized PEF energy is delivered to isolated, operable NSCLC lesions via either an endoluminal (bronchoscopy) or percutaneous approach prior to surgical resection. The patient population includes adult patients with suspected or confirmed 8th Edition NSCLC stage IA2, IA3, or IB (isolated lesions >1 cm to ≤4 cm), lesions noted for higher recurrence rates. Subjects are surgical candidates and have not undergone oncologic treatment within the past two years. Computed tomography (CT) scans are performed prior to PEF delivery and prior to surgical resection. Safety is assessed by evaluation of device- and / or procedure-related serious adverse events (SAEs) from initial PEF delivery through surgical resection. Safety is also assessed through gross and histologic evaluation of the resected specimen, including impact on any adjacent bronchi and vasculature. Changes in tumor size, planned surgical approach as a result of the PEF procedure, 30-day surgical mortality, and percentage of surgical complications due to PEF delivery are also evaluated. Clinical efficacy is assessed by evaluation of the treatment zone from resected specimens. Technical success of the PEF procedure is defined and assessed as the frequency with which clinicians can access the tumor and deliver PEF energy. Additionally, immune response assessment is assessed by comparing pre- and post-treatment samples from blood, BAL fluid (if the procedure is performed intraluminally), and tumor tissue.

[0222] Preliminary results: Four subjects (average 100%) had lesions that abutted multiple surfaces and a previously performed lesion that abutted a large crack. + Standard deviation age 65.0 + Specialized PEF delivery was successfully performed on all subjects (7.4 years old, 50% male). Three of the four procedures were completed percutaneously. Each subject received a single PEF treatment, which reduced the longest diameter of the tumor by 0.7 mm, as measured before surgical resection. +The study demonstrated a 3.6% reduction in PEF-related adverse events (AEs) in the tumor tissue samples taken before treatment, including increased PD-L1 expression on tumor cells and increased CD8+ / CD4+ inflammatory cells in the treated area.

[0223] Results: The current enrollment includes 26 subjects (21 in the treatment group and 5 in the control group) with a mean age of 67 years (SD 5.7 years), 80% of whom were male. A single PEF treatment (refined dose) was successfully performed in all 21 subjects, including one lesion abutting the pleural surface and one lesion abutting a large fissure. A single PEF treatment was delivered to at least each of the first four subjects, resulting in a 14 ± 7% reduction in tumor diameter, as measured before surgical resection. Figures 13A–13D provide (1) a 3.4 cm lesion before PEF delivery (Figure 13A), (2) the same lesion before surgery with a longest diameter of 2.8 cm (Figure 13B); (3) a macroscopic section of the resected tumor 20 days after PEF delivery (Figure 13C); and (4) a CT image of the chest with the corresponding hematoxylin and eosin (H&E) (Figure 13D). Immune responses were assessed by comparing pre- and post-treatment blood, BAL samples, and via transcriptome changes in tumor tissue according to the schedule shown in Figure 14. Figure 16 provides a table of H&E and immunohistochemistry (IHC) performed. Additionally, flow cytometry, ssRNAseq (Rhapsody™), and Luminex Multiplex 71-plex panel (Figure 16) were performed. Figure 17 shows details of baseline characteristics and treatment.

[0224] The procedure was successful in all 27 subjects (technical success = 100%). No changes to the planned surgical approach or surgical complications were observed, and no adverse events related to the device or PEF procedure were observed from the initial PEF delivery to surgical resection (19–21 days later). Preliminary results demonstrated changes in the immune response within the tumor at the time of resection compared to pretreatment tumor tissue samples, including increased PD-L1 expression on tumor cells and an increased CD8+ / CD4+ T cell ratio near the treatment zone, with a greater number of CD8+ T cells interacting with tumor cells. Increased cytotoxic CD8+ T cells were found to interact with tumor cells in tumor "islands," and increased CD4+ helper T cells were observed outside the tumor "islands." Lesions were evaluated, including the treated zone (cell-depleted zone) and surrounding untreated tissue in resected specimens. The cell-depleted zone (CDZ) is an area primarily devoid of cancerous cells. The CDZ is visible upon gross dissection and consistently identified by PanCK staining. In this study, the minimum CDZ dimensions ± SD (n = 8) on day 20 ± 3 were D1 (longest dimension) = 0.9 ± 0.3 cm and D2 (perpendicular to D1) = 0.6 ± 0.3 cm. Changes in tumor size were assessed by radiological evaluation.

[0225] It can be seen that, with the exception of one mucinous adenocarcinoma and the control, all tumors treated with specialized PEF energy contained multiple tertiary lymphoid structures (TLS). TLS are organized aggregates of immune cells that form non-lymphoid tissues after birth. TLS are not found under physiological conditions but arise in chronic inflammatory situations, such as autoimmune diseases, chronic infections, and cancer. In the tumor context, TLS promote the influx of immune cells into the tumor site, thus improving anti-cancer immunity and favorable treatment responses to immunotherapy in patients. The presence of TLS in tumors appears to correlate with better prognosis and clinical outcomes. Lymphoid aggregates and TLS are consistently observed near the periphery of PEF-treated tumors. Figure 18 shows various tissue samples with TLS (dark spots).

[0226] Preliminary serum cytokine analysis (days 0-18) showed that pathways affected by PEF included upregulation of lymphatic pathways (e.g., lymphoid organ formation and growth) and downregulation of regulatory T lymphocytes. Figures 19A-19B show the results of the preliminary serum cytokine analysis, i.e., the expression of various cytokines on days 3, 10, and 18.

[0227] This initial case series demonstrated the feasibility of delivering specialized PEF energy with a single electrode in NSCLC tumors. No PEF-related adverse events were observed, and treatment of adjacent critical structures, including pleural surface lesions and large fissures, was feasible without impacting the planned surgical resection. Preliminary analysis of INCITE ES data illustrates the impact of specialized PEF energy on the immune system (innate and adaptive immune responses). Combination with immunotherapy

[0228] Over the past decade, a paradigm shift has occurred in our understanding of the relationship between the immune system and cancer initiation and subsequent disease progression. Immune evasion, which allows malignant cells to escape detection by the immune system, is now recognized as the basis of tumorigenesis. Tumors induce a complex network of immunosuppressive or immunological pressures, making it difficult for immune effectors to mount meaningful antitumor responses. In recent years, a vast amount of research has investigated the immunosuppressive network defined by the tumor microenvironment (TME). One of the most important discoveries in oncology is the understanding of the role of immune checkpoints in cancer. Immune checkpoints are inhibitory or stimulatory molecules present on the surface of T cells and antigen-presenting cells (APCs), and their direct interaction regulates the duration and response of T cells to foreign substances. This is particularly important for maintaining self-tolerance, as it prevents T cells from damaging healthy tissue and generating an autoimmune response.

[0229] In the context of cancer, tumor cells can upregulate immune checkpoints to bind to partner receptors on T cells and evade detection. One such immune checkpoint, programmed cell death protein 1 (PD-1) and its ligand PD-L1, have been identified as key regulators of immune evasion in numerous cancers. PD1 is a transmembrane protein that is transcriptionally upregulated in activated T cells, B cells, and myeloid cells. Therefore, activated T cells that infiltrate tumors due to the immune system's inflammatory response often express PD-1 on their surface. PD-L1 is its ligand, and it is found on several tumor cells, including various head and neck carcinomas, lung cancers, and melanomas, to name a few. When PD-1 binds to PD-L1, it prevents T cells from killing tumor cells and induces T cell suppression upon their interaction. Specifically, PD-1 / PD-L1 interactions have been found to mediate T cell immunosuppression through the following mechanisms: inducing apoptosis in activated T cells, promoting T cell anergy and exhaustion, enhancing the immunosuppression of regulatory T cells (Tregs), limiting T cell proliferation, and suppressing T cell activation and interleukin-2 (IL-2) production.

[0230] Recently, antibodies targeting the PD-1 / PD-L1 interaction (nivolumab, pembrolizumab, atezolizumab, and durvalumab) have been approved by the FDA. For example, nivolumab is a human immunoglobulin G4 (IgG4) monoclonal antibody that binds to the PD-1 receptor and blocks its interaction with PD-L1, resulting in PD-1 pathway-mediated inhibition of immune responses. This results in reduced tumor growth. Atezolizumab is a fully humanized engineered monoclonal antibody of the IgG1 isotype directed against PD-L1. Thus, atezolizumab blocks the interaction between PD-1 and PD-L1. Although promising, these drugs have limited efficacy as monotherapy. For example, in non-small cell lung cancer (NSCLC), e.g., any type of epithelial lung cancer other than small cell lung cancer (SCLC), only 20% of patients achieve an objective clinical response to PD-1 blockade. Furthermore, for those patients who respond, the antitumor response is transient as tumors develop drug resistance. This low response rate is due to a combination of factors, including PD-L1 expression and the fraction of tumor immune cell infiltrates.

[0231] Tumors that are PD-L1 negative and have low tumor immune cell infiltration are known as "cold" tumors. Tumors that are PD-L1 positive and have high tumor immune cell infiltration are known as "hot" tumors. Patients with cold tumors are non-responsive to checkpoint therapies because the PD-1 / PD-L1 interaction they are designed to block is absent. As mentioned above, patients with hot tumors often develop resistance to therapy over time. This is because PD-L1-expressing tumor cells are eliminated by blocking their interaction with PD-1. The remaining tumor cells represent a remaining, more resistant cancer clone population.

[0232] Therefore, improved treatments are desirable, such as increasing the number of patients who respond to immunotherapy and increasing the clinical response of such patients. Such treatments should be safe, effective, and provide improved outcomes. Combining immunotherapy with the special PEF energy described herein meets these goals.

[0233] The devices, systems, and methods described herein utilize specialized pulsed electric field energy to treat patients in a manner that utilizes aspects of the immune system, such as aspects of the immune checkpoint system. In some examples, improved outcomes are achieved in cancer immunotherapy patients, particularly those who have limited response to some immunotherapy treatments or who develop acquired resistance over time. In some embodiments, the PD-1 / PD-L1 interaction is targeted for treatment. In other examples, other pathways of the immune system are involved in treatment in addition to or independently of PD-1 / PD-L1.

[0234] In some embodiments, specialized pulsed electric field (PEF) energy is delivered to a target treatment area, such as a tumor or malignant tissue, to cause tissue destruction. Specialized PEF energy is an energy-directed focal therapy that relies on the application of brief, high-amplitude pulsed energy, ultimately resulting in a cascade of local events, including cell death, inflammatory signaling, and the generation of viable antigen presentation within the tumor microenvironment. Specifically, in some instances, specialized PEF energy promotes the release of tumor antigens, which, when taken up by dendritic cells (DCs) via macropincytosis, can process them into peptide-MHC class I complexes for cross-presentation to T cells. Once the antigen is cross-presented, cytotoxic T cells bearing T cell receptors that recognize this epitope in the peptide-MHC class I complex proliferate and initiate an anti-tumor response (cross-priming), as shown in Figure 20. In particular, Figure 20 shows a tumor T treated with specialized PEF energy, which kills the cells in a manner meaningful to the immune system (e.g., immunogenic cell death). Here, treatment with specialized PEF energy promotes the release of tumor antigens (TA) from the tumor T. Antigen-presenting cells (APCs), such as dendritic cells, internalize antigens and process them into peptide-complexed PCs that are recognized by the T cell receptors of T cells (e.g., CD8+ T cells). T cells have unique T cell receptors that recognize a single peptide epitope. Upon activation, they clonally expand and mount an immune response against their specific target (cross-priming). Importantly, this feature of PEF therapy increases the proportion of T cells in the tumor immune infiltrate, leading to more beneficial outcomes.

[0235] In some embodiments, specialized PEF energy is delivered to a target treatment area, such as one containing a tumor, in a manner that promotes upregulation of PD-L1 by tumor cells, as shown in FIG. 21. In this embodiment, tumor T cells are stressed due to inflammatory insults induced by PEF energy, which transcriptionally activates PD-L1. It can be appreciated that PD-L1 activation may typically be considered harmful in tumor treatment, in that such upregulation of PD-L1 increases the immunosuppressive PD-1 / PD-L1 interaction, thereby inhibiting T cells (e.g., CD8+ T cells). However, such upregulation has the potential to cause cold tumors (i.e., tumors that are PD-L1 negative and have low tumor immune cell infiltration) to become PD-L1 positive, thereby becoming hot. Hot tumors are PD-L1 positive and have high tumor immune cell infiltration. Patients with hot tumors may be responsive to checkpoint therapy targeting the PD-1 / PD-L1 interaction. Thus, patients with cold tumors, either naturally or by developing resistance, can be converted to have hot tumors by appropriate treatment with PEF energy.

[0236] Thus, in some embodiments, an antibody targeting PD-1 (αPD-1) is delivered to a patient at a predetermined time or multiple times in combination with or in conjunction with PEF energy. αPD-1 disrupts the PD-1 / PD-L1 interaction, allowing tumor cells to be identified and killed by T cells. Thus, transformed patients are now responsive to immunotherapy, such as αPD-1 therapy.

[0237] The effects of PEF therapy and checkpoint inhibition on tumor growth rate, survival, and expression of pro- and anti-inflammatory cytokines were tested in Balb / c mice bearing two syngeneic models of breast carcinoma: EMT6 and 4T-1 tumors. Because mouse models of cancer have varying levels of immunogenicity (i.e., the ability of the implanted cancer to elicit an immune response), it was desirable to validate the therapy across multiple tumor models to accurately determine immunological outcomes.

[0238] In the EMT6 tumor model, a combination treatment protocol involving both a unique PEF therapy and checkpoint inhibition (delivery of αPD-1), known as "PEF + αPD-1," was attempted. This was compared with PEF delivery alone and αPD-1 alone. Each cohort had 10–12 subjects. In this tumor model, each female mouse was challenged with EMT6 cells in the fourth / fifth mammary fat pad (on the left side of the animal). Eight days after challenge, tumors (e.g., 4–5 mm in length) were directly treated with PEF energy. In one embodiment, the PEF energy waveform consisted of multiple biphasic pulses grouped into packets, each containing 40 cycles (i.e., biphasic pulses) separated by a 1000-microsecond delay. In this embodiment, the treatment included 100 packets, each separated by a 3-second interpacket delay. In this embodiment, the voltage was 2500 V and the fundamental frequency was 400 kHz. In some cases, mice were administered αPD-1 once a week, starting either on the day of initial challenge with EMT6 cells or on the day of specific PEF energy delivery. In other cases, mice were administered αPD-1 multiple times a week, starting on the day of initial challenge with EMT6 cells or on the day of PEF energy delivery. Three days after PEF treatment, all animals were challenged with EMT6 cells in the contralateral side (the fourth / fifth mammary fat pad on the right side of the animal). Tumor size was measured multiple times a week and tumors were allowed to grow until they reached the predetermined maximum allowable volume.

[0239] Directly treated contralateral tumors in the PEF + αPD-1 cohort experienced significant tumor growth suppression by 24 days post-treatment compared with all groups. As shown in Figure 22, a total of 41% of animals treated with PEF + αPD-1 achieved a complete response (compared to 10% of mice for αPD-1 and 18% for PEF monotherapy). Thus, delivery of αPD-1 alone had the lowest response, similar to traditional antibody immunotherapy. Delivery of specialized PEF energy alone had an improved response due to the effects of PEF energy described herein above. Not only did combined delivery of PEF + αPD-1 significantly (P < 0.001) improve survival outcomes compared with PEF or αPD-1 monotherapy, but PEF energy and αPD-1 worked together to produce a synergistically enhanced antitumor response. This outcome exceeded additive responses.

[0240] Mice that achieved a complete response were then rechallenged with EMT6 tumor cells by implanting new EMT6 tumors. This was to determine whether the initial treatment was sufficient to induce a longer-lasting immune response against EMT6 cells in other areas of the body, a phenomenon known as the abscopal effect. Immune system activation was assessed by measuring lymphocyte (CD8+) counts in treated primary tumors, untreated secondary tumors, and peripheral blood. As a control cohort, naive mice were challenged with EMT6 cells and administered IgG three times a week. Figure 23 shows that all control animals developed tumors. Day 0 is the day of EMT6 challenge.

[0241] Figure 24 shows the results for three groups: PEF + αPD-1, PEF energy alone, and αPD-1 alone. Re-challenged mice administered αPD-1 alone grew tumors. Of the re-challenged mice receiving PEF energy alone, one developed a tumor and one did not. Of the re-challenged mice receiving the combination therapy PEF + αPD-1, one developed a tumor and two did not. This data demonstrates that specific PEF energy is sufficient to promote long-term immunity against specific cancer subsets.

[0242] A combination treatment protocol (PEF + αPD-1) was also utilized in the 4T-1 tumor model, a more aggressive metastatic tumor model. Again, this was compared with specific PEF delivery alone, αPD-1 alone, and a control group, where IgG was administered as a control. In this model, we sought to determine the effects of specific pulsed electric fields (PEF) and checkpoint inhibition (αPD-1) on directly treated and distant disease and to identify the mechanism of the enhanced response obtained by the combination treatment.

[0243] Cytokine analysis was performed on untreated tumors and lung tissues bearing metastatic disease 18 days after treatment. The cytokines analyzed were: A) the T cell activating factor, IL-2; B) the neutrophil-recruiting chemokine, CXCL1; C) the tumor viability marker Ki67; and D) the cytotoxic T cell CD8 in immune cell infiltrates. Example results are shown in Figures 25A-25D. As shown in Figure 25A, delivery of specialized PEF energy and checkpoint blockade (i.e., PEF + αPD-1) significantly elevated the expression of the T cell activating factor IL-2 in untreated tumors, whereas all other biological cohorts (i.e., control, specialized PEF energy only, αPD-1 only) did not. As shown in Figure 25B, delivery of PEF + αPD-1 significantly reduced the neutrophil-recruiting chemokine CXCL1 in both tumors and lungs compared to the IgG control group. As shown in Figure 25C, a marker for tumor viability (Ki67) was quantified in whole lung tissue by ELISA to characterize metastatic burden in treated animals. Importantly, only the PEF energy and immunotherapy cohort (i.e., PEF + αPD-1) reduced total Ki67 expression in the lung compared to the IgG control group. As shown in Figure 25D, cytotoxic T cells (CD8) in immune cell infiltrates were quantified in whole lung tissue by ELISA. Delivery of PEF energy alone and PEF energy in combination with immunotherapy (i.e., PEF + αPD-1) increased CD8 expression in the lung compared to both the IgG control and αPD-1 alone. Collectively, these data suggest that including a specialized PEF (PEF + αPD-1) in a protocol using checkpoint inhibitors induces a robust systemic antitumor response in the 4T-1 "cold" tumor model. Thus, combined treatment alleviated systemic immunosuppression.

[0244] The devices, systems, and methods typically deliver specialized PEF energy to cells using a system including a specialized energy delivery device, a waveform generator, and at least one separate energy delivery algorithm, as described above. Additional accessories and equipment may be utilized. The energy delivery device delivers energy provided by the waveform generator according to at least one separate energy delivery algorithm. It will be understood that in some embodiments, the energy delivery device also delivers one or more agents, such as an antibody (e.g., αPD-1). However, in other embodiments, the agents are delivered by a separate device, such as an IV, catheter, or needle injection. If desired, the agents may be delivered by both the energy delivery device and a separate device. While exemplary embodiments of specialized energy delivery devices are provided herein primarily focusing on monopolar energy delivery, it will be understood that bipolar or multipolar configurations may also be used.

[0245] 26 shows an embodiment of an energy delivery system 100 comprising a specialized energy delivery device or instrument 102, a return electrode 140, and a waveform generator 104. In this embodiment, the target tissue is located within the liver L of a patient P. Target tissue cells may include cells of the digestive system (e.g., mouth, glands, esophagus, stomach, duodenum, jejunum, ileum, intestines, colon, rectum, liver, gallbladder, pancreas, anal canal, etc.), cells of the respiratory system (e.g., nasal cavity, pharynx, larynx, trachea, bronchi, lungs, etc.), cells of the urinary system (e.g., kidney, ureter, bladder, urethra, etc.), cells of the reproductive system (e.g., genitalia, ovaries, fallopian tubes, uterus, cervix, vagina, testes, epididymis, vas deferens, etc.), to name a few. It will be understood that cells may be treated anywhere throughout the body, including cells of the esophagus, seminal vesicles, prostate, glands, penis, scrotum, breasts, etc.), cells of the endocrine system (e.g., pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal glands), cells of the circulatory system (e.g., heart, arteries, veins, etc.), cells of the lymphatic system (e.g., lymph nodes, bone marrow, thymus, spleen, etc.), cells of the nervous system (e.g., brain, spinal cord, nerves, ganglia, etc.), cells of the muscular system, and cells of the skin.

[0246] In this embodiment, instrument 102 includes a flexible, elongate shaft having a distal end that can be advanced intraluminally to target tissue within the liver L. As shown, the distal end of instrument 102 is advanced through the mouth M, down the esophagus E, and into the stomach S, where it passes through the stomach wall and into the liver L. In some embodiments, the distal end has a distal tip 103 configured to penetrate the stomach wall and / or liver L. In other embodiments, a passage through the stomach wall is formed using a separate instrument, which is then removed to allow instrument 102 with an atraumatic tip to pass through the passage.

[0247] In this embodiment, the drug 111 is delivered systemically intravenously using an IV bag 112. This typically distributes the drug throughout the patient P's body, including the target tissue in the liver L. In other embodiments, the drug 111 is delivered locally. In such embodiments, the drug 111 may be delivered to the upstream vasculature of the arterial system leading to the target organ or tissue area. The drug 111 then travels downstream through the arterial circulation to the target region. If a bolus injection of the drug 111 is provided, the drug 111 suddenly enters the target tissue. However, if the drug 111 is delivered over time, such as by use of an infusion pump, a steady, sustained level of the drug 111 may be achieved in the target tissue. In other embodiments, the drug 111 may be delivered by direct injection into the target tissue. In such embodiments, an injection device is inserted into or near the target tissue, such as into the parenchyma of the target organ region, and a solution containing the drug is injected. The drug solution may be allowed a period of time for its distribution through the parenchyma and interstitial space to reach the target area or volume. It will be appreciated that any combination of systemic, local and topical delivery may alternatively be used.

[0248] Specialized pulsed electric field (PEF) energy is delivered to the target tissue through the distal end of the delivery device 102. The proximal end of the delivery device 102 is electrically connected to a waveform generator 104. In some embodiments, the generator 104 is also connected to an external cardiac monitor (not shown) to enable coordinated delivery of energy with cardiac signals sensed from the patient P.

[0249] In this embodiment, the energy delivery device 102 is designed to be monopolar, with the distal end of the instrument 102 having a delivery electrode and a return electrode 140 placed on the skin on the outside of the body, typically on the thigh (as shown), lower back or back.

[0250] A specialized pulsed electric field (PEF) is provided by generator 104 and delivered to tissue via energy delivery bodies 108 positioned on, within, or near the target tissue area. It will be appreciated that in some embodiments, energy delivery body 108 is placed in contact with a conductive material that also contacts the target tissue. Such a solution may include an isotonic or hypertonic solution. Electrical pulses are then delivered through energy delivery body 108 in the vicinity of the target tissue. These electrical pulses are provided by at least one energy delivery algorithm 152. In such embodiments, algorithm 152 specifies signal parameters such as energy amplitude (e.g., voltage) and duration of applied energy, consisting of the number of pulses, pulse width, and delay between pulses, to name a few. In some embodiments, one or more of the energy delivery bodies are small and tend to dissipate a large amount of energy around the electrodes. Therefore, optimal delivery of energy is desired.

[0251] In some embodiments, biphasic pulses may be used. In such embodiments, additional parameters may include a switch time between biphasic pulse polarities and a dead time between biphasic cycles. Feedback loops based on sensor information, automatic shutoff features, and the like may also be included. Biphasic waveforms are useful for reducing muscle stimulation in patients. This is particularly important in applications where slight movement of the energy delivery device can easily result in ineffective therapy. Biphasic waveforms involve rapid changes in signal phase / polarity to minimize neural activation during transitions between polarities.

[0252] The specialized PEF energy may be delivered by a variety of energy delivery devices or instruments 102. Typically, the instrument 102 includes a flexible, elongated shaft having a distal end that can be advanced with the main body to the target tissue, and at least one energy delivery body 108 disposed near the distal end. The energy delivery body 108 includes one or more electrodes that deliver the PEF energy to the target tissue. As previously described, in some embodiments, the energy delivery device 102 delivers the PEF energy, and the pharmaceutical agent 111 is delivered by a separate device, such as an IV, catheter, or needle injection. Such delivery of the pharmaceutical agent 111 can occur at various times, such as before, during, or after the delivery of the PEF energy. If delivered at a time separate from the PEF energy delivery, such delivery can be separated from the PEF energy delivery by a few seconds or less, a few seconds, minutes, hours, days, weeks, or months.

[0253] FIG. 27A shows the direct injection of an agent 111 into a target tissue via a needle or probe 500. The target tissue is shown as cell C (not to scale). The needle or probe 500 is inserted into or near the target tissue so that the injected agent 111 can bathe the target tissue. In this embodiment, the probe 500 is then removed, leaving the agent 111 to dwell for biodistribution. Referring to FIG. 27B, the distal end of the instrument 102 is then inserted into the target tissue at a desired time, thereby desirably positioning the energy delivery body 108 within or near the target tissue. In this embodiment, the energy delivery body 108 consists of a single electrode. Specialized PEF energy is then delivered from the energy delivery body 108 to the target tissue, as indicated by the wavy line 502. In this embodiment, the PEF energy upregulates PD-L1 in cell C, among other effects, and the agent 111 includes αPD-1, which disrupts the PD-1 / PD-L1 interaction. This allows T cells within patient P to respond by identifying and killing target cells C.

[0254] In some embodiments, the agent 111 and energy are delivered close enough in time that the agent 111 is delivered by the energy delivery device or instrument 102. FIGS. 28A-28B show an energy delivery device or instrument 102 having a needle-shaped energy delivery body 108. The needle-shaped tip can penetrate like a needle and deliver the agent 111 through its internal lumen. The energy delivery body 108 is also electrically insulated by an insulating layer 504, except for the needle-shaped tip, which acts as an electrode. FIG. 28A shows direct injection of the agent 111 into target tissue via the energy delivery body 108. Again, the target tissue is shown as cell C (not to scale). The tip is inserted into or near the target tissue so that the injected agent 111 can bathe the target tissue and, if necessary, reside for biodistribution. Referring to FIG. 28B, PEF energy is then delivered from the energy delivery body 108 to the target tissue, as indicated by the wavy line 501. In this embodiment, PEF energy, among other effects, upregulates PD-L1 in cell C, and agent 111 includes αPD-1, which interferes with the PD-1 / PD-L1 interaction, thereby enabling T cells in patient P to identify and kill target cell C in response.

[0255] FIG. 29 shows a drug 111 being delivered locally while energy is being delivered locally (optionally, the drug 111 can also be delivered locally). Here, the drug 111 is delivered by a separate device, such as a catheter 503, placed in the vasculature V supplying the target tissue area. Thus, the drug 111 is delivered locally to the target tissue area. An energy delivery device or instrument 102 is inserted into the target tissue area from a different approach. Here, the instrument 102 includes an energy delivery body 108 having a needle shape. The needle-like tip is penetrable similar to a needle. In some embodiments, the drug 111 can be delivered through its internal lumen. In this embodiment, the energy delivery body 108 is electrically insulated by an insulating layer 504, except for the needle-shaped tip, which acts as an electrode. The tip is inserted into or near the target tissue, and then PEF energy is delivered from the energy delivery body 108 to the target tissue, as indicated by the wavy line 501. In this embodiment, PEF energy, among other effects, upregulates PD-L1 in cell C, and agent 111 includes αPD-1, which interferes with the PD-1 / PD-L1 interaction, thereby enabling T cells in patient P to identify and kill target cell C in response.

[0256] FIG. 30 illustrates an energy delivery device or instrument 102 including a shaft 106 having an energy delivery body 108 near its distal end, the energy delivery body 108 including a plurality of tines 600. Typically, the tines 600 have a pointed shape to penetrate tissue. Similarly, the tines 600 typically extend laterally outward from the shaft 106, e.g., at an oblique angle or perpendicular to the shaft. In some embodiments, the tines 600 are deployed circumferentially around the shaft 106, while in other embodiments, the tines 600 are deployed from the sides of the shaft 106, such as in a row. In some embodiments, the tines 600 extend the same distance from the shaft 106, while in other embodiments, the tines 600 extend varying distances. It will be appreciated that in some embodiments, the extension of at least some of the tines 600 from the shaft 106 is adjustable. It will also be appreciated that in some embodiments, the tines 600 extend from the distal tip of the shaft 106, e.g., through a central lumen.

[0257] Typically, each tine 600 delivers a drug 111 and / or energy therefrom. In some embodiments, the drug 111 is delivered from the tip 601 of the tine 600, while in other embodiments, the drug 111 is delivered from a delivery port 602 along the tine 600. In some embodiments, the tines 600 are energizable together (e.g., acting as a single electrode), or at least some of the tines 600 are individually energizable (e.g., acting as a bipolar pair or as a selectable single electrode, including acting in a group). In some embodiments, one or more tines 600 deliver different energies (e.g., generated from different energy delivery algorithms 152) and / or different types of drug 111.

[0258] In this embodiment, the shaft 106 has three sections: a first section 106a, a second section 106b, and a third section 106c. As shown in FIG. 30, the first section 106a is distal to the second section 106b, which is distal to the third section 106c. Each section 106a, 106b, 106c may be insulated or non-insulated to create a variety of different electrode combinations. This allows for a variety of electric field shapes and / or directing the electric field in a desired direction. It will also be appreciated that in some embodiments, at least a portion of at least one tine 600 is insulated to direct the energy emanating therefrom. Overall, the tines 600 often allow for delivery of agent 111 and / or energy to a larger volume of target tissue with a single positioning of the instrument 102 than with an instrument 102 having an energy delivery device 108 that includes a single needle.

[0259] In some embodiments, the first section 106a acts as the energy delivery body 108, and one or more tines 600 act as energy delivery bodies 108. Each of the different energy delivery bodies 108 may deliver the same or different types of energy. Similarly, the energy delivery bodies 108 may act in groups. In some embodiments, the tines 600 extend beyond the first section 106a. In some embodiments, the tines 600 extend the same distance from the shaft 106 (relative to the first section 106a), while in other embodiments, the tines 600 extend various distances (relative to the first section 106a). In some embodiments, the first section 106a acts as the energy delivery body 108, and one or more tines 600 act as conduits for delivering the agent 111.

[0260] It may be appreciated that in some embodiments, the PEF energy is delivered to a conductive fluid (e.g., blood, saline, etc.) in contact with the target tissue, and thus the energy can pass through the conductive fluid to the target tissue for the effects described herein above.

[0261] In summary, the specialized PEF treatment described herein elicits adaptive immune responses that benefit treatment outcomes. To verify that including checkpoint blockade (CPB) in a PEF treatment protocol improves outcomes over PEF alone, two syngeneic orthotopic models of triple-negative breast cancer (EMT6 and 4T-1 mouse tumor models) were used in this study. To verify that including checkpoint blockade in a PEF treatment protocol with PEF treatment can improve outcomes over PEF alone, a syngeneic orthotopic model of triple-negative breast cancer (EMT6 tumor model) was used in this study. Next, to verify that the combined treatment cohort provides long-term tumor-specific immunity, animals that achieved a CR with EMT6 tumor cell injection were re-challenged 60 days after treatment. To verify how the combined treatments synergize to generate anti-tumor immune responses, pathway analysis was performed on systemic changes in cytokine expression in the blood before and after treatment. Next, tumor suppression mediated by PEF treatment in combination with checkpoint blockade in a syngeneic model of metastatic breast cancer was evaluated. To determine that the combination therapy improved the antitumor immune response in lung metastases, cytokine analysis was performed to quantify nuclear growth factor (Ki67) in 4T-1 lung tissue 18 days after treatment.

[0262] In some embodiments described herein, it can be seen that PEF energy delivery and CPB synergize to achieve immunogenic cell death, release of tumor antigens and DAMPs, driving pro-inflammatory TME by the innate arm, Skew Th1 / Th17, DC activation, NK activation, TLR signaling, cytotoxic T cell activation, and wound healing responses, to name a few. Example 3

[0263] The inclusion of checkpoint inhibitor therapy enhances the immunogenicity of specialized pulsed electric fields

[0264] Background: Checkpoint blockade monotherapy has produced impressive clinical results across several cancer subsets; however, responses remain limited in patients whose tumors lack robust T-cell infiltrates. Furthermore, in patients who achieve clinically meaningful responses, these antitumor effects are generally transient as tumors transition to a more immunosuppressive phenotype. Therefore, there is growing interest in improving disease outcomes by combining checkpoint blockade with localized therapy. Localized therapy has the potential to rapidly promote immunogenic cell death and subsequent tumor antigen release to drive systemic antitumor responses. PEF treatment is a non-thermal, needle-guided, locoregional therapy that uses high-frequency biphasic electrical pulses to destabilize cell membranes and promote cell death. Importantly, PEF preserves the extracellular matrix (ECM), allowing for the regeneration of blood and lymphatic vessels. Here, we set out to determine whether combining PEF with programmed death receptor-1 (anti-PD-1) blockade enhances systemic antitumor responses in a mouse model of triple-negative breast cancer.

[0265] Objective: This preclinical study evaluated immune responses to a specialized pulsed electric field (PEF) tumor treatment that included checkpoint blockade with αPD-1. If an adequate PEF treatment dose does not denature proteins, tumor-specific and tumor-associated antigens remain intact and available for antigen-presenting cells to interact with them in treatment resolution and repair processes, including the abscopal effect. This study identified immunogenic mechanisms underlying the abscopal effect and enhanced responses obtained by the combined treatment.

[0266] Methods: Immunocompetent female Balb / c mice were orthotopically inoculated with 1,200,000 4T1 breast cancer cells (representing a "cold" tumor microenvironment) in the fifth mammary fat pad (primary tumor). Mice were divided into four groups: IgG control, anti-PD1 alone, PEF alone, and anti-PD1 + PEF. Anti-PD1 administration was 200 μg administered three times weekly from the day of tumor inoculation. Once tumors reached a diameter of 5 mm (days 8–10), tumors in the PEF group were treated with a single application of specialized PEF delivered through a single needle placed at the center of the tumor. The PEF dose was established to target approximately 80% of the tumor volume. The specialized PEF dose was 2500 V, 400 kHz, 40 cycles, and 100 packets. Three days after the PEF treatment delivery date, a second 4T-1 tumor was challenged contralaterally in the fifth mammary fat pad (secondary tumor). Eighteen days after treatment, contralateral tumors and lungs were harvested for analysis. Enzyme-linked immunosorbent assay (ELISA) was used to identify and characterize the biological immune response to the different treatment cohorts.

[0267] Results: Cytokine analysis performed 18 days after treatment in untreated tumors and lung tissue bearing metastatic disease confirmed that combination treatment alleviated systemic immunosuppression. The cytokine panel included GM-CSF, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-17, CXCL1, CXCL2, CXCL5, CCL2, and TGF-α. PEF and checkpoint blockade significantly elevated the expression of the T cell activating factor IL-2 in secondary untreated tumors, whereas all other test cohorts did not provide a heatmap showing cytokine expression from contralateral whole tumor lysates 18 days after treatment (Figure 31). Biological groups included: control (IgG, n=4), anti-PD-1 checkpoint monotherapy (αPD-1, n=4), direct treatment with pulsed radiation field (PEF, n=3), and combination therapy (PEF+αPD-1, n=4). Heat maps represent the mean cytokine concentrations (pg / mL) for each biological group. Multiplex cytokine quantification for IL-α (FIG. 32A), IL-β (FIG. 32B), IL-2 (FIG. 32C), and IL-13 (FIG. 32D) was used to construct the heat maps in FIG. 31. *p<0.05, **p<0.01 (ANOVA with Fisher's test). All data are averaged. + Plotted with standard deviation. Figures 32A-32D indicate the presence of cytokines in secondary (untreated) tumors.

[0268] Furthermore, treatment with PEF and immunotherapy significantly reduced the neutrophil-recruiting chemokine CXCL1 in both the secondary untreated tumor and lung compared with the IgG control group. Thus, immunotherapy combined with PEF induced an antitumor immune response in lung metastases. Figure 33A provides a heatmap showing cytokine expression from contralateral whole tumor lysates 18 days after treatment. Figure 33B shows an analysis of how cytokine expression from lung tissue affects cancer-related pathways, as determined by Ingenuity Pathway Analysis (IPA) software.

[0269] To characterize metastatic burden in treated animals, markers of tumor viability (Ki67) and cytotoxic T cells (CD8) in immune cell infiltrates were quantified in whole lung tissue by ELISA. PEF monotherapy and PEF in combination with immunotherapy increased CD8 expression in the lung compared to both the IgG control monotherapy cohort and the checkpoint monotherapy cohort. Figures 34A-34B provide ELISA quantification of Ki-67 from whole lung tissue lysates 18 days after treatment. Biological groups included: control (IgG, n=7), anti-PD-1 checkpoint monotherapy (αPD-1, n=8), direct treatment with pulsed radiation field (PEF n=3), and combination therapy (PEF + αPD-1, n=7). *p<0.05, **p<0.01, ***p<0.001 (ANOVA with Fisher's test). All data are averaged. + Plotted with standard deviation. Importantly, only the PEF and immunotherapy cohorts reduced total Ki67 expression in the lungs compared to the IgG control group. Figure 34C provides ELISA quantification of CD8 from whole lung tissue lysates after 18 days of treatment. Biological groups included: control (IgG, n=7), anti-PD-1 checkpoint monotherapy (αPD-1, n=8), direct treatment with pulsed radiation field (PEF n=3), and combination therapy (PEF + αPD-1, n=7). *p<0.05, **p<0.01, ***p<0.001 (ANOVA with Fisher's test). All data are averaged. + Plot with standard deviation.

[0270] Summary: Cytokine analysis performed in untreated tumors and lung tissue with metastatic disease 18 days after treatment confirmed that the combination treatment alleviated systemic immunosuppression. PEF and checkpoint blockade significantly elevated the expression of the T cell-activating factor IL-2 in secondary untreated tumors, but not in any of the other study cohorts. Furthermore, treatment with PEF and immunotherapy significantly reduced the neutrophil-recruiting chemokine CSCL-1 in both secondary untreated tumors and lungs compared with the IgG control group. To characterize PEF-mediated changes to metastatic burden in the lung, we first used Ingenuity Pathway Analysis (IPA) software to identify biological functions and gene networks affected by treatment. Combination therapy resulted in a significant reduction in cancer-related pathways, including tumor growth and metastasis. Next, we quantified tumor viability (K167) and markers of cytotoxic T cells (CD8) in immune cell infiltrates in whole lung tissue by ELISA. PEF monotherapy increased CD8 expression in the lung compared with both the IgG control and checkpoint monotherapy cohorts. Importantly, only the PEF and immunotherapy cohort decreased total K167 expression in the lung compared with the IgG control group.

[0271] Conclusions: Combining PEF with αPD-1 checkpoint blockade induces robust local and systemic antitumor immune responses in the immunosuppressive 4T-1 tumor model. Example 4

[0272] Primary and secondary tumor responses to combined delivery of specialized PEF and αPD-1 within 4T-1 and EMT-6 orthotopic tumor models

[0273] Background: Although treatment options for cancer patients have improved for many forms of cancer, the development of acquired drug resistance, high incidence of tumor recurrence, and systemic spread remain persistent negative outcomes. However, recent efforts have demonstrated that the combination of pulsed electric fields (PEF) with coordinated immunotherapy (i.e., checkpoint inhibitors such as αPD-1) can improve overall outcomes. PEF is an energy-directed focal therapy that relies on the application of brief, high-amplitude pulsed energy to induce a cascade of local events, including cell death, inflammatory signaling, and the generation of viable antigen presentation within the tumor microenvironment, resulting in a measurable systemic response.

[0274] Objective: The aim of this preclinical study was to evaluate the overall synergistic effect of combining specialized PEF with checkpoint blockade (CPB).

[0275] Methods: Immunocompetent female Balbc mice were anesthetized and orthotopically inoculated with 200,000 cells (EMT-6 or 4T-1, representing immune "hot" and "cold" tumor microenvironments, respectively) into the fifth mammary fat pad (primary tumor). Mice were divided into four groups: IgG control, αPD1 monotherapy, PEF monotherapy, and αPD-1 + PEF. αPD-1 administration was 200 μg administered three times weekly from the day of tumor inoculation. Once tumors reached a diameter of 5 mm (8–10 days), tumors in the PEF group were treated with a single application of PEF delivered through a single needle placed at the center of the tumor. The PEF dose was established to target approximately 80% of the tumor volume. Three days after the PEF treatment delivery date, a second inoculation was administered into the contralateral fat pad (secondary tumor). Mice were monitored for health and tumor size three times weekly. Mice were euthanized at the experimental time point or at the end of the study, or when considered clinically moribund.

[0276] Results: Directly treated contralateral tumors in the PEF + αPD-1 cohort experienced significant suppression of tumor growth by 24 days post-treatment compared to all other groups in the EMT-6 mouse tumor model (Figure 35). Furthermore, 58% of the PEF + αPD-1 cohort did not grow contralateral tumors (Figure 35). Overall, the combination treatment protocol significantly improved survival outcomes compared to PEF or αPD-1 monotherapy (P < 0.001). More specifically, 41% of animals treated with PEF + αPD-1 achieved a complete response, compared to 10% and 18% of animals treated with αPD-1 and PEF monotherapy, respectively (Figure 36). Figure 35 shows tumor growth curves for EMT6 tumors treated with PEF, anti-PD1, and both therapies compared to untreated tumors (IgG). The PEF-treated group showed significant growth delay compared to IgG and αPD-1. The combination of PEF and anti-PD-1 further inhibited tumor growth. Figure 35 shows tumor growth of contralateral untreated tumors. Contralateral tumors in mice receiving systemic anti-PD1 and local PEF on the primary tumor failed to develop satellite tumors in 7 / 12 mice. Figure 36 shows that mice treated with PEF and systemic anti-PD1 therapy on the primary tumor had a 41% survival rate compared to PEF alone (18%), anti-PD1 alone (10%), and no treatment (0%). Thus, Figures 34-36 show primary tumor growth curves (Figure 35) and secondary tumor growth curves (Figure 36) at 14 days post-PEF for the EMT-6 mouse tumor model, along with survival plots over 24 days (Figure 37).

[0277] Similarly, the 4T-1 tumor model showed a significant reduction (P value < 0.05) in primary and contralateral tumor growth for both the PEF monotherapy and PEF + αPD-1 groups compared with IgG and αPD-1 monotherapy at 19 days post-PEF (Figure 38A). Figures 38A-38B show the primary tumor growth curve (Figure 38A) and secondary tumor growth curve (Figure 38B) for the 4T-1 mouse tumor model at 19 days post-PEF. Contralateral tumor growth was not achieved in 29% of mice receiving the combination therapy and 13% of mice receiving PEF monotherapy.

[0278] Conclusions: The data suggest that checkpoint blockade (αPD-1) and focal PEF therapy act synergistically to produce enhanced antitumor responses in both immunogenic (EMT-6) and immunosuppressive (4T-1) mouse tumor models. Example 5

[0279] A similar study comparing two different dosing schedules was conducted. To optimize the improvement of combining CPB with PEF therapy compared with CPB alone, two separate CPB dosing schemes were evaluated by their impact on survival. These dosing schemes included a high-frequency anti-PD-1 dosing schedule initiated at the time of primary tumor inoculation (Figure 39A) and a low-frequency, late-stage anti-PD-1 dosing schedule in which CPB was initiated at the time of PEF treatment (Figure 39B). Each of the combined treatment dosing schemes significantly (p<0.001) improved survival outcomes compared with PEF or anti-PD-1 monotherapy. Complete responses (CRs) were achieved in 36% and 85% of combined PEF + anti-PD-1 mice in the high-frequency and low-frequency, late-stage anti-PD-1 dose groups, respectively. This compares to 10% and 18% of animals treated with anti-PD-1 and PEF monotherapy, respectively (Figure 40). Figure 40 shows Kaplan-Meier curves showing survival 60 days after treatment. Biological groups included control (IgG, n = 11), anti-PD-1 checkpoint monotherapy (αPD-1, n = 11), direct treatment with pulsed radiation field (PEF, n = 11), high-frequency CPB + PEF (PEF + αPD-1) (HF), n = 22), and low-frequency late CPB + PEF (PEF + αPD-1) (LFLS), n = 7.

[0280] Next, to identify a combination therapy cohort that would provide long-term tumor-specific immunity, we rechallenged animals that achieved a CR by EMT6 tumor cell injection 60 days after treatment in the second mammary fat pad, the site of the native tumor (Figures 41A-41B). Figure 41A shows a naive control mouse that had not previously been challenged with EMT6 tumor cell injection. Figure 41B shows an animal from the PEF and CPB combination group that had previously achieved a complete response. For naive control mice, tumors were inoculated into the fifth mammary pad (left and right sides) and the second mammary fat pad (left side only). For PEF + anti-PD-1 mice, tumors were challenged only in the second mammary fat pad. All tumors grew readily in the control cohort. In summary, 94% (17 / 18) of animals treated with the combination therapy were tumor-free by 20 days post-inoculation.

[0281] With reference to Figure 42, Balb / c mice were orthotopically challenged with 200,000 cells (4T-1) in the fifth mammary fat pad (bilateral, bilateral tumor model). Mice were divided into four groups: control (IgG, n = 8), anti-PD-1 checkpoint monotherapy (αPD-1, n = 8), direct treatment with a pulsed radiation field (PEF, n = 8), and combination therapy (PEF + αPD-1, n = 7). Once tumors in the PEF group reached 5 mm in diameter (10 days), they were treated with a single application of PEF delivered through a single needle placed in the center of the tumor.

[0282] Similarly, the 4T1 tumor model showed a significant reduction (p<0.05) in primary and contralateral tumor growth for both the PEF monotherapy and PEF + anti-PD-1 groups compared with IgG and PD-1 monotherapy at 19 days post-PEF. Contralateral tumors were significantly smaller in the PEF + anti-PD-1 group (Figure 43). Contralateral tumor growth was not achieved in 43% of mice receiving combination therapy and 12% of mice receiving monotherapy (PEF alone).

[0283] Figure 43 shows growth plots of primary or directly treated 4T-1 tumors. Biological groups included: control (IgG, n=8), anti-PD-1 checkpoint monotherapy (αPD-1, n=8), direct treatment with pulsed radiation field (PEF, n=8), and combination therapy (PEF+αPD-1) (LFLS), n=7. *p<0.05 (repeated measures ANOVA with Tukey's test). All data are means + The SEM was plotted.

[0284] Figure 44 shows a comparison of contralateral or untreated 4T-1 tumor volumes between biological groups at 18 days after PEF or 13 days after inoculation. All data are averaged. + Plot with standard deviation.

[0285] Conclusions: The data suggest that checkpoint inhibition (αPD-1) and focal PEF therapy act synergistically to produce enhanced and durable antitumor responses in both immunogenic (EMT-6) and immunosuppressive (4T-1) mouse tumor models. Change in PEF dose

[0286] As previously mentioned, the specific treatment dose is provided by a waveform generated from a combination of parameter values ​​including: voltage = 3000 V, fundamental frequency = 400 kHz, number of biphasic pulses (i.e., cycles) per packet = 40 cycles, inter-cycle delay = 1000 microseconds, number of packets = 100 packets, and inter-packet delay = 3 seconds. It will be understood that deviations from this specific dose will typically produce different results.

[0287] Referring back to Figure 18, various tissue samples are shown to contain tertiary lymphoid structures (TLS), the presence of which correlates with better prognosis and clinical outcomes. When treated with the specific treatment dose, lymphoid aggregates and TLS are consistently observed near the periphery of PEF-treated tumors. However, when the dose is changed (tissue samples T1, T2, and T3 in Figure 18), the number of TLS is dramatically reduced or absent. The tumor in tissue sample T1 received the specific dose, but energy delivery was asynchronous with the heartbeat, resulting in a brief interruption of energy delivery. The tumor in tissue sample T2 received energy in two doses with 50 packets each, rather than a single dose with 100 packets. The tumor in tissue sample T3 also received energy in two doses with 50 packets each, rather than a single dose with 100 packets, to generate a CT scan to evaluate needle placement and packet delivery. In these cases, energy delivery was interrupted prematurely, but with a pause long enough to prevent further accumulation of treatment effects and thus had a functionally different dose than subsequent samples. The average number of TLS in the tissues in Figure 18 (T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, and T12) is 54.8, while the average number of TLS in tissues receiving altered doses (T1, T2, and T3) is only 4. The number of TLS is comprised of mature TLS (with germinal centers containing mature CD20-B cells exhibiting the characteristic morphology of proliferating centroblasts, often with pigmented macrophages and follicular dendritic cells within the germinal centers) and immature TLS (without germinal centers). The average number of mature TLS in the tissues in Figure 18 (T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12) is 5.6, while the average number of TLS in the tissues that received the modified dose (T1, T2, T3) is 0. The average number of immature TLS in the tissues in Figure 18 (T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12) is 49.2, while the average number of TLS in the tissues that received the modified dose (T1, T2, T3) is 4. It can be seen that the average for the tissues in Figure 18 includes T1, T2, and T3, and therefore the average would be even higher if these three tumors were excluded.Therefore, the difference in the effect of the modified dose is greater than calculated, indicating that changes in specific doses may result in a weaker immunological response, reduced efficacy, and less favorable clinical outcomes.

[0288] Nevertheless, it will be appreciated that different parameters may be used, typically with different outcomes. The different outcomes may be different in some embodiments and similar in other embodiments. Similarly, different outcomes may be preferred in some circumstances and not preferred in others. Energy waveform parameters are further described below. A. Voltage

[0289] The voltages used and considered may be the top of a square waveform, the peak of a sine wave or sawtooth waveform, or the RMS voltage of a sine wave or sawtooth waveform. In some embodiments, energy is delivered in a unipolar manner and each high voltage pulse or set voltage 416 is between about 500V and 10,000V, particularly between about 3000V and 3300V, 3000V and 3500V, 3300 and 3500V, 3500V and 4000V, between about 3500V and 5000V, between about 3500V and 6000V, including all values ​​and subranges therebetween, including between about 3000V, 3300V, 3500V, 4000V, 4500V, 5000V, 5500V, and 6000V, to name a few. The voltage delivered to the tissue can be based on the set point of the generator 104, with or without taking into account electrical losses along the length of the instrument 102 due to the intrinsic impedance of the instrument 102, i.e., the voltage delivered can be measured at the tip of the generator or instrument.

[0290] It will be appreciated that the set voltage 416 may vary depending on whether the energy is delivered in a monopolar or bipolar fashion. In bipolar delivery, a lower voltage may be used due to a smaller, more directional electric field. The bipolar voltage selected for therapy depends on the electrode separation distance, but a monopolar electrode configuration using one or more remote dispersive pad electrodes may be delivered without significant consideration for the precise placement of the catheter electrode and dispersive electrode on the body. In monopolar electrode embodiments, with effective separation distances on the order of 10 cm to 100 cm, higher voltages are typically used due to the dispersive behavior of the delivered energy through the body to reach the dispersive electrode. Conversely, in bipolar electrode configurations, the relatively close active area of ​​the electrodes, on the order of 0.5 mm to 10 cm, including 1 mm to 1 cm, has a greater impact on the electrical energy concentration and effective dose delivered to the tissue from a separation distance. For example, if the target voltage-to-distance ratio is 3000 V / cm to induce the desired clinical effect at the desired tissue depth (1.3 mm), if the separation distance is changed from 1 mm to 1.2 mm, this results in a required increase in treatment voltage from 300 to approximately 360 V, a change of 20%. B.Frequency

[0291] The number of biphasic cycles per second can be understood to be the fundamental frequency when the signal is continuous. Because specialized PEF waveforms are not continuous throughout the dose (e.g., the waveforms include packets and delays), the fundamental frequency is used as a way to describe the pulse width of the biphasic pulses of specialized PEF waveforms. It will be understood that pulse width is a measure of the elapsed time between the leading and trailing edges of a single biphasic cycle and can be derived from the value of the fundamental frequency. For example, a waveform with a fundamental frequency of 400 Hz has a biphasic pulse width of 2.5 microseconds.

[0292] In some embodiments, biphasic pulses are utilized to reduce unwanted muscle stimulation, particularly myocardial stimulation. In other embodiments, the pulse waveform is monophasic and does not have a distinct inherent frequency. Instead, a fundamental frequency can be considered by doubling the monophasic pulse length to derive the frequency. In some embodiments, the signal has a frequency in the range of 100 kHz to 1 MHz, more specifically 100 kHz to 1000 kHz, including 100 kHz, 200 kHz, 300 kHz, 400 kHz, 500 kHz, 100-500 kHz, etc. In some embodiments, the signal has a frequency in the range of approximately 100-600 kHz, which typically penetrates the luminal wall, to treat or affect specific cells located at a greater depth, such as submucosal or smooth muscle cells. In some embodiments, the signal has a frequency in the range of approximately 600 kHz to 1000 kHz or 600 kHz to 1 MHz, which typically penetrates the luminal wall, to treat or affect specific cells located at a greater depth, such as epithelial or endothelial cells. It will be appreciated that at some voltages, frequencies below 100-250 kHz may cause undesired muscle stimulation. Thus, in some embodiments, the signal has a frequency in the range of 400-800 kHz or 500-800 kHz, such as 400 kHz, 450 kHz, 500 kHz, 550 kHz, 600 kHz, 650 kHz, 700 kHz, 750 kHz, or 800 kHz. In particular, in some embodiments, the signal has a frequency of 600 kHz. Furthermore, cardiac synchronization is typically utilized to reduce or avoid undesired myocardial stimulation during susceptible rhythmic periods. It will be appreciated that even higher frequencies can be used with components that minimize signal artifacts. C. Voltage-Frequency Balancing

[0293] The frequency of the delivered waveform can be varied in synchronization with the treatment voltage to maintain the appropriate treatment effect. Such synergistic variations include a decrease in frequency that produces a stronger effect, combined with a decrease in voltage that produces a weaker effect. For example, in some cases, treatment can be delivered using 3000V in a monopolar manner with an 800kHz waveform frequency, while in other cases, treatment can be delivered using 2000V with a 400kHz waveform frequency.

[0294] When used in the opposite direction, treatment parameters can be manipulated to make it highly effective, which may increase the likelihood or risk of muscle contraction in undesired tissues, such as cartilage, for airway treatment. For example, increasing the frequency and decreasing the voltage, such as using 2000 V at 800 kHz, may result in treatment with insufficient clinical benefit. Conversely, increasing the voltage to 3000 V and decreasing the frequency to 400 kHz may result in undesired treatment effects in collateral-sensitive tissues. In some cases, overtreatment of these undesired tissues may result in patient morbidity or safety concerns, such as sufficient destruction of cartilage tissue in the airway to cause airway collapse or sufficient destruction of smooth muscle in the GI tract to cause disruption of normal peristalsis. In other cases, overtreatment of untargeted or undesired tissues may have a benign clinical outcome and not affect patient response or morbidity if overtreated. D. Cycles and Packets

[0295] As described above, algorithm 152 defines a signal having a waveform that includes a series of energy packets, each of which includes a series of high-voltage pulses. Typically, the pulses are biphasic, so they are referred to as cycles. Referring to FIG. 4, the first packet 402 has a cycle count 420 of 2. In some embodiments, the cycle count 420 is set between 1 and 100 per packet, including 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, and all values ​​and subranges therebetween. In some embodiments, the cycle count 420 is between 1 and 5 cycles, between 1 and 10 cycles, between 1 and 25 cycles, between 10 and 20 cycles, between 1 and 40 cycles, between 40 and 50 cycles, between 1 and 60 cycles, between 50 and 60 cycles, between 1 and 80 cycles, between 1 and 100 cycles, between 50 and 100 cycles, between 1 and 1,000 cycles, or between 1 and 2,000 cycles, including all values ​​and subranges therebetween.

[0296] Packet duration is determined by, among other factors, the cycle count. Typically, the higher the cycle count, the longer the packet duration and the greater the amount of energy delivered. In some embodiments, the packet duration is in the range of approximately 50-1000 microseconds, e.g., 50 μs, 60 μs, 70 μs, 80 μs, 90 μs, 100 μs, 125 μs, 150 μs, 175 μs, 200 μs, 250 μs, 100-250 μs, 150-250 μs, 200-250 μs, or 500-1000 μs, to name a few. In other embodiments, the packet duration is in the range of approximately 100-1000 microseconds, such as 150 μs, 200 μs, 250 μs, 500 μs, or 1000 μs.

[0297] In some embodiments, the number of packets delivered during treatment, or packet count, may include 50-280 packets, including all values ​​and subranges therebetween, such as 50, 60, 70, 80, 90, 100, 50-100, 80-100, 100-110, 110, 120, 130, 140, 150, 100-150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, and 280 packets.

[0298] [Table 3] E. Quiet Periods / Inter-Packet Delay

[0299] In some embodiments, the time between packets, referred to as the pause period or inter-packet delay 406, is set between approximately 0.1 seconds and approximately 5 seconds, including all values ​​and subranges therebetween. In other embodiments, the pause period 406 ranges from approximately 0.001 seconds to approximately 10 seconds, including all values ​​and subranges therebetween, such as 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, and 10 seconds. In some embodiments, the pause period 406 is approximately 3 to 5 seconds. Notably, in some embodiments, the signal is synchronized with the cardiac rhythm so that each packet is delivered synchronously within a specified period relative to the heartbeat, and therefore the pause period coincides with the heartbeat. In other embodiments where cardiac synchronization is utilized, the pause period 406 may vary, as the pause period between packets may be affected by cardiac synchronization, as described in a later section. F. Switch Time and Dead Time / Intercycle Delay

[0300] The switch time is the delay or period of time during which no energy is delivered between the positive and negative peaks of a biphasic pulse. In some embodiments, the switch time is in the range of about 0 to about 1 microsecond, including all values ​​and subranges therebetween. In other embodiments, the switch time is in the range of 1 to 20 microseconds, including all values ​​and subranges therebetween. In other embodiments, the switch time is in the range of about 2 to about 8 microseconds, including all values ​​and subranges therebetween.

[0301] A delay, called "dead time" or inter-cycle delay, may also be inserted between each cycle of a biphasic pulse. The inter-cycle delay occurs within a packet, but not between cycles or biphasic pulses. This contrasts with the rest period or inter-packet delay, which occurs between packets. In other embodiments, the inter-cycle delay 412 is within the range of 0.01 to 0.5 microseconds, 1 to 10 microseconds, 2 to 5 microseconds, 10 to 20 microseconds, 50 to 100 microseconds, 1000 microseconds, 1000 to 1500 microseconds, or 1000 microseconds to 100 milliseconds, including all values ​​and subranges therebetween. In some embodiments, the inter-cycle delay 412 is within the range of 0.2 to 0.3 microseconds. The inter-cycle delay may also be used to define the period between separate monophasic pulses within a packet.

[0302] This type of delay is typically introduced into the packets to reduce the effects of biphasic cancellation within the waveform. Biphasic cancellation is a term used to refer to the reduction in the induction of cellular modulation in response to biphasic versus monophasic waveforms, particularly when the switch time and dead time are small (e.g., less than 10 μs). In some embodiments, the effects of biphasic cancellation are reduced by introducing a switch time delay and dead time. In some cases, both the switch time and dead time are increased together to enhance the effect. In other instances, only the switch time or only the dead time is increased to induce this effect.

[0303] In some embodiments, the switch time duration is adjusted to optimize the degree of therapeutic effect relative to the distant cellular effect for the therapeutic target. In some embodiments, the switch time duration or dead time duration is minimized to reduce distant muscle cell contraction, resulting in a lower local therapeutic effect. In other embodiments, the switch time duration is extended to increase the local therapeutic effect, allowing for additional distant muscle cell contraction. In some embodiments, the switch time or dead time duration is extended to enhance the local therapeutic effect, and a neuromuscular paralytic drug is used to control the resulting increase in muscle contraction. In some embodiments, the switch time duration is 10 ns to 2 μs, while in other embodiments, the switch time duration is 2 μs to 20 μs. In some instances, when cellular modulation is targeted in a manner where transmembrane potential manipulation is not the primary mechanism required to elicit the targeted therapeutic effect, the switch time delay and dead time delay are minimized to less than 0.1 μs or 0 μs. Eliminating this delay minimizes peripheral, non-targeted therapeutic effects, such as skeletal muscle contraction or cardiac action potential and contraction.

[0304] Another advantage of utilizing switch and dead time delays to enhance the therapeutic effect of biphasic waveforms is reduced generator demand, whereby the introduction of pauses allows for a stronger therapeutic effect without the need for asymmetric / unbalanced pulse waveforms. In this case, the unbalanced waveform is described as being monophasic or having a duration, voltage, or combination in which one polarity is unbalanced relative to the other. In some cases, imbalance means that the integral of the positive portion of the waveform is not equal to the integral of the negative portion of the waveform. Generators capable of delivering unbalanced waveforms have a separate set of design considerations that are taken into account, thereby increasing the potential generator complexity. G. Waveform

[0305] In some embodiments, the waveform has symmetric pulses, whereby the voltage and duration of the pulse in one direction (i.e., positive or negative) are equal to the voltage and duration of the pulse in the other direction. In some embodiments, the waveform has pulses of unbalanced voltage. Because a predominantly positive or negative amplitude results in a longer duration of the same charge cell membrane charging potential, an unbalanced waveform may result in a more pronounced treatment effect. In some embodiments, the imbalance includes pulses with pulse widths of unequal duration. In some embodiments, a biphasic waveform is unbalanced, whereby the voltage in one direction is equal to the voltage in the other direction, but the duration of one direction (i.e., positive or negative) is longer than the duration of the other direction, such that the area under the curve of the positive portion of the waveform is not equal to the area under the negative portion of the waveform. In some embodiments, an unbalanced waveform is achieved by delivering more than one pulse in one polarity before reversing to an unequal number of pulses of the opposite polarity. H. Waveform shape

[0306] In some embodiments, the pulses are sinusoidal rather than square. One advantage of a sinusoidal waveform is that it is balanced or symmetrical, so that the shape of each phase is equal. This balancing can help reduce unwanted muscle stimulation. It will be appreciated that in other embodiments, the pulses have a decaying waveform.

[0307] Larger lesions (e.g., approximately 1 × 1 × 1 cm 3 It will be appreciated that the generation of a larger (larger) may involve different parameter values ​​to obtain the same or similar lesion characteristics and / or immune response. In some embodiments, the size of the tumor is approximately 1.8 x 1.8 x 2.4 cm. 3 A lesion having a size of approximately 3x3x4 cm can be achieved with a waveform generated from a combination of parameter values ​​including: voltage = 3300 V, fundamental frequency = 100 kHz, number of biphasic pulses (i.e., cycles) per packet = 10 cycles, inter-cycle delay = 1000 microseconds, number of packets = 100 packets, and inter-packet delay = 3 seconds. In some embodiments, a lesion having a size of approximately 3x3x4 cm can be achieved with a waveform generated from a combination of parameter values ​​including: voltage = 3300 V, fundamental frequency = 100 kHz, number of biphasic pulses (i.e., cycles) per packet = 10 cycles, inter-cycle delay = 1000 microseconds, number of packets = 100 packets, and inter-packet delay = 3 seconds.3 Lesions having a size of can be achieved with waveforms generated from combinations of parameter values ​​including: voltage = 6000 V, fundamental frequency = 100 kHz, number of biphasic pulses (i.e., cycles) per packet = 10 cycles, inter-cycle delay = 1000 or 2000 microseconds, number of packets = 100 packets, and inter-packet delay = 1, 3, 5, or 10 seconds.

[0308] It will be appreciated that in some embodiments, the waveform is generated as a function of current rather than voltage. For example, a dose generated using 3000V or 3300V is delivered into a 150-300 ohm environment (producing 10-20 A at 3000V and 11-22 A at 3300V). In some embodiments, the current is 10 A, 15 A, 20 A, 25 A, 30 A, 35 A, 40 A, 45 A, 50 A, 55 A, 60 A, 65 A, 70 A, etc., and all ranges therebetween. It will be appreciated that in some instances, for example, when using a 100 kHz treatment that may be delivered with paralysis, 70 A may be the upper limit.

[0309] Energy delivery can be activated by various mechanisms, such as the use of an actuator 132 on the instrument 102 or a footswitch operably connected to the generator 104. Such activation typically provides a single energy dose. The energy dose is defined by the number of packets delivered and the voltage of the packets. Each energy dose delivered to the target tissue maintains the temperature at or within the target tissue below the threshold for thermal ablation, particularly thermal ablation or denaturation of interstitial proteins in the basement membrane or deeper submucosal extracellular protein matrix. Furthermore, the dose can be titrated or relaxed over time to further reduce or eliminate heat accumulation during the treatment procedure. Instead of inducing thermal damage, defined as protein coagulation at sites at risk for therapy, the energy dose provides a level of energy that induces treatment of conditions such as cancer without damaging sensitive tissue.

[0310] Although the examples described herein relate to tumors, such as cancer treatment, it will be understood that the devices, systems, and methods are not so limited. For example, signaling regulated by the PD-1 / PD-L pathway is also associated with substantial inflammatory effects that may resemble those in autoimmune responses, chronic infections, and sepsis, consistent with the pathway's role in the balance between protective immunity and immunopathology, as well as homeostasis and tolerance. Thus, devices, systems, and methods can be used to target this pathway in autoimmune and inflammatory disorders, chronic infections, and sepsis. Other conditions and disorders that can be treated include ischemia-reperfusion injury (IRI), stroke, Alzheimer's disease (AD), and pain due to the immunoregulatory role of the PD-1 / PD-L pathway in these disorders. Similarly, inflammation after stroke can be treated due to the involvement of PD-1 / PD-L.

[0311] As used herein, the terms "about" and / or "approximately," when used in conjunction with a numerical value and / or range, generally refer to a numerical value and / or range that is close to the recited numerical value and / or range. In some cases, the terms "about" and "approximately" can mean within ±10% of the recited value. For example, in some cases, "about 100 [units]" can mean within ±10% of 100 (e.g., 90 to 110). The terms "about" and "approximately" can be used interchangeably.

[0312] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A system for treating tissue within a patient's body, comprising: an instrument having at least one energy delivery body configured to be positioned to direct pulsed electric field energy to the tissue creating a lesion; and a generator in electrical communication with the at least one energy delivery entity, the generator comprising at least one energy delivery algorithm configured to provide the pulsed electric field energy in a manner that produces multiple zones within the lesion that elicit an increased adaptive immune response in the patient.

2. 10. The system of claim 1, wherein the plurality of zones includes at least a pulsed electric field zone and one or more of an immune response zone, a thermal zone, and an inflammation zone.

3. 2. The system of claim 1, wherein the plurality of zones includes at least a pulsed electric field zone and a thermal zone, the pulsed electric field zone and the thermal zone together covering an area within the lesion, the thermal zone not exceeding 25% of the area.

4. 4. The system of claim 3, wherein the at least one energy delivery algorithm includes an inter-cycle delay in a waveform of the pulsed electric field energy, the inter-cycle delay configured to control a size of the thermal zone.

5. The system of claim 4 , wherein the inter-cycle delay is at least 10 microseconds.

6. The system of claim 4 , wherein the inter-cycle delay is configured to minimize or eliminate cavitation zones.

7. 7. The system of claim 6, wherein the at least one energy delivery algorithm includes an inter-packet delay in a waveform of the pulsed electric field energy, the inter-packet delay configured to control a size of the thermal zone.

8. The system of claim 7, wherein the inter-packet delay is between 3 and 5 seconds.

9. The system of any one of claims 1 to 8, wherein the adaptive immune response comprises an increase in the ratio of CD8+ / CD4+ T cells.

10. The system according to any one of claims 1 to 8, wherein the adaptive immune response comprises an increase in PDL1 expression.

11. The system of any one of claims 1 to 8, wherein the adaptive immune response comprises upregulation of lymphatic pathways.

12. The system of any one of claims 1 to 8, wherein the adaptive immune response comprises the generation of tumor antigen release.

13. The system of any one of claims 1 to 8, wherein the adaptive immune response comprises downregulation of regulatory T cells.

14. The system of any one of claims 1 to 8, wherein the adaptive immune response comprises the production or increased production of tertiary lymphoid structures in the tissue.

15. 9. The system of any one of claims 1-8, wherein the at least one energy delivery algorithm produces a synergistic effect when the pulsed electric field energy is combined with an immune checkpoint inhibitor.

16. The system of claim 15, wherein the immune checkpoint inhibitor comprises an anti-PD1 therapy, and optionally the synergistic effect increases PDL-1 expression.

17. The system of claim 15 , wherein the synergistic effect increases the abscopal effect.

18. The system of claim 15, wherein the synergistic effect increases PDL-1 expression.

19. A system described in any one of claims 1 to 8, wherein the at least one energy delivery algorithm generates a waveform of the pulsed electric field energy having a voltage of 3000V to 5000V.

20. A system described in any one of claims 1 to 8, wherein the at least one energy delivery algorithm generates a waveform of the pulsed electric field energy having a fundamental frequency of 100 to 600 kHz.

21. The system of any one of claims 1 to 8, wherein the at least one energy delivery algorithm generates a waveform of the pulsed electric field energy having packets of biphasic pulse cycles.

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