WO2024206710A1 - Method for inducing tumor regression - Google Patents

Abstract

Methods for treating cancer and/or inducing tumor regression in mammals (e.g., humans) by administering a BA dye to tumor cells isolated from the tumor bearing mammal, and thereafter exposing the isolated tumor cells to actinic light for activation of the BA dye, culturing subfractions of the treated tumor cells with antigen presenting cells isolated from the tumor bearing mammal and reintroducing the antigen presenting cells or the BA –actinic light treated tumor cells to the tumor bearing mammal. In a further embodiment the circadian neuroendocrine axis that regulates immunity is reset towards normal by timed daily administration of neuroendocrine modulators in vivo to the mammal and/or in vitro to the cultured antigen presenting cells.

Description

METHOD FOR INDUCING TUMOR REGRESSION

TECHNICAL FIELD

Disclosed herein are methods for treating cancer in a subject (e.g., an animal such as a human) in need thereof. In particular, the disclosure relates to methods for inducing malignant tumor regression using photodynamic therapy combined with one or more additional treatments.

BACKGROUND

Photodynamic therapy (PDT) is a technique useful for the treatment of cancer, in tumorbearing organisms (e.g., animals including humans). PDT generally involves the systemic administration of a light-absorbing compound (i.e., a photosensitizer) to a tumor-bearing organism that sequesters within the tumor cell mass, followed by irradiation of the photosensitizer-laden tumor mass with light of an appropriate wavelength (i.e., in the therapeutic window of wavelengths between 620 to 700 nm, actinic light). This irradiation transfers energy to the photosensitizer in a manner that causes its conversion into a phototoxin (i.e., the excited state of the photosensitizer). The phototoxin is then able to chemically interact with surrounding molecules and alter them, e.g., via oxidation-reduction reactions (termed a Type I reaction that generates highly reactive molecular species such as free radicals) or via the transfer of its energy to nearby oxygen, to generate singlet (excited state) oxygen (termed a Type II reaction). In turn, the singlet oxygen transfers its energy to surrounding biomolecules, damaging and/or degrading them. This damage is useful in treatments focused on ablating malignant tumor tissue as it impairs cellular organization and/or function, and thus reduces the viability of the (tumor) cell in which the phototoxin is present. These reactions can result in tumor cell death via tumor cell necrosis and/or various tumor cell programmed cell death mechanisms including apoptosis, necrosis, necroptosis, pyroptosis, and ferroptosis (collectively termed PANoptosis). This mechanism of PDT-induced cell death, including the magnitude of success of such an event, is highly dependent upon the particular photosensitizer used to cause such a biochemical reaction. Particular photosensitizers vary in their aqueous and lipid solubility, intracellular localization within areas and organelles of the tumor cell, ability and efficiency to absorb light, i photochemistry, and intracellular mobility among other photophysical and biochemical attributes.

A particular class of photosensitizers, the benzophenoxazinium, benzophenothiazinium [for example 2-iodo-5-ethylamino-9-diethylamino-benzo[a]phenothiazine-(2I-EtNBS)], and benzophenoselenazinium dyes, and their substituents [e.g., amines, halogens, alcohols, or alkyls], (collectively referred to as “BAs”), possess several properties that make them uniquely favorable for use as effective PDT photosensitizers for cancer therapy versus all other classes of photosensitizers (particularly as applied in the current invention). These properties include, e.g., a positive delocalized charge, very high lipophilicity yet water soluble, unique tumor cell distribution dependent upon local intracellular pH and redox state of the cell, high efficiency for absorbing light with a wavelength > 650 nm (correlating with the light wavelengths in the “therapeutic window” wherein penetration of light into biological tissue is optimal), rapid absorption within a tumor cell or tumor cell mass, and ability to directly kill tumor cells upon photo irradiation (generally about 50 - 500 mW/cm² and 50 -500 J/cm²) (instead of only inflicting indirect damage, e.g., to the vasculature of and/or surrounding the tumor). See, e.g., Cincotta et al., PhotoChem PhotoBiol 46:751-758, 1987; Cincotta et al., Cancer Res 53:2571- 2580, 1993; Cincotta et al., Cancer Res 54: 1249-1258, 1994; United States Patent 4,962,197; and United States Patent 5,952,329 for further description and method of synthesis of BA’ s and their analogs, derivatives and conjugates. Additional examples of BA dyes and their synthesis are described in United States Patents 5,952,329 and 4,962,197, incorporated herein by reference in their entirety.

Further, BAs can undergo specific reactions within tumor cells and normal cells (including, e.g., different reactions within tumor versus normal cells) that influence their ability to absorb light in the therapeutic window. BAs are the only presently known photosenitizers that can exist in, and reversibly transition between, an active cationic form (where they are able to absorb light in the therapeutic window and thus induce phototoxin production) and a neutral inactive form (where they are unable to absorb light in the therapeutic window). The main mechanisms involved in the inactivation of the BAs (rendering them unable to absorb light in the therapeutic window) are deprotonation based upon the pH of the environment (deprotonation in basic environment) and/or reduction of the BAs (particularly in the presence of a low oxygen concentration), induced for example by either cellular enzymatic processes, biochemical processes, and/or light (e.g., prolonged exposure to high-intensity light). The photo toxicity of the BAs also depends upon the intracellular location of the BAs, which is also a function of their redox or protonation state. Therefore, the reactivity of the BAs to function as optimal PDT agents for cancer therapy depends upon their structure and chemical state within the tumor cell as well as the cellular biochemical environment within which they exist. For any given BA, a goal of PDT using these agents is to create an appropriate local intracellular environment that potentiates the appropriate level of photoactive (cationic) form of the BA at the time of photoirradiation in a cellular location that most favorably facilitates cell killing and ultimate tumor eradication. This mechanism of BA-PDT directed tumor eradication may include and/or involve the appropriate stimulation of anti-tumor immune responses. (As used herein, the term “BA- PDT” refers to treating a tumor mass in vivo or tumor cell in vitro with a BA followed by exposure of the tumor to actinic light). BAs represent the only photosensitizers that have the ability to exist in active and inactive forms within the cell as a function of the local pH and redox potentiality within the cell. This unique effect ultimately determines the cell death response to PDT with these BA agents. Consequently, cellular PDT reactions with BAs, including mechanisms of cell death - and most importantly, the overall PDT response, cannot be predicted or deduced from PDT effects with other photosensitizers-

Tumor eradication with BA-PDT also involves more than just the direct photoactive reaction of light with the BA and resultant cell killing via necrosis or programmed cell death mechanisms. Indeed, shortly following a PDT “event” (defined as irradiation of the tumor mass with actinic light following the administration of one or more BAs), much of the tumor mass generally remains intact and viable. However, during the ensuing days following the BA-PDT, the tumor mass can shrink and on certain occasions become completely resolved. This process involves the BA-PDT-initiated activation of a complex, innate and adaptive immune response against BA-PDT-treated tumor cells. See, e g., Hendrzak-Henion et al., PhotoChem PhotoBiol 69:575-582, 1999. It is believed that BA-PDT treatment induces the activation of this immune cell death response in theory by initiating what is now appreciated to be the presentation of damage-associated molecular patterns (DAMPs) that arise from chemical interactions between photo-irradiated active BAs and local molecules within the cell (e.g., redox reactions with proteins, lipids, and carbohydrates, and/or energy transfer from singlet oxygen to such biomolecules) that alter the local molecules’ structure and function. These damage associated molecular pattern (DAMP) biomolecules then participate in presentation of tumor associated antigens to the immune system through a complex series of cellular processes. It is critically important to appreciate that not all programmed cell death processes lead to the production of DAMPs and subsequent immunologic cell death - a cellular process that results in the presentation of tumor-associated antigens within certain intracellular vesicles and the plasma membrane capable of eliciting (via immune antigen presenting cells, NK ells, and T cells) activation of an immune cytotoxic reaction against the cell. A certain set of biochemical criteria must be realized before programmed cell death can lead to immunologic cell death (ICD). In general, these criteria minimally include generation of a critical level of specific-type DAMP production in the appropriate organelles of the cell and appropriate presentation and vesicular packaging of such DAMPs to the immune system that in turn activates the immune system response to destroy the tumor cell (i.e., becomes sensitized against the tumor cell) is required to elicit an immune cell death reaction.

We have now discovered a new methodology by which BA-PDT of cancer cells is capable of eliciting all the required steps of ample DAMPs generation including generation of tumor antigens and their subsequent appropriate packaging into cellular vesicles necessary to elicit a substantial anti-tumor immune response. Following BA-PDT induced endoplasmic reticulum and whole cell redox stress, resultant damaged cellular biomolecules (particularly proteins) (i.e., DAMPs) may be processed by the cell via a series of organized steps to present either a) on areas of the plasma membrane with MHC-1 or in association with chaperone proteins as tumor cell antigen in membrane outcroppings (outward buddings) termed blebs (or blebosomes) (generally 100 - 1000 nm diamater) - that are destined to become extracellular vesicles carrying a variety of BA-PDT -induced tumor antigens or b) within intracellular vesicles generated from inward budding of endosomal membranes during their maturation into multivesicular endosomes termed exosomes (about 30 -150 nm diameter) that are destined to be released from the cell as tumor antigen packages. Because BA-PDT may generate a very large number of DAMPs due to BAs ubiquitous distribution within the tumor cell, its intracellular conversion between active and inactive forms, and its high efficiency of absorbing light in the 500-700 nm window, BA-PDT is uniquely capable of generating a wide variety of tumor associated antigens. Moreover, BA-PDT is also capable of inducing the packaging of such a copious and diverse range of tumor antigens upon/within appropriate antigen-presenting bioscaffolds, primarily cellular blebs and exosomes that can then be highly immunogenic.

This reaction between the BA-PDT treated tumor cells and the immune system is highly complex and to elicit a functional interaction between these two processes to enhance in vivo tumor stasis or regression requires a precise set of physiological and biochemical circumstances that itself is BA photosensitizer-dependent (i.e., not possible with other PDT photosensitizers). That is, the effect of BA-PDT treatment to induce a specific immune response against tumor cells is BA-photosensitizer specific (as opposed to PDT exposure of other photosensitizers) and the mechanisms required for BA-PDT treatment to induce an operative, functional, and clinically useful anti -tumor immunity also require a specific set of introduced physiological and biochemical steps that affect both the individual’s tumor cells and the individual’s immune system to achieve this goal and elicit tumor stasis and/or regression. This disclosure provides for a tumor cell directed BA-PDT - immuno-stimulatory coupled process to induce tumor stasis and/or tumor regression.

While the direct effect of BA-PDT treatment combined with its indirect, long-term immunogenic effect can produce reductions in tumor mass and may even result in tumor eradication in certain cases, the rate of such eradications (complete tumor response) has been and is generally both low and highly variable, both within a particular tumor type as well as between different tumor types. BA-PDT induced rates of immunogenic eradication of immunogenic tumors (tumors that elicit an immune response against the tumor that result in its inhibited growth)-can, for example, vary from about 0% to about 40-60% within a given tumor type in a given species, with only an occasional rare complete response (tumor eradication). Similarly, BA-PDT rates of eradication can vary between different tumor types in a given species, e.g., from about 0% to about 40%. Rates of eradication of non-immunogenic tumors, although originally thought to be similar to that of immunogenic tumors, upon further investigation have been found to be generally much lower if at all however. This is a problem for effective in vivo treatment of cancer with BA-PDT in that many tumor types are generally non-immunogenic, defined as a tumor type that does not elicit a strong immune response against the tumor resulting in halt of tumor growth or tumor eradication. Responses to such BA-PDT treatments vary depending upon the method of presentation of the BA to the body (i.e., pharmacokinetics and pharmacodynamics) as well as the whole body and tumor metabolic and immune status at the time of BA-PDT treatment. Moreover, PDT of non-solid tumors (e.g., circulating tumor cells such as leukemia or peritoneal tumor cells such as ovarian cancer) is generally impractical and ineffective.

It is known that PDT of cancer cells either in vivo or in vitro with other non-BA photosensitizers (e.g., porphyrins, chlorins, phthalocyanines, purpurins) can induce immunogenicity against these tumor cells and subsequent anti-tumor activity in vivo, however none of these approaches have been shown to induce all the necessary molecular determinants of ICD simultaneously to a necessary level to elicit clinically meaningful reduction of tumor growth and progression on a routine basis and across tumor types in vivo. Major aspects missing from the current methods available to treat cancer with this approach that result in sub-optimal clinical reduction of tumor growth include 1) lack of a photosensitizer that is able to rapidly sequester within tumor cells, move to specific organelles based upon the protonation or redox state of the photosensitizer, and rapidly (within seconds to minutes) generate a major shift in oxidation-reduction balance within the cell to initiate sufficient immune cell death (ICD) within this brief time frame to generate specific tumor antigens, 2) inability to safely overcome the tumor-generated immune-suppression of leukocytes including antigen presenting cells (APCs - defined as a group of different immunocyte cell types including dendritic cells, macrophages, and B cells) induced by the tumor and enhance the anti-tumor immunity of the isolated APCs in vitro and 3) inability to safely overcome the tumor-generated immune-suppression of the systemic and local tumor immune system induced by the tumor (and by most chemotherapeutic agents) and enhance the anti-tumor immunity of the entire immune system in vivo. Therefore, what is needed is a method that addresses these treatment inadequacies and provides a consistent and high eradication rate or similar successful tumor treatment rate (i.e., long-term stasis, longterm remission post-treatment or tumor free for a defined extended period of time post- treatment) for the treatment of tumors, particularly non-immunogenic tumors. The current disclosure provides for such a method.

SUUMMARY OF INVENTION

In contradistinction to previously known methods of BA-PDT in vivo to treat cancer, the present invention relates to a unique process for BA-PDT in vitro to induce a potent anti-cancer effect. The present disclosure provides methods for treating cancer and/or inducing tumor regression in animals (e.g., humans) by

(i) exposing tumor cells that were previously isolated from the subject (patient) to treatment with BA-PDT and

(ii) contacting in vitro sub-cellular fractions of the BA-PDT treated tumor cells comprising cellular blebs and exosomes (so-called extracellular vesicles, or small bubbles, released from cells), with antigen presenting cells (APCs) including by way of non-limiting example , dendritic cells [DCs], macrophages, and B cells, isolated from the patient, with or without T cells from the patient.

(iii) Introducing the subject’s BA-PDT tumor cell fraction comprising blebs and exosomes primed - APCs (i.e, APCs that have been exposed to the BA-PDT generated tumor cell blebs and/or exosomes) with or without T cells or other immunocytes back into the subject.

Two important points should be noted here regarding the novelty of this approach to treat cancer: 1) using exosomes to treat cancer is not a well-accepted methodology for doing so inasmuch as exosomes under certain circumstances often have the capacity to provide a potent stimulus for tumor growth by acting as metastasis stimulators and pro-tumor immunity inducers (stimulate the immune system to support tumor growth and inhibit anti-tumor immunity) and by inducing anti-cancer drug resistance and 2) the blebosomes an exosomes of the current methodology are not at all regular exosomes but rather unique BA-PDT transformed blebosomes and exosomes that are generated to now carry BA-PDT-induced tumor antigens to uniquely activate the immune system against the cancer. When these BA-PDT generated tumor derived blebosomes and exosomes are presented to APCs they activate the APCs to a) kill naive tumor cells (those never exposed to BA-PDT) and b) cross prime T cells for their activation against naive tumor cells. The specific molecular components and structure of these blebosomes and/or exosomes using this specific procedure cannot be duplicated by PDT with other photosensitizers. Moreover, it should be noted that this method does not employ introduction of the photosensitizer into tumor-derived exosomes for re-introduction into the tumor bearing subject for subsequent PDT at the tumor site. In other words, in the method described herein, the exosomes are not carriers of the PDT agent to be used to drive the in vivo administered PDT agent-exosome unit into the tumor for subsequent photoirradiation. The present methodology is effective against solid tumors and non-solid tumors such as circulating tumor cells such as leukemia types and peritoneal cavity tumor cells such as ovarian cancer.

In one embodiment the BA-PDT treatment of tumor cells step (i) may be at 0.5 to 10 mW/cm² and 1.0 to 10.0 J/cm² and the contacting step of (ii) may be with APCs from the patient that have been previously cultured in vitro for 2 to 14 days. The tumor cell fraction comprising blebs and exosomes from such cells previously exposed to BA PDT are preferably added to the cultured APC’s, preferably within 12 hours of the PDT. The APCs are cultured with the BA- PDT exosome/bleb fraction for between about 1 to about 96 hours of time followed by (iii) administering the BA-PDT treated tumor cell fraction - primed APCs (with or without the subject’s T cells or other immunocytes) to the patient to enhance whole body anti-tumor immunity and to induce tumor stasis and/or regression.

The treatment methods disclosed herein incorporate the use of specific BA photosensitizers in vitro to induce tumor antigen via BA-PDT of tumor cells followed by presentation of such cellular material (blebs containing tumor antigen associated with or without MHC-1 and/or exosomes (multivesicular endosomes) that may contain chaperone protein molecules such as calreticulin, HSP70, HSP90, GRP94 to APCs in vitro. These specific tumor cell fractions generated by BA-PDT are able to stimulate the maturation and activation of the APCs. The BA-PDT treated tumor cell fraction[s]) - primed APCs are subsequently 1) stimulated to recognize and kill naive tumor cells never exposed to BA-PDT and also 2) able to activate T cells and other immunocytes to kill tumor cells never exposed to BA-PDT, a process termed cross-priming. This methodology, owing to the uniqueness of the BA photophysical and biochemical characteristics within tumor cells, thus is capable of inducing a strong systemic immunity against the tumor following re-introduction of the primed APCs into the subject. This methodology is particularly effective when the BA-PDT treated tumor cell fraction contains isolated plasma membrane blebs and cellular exosomes that can be obtained for example from the 100,000 g centriguation pellet from the supernatant of the 300 g centrifugation or any similar fraction isolation method known in the art for isolating blebs and exosomes, including for instance the 16,000 g centrifugation pellet from the 300 to 900 g supernatant (that is processed in/ contains polyethylene glycol) centrifugation to stimulate the APCs. It also carries the advantage of excluding whole tumor cells from the isolate to be re-introduced into the subject following priming of the APCs. Upon re-introduction to the subject, activated APCs (and T cells or other immunocytes) from this process are able to induce systemic immunity against the tumor in vivo and reduce tumor size and/or halt its growth significantly. This entire process is dependent upon the ratio of protonated or oxidized active versus unprotonated or reduced inactive state of the BA in the tumor cell, a biochemical process which does not occur and which is not possible with other available photosensitizers, and is not produced by PDT exposure to other photosensitizers.

In another embodiment BA-PDT is combined with methods for resetting towards normal the circadian profiles of neuronal activities within the central nervous system that control physiological events regulating immunity in vertebrates, so as to treat abnormalities of functions within immunological systems that impede immunity against the tumor.

In another embodiment the biological clock of the APC’s is reset in vivo prior to removal of the APC’s from the tumor bearing subject and/or in vitro following their removal from the tumor bearing subject. This optimizes response to tumor antigen presented by BA-PDT followed by re-presentation of such APCs back into the tumor bearing individual (in whom optionally the e biological clock has been reset by circadian timed administration of neuroendocrine agents such as dopamine, dopamine agonists, prolactin, prolactin stimulators, melatonin and Thyrotropin Releasing Hormone (TRH) This enhances overall immune function, anti-tumor immunity, and responsiveness to the re-presented APCs to induce tumor stasis or regression.

In another embodiment the biological clock of the APC’s is reset in vitro prior to introducing BA-PDT generated tumor cell blebs and exosomes to the APC’s from the tumor bearing subject following their removal from the tumor bearing subject. This optimizes APC response to tumor antigen presented by BA-PDT followed by re-presentation of such APCs back into the tumor bearing individual (in whom optionally the biological clock has been reset by circadian timed administration of neuroendocrine agents such as dopamine, dopamine agonists, prolactin, prolactin stimulators, melatonin and Thyrotropin Releasing Hormone (TRH) This enhances overall immune function, anti-tumor immunity, and responsiveness to the re-presented APCs to induce tumor stasis or regression.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Introduction to Figures 1A - 1C and Figures 2A- 5Cii

BA-PDT of Tumor Cells with Benzophenothiazinium (2-iodo-5-ethylamino-9- diethylamino-benzo[a]phenothiazine)-(2I-EtNBS) Initiates Gene Transcription - Driving Immune Cell Death via Apoptosis, Necroptosis, Pyroptosis, and Necrosis and Concurrent Tumor Antigen Presentation in Human Pancreatic Cancer Cells

General Study Design: 2I-EtNBS is administered to tumor cells (0.7 uM) in vitro for ~10 minutes and then photoirradiated with red light for ~10 minutes (~4 J/cm²) and then harvested at 4 hours after PDT for assessment of gene expression associated with ER stress, PANoptosis, and antigen generation and processing for immune reactivity (and assessment of DC activation and maturation following coculture with blebosomes and exosomes harvested from BA-PDT treated tumor cells as described in the examples 1-5). BA-PDT of Tumor Cells with Benzophenothiazinium (2-iodo-5-ethylamino-9- diethylamino-benzo[a]phenothiazine)-(2I-EtNBS) Initiates Gene Transcription - Driving Immune Cell Death via Apoptosis, Necroptosis, Pyroptosis, and Necrosis and Concurrent Tumor Antigen Presentation in Human Pancreatic Cancer Cells

General Study Design: 2I-EtNBS is administered to tumor cells (0.7 uM) in vitro for ~I0 minutes and then photoirradiated with red light for ~10 minutes (~4 J/cm²) and then harvested at 4 hours after PDT for assessment of gene expression associated with ER stress, PANoptosis, and antigen generation and processing for immune reactivity (and assessment of DC activation and maturation following coculture with blebosomes and exosomes harvested from BA-PDT treated tumor cells as described in the examples 1-5).

Fig. 1A shows the effect of BA-PDT of BxPC3 tumor cells to induce specific apoptosis pathway gene expressions that facilitate and induce enhanced tumor-associated antigen expression and antigenic bleb and exosome formation for activation of DCs and other immunocytes against BxPC3 tumor cells.

Fig. IB shows the effect of BA-PDT of BxPC3 tumor cells to induce specific necroptosis and pyroptosis pathway gene expressions that facilitate and induce enhanced tumor-associated antigen expression and antigenic bleb and exosome formation for activation of DCs and other immunocytes against BxPC3 tumor cells.

FIG. 1C shows the effect of BA-PDT of BxPC3 tumor cells to induce specific ER-stress pathway associated immunogenic gene expressions that generate tumor-associated antigen expression that are coupled to tumor antigenic bleb and exosome formation for activation of DCs and other immunocytes against BxPC3 tumor cells.

FIG. 2 A shows the effect of BA-PDT of PL-45 tumor cells to induce tumor-cell expression of exosomes (measured by increased expression of exosome markers CD9, CD81, and CD 63) that can contain anti-PL-45 tumor antigens

FIG. 2B shows the effect of BA-PDT of PL-45 tumor cells to induce tumor-cell expression of PL-45 specific exosomes that can contain anti -PL-45 tumor antigens FIG. 2C-i shows the effect of blebosomes and exosomes derived from BA-PDT of PL-45 tumor cells to induce activation and maturation of HLA-DR+CD1 lc+ dendritic cells (DCs) capable of activating T cells against the tumor antigens expressed by these DCs.

FIG. 2C-ii shows the effect of blebosomes and exosomes derived from BA-PDT of PL-45 tumor cells to induce activation and maturation of HLA-DR+ and CCR7+HLA-DR+ dendritic cells (DCs) capable of activating T cells against the tumor antigens expressed by these DCs.

FIG 2D-i shows the effect of T cells activated by DCs that were pulsed with blebosomes and exosomes isolated from BA-PDT treated PL45 tumor cells to kill naive PL45 tumor cells.

FIG. 2D-ii shows the effect of T cells that were stimulated with DCs that were pulsed with blebosomes and exosomes isolated from BA-PDT treated PL45 tumor cells to kill naive PL45 tumor cells.

FIG. 3 A shows the effect of BA-PDT of BxPC3 tumor cells to induce tumor-cell expression of exosomes (measured by increased expression of exosome markers CD9, CD81, and CD 63) that can contain anti-BxPC3 tumor antigens.

FIG. 3B shows the effect of BA-PDT of BxPC3 tumor cells to induce tumor-cell expression of BxPC3 specific exosomes that can contain anti-BxPC3 tumor antigens .

FIG 3C-i shows the effect of blebosomes and exosomes derived from BA-PDT of BxPC3 tumor cells to induce activation and maturation of HLA-DR+CD1 lc+ dendritic cells (DCs) capable of activating T cells against the BxPC3 tumor antigens expressed by these DCs.

FIG. 3C-ii shows the effect of blebosomes and exosomes derived from BA-PDT of BxPC3 tumor cells to induce activation and maturation of HLA-DR+, CD80+ HLA-DR+, CD83+ HLA-DR+, and CCR7+ HLA-DR+ dendritic cells (DCs) capable of activating T cells against the BxPC3 tumor antigens expressed by these DCs .

FIG. 4A shows the effect of BA-PDT of Pane- 1 tumor cells to induce tumor-cell expression of exosomes (measured by increased expression of exosome markers CD9, CD81, and CD 63) that can contain anti -Pane- 1 tumor antigens .

FIG. 4B shows the effect of BA-PDT of Panc-1 tumor cells to induce tumor-cell expression of Panc-1 specific exosomes that can contain anti-Panc-1 tumor antigens .

FIG. 4C-i shows the effect of blebosomes and exosomes derived from BA-PDT of Panc-1 tumor cells to induce activation and maturation of HLA-DR+, CD80+ HLA-DR+, and CCR7+ HLA-DR+ dendritic cells (DCs) capable of activating T cells against the Panc-1 tumor antigens expressed by these DCs .

FIG. 4Cii shows the effect of blebosomes and exosomes derived from BA-PDT of Panc-1 tumor cells to induce activation and maturation of HLA-DR+ CD1 lc+, CD80+, CD83+ and CCR7+ dendritic cells (DCs) capable of activating T cells against the Panc-1 tumor antigens expressed by these DCs.

FIG. 5 A. shows the effect of BA-PDT of Kasumi-1 tumor cells to induce tumor-cell expression of exosomes (measured by increased expression of exosome markers CD9, CD81, and CD 63) that can contain anti -Panc-1 tumor antigens.

FIG. 5B shows the effect of BA-PDT of Kasumi-1 tumor cells to induce tumor-cell expression of Kasumi-1 specific exosomes (expressing CD117, CD34, HSP 70, and HSP 90) that can contain anti -Kasumi-1 tumor antigens.

FIG. 5C-i shows the effect of blebosomes and exosomes derived from BA-PDT of Kasumi-1 tumor cells to induce activation and maturation of HLA-DR+, CD80+ HLA-DR+, and CCR7+ HLA-DR+ dendritic cells (DCs) capable of activating T cells against Kasumi-1 tumor antigens expressed by these DCs.

FIG. 5C-ii shows the effect of blebosomes and exosomes derived from BA-PDT of Kasumi-1 tumor cells to induce activation and maturation of HLA-DR+ CDl lc+, CD80+, and CCR7+ dendritic cells (DCs) capable of activating T cells against the Kasumi-1 tumor antigens expressed by these DCs.

DETAILED DESCRIPTION

As used herein, the terms “about” and “approximately” are defined as being within plus or minus (±) 10% of a given value or state, preferably within ±5% of said value or state.

The terms “tumor” and “solid tumor,” as used herein, refer to an abnormal tissue growth or mass (i.e., a neoplasm) comprising cells having one or more mutations and supportive cells (e g., non-cancerous stromal cells surrounding the mutated cancer cells). Tumors can be, e.g., benign, in situ, and/or malignant. A “cancer,” as used herein, refers to one or more malignant tumors. Tumor types treatable by the current invention include tumors of endodermal, mesodermal, and ectodermal origins and a non-limiting list of treatable tumors includes tumors of the brain, colon, breast, pancreas, lung, prostate, muscle, liver, and blood. The term “basal or baseline level” as used herein refers either 1) to the tumor status in a patient before the patient received treatment with APCs that were exposed to BA-PDT treated tumor cells or 2) to the condition of the APC before BA-PDT generated bleb/exosome treatment or 3) to the stable concentration following an overnight fast of humoral or neuronal factors in the subject’s plasma prior to treatment with the methods disclosed herein.

The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more compounds or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment of cancer).

The term “photosensitizer” refers to a molecule which when exposed to light of the appropriate wavelength absorbs the light and generates a cytotoxic reaction within a living cell.

The overall method for inducing regression of a mammalian tumor includes the following steps:

1. Isolating tumor cells from a tumor bearing individual by surgical removal using standard sterile surgical procedures and culturing such tumor cells in an art recognized cell culture growth medium for the isolated tumor cells.

2. Optionally administering a neuroendocrine immune-resetting therapy daily to the individual for a period of time from a few days to several weeks to overcome the systemic immunosuppression induced by the tumor and provide for a normalized or enhanced immune function against tumor. The resetting therapy can comprise at least one of the following administration procedures: a. administration of a dopamine agonist (e.g., a dopamine D2 receptor agonist such as bromocriptine or a dopamine DI receptor agonist such as a benzazapine analog [such as SKF38393 or its derivatives] or apomorphine within 4 hours of the time of daily waking from the natural nocturnal sleep cycle b. administering prolactin or a prolactin stimulating agent such as (a) dopamine receptor antagonists (e.g., domperidone); (b) prolactin (c) drugs enhancing serotoninergic neurotransmission (e.g. the serotoninergic precursors tryptophan and 5-hydroxytryptophan) within about 2 hours of the daily sleep time in a formulation and a manner to produce a circadian plasma pharmacokinetic profile that mimics that of a healthy individual of the same species and sex. c. administering melatonin within about 2 hours of the daily sleep time in a formulation and a manner to produce a circadian plasma pharmacokinetic profile that mimics that of a healthy individual of the same species and sex . d. administering thyrotropin releasing hormone within about 4 hours before the onset of daily sleep time.

The preferred resetting therapy is timed administration of TRH, melatonin, prolactin, prolactin stimulator, or a dopamine agonist alone or in combination in a manner that effectuates a bioavailability profile that mimics their circadian plasma or brain profile of these agents (TRH, prolactin, dopamine, and/or melatonin) of a healthy individual of the same species and sex.

3. Isolating APCs (e.g., dendritic cells (DCs)) from the tumor bearing individual a.APC’s are isolated from the subject in the conventional manner (e.g., isolated from plasmacytoid DCs or from blood monocytes that are then induced to transform into DCs in culture, or from bone marrow or spleen) and the isolated APC’s are maintained in culture medium for, between about 4 to 6 days, optionally with a dopamine agonist (10'¹⁰to 10'⁵ M) and/or prolactin (0.1 ng/ml to 10 ug/ml) pulsed into the culture medium for a brief period of time, generally for 1 to 240 minutes, at times of day that mimic their circadian peak stimulatory immune activity on APCs [DCs] - generally within 4 hours of the onset of daily waking for dopamine and within about 4 hours of the onset of daily sleep for prolactin.

4. Administering BA photosensitizer(s) to the tumor cells in vitro and then photoirradiating tumor cells with actinic light within the absorption peak range of the BA.

5. Presenting to the APCs of the tumor bearing individual the BA-PDT treated tumor cell fractions of such whole tumor cell aggregate as from post centrifugation of cellular sub-fractions to isolate for use certain cellular components consisting of exosomes, multivesicular endosomes, blebs via: a. a 300 g centrifugation pellet [nuclei, large cellular particulates] for use to stimulate APCs, or b. preferably, isolation of the 300 g centrifugation supernatant [containing cellular membranes, organelles, plasma membrane blebs and exosomes] for use to stimulate APCs, or c. more preferably, isolation of the 100,000 g centrifugation supernatant [cellular proteins, lipids, carbohydrates] for use to stimulate APCs to the cultured APCs within about 48 hours (preferably within 12 hours) following the BA-PDT and for a period of time thereafter, generally from 4 hours to 48 hours. The 100,000 g centrifugation pellet derived from the 300 g centrifugation supernatant is the preferred BA-PDT treated tumor cell subfraction for stimulation of the APCs in that it is cell-free, reducing contamination risk of re-introduction to the subject and also concentrates the BA-PDT treated tumor cell antigens in a form most responsive to by the APCs. d. most preferably, isolation of the 100,000 g centrifugation pellet from the 300 g centrifugation supernatant [containing exosomes and plasma membrane blebs] for use to stimulate APCs, or isolation of the 16,000 g centrifugation pellet from the 300 to 900 g centrifugation supernatant (processed in/containing polyethylene glycol) [containing exosomes and plasma membrane blebs] for use to stimulate APCs, or any methodology known in the art to isolate the cellular subfraction of blebs and exosomes from whole cell material.

6. Re-administering the APCs that have been primed with BA-PDT generated sub- cellular components (e.g., blebs and exosomes) back into the tumor bearing individual

7. Optionally, continuing the neuroendocrine-immuno- resetting therapy from step 2 continuously for between 2 to 30 days after step 6. In certain instances, this therapy can be administered for longer periods of time if necessary.

8. Optionally re-executing steps 1 through 6 or 7 periodically about every 2 to 24 weeks or more for as long as necessary to induce tumor regression or stasis, utilizing the subject’s cultured tumor cells from the first or subsequent isolation. 9. Optionally administration of in vivo BA-PDT either prior to and/or subsequent to the administration of the BA-PDT cell fraction primed APCs to the individual.

The in vivo BA-PDT method optionally includes the steps of optionally increasing the metabolic activity level of a subject having a malignant tumor, and more specifically increasing metabolic activity of the tumor within the subject to a level above its basal metabolic activity level of the tumor cells (see US patent 11,607,455 for details) (metabolic activity level is measured by, for example, MRI of the tumor mass or plasma metabolite levels), administering a BA dye to the subject, and thereafter exposing the tumor to actinic light for the BA dye.

In one embodiment the method additionally includes the steps of increasing the metabolic activity level (e g., rate of glucose, carbohydrate, lipid, amino acid, or protein utilization [anabolism or catabolism] that generates or consumes energy) in a subject having a malignant tumor to a level that is at least 10% above the basal metabolic activity level, administering a BA dye to the subject, and thereafter exposing the tumor to actinic light for the BA dye.

In another embodiment the method includes the steps of increasing the metabolic activity level (e.g., rate of glucose, carbohydrate, lipid, amino acid, or protein utilization) of the tumor of a subject having a malignant tumor to a level that is at least 10% above the basal metabolic activity level of the tumor of the subject, administering a BA dye to the subject, and thereafter exposing the tumor to actinic light for the BA dye.

Another embodiment includes the steps of raising the subject’s glucose and/or glutamine level by more than 10% above the subject’s basal glucose and/or glutamine level, administering a BA dye to the subject, and thereafter exposing the tumor to actinic light for the BA dye. (See US patent 11,607,455)

Yet another embodiment includes the step of increasing the plasma glucose level of the subject by administering one or more hyperglycemic agents to the subject. Suitable hyperglycemic agents for use in the method disclosed herein include glucose, carbohydrate, short chain saccharides such as sucrose, carbohydrates such as starch, or, glycogen, and glucagon for example.

In another embodiment the method includes the steps of administering BA dyes to a subject, comprising intravenously infusing into the subject a BA solution in a volume equal to between about one-tenth and one-third of the total blood volume of the subject at a rate that creates a plasma BA Tmax within a time frame extending to about 240 minutes following termination of infusion, followed by a plasma level less than about 50% of the Tmax level within about 60-240 minutes following the initial T_max time.

In another aspect, the method of treating a tumor includes the steps of increasing the ketone, lactate, lipid, glutamine, and/or free fatty acid (FFA) plasma level of a subject in need of such treatment to a level above the basal ketone, lactate, lipid, glutamine, and/or free fatty acid (FFA) plasma level, respectively of the subject, administering at least one BA to the subject, and thereafter exposing the tumor to actinic light for the BA.

1. In another embodiment the BA-PDT and metabolic adjustment technique of increasing the plasma glucose level above basal level of the subject by administering one or more hyperglycemic agents to the subject, is combined with stimulating or enhancing immune function in a vertebrate in need of such treatment, by administering thyrotropin releasing hormone (TRH), prolactin stimulating agent, melatonin, and/or dopamine agonist to the mammal to effectuate a plasma or brain peak of their levels that mimic the natural circadian peak of these agents in a healthy individual of the same species and sex. These agents can be administered to the vertebrate by any of intravenous administration, sublingual administration, subcutaneous administration, nasal administration, transmucosal administration, transdermal administration, and oral administration in a formulation that protects the TRH, prolactin, dopamine agonists or other resetting agents from gastrointestinal degradation.

The procedures employed in practicing the tumor regression technique of this disclosure are set forth below.

Resetting Therapy

It is known that circadian rhythms of neuroendocrine activity can influence immune function in the body. Several aspects of immune function themselves exhibit circadian or diurnal activity. Cancer (tumor cell growth) has the capacity to disrupt both neuroendocrine and immune circadian organization of immune function thus leading to a diminished ability of the immune system to combat or attenuate cancer growth. It has now been found that the metabolism altering activity-BA-PDT approaches described herein can be further enhanced to inhibit tumor growth by appropriate circadian timed administration of dopaminergic agonists (e.g., bromocriptine, dihydroergocriptine, dihydroergotoxine [hydergine], dopamine DI agonists such benzazepine analogs) at 0.001 to 1 mg/kg BW, and prolactin stimulating compounds (e.g., prolactin [at 0.01 to 100 ug/kg BW], or melatonin, tryptophan, 5-hydroxy- tryptophan [at 0.01 ug to 10 mg/kg/BW]). The dopamine agonist is administered at a time of day so as to effectuate a daily peak in brain dopaminergic activity that coincides with the natural circadian peak in such activity in healthy individuals without cancer (generally within about 4 hours of waking from the daily sleep cycle, preferably within about 2 hours of waking). The prolactin stimulating compound is administered at a time of day so as to effectuate a daily peak in plasma prolactin level that coincides with the natural circadian peak in such activity in healthy individuals without cancer (generally within about 4 hours of onset of daily sleep cycle, preferably just before the onset of sleep).

The natural circadian peak in such activity in healthy indi- vi duals without cancer (generally within about 4 hours of waking from the daily sleep cycle, preferably within about 2 hours of waking). The prolactin stimulating compound is administered at a time of day so as to effectuate a daily peak in plasma prolactin level that coincides with the natural circadian peak in such activity in healthy individuals without cancer (generally within about 4 hours of onset of daily sleep cycle, preferably just before the onset of sleep).

Photo irradiation of tumor cells with BAs to generate tumor antigens

BAs are first solubilized in acidified aqueous simple sugar solution (e.g., glucose, sucrose, monosaccharide, disaccharide, polysaccharide, with addition of acid [0.01 - 1.0% HC1 or acetic acid]) at 10‘³ to 10'⁵M. BAs are added to in vitro cultured tumor cells in phosphate buffered saline solution pH 7.4 to give a final concentration in the cell culture medium of between about 0.01 to about 10 uM and incubated for about 1 to 30 minutes at 37°C. Following this incubation period, (the cells are washed twice with buffered saline and then within about 120 minutes therefrom they are irradiated with actinic light in the 590-700 nm range, [preferably at or mostly at the lambamax for the BA (usually between 600 to 700 nm)] to give an energy density of 1-15 J/cm² and power density of about 0.1 - 30 mW/cm² (i.e, preferably for about 1 to 15 minutes at about 4 mW/cm²) and then returned to the culture medium for a period of about 1 to 12 hours. The BA-PDT treated tumor cells are then fractionated using one of the methods described above (preferably using the 100,000 g centrifugation pellet derived from the 300 g spin supernatant of whole BA-PDT treated tumor cells) for presenting such treated BA-PDT tumor cell fraction(s) to APCs and transferred to the standard APC growth culture medium following the BA-PDT procedure.

Co-culture of the BA-PDT treated tumor cells with APCs.

APCs are isolated from the individual by any of several means well known in the art, such as from bone marrow or obtaining blood samples containg circulating monocytes and, following removal of red blood cells, culturing the monocytes in nutrient medium to stimulate their conversion into dendritic cells (e.g., nutrient medium containing GMCSF plus IL-4 at about lOOOU/ml each with or without other growth factors). After several days in such culture, dendritic cell viability and expression can be determined by presence of dendritic cell surface markers. The APCs (e.g., dendritic cells) may be pulsed with dopamine agonist (0.01 nM to 1.0 uM) and/or prolactin (0.01 ng to lug/ml) each for a period of 1 to 240 minutes at the times of day that mimic the natural circadian peak of dopamine and prolactin in the circulation of healthy individuals of the same species and sex and this treatment may be continued for several days at which time the APCs or dendritic cells are pulsed with BA-PDT treated tumor cell fractions as described above.

At 1 to 12 hours following the BA-PDT event, either 1) whole BA-PDT treated tumor cells plus the culture medium that are removed from the culture flask container or 2) subfractions thereof constituting either a) supernatant from a 300 g centrifugation spin or b) the 100,000 g centrifugation pellet from the 300 g supernatant and/or such 100,000 g centrifugation supernatant) or c) the 16,000 g centrifugation pellet of the 300 to 900 g centrifugation supernatant (processed in/containing polyethylene glycol) or d) blebs and/or exosomes isolated from the BA-PDT treated tumor cells by any methodology known in the are - are transferred to the dendritic cells and co-cultured for a period of up to 96 hours. Such co-culture is sufficient to induce the activation and maturation of dendritic cells, to induce their processing of BA-induced tumor-associated antigen(s), and to allow for APC activation of an anti-tumor immune response via their own anti-tumor activation as well as stimulation of other immunocytes (e.g., T cells via cross priming) to destroy naive tumor cells (never exposed to BA-PDT) in vitro and in vivo. It should be noted that the BA-PDT treated cells or described sub-fractions thereof are now tumor- antigenic and capable themselves of inducing anti-tumor immunity. As such, the BA-PDT treated tumor cell fraction containing the tumor cell blebs and exosomes (for example from the 300 g supernatant or preferably from the 100,000 g pellet derived from the 300 g supernatant or from the 16,000 g pellet from the 300 to 900 g supernatant [processed in/containing polyethylene glycol]) may be introduced into the subject to induce anti -tumor immunity. However, the APC processing of such antigens is a safer and more robust method of inducing anti-tumor immunity with BA-PDT treated tumor cells or described cell fractions, particularly those containing the cellular blebs and exosomes.

It has now been found that tumor stasis and/or tumor regression including establishment of anti-tumor immunity and methods of preparing anti-tumor vaccines can be realized by the following methods.

1. Obtaining tumor cells from a tumor bearing individual by surgical removal using standard sterile surgical procedures and culturing such tumor cells in an appropriate cell culture growth medium (e.g., DMEM or RPMI 1640) for the isolated tumor cells. Excised tumor tissue is collected under sterile conditions , maintained in tissue culture medium (e.g., DMEM or RPMI 1640) at 4°C for a period of time, preferably from 1 to 48 hours until isolated tumor cells are harvested by simple enzyme (e.g., collagenase) digestion of extracellular matrix tissue, washed in cell culture medium (e.g., RPMI 1640 or other similar cell growth medium) by repeated centrifugation at 300 g and finally re-suspended in cell culture medium. Such cells are plated adherent to cell culture flasks or grown in suspension. The cells may be maintained in such medium at 37°C by scheduled subpopulation dilution (splitting) of cells in culture to prevent overgrowth and/or by preparing cells in glycerol medium for freezing storage at -80°C for future use in culture medium at 37°C and subsequent BA-PDT cell fraction isolation procedures. The APCs cells may be co-cultured with BA-PDT treated tumor cell fractions to create APC’ s that are activated against naive tumor cells as described above.

2. Optionally administering [either orally or non-orally-e.g. parenteral, sub-lingual, intranasal, topical,] a neuroendocrine immune-resetting therapy to the tumor bearing individual daily for a period of time from a few days to several weeks consisting of at least one of the following: a. administration of dopamine or a dopamine agonist (e.g., a dopamine D2 receptor agonist such as bromocriptine and/or a dopamine DI receptor agonist such as a benzazapine analog [such as SKF38393] within 4 hours of the time of daily waking within a dose range of 0.001 to 1 mg/kg body weight. b. administering prolactin (0.01 ng/kg to 500 ug/kg body weight) or a prolactin stimulating agent within 2 hours of the daily sleep time c. administering melatonin (0.01 ug/kg to 0.5 mg/kg body weight) within 2 hours of the daily sleep time in a formulation and manner to produce a circadian pharmacokinetic profile that mimics that of a healthy individual of the same species and sex. d. administering thyrotropin releasing hormone (0.01 to 300 ug/kg body weight) within about 4 hours before the onset of sleep time

3. Obtaining APCs (e.g., DCs) from the tumor bearing individual (for example by isolation of circulating monocytes followed by cell culture in medium necessary to facilitate their conversion to dendritic cells typically in the presence of GMCSF and IL-4 for several days) and concurrently and/or thereafter culturing them in appropriate culture medium for a period of time, generally between 1 to 6 days, optionally with dopamine/dopamine agonist and/or prolactin each circadian-timed -pulsed into the culture medium for a brief period of time, generally for 1 to 240 minutes, at times of day that mimic their circadian peak stimulatory immune activity on APCs [DCs] which would be at about the daily waking time (0500 to 0900 hours) for the dopamine/dopamine agonist and at about the daily sleep time for the prolactin (2000 to 2400 hrs).

3a. Optionally, T cells from the tumor bearing individual may be obtained from the blood (or if need be from a lymph node or spleen) and co-cultured along with the APCs.

4. Administering BA photosensitizer(s) to tumor cells in vitro (0.1 nM to 5 uM) and then photo-irradiating tumor cells with actinic light (0.1 to 15 J/cm²) within the absorption peak range of the BA (usually between 500 to 700 nm, preferentially between 600 to 700 nm of light).

5. At 4 hours post BA-PDT, contacting the BA-PDT treated tumor blebosome and exosome fractions isolated from such treated whole tumor cells (created by post centrifugation of cellular subfractions e.g., supernatant of a 300 g centrifugation, preferably 100,000 g pellet of the 300 g centrifugation supernatant, , or the 16,000 g centrifugation pellet of the 300 to 900 g centrifugation supernatant (processed with/containing polyethylene glycol)) to remove certain cellular components (nuclei, whole cell membranes, mitochondria, etc.). These isolated blebs and exosomes are then , concentrated and contacted with the individual’s cultured APCs within 48 hours of BA-PDT treatment, and co-cultured with the APCs for a period of time generally between about 12 to 96 hours.

6. Re-administering such APCs (usually 10⁵ to 10⁸ cells) that have now been primed with BA-PDT treated tumor cellular components with concentrated cellular blebs and exosomes back into the tumor bearing individual with or without such APC-primed T cells previously isolated from the subject.

7. Optionally, continuing the neuroendocrine-immuno- resetting therapy from step 2 continuously through to several days to a few weeks beyond the end of step 6.

8. Optionally administration of in vivo BA-PDT according to the in vivo BA-PDT steps set forth below at any point after step 2 but preferably after step 7 in this process.

9. If so desired, tumor re-immunization therapy can be continued at later dates following step 7 or 8 by redoing steps 1 through 6 or steps 3 through 6.

EXAMPLES

Example 1. Human pancreatic tumor cells (BxPC3) were cultured in vitro and exposed to BA-PDT as follows. BA-PDT with 2I-EtNBS in aqueous solution was administered to tumor cells (0.7 uM) in vitro for ~10 minutes and then cells were photoirradiated with red light for ~10 minutes (~3-4 J/cm²) and then cells and cellular debris was harvested at 4 hours after PDT for assessment of gene expression associated with ER stress, PANoptosis, and antigen generation and processing genes used for induction of immune reactivity. BA-PDT induces marked intracellular (organellar and cytoplasmic) reactive oxygen species generation that damages intracellular proteins that become antigenic when further processed during the induced ER stress event that is coupled to TAP processing of BA-PDT generated peptides for the production of MHC1 antigen generation - with surface antigen presentation to immune system and immune cell death (with or without PANoptosis) - and subsequent immunization against tumor. BA- PDT induction of specific gene expressions within tumor cells was assessed via standard techniques for real time qPCR using appropriate house-keeping gene as a base control for standardization and primers for each studied gene that were identified from the scientific literature as valid. Data are presented as change from basal (baseline) value of expression (i.e., without PDT stimulation). This BA-PDT of the BxPC3 cells induced marked induction of gene expression of several genes in the antigen processing and presentation pathways (see Figures 1A - 1C). This demonstrates that in vitro BA-PDT of tumor cells can initiate specific intracellular pathways that sequester specific antigens within specific cellular compartments for presentation to the immune system. Such BA-PDT of BxPC3 tumor cells increased the cellular gene expression of apoptosis/ER stress markers GRP78, PERK, ATF4, XBP1, CHOP, Caspase 3, and Caspase 9 by 2 to 16 fold (See Figures 1A for detailed presentation of these gene amplifications following BA-PDT of BxPC3 cells). Such BA-PDT of BxPC3 tumor cells increased the cellular gene expression of necrosis and pyroptosis markers Caspase 1, RIPK1, MLKL, GSDMD, HMGB1, by 3 to 4.5 fold (See Figure IB for detailed presentation of these gene amplifications following BA-PDT of BxPC3 cells). Such BA-PDT of BxPC3 tumor cells increased the cellular gene expression of ER stress - antigen processing pathway markers TAPI, TAP2, TAPBP, ERAP1, ERp57, CANX, CALR, B2M by 2 to 16 fold (See Figure 1C for detailed presentation of these gene amplifications following BA-PDT of BxPC3 cells).

Example 2. APCs [DCs] were isolated from human plasma monocytes by standard isolation procedures and transferred into culture medium containing GMCSF (lOOOU/ml) plus IL-4 (1000 U/ml). After 4-6 days the APCs were combined with specific 100,000 g centrifugation pellet derived from the 300 g centrifugation spin isolates of BA-PDT treated human pancreatic tumor cells containing plasma membrane blebs and cellular exosomes prepared as follows. Human pancreatic tumor cells (PL45) were grown in monolayer in culture medium to a confhiency of about 1-2 X 10⁶ cells/ 10 cm² transferred into phosphate buffered salt solution pH 7.4 and incubated with 0.7 uM BA for 15 minutes then washed and irradiated in APC culture medium or phosphate buffered salt solution pH 7.4 with 590-700 nm light for 10 minutes providing 3.3J/cm². Four hours after the BA-PDT irradiation a 100,000 g centrifugation isolate (pellet) from a prior 300 g centrifugation supernatant of the tumor cell composite including medium was transferred onto the APC cultured cells and co-cultured for 48 hours. At the end of the co-culture period, tumor exosome markers and various DC cell surface markers for dendritic cell activation and maturation were identified by flow cytometry using appropriate fluorescently tagged antibodies to the cell surface markers of interest in a MACSQuant flow cytometer. Changes in the detected levels of targeted markers were assessed and quantified using MACSQuant associated software version 2.11.1907.19925. Such experimental design resulted in 1) increased tumor cell production of vesicles expressing exosome markers CD9, CD81, CD63 by 2 to 4 fold (See Figure 2A for details of individual marker results) , 2) an increase in the production of exosomes/blebs expressing pancreatic tumor cell marker GPC1 by over 50 fold (See Figure 2B), and 3) a 2 to 50 fold increase in markers of the activation and maturation of dendritic cells HLA-DR+. CCR7+ HLA-DR+, (See Figure 2Ci for specific details) and HLA-DR+ CD1 lc+ and CCR7+ (See Figure 2Cii for specific details) relative to control dendritic cells not exposed to such BA-PDT treated tumor cell isolates. Moreover, co-culture of BA-PDT tumor cell isolate treated DCs with naive T cells (never exposed to tumor cells) activated such T cells with the induced ability to kill naive (non-BA-PDT treated) tumor cells (approximate 50 - 60% reduction in viable cells versus control group) (see figures 2Di - 2Dii for details).

Example 3. APCs [DCs] were isolated from human plasma monocytes by standard isolation procedures and transferred into culture medium containing GMCSF (lOOOU/ml) plus IL-4 (1000 U/ml). After 4-6 days the APCs were combined with specific 100,000 g centrifugation pellet derived from the 300 g centrifugation spin isolates of BA-PDT treated human pancreatic tumor cells containing plasma membrane blebs and cellular exosomes prepared as follows. Human pancreatic tumor cells (BxPC3) were grown in monolayer in culture medium to a confluency of about 1-2 X 10⁶ cells/ 10 cm² transferred into phosphate buffered salt solution pH 7.4 and incubated with 0.7 uM BA for 15 minutes then washed and irradiated in APC culture medium or phosphate buffered salt solution pH 7.4 with 590-700 nm light for 10 minutes providing 3.3J/cm². Four hours after the BA-PDT irradiation a 100,000 g centrifugation isolate (pellet) from a prior 300 g centrifugation supernatant of the tumor cell composite including medium was transferred onto the APC cultured cells and co-cultured for 48 hours. At the end of the co-culture period, tumor cell exosome markers and DC cell surface markers for dendritic cell activation and maturation were identified by flow cytometry using appropriate fluorescently tagged antibodies to the cell surface markers of interest in a MACSQuant flow cytometer. Changes in the detected levels of targeted markers were assessed and quantified using MACSQuant associated software version 2.11.1907.19925. . Such experimental design resulted in 1 increased tumor cell production of vesicles expressing exosome markers CD9, CD81, CD63 by approximately 10% to 4 fold (See Figure 3 A for details), 2) an increase in the production of exosomes/blebs expressing pancreatic tumor cell marker GPC-1 by about 100 fold (See Figure 3B for details), and 3) a 4 to 45fold increase in markers of the activation and maturation of dendritic cells CD80, CD83, HLA-DR+ CD1 lc+, HLA-DR+ relative to control dendritic cells not exposed to such BA-PDT treated tumor cell isolates (see Figures 3 Ci and 3Cii for details).

Example 4. APCs [DCs] were isolated from human plasma monocytes by standard isolation procedures and transferred into culture medium containing GMCSF (lOOOU/ml) plus IL-4 (1000 U/ml). After 4-6 days the APCs were combined with specific 100,000 g centrifugation pellet derived from the 300 g centrifugation spin isolates of BA-PDT treated human pancreatic tumor cells containing plasma membrane blebs and cellular exosomes prepared as follows. Human metastatic tumor cells (PANCI) were grown in monolayer in culture medium to a confluency of about 1-2 X 10⁶ cells/ 10 cm² transferred into phosphate buffered salt solution pH 7.4 and incubated with 0.7 uM BA for 15 minutes then washed and irradiated in APC culture medium or phosphate buffered salt solution pH 7.4 with 590-700 nm light for 10 minutes providing 3.3J/cm². Four hours after the BA-PDT irradiation a 100,000 g centrifugation isolate (pellet) from a prior 300 g centrifugation supernatant of the tumor cell composite including medium was transferred onto the APC cultured cells and co-cultured for 48 hours. At the end of the co-culture period, tumor exosome markers and DC cell surface markers for dendritic cell activation and maturation were identified by flow cytometry using appropriate fluorescently tagged antibodies to the cell surface markers of interest in a MACSQuant flow cytometer. Changes in the detected levels of targeted markers were assessed and quantified using MACSQuant associated software version 2.11.1907.19925. Such experimental design resulted in 1) increased tumor cell production of vesicles expressing exosome markers CD9, CD81, CD63 by approximately 10% to 3 fold (see Figure 4A for details), 2) an increase in the production of exosomes/blebs expressing pancreatic tumor cell marker GPC-1 by approximately 15 fold (see Figure 4B for details), and 3) a 3 to 1000 fold increase in markers of the activation and maturation of dendritic cells HLA-DR+, CD80+HLA-DR+,CCR7+ HLA-DR+,(see Figure 4Ci for details) and HLA-DR+ CD1 lc+, CD83+, CD80+, and CCR7+ (see Figure 4Cii for details) relative to control dendritic cells not exposed to such BA-PDT treated tumor cell isolates (see Figures 4ci - 4Cii).

Example 5. APCs [DCs] were isolated from human plasma monocytes by standard isolation procedures (give brief description) and transferred into culture medium containing GMCSF (lOOOU/ml) plus IL-4 (1000 U/ml). After 4-6 days the APCs were combined with specific 100,000 g centrifugation pellet derived from the 300 g centrifugation spin isolates of BA-PDT treated human leukemia tumor cells containing plasma membrane blebs and cellular exosomes prepared as follows. Human leukemia cancer cells (Kasumi-1) were grown in monolayer in culture medium to a confluency of about 1-2 X 10⁶ cells/ 10 cm² transferred into phosphate buffered salt solution pH 7.4 and incubated with 0.7 uM BA for 15 minutes then washed and irradiated in APC culture medium or phosphate buffered salt solution pH 7.4 with 590-700 nm light for 10 minutes providing 3.3J/cm². Four hours after the BA-PDT irradiation a 100,000 g centrifugation isolate (pellet) from a prior 300 g centrifugation supernantant of the tumor cell composite including medium was transferred onto the APC cultured cells and cocultured for 48 hours. At the end of the co-culture period cell, tumor exosome markers and DC surface markers for dendritic cell activation and maturation were identified by flow cytometry using appropriate fluorescently tagged antibodies to the cell surface markers of interest in a MACSQuant flow cytometer. Changes in the detected levels of targeted markers were assessed and quantified using MACSQuant associated software version 2.11.1907.19925. Such experimental design resulted in 1) increased tumor cell production of vesicles expressing exosome markers CD9, CD81, CD63 by 40% to 50 fold (see Figure 5 A for details), 2) an increase in the production of exosomes/blebs expressing kasumi-1 leukemia tumor cell markers, and 3) a 5 to 800 fold increase in markers of the activation and maturation of dendritic cells HLA-DR+ CD1 lc+, CD80+, CCR7+ (see Figure 5Ci for details) and HLA-DR+, CD80+ HLA- DR+, CD83+ HLA-DR+, and CCR7+ HLA-DR+ (see Figure 5Cii for details) relative to control dendritic cells not exposed to such BA-PDT treated tumor cell isolates.

Example 6. Rodents are injected with living tumor cells (0.1 - 1.0 million cells) and divided into four groups as follows: Group A: animals receiving only APC culture media with no APC exposure; Group B: animals receiving APCs with or without T cells from a donor animal; Group C: animals receiving syngeneic APCs with or without T cells that were previously cocultured with tumorcells; Group D: animals receiving syngeneic APCs previously co-cultured with BA-PDT-treated tumor cell bleb and exosome fraction isolates (that were obtained as follows): bone marrow is removed from sacrificed animals and following removal of red blood cells, bone marrow cells are cultured in RPMI 1640 for 6 days in the presence of GMCSF and IL-4 to promote differentiation of dendritic cells. On day 6 of culture the DCs are co-cultured with BA-PDT treated tumor cell isolates (10⁶ cells given 0.7uM 2- iodo-benzophenothiazine administration for 15 minutes at 37C followed by washing and irradiation with 590-700nm light for 10 minutes and 3.3J/cm² and 4 hours later isolated cellular subfractions [exosomes and membrane blebs) of these PDT treated cells were isolated, harvested, and added to the cultured APCs [including the DCs]) for 24 to 48 hours,. Tumor growth is subsequently monitored over the ensuing 28 day period. Relative to tumor growth within animals from Group A (exposed only to APC culture medium with no APCs (absolute control group), the growth of tumor among Group B animals treated with naive APCs (never exposed to tumor cells) or Group C animals administered APCs previously co-cultured with tumor cells is unchanged. However the tumor growth within Group D animals receiving the APCs previously co-cultured with blebs and exosomes from BA-PDT treated tumor cells according to the methods described herein demonstrates a significant reduction in tumor growth rate relative to Group A, B, and C animals.

Example 7. Rodents were injected with tumor cells and the tumor was allowed to grow to a volume of about 50 -100 mm³ at which point the tumor was subjected to BA-PDT by intravenous injection of BA at a concentration of 8 - 12 mg/kg BW followed 5 - 7 hours later by photoirradiation of the tumor with 600 - 670 nm wavelength light at an energy of about 130 to 200 J/cm² (and a fluence of about 130 to 200 mW/cm²). Several (4 to 8) months after this BA- PDT treatment, the animals were sacrificed and spleens from these BA-PDT treated animals containing immunocytes including DCs and T cells were removed, maintained viable, and administered to tumor naive animals that were subsequently administered live tumor cells (approximately 100,000 cells). Control tumor bearing animal groups received either no splenocytes or splenocytes from non-BA-PDT treated animals. Tumor growth was subsequently monitored in all three experimental groups. Relative to the control groups, tumor growth was inhibited by at least 50% in the group that received the administration of splenocytes from tumor-bearing animals that received BA-PDT.

Example 8. Tumor cells from individuals bearing either solid or non-solid tumors (e.g., circulating such as leukemias or intraperitoneal such as ovarian tumor cells) are harvested, maintained viable and cultured in growth medium until they are exposed to BA-PDT (by photoirradiation of the tumor with 600 - 670 nm wavelength light at an energy of about 130 to 200 J/cm² [and a fluence of about 130 to 200 mW/cm²]). The blebs and exosomes generated from this BA-PDT are harvested and subsequently cultured with the individual’s APCs with or without T cells (at a ratio of 1 tumor cell to 1-5 unit cell derived bleb/exosome) for a period of 24 to 48 hours. The APCs with or without T cells are then re-administered to the individual. This procedure is repeated every 2 to 12 weeks. Over time, the tumor growth rate within the individual is reduced.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

All patent applications, patents, and other publications cited herein are incorporated by reference in their entireties.

GENE DESCRIPTIONS 1. GRP78 (Glucose-Regulated Protein, 78kDa)

HSPA5 (Heat Shock Protein Family A Member 5)

Heat Shock Protein Family A (Hsp70) Member

BiP

Heat Shock 70kDa Protein 5 (Glucose-Regulated Protein, 78kDa)

Endoplasmic reticulum chaperone that plays a key role in protein folding and quality control in the endoplasmic reticulum lumen. Increases in its expression are associated with ER stress and can drive antigen presentation pathways after BA-PDT of tumor cells.

2. PERK (PRKR-Like Endoplasmic Reticulum Kinase)

EIF2AK3 (Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3)

Metabolic-stress sensing protein kinase that phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (EIF2Sl/eIF-2-alpha) in response to various stress conditions. Key activator of the integrated stress response (ISR) required for adaptation to various stress, such as unfolded protein response (UPR). The protein encoded by this gene phosphorylates the alpha subunit of eukaryotic translation-initiation factor 2, leading to its inactivation, and thus to a rapid reduction of translational initiation and repression of global protein synthesis. Increases in its expression are associated with ER stress and can drive antigen presentation pathways after BA-PDT of tumor cells.

3. ATF4 (Activating Transcription Factor 4)

CREB-2

Cyclic AMP-Dependent Transcription Factor ATF-4

Transcription factor that binds the cAMP response element (CRE) and displays two biological functions, as regulator of metabolic and redox processes under normal cellular conditions, and as master transcription factor during integrated stress response (ISR) and can drive antigen presentation pathways after BA-PDT of tumor cells.

4. XBP1 (X-Box Binding Protein 1)

Tax-Responsive Element-Binding Protein 5

Functions as a transcription factor during endoplasmic reticulum (ER) stress by regulating the unfolded protein response (UPR) and can drive antigen presentation pathways after BA-PDT of tumor cells.

5. CHOP (C/EBP Homologous Protein)

DDIT3 (NA Damage Inducible Transcript 3)

Multifunctional transcription factor in endoplasmic reticulum (ER) stress response. Plays an essential role in the response to a wide variety of cell stresses and induces cell cycle arrest and apoptosis in response to ER stress and can drive antigen presentation pathways after BA-PDT of tumor cells. 6. Caspase3 (CASP3)

Caspase 3, Apoptosis-Related Cysteine Peptidase

PARP Cleavage Protease

Thiol protease that acts as a major effector caspase involved in the execution phase of apoptosis. Following cleavage and activation by initiator caspases (CASP8, CASP9 and/or CASP10), mediates execution of apoptosis by catalyzing cleavage of many proteins and can drive antigen presentation pathways after BA-PDT of tumor cells.

7. Caspase9 (CASP9)

Caspase 9, Apoptosis-Related Cysteine Peptidase

Protein Phosphatase 1, Regulatory Subunit 56

Apoptotic Protease Activating Factor 3

Involved in the activation cascade of caspases responsible for apoptosis execution.

Binding of caspase-9 to Apaf-1 leads to activation of the protease which then cleaves and activates effector caspases caspase-3 (CASP3) or caspase-7 (CASP7). Promotes DNA damage-induced apoptosis in a ABLl/c-Abl-dependent mannre and can drive antigen presentation pathways after BA-PDT of tumor cells.

8. Caspasel (CASP1)

ICE (IL-1 Beta-Converting Enzyme)

Thiol protease involved in a variety of inflammatory processes by proteolytically cleaving other proteins, such as the precursors of the inflammatory cytokines interleukin- 1 beta (IL1B) and interleukin 18 (IL 18) as well as the pyroptosis inducer Gasdermin-D (GSDMD), into active mature peptides. Plays a key role in cell immunity as an inflammatory response initiator: once activated through formation of an inflammasome complex, it initiates a pro-inflammatory response through the cleavage of the two inflammatory cytokines IL1B and IL 18, releasing the mature cytokines which are involved in a variety of inflammatory processes and can drive antigen presentation pathways after BA-PDT of tumor cells.

9. RIPK1 (Receptor Interacting Serine/Threonine Kinase 1)

Receptor (TNFRSF)-Interacting Serine-Threonine Kinase 1

Serine-threonine kinase which is a key regulator of TNF-mediated apoptosis, necroptosis and inflammatory pathways.. Exhibits kinase activity-dependent functions that regulate cell death and kinase-independent scaffold functions regulating inflammatory signaling and cell survival and can drive antigen presentation pathways after BA-PDT of tumor cells. MLKL (Mixed Lineage Kinase Domain Like Pseudokinase)

HMLKL

Pseudokinase that plays a key role in TNF-induced necroptosis, a programmed cell death process. Does not have protein kinase activity. Activated following phosphorylation by RIPK3, leading to homotrimerization, localization to the plasma membrane and execution of programmed necrosis characterized by calcium influx and plasma membrane damage and can drive antigen presentation pathways after BA-PDT of tumor cells. GSDMD (Gasdermin D)

GSDMDC1

Precursor of a pore-forming protein that plays a key role in host defense against pathogen infection and danger signals (PubMed:26375003, 26375259, 27281216). This form constitutes the precursor of the pore-forming protein: upon cleavage, the released N- terminal moiety (Gasdermin-D, N-terminal) binds to membranes and forms pores, triggering pyroptosis. [Gasdermin-D, N-terminal]: Promotes pyroptosis in response to microbial infection and danger signals. Produced by the cleavage of gasdermin-D by inflammatory caspases CASP1, CASP4 or CASP5 in response to canonical, as well as non-canonical (such as cytosolic LPS) inflammasome activator and can drive antigen presentation pathways after BA-PDT of tumor cells. HMGB1 (High Mobility Group Box 1)

Amphoterin

SBP-1

HMG3

HMG1

Multifunctional redox sensitive protein with various roles in different cellular compartments. In the nucleus is one of the major chromatin-associated non-histone proteins and acts as a DNA chaperone involved in replication, transcription, chromatin remodeling, V(D)J recombination, DNA repair and genome stability. Proposed to be an universal biosensor for nucleic acids. Promotes host inflammatory response to sterile and infectious signals and is involved in the coordination and integration of innate and adaptive immune responses. In the cytoplasm functions as sensor and/or chaperone for immunogenic nucleic acids implicating the activation of TLR9-mediated immune responses, and mediates autophagy. Acts as danger associated molecular pattern (DAMP) molecule that amplifies immune responses during tissue injury and can drive antigen presentation pathways after BA-PDT of tumor cells. TAPI (Transporter 1, ATP Binding Cassette Subfamily B Member) RING4

PSF1

ABC transporter associated with antigen processing. In complex with TAP2 mediates unidirectional translocation of peptide antigens from cytosol to endoplasmic reticulum (ER) for loading onto MHC class I (MHCI) molecules.. Uses the chemical energy of ATP to export peptides against the concentration gradient and can drive antigen presentation pathways after BA-PDT of tumor cells. TAP2 (Transporter 2, ATP Binding Cassette Subfamily B Member)

RING11

PSF2

ABC transporter associated with antigen processing. In complex with TAPI mediates unidirectional translocation of peptide antigens from cytosol to endoplasmic reticulum (ER) for loading onto MHC class I (MHCI) molecules (PubMed:25656091, 25377891). Uses the chemical energy of ATP to export peptides against the concentration gradient and can drive antigen presentation pathways after BA-PDT of tumor cells. TAPBP (TAP Binding Protein)

TAPA

Tapasin

NGS17

This gene encodes a transmembrane glycoprotein which mediates interaction between newly assembled major histocompatibility complex (MHC) class I molecules and the transporter associated with antigen processing (TAP), which is required for the transport of antigenic peptides across the endoplasmic reticulum membrane. This interaction is essential for optimal peptide loading on the MHC class I molecule and can drive antigen presentation pathways after BA-PDT of tumor cells. ERAP1 (Endoplasmic Reticulum Aminopeptidase 1)

Endoplasmic Reticulum Aminopeptidase Associated With Antigen Processing

APPILS

Aminopeptidase that plays a central role in peptide trimming, a step required for the generation of most HLA class I-binding peptides. Peptide trimming is essential to customize longer precursor peptides to fit them to the correct length required for presentation on MHC class I molecules and can drive antigen presentation pathways after BA-PDT of tumor cells. Erap2 (Endoplasmic Reticulum Aminopeptidase 2

LRAP

Leukocyte-Derived Arginine Aminopeptidase

Aminopeptidase that plays a central role in peptide trimming, a step required for the generation of most HLA class I-binding peptides. Peptide trimming is essential to customize longer precursor peptides to fit them to the correct length required for presentation on MHC class I molecules and can drive antigen presentation pathways after BA-PDT of tumor cells. ERp57 (Endoplasmic Reticulum Resident Protein 57)

PDIA3 (Protein Disulfide Isomerase Family A Member 3) Endoplasmic Reticulum Resident Protein 60

GRP57

Glucose Regulated Protein, 58kDa

Protein disulfide isomerase that catalyzes the formation, isomerization, and reduction or oxidation of disulfide bonds in client proteins and functions as a protein folding chaperone. Core component of the major histocompatibility complex class I (MHC I) peptide loading complex where it functions as an essential folding chaperone for TAPBP and can drive antigen presentation pathways after BA-PDT of tumor cells.

19. CANX (Calnexin)

IP90

Major Histocompatibility Complex Class I Antigen-Binding Protein P88 Calcium-binding protein that interacts with newly synthesized monoglucosylated glycoproteins in the endoplasmic reticulum. It may act in assisting protein assembly and/or in the retention within the ER of unassembled protein subunits. It seems to play a major role in the quality control apparatus of the ER by the retention of incorrectly folded protein and can drive antigen presentation pathways after BA-PDT of tumor cells s. 0. CALR (Calreticulin)

CClqR

CRT

ERp60 (Endoplasmic Reticulum Resident Protein 60)

Calcium-binding chaperone that promotes folding, oligomeric assembly and quality control in the endoplasmic reticulum (ER) via the calreticulin/calnexin cycle. This lectin interacts transiently with almost all of the monoglucosylated glycoproteins that are synthesized in the ER and can drive antigen presentation pathways after BA-PDT of tumor cells. 1. B2M (Beta-2 -Microglobulin)

Beta Chain Of MHC Class I Molecules

IMD43

Component of the class I major histocompatibility complex (MHC). Involved in the presentation of peptide antigens to the immune system and can drive antigen presentation pathways after BA-PDT of tumor cells.

Claims

CLAIMS What is claimed is:

1. A method of inducing tumor regression in an animal afflicted with a malignant tumor which comprises

(i) treating tumor cells isolated from the tumor bearing animal with a BA photosensitizer

(ii) thereafter exposing the BA- treated tumor cells to actinic light

(ii) contacting a BA-PDT treated tumor cell fraction that contains tumor cell blebs and exosomes from the whole tumor cell and medium culture isolate with antigen presenting cells (APC) obtained from the tumor bearing mammal and

(iii) reintroducing the APC’s and the treated tumor cells to the mammal afflicted with the tumor.

2. The method of claim 1 wherein the BA-PDT treated tumor cell fraction comprises tumor cell plasma membrane blebs.

3. The method of claim 1 wherein the BA-PDT treated tumor cell fraction comprises tumor cell exosomes.

4. The method of claim 1 wherein the tumor cell fraction is the supernatant from a 300 g centrifugation of BA-PDT treated tumor cells.

5. The method of claims 1-3 wherein the tumor cell fraction comprises at least one of tumor cell plasma membrane blebs, exosomes and multivesicular endosomes.

6. The method of claim 1 wherein the blebs and exosomes are isolated from the BA- PDT treated tumor cells by differential centrifugation.

7. The method of claim 6 wherein the bleb and exosome isolation process comprises isolation of the 100,000 g centrifugation of supernatant from a preceding 300g centrifugation step of whole tumor cell isolate following BA-PDT.

8. The method of claim 6 wherein the bleb and exosome isolation process comprises isolation of the 16,000 g centrifugation of supernatant (processed in/containing polyethylene glycol) from a preceding 300 to 900 g centrifugation step of whole tumor cell isolate following BA-PDT.

9. The method of claims 1-5 which comprises administering a neuroendocrine immune-resetting therapy daily to the tumor bearing mammal prior to treating the isolated tumor cells in vitro with BA=PDT.

10. The method of claim 1 which comprises maintaining the isolated APC’s in culture medium for between about 2 to about 10 days prior to contact with the BA-PDT treated cells.

11. The method of claim 6 which comprises pulsing at least one of the dopamine agonist or into the culture medium within 4 hours of the onset of daily waking and/or prolactin at the onset of daily sleep of a healthy subject of the same species and sex.

12. The method of claim 8 comprising pulsing at least one of a dopamine agonist (0.1 nM to 1 uM) or prolactin (0.1 ng to 10 ug) into the culture medium for between about 1 to about 240 minutes.

13. The method of claim 1 which comprises administering the BA to the tumor cells in vitro and then photo-irradiating tumor cells with actinic light within the absorption peak range of the BA.

14. The method of claim 6 comprising continuing the neuroendocrine resetting step continuously for between about 2 to about 10 days.

15. The method of claim 1 which further comprises administration of in vivo BA- PDT treatment.

16. A method of treating a tumor in a subject in need of such treatment which comprises the following steps:

(a) isolating tumor tissue from the subject;

(b) culturing the isolated tumor cells in vitro;

(c) optionally administering at least one, member selected from the group consisting of prolactin, a prolactin stimulator, a dopamine agonist, melatonin and TRH to the subject for between 1 and about 20 days so that such administration produces a peak plasma level of such factors that mimics or amplifies the natural circadian peak levels of these natural factors in the brain or blood of a healthy individual;

(d) removing circulating immunocytes including monocytes from the blood of the tumor bearing subject;

(e) growing the removed immunocytes comprising monocytes in culture medium to induce transformation of the monocytes into dendritic cells (APC population);

(f) optionally pulsing the APC population comprising dendritic cells for between about 1 to about 360 minutes per day for one or more days with dopamine and/or prolactin at times of day that dopamine and prolactin normally peak in the circulation of a healthy individual of the same species;

(g) exposing the BA-PDT cultured tumor cells to actinic light of 590 to 700 nm wavelength plus BA;

(h) co-culturing the APC population comprising dendritic cells with the BA-PDT cell sub-fraction(s) therefrom comprising cellular blebs and/or exosomes re-administering the BA-PDT tumor cell primed dendritic cells to the subject in a dose of about 10⁵ to 10⁸ cells;

(j) optionally repeating steps a through i every 2 to 24 weeks after initiation of step a until a tumor stasis or regression response is obtained; and

(k) optionally continuing the circadian-timed daily administration of at least one member selected from the group consisting of prolactin, a dopamine agonist, melatonin and/or TRH for a period of 14 to 60 days in a manner that mimics or amplifies the natural circadian peak levels of these factors (prolactin, dopamine, melatonin, TRH)in the brain or plasma of a healthy individual.

17. The method of claim 13 which comprises administering to the subject at least one BA, and thereafter exposing the tumor to actinic light subsequent to step (i) or before step (a).

18. The method of claim 13 which comprises increasing the plasma glucose level of the subject to a level above the basal plasma glucose level of the subject subsequent to step (i) or before step (a), thereafter administering at least one BA to the subject, and exposing the tumor to actinic light.

19. A method of inducing tumor regression in patient afflicted with a malignant tumor which comprises collecting a sample of malignant tumor cells from the patient; treating the tumor cells with a BA photosensitizer; exposing the BA treated cells to actinic light; contacting a BA-PDT treated tumor cell fraction containing blebs and exosomes with antigen presenting cells (APC) obtained from the tumor bearing mammal; and reintroducing the APC’s and the treated tumor cells to the mammal afflicted with the tumor.

20. The method of claim 15 wherein the treated tumor cell fraction comprises cellular blebs or exosomes. The method of claim 13 or 16 wherein the tumor cellular blebs or exosomes are obtained from the 16,000 g pellet of the 300 to 900 g centrifugation supernatant that contains polyethylene glycol or from the 100,000 g pellet of the 300 g centrifugation supernatant.

21. The method of claim 1 or 13 wherein the BA is a member of the group consisting of benzophenoxazinium, benzophenothiazinium and benzophenoselenazinium compounds.

22. The method of claim 21 wherein the BA is 2-iodo-5-ethylamino-9-diethylamino- benzophenothiazine-(2I-EtNBS).

23. A method of treating a tumor in a subject in need of such treatment which comprises: (a) isolating tumor tissue from the subject;

(b) culturing tumor cells isolated from the tumor tissue in vitro;

(c) treating the cultured tumor cells with BA;

(d) exposing the BA treated cells to actinic light of 590 to 700 nm wavelength;

(e) optionally administering at least one member selected from the group consisting of prolactin, a prolactin stimulator, a dopamine agonist, melatonin and TRH to the subject for between 1 and about 20 days so that such administration produces a peak plasma level of such factors that mimics or amplifies the natural circadian peak levels of these natural factors in the brain or blood of a healthy individual;

(f) removing circulating immunocytes including T cells and monocytes from the blood of the tumor bearing subject;

(g) growing the removed immunocytes comprising monocytes in culture medium to induce transformation of the monocytes into dendritic cells (and growing the T cells in culture;

(h) Pulsing the dendritic cells and the T-cells for between about 1 to about 360 minutes per day for one or more days with dopamine and/or prolactin at times of day that dopamine and prolactin normally peak in the circulation of a healthy individual of the same species;

(i) co-culturing the dendritic cells with the BA-PDT cell sub-fraction(s) therefrom comprising cellular blebs and/or exosomes;

(j) co-culturing the dendritic cells previously exposed to BA-PDT cell sub-fraction(s) comprising cellular blebs and/or exosomes with the subject’s T cells;

(k) re-administering the BA-PDT tumor cell primed dendritic cells plus co-cultured T cells to the subject in a dose of about 10⁵ to 10⁸ DC cells and about 10⁵ to 10⁸ T cells or -readministering the co-cultured T cells to the subject in a dose of about 10⁵ to 10⁸ T cells; and

(l ) optionally repeating steps a through 1 every 2 to 24 weeks after initiation of step a until a tumor stasis or regression response is obtained.