JP6538647B2 - Ablation immunotherapy - Google Patents

Description

  The present disclosure relates generally to immunotherapy, and more specifically to therapeutic methods and compositions for treating tumor and pathogen infected tissue.

  Utilizing the power of the immune system to treat chronic infectious diseases or cancer is the main goal of immunotherapy. Active immunotherapeutic treatment is a method designed to activate the immune system to specifically recognize and destroy tumor or pathogen infected cells. For over 200 years, active immunotherapy approaches have been used to treat smallpox, rabies, typhoid fever, cholera, plague, measles, varicella, mumps, polio virus infection, hepatitis B and tetanus It has been able to prevent numerous infectious diseases including diphtheria toxin.

  The concept of active immunotherapy is currently to treat the existing tumor or to develop the therapeutic cancer vaccine of the present invention to prevent the recurrence of the tumor, and for the treatment and prevention of chronic viral infection Has been applied. Many of these techniques have been shown to successfully develop the increased impact of circulating immune cells with the ability to specifically kill tumor or pathogen infected cells. However, regardless of the ability to generate immune cells reactive to tumor antigens, the escape mechanism of the tumor can overwhelm this immune response, resulting in eventual tumor progression.

  Active immunotherapy of cancer has proven to be extremely effective in a number of rodent models. However, due to the disappointing results in clinical trials of various types of immunotherapy in humans for several decades, the human immune system does not perceive the threat / risk of human cancer cells, and does the same It is also shown that the immune system of rodent models of disease is also not perceived.

  The same applies to chronic viral infections. The innate immune response can slow down viral replication and activate cytokines that trigger the synthesis of antiviral proteins. The adaptive immune system neutralizes viral particles and destroys infected cells. However, viruses develop numerous counter measures to avoid the immune attack and are a moving target for the immune system.

U.S. Patent No. 6,875,849 U.S. Patent No. 6,797,480 U.S. Patent No. 6,187,312 U.S. Patent No. 6,162,436 U.S. Patent No. 6,139,841 U.S. Patent No. 6,136,315 U.S. Patent No. 5,837,251

  Provide an active immunotherapy that can overcome the tumor and virus immune evasion mechanisms and train the human immune system to perceive the threat / risk of human cancer cells and virus infected cells, tumor or pathogen infection It is necessary to generate an immune response that can eradicate the cells wherever they are located in the body.

SUMMARY The present disclosure relates to methods and compositions for eliciting a systemic adaptive immune response against a tumor or pathogen. The method uses a combination of allogeneic cell therapy and a method for subjecting a tumor or pathogen infected tissue to cell damage, a tumor specific antigen (s) or a pathogen specific antigen (s) Results in the release of

  In one aspect, the disclosure is a method for inducing an adaptive immune response against a tumor or pathogen in a subject. The method comprises the following steps: (1) For a subject with cancer or infectious disease, an aliquot of allogeneic cells designed to be rejected by the subject's immune system, Administering in a manner that induces anti-allogeneic Th1 immunity; and (2) after a time (about 7 to 14 days) allowing to form anti-allogeneic Th1 immune memory in the same subject The ablation method is used to perform in situ ablation of accessible tumor lesions or pathogen infected tissue, whereby at least a portion of the tumor or infected tissue is preferably by necrosis, such as, but not limited to, electropo , Cryoablation, chemotherapy, radiation therapy, ultrasound therapy, ethanol chemoablation, microwave thermal ablation And (3) preferably, a second aliquot of the same allogeneic cell is lesioned after 2-24 hours of ablation step. Subsequent injection of host antigen presenting cells (ie dendritic cells), which are injected inside (the same cells as used for priming) and respond to uptake of antigen (s) and necrosis or apoptotic tissue Creating an immune response that functions as an adjuvant for maturation. The mature antigen presenting cells from the lesion then migrate to the lymph nodes to stimulate systemic anti-tumor or anti-pathogenic immunity. In another aspect, the priming step is omitted. Tumor-derived or pathogen-infected tissue is ablated in situ and aliquots of allogeneic cells are injected after ablation to generate the desired immune response.

  In another aspect, the present disclosure includes a vaccine for a patient against a tumor or pathogen. The vaccine comprises an antigenic substance from a tumor or pathogen and an antigenic composition comprising an aliquot of allogeneic cells, wherein administration of the antigenic composition to the patient produces rejection and is directed against the antigen It stimulates a delayed type of hypersensitivity response, thereby acting as an adjuvant for the stimulation of systemic anti-tumor or anti-pathogen immunity in the patient. The vaccine may also comprise a priming composition, which comprises an aliquot of allogeneic cells.

  In still another aspect, the present disclosure includes a method of vaccinating a patient. The method is designed to be rejected by the subject's immune system in a manner that induces anti-allogeneic Th1 immunity to the subject having the following steps: (1) cancer or an infectious disease Administering a priming composition to induce an aliquot of the allogeneic cells; and (2) time to allow development of anti-allogeneic Th1 immune memory in the same subject (approximately 7 to 14 days) After that, preferably from the same individual autologous transplant lysate of infected or cancerous tissue, preferably a lysate containing chaperone protein, and an aliquot of allogeneic cells (the same cells used to prime the patient) A source of tumor antigen or pathogen antigen (eg, attenuated virus, tumor lysate, heat shock protein) comprising such a lysate formulated in The antigenic composition is preferably injected percutaneously to produce a delayed response to the alloantigen which causes rejection and which functions to agonize the stimulation of systemic anti-tumor or anti-pathogen immunity. Stimulating. In a further aspect, the method may be performed without a priming step.

In another aspect, the present disclosure includes a therapeutic composition for treating a tumor or pathogen in a patient, comprising an in situ generated tumor or pathogen antigen and allogeneic cells, wherein: The allogeneic cells and in situ generated antigens elicit an immune response whereby subsequent maturation of the patient's antigen presenting cells systemically stimulates anti-tumor or anti-pathogenic immunity. The tumor antigen or pathogen antigen is generated in situ from tumor necrosis or pathogen infected tissue. The therapeutic composition may also include a priming composition comprising allogeneic cells. The allogeneic cells in the priming composition and in the antigenic composition may comprise about 1 × 10 ⁸ to about 1 × 10 ¹⁰ cells.

In yet a further aspect, the present description is a vaccine composition for treating a patient's tumor or pathogen comprising an in situ generated antigen comprising a tumor antigen or a pathogen antigen, and allogeneic cells. Here, injecting the allogeneic cells into the patient in the presence of the in situ generated antigen produces an immune response, whereby the subsequent maturation of the patient's antigen presenting cells is systemically antitumor or Included are vaccine compositions that stimulate anti-pathogenic immunity. Tumor antigens in the composition are derived from in situ necrosis of the tumor. The pathogen antigens in the composition are derived from the in situ necrosis of pathogen infected tissue. The composition may also include a priming composition comprising allogeneic cells. The allogeneic cells in the priming composition and in the vaccine composition may comprise about 1 × 10 ⁸ to about 1 × 10 ¹⁰ cells.

In a further aspect, the disclosure includes a method of vaccinating a patient. The method comprises the following steps: (1) to be rejected by the subject's immune system in a manner that induces anti-allogeneic Th1 immunity to the subject with cancer or infectious disease. Administering a priming composition comprising an aliquot of the allogeneic cell being designed; and (2) time to allow development of anti-allogeneic Th1 immune memory in the same subject (approximately 7 to 14). After day) ablating the tumor or pathogen infected tissue in situ to release the tumor antigen or pathogen antigen, and (3) the composition of allogeneic cells (the same cells used to prime the patient) Administering a rejection response to the alloantigen which acts to adjuvant the stimulation of systemic anti-tumor or anti-pathogenic immunity and stimulate a delayed type hypersensitivity reaction. In a further aspect, the method may be performed without a priming step.
The present application provides, in particular embodiments, the following items:
(Item 1)
Therapeutic compositions for treating a tumor or pathogen in a patient:
With allogeneic cells;
An antigen comprising the product of tumor necrosis or necrosis of a pathogen infected tissue, wherein the antigen is generated in situ whereby allogeneic cells and antigens generated in situ elicit an immune response by the patient to produce an anti With antigens that create tumor or antipathogenic immunity;
Therapeutic composition comprising:
(Item 2)
A vaccine composition:
An adjuvant comprising allogeneic cells;
An antigen generated in situ by necrosis of cells or tissues of a disease, wherein the antigen generated in situ with the presence of the adjuvant at the site of necrosis induces an immune response by the patient to the disease An antigen that produces immunity,
Vaccine compositions, including.
(Item 3)
The composition according to item 1 or 2, further comprising a priming composition, wherein the priming composition comprises allogeneic cells.
(Item 4)
3. A composition according to item 1 or 2, wherein said antigen is generated in situ by ablation of a tumor or pathogen infected tissue.
(Item 5)
5. The composition according to item 4, wherein the ablation is cryoablation.
(Item 6)
5. The composition according to item 4, wherein the ablation is electroporation.
(Item 7)
The composition according to item 1 or 2, wherein the antigen comprises a tumor or pathogen antigen, a heat shock protein and a combination thereof.
(Item 8)
The composition according to item 1 or 2, wherein the allogeneic cell is an activated T cell.
(Item 9)
The composition according to item 1 or 2, wherein the allogeneic cells express high levels of type 1 cytokines.
(Item 10)
The composition according to item 1 or 2, wherein the allogeneic cells express high density of CD40L, TRAIL, FasL and a combination thereof on the cell surface.
(Item 11)
A method of inducing an adaptive immune response against a tumor or pathogen in a patient, comprising:
Producing an antigen from a disease or pathogen infection in situ;
Administer allogeneic cells to the site of the in situ generated antigen, thereby producing an immune response that functions as an adjuvant for antigen uptake from the ablated tissue, and thereby subsequent maturation of the patient's antigen presenting cells Stimulates anti-tumor immunity or anti-pathogen immunity systemically;
Method, including
(Item 12)
The method according to item 11, further comprising:
Administering to said patient a priming composition in situ prior to production of the antigen, wherein said priming composition is rejected by the patient's immune system in a manner that induces allogeneic Th1 immunity To contain allogeneic T cells, and
Allowing the patient's immune system to form anti-allogeneic Th1 immune memory prior to generation of in situ antigen;
Method, including
(Item 13)
12. A method according to item 11, wherein the antigen is generated in situ by ablation of tumor or pathogen infected tissue.
(Item 14)
14. A method according to item 13, wherein the ablation is by cryoablation.
(Item 15)
14. The method according to item 13, wherein the ablation is by electroporation.
(Item 16)
The method according to claim 11, wherein the antigen comprises a tumor or pathogen antigen, a heat shock protein and a combination thereof.
(Item 17)
12. The method according to item 11, wherein the allogeneic cells are allogeneic T cells.
(Item 18)
12. The method according to paragraph 11, wherein the allogeneic cells express high levels of type 1 cytokines.
(Item 19)
12. The method according to item 11, wherein the allogeneic cells express high density of CD40L, TRAIL, FasL and their combination on the cell surface.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS The present disclosure includes methods for stimulating anti-tumor or anti-pathogen immunity in a patient. This method involves the initial "priming" of a patient to develop Th1 anti-alloantigen immune memory by infusion of aliquots of allogeneic cells. It is desirable that infusion of allogeneic cells stimulate the patient's immune system to respond to allogeneic cells. A time interval is passed until the patient's immune system is able to form anti-allogenic memory. In some embodiments, the patient may need a booster of allogeneic cells to develop appropriate Th1 immune memory.

  Patients as used herein include not only mice but also humans. A Th1 response, as used herein, refers to the generation of cytokine profiles that activate T cells and macrophages. The Th1 response should be distinguished from the Th2 response, which mainly activates the immune response depending on the antibody and competes with the Th1 response.

  The next step involves damage and / or death of cells in the tumor bed or pathogen infected tissue after the patient has developed sufficient anti-allogeneic Th1 immune memory. Tissue injury or death releases cellular components and recruits scavenger cells to the site of injury. The release of cellular components during tissue damage or tissue death by necrosis is important for the in situ generation of tumor or pathogen antigens. Various methods are known in the art to cause tissue damage or death in a tumor bed or pathogen infected tissue. Preferably, the death is due to necrosis, which results in the recruitment of scavenger cells to the site of injury. In some preferred embodiments, the death or damage of the tissue is by cryoablation or by irreversible electroporation. Alternatively, the tissue is ablated ex vivo and the released component is infused into the patient.

  Scavenger cells, including immature dendritic cells, can pick up antigens released from damaged or dead tissue. A second aliquot of allogeneic cells is injected into the lesion resulting in maturation of dendritic cells (DV) for priming of Th1 immunity to the antigen. Intralesion means administration of a composition of the present invention through injection or otherwise directly to the cancerous area or tumor or pathogen infected tissue. In a preferred embodiment, all of the allogeneic cells administered to the patient are from the same source. Preferably, the allogeneic cells are administered approximately 2 to 24 hours after ablation of the tissue. This method is particularly useful in the treatment of solid or metastatic tumors, particularly in patients with tumor lesions in the prostate, breast, bone, liver, lung or kidney.

  Introduction of a second aliquot of allogeneic cells resulting in rejection of allogeneic cells due to the fact that the patient was primed for Th1 immunity against allogeneic cells induced by the first aliquot At the same time, it is desirable for patients to develop a strong delayed type hypersensitivity (DTH) response. The bystander effect of the anti-allogeneic DTH response can result in "danger signals" that act to generate DCs, collecting, processing, and maturation of the antigen from the damaged tissue, draining lymph nodes Migrate to The combination of induction of tissue damage and DTH rejection can result in an inflammatory environment, which leads to Th1 immunity to antigens released from the damaged tissue.

  The general state of inflammation produced by the treatment process is that DCs can function to program T cells to Th1 immunity to antigens in the damaged tissue, and a systemic adaptive immune response to tumor or pathogen infected cells, And incapacitation of tumor and pathogen-mediated immune evasion mechanisms. Adaptive immunity means that the protection of the patient is mediated by B and T cells after exposure to antigen, and that such protection exhibits specificity, diversity, memory and self / non-self recognition. Do. Such adaptive immunity is systemic within the patient. Adaptive immunity should be distinguished from innate immunity, which is nonspecific and present prior to exposure to the antigen.

  In some embodiments, following ablation, administration of allogeneic cells may be sufficient to produce a desired response. In other words, priming of the patient with a first aliquot of allogeneic cells may be omitted. In these embodiments, tissue from a tumor or tissue infected with a pathogen is ablated and subsequently injected with an aliquot of allogeneic cells.

  The invention also encompasses methods of vaccinating a patient having cancer cells or infected tissue. This method may be used to vaccinate patients with in situ generated antigens from cancer cells or infected tissues in combination with the administration of allogeneic cells. The method also included hematologic malignancies (eg, chronic lymphocytic leukemia, multiple myeloma, and non-Hodgkin's lymphoma) or viral infectious diseases (eg, hepatitis B or C, HIV C) and diseased It may also be used in patients with other disorders where the lesion is not easily accessible for ablation.

  This method involves the first "priming" of a patient to develop Th1 anti-allogeneic immune memory by infusion of a first aliquot of allogeneic cells. Infusion of allogeneic cells is desirable to stimulate the patient's immune system to respond to allogeneic cells. A period of time is allowed to elapse until the patient's immune system is able to form anti-allogenic memory. In some embodiments, the patient may need a booster of allogeneic cells to develop appropriate Th1 immune memory.

  The next step involves exposing the patient's immune system to antigens derived from cancerous cells or pathogen infected tissue in addition to the activated allogeneic cells described herein. In a preferred embodiment, the antigen is generated in situ by ablation of cancerous cells / tumor or pathogen infected tissue. In situ ablation of tumor or pathogen infected tissue results in the release of tumor antigen or pathogen antigen in the patient. Subsequent administration of allogeneic cells at the site of the lesion produces the desired immune response by producing rejection in the patient and stimulating delayed type hypersensitivity to the antigen. Ablation may also be by cryoablation, electroporation or other means that result in necrotic killing of the tumor or pathogen infected tissue.

  In another embodiment, following the priming step, the method may include the step of administering to the patient an antigenic composition comprising an autologous lysate comprising an antigen from a cancerous cell or infected tissue. . The composition also comprises an aliquot of allogeneic cells, ie allogeneic cells from the same source as the allogeneic cells used in the priming step. Injection of the antigenic composition can result in rejection in the patient and can stimulate a delayed type hypersensitivity reaction to the antigen.

  Scavenger cells, including immature dendritic cells, can pick up antigen resulting from in situ ablation or from autologous lysates. Allogeneic cells can result in maturation of dendritic cells for priming of Th1 immunity to antigen. Due to the fact that the patient primed Th1 immunity against allogeneic cells induced by the first aliquot of allogeneic cells during the priming step, the allogeneic cells at the site of necrotic lesions It is desirable for the patient to develop a strong DTH response upon induction.

  The general state of inflammation produced by the treatment process is that DCs can function to program T cells to Th1 immunity to antigens at the ablation site, and a systemic adaptive immune response against tumor or pathogen infected cells, as well as This results in the disabling of the tumor and pathogen-mediated immune evasion mechanism.

  The disclosure also describes methods for enhancing the immunogenicity of weakly immunogenic or non-immunogenic tumors, and immune responses (eg, Th1) that are protective against non-protective immune responses (eg, Th2 responses). Provide a way to bias the immune response). Such diseases include, for example, all types of cancer and infections with various pathogens (eg hepatitis virus, fungal infections such as Aspergillus, HIV, malaria, typhoid fever, cholera, herpes virus, chlamydia and HPV) And diseases caused by

  The present disclosure also encompasses therapeutic compositions for treating tumors or pathogens in patients. The therapeutic composition preferably comprises in situ generated allogeneic cells and an antigen, which comprises the product of tumor necrosis or necrosis of pathogen infected tissue. The therapeutic composition may also include a priming composition. The priming composition generally comprises allogeneic cells infused into the patient to generate rejection by the patient's immune system in a manner that induces allogeneic Th1 immunity.

  The therapeutic composition comprises aliquots of antigenic material and allogeneic cells from tumor or pathogen infected tissue. In a preferred embodiment, the antigenic substance is an in situ released antigenic substance comprising an antigen from a cancerous cell or from an infectious tissue. The antigenic material can be derived in situ from necrosis of tumor or pathogen infected tissue. Preferably, the antigenic substance is derived from ablation of a tumor or pathogen infected tissue.

  Ablation may be performed in vivo, in situ, or ex vivo. In some embodiments, the antigenic substance comprises a heat shock protein released upon ablation of a tumor derived tissue or pathogen infected tissue.

  Therapeutic compositions also include allogeneic cells. In embodiments with in situ generated antigenic material, the therapeutic composition comprises allogeneic cells administered directly to the lesion site containing the in situ generated antigen. The allogeneic cells, when injected into a patient, cause rejection and stimulate a delayed type hypersensitivity reaction to antigens, thereby acting as an adjuvant for stimulation of systemic anti-tumor or anti-pathogenic immunity in the patient. Do.

  Therapeutic compositions may include priming compositions and other compositions that act as adjuvants to the reaction produced by the allogeneic cells. The priming composition and allogeneic cells may also contain other components commonly found in therapeutic compositions, such as preservatives. The addition of these components is within the scope of the present invention.

  In some embodiments, the therapeutic composition may only include allogeneic cells and antigens generated in situ, but not the priming composition. The priming composition may not be necessary to obtain the desired immune response.

  The invention also encompasses vaccine compositions for patients against tumors or pathogens. The vaccine preferably comprises a priming composition and an antigenic composition. Priming compositions generally comprise allogeneic cells that are injected into a patient to cause rejection by the patient's immune system in a manner that induces allogeneic Th1 immunity.

  The antigenic composition comprises an antigenic substance from a tumor or pathogen infected tissue, and an aliquot of allogeneic cells. In some embodiments, the antigenic substance is an autolytic solution comprising an antigen from a cancerous cell or from an infected tissue. The antigenic material may be from tumor necrosis of a tumor or necrosis of pathogen infected tissue. Preferably, the antigenic substance is from ablation of a tumor or pathogen infected tissue. The ablation may be in vivo or ex vivo. In some embodiments, the antigenic substance comprises a heat shock protein released upon ablation of a tumor derived tissue or pathogen infected tissue. The antigenic composition also comprises allogeneic cells. The antigenic substance and the allogeneic cells may be combined together or packaged separately.

  The present disclosure also encompasses vaccine compositions generated in situ. The antigenic substance in this vaccine is generated in situ and allogeneic cells are administered as an adjuvant to the patient, preferably within the lesion. In situ generation of antigenic material may be achieved by ablation of tumor or pathogen infected tissue in a patient that releases the antigenic material. The ablation may be by cryoablation, electroporation or other methods that result in necrotic death of the tumor or pathogen infected tissue.

  In embodiments with the in situ generated antigen, the antigenic composition administered to the patient comprises allogeneic cells, which are administered at the necrotic lesion site containing the released antigen. Ru. The antigenic composition, when injected into a patient, causes rejection and stimulates a delayed type hypersensitivity reaction to the antigen, thereby acting as an adjuvant for stimulation of systemic anti-tumor or anti-pathogenic immunity in the patient. Do.

  The vaccine may comprise a priming composition and other components that act as an adjuvant to the reaction generated by the antigenic composition. The priming composition and the antigenic composition may comprise other ingredients commonly found in vaccines, such as preservatives. The addition of all of these components is within the scope of the present invention.

  In some embodiments, the vaccine may only comprise an antigenic composition and not a priming composition. The antigenic composition may be sufficient to obtain the desired immune response.

  The therapeutic vaccine of the present invention is useful for the prevention and treatment of diseases such as cancer or chronic viral diseases that develop and / or persist by suppressing or avoiding the immune response.

  The purpose of the priming step is to generate anti-allogeneic Th1 immunity in patients that can be recalled upon subsequent exposure to alloantigen. Priming results from exposing the patient to an aliquot of allogeneic cells and, when a second aliquot is administered to the patient, the subsequent rejection of these allogeneic cells by the patient's immune system arising from immune memory. Preferably, the patient is not immunosuppressed prior to priming. This is because it inhibits the ability of the patient to reject infused allogeneic cells and also inhibits the development of anti-allogeneic Th1 immunity.

  In one embodiment of the invention, the patient's immune system is distorted to generate Th1 immunity. It is preferred to engineer allogeneic cells so that Th1 immunity develops in response to allogeneic cell rejection but not Th2 immunity. In one embodiment, the patient's immune system may be skewed to produce a Th-1 response by administering allogeneic cells that, when injected, produce Th1 cytokines (eg, IFNγ and TNFα) . Th1 cytokines can help to distort the immune response to alloantigens to Th1-type immunity. Other methods of distorting the patient's immune system to generate Th1- immunity are also within the scope of the present invention.

  The allogeneic cells used to initially prime the patient and then later used as an adjuvant to the intralesional administration (after induction of cell death) or to the source of the pathogenic or tumoral substance are preferably allogeneic Heterologous activated T cells, more preferably allogeneic activated CD4 + Th1 cells, more preferably allogeneic CD4 + T cells (differentiated into effector or memory cells, high level of type 1 Cytokines such as IL-2, IL-15, IFN-γ, TNF-α, and preferably at high density, express effector molecules such as CD40L, TRAIL, and FasL on the cell surface, but not IL Neither -4 nor other type 2 cytokines). CD40 ligation of innate immune cells (e.g. dendritic cells, macrophages and NK cells) results in high levels of the cytokine IL-12 (which polarizes CD4 + T cells towards Th1-type immunity and proliferation of CD8 + T cells And have the ability to induce NK cells). These proinflammatory events may allow for consistent development of Th1 immunity to alloantigens on allogeneic cells upon rejection by the patient's immune system.

In the priming step, activated allogeneic T cells are preferably administered intravenously to the patient, but may be administered transdermally. The allogeneic cells are preferably intentionally derived from HLA mismatched donors. Preferred dosages in aliquots of allogeneic cells for intravenous infusion are at least about 1 × 10 ⁷ cells, more preferably about 1 × 10 ⁸ to 1 × 10 ¹⁰ cells. Doses of allogeneic cells outside this range that can primarily produce an immune response are also within the scope of the invention.

  It is desirable to test a patient for the development of Th1 anti-allogeneic immunity prior to ablation of the affected tissue or administration of the antigenic composition. The development of Th1 anti-allogeneic immunity may take at least about 7 days. Preferably, the patient takes about 7 days to about 14 days to develop Th1 anti-allogeneic immunity. The development of Th1 anti-alloantigen immunity can be measured, for example, by the ELISPOT assay. Other methods of testing a patient for the development of Th1 anti-alloantigen immunity are also within the scope of the invention. If Th1 anti-allogeneic immunity is weak, additional booster injections of allogeneic cells may be administered. Booster injections are preferably given intradermally to produce a delayed type hypersensitivity (DTH) response in the skin.

Generation of Allogeneic T Cells It is desirable that allogeneic T cells can be generated upon activation and infusion into a patient such that Th-1 immunity can be generated by that patient. Preferred methods for generating allogeneic cells with properties necessary for stimulation of anti-allogeneic Th1 immunity include: (1) mononuclear cell delivery by leukapheresis from a normal screened donor (2) isolation of CD4 T cells from source material; (3) activation of CD4 + cells by anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) fixed on days 0, 3 and 6; 4) Cell reactivation on day 9 with immobilized anti-CD3 and anti-CD28 mAbs, and injection of cells within 24 hours of activation.

Cell death phase Cell death or cell damage in situ results in recruitment of DC to the lesion and can provide a source of antigen for uptake by DC. The target tissue is preferably destroyed by a process in which necrosis results in cell death. Necrosis means the death of an individual cell or group of cells such that the amount of components within the cell is released into the environment. For the purpose of the present application, necrosis includes cell death by various methods, such as cryoablation, irreversible electroporation, chemotherapy, radiation therapy, ultrasound therapy, ethanol chemoablation, microwave Thermal ablation, radio frequency energy or combinations thereof. Necrottically killed cells activate the intrinsic signal of injury responsible for DC recruitment and maturation, a stimulus that does not occur in healthy or apoptotic dying cells. Furthermore, exposure of immature DCs to these stimuli provides maturation signals that are important for the initiation of local and systemic Th1 immunity.

In one preferred embodiment, it is preferable to freeze the target tissue in situ to cause death due to necrosis. Cryosurgery (cryosurgery) is a well-known, controlled procedure that can induce tissue necrosis by the application of liquid N ₂ or argon gas. The biological changes that occur during and after cryosurgery have been studied in vitro and in vivo. Tissue damage and necrosis are induced by cell freezing and by blood stasis that develops after thawing. Cryosurgery (in-situ freezing) is known to elicit an antigenic stimulus (comparable to the stimulus obtained through parenteral administration of the antigen) which can produce a specific immunogenic response to the frozen tissue's autologous antigen. there were.

  Cryoablation can release the peptide from lysed tumor or pathogen infected cells for antigen processing by DCs and can create an environment of proinflammatory cytokines. Cytokine released after cryoablation, such as IL-1, IL-2, TNF-α, IFN-γ, and GM-CSF are essential for the immune response that can destroy cancer or pathogen infected cells T cells, NK cells And langerhans cells can be activated.

  In another preferred embodiment, it is preferable to subject the target tissue to irreversible electroporation in order to cause death by necrosis. Irreversible electroporation is a tissue ablation technique in which micro-to-millisecond electrical parl is delivered to tissues to cause cell death by irreversible cell membrane permeabilization. In irreversible electroporation, the cell membrane of the cells between the electrodes is disrupted resulting in cell necrosis. Irreversible electroporation can release antigen from lysed tumor or pathogen infected cells for antigen processing by DCs and can create an environment of proinflammatory cytokines.

  Another preferred method for generating a source of antigen is to isolate the autologous chaperone protein, also known as heat shock protein (HSP), from dead infected tissue or tumor. HSPs are among the main targets of the immune response to bacterial, fungal and parasitic pathogens. Certain chaperones in the extracellular environment also modulate innate and adaptive immunity due to their ability to chaperone polypeptides and interact with the host's immune system, particularly the specialized antigen presenting cells. obtain. Vaccination with heat shock proteins from tumors has been shown to elicit an anti-tumor response. Current research indicates that the immunogenicity of HSPs is derived from the antigenic peptides with which they associate.

  A preferred method for the isolation of chaperone proteins for use as a source of antigen is described by Katsantis in US Pat. Additional methods are described by Srivastava in US Pat.

Adjuvant Step The purpose of the adjuvant step is to mature DC and stimulate Th1 immunity against antigens harvested at lesions containing dead target tissue. This may be achieved by injection of the same allogeneic cells, ie allogeneic cells of the same origin as were used to prime the patient. This aliquot of allogeneic cells is preferably injected directly into the lesion, ie, into a necrotic lesion resulting from cryoablation or other method of cell death. Alternatively, where chaperone proteins are used as a source of antigen, the same allogeneic cells used to prime the patient are injected, preferably transdermally, with the chaperone proteins. The dose of allogeneic cells that produce the desired immune response is generally at least about 1 × 10 ⁷ cells, more preferably about 1 × 10 ⁸ to 1 × 10 ¹⁰ cells. Allogeneic cells outside this range that are capable of generating the desired immune response are also within the scope of the present invention. Preparation of allogeneic cells is the same as described above.

  In order to mount an immune response and to overcome natural tolerance, the immune system has a self tissue, and antigens released after necrotic cell death or associated with chaperone proteins are taken up by DC It must be presented with an immunostimulatory component that signals "danger". Memory immune responses to allogeneic cells create this "risk".

This tissue resident DC (referred to as immature DC) is capable of capturing in situ generated antigens from the environment but lacks stimulation of T cells. In response to pathogen infection and the resulting inflammatory response, DCs undergo a differentiation process called maturation, thereby upregulating their ability to migrate to the draining lymph nodes to capture the captured antigen into T cells. To present. In order to activate Th1 CD4 ⁺ T cells and CTLs, DCs have to integrate multiple maturation / differentiation stimuli. At the site of pathogen or tumor encounter, exposure to pathogen or tumor derived determinants, proinflammatory cytokines, and / or cell debris induce the first stage of the maturation process. This involves upregulation of costimulatory molecules and chemokine receptors, whereby DCs each present antigen to T cells and gain the ability to migrate to lymph nodes. In the lymph node, the encounter of cognate CD4 ⁺ T cells provides an additional differentiation stimulus for DCs, which regulates the survival of activated T cells, and the polarization of CD4 + T cells.

  DC maturation occurs at the site of antigen uptake and recall rejection provides appropriate inflammatory danger signals necessary for DC maturation, migration to lymph nodes, and programming of Th1 immunity to antigens taken at the lesion Act as an adjuvant for

Animals Balb / c mice were the host and C57B1 / 6 (B6) mice were used as a source of Th1 cells. All mice were 6-10 weeks old and were housed in a specific sterile facility at Hadassah-Hebrew University Medical Center and treated with a certified animal protocol.

Preparation of Allogeneic Th1 Memory Cells Spleen cells from male C57BL / 6 mice were harvested and treated with ammonium-potassium chloride (ACK) buffer for lysing red blood cells. Approximately 70 million to 100 million cells were isolated per spleen. The CD4 + T cells are then purified by positive selection (purity> 98%) using CD4 immunomagnetic particles on MS columns (Miltenyi Biotec, Germany), approximately 80 million to 12 million CD4 cells by 50 to 60 Isolated in% yield. Th1 memory cells are grown at anti-CD3 and anti-CD28-coated paramagnetic beads (CD3 / CD28 T cell expander beads, Dynal / Invitrogen) at an initial bead: CD4 cell ratio of 3: 1. Generated. Purified CD4 cells were injected with 20 IU / mL recombinant mouse (rm) IL-2, 20 ng / mL rmIL-7, and 10 ng / mL rmIL-12 (Peprotech, New Jersey), and 10 μg / mL anti-mouse. IL-4 mAb (Becton Dickenson) (10% FBS, penicillin-streptomycin-glutamine, non-essential amino acids (NEAA) (Biological Industries, Israel) and 3.3 mM N-acetyl-cysteine (NAC; Sigma) (In complete medium)). Additional cytokine-containing complete medium (containing rmIL-2 and rmIL-7) is added daily to CD4 cultures for up to 3 to 6 days to maintain cell concentrations between 0.5-1 x 10 ⁶ cells / mL did. Additional CD3 / CD28 beads were added daily for 3-6 days. The number of beads added was calculated to maintain a bead: cell ratio of 1: 1 as expanded cells. After 6 days in culture, CD4 cells were grown approximately 80-100 times, harvested, and de-beaded by physical disruption and passage over a magnet. Phenotype of the recovered cells were used in the experiments,> 95% of ^{CD4 +, CD45RO +, CD62L 10} , IFN-α + and were IL-4-a.

CD3 / CD28 NanoBeads Preparation Biotinylated mouse anti-CD3 and anti-CD28 mAb (BD Pharmingen) are each diluted to a final concentration of 25 μg / ml in 400 μl PBS, and then a 1: 1 ratio to a final volume of 800 μl Mixed. Twenty μl of streptavidin-coated nanobeads (Miltenyi, Germany) were washed and diluted in PBS to a final volume of 200 μl. 800 μl of CD3 / CD28 mAb solution and 200 μl of diluted nanobeads were then mixed such that the final concentration of each mAb was 10 μg / ml in a total volume of 1 ml. The mixture was placed in a mixing device rotating at RT for 30 minutes. The mAb-conjugated nanobeads were then passed over an MS column (Miltenyi, Germany) on a magnet and washed thoroughly. The retained nanobeads were then released from the column and resuspended in 200 μl PBS. The nanobeads were unable to activate naive T cells. Thus, the nanobeads were titted on recovered Th1 memory cells that had been pre-activated 6 days before using CD3 / CD28 T cell amplification beads (Dynal, Norway). Generally, 20 μl / 10 ⁷ cells were found to provide optimal activation of previously activated Th1 memory cells, although there was slight variation per batch.

CD3 / CD28 Crosslinking In experiments that required injection of activated Th1 memory cells, harvested Th1 cells were incubated with a pre-tittered concentration of CD3 / CD28 conjugated nanobeads prior to injection. For optimal activation, cells had to be incubated with nanobeads for a minimum of 4 hours and a maximum of 18 hours. Optimal activation resulted in the production of IFN-α and upregulation of CD40L and FasL on the cell surface. For these experiments, all injections of CD3 / CD28 cross-linked Th1 memory cells were performed 4 to 8 hours after preincubation. Cells were thoroughly washed prior to injection to remove unbound beads. Cross-linked Th1 memory cells used in these experiments express FasL and CD40L on the cell surface, with excess 2000 ng / ml / 10 ⁶ cells / ⁶ h IFN-α and less than 20 pg / ml IL-4. It occurred at 10 ⁶ cells / ⁶ h. Th1 memory cells without CD3 / CD28 crosslinks did not produce cytokines nor express FasL or CD40L.

Cryotherapy Cryotherapy was performed with a spherical nitrous oxide cryoprobe (cryoprobe) 3 mm in diameter. The gas was maintained at a pressure of 50 bar and the Joule Thomson effect made it possible to obtain temperatures in the tissue ranging from -30 to -40.degree. An incision is made in the center of the tumor and the cryoprobe is placed in contact with the tumor (this was inserted 1 to 2 mm deep): the purpose is to affect the tumor by freezing it It was not to destroy it completely. Three cycles of rapid freezing (20 seconds lasting) were followed by slow thawing. An ice ball was created at the center of the lesion to reach approximately 2/3 of the total tumor volume.

The following protocol was tested to test the ability of allogeneic Th1 cells to stimulate systemic anti-tumor immunity during extensive metastatic disease. A lethal dose of tumor cells, including BCL1 leukemia, 4T1 breast cancer and 3LL lung cancer, was injected intravenously into mice on day 0, and tumor cells were also injected percutaneously to establish a solid tumor mass. On day 7, mice received 1 × 10 ⁵ doses of allogeneic Th1 cells. On day 14, mice are: (a) saline; (b) saline + partial cryoablation of tumors; (c) doses of 10 ³ cells of allogeneic Th1 cells, or (d ) Treated within the tumor by any injection of allogeneic Th1 cells + partial cryoablation of the tumor. The results for surviving animals on day 90 are shown below (n = 10).

  To investigate whether treatment of patients with solid tumors may be beneficial from the present invention, the above experimental design was repeated with animals that received only intradermal injection of tumors that produce solid tumor masses. The results were similar to those obtained with animals with metastatic disease.

  The combination of Th1 cells with cryotherapy produces high cure rates. Cryotherapy kills the tumor by necrosis, which is considered to be a pathological type of cell death rather than apoptotic death (type of death caused by chemotherapy). Cryotherapy makes the tumor more immunogenic, so it is believed that the combination of allogeneic Th1 cells and necrotic tumor death results in certain tumor vaccines that lead to systemic anti-tumor immunity. Be

  Although the invention has been described with reference to the preferred embodiments, those skilled in the art will understand that changes may be made in form and detail without departing from the spirit and scope of the invention.

Claims (13)

Therapeutic compositions for treating a tumor or pathogen in a patient:
With allogeneic cells;
An antigen comprising a product of tumor necrosis or necrosis of a pathogen infected tissue, wherein the antigen is generated in situ by cryoablation and combined with the allogeneic cells at the cryoablation site , whereby the allogeneic cells And said antigen generated in situ elicits an immune response by the patient to produce anti-tumor or anti-pathogenic immunity;
A therapeutic composition, wherein said allogeneic cells are Th1 cells. A vaccine composition:
An adjuvant comprising allogeneic cells;
An antigen generated in situ by necrosis of diseased cells or tissue, wherein the antigen is generated in situ by cryoablation, wherein the antigen generated in situ with the presence of the adjuvant at the site of necrosis An antigen that elicits an immune response by the
A vaccine composition, wherein said allogeneic cells are Th1 cells.   The composition according to claim 1 or 2, further comprising a priming composition, wherein the priming composition comprises allogeneic cells.   The composition according to claim 1 or 2, wherein the antigen comprises a tumor or pathogen antigen, a heat shock protein and a combination thereof.   The composition according to claim 1 or 2, wherein the allogeneic cell is an activated T cell.   3. The composition of claim 1 or 2, wherein the allogeneic cells express high levels of type 1 cytokines.   The composition according to claim 1 or 2, wherein the allogeneic cells express high density of CD40L, TRAIL, FasL and their combination on the cell surface. A combination for inducing an adaptive immune response against a tumor or pathogen in a patient:
Antigens generated in situ from disease or pathogen infection;
An allogeneic cell, wherein the allogeneic cell is administered to the site of the antigen generated in situ, thereby producing an immune response that functions as an adjuvant for antigen uptake from the ablated tissue, thereby causing the patient to Allogeneic cells, with subsequent maturation of antigen-presenting cells systemically stimulating anti-tumor immunity or anti-pathogen immunity,
It encompasses, where a of identity species allogeneic cells Th1 cells, antigen is generated in situ by cryo-ablation, Ru combined with of identity species allogeneic cells in the cryo-ablation site combination. 9. A combination according to claim 8 , further comprising a priming composition, wherein said priming composition is administered prior to generation of the antigen in situ, said priming composition being allogeneic. Containing allogeneic T cells to be rejected by the patient's immune system in a manner that elicits Th1 immunity in the system, the patient's immune system producing anti-allogeneic Th1 immune memory prior to in situ antigen production It is possible to form a combination of things. 9. The combination of claim 8 , wherein the antigen comprises a tumor or pathogen antigen, a heat shock protein and combinations thereof. 9. The combination of claim 8 , wherein the allogeneic cells are allogeneic T cells. 9. The combination of claim 8 , wherein the allogeneic cells express high levels of type 1 cytokines. 9. The combination of claim 8 , wherein the allogeneic cells express high density of CD40L, TRAIL, FasL and combinations thereof on the cell surface.