Patent Application
20150202229
This invention relates to the use of cellular-based preparations using non-proliferative allogeneic leukocytes to decrease the level of regulatory T (Treg) cells and/or decrease the level of pro-inflammatory T cells for inducing of an immune stimulation or a pro-inflammatory state in the treated subject. These cellular-based preparations are useful for the treatment of various conditions associated with decreased or inappropriate immune responses, such as proliferation-associated diseases and infections.
Failure of an animal's immune system to recognize and destroy abnormal cells arising from normal progenitor cells can result in uncontrolled growth and formation of tissue masses that may cause significant morbidity and mortality in the absence of ineffective therapeutic interventions. This is commonly exemplified, but not limited to, cancer cells caused by spontaneous genetic mutation and deletions or exposure to environmental agents leading to similar genetic and cellular changes. Currently, most therapeutic drugs consist of chemical cytotoxic agents targeting proliferating cells, of which the cancer cells are preferentially affected due to their higher mitotic rate, but with limited direct specificity towards the cancer cells.
New cellular based approaches attempt to overcome the lack of specificity of cytotoxic drugs by inducing (in vivo or ex vivo) a target cell antigen-specific response by clonal expansion of a subset of reactive leukocytes from the affected individual or animal. This can be done by isolating either the “target cell” (e.g. cancer cell) itself or by molecular mimicking of a target cell antigen. However, this approach is expensive and may or may not effectively stimulate the desired pro-inflammatory state in vivo. Moreover, in some cases, infective agents (e.g. viruses and parasites) may be established and persist in a subject due to a failure of the subjects immune system to effectively respond to the infective agent/organism via a pro-inflammatory mechanism. Indeed, in many cases the infective organism may actively exert an anergic effect yielding a decreased ratio of Treg to pro-inflammatory cells.
It would be highly desirable to be provided with a cellular-based preparation capable of inducing a state of immune stimulation by decreasing the ratio of the level of regulatory T cells (such as Treg) to pro-inflammatory T cells (such as Th1 and Th17). The cellular-based preparation could induce an immune stimulation either by decreasing Treg levels, increase pro-inflammatory T cell levels or both. These preparations could be useful for treating, preventing and/or alleviating the symptoms associated to a condition associated with a low or inappropriate immune response (e.g. anergy or tolerance for example), such as a proliferation-associated disorder (cancer for example) or an infection (a parasitic infection for example).
One aim of the present invention is to provide cellular-based preparations capable of inducing a state of immune stimulation by decreasing the ratio of the level of regulatory T cells (such as Treg) to the level of pro-inflammatory T cells (such as Th1 and Th17). The cellular-based preparations could induce immune stimulation either by decreasing Treg levels, increasing pro-inflammatory T cell levels or both. These cellular-based preparations are useful for treating, preventing and/or alleviating the symptoms associated to a condition caused/exacerbated by a low or inappropriate immune response. The cellular-based preparations and therapies presented herewith are derived from the contact of at least two distinct leukocyte populations which are considered allogeneic with respect to one another and wherein one of the leukocyte population is non-proliferative. The two leukocyte populations are contacted under conditions so as to allow pro-inflammatory allo-recognition and ultimately immune stimulation. The two leukocyte populations can be contacted in vitro, ex vivo or in vivo to induce immune stimulation and/or a pro-inflammatory state.
In accordance with the present invention, there is provided a method of decreasing a ratio of the level of regulatory T (Treg) cells to the level of pro-inflammatory T cells in a subject in need thereof. Broadly, the method comprises administering: a cellular preparation comprising a first leukocyte being allogeneic to the subject as well as being non-proliferative; a cultured cellular preparation comprising a leukocyte from the subject which has been obtained by culturing it with the first leukocyte and/or a supernatant of a cell culture of a second leukocyte and a third leukocyte wherein the second leukocyte is allogeneic to the third leukocyte and one of the second or the third leukocyte is non-proliferative. The method is designed to provide a decrease in the ratio of the level of Treg cells to the level of pro-inflammatory T cells in the treated subject. In an embodiment, the leukocyte is irradiated to prevent it from proliferating. In another embodiment, the leukocyte is modified with a biocompatible polymer. In such embodiment, it is contemplated that the cytoplasmic membrane of the leukocyte has a membrane-associated protein covalently bound to the biocompatible polymer. In some embodiment, the biocompatible polymer is a polyethylene glycol (PEG) or 2-alkyloxazoline (POZ). In yet another embodiment, the leukocyte described herein is a T cell (such as, for example, a CD4-positive or a CD8-positive T cell). In another embodiment, in the cultured cellular preparation, the leukocyte from the subject is expanded in vitro (or ex vivo) prior to administration to the subject. In yet another embodiment, in the cultured cellular preparation, the first leukocyte is removed prior to administration to the subject. In an embodiment of the cell culture supernatant, the second leukocyte or the third leukocyte is from the subject. In still another embodiment, the decreased ratio between the level of Treg cells and the level of pro-inflammatory T cells is for treating, preventing and/or alleviating the symptoms associated to a condition caused/exacerbated by a reduced immune response (e.g. for example, a state of anergy or tolerance, a proliferation-associated disorder such as cancer or an infection such as a parasitic infection).
In accordance with the present invention, there is provided a cellular-based preparation for decreasing a ratio of regulatory T (Treg) cells to pro-inflammatory T cells in a subject. The cellular-based preparation comprises a first cellular preparation comprising a first leukocyte being allogeneic to the subject as well as being non-proliferative; a cultured cellular preparation comprising a leukocyte from the subject which has been obtained by culturing it with the first allogeneic and non-proliferative leukocyte and/or a supernatant of a cell culture of a second leukocyte and a third leukocyte, wherein the second leukocyte is allogeneic to the third leukocyte and one of the second or the third leukocyte is non-proliferative. The cellular-based preparation can be admixed with an appropriate excipient prior to administration to the subject. Embodiments with respect to the type of non-proliferative cells, the type of polymer modifications that can be made to the leukocyte, the first leukocyte, the leukocyte from the subject, the second leukocyte, the third leukocyte as well as the various uses of the preparations have been described above and do apply herein.
In accordance with the present invention, there is provided the use of the cellular-based preparation described herein for decreasing a ratio of regulatory T (Treg) cells to pro-inflammatory T cells in a subject. There is provided the use of the cellular-based preparation described herein for the preparation of a medicament for decreasing a ratio of regulatory T (Treg) cells to pro-inflammatory T cells in a subject. The cellular-based preparation comprises a first cellular preparation comprising a first leukocyte being allogeneic to the subject as well as being non-proliferative; a cultured cellular preparation comprising a leukocyte from the subject which has been obtained by culturing it with the first leukocyte and/or a supernatant of a cell culture of a second leukocyte and a third leukocyte wherein the second leukocyte is allogeneic to the third leukocyte and one of the second or the third leukocyte is non-proliferative. The cellular-based preparation can be admixed with an appropriate excipient prior to administration to the subject. Embodiments with respect to the type of non-proliferative cells, the type of polymer modifications that can be made to the leukocyte, the first leukocyte, the leukocyte from the subject, the second leukocyte, the third leukocyte as well as the various uses of the preparations have been described above and do apply herein.
In accordance with the present invention, there is provided a process for increasing and/or for providing the ability of a cellular-based preparation to decrease a ratio of regulatory T (Treg) cells to pro-inflammatory T cells in a subject. Broadly, the process comprises (i) at least one of 1/ preventing a first leukocyte from proliferating to obtain a first modified leukocyte (wherein the first leukocyte is allogeneic to the subject), 2/ culturing the first modified leukocyte with a leukocyte from the subject to obtain a cultured cellular preparation and/or 3/ preventing one of a second leukocyte or a third leukocyte from proliferating, culturing the second leukocyte with the third leukocyte (wherein the second leukocyte is allogeneic to the third leukocyte), isolating the cell culture supernatant to obtain a cell culture supernatant; and (ii) formulating the first modified leukocyte, the cell cultured cellular preparation or the cell culture supernatant for administration to the subject (such as, for example, intravenous administration). The formulating step can also encompass formulating the first modified leukocyte, the cell cultured cellular preparation or the cell culture supernatant in a vaccine. In an embodiment, the method can comprise modifying the first leukocyte, the leukocyte from the subject, the second leukocyte and/or the third leukocyte with a biocompatible polymer. For example, the method can comprise covalently binding the biocompatible polymer to a membrane-associated protein of the cytoplasmic membrane of the first leukocyte, the leukocyte from the subject, the second leukocyte and/or the third leukocyte. In a further embodiment, the biocompatible polymer is a polyethylene glycol (PEG) or 2-alkyloxazoline (POZ). Embodiments with respect to type of non-proliferative cells, the first leukocyte, the leukocyte from the subject, the second leukocyte, the third leukocyte as well as the various uses of the preparations have been described above and do apply herein.
Throughout this text, various terms are used according to their plain definition in the art. However, for purposes of clarity, some specific terms are defined below.
Allogeneic cell. A cell is considered “allogeneic” with respect to another cell if both cells are derived from the same animal species but presents sequence variation in at least one genetic locus. A cell is considered “allogeneic” with respect to a subject if the cell is derived from the same animal species as the subject but presents sequence variation in at least one genetic locus when compared to the subject's respective genetic locus. Allogeneic cells induce an immune reaction (such as a cell-based immune reaction, a rejection for example) when they are introduced into an immunocompetent host. In an embodiment, a first cell is considered allogeneic with respect to a second cell if the first cell is HLA-disparate (or HLA-mismatched) with the second cell.
Allo-recognition. As it is known in the art, the term “allo-recognition” (also spelled allorecognition) refers to an immune response to foreign antigens (also referred to as alloantigens) from members of the same species and is caused by the difference between products of highly polymorphic genes. Among the most highly polymorphic genes are those encoding the MHC complex which are most highly expressed on leukocytes though other polymorphic proteins may similarly result in immune recognition. These polymorphic products are typically recognized by T cells and other mononuclear leukocytes. In the context of the present invention, the term “pro-inflammatory allo-recognition” refers to an immune response associated with the expansion of pro-inflammatory T cells and/or the differentiation of naïve T cells into pro-inflammatory T cells. Pro-inflammatory allo-recognition in vivo mediates cell or tissue injury and/or death and loss of cell or tissue function. Still in the context of the present invention, the term “pro-tolerogenic allo-recognition” refers to an immune response associated with the expansion of Treg cells and/or the differentiation of naïve T cells into Treg cells. A pro-tolerogenic allo-recognition is usually considered weaker than a pro-inflammatory allo-recognition. Further, an in vivo pro-tolerogenic allo-recognition does not lead to significant cell or tissue injury and/or death nor loss of cell or tissue function.
Anergy and Tolerance. In the present context, the term “anergy” refers to a non-specific state of immune unresponsiveness to an antigen to which the host was previously sensitized to or unsensitized to. It can be characterized by a decrease or even an absence of lymphokine secretion by viable T cells when the T cell receptor is engaged by an antigen. In the present context, the term “tolerance” (also referred to as a pro-tolerogenic state) refers to an acquired specific failure of the immunological mechanism to respond to a given antigen, induced by exposure to the antigen. Tolerance refers to a specific nonreactivity of the immune system to a particular antigen, which is capable, under other conditions, of inducing an immune response. However, in the present context, the terms “anergy” and “tolerance” are used interchangeably since the compositions and methods presented herewith can be used to achieve both anergy and tolerance.
Autologous cell. A cell is considered “autologous” with respect to another cell if both cells are derived from the same individual or from genetically identical twins. A cell is considered “autologous” to a subject, if the cell is derived from the subject or a genetically identical twin. Autologous cells do not induce an immune reaction (such as a rejection) when they are introduced into an immuno-competent host.
Conditions associated with a reduced (low or inappropriate) immune response. In the context of the present invention, the subjects afflicted by these conditions have increased ratio of Treg to pro-inflammatory T cells when compare to the ratio observed in sex- and age-matched healthy subjects. In some embodiments, the immune system of subjects afflicted by a condition associated with a low, repressed or inappropriate immune response is in a state of anergy. The immune system of some of the subjects afflicted by these conditions fails to produce target specific pro-inflammatory cell (T and B lymphocytes) capable of recognizing and destroying abnormal cells (e.g., cancer cells or infected cells). Alternatively, the immune system of some of the subjects afflicted by these conditions exhibit elevated levels of regulatory T and B cells that inhibit normal pro-inflammatory T and B cells from exerting their function (i.e. inducing a partial or complete immune suppression) thereby preventing destruction of an abnormal cell of cell aggregates. One of these conditions is a proliferation-associated disorder (such as, for example, cancer). Another of these conditions is an infection (such as for example a parasitic infection).
Proliferation-associated disorders. These disorders (also referred to as hyperproliferative disorders) form a class of diseases where cells proliferate more rapidly, and usually not in an ordered fashion, than corresponding healthy cells. The proliferation of cells cause an hyperproliferative state that may lead to biological dysfunctions, such as the formation of tumors (malignant or benign). One of the proliferation-associated disorder is cancer. Also known medically as a malignant neoplasm, cancer is a term for a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. In an embodiment, the cancer is a carcinoma (e.g. a cancer of the epithelial cells). Other types of cancer include, but are not limited to sarcoma, lymphoma, leukemia, germ cell tumor and blastoma.
Immune stimulation. In the present context, the term “immune stimulation” or “pro-inflammatory state” refers to a state of immune responsiveness to an antigen independent of the host previously sensitization to the antigen. It can be characterized by an increase or a modulation in the level of lymphokine secretion by viable T cells when the T cell receptor is engaged by an antigen. In the present context, the term “stimulation” refers to an acquired specific activation of the immunological mechanism to respond to a given antigen, induced by exposure to the antigen. In the context of the present invention, the immune stimulation is considered therapeutic and specifically excludes inflammatory diseases, conditions and/or disorders.
Immunogenic cell. A first cell is considered immunogenic with respect to a second cell when it is able to induce an immune response in the latter cell. In some embodiment, the immune response is in vitro (e.g. a mixed lymphocyte reaction) or can be observed in vivo (e.g. in a subject having the second cell and having received the first cell). The second cell can be located in an immunocompetent subject. Preferably, the immune response is a cell-based immune response in which cellular mediator can be produced. In the context of this invention, the immunogenic cells are immune cells, such as white blood cells or leukocytes.
Immunogenic cell culture conditions. A cell culture is considered to be conducted in immunogenic conditions when it allows the establishment of a pro-inflammatory immune response between two distinct and unmodified leukocytes (and, in an embodiment, allo-recognition). Preferably, the pro-inflammatory immune response is a cell-based immune response in which cellular mediator can be produced. For example, the cell culture conditions can be those of a mixed lymphocyte reaction (primary or secondary).
Infection. As used in the context of the present invention, the term “infection” or “infective disease” is a condition caused by the presence and proliferation of an infectious agent which induces a state of low or repressed immune response (e.g. anergy). In some embodiments, the infection is caused by a parasite and in such instances, it is referred to as a “parasitic” infection. There are mainly three classes of parasites which can cause infections, at least in humans, protozoa (causing protozoan infection), helminths (causing an helminthiasis) and ectoparasites. As it is known in the art, parasites have the intrinsic ability, upon infecting their host, to upregulate or enhance Treg's levels and/or activity and thereby induce a state of immune tolerance. This is exemplified by filarial nematodes in which the nematode secretes substances that cause an increase in the host's Treg lymphocytes levels. The increase in Tregs actively down-regulate the Th1 and Th2 responses necessary for eradication of the parasite. Administration of an agent that can reverse the parasite's induced Treg increase would enhance the ability of the subjects immune system to eradicate the parasitic infection.
Leukocyte. As used herein, a leukocyte (also spelled leucocyte) is defined as a blood cell lacking hemoglobin and having a nucleus. Leukocytes are produced and derived from hematopoietic stem cells. Leukocytes are also referred to as white blood cells. Leukocytes include granulocytes (also known as polymorphonuclear leucocytes), e.g. neutrophils, basophils and eosoniphils. Leukocytes also include agranulocytes (or mononuclear leucocytes), e.g. lymphocytes, monocytes and macrophages. Some of the lymphocytes, referred to as T cells (or T-cell), bear on their surface a T-cell receptor. T cell are broadly divided into cells expressing CD4 on their surface (also referred to as CD4-positive cells) and cells expressing CD8 on their surface (also referred to as CD8-positive cells). Some of the lymphocytes, referred to as B cells (or B-cells), bear on their surface a B-cell receptor.
Non-proliferative leukocyte. As used herein, the term “non-proliferative leukocyte” refers to a leukocyte which has been modified to no longer being capable of cellular proliferative (e.g. performing at least one complete division cycle). In some embodiments, this modification may be temporary and the non-proliferative properties of a leukocyte may be limited in time. For example, when a leukocyte is modified from a contact with a pharmacological agent capable of limiting its proliferation, the removal of the pharmacological agent from the cell culture can allow the leukocyte to regain its proliferative properties. In other embodiments, the modification is permanent and the modified leukocyte cannot regain its proliferative properties. For example, when a leukocyte is irradiated, it is not possible for it to regain its proliferative properties. In the context of the present application, the expressions “non-proliferative leukocyte” or “leukocyte limited from proliferating” are used interchangeably.
Peripheral blood mononuclear cells (PBMC). This term refers to the cell population recuperated/derived from the peripheral blood of a subject (usually a mammal such as a human). PBMC usually contains T cells, B cells and antigen presenting cells.
Pharmaceutically effective amount or therapeutically effective amount. These expressions refer to an amount (dose) of a cellular preparation effective in mediating a therapeutic benefit to a patient (for example prevention, treatment and/or alleviation of symptoms of an immune-associated disorder or infection in which the ratio of Tregs to pro-inflammatory T cells is high when compared to sex- and aged-matched healthy subjects). It is also to be understood herein that a “pharmaceutically effective amount” may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents.
Prevention, treatment and alleviation of symptoms. These expressions refer to the ability of a method or cellular preparation to limit the development, progression and/or symptomology of a immune-associated disorder associated to conditions caused/exacerbated by a low or inappropriate immune response (also known as a state of anergy or tolerance). The subjects being afflicted with these conditions/disorders s ratio of Tregs to pro-inflammatory T cells which is considered high when compared to sex- and aged-matched healthy subjects. Broadly, the prevention, treatment and/or alleviation of symptoms encompasses decreasing the levels of Treg cells and/or increasing the levels of pro-inflammatory T cells. A method or cellular-based preparation is considered effective or successful for treating and/or alleviating the symptoms associated with the disorder when a reduction in the pro-tolerogenic state (when compared to an untreated and afflicted individual) in the treated individual (previously known to be afflicted with the disorder) is observed. A method or cellular-based preparation is considered effective or successful for preventing the disorder when a reduction in the pro-tolerogenic state (when compared to an untreated and afflicted individual) in the treated individual is observed upon an immunological challenge (such as, for example, an antigenic challenge). In instances where the conditions to be treated is cancer, exemplary symptoms which can be alleviated with the cellular-based preparations described herewith include, but are not limited to, number and/or size of metastasic tumors, presence and/spread of metastatic tumors and/or size of primary tumor. In instances where the conditions to be treated is an infection, exemplary symptoms which can be alleviated with the cellular-based preparations described herewith include, but are not limited to, infectious agent's burden, infectious agent's presence and fever.
Pro-inflammatory T cells. In the present context, pro-inflammatory T cells are a population of T cells capable of mediating an inflammatory reaction. Pro-inflammatory T cells generally include T helper 1 (Th1 or Type 1) and T helper 17 (Th17) subsets of T cells. Th1 cells partner mainly with macrophage and can produce interferon-γ, tumor necrosis factor-β, IL-2 and IL-10. Th1 cells promote the cellular immune response by maximizing the killing efficacy of the macrophages and the proliferation of cytotoxic CD8+ T cells. Th1 cells can also promote the production of opsonizing antibodies. T helper 17 cells (Th17) are a subset of T helper cells capable of producing interleukin 17 (IL-17) and are thought to play a key role in autoimmune diseases and in microbial infections. Th17 cells primarily produce two main members of the IL-17 family, IL-17A and IL-17F, which are involved in the recruitment, activation and migration of neutrophils. Th17 cells also secrete IL-21 and IL-22.
Regulatory T cells. Regulatory T cells are also referred to as Treg and were formerly known as suppressor T cell. Regulatory T cells are a component of the immune system that suppress immune responses of other cells. Regulatory T cells usually express CD3, CD4, CD8, CD25, and Foxp3. Additional regulatory T cell populations include Tr1, Th3, CD8+CD28−, CD69+, and Qa-1 restricted T cells. Regulatory T cells actively suppress activation of the immune system and prevent pathological self-reactivity, i.e. autoimmune disease. The critical role regulatory T cells play within the immune system is evidenced by the severe autoimmune syndrome that results from a genetic deficiency in regulatory T cells. The immunosuppressive cytokines TGF-β and Interleukin 10 (IL-10) have also been implicated in regulatory T cell function. Similar to other T cells, a subset of regulatory T cells can develop in the thymus and this subset is usually referred to as natural Treg (or nTreg). Another type of regulatory T cell (induced Treg or iTreg) can develop in the periphery from naïve CD4+ T cells. The large majority of Foxp3-expressing regulatory T cells are found within the major histocompatibility complex (MHC) class II restricted CD4-expressing (CD4+) helper T cell population and express high levels of the interleukin-2 receptor alpha chain (CD25). In addition to the Foxp3-expressing CD4+CD25+, there also appears to be a minor population of MHC class I restricted CD8+ Foxp3-expressing regulatory T cells. Unlike conventional T cells, regulatory T cells do not produce IL-2 and are therefore anergic at baseline. An alternative way of identifying regulatory T cells is to determine the DNA methylation pattern of a portion of the foxp3 gene (TSDR, Treg-specific-demthylated region) which is found demethylated in Tregs.
Splenocytes. This term refers to the cell population obtained from the spleen of a subject (usually a mammal such as a rodent). Splenocytes usually comprise T cell, B cell as well as antigen presenting cells.
Syngeneic cell. A cell is considered “syngeneic” with respect to a subject (or a cell derived therefrom) if it is sufficiently identical to the subject so as to prevent an immune rejection upon transplantation. Syngeneic cells are derived from the same animal species.
Viable. In the present context, the term “viable” refers to the ability of a cell to complete at least one cell cycle and, ultimately proliferate. A viable cell is thus capable of proliferating. By opposition, the term “non-viable” or “non-proliferative” both refer to a cell which is no longer capable of completing at least one cell cycle. By comparison, the term “cycle arrest” refers to a cell which has been treated to halt its cell cycle progression (usually with a pharmacological agent) but which is still capable of re-entering the cell cycle (usually when the pharmacological agent is removed).
Xenogeneic cell. A cell is considered “xenogeneic” with respect to a subject (or a cell derived from the subject) when it is derived from a different animal species than the subject. A xenogeneic cell is expected to be rejected when transplanted in an immunocompetent host.
Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof.
In accordance with the present invention, there is provided cellular-based preparations for decreasing the level of regulatory T cells and/or increasing the level of pro-inflammatory T cells for inducing immune stimulation and/or a pro-inflammatory state in a subject in need thereof. The cellular-based preparations and therapies presented herewith concern the contact of at least two distinct leukocyte populations which are considered allogeneic with respect to one another and wherein at least one of the leukocyte population is considered non-proliferative. The contact between the two leukocyte populations occurs under conditions to allow pro-inflammatory allo-recognition but limit or prevent pro-tolerogenic recognition. The cellular-based preparations can be a first leukocyte (which is allogeneic to the treated subject) which has been prevented from proliferating. The cellular-based preparation can also be a cultured cellular preparation obtained by culturing the first leukocyte with a leukocyte from the subject (or syngeneic to the subject). Alternatively, the cellular-based preparation can be a cell culture supernatant (or a sample thereof) obtained by isolating the cell culture supernatant of a co-culture a second and a third leukocytes, wherein the second leukocyte is allogeneic to the third leukocyte and one of the second or third leukocyte is considered non-proliferative.
As it will be shown below, the modification of allogeneic leukocyte to prevent them from proliferating provides a significant opportunity to modulate the responsiveness (i.e., immunoquiescent versus pro-inflammatory) of the recipient's immune system. Of importance, the allogeneic leukocyte, besides being prevented from proliferating, does not need to be further manipulated to mediate its therapeutic effect. However, in some embodiments, the surface of the allogeneic leukocyte can be further modified (for example cross-linked and/modified with a polymer) to increase its antigenicity. The allogeneic leukocyte can be expanded in vitro prior to a co-culture step or its administration to the subject in need thereof.
As shown herein, the contact between two leukocyte populations (wherein one population has been refrained from proliferating) which are considered allogeneic to one another induces an immune stimulation (e.g. a pro-inflammatory state). More specifically, the contact between two leukocyte populations decreases the level of Treg cells and especially the levels of CD69+ Treg cells. In addition, the contact between two leukocyte populations potentiates natural killer (NK) cells. Taken together, this indicates that the contact between the two leukocyte populations can induce therapeutic effects in subjects experiencing a low or inappropriate immune response (for example having elevated levels of Treg cells (especially CD69+ Treg cells) and/or having a low level or inactive NK cells) by favoring immune stimulation via the induction of a pro-inflammatory state in subjects experiencing anergy.
As it is known in the art, the administration of a population of viable allogeneic leukocyte preparation can induce the onset of graft-vs.-host disease. As shown herein, the administration of non-viable/non-proliferative allogeneic leukocytes (or products derived therefrom) can induce an immune stimulation (in vivo as well as in vitro) and can be used to shift the recipient's immune system from a pro-tolerogenic state to a pro-inflammatory state while preventing graft-vs.-host disease. These cellular preparations provide therapeutic tools for the treatment of conditions associated with a pro-tolerogenic state (e.g. anergy), such as proliferation-associated disorders as well as infections. These cellular preparations provide tools for shifting the immune system in a non-specific manner and to bolster the immune system.
Methods for Modulating the Treg/Pro-inflammatory T Cells Ratio
The present invention provides methods and cellular preparations for decreasing the ratio of the level of regulatory T cells with respect to the level of pro-inflammatory T cells. In the present invention, the ratio can be decreased either by lowering the level of regulatory T cells in the subject or increasing the level of pro-inflammatory T cells in the subject. Alternatively, the ratio can be decreased by lowering the level of regulatory T cells in the subject and increasing the level of pro-inflammatory T cells in the subject. When the Treg/pro-inflammatory T cells ratio is decreased in a subject, it is considered that a state of immune stimulation is induced or present in the subject. The induction of a state of immune stimulation in subjects experiencing an abnormally decreased immune state can be therapeutically beneficial for limiting the symptoms or pathology associated with the abnormally low immune reaction or an acquired state of anergy. In some embodiments, it is not necessary to induce a state of complete immune stimulation, a partial induction of immune stimulation can be beneficial to prevent, treat and/or alleviate the symptoms of a disorder associated with a pro-tolerogenic state (such as, for example, a proliferation-associated disorder or an infection).
In order to decrease the Treg/pro-inflammatory T cells ratio, an allogeneic cellular preparation can be administered to the subject in a therapeutically effective amount. In such instance, the cellular preparation can comprise a first leukocyte that has been modified to be considered to be non-proliferative. Prior to its modification, the first leukocyte is considered immunogenic (e.g. allogeneic for example) with respect to the subject because it is able to induce an immune response (e.g. a cell-mediated immune response) when administered to the subject. As indicated above, it is possible to determine if two cells are considered immunogenic with respect to one another by conducting conventional in vitro assays, such as a mixed lymphocyte reaction. It is also expected that MHC-disparate cells would be considered immunogenic with respect to one another. In an embodiment, the first leukocyte can be xenogeneic to the subject. However, the first leukocyte cannot be autologous or syngeneic to the subject. Importantly, the first leukocyte, prior to its modification, is also considered viable and capable of cellular proliferation. The first leukocyte can even be optionally expanded in vitro (preferably under conditions favoring the expansion of pro-inflammatory T cells or the differentiation of naïve T cells in pro-inflammatory T cells), however, in such embodiment, the first leukocyte is modified to become non-proliferative prior to its administration to the subject. In an embodiment, the first allogeneic leukocyte can be modified to bear on its surface a polymer. However, the polymer, when present, must be selected or grafted at a density so as to allow the pro-inflammatory allo-recognition of the first leukocyte by the recipient. When the first leukocyte is modified to bear on its surface a polymer, it can be modified to be non-proliferative either prior to or after the polymer modification.
Alternatively, in order to decrease the Treg/pro-inflammatory T cells ratio, a cultured cellular preparation can be administered to the subject in a therapeutically effective amount. In order to do so, the first leukocyte is placed in contact in vitro with a leukocyte from the subject or a leukocyte syngeneic to the subject. One of the two leukocyte population is prevented from proliferating. In some embodiments, the first leukocyte is refrained from proliferating while the leukocyte from the subject (or syngeneic to the subject) is viable and capable of proliferation. The two cell populations are cultured under immunogenic conditions to provide a cultured cellular preparation in which a pro-inflammatory allo-recognition occurs. In an embodiment, the two cells populations are cultured under conditions favoring the expansion (e.g. proliferation) and/or differentiation (e.g. naïve to pro-inflammatory T cells) of the cultured cells (preferably the leukocytes from the subject). This expansion/proliferation can occur before, during or after the co-culture of the two leukocyte populations. In some embodiments, it is preferable to remove the first leukocyte from the cultured cellular preparation prior to the administration of the cultured cellular preparation to the subject. Methods of separating the two cellular populations are known to those skilled in the art and include, without limitation, cell sorting and magnetic beads. In an embodiment, the first allogeneic leukocyte and/or the leukocyte from the subject can be modified to bear on its surface a polymer. However, the polymer, when present, must be selected or grafted at a density so as to allow the pro-inflammatory allo-recognition of the first leukocyte by the leukocyte from the subject (or syngeneic to the subject). When the leukocyte is modified to bear on its surface a polymer, it can be modified to be non-proliferative either prior to or after the polymer modification.
An alternative way of decreasing the Treg/pro-inflammatory T cell ratio concerns the administration of the supernatant of a cell culture of a second leukocyte and a third leukocyte. In such embodiment, one of the second or third leukocyte is limited from proliferating when both leukocyte populations are cultured together under immunogenic conditions. In some embodiments, the cell culture supernatant can comprise leukocytes or leukocyte fractions (for example a part of the cytoplasmic membrane) and/or even cellular products present in the cell culture. In such embodiment, the second leukocyte is considered immunogenic (e.g. allogeneic) with respect to the third leukocyte because when the second leukocyte is placed into contact with the third leukocyte, an immune response (e.g. a cell-mediated immune response) occurs (provided that the cell culture is performed under immunogenic conditions). It is possible to determine if two cells are considered immunogenic with respect to one another by conducting conventional in vitro assays, such as the mixed lymphocyte reaction. It is also expected that MHC-disparate cells would be considered immunogenic with respect to one another. In another embodiment, the second leukocyte cell can be xenogeneic to the third leukocyte However, the second leukocyte cannot be autologous or syngeneic to the third leukocyte. In the methods and cellular compositions described herein, it is possible that one of the second or third leukocyte be syngeneic or derived from the subject which will be treated with the cell culture supernatant. In addition, in other embodiments, both the second and/or third leukocytes can be considered allogeneic or xenogeneic to the subject which will be treated. In some embodiment, the leukocytes are being cultured in conditions favoring in vitro expansion and/or differentiation of naïve T cells to pro-inflammatory cells of the leukocyte population that is not refrained from proliferating. Such expansion/differentiation can occur prior to, during or after the co-culture of the two leukocyte populations. Importantly, the cell culture supernatant, apart from being optionally filtered to remove cells and cellular debris, is not submitted to further extraction/size fractionation or specific enrichment of one of its components prior to its administration to the subject. The cell culture supernatant thus comprises the conditioned media from the cell culture (e.g. cellular by-products such as cytokines for example). In an embodiment, the second leukocyte and/or the third leukocyte can be modified to bear on its surface a polymer. However, the polymer, when present, must be selected or grafted at a density so as to allow the pro-inflammatory allo-recognition of the second leukocyte by the third leukocyte. When the leukocyte is modified to bear on its surface a polymer, it can be modified to be non-proliferative either prior to or after the polymer modification.
An alternative way of decreasing the Treg/pro-inflammatory T cell ratio in a subject to be treated, is to administer the conditioned blood (or fraction thereof such as plasma or serum) of a test subject that has been administered with a first non-proliferative allogeneic leukocyte. The animal is transfused with in conditions so as to allow a pro-inflammatory allo-recognition but to prevent the onset of GVHD. In some embodiments, this conditioned blood can comprise the first leukocyte or a derivative thereform (a part of the cytoplamsic membrane from the first leukocyte for example). The first leukocyte is considered immunogenic (e.g. allogeneic) with respect to the test subject because when the first leukocyte is transfused into the animal, an immune response (e.g. a cell-mediated immune response, preferably a pro-inflammatory allo-recognition) occurs. In another embodiment, the first leukocyte can be xenogeneic with respect to the animal. However, the first leukocyte cannot be autologous or syngeneic to the animal. In some embodiments, the first leukocyte can be allogeneic or xenogeneic to the subject which will be treated with the conditioned blood. In alternative embodiment, the first leukocyte can be syngeneic or derived from the subject which will be treated with the conditioned blood. In an embodiment, the first allogeneic leukocyte can be modified to bear on its surface a polymer. However, the polymer, when present, must be selected or grafted at a density so as to allow the pro-inflammatory allo-recognition of the first leukocyte by the recipient. When the first leukocyte is modified to bear on its surface a polymer, it can be modified to be non-proliferative either prior to or after the polymer modification.
In the context of the present invention, some of the leukocytes used in the cellular preparations are both modified for bearing a low-immunogenic biocompatible polymer and being modified to no longer be capable of proliferation. The order in which the leukocytes are modified (modification with polymer and modification to prevent proliferation) is not important. Leukocytes can be first modified to bear the polymer and then modified to refrain from proliferating. Alternatively, the leukocytes can be first modified to refrain from proliferating and then modified to bear the polymer.
The leukocytes described herein can be derived from any animals, but are preferably derived from mammals (such as, for example, humans and mice).
In the methods and cellular preparations provided herewith, the surface of the leukocyte can be modified with a low-immunogenic biocompatible polymer. The polymer must be grafted at concentrations or polymer size that will allow pro-inflammatory allo-recognition while preventing or limiting pro-tolerogenic allo-recognition. For some specific applications, it may be preferable to modify the surface of the leukocyte with a single type of low-immunogenic biocompatible polymer. However, for other applications, it is possible to modify the surface of the leukocyte with at least two different types of low-immunogenic biocompatible polymers.
In order to achieve these modifications, the low-immunogenic biocompatible polymer can be covalently bound to the cytoplasmic membrane of the leukocyte and, in a further embodiment, a membrane-associated protein of the surface of the leukocyte or inserted, via a lipophilic tail, in the cytoplasmic membrane of the leukocyte. When the polymer is bound to a membrane-bound protein, the membrane-associated protein must have at least a portion which is accessible on the external surface of the cytoplasmic membrane of the leukocyte for being covalently attached to the polymer. For example, the membrane-associated protein can be partially embedded in the cytoplasmic membrane or can be associated with the external surface of the membrane without being embedded in the cytoplasmic membrane. The low-immunogenic biocompatible polymer can be covalently bound to a plurality of membrane-associated proteins. In an alternative or complementary embodiment, the low-immunogenic biocompatible polymer can be inserted in the cytoplasmic membrane by using a lipid-modified polymer.
In some embodiment, the low-immunogenic biocompatible polymer can be polyethylene glycol (methoxy polyethylene glycol for example). The polyethylene glycol can be directly and covalently bound to a membrane-associated protein or, alternatively, a linker attaching the low-immunogenic biocompatiable polymer can be used for attaching the polymer to the protein. Exemplary linkers are provided in U.S. Pat. No. 8,007,784 (incorporated herewith in its entirety). In alternative embodiments, the low-immunogenic polymer can be POZ or HPG.
In the methods and cellular preparations provided herewith, the leukocytes can be mature leukocytes or be provided in the form of stem cells. For example, leukocytes can be obtained from isolating peripheral blood mononuclear cells (PBMC) from the subject. Optionally, the PBMCs can be differentiated in vitro into DC or DC-like cells. Alternatively, the leukocytes can be obtained from the spleen (e.g. splenocytes). Leukocytes usually include T cells, B cells and antigen presenting cells. In the methods and cellular preparations provided herewith, the leukocytes are not erythrocytes. However, traces of erythrocytes in the leukocytic preparations are tolerated (for example, less than about 10%, less than about 5% or less than about 1% of the total number of cells in the preparation).
Even though it is not necessary to further purify the leukocytes to conduct the method or obtain the cellular preparations, it is possible to use a pure cell population or a relatively homogenous population of cells as leukocytes. This pure cell population and relative homogenous population of cells can, for example, essentially consist essentially of a single cell type of T cells, B cells, antigen presenting cells (APC) or stem cells. Alternatively, the population of cells can consist essentially of more than one cell type. The population of cells can be obtained through conventional methods (for example cell sorting or magnetic beads). In an embodiment, when the population of cells consist of a single cell type (for example, T cells), the percentage of the cell type with respect to the total population of cells is at least 90%, at least 95% or at least 99%. The relatively homogenous population of cells are expected to contain some contaminating cells, for example less than 10%, less than 5% or less than 1% of the total population of cells.
The cell culture supernatant used in the method or in the cultured cellular preparation can be obtained by co-culturing a second leukocyte population with a third leukocyte population. It is also possible to co-culture a second leukocyte homogenous cell population (such as, for example, a T pure cell population or a substantially pure T cell population) with a third leukocyte preparation. It is also contemplated to culture a second leukocyte population with a third leukocyte population (such as, for example, a pure T cell population or a substantially pure T cell population).
In the methods and preparations presented herewith, it is required to inhibit/limit the proliferation of one of the two leukocyte populations. For example, a leukocyte can be treated/modified prior to cell culture or its administration into the subject in order to inhibit/limit the cell from proliferating in the subject. For example, the cell can be irradiated (e.g. γ-irradiation) prior to its introduction in the subject or its introduction into a culture system. Upon irradiation, the leukocyte is not considered viable (e.g. capable of proliferation). In an embodiment, polymer grafting can be used to affect the leukocyte viability and utlimately refrain the leukocyte from proliferating. In a further embodiment of irreversible non-proliferation, a cell can be treated with a fixation agent (e.g. glutaraldehyde). Alternatively, leukocyte can be treated with a pharmacological agent which halts cell cycle progression. Upon the administration of such pharmacological agent, the leukocyte is considered viable since it can resume cellular proliferation when the agent is removed from the cell-containing medium. When the first leukocyte is administered to the subject in need thereof, it is preferable that the leukocyte is modified in order to permanently being refrained from proliferating.
When the cell culture supernatant is used in the method or in the cellular preparations, it is required to inhibit/limit the proliferation of one of the two or the two leukocyte populations. As indicated above, the inhibition of cellular proliferation can be achieved by various means, including irradiation and the use of a polymer, a fixation agent or a pharmacological agent. In this particular embodiment, it is important that only one of the two cell populations be inhibited/limited from proliferating and that the other cell population be able to proliferate.
The conditioned blood that can be used in the method can be obtained by administering (preferably transfusing or intravenously administering) to a test subject (such as a rodent), a first leukocyte which has been modified so as to limit, preferably permanently, its ability to proliferate. It is also possible to transfuse a first leukocytic homogenous cell population (such as, for example, a T pure cell population or a substantially pure T cell population) to the test subject. The blood (or a fraction thereof) is recuperated from the test subject after a time sufficient to induce in the subject a state of immune stimulation or a pro-inflammatory state. In order to obtain a blood fraction (such as serum or plasma) from the animal, it is possible to submit the blood of the animal to a centrifugation step and, optionally, eliminate red blood cells via cellular lysis.
As shown herein, the administration of the cellular preparations induce a state of immune stimulation in the treated subject. In some embodiments, the state of stimulation can persist long after the administration of the cellular preparation or the cell culture supernatant (as shown below, at least 270 days in mice). In an optional embodiment, the state of stimulation does not revert back to a pro-tolerogenic state. Consequently, the methods and cellular preparations described herein are useful for the treatment, prevention and/or alleviation of symptoms associated with conditions caused/exacerbated by a low or inappropriate immune response.
A state of immune stimulation can be considered therapeutically beneficial in subjects experiencing (or at risk of experiencing) a repressed immune response (anergy or tolerance), such as for example those observed upon the induction and maintenance of an proliferation-associated disorder (such as cancer). Some of these conditions are associated with either a high level of Tregs and/or a low level of pro-inflammatory T cells (such as Th17 and/or Th1) when compared to sex- and aged-matched healthy subjects. Because it is shown herein that the cellular-based preparations are beneficial for decreasing the ratio Tregs/pro-inflammatory T cells, it is expected that administration of the cellular-based preparations to afflicted subjects will treat, prevent and/or alleviate symptoms associated with the proliferation-associated disorder.
A state of immune stimulation can also be considered therapeutically beneficial in subjects at risk of developing an abnormally repressed immune response, a state or anergy or a pro-tolerogenic state. Such abnormally repressed immune responses can be observed in subjects being afflicted by or susceptible to be afflicted by a proliferation-associated disorder such as cancer. In this embodiment, the methods and cellular preparations can be applied to prevent or limit the onset or maintenance of a repressed immune response. The cellular-based preparation can be co-administered with the other therapeutics currently used to managed the proliferation-associated disorder. The cellular-based preparation can be administered to any subjects in need thereof, including humans and animals.
Such abnormally repressed immune responses can be also observed in subjects being infected, especially by a parasite or a virus. In these conditions, the methods and cellular preparations can be applied to prevent or limit the onset or maintenance of a repressed immune response. The cellular-based preparation can be co-administered with the other therapeutics currently used to managed the infection.
The cellular-based preparation can be administered to any subjects in need thereof, including humans and animals.
In an embodiment, the state of abnormal repression of the immune system is not caused by an infection of the immune cells themselves (e.g. EBV or HIV for example). However, in other embodiment, in instances where an infection of the immune cells is afflicting the subject, it is possible to use cellular preparations described to treat or alleviate the symptoms of the viral infection. For example, a leukocyte from the subject (preferably a cytotoxic T cell which is specific to the infectious agent) can be co-cultured, under immunogenic conditions, with a first allogeneic and non-proliferative leukocyte. After the co-culture, the cultured leukocyte can be reintroduced in the infected subject to treat and/or alleviate the symptoms associated to the infection (a viral infection, for example, an EBV or HIV infection).
In the methods and cellular preparations described herein, it is contemplated that the cellular-based preparations be optionally administered with other therapeutic agents known to be useful for the treatment, prevention and/or alleviation of symptoms of conditions associated to a condition caused/exacerbated by a low or inappropriate immune response, such as, for example, IL-2, IL-4, TNF-α and/or INF-γ.
Processes for Obtaining Cellular Preparations
The cellular-based preparations described herein are obtained by contacting two distinct and allogeneic leukocyte populations. One of the two leukocyte population is non-proliferative or modified to become non-proliferative. In this first step, it is important that this modification is made without interfering substantially with the intrinsic ability of the first leukocyte to induce a pro-inflammatory allo-recognition by the leukocyte (either in vivo or in vitro). In order to determine if pro-inflammatory allo-recognition occurs (or alternatively is substantially reduced), various techniques are known to those skilled in the art and include, but are not limited to, a standard mixed lymphocyte reaction (MLR), high molecular weight mitogen stimulation (e.g. PHA stimulation) as well as flow cytometry (Chen and Scott, 2006, Wang et al. 2011).
In order to prevent a leukocyte from proliferating, the cell can be irradiated (e.g. γ-irradiation) prior to its introduction in the subject or its introduction into a culture system. Upon irradiation, the leukocyte is not considered viable (e.g. capable of proliferation). In a further embodiment, the surface of the leukocyte can be modified by a polymer to alter or limit its viability. In another embodiment, the leukocyte can be treated with a fixation agent to prevent it from proliferating. Alternatively, leukocyte can be treated with a pharmacological agent which halts cell cycle progression. Upon the administration of such pharmacological agent, the leukocyte is considered viable since it can resume cellular proliferation when the agent is removed from the cell-containing medium.
To provide the cellular preparations described herewith, the leukocytes used can be mature leukocytes or be provided in the form of stem cells. For example, leukocytes can be obtained from isolating peripheral blood mononuclear cells (PBMC) from the subject. Optionally, the PBMCs can be differentiated in vitro into dendritic (DC) or DC-like cells. Alternatively, the leukocytes can be obtained from the spleen (e.g. splenocytes). Leukocytes usually include T cells, B cells and antigen presenting cells. For providing the cellular preparations, the leukocytes are not erythrocytes. However, traces of erythrocytes in the leukocyte population used are tolerated (for example, less than about 10%, less than about 5% or less than about 1% of the total number of cells in the preparation).
Even though it is not necessary to further purify the leukocytes to provide the cellular preparations, it is possible to use a pure cell population or a relatively homogenous population of cells as leukocytes. This “pure” cell population and “relative homogenous population” of cells can, for example, essentially consist essentially of a single cell type of T cells, B cells, antigen presenting cells (APC) or stem cells. Alternatively, the population of cells can consist essentially of more than one cell type. The population of cells can be obtained through conventional methods (for example cell sorting or magnetic beads). In an embodiment, when the population of cells consist of a single cell type (for example, T cells), the percentage of the cell type with respect to the total population of cells is at least 90%, at least 95% or at least 99%. The relatively homogenous population of cells are expected to contain some contaminating cells, for example less than 10%, less than 5% or less than 1% of the total population of cells.
The leukocytes can be obtained from any animals, but are preferably derived from mammals (such as, for example, humans and mice). In an embodiment, the leukocytes can be obtained from a subject intended to be treated with the cellular preparations.
In the embodiment where an allogeneic leukocyte population is administered to the treated subject (optionally to recuperate the conditioned blood), it is contemplated that it can be expanded/differentiated (e.g. from naïve to pro-inflammatory) prior to the administration.
In embodiments where two leukocyte populations are co-cultured in vitro, the step of preventing the leukocyte from proliferating occurs prior to the co-culture. However, it is contemplated, in this embodiment, that the leukocyte population which is going to be prevented from proliferating can be expanded/differentiated (e.g. from naïve to pro-inflammatory) prior to the co-culture. The co-culture of the two leukocyte populations is performed in immunogenic conditions so as to allow a pro-inflammatory allo-recognition in the leukocyte population which has not been modified (e.g. which can exhibit cellular proliferation). Since a physical contact between the two leukocyte populations is important for allowing pro-inflammatory allo-recognition, it is important that the two leukocyte population be cultured into conditions allowing for such physical contact (for example in a culture vessel which does allow physical contact between the two leukocyte populations).
When a co-culture system is used, it is possible to culture a first leukocytic population (such as, for example a PBMC or splenocyte) with a leukocytic population from a subject (such as, for example a PBMC or splenocyte). It is also possible to culture a first leukocytic relatively homogenous cell population (such as, for example, a T cell population) with a leukocytic population from a subject (such as, for example a PBMC or splenocyte). It is also contemplated to culture a first leukocytic population (such as, for example a PBMC or splenocyte) with a leukocytic relatively homogenous population of cells from the subject (such as, for example, a T cell population). It is further completed to culture a first leukocytic relatively homogenous cell population (such as, for example, a T cell population) with a leukocytic relatively homogenous population of cells from the subject (such as, for example, a T cell population).
Usually, the cultured cellular preparation (between the first leukocyte and the leukocyte from the subject or syngeneic to the subject) is obtained at least 24 hours after the initial contact between the first leukocyte and the leukocyte from the subject. In some embodiments, the cultured cellular preparation is obtained at least 48 hours or at least 72 hours after the initial contact between the first leukocyte and the leukocyte from the subject. When the incubation takes place in a 24-well plate, the concentration of each leukocyte population can be at least 1×106 cells.
In yet a further optional embodiment, the modified second leukocyte can be placed in a cell culture with the a third leukocyte and the supernatant of this cell culture can be administered to the subject in need thereof. The supernatant can be modified (e.g. filtered) to remove the second and/or third leukocyte and the cellular debris associated thereto. However, no specific size fractionation nor enrichment of a specific fraction of the supernatant is applied to the cell culture supernatant prior to administering it to the subject. The second and third leukocytes are cultured in the same medium (or in the same culture system), one of the two cell populations is inhibited/limited from proliferating (as long as the other cell populations remains capable of proliferating). In an embodiment, the modified second leukocyte can first be expanded/differentiated and then inhibited/limited from proliferating prior to its co-culture with the third leukocyte. Alternatively, the third leukocyte can be expanded/differentiated prior to its co-culture with the modified second leukocyte or afterwards. As indicated above, in the cell culture system, the second leukocyte is allogeneic to the third leukocyte. In some embodiments, the second leukocyte can be allogeneic to the subject and to third leukocyte. Alternatively, the second leukocyte can be xenogeneic to the subject and/or to the third leukocyte. Optionally, one of the second or third leukocyte can be syngeneic or derived from the subject.
When a co-culture system is used, it is possible to culture a second leukocytic population (such as, for example a PBMC or splenocyte) with a third leukocytic population (such as, for example a PBMC or splenocyte). It is also possible to culture a second leukocytic relatively homogenous cell population (such as, for example, a T cell population) with a third leukocytic population (such as, for example a PBMC or splenocyte). It is also contemplated to culture a second leukocytic population (such as, for example a PBMC or splenocyte) with a third leukocytic relatively homogenous population of cells (such as, for example, a T cell population). It is further completed to culture a second leukocytic relatively homogenous cell population (such as, for example, a T cell population) with a third leukocytic relatively homogenous population of cells (such as, for example, a T cell population).
Usually, the cultured cellular preparation is obtained at least 24 hours after the initial contact between the second leukocyte and the third leukocyte. In some embodiment, the cultured cellular preparation is obtained at least 48 hours or at least 72 hours after the initial contact between the second leukocyte and the third leukocyte. When the incubation takes place in a 24-well plate, the concentration of each leukocyte population can be at least 1×106 cells.
In other embodiments, a conditioned blood can be used. The conditioned blood used can be obtained by administering a first leukocyte, a first leukocyte population or a first leukocytic relatively homogeneous population (e.g. all modified to be refrained or inhibited from proliferating) to the test subject (usually an animal, such as a mouse). The blood (or a fraction thereof) is recuperated from the subject after a time sufficient to induce in the test subject a state of immune stimulation. It is important that the first leukocyte be administered to an immune competent test subject and that the blood or blood fraction be obtained at a later a time sufficient to provide a conditioned blood. The test subject is a subject being immune competent and having a Treg/pro-inflammatory T cell ratio which is substantially similar to age- and sex-matched healthy subjects. As used herein, the conditioned blood refers to physical components present in the blood and obtained by administering the first leukocyte to the immune competent test subject and having the pro-inflammatory properties described herein. It is recognized by those skilled in the art that the conditioned blood may be obtained more rapidly by increasing the amount of leukocytes being administered or administering more than once (for example one, twice or thrice) the modified leukocyte. Usually, the conditioned blood is obtained at least one day after the administration of the first leukocyte. In some embodiment, the conditioned blood is obtained at least 2, 3, 4, 5, 6, 7 or 8 days after the administration of the first leukocyte. In an embodiment, the conditioned blood can be obtained by administering at least 5×106 leukocytes to the test subject (e.g. a mice) and recuperating the plasma five days later. In some embodiment, the conditioned blood can be obtained by administering at least 20×106 polymer-modified leukocytes. Methods for obtaining the blood or its fractions (such as serum or plasma) are known to those in the art and usually involve centrifugation and cell lysis.
As indicated herein, it is possible to modify the surface of the leukocyte with a biocompatible polymer. It is important that the polymer used exhibits biocompatibility once introduced into a cell culture system or administered to the test subject. Such biocompatible polymer include, but are not limited to polyethylene glycol (particularly methoxypoly(ethylene glycol)), POZ and hyperbranched polyglycerol (HPG). In some embodiments, it is preferable to use a single type of polymer to modify the surface of leukocytes. In other embodiments, it is possible to use at least two distinct types of polymers to modify the surface of the leukocyte.
In an embodiment, the biocompatible polymer can be covalently associated with the membrane-associated protein(s) of the leukocyte by creating a reactive site on the polymer (for example by deprotecting a chemical group) and contacting the polymer with the leukocyte. For example, for covalently binding a methoxypoly(ethylene glycol) to the surface of a leukocyte, it is possible to incubate a methoxypoly(-ethylene glycol) succinimidyl valerate (reactive polymer) in the presence of the leukocyte. The contact between the reactive polymer and the leukocyte is performed under conditions sufficient for providing a grafting density which will prevent/limit pro-tolergenic allo-recognition and allow pro-inflammatory allo-recognition. In an embodiment, the polymer is grafted to a viable leukocyte and under conditions which will retain the viability of the leukocyte. A linker, positioned between the surface of the leukocyte and the polymer, can optionally be used. Examples of such polymers and linkers are described in U.S. Pat. Nos. 5,908,624; 8,007,784 and 8,067,151. In another embodiment, the biocompatible polymer can be integrated within the lipid bilayer of the cytoplasmic membrane of the leukocyte by using a lipid-modified polymer.
As indicated above, it is important that the biocompatible polymer be grafted at a density sufficient for preventing/limiting pro-tolerogenic allo-recognition and allow pro-inflammatory allo-recognition. In an embodiment, the polymer is polyethylene glycol (e.g. linear) and has an average molecular weight between 2 and 40 KDa as well as any combinations thereof. In a further embodiment, the average molecular weight of the PEG to be grafted is at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 kDa. In another embodiment, the average molecular weight of the PEG to be granted is no more than 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, or 2 kDa. In another embodiment, the grafting concentration of the polymer (per 20×106 cells) is no more than 2.4, 2.0, 1.0, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 or 0.005 mM. In still another embodiment, the grafting concentration of the polymer (per 20×106 cells) is equal to or lower than 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.4 mM. In embodiments where the polymer is grafter to affect the viability of the leukocyte (for example by creating cellular instability, cellular fragmentation or vesiculization, the concentration of the polymer (per 20×106 cells) is equal to or higher than 10 mM. In order to determine if pro-inflammatory allo-recognition occurs (or is prevented), various techniques are known to those skilled in the art and include, but are not limited to, a standard mixed lymphocyte reaction (MLR), high molecular weight mitogen stimulation (e.g. PHA stimulation) as well as flow cytometry (Chen and Scott, 2006). In order to determine if a weak pro-tolerogenic allo-recognition occurs (or is prevented), various techniques are known to those skilled in the art and include, but are not limited to, the assessment of the level of expansion and differentiation of Treg cells and or prevention of Th17 expansion/differentiation.
Once the cellular preparations have been obtained, they can be formulated for administration to the subject. The formulation step can comprise admixing the cellular preparations (at a therapeutically effective dose) with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, and/or carriers. The formulations are preferably in a liquid injectable form and can include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces. The formulations can comprise pharmaceutically acceptable solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol).
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
Human PBMC and dendritic cell preparation. Human whole blood was collected in heparinized vacutainer blood collection tubes (BD, Franklin Lakes, N.J.) from healthy volunteer donors following informed consent. PBMC were isolated from diluted whole blood using FicollePaque PREMIUM™ (GE Healthcare Bio-Sciences Corp, Piscataway, N.J.) as per the product instructions. The PBMC layer was washed twice with 1× Hank's Balanced Salt Solution (HBSS; without CaCl2 and MgSO4; Invitrogen by Life Technologies, Carlsbad, Calif.) and resuspended in the appropriate media as needed for mixed lymphocyte reactions and flow cytometric analysis of Treg and Th17 phenotypes. Dendritic cells (DC) were prepared from isolated PBMC as described by O'Neill and Bhardwaj (O'Neill et al., 2005). Briefly, freshly isolated PBMC were overlaid on Petri dishes for 3 h in AIM V serum free culture medium (Invitrogen, Carlsbad, Calif.). Non-adherent cells were gently washed off the plate. The adherent cells (monocyte rich cells) were treated with IL-4 and GM-CSF (50 and 100 ng/mL respectively; R&D Systems, Minneapolis, Minn.) in AIM V medium. Cells were again treated with IL-4 and GM-CSF on days 2 and 5. On day 6, cells were centrifuged and resuspended in fresh media supplemented with DC maturation factors (TNF-α, IL-1β, IL-6; R&D Systems, Minneapolis, Minn.) and prostaglandin E2 (Sigma Aldrich, St. Louis, Mo.). The mature DC-like cells were harvested on day 7 and CD80, CD83, CD86 and HLA-DR expressions were determined to confirm DC maturation via flow cytometry (FACSCalibur™ Flow Cytometer, BD Biosciences, San Jose, Calif.).
Murine splenocyte and tissue harvesting. All murine studies were done in accordance with the Canadian Council of Animal Care and the University of British Columbia Animal Care Committee guidelines and were conducted within the Centre for Disease Modeling at the University of British Columbia. Murine donor cells used for the in vivo donation and in vitro studies were euthanized by CO2. Three allogeneic strains of mice were utilized for syngeneic and allogeneic in vitro and in vivo challenge: Balb/c, H-2d; C57Bl/6, H-2b; and C3H, H-2k. Murine spleens, brachial lymph nodes, and peripheral blood were collected at the indicated days. Mouse spleens and brachial lymph nodes were dissected and placed into cold phosphate buffered saline (PBS; 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, and 154 mM NaCl, pH 7.3) containing 0.2% bovine serum albumin (BSA; Sigma Aldrich, St. Louis, Mo.) and kept on ice until ready to process. Whole blood was collected in heparinized tubes via cardiac puncture. Murine donor splenocytes were prepared from freshly harvested syngeneic or allogeneic spleens via homogenization into a cell suspension in PBS (0.2% BSA) using the frosted end of two microscope slides. The resultant cell suspension was spun down at 500×g. The splenocyte pellet was resuspended in 1 mL of 1× BD Pharm LYSE™ lysing buffer (BD Biosciences, San Diego, Calif.) and incubated for 1 min at room temperature. Lymph node cells were harvested via tissue homogenization as described above, washed twice and resuspended in PBS (0.2% BSA) for flow cytometric analysis of Th17, Treg and murine haplotype. Recipient peripheral blood lymphocytes were prepared via lysis of the red cells (BD Pharm Lyse lysing buffer; BD Biosciences, San Diego, Calif.) at 1× concentration, followed by washing (1×) and resuspension in PBS (0.2% BSA) for flow analysis of Th17, Treg and murine haplotype.
mPEG modification (PEGylation) of PBMCs and splenocytes. Human PBMC and murine splenocytes were derivatized using methoxypoly(-ethylene glycol) succinimidyl valerate (mPEG-SVA; Laysan Bio Inc. Arab, Ala.) with a molecular weight of 5 or 20 kDa as previously described (Scott et al., 1997; Murad et al, 1999A; Chen et al., 2003; Chen et al., 2006). Grafting concentrations ranged from 0 to 5.0 mM per 4×106 cells/mL. Cells were incubated with the activated mPEG for 60 min at room temperature in isotonic alkaline phosphate buffer (50 mM K2HPO4 and 105 mM NaCl; pH 8.0), then washed twice with 25 mM HEPES/RPMI 1640 containing 0.01% human albumin. Human PBMC were resuspended in AIM V media at a final cell density of 2.0×106 cells/mL for use in the MLR. Murine splenocytes used for in vivo studies were resuspended in sterile saline at a final cell density of 2.0×108 cells/ml for i.v. injection. To determine if the simple presence of the mPEG polymer itself altered the immune response either in vitro and in vivo, additional studies were done with unactivated polymer incapable of covalent grafting to the cell surface. For these studies, allogeneic human (in vitro studies) or syngeneic and allogeneic murine splenocytes (in vivo studies) were treated with non-covalently bound mPEG (soluble mPEG) under the same reaction conditions described for the covalent grafting studies. For clarity, “soluble mPEG” refers to cells treated with non-covalently grafted polymer while “mPEG-modified” refers to treatment with activated polymer resulting in the covalent grafting of the mPEG to the cell membrane.
In vitro and in vivo cell proliferation. Cell proliferation (both in vitro and in vivo) was assessed via flow cytometry using the CELLTRACE™ CFSE (Carboxyfluorescein diacetate, succinimidyl ester) Cell Proliferation Kit (Invitrogen by Life Technologies e Molecular probes, Carlsbad, Calif.). Human and murine cells labeling was done according to the product insert at a final concentration of 2.5 mM CFSE per 2×106 cells total. Donor and recipient cell proliferation was differentially determined by haplotype analysis. In some experiments, cell proliferation was measured by 3H-thymidine incorporation. In these experiments, donor splenocytes (5.12×106 cells per well) were co-incubated in triplicate in 96-well plates at 37° C., 5% CO2 for 3 days. On day 3, all wells were pulsed with 3H-thymidine and incubated for 24 h at 37° C., 5% CO2. Cellular DNA was collected on filter mats using a Skatron cell harvester (Suffolk, U.K.) and cellular proliferation was measured by 3H-thymidine incorporation.
Mixed lymphocyte reaction (MLR)—control and conditioned media. The effects of polymer grafting (5 kDa SVAmPEG) on allorecognition in vitro were assessed using two-way MLR (Murad et al, 1999A; Chen et al., 2003; Chen et al., 2006). PBMC from two MHC-disparate human donors were label with CFSE as described. Each MLR reaction well contained a total of 1×106 cells (single donor for resting or mitogen stimulation or equal numbers for disparate donors for MLR). Cells were plated in multiwell flat-bottom 24-well tissue culture plates (BD Biosciences, Discovery Labware, Bedford, Mass.). PBMC proliferation, cytokine secretion, as well as Treg and Th17 phenotyping was done at days 10 and 14. For flow cytometric analysis, the harvested cells were resuspended in PBS (0.1% BSA). While time course studies (Days 4, 7, 10 and 14) were done, the presented studies show days 10 and 14. These extended studies are, in fact, the most stringent test of the immunomodulatory effects of the grafted polymer as membrane remodeling over this time could have resulted in a latter onset of proliferation. To investigate in vitro whether polymer grafting to allogeneic PBMC gave rise to tolerance or anergy, secondary (2°) MLR studies were conducted using conditioned media. Conditioned media from a primary (1°) 2 way-MLR was collected at 72 h for conducting a secondary (2°) MLR as schematically shown in
Immunophenotyping by flow cytometry. The T lymphocytes populations (double positive for CD3+ and CD4+) in both the in vitro and in vivo studies were measured by flow cytometry using fluorescently labeled CD3 and CD4 monoclonal antibodies (BD Pharmingen, San Diego, Calif.). Human and mouse Regulatory T lymphocytes (Treg) were CD3+/CD4+ and FoxP3+ (transcription factor) while inflammatory Th17 lymphocytes cells were CD3+/CD4+ and IL-17+ (cytokine) as measured per the BD Treg/Th17 Phenotyping Kit (BD Pharmingen, San Diego, Calif.). After the staining, the cells (1×106 cells total) were washed and resuspended in PBS (0.1% BSA) prior to flow acquisition. Isotype controls were also used to determine background fluorescence. All samples were acquired using the FACSCalibur™ flow cytometer (BD Biosciences, San Jose, Calif.) and CellQuest Pro™ software for both acquisition and analysis.
Cytokine quantitation. Cell culture supernatants were collected from the 1° two-way MLR plate and stored at −80° C. prior to analysis. Conditioned media aliquots from a minimum of four independent experiments were used for quantification of supernatant cytokine levels using the BD Cytometric Bead Array (CBA) system (BD Biosciences, San Diego, Calif.) for flow cytometry. The CBA system is a multiplexed bead based immunoassay used to quantitate multiple cytokine levels in a single sample simultaneously by fluorescence-based emission and flow cytometry. Cytokine measured included: IFNγ, TNF-α, IL-10, IL-5, IL-4, and IL-2 using the BD Human Th1/Th2 Cytokine Kit I™. The IL-6 and IL-17A levels were measured using the BD CBA Human Soluble Protein Flex Set™. Both assays were performed following the manufacturer's product instruction manual. Briefly, cell culture supernatants of resting unmodified PBMC, unmodified MLR, PEGylated (5 kDa SVAmPEG; one donor) resting PBMC, PEGylated MLR, and mitogen (PHA) stimulated PBMC were incubated at room temperature in the dark with a mixture of each cytokine antibody-conjugated capture bead and the PE-conjugated detection antibody. Following the incubation, the samples were washed and acquired using a FACSCalibur™ flow cytometer and analyzed using Cell-Quest Pro™ software. Cytokine protein levels were determined using the BD Cytometric Bead Array™ and FCAP Array™ analysis software (BD Biosicences, San Diego, Calif. and Soft Flow Inc, St. Louis Park, Minn.).
In vivo murine studies. To investigate whether mPEG grafting to leukocytes would have systemic in vivo effects, a murine adoptive transfer system was employed using three genetically different strains: Balb/c, H-2d; C57Bl/6, H-2b; and C3H, H-2k (Chen et al., 2003; Chen et al., 2006). All mice (donors and recipients) were 9-11 weeks old. Donor splenocytes were prepared and CSFE labeled as described. control and mPEG-grafted (1 mM, 20 kDa SVAmPEG) syngeneic or allogeneic cells (20×106 splenocytes) were transfused intravenously (i.v.) via the tail vein into recipient animals. BALB/c and C57BL/6 mice injected with sterile saline served as control animals. Animals were euthanized by CO2 at predetermined intervals at which time blood, brachial lymph nodes and spleen were collected and processed for Th17/Treg phenotyping analysis and splenocyte proliferation studies by flow cytometry. Donor cell engraftment and proliferation were assessed via flow cytometry using murine haplotype (H-2Kb vs. H-2Kd) analysis. To determine the persistence of the immunomodulation, mice were re-challenged (2° challenge) 30 days after the initial transfer of allogeneic or mPEGallogeneic splenocytes with unmodified allogeneic cells. At 5 days post 2° challenge, Treg and Th17 phenotyping of murine splenocytes isolated from the spleen, lymph node and peripheral blood was again assessed via flow cytometry.
Statistical analysis. Data analysis was conducted using SPSS™ (v12) statistical software (Statistical Products and Services Solutions, Chicago, Ill., USA). For significance, a minimum p value of <0.05 was used. For comparison of three or more means, a one-way analysis of variance (ANOVA) was performed. When significant differences were found, a post-hoc Tukey test was used for pair-wise comparison of means. When only two means were compared, student-t tests were performed.
The material and methods used in this example are provided in Example I.
To determine the effects of polymer-grafting on the immune response, initial in vitro experiments examined the cytokine burst characterizing control and polymer modified MLR. The polymer-mediated immunocamouflage of human PBMC resulted in significant changes in the cytokine profile of the conditioned media obtained from the 1° MLR plate (
The conditioned media produced from the initial 72 h MLR exerted a significant effect on the 2° MLR as demonstrated in
Furthermore, as shown in
Hence, the in vitro experiments demonstrated that allogeneic PBMC results in a pro-inflammatory effect governed in part by changes in the Th17 and Treg populations and subsequent ratio of these cell populations. Moreover, these conditioned media experiments demonstrated that this immunomodulatory effect arises from soluble factors that might be able to induce a systemic effect in vivo. To determine if similar effects would be observed in vivo, a murine splenocyte adoptive transfer model was utilized. As demonstrated in
As foreshadowed by our in vitro human PBMC findings (Example II), murine Th17 lymphocyte levels were differentially influenced by the administration of unmodified or mPEG-modified allogeneic donor cells (
As also shown on
As might be anticipated, the allogeneic splenocyte mediated increase in Th17 cells in the peripheral blood samples occurred later in the studied time course (96 h) compared to either of the lymphatic tissues (spleen and lymph nodes; 48 h). This clearly suggests that T cell proliferation initially occurred within the lymphatic tissues and secondarily migrated into the peripheral blood. A similar time dependency was noted with the Treg proliferation induced by the mPEG-modified splenocyte populations. Proliferation initially occurred within lymphatic tissue within ˜48 h and only appeared within the peripheral blood after ˜96 h.
Of importance was the observation that the immunomodulatory effects of the allogeneic splenocytes were long lived. As shown in
To determine if the observed in vivo murine findings gave rise to a tolerance to a specific H-2 haplotype or a more generalized anergy to allogeneic tissues, in vitro two-way murine MLR studies of three allogeneic splenocyte populations (Balb/c, H-2d; C57Bl/6, H-2b; and C3H, H-2k) were done. As demonstrated in
As demonstrated herein, allogeneic lymphocytes (human PBMC or murine splenocytes) relative to mPEG-modified allogeneic cells dramatically increase allorecognition and pro-inflammatory effects at both the local (cell:cell; MLR) and systemic (in vivo murine models) levels. Importantly, as demonstrated in our in vivo studies, it is not the donor cells that differentiate into Th17 (or other pro-inflammatory subpopulations) or Treg cells, rather it is the recipients immune system that responds to the unmodified or PEGylated splenocytes and upregulates production of either Th17 (upon challenge with unmodified splenocytes) or Treg (upon challenge with mPEG-splenocytes) populations. This was noted by both the absence of CFSE-staining (only donor cells were stained) and H-2 phenotyping of the Th17 and Treg cell populations.
The observed proinflammatory state induced by unmododified lymphocyte preparations is surprisingly long lasting in vivo. As noted in
The balance between Treg and Th17 cells has been identified as a key factor that orchestrates the tolerance/inflammation level of human immune system. Regulatory T cells provide suppressor effect and maintain tolerance, while Th17 cells mediate and are indicative of a pro-inflammatory state. Hence, modulation of this balance (either increasing or decreasing the Treg:Th17 ratio) may be clinically useful. Recent findings have shown that cyclosporine, a clinically used immunosuppressive agent, has substantial effects on the Treg/Th17 cell response; though this may be mediated by Th17 cytotoxicity as Treg cells cultured in the presence of rapamycin, but not cyclosporine A, are found to suppress ongoing alloimmune responses. Additionally, mycophenolic acid, another immunosuppressive agent, was found to shift the lymphocyte polarization by inhibiting IL-17 expression in activated PBMC in vitro. Of clinical importance, all of these pharmacologic agents exert significant systemic toxicity and their ongoing use requires substantial monitoring.
While induction of tolerance or anergy in transfusion and transplantation medicine by the polymer-mediated immunocamouflage of allogeneic leukocytes may provide a less toxic approach than current conventional pharmacologic agents, other situations exist in which enhancing the pro-inflammatory state of a subject would be beneficial. Indeed, increased Treg levels (or Treg:Th17/Th1 ratio) may prevent desired immunological responses to cancer cells, parasitic infections, or viral infection. While an abundant number of approaches are being investigated to prevent and/or regulate the consequences of allorecognition exist (as exemplified by phenotype matching (ranging from blood group to HLA matching) and the use of immunosuppressive agents;
Thus, while pharmacological interventions have been employed to enhance the probability of successful donor tissue engraftment (
Conversely to the induction of tolerance, the administration of immunogenic allogeneic or xenogeneic cells (e.g., leukocytes) that retain all or part (e.g. a partial modification via low levels of grafted polymer) of their inherent immunogenicity can be used to stimulate the immune system as evidenced by a reduced Treg:Th1/Th17 ratio. This approach can be used to counter a pre-existing state of anergy or tolerance arising either inherently or due to an infective agent (e.g. parasite). As shown by administration of allogeneic, non-viable or viable, leukocytes, or preparations thereof, a proinflammatory state can be induced. Said state will provide an enhanced systemic pro-inflammatory response allowing for overcoming an immunosuppressive state and a more effective response abnormal cells or cell aggregates (e.g. cancer cells) can be achieved as well as an enhanced response to infective agents (e.g. nematodes) that induce an immunosuppressive state.
In summary, administration of viable or non-viable allogeneic donor lymphocytes can be used to induce or enhance a pro-inflammatory state in subjects exhibiting a spontaneous or induced immunosuppressive state. The enhanced proinflammatory state exists at the cell:cell level and also gives rise to systemic immunomodulation. The systemic immunomodulation is evidenced by a significant up-regulation of pro-inflammatory Th17 cells and/or a significant down-regulation of Treg cells. This immunomodulation is persistent (˜30 days). The clinical use of unmodified or partially modified (e.g., PEG or other covalently grafted polymers) allogeneic leukocytes may be useful in inducing a pro inflammatory state and enhancing the destruction of abnormal cells or cell aggregates (e.g. cancer cells and cancer tumors) and/or enhancing the immunological response to infective agents (e.g., parasitic nematodes).
Some of the material and methods referred to in this example are provided in Example I.
In the NOD mice, autoimmune destruction of the pancreatic islets occurs within approximately 16 weeks and was confirmed with elevated blood glucose measures. The lymphocytes from pre-diabetic and diabetic animals has been obtained from the spleen, the brachial lymph node and the pancreatic lymph node. These lymphocytes have been submitted to flow cytometry using anti-IL-17A (PE) and anti-FoxP3 (Alexa 697) antibodies. As shown in
The NOD mice (8 to 10 week-old) have been treated with allogeneic leukocytes (as described in Example I) and mPEG-allogeneic leukocyte (as described in Example I) and were compared to untreated control mice (naïve or NOD in Table 1). Th17 levels have been measured in various tissues (as described in Example I). Peripheral blood samples of the groups were pooled for analysis, all other samples were measured individually. Five male NOD mice per group were used. The results are shown in Table 1 provided below. As noted in Table 1, the level of Th17 (pro-inflammatory) cells can be increased by treatment with unmodified allogeneic cells. This pro-inflammatory state results in increased tissue destruction. While the use of unmodified or partially modified allogeneic cells would not be used therapeutically in this disease model, the finding provide evidence of an increased pro-inflammatory state and increased killing of specific cells (i.e., pancreatic islets of Langerhans) types.
Some of the material and methods referred to in this example are provided in Example I.
Human PBMC and dendritic cell preparation. Human whole blood was collected in heparinized vacutainer blood collection tubes (BD, Franklin Lakes, N.J.) from healthy volunteer donors following informed consent. PBMC were isolated from diluted whole blood using FicollePaque PREMIUM™ (GE Healthcare Bio-Sciences Corp, Piscataway, N.J.) as per the product instructions. The PBMC layer was washed twice with 1× Hank's Balanced Salt Solution (HBSS; without CaCl2 and MgSO4; Invitrogen by Life Technologies, Carlsbad, Calif.) and resuspended in the appropriate media as needed for mixed lymphocyte reactions and flow cytometric analysis of Treg and Th17 phenotypes. Dendritic cells (DC) were prepared from isolated PBMC as described by O'Neill and Bhardwaj (O'Neill et al., 2005). Briefly, freshly isolated PBMC were overlaid on Petri dishes for 3 h of in AIM V serum free culture medium (Invitrogen, Carlsbad, Calif.). Non-adherent cells were gently washed off the plate. The adherent cells (monocyte rich cells) were treated with IL-4 and GM-CSF (50 and 100 ng/mL respectively; R&D Systems, Minneapolis, Minn.) in AIM V medium. Cells were again treated with IL-4 and GM-CSF on days 2 and 5. On day 6, cells were centrifuged and resuspended in fresh media supplemented with DC maturation factors (TNF-a, IL-1b, IL-6; R&D Systems, Minneapolis, Minn.) and prostaglandin E2 (Sigma-Aldrich, St. Louis, Mo.). The mature DC-like cells were harvested on day 7 and CD80, CD83, CD86 and HLA-DR expressions were determined to confirm DC maturation via flow cytometry (FACSCalibur™ Flow Cytometer, BD Biosciences, San Jose, Calif.).
mPEG modification (PEGylation) of PBMCs and splenocytes. Human PBMC and murine splenocytes were derivitized using methoxypoly(-ethylene glycol) succinimidyl valerate (mPEG-SVA; Laysan Bio Inc. Arab, Ala.) with a molecular weight of 20 kDa as described in Example I. Grafting concentrations ranged from 0 to 3.0 mM per 4×106 cells/mL.
POZ modification (POZylation) of PBMCs and splenocytes. N-hydoxysuccinimidyl ester of polyethyloxazoline propionic acid (SPA-PEOZ; Serina Therapeutics, Huntsville, Ala.) with a molecular weight of 20 kDa were grafted on the cells as described in Example I. Grafting concentrations ranged from 0 to 3.0 mM per 4×106 cells/mL.
In vitro and in vivo cell proliferation. Cell proliferation (both in vitro and in vivo) was assessed via flow cytometry using the CellTrace™ CFSE (Carboxyfluorescein diacetate, succinimidyl ester) Cell Proliferation Kit (Invitrogen by Life Technologies e Molecular probes, Carlsbad, Calif.) as described in Example I.
Mixed lymphocyte reaction (MLR)—control and conditioned media. The effects of polymer grafting (20 kDa SVAmPEG or 20 kDa POZ) on allorecognition in vitro were assessed using two-way MLR (Murad et al, 1999A; Chen et al., 2003; Chen et al., 2006) as described in Example I.
A 2-way MLR was conducted using either PEGylated or POZylated human cells. As shown on
Some of the material and methods referred to in this example are provided in Example I.
Non-modified allogeneic splenocytes (20×106) and mPEG-modified allogeneic splenocytes (20×106) have been intravenously administered to mouse (naïve 8-week old Balb/c mouse; 10 mice per treatment condition). After 5 days, the spleen and the lymph nodes were harvested and the CD4-positive cells they contained were further analyzed by flow cytometry. As shown in
Non-modified allogeneic splenocytes (either 5, 20 or 40×106 C57BL/6 cells) and mPEG-modified allogeneic splenocytes (either 5, 20 or 40×106 C57BL/6 cells grafted at a density of 0.5 mM, 1 mM or 4 mM) have been intravenously administered to mouse (5 Balb/c mice/treatment condition). After 5 days, the spleen and the lymph nodes were harvested and the CD4-positive cells they contained were further analyzed by flow cytometry. As shown in
Saline, syngeneic splenocytes, non-modified allogeneic splenocytes (20×106 C57BL/6 cells) and mPEG-modified allogeneic splenocytes (20×106 C57BL/6 cells grafted at a density of 1 mM PEG) have been intravenously administered to mouse either once (at day 0, e.g. condition 1) or thrice (at days 0, 2 and 4, e.g. condition 3) (20×106 C57BL/6 cells grafted at a density of 1 mM PEG). After 5 or 10 days, the spleen and lymph nodes were harvested and the CD4-positive cells they contained were further analyzed by flow cytometry with an anti-CD279 antibody. As shown in
The thymus of these animals has also been harvested and the thymic cells characterized. As shown in
Some of the material and methods referred to in this example are provided in Example I.
Conditioned serum. Conditioned serum was obtained (by bleeding the animal and separating the cellular components of blood from the serum via centrifugation) five days after mice (Balb/c; N=5) received saline, unmodified syngeneic splenocytes (Balb/c), unmodified allogeneic splenocytes (20×106 C57BL/6 cells) or mPEG-modified allogeneic splenocytes (20×106 C57BL/6 cells grafted at a density of 1 mM PEG). The serum from naïve animals was also obtained as a control. The conditioned or naïve serum (100 μl) was then administered (i.v. tail vein injection) once (at day 0) or thrice (at days 0, 2 and 4) to recipient mice (Balb/c; N=5). Five days after the last administration, a blood sample, the spleen and the brachial lymph nodes were obtained from the treated animals and the leukocytes they contained were analyzed.
As shown on
This modulation in Treg/Th17 ratio was also shown to be associated in the long term modification of the expression of pro-/anti-inflammatory cytokine positive CD4+ lymphocytes. As shown on
The administration of the conditioned medium also caused a shift in the Treg subsets. As shown on
As shown on
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While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2782942 | Jul 2012 | CA | national |
This application claims priority from CA patent application 2782942, U.S. provisional patent application 61/670636 and U.S. provisional patent application 61/670694 all filed on Jul. 12, 2012. Their content is incorporated herewith in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2013/050546 | 7/12/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61670636 | Jul 2012 | US | |
| 61670694 | Jul 2012 | US |
