US20050202098A1 - Disease therapy using dying or dead cells - Google Patents

Disease therapy using dying or dead cells Download PDF

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US20050202098A1
US20050202098A1 US11/121,048 US12104805A US2005202098A1 US 20050202098 A1 US20050202098 A1 US 20050202098A1 US 12104805 A US12104805 A US 12104805A US 2005202098 A1 US2005202098 A1 US 2005202098A1
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leukocytes
apoptosis
disease
monocytes
cells
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Dror Mevorach
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Enlivex Therapeutics Ltd
Tolaren
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Tolaren
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Priority claimed from US10/470,536 external-priority patent/US20050031618A1/en
Priority to US11/121,048 priority Critical patent/US20050202098A1/en
Assigned to TOLAREN reassignment TOLAREN ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MEVORACH, DROR
Application filed by Tolaren filed Critical Tolaren
Publication of US20050202098A1 publication Critical patent/US20050202098A1/en
Priority to JP2008509575A priority patent/JP2008540400A/ja
Priority to CA002606803A priority patent/CA2606803A1/fr
Priority to PCT/IL2006/000527 priority patent/WO2006117786A1/fr
Priority to EP06728323.4A priority patent/EP1879601B1/fr
Priority to EP10183710A priority patent/EP2283847A3/fr
Priority to IL187122A priority patent/IL187122A/en
Assigned to TOLAREX LTD. reassignment TOLAREX LTD. CORRECTIVE ASSIGNMENT TO CORRECT ERRORS IN A PREVIOUSLY RECORDED ASSIGNMENT. DOCUMENT RECORDED AT REEL 016537 FRAME 0619. Assignors: MEVORACH, DROR
Assigned to ENLIVEX THERAPEUTICS LTD. reassignment ENLIVEX THERAPEUTICS LTD. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: TOLAREX LTD.
Priority to US14/499,279 priority patent/US9567568B2/en
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Definitions

  • the present invention relates to methods of using dying cells for treating diseases characterized by pathological immune responses, and to devices for preparing such dying cells. More particularly, the present invention relates to methods of using apoptotic leukocytes for treating diseases characterized by pathological immune responses, such as autoimmune diseases and transplantation related diseases, and to devices for preparing such apoptotic leukocytes.
  • Diseases characterized by pathological immune responses include a large number of diseases which are associated with significant mortality and morbidity, and for which no satisfactory/optimal treatments are available.
  • diseases particularly include autoimmune diseases, such as systemic lupus erythematosus, (SLE), transplantation-related diseases such as graft-versus-host disease (GVHD).
  • SLE systemic lupus erythematosus
  • GVHD transplantation-related diseases
  • GVHD graft-versus-host disease
  • the immune system is a complex network comprising cells, antibodies, tissues, and chemical messenger molecules which allow for communication between these structures.
  • a hallmark of a healthy immune system is the ability to recognize bacteria, viruses, and other foreign bodies and to effectively attack such pathogens while continuing to distinguish between the foreign bodies and the molecules, cells, tissues and organs of the body.
  • the immune system has other roles in maintaining the normal state of health and function of the body. Throughout the life span of an organism, tissues become reshaped with areas of cells being removed. This is accomplished by a process termed programmed cell death or apoptosis, the apoptotic cells disintegrating in an orderly and harmless fashion and being phagocytosed.
  • a certain percentage of the cells die off every day while different branches of the immune system are typically called in to remove the dead cells and parts thereof to make room for the new cells which arise to replace them.
  • the process of apoptosis is furthermore considered to be particularly important in the development and maintenance of the immune system itself, where the immune cells which recognize or attack normal cells of the body are destroyed and removed by this process.
  • the number of monocytes, neutrophils, and lymphocytes that are produced, circulating, dying, and extravasating in the body is controlled at various levels, including via apoptosis.
  • monocytes CFU-GM
  • CFU-GM the earliest identified cell committed to differentiate along the myeloid pathway, develops into monocyte in the bone marrow, mainly in the presence of M-CSF, IL-3, and low levels of GM-CSF.
  • Monocytes comprise 1-6 percent of peripheral leukocytes, and it is estimated that 5.7 ⁇ 10 6 monocytes/kg are produced every day. Monocytes can survive in tissues as macrophages for long periods, but a substantial portion of monocytes are constantly undergoing apoptosis, either in the absence of anti-apoptotic factors or following infection or activation.
  • Monocytes express Fas and Fas: ligand irrespective of their state of activation [2, 3], and were shown to undergo Fas-dependent apoptosis upon culture [3], activation [4], or infection [5]. Monocytes can be rescued from apoptosis upon exposure to growth factors; differentiating factors (GM-CSF and IL-4), or activation factors [3, 6-8]. Upon differentiation to macrophages, monocytes are rescued from Fas-dependent apoptosis by the expression of Fas-associated death domain-like IL-1beta-converting enzyme-inhibitory protein (FLIP) [3, 9].
  • FLIP Fas-associated death domain-like IL-1beta-converting enzyme-inhibitory protein
  • Neutrophils constitute the most abundant population of leukocytes. In humans, the daily turnover of neutrophils is about 1.6 ⁇ 10 9 cells/kg body weight (Klebanoff S J, Clark R X: The Neutrophil: Function and Clinical Disorders. Amsterdam, North-Holland Publishing, 1978, p 313), which keeps the number of mature neutrophils within defined limits despite the tremendous proliferative potential of the bone marrow precursor cells. This large turnover is mediated by the continuous egress of neutrophils from the circulation. Neutrophils do not return to the circulation but are eliminated by secretion in mucosa or die in the tissues within 1-2 days (Klebanoff S J, Clark R X: The Neutrophil: Function and Clinical Disorders.
  • apoptosis is a process used by the immune system in protecting the body, it is also used to maintain tolerance to self-antigens and therefore allowing the immune system to fulfill its role in distinguishing the body's own cells from foreign bodies.
  • Immature dendritic cells have the capacity to engulf apoptotic cells and to acquire and immunologically present their antigens. Immature dendritic cells that capture apoptotic macrophages exposed to killed influenza-virus, mature and activate lymphocytes to mount virus-specific CTL responses in the presence of conditioned media. However, in the absence of infection and conditioned media, immature dendritic cells do not mature following uptake of apoptotic cells and as a consequence are less able to efficiently present acquired antigens.
  • immature dendritic cells may have a role in maintaining, peripheral tolerance to self-antigens that are permanently created at different sites.
  • autoimmunity or SLE-like disease has been observed in mice and humans deficient in receptors important for uptake of apoptotic cells such as ABC cassette transporter, Mer, and complement deficiencies, as further described hereinbelow. Clearance via specific receptors may dictate specific immune response or tolerance as demonstrated by TGF-beta and IL-10 secretion by macrophages following uptake of apoptotic cells by macrophages.
  • cytokines, chemokines, eicosanoids, and additional mediators present in the milieu of the interaction may polarize the immune response.
  • autoimmune disease When the immune system is deficient in recognition between self- and non-self-antigens, the result is a: state of disease, this may result in the immune system attacking one or more specific self molecules or cells leading to tissue and organ damage, resulting in autoimmune disease. Immunopathology of non-targeted tissues also may be indirectly caused non-specifically as a consequence of inflammation resulting from immune rejection of neighboring cells and tissues. Other than classical autoimmune diseases such as those mentioned hereinabove, it is becoming increasingly apparent that many vascular disorders, including atherosclerotic forms of such disorders, have an autoimmune component, and a number of patients with vascular disease have circulating autoantibodies.
  • Autoimmune diseases may be divided into two general types, namely systemic autoimmune diseases, such as SLE and scleroderma, and organ specific autoimmune diseases, such as multiple sclerosis, and diabetes. Many clinically different types and subtypes of autoimmune disease occur. Although each type of autoimmune disease is associated with a spectrum of clinical symptoms and aberrant laboratory parameters, signs and symptoms of autoimmune diseases frequently overlap so that one or more are diagnosed in the same patient. The vast majority cases in which one or more autoimmune disease has been diagnosed are characterized by the presence in the affected subject of antibodies directed against self-antigens, termed autoantibodies.
  • Such autoantibodies are often present in tissues at ten to one hundred times the normal level in healthy individuals and give rise to a significant proportion of the organ and tissue damage associated with the particular autoimmune disease.
  • myasthenia gravis autoantibodies against a receptor in neuromuscular junction are associated with muscle weakness
  • SLE anti-dsDNA antibodies are associated with nephritis in human patients and: can cause nephritis upon injection to normal mice.
  • the tissue and organ damage is attributed to the presence of autoantibodies and to the inflammation, which arises due inflammatory immune responses set off by autoantibodies.
  • Systemic lupus, erythematosus is a model disease for understanding and developing inventive treatments for autoimmune disease in general. While it has long been appreciated that DNA and histones are major autoantigens SLE, only recently has evidence been provided that the DNA-histone complex, i.e., nucleosomes, are the preferred targets of autoantibodies in SLE.
  • DNA-histone complex i.e., nucleosomes
  • the membrane of cells undergoing apoptosis form cytoplasmic blebs, some of which are shed as apoptotic bodies.
  • the immunopathology of SLE appears to further involve defective uptake of apoptotic material by macrophages, as observed in the reduced uptake/clearance of apoptotic cells by macrophages from SLE patients in-vitro, and by the high incidence of SLE in patients deficient in the C1q and C4 components' of the complement system, which is involved in uptake of targeted antigens.
  • Lymphocytes i.e. T-cells and B-cells
  • T-cells and B-cells are relatively resistant to apoptosis.
  • B-cells and T-cells proliferate and some will differentiate into effector cells.
  • Plasma cells secrete antibodies that immobilize pathogens and promote their complement-mediated destruction and Fc (Ig constant region)-receptor-mediated ingestion by certain myeloid cells.
  • Fc Ig constant region
  • This armory has the potential to destroy healthy cells and tissues because many of the effector molecules, such as pro-inflammatory cytokines, act in a non-antigen-specific manner and also because certain pathogen-specific receptors, such as B-cell receptors (BCRs) and T-cell receptors (TCRs) may cross-react with host antigens.
  • BCRs B-cell receptors
  • TCRs T-cell receptors
  • Immune responses to pathogens therefore pose a potential danger to the host and immunopathology occurs with many types of infection.
  • chronically activated lymphocytes that are rapidly proliferating, particularly B-cells in germinal centers undergoing Ig-variable gene hyper-mutation, are at risk of sustaining mutations in proto-oncogenes or tumor suppressor genes that could lead to the development of lymphoma and/or leukaemia.
  • Multiple regulatory mechanisms have evolved to prevent immunopathology. These include functional inactivation of cells of the immune system, a process that is potentially reversible and therefore does not eliminate the danger, and killing of no-longer needed and/or potentially dangerous cells by apoptosis [Marsden and A. Strasser, 2003. Annu. Rev. Immunol. 21:71-105].
  • autoimmune diseases such as SLE
  • transplantation-related diseases such as GVHD
  • immunosuppressive drugs such as corticosteroids, azathioprine, cyclophosphamide, and cyclosporine. While such drug-induced immunosuppression has resulted, for example, in improvement of the 5-year survival rate of SLE patients in the last three decades, it is far from being an ideal treatment since no cure is achieved, since such treatment is associated with very serious side-effects, including general immune suppression, leading to high rates of morbidity, and is the primary cause of premature mortality.
  • apoptotic donor cells, such as apoptotic donor leukocytes, to facilitate engraftment of donor hematopoietic grafts transplanted into an allogeneic recipient [Perruche S. et al., 2004. Am J Transplant. 4:1361-5; Kleinclauss F. et al., 2003. Transplantation 75(9 Suppl):43S-45S].
  • extracorporeal photopheresis involves administering to a patient a photoactivatable pigment which can be specifically taken up by specific hematopoietic cells, such as T-cells, and following such uptake harvesting blood, isolating the specific hematopoietic cells, triggering their apoptosis via UV irradiation, and infusing them back into the patient (U.S. Pat. No. 6,219,584).
  • This approach has been advocated for treatment of hypersensitivity, graft rejection, or SLE (U.S. Pat. No.
  • Anaphylactoid reactions during dextran apheresis may occur even in the absence of ACE-inhibitor administration.
  • the present invention discloses the use of dying or dead leukocytes for treatment of diseases associated with pathological immune responses, and discloses devices for generating such dying or dead leukocytes. This use can be effected in a variety of ways, and these devices can be configured in a variety of ways, as further described and exemplified hereinbelow.
  • a method of treating a disease characterized by a pathological immune response in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a cell preparation which comprises dying or dead leukocytes, the dying or dead leukocytes being capable of suppressing the pathological immune response, thereby treating the disease in the subject.
  • the method further comprises subjecting live leukocytes to a cytocidal treatment selected from the group consisting of in-vitro serum deprivation, treatment with a steroid or steroid derivative, irradiation, and a pro-apoptotic treatment, thereby generating the dying or dead leukocytes.
  • a cytocidal treatment selected from the group consisting of in-vitro serum deprivation, treatment with a steroid or steroid derivative, irradiation, and a pro-apoptotic treatment, thereby generating the dying or dead leukocytes.
  • the method of treating the disease further comprises inducing live leukocytes to adhere to a surface, thereby generating the dying or dead leukocytes.
  • the pathological immune response is directed against at least one antigen
  • the dying or dead leukocytes comprise the at least one antigen
  • the dying or dead leukocytes are derived from the subject.
  • the dying or dead leukocytes comprise dying or dead splenocytes and/or dying or dead thymocytes.
  • the dying or dead leukocytes comprise dying or dead lymphocytes.
  • the dying or dead leukocytes comprise dying or dead monocytes.
  • the dying or dead leukocytes comprise dying or dead neutrophils.
  • the dying or dead leukocytes comprise apoptotic leukocytes.
  • the disease is a systemic autoimmune disease.
  • the disease is an antibody-mediated autoimmune disease.
  • the disease is lupus erythematosus:
  • the disease is a transplantation-related disease.
  • the disease is graft-versus-host disease.
  • administering the cell preparation comprises administering to the subject a total number of the dying or dead leukocytes selected from a range of about 20 million to about 2 billion cells per kilogram body weight of the subject.
  • administering the cell preparation comprises administering to the subject at least one unit dose of the dying or dead leukocytes, wherein the unit dose comprises a number of the dying or dead leukocytes selected from a range of about 4 million to about 2 billion cells per kilogram body weight of the subject.
  • a device for treating a disease characterized by a pathological immune response comprising: (a) a pump for pumping blood from a subject into the device and returning blood to the subject from the device; (b) a leukocytes separator in communication with the pump for separating circulating-leukocytes from whole blood; and (c) an apoptosis-inducing chamber or chambers in communication with the leukocytes separator for inducing apoptosis of the leukocytes to thereby obtain apoptotic leukocytes, and further in communication with the pump for administering the apoptotic leukocytes to the subject.
  • the apoptosis-inducing chambers comprise a first chamber for inducing apoptosis of monocytes, a second chamber for inducing apoptosis of neutrophils, and a third chamber for inducing apoptosis of lymphocytes.
  • a device for inducing apoptosis of leukocytes comprising an apoptosis-inducing chamber or chambers for inducing apoptosis of leukocytes to thereby obtain apoptotic leukocytes, wherein the apoptosis-inducing chamber or chambers is selected from the group consisting of a first chamber for inducing apoptosis of monocytes, a second chamber for inducing apoptosis of neutrophils, and a third chamber for inducing apoptosis of lymphocytes.
  • the first chamber comprises a surface for enhancing adherence of monocytes thereto.
  • the device further comprises a first reservoir for containing a monocyte medium, wherein the monocyte medium is for inducing apoptosis of monocytes.
  • the device further comprises a second reservoir for containing a neutrophil medium, wherein the neutrophil medium is for inducing apoptosis of neutrophils.
  • the device further comprises a third reservoir for containing a lymphocyte medium, wherein the lymphocyte medium is for inducing apoptosis of lymphocytes.
  • the device further comprises mechanism for resuspending surface-adherent monocytes.
  • the mechanism for resuspending the surface adherent monocytes is selected from the group consisting of: a reservoir for containing a protease and a mechanism for introducing the protease into the first chamber; a flow-generating mechanism for generating in the first chamber a flow of sufficient force and direction for resuspending the surface-adherent monocytes; and a scraping mechanism for scraping the surface-adherent monocytes off the surface of the first chamber.
  • the apoptosis-inducing chamber or chambers comprises an apoptosis-inducing mechanism selected from the group consisting of an irradiating mechanism for inducing apoptosis; a mechanical mechanism for inducing apoptosis; and a chemical or biochemical substance or environment for inducing apoptosis.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing a method of treating with improved safety and effectiveness diseases associated with pathological immune responses, such as autoimmune diseases and GVHD, by administration of dying or dead leukocytes, by providing a device for generating such leukocytes, and by providing a device for practicing such methods.
  • FIG. 1 is a histogram depicting reduction of serum anti-single-stranded DNA antibodies in MRL/MpJ-Fas Ipr mice following treatment with syngeneic apoptotic cells. Filled circles, control group of 6 week-old MRL/Ipr/Ipr-mice immunized with vehicle only; open circles, experimental group of 6 week-old MRL/Ipr/Ipr mice immunized with syngeneic apoptotic cells; filled triangles, control group after 10 weeks of treatment; open triangles, experimental group after 10 weeks of treatment.
  • FIG. 2 is a histogram depicting reduction of serum anti-double-stranded DNA antibodies in MRL/MpJ-Fas Ipr mice following treatment with syngeneic apoptotic cells. Filled circles, control group of 6 week-old MRL/Ipr/Ipr mice immunized with vehicle only; open circles, experimental group of 6 week-old MRL/Ipr/Ipr mice immunized with syngeneic apoptotic cells; filled triangles, control group after 10 weeks of treatment; open triangles, experimental group after 10 weeks of treatment.
  • FIG. 3 is a set of fluorescence activated cell sorting (FACS) dot plots depicting induction of monocyte apoptosis by serum withdrawal and substrate-adherence. More than 70 percent of monocytes were annexin V-positive PI-negative by 12 hours indicating early apoptosis. Secondary necrotic cells represented less than 5 percent of the cells as indicated by annexin V-positive, propidium iodide: (PI) positive cells. The specificity of the apoptotic process was further shown by marked inhibition in the presence of 20 mM zVAD-fmk. The percentage of early apoptotic and secondary necrotic cells is indicated within each histogram. Data is representative of six different experiments.
  • FACS fluorescence activated cell sorting
  • FIG. 4 is a set of fluorescence activated cell sorting (FACS) dot plots depicting that suspension+serum-withdrawal-induced death of monocytes is non-apoptotic and shows features of necrosis. Prevention of contact in addition to serum withdrawal switched the mechanism of death. Cell numbers were reduced progressively whereas the percentage of annexin+PI ⁇ remain constant and low. Cells were becoming directly annexin+PI+ and 20 mM zVAD-fmk did not reduce the rate of death (not shown).
  • FACS fluorescence activated cell sorting
  • FIGS. 5 a - c depict de-novo transcription of pro-inflammatory cytokine/chemokine mRNAs by monocytes subjected to suspension+serum deprivation.
  • FIGS. 5 a - b are gene expression array analyses depicting de-novo transcription of pro-inflammatory cytokine/chemokine mRNAs by monocytes subjected to suspension+serum deprivation at 0 time and 30 minutes, respectively.
  • Coordinates (A2, B2), which represent IL-1-beta and coordinates (E3, F3), representing IL-8, show no visible fluorescence at time zero and a marked fluorescence at 30 minutes following apoptosis induction.
  • cDNA of IL-6 C3-D3
  • IL-1-alpha E1-F1
  • Other cDNAs that are present with viable cells and did not change much upon death induction are TGF-beta-1 (A7-B7), IL-2 (C2-D2), and TNF-alpha (A8-B8).
  • MIF E6-F6 shows down regulation.
  • A1-B1 for G-CSF (C1-D1) for GM-CSF, (E2-F2) for IL-4, (A3-B3) for IL-5, (A4 B4) for IL-10, (C4-D4) for IL-12-alpha (E4-F4) for IL-12-beta, (A5-B5) for IL-16, (C5-D5) for IL-17, (A6-B6) for LT-beta, (C6-D6) for MCP-1, (C7-D7) for TGF-beta-2, (E7-F7) for TGF-beta-3, and (C8-D8) for TNF-beta.
  • Coordinates that represent negative controls are (G1-G2, PUC18); and as positive controls (G3-G4, beta-actin) and (G5-G6, G7-G8, E8-F8, GAPDH).
  • Chemokine membrane screening showed only IL-8, MIP-1-alpha and MIP-1-beta upregulation (not shown).
  • Membranes contained eotaxin, fractalkine, GROa/MGSA, HCC-4, MCP-3, SDF2, PF-4, MDC, HCC-1, I-309, I-TAC, lymphotactin, MCP-1, MCP-4, MIG, MIP-2, MIP-3-alpha, P10, SDF-1, RANTES.
  • 5 c is a data plot depicting representative cytokine and chemokine cDNA level changes as a function of time following induction of cell death. Note that only IL-1-beta, IL-8, and MIP-1-alpha are produced de-novo.
  • FIG. 6 a is a data plot depicting that the pro-inflammatory cytokine IL-1-beta is produced by monocytes subjected to suspension+serum withdrawal.
  • Control PBMCs exhibit elevation of IL-1-beta secretion following suspension+serum withdrawal (open triangles).
  • FIG. 6 b is a histogram depicting that pro-inflammatory cytokine/chemokine mRNA and protein: are transcribed and translated de-novo by monocytes subjected to suspension+serum withdrawal. Inhibition of transcription activity with actinomycin D and translational activity with cycloheximide shows marked inhibition in cytokine secretion.
  • FIG. 7 a is an ELISA data plot depicting that secretion of IL-1-beta is neither caspase 3—nor caspase 1-dependent and is specific to monocytes subjected to suspension+serum deprivation.
  • IL-1-beta is secreted by monocytes subjected to suspension-induced death but not from viable monocytes, monocytes subjected to hyperthermia-induced necrosis, or apoptotic monocytes.
  • IL-1-beta levels of IL-1-beta were measured by ELISA at 0, 1, 4, and 24 hours following incubation of viable monocytes (closed circles), monocytes rendered necrotic via hyperthermia (open circles), monocytes rendered apoptotic via serum deprivation (open triangles), or monocytes subjected to suspension-induced death (closed triangles).
  • FIG. 7 b is an ELISA data plot depicting secretion of IL-1-beta by monocytes subjected to suspension-induced death (closed triangles, in 20 micromolar DMSO) was neither inhibited with the caspase 1 inhibitor, Z-WEHD (20 micromolar, closed circles), nor with the pan-caspase inhibitor ZVAD-fink (20 micromolar, open circles). In fact, ZVAD-fink significantly increased IL-1-beta secretion (p ⁇ 0.001).
  • FIGS. 8 a - c depict that pro-inflammatory cytokine secretion during monocyte apoptosis is not NFkappaB-dependent.
  • FIG. 8 a is a photograph of a Western immunoblotting assay depicting that pro-inflammatory cytokine secretion during monocyte apoptosis is not NFkappaB-dependent. Shown is 37 kDa IkappaB and phosphorylated IkappaB (black arrow). Viable monocytes (lanes a and b), were incubated for 2 hours in the presence of 1 mg/ml zymosan with (lane a) or without (lane b) MG132 (a proteasome inhibitor).
  • FIG. 8 b is a bar-graph depicting that IL-1-beta secretion in the presence of MG132 is slightly elevated (3 experiments).
  • FIG. 8 c is a histogram depicting transcriptional activity in the presence of MG132 (representative of 33 experiments). Note that fold increases in the levels of mRNA (filled bars) are not changed in the presence of MG132 (empty bars).
  • FIGS. 9 a - e depict that pro-inflammatory cytokine secretion during monocyte apoptosis is p38-dependent.
  • FIG. 9 a is a bar-graph depicting that after 24 hours in the presence of anti-Fas inhibitory antibodies (BD29 or ZB4), monocyte apoptosis was only slightly decreased (BD29 is shown) compared to the significant * (p ⁇ 0.001) decrease in apoptosis seen in the presence of p38 inhibitor or 38 and anti-fas (ZB4).
  • FIG. 9 b is a Western immunoblotting assay depicting that P38 is expressed at comparable levels in monocytes exposed to LPS or induced to undergo apoptosis.
  • FIG. 9 a is a bar-graph depicting that after 24 hours in the presence of anti-Fas inhibitory antibodies (BD29 or ZB4), monocyte apoptosis was only slightly decreased (BD29 is shown) compared to the significant * (p ⁇ 0.001) decrease in a
  • FIG. 9 c is a Western immunoblotting assay depicting that phosphorylated p38 is transiently increased upon LPS stimulation but shows sustained increase upon apoptosis. No phosphorylation of JNK was found (not shown). Representative of six experiments.
  • FIG. 9 d is a bar-graph depicting that IL-1-beta secretion by apoptotic monocytes is completely abrogated by specific p38 inhibitor (p381N) but not in p38 control (DMSO). No inhibition is seen in the presence of JNK inhibitor (JNKIN) or its control (LTAT).
  • FIG. 9 e is a bar-graph depicting the marked decrease in IL-8 secretion from apoptotic monocytes in the presence of p38 inhibitor (p381N) but not in control (DMSO) or JNK inhibitor (JNKN).
  • FIG. 10 is a schematic diagram depicting a device for inducing apoptosis of leukocytes. Arrows indicate direction of fluid flow.
  • FIG. 11 is a schematic diagram depicting a device for treating a disease characterized by a pathological immune response. Arrows indicate direction of fluid flow.
  • the present invention is of methods of treating diseases associated with pathological immune responses using dying or dead leukocytes, of devices for generating such cells, and of devices for practicing such methods.
  • the prior art fails to provide satisfactory methods of using administration dying leukocytes for treating diseases characterized by pathological immune responses.
  • Example 1 of the Examples section which follows While reducing the present invention to practice, as is described in Example 1 of the Examples section which follows, effective treatment of a systemic autoimmune disease in mammalian subjects by administration of autologous apoptotic lymphocytes was achieved for the first time relative to the prior art.
  • the present invention can be used to treat an autoimmune disease with no or minimal administration of harmful and suboptimally effective anti-inflammatory drugs, as is standard practice in the art.
  • Example 2 of the Examples section which follows While further reducing the present invention to practice, as is described in Example 2 of the Examples section which follows, primary monocytes subjected to suspension conditions ex-vivo were found for the first time to undergo necrosis and to produce pro-inflammatory mediators, whereas, in sharp contrast, such cells subjected to substrate-adherent conditions were found for the first time to undergo apoptosis in the absence of production of pro-inflammatory mediators.
  • prior art procedures involving ex-vivo manipulation of blood such as apheresis procedures, which inherently involve subjecting primary monocytes to suspension conditions, in fact involve induction of monocyte necrosis and concomitant secretion of pro-inflammatory mediators by such necrotic cells, and hence in fact involve introduction of potentially harmful pro-inflammatory mediators into recipients of therapeutic-blood fractions obtained by apheresis.
  • prior art apheresis procedures which are employed in numerous therapeutic applications, including treatment of diseases associated with pathological immune responses, such as GVHD and autoimmune diseases, may be associated with suboptimal efficacy, and harmful side-effects, such as inflammatory side-effects.
  • the present invention can be used to practice apheresis to as to produce blood fractions which are depleted of pro-inflammatory mediators relative to blood fractions produced via prior art apheresis methods. Therefore, the present invention can be used to treat, via apheresis-based methods, diseases associated with pathological immune responses, such as GVHD and autoimmune diseases, with improved safety and effectiveness relative to the prior art.
  • diseases associated with pathological immune responses such as GVHD and autoimmune diseases
  • a method of treating a disease characterized by a pathological immune response in a subject in need thereof is effected by administering to the subject a therapeutically effective amount of a cell preparation which comprises dying or dead leukocytes which are capable of suppressing the pathological immune response.
  • the method of the present invention can be used to treat in any of various types of subject, any of various diseases associated with a pathological immune response.
  • diseases particularly include autoimmune diseases, transplantation-related diseases, and inflammation-associated diseases. Examples of diseases characterized by pathological immune responses which can be effectively treated according to embodiments of the present invention are described hereinbelow.
  • treating when relating to a disease of the present invention refers to preventing onset of the disease, alleviating, attenuating, palliating or eliminating the symptoms of a disease, slowing, reversing or arresting the progression of the disease, or curing the disease.
  • disease refers to any medical disease, disorder, condition, or syndrome, or to any undesired and/or abnormal physiological morphological, cosmetic and/or physical state and/or condition.
  • the method of the present invention is used to treat the disease in a mammalian subject, such as a human subject.
  • a mammalian subject such as a human subject.
  • the method can be used to treat a human subject in view of its successful use in treating a systemic autoimmune disease in mice, as is described and illustrated in Example 2 of the following Examples section, and in view of the well-established extensive and relevant homologies between the human and the murine immune systems.
  • the dying or dead leukocytes may be dying or dead as a result of any of various types of suitable cell death processes, according to this aspect of the present inventions the therapeutic leukocytes are preferably undergoing apoptosis. Leukocytes undergoing apoptosis are referred to herein as “apoptotic” leukocytes.
  • Apoptosis which is a distinct cell death process from necrosis; is the programmed and orderly physiological elimination of cells, occurring, for example, during normal cell and tissue development, T-lymphocyte killing of pathogen-infected cells, and self-elimination of mutationally damaged cells.
  • Apoptotic cells are characterized by distinct morphologic alterations in the cytoplasm and nucleus, chromatin cleavage at regularly spaced sites, and endonucleolytic cleavage of genomic DNA at internucleosomal sites.
  • Necrosis on the other hand; is an inherently pathological and pro-inflammatory process of cell death caused, typically but not exclusively, by the uncontrolled, progressive degradative action of enzymes following lethal cellular injury.
  • Necrotic cells are typically characterized by mitochondrial swelling, nuclear flocculation, cell lysis, loss of membrane integrity, and ultimately cell death. Necrosis can be identified, by light, fluorescence or electron microscopy techniques, or via uptake of the dye trypan blue.
  • the present inventors are of the opinion that cell death may be suitably induced, as in apoptosis, so as to provide signals for suppressing immune responses, and that the method of the present invention harnesses such properties of processes to achieve effective treatment of a disease of the present invention by suppressing the pathological immune response associated therewith.
  • the present inventors are of the opinion that therapeutic leukocytes of the present invention can suppress immune responses directed against antigens which are included in the therapeutic leukocytes.
  • necrotic cells stand in sharp contrast to those of necrotic cells, since necrosis is inherently a pathological process that is associated with generation of pro-inflammatory “danger” signals serving to insulate as opposed to suppress—immune responses for the body's defense.
  • the term “suppressing” when relating to an immune response refers to preventing or reducing the immune response.
  • the method of the present invention can be used to treat diseases which are characterized by pathological non-antigen-specific immune responses, such as non-antigen-specific inflammation in general.
  • the therapeutic leukocytes may advantageously include one or more of the targeted antigens.
  • therapeutic leukocytes which include such targeted antigens can be administered so as to suppress such a pathological immune response, to thereby achieve treatment of such a disease of the present invention.
  • suitable therapeutic leukocytes which include targeted antigens are preferably derived from leukocytes selected endogenously expressing such targeted antigens, depending on the application and purpose, these may be alternately derived from leukocytes genetically modified to express such targeted antigens. It is well within the purview of the ordinarily skilled artisan to genetically modify leukocytes so as to induce these to include a polypeptide or nucleic acid targeted antigen. Ample guidance for genetically modifying leukocytes so as to induce such cells to include desired polypeptides or nucleic acids is provided in the literature of the art (refer, for example, to: Rossig C, Brenner M K., 2004. Genetic modification of T lymphocytes for adoptive immunotherapy. Mol Ther.
  • the therapeutic leukocytes may have any one of various genotypes, depending on the application and purpose.
  • the therapeutic leukocytes are syngeneic with the subject, more preferably are derived from the subject.
  • subject-derived/syngeneic leukocytes will be optimal for treating a: disease characterized by immune responses directed against particular subject-specific variants, or a combination of variants, of targeted autoantigens (e.g. allelic glycosylation, and/or splice variants of polypeptide autoantigens; or sequence variants of nucleic acid autoantigens; etc.), since such combinations may be highly specific to the individual.
  • syngeneic therapeutic leukocytes will avoid the risk of pro-inflammatory immune alloreactivity or xenoreactivity and concomitant stimulation of pathological immune responses inherent to 6 the use of non-syngeneic therapeutic leukocytes, such as allogeneic or xenogeneic therapeutic leukocytes, respectively.
  • the therapeutic leukocytes may be advantageously non-syngeneic with the subject, for example, where sufficient quantities of autologous therapeutic leukocytes are not available, or for treating a disease, such as allograft rejection, or alloimmune spontaneous abortion (Pandey M K. et al., 2004. Arch Gynecol Obstet. 269:161-72), involving pathological immune responses against allogeneic antigens from an allogeneic individual.
  • the therapeutic leukocytes are preferably derived from the allogeneic individual, i.e. the graft donor or the father of the fetus for treatment of allograft rejection or alloimmune spontaneous abortion, respectively.
  • non-syngeneic therapeutic leukocytes are allogeneic leukocytes, most preferably allogeneic leukocytes which are haplotype-matched with the subject.
  • Haplotype-matching of human subjects, with allogeneic cells is routinely practiced in the art in the context of therapeutic transplantation and usually involves of HLA-A, HLA-B, and HLA-DR alleles.
  • the therapeutic leukocytes used to practice the method of the present invention may be derived from leukocytes of any one of various lineages, depending on'the application and purpose.
  • the therapeutic leukocytes are dying or dead lymphocytes (referred to hereinafter as “Therapeutic lymphocytes”).
  • lymphocytes can be used according to the present teachings to effectively treat, without or with minimal requirement for harmful prior art administration of toxic immunosuppressive agents, a disease characterized by a pathological immune response, such as an autoimmune disease, such as a systemic autoimmune disease, such as systemic lupus erythematosus.
  • a pathological immune response such as an autoimmune disease, such as a systemic autoimmune disease, such as systemic lupus erythematosus.
  • the therapeutic leukocytes are dying or dead monocytes (referred to hereinafter as “therapeutic monocytes”).
  • the method of the present invention can employ administration of a cell preparation comprising therapeutic monocytes to treat with enhanced safety and effectiveness relative to the prior art a disease of the present invention which is amenable to treatment by administration of dying or dead cells generated via an apheresis procedure involving suspension of monocytes.
  • the phrase “suspension conditions” refers to any culturing conditions which do not involve adhesion of cultured cells to a surface, such as static culturing conditions in a culture recipient having an underlying substrate with a non-cell adherent surface (e.g. non-tissue culture-treated petri dishes), or dynamic culturing conditions, such as those involving shaking, which do not allow for static contact of cultured cells with a surface/substrate.
  • the therapeutic leukocytes are dying or dead neutrophils (referred to hereinafter as “therapeutic neutrophils”).
  • therapeutic neutrophils can be used according to the present teachings to effectively treat, any of various diseases which are associated with a pathological immune response.
  • the therapeutic leukocytes used to practice the method of the present invention may be derived from any lineage, or sub-lineage, of nucleated cells of the immune system and/or hematopoietic system, including but not limited to dendritic cells, macrophages, mast cells, basophils, hematopoietic stem cells, bone marrow cells, natural killer cells, and the like.
  • Source leukocytes Leukocytes from which therapeutic leukocytes of the present invention may be derived (referred to hereinafter as “source leukocytes”) may be obtained in any of various suitable ways, from any of various suitable anatomical compartments, according to any of various commonly practiced methods, depending on the application and purpose, desired leukocyte lineage, etc.
  • the source leukocytes are primary leukocytes, more preferably primary peripheral blood leukocytes.
  • Peripheral blood leukocytes include 0.60 percent neutrophils, 30 percent lymphocytes, and 7 percent monocytes.
  • source lymphocytes can be achieved, for example, by harvesting blood in the presence of an anticoagulant, such as heparin or citrate. The harvested blood is then centrifuged over a Ficoll cushion to isolate lymphocytes and monocytes at the gradient interface, and neutrophils and erythrocytes in the pellet.
  • Leukocytes may be separated from each other via standard immunomagnetic selection or immunofluorescent flow cytometry techniques according to their specific surface markers, or via centrifugal elutriation.
  • monocytes can be selected as the CD14+fraction
  • T-lymphocytes can be selected as CD3+ fraction
  • B-lymphocytes can be selected as the CD19+ or CD22+fraction
  • neutrophils can be selected as the CD15+fraction.
  • Lymphocytes and monocytes may be isolated from each other by subjecting these cells to substrate-adherent conditions, such as by static culture in a tissue culture-treated culturing recipient, which results in selective adherence of the monocytes, but not the lymphocytes, to the cell-adherent substrate.
  • Neutrophils may be isolated from other blood cells via standard counterflow centrifugal elutriation protocols.
  • Isolation of source monocytes is preferably performed via immunomagnetic or substrate-adherence-based selection, according to the protocols provided in the Materials and Methods section of Example 2 of the Examples section which follows.
  • Therapeutic lymphocytes may suitably be derived from lymphoid tissues, such as spleen, or thymus.
  • lymphoid tissues such as spleen, or thymus.
  • therapeutic leukocytes derived from source splenocytes or thymocytes may be used according to the present teachings to effectively treat a disease of the present invention, such as an autoimmune disease, such as a systemic autoimmune disease, such as systemic lupus eryhematosus.
  • the therapeutic leukocytes may be derived from cultured primary source leukocytes, or may be derived from suitable established cell lines.
  • Culturing of suitable source leukocytes such as leukocytes of human origin, may be performed in-vivo, for example in immune deficient hosts, such as in lines of severe combined immunodeficiency (SCID) animals.
  • SCID severe combined immunodeficiency
  • Suitable leukocyte cell lines may be obtained from commercial suppliers, such as the American Tissue Type Collection (ATCC).
  • ATCC American Tissue Type Collection
  • Established leukocyte cell lines may be particularly amenable to genetic, modification, for example, to thereby include an antigen targeted by a pathological immune response of a disease of the present invention, as described hereinabove, for treatment of a disease of the present invention characterized by a pathological immune response targeted against such an antigen.
  • source leukocytes should not be obtained via a technique which will significantly interfere with their capacity to produce the therapeutic leukocytes.
  • Source leukocytes may treated in any of Various ways, in accordance with known prior art methods, so as to produce the therapeutic leukocytes, depending on the application and purpose.
  • Apoptosis of leukocytes may be induced according to a wide variety of treatments which are well known and commonly practiced in the art. Such treatments include, but are not limited to culturing under conditions of growth factor and/or nutrient deprivation; culturing under conditions of cellular substrate-adherence, culturing tinder conditions of serum-withdrawal; irradiation; for example with UV or gamma rays; treatment with: a biological apoptosis-inducing mediator, such as an activating death receptor ligand such as perforin treatment with apoptosis-inducing cells, such as immunoreactive cytotoxic T-lymphocytes (CTLs); treatment with immunosuppressive drugs such as steroids, corticosteroids, dexamethasone, cyclophosphamide, methotrexate, azathioprine, cyclosporine, staurosporine, and the like; cryotreatment; hyperthermal treatment; culturing under:
  • apoptosis of lymphocytes is induced by treating the primary lymphocytes with serum deprivation, a corticosteriod, or irradiation.
  • inducing apoptosis of primary lymphocytes via treatment with a corticosteroid is effected by treating the primary lymphocytes with dexamethasone, more preferably with dexamethasone at a concentration of about 1 micromolar.
  • inducing apoptosis of primary lymphocytes via irradiation is effected by treating the primary lymphocytes with gamma-irradiation, more preferably with a dosage of about 66 rad.
  • apoptosis-inducing treatments can be used to generate therapeutic leukocytes which may be used according to the present teachings to effectively treat a disease of the present invention, such as an autoimmune-disease, such as a systemic autoimmune disease, such systemic lupus erythematosus.
  • apoptosis of monocytes is induced by subjecting the monocytes to in-vitro conditions of substrate/surface-adherence, as is taught for the first time in the present specification, more preferably concomitantly under conditions of serum deprivation.
  • Subjecting the monocytes to in-vitro substrate/surface-adherent conditions suitable to produce therapeutic monocytes of the present invention may be suitably effected, for example, by culturing primary monocytes in tissue culture-coated tissue culture flasks under conditions of serum deprivation for a period of 40 minutes. As is described and illustrated in Example 2 of the Examples section below, such treatment will generate non-pro-inflammatory apoptotic monocytes suitable for practicing the method of the present invention.
  • Adherent leukocytes such as adherent monocytes
  • cell-releasing compound capable of facilitating release of surface-adherent cells, such as surface-adherent monocytes, and application of fluid shear flow or scraping with a suitable instrument, such as a rubber policeman, serving to release the adherent cells from the surface.
  • Such suitable cell-releasing compounds and appropriate methods of their use (compound concentration, duration of exposure to compound, termination of exposure of compound, etc.), are well known and widely employed in the art.
  • Such compounds include, for example, proteases, such as trypsin; and divalent cation chelators, such as EDTA. It will be appreciated that methods of releasing adherent cells which would normally harm or disrupt viable cells may be employed since the cells are already apoptotic and do not necessarily need to be administered as intact cell structures so as to enable disease treatment according to the method of the present invention.
  • Apoptosis of source leukocytes so as to generate the therapeutic leukocytes is preferably effected in-vitro.
  • apoptosis of the source leukocytes is preferably effected outside the body, i.e. ex-vivo.
  • apoptosis of source leukocytes may be induced in-vivo in-situ.
  • Apoptosis of a cell can be confirmed by any of various commonly employed methods. Such methods include gel electrophoresis of cellular DNA to detect apoptosis-specific ladder-like DNA fragment patterns, TUNEL-staining to detect apoptosis-specific DNA fragmentation, staining with an annexin fluorophore conjugate to detect apoptosis-specific reversal of cell membrane orientation, staining with anti-cleaved caspase-3 antibody for detection of apoptosis-specific caspase activation, microscopic; inspection to detect apoptosis-specific cellular fragmentation and blebbing and the like.
  • Example 2 of the Examples section below primary monocytes were induced to undergo apoptosis by incubation in a tissue culture dish having a cell-adherent substrate.
  • the present inventors have devised and implemented a novel device for inducing apoptosis of source leukocytes in-vitro.
  • an apoptosis-inducing device for inducing apoptosis of leukocytes ( FIG. 10 ).
  • the device 10 comprises an apoptosis-inducing chamber or chambers (each indicated by 12 ) selected from a chamber 14 for inducing apoptosis of monocytes (referred to hereinafter as “monocyte chamber”), a chamber 16 for inducing apoptosis of neutrophils (referred to hereinafter as “neutrophil chamber”), and/or a chamber 18 for inducing apoptosis of lymphocytes (referred to hereinafter as “lymphocyte chamber”).
  • monocyte chamber a chamber 14 for inducing apoptosis of monocytes
  • neutral chamber a chamber 16 for inducing apoptosis of neutrophils
  • lymphocyte chamber referred to hereinafter as “lymphocyte chamber”.
  • the device may comprise any of various combinations of apoptosis-inducing chambers, depending on which lineages of apoptotic leukocytes are desired.
  • the monocyte chamber preferably comprises a surface 20 for enhancing adherence of monocytes thereto, a reservoir 22 for containing a medium for inducing apoptosis of monocytes (referred to hereinafter as “monocyte medium”), and/or a mechanism 28 for resuspending surface-adherent monocytes (referred to hereinafter as “cell-adherent surface”), more preferably all of which.
  • monocyte medium a medium for inducing apoptosis of monocytes
  • cell-adherent surface resuspending surface-adherent monocytes
  • the cell-adherent surface is hydrophilic and negatively charged and may be obtained in any of various ways known in the art, preferably by modifying a polystyrene surface using, for example, corona discharge, or gas-plasma. These processes generate highly energetic oxygen ions which graft onto the surface polystyrene chains so that the surface becomes hydrophilic and negatively charged thereby facilitating cellular adherence thereto.
  • Suitable cell-adherent surfaces for inducing leukocyte apoptosis according to the present invention may be provided by any one of various tissue-culture-treated tissue culture recipients designed for facilitating cell-adherence thereto which are available from various commercial suppliers (e.g. Corning, Perkin-Elmer, Fisher Scientific, Evergreen Scientific, Nunc, etc.).
  • the monocyte chamber may comprise, any of various suitable mechanisms for resuspending surface-adherent monocytes, so as to enable the harvesting thereof.
  • Suitable mechanisms for such purpose include any combination of a reservoir for containing a cell-releasing compound of the present invention, and a mechanism for introducing the cell-releasing compound into the monocyte chamber; a flow generating mechanism for generating in the monocyte chamber a flow of sufficient force and direction for resuspending the surface-adherent monocytes, and a mechanism of controlling the operation of the flow-generating mechanism; and a scraping mechanism for scraping the surface-adherent monocytes off the cell-adherent surface of the monocyte chamber, and a mechanism for controlling the operation of the scraping mechanism.
  • Suitable flow-generating mechanisms for facilitating resuspension of surface-adherent cells include for example, magnetic stirrers, and fluid mixing mechanisms based on rotating propeller blades.
  • a suitable scraping mechanism for scraping the surface-adherent monocytes off the cell-adherent surface of the monocyte chamber is an automated rubber policeman.
  • the neutrophil chamber comprises a reservoir 24 for containing a medium for inducing apoptosis of neutrophils (referred to hereinafter as “neutrophil medium”).
  • neutrophil medium a medium for inducing apoptosis of neutrophils
  • the lymphocyte chamber comprises a reservoir 26 for containing a medium for inducing apoptosis of lymphocytes (referred to hereinafter as “lymphocyte medium”).
  • lymphocyte medium a medium for inducing apoptosis of lymphocytes
  • each apoptosis-inducing chamber may be configured so as, to comprise an apoptosis-inducing mechanism 30 selected from the group consisting of an irradiating mechanism for inducing apoptosis, a mechanical mechanism for inducing apoptosis, and a chemical or biochemical substance or environment for inducing apoptosis.
  • an apoptosis-inducing mechanism 30 selected from the group consisting of an irradiating mechanism for inducing apoptosis, a mechanical mechanism for inducing apoptosis, and a chemical or biochemical substance or environment for inducing apoptosis.
  • each apoptosis inducing chamber is preferably equipped with a temperature control mechanism enabling maintenance of leukocytes at a desired temperature, and is further preferably equipped with a mechanism for maintenance of carbon dioxide air levels appropriate to the particular cell medium employed.
  • each apoptosis-inducing chamber is preferably equipped with a fluid inlet 32 and a valve for controlling fluid flow therethrough, and a fluid outlet 34 and a valve for controlling fluid flow therethrough.
  • the device according to this aspect of the present invention is configured so as to enable introduction of each lymphocytes, monocytes, and/or neutrophils into respective chambers configured so as to induce apoptosis thereof according to the teachings of the present invention, and is configured so as to enable harvesting of such leukocytes from such chambers for administration for disease treatment according to the method of the present invention.
  • Treatment of a disease characterized by a pathological immune response according to the method of the present invention may be effectively practiced, depending on the application and purpose, by administering to the subject according to any of various suitable administration regimens a therapeutically effective, amount of any of various suitable types of cell preparation which comprise therapeutic leukocytes of the present invention.
  • disease treatment may be effectively practiced by administering to the subject a therapeutically effective amount of a cell preparation which may comprise any of various combinations of therapeutic leukocyte lineages.
  • Examples of specific treatment protocols which may be used for treatment of various diseases via administration of therapeutic lymphocytes, therapeutic monocytes, and/or therapeutic neutrophils of the present invention are provided in Examples 3, 4 and 5 of the Examples section which follows, respectively.
  • administration of therapeutic lymphocytes is used to treat an autoimmune disease.
  • the autoimmune disease is a systemic autoimmune disease, more preferably systemic lupus erythematosus.
  • administration of combined therapeutic lymphocytes, monocytes, and neutrophils may be used to treat graft-versus-host disease.
  • treatment of a disease of the present invention is effected by administering to the subject a cell preparation which comprises a total dose of about 200 million therapeutic leukocytes per kilogram body weight.
  • a total dose is administered as unit doses of about 40 million cells per kilogram body weight, and/or is administered as unit doses at weekly intervals, more preferably both of which.
  • Suitable total doses according to this embodiment include total doses of about 20 million to about 2 billion cells per kilogram body weight, more preferably about 40 million to about 1 billion cells per kilogram body weight, more preferably about 80 million to about 500 million cells per kilogram body weight, and more preferably about 160 million to about 250 million cells per kilogram body weight.
  • Suitable unit doses according to this embodiment include unit doses of about 4 million to about 400 million cells per kilogram body weight, more preferably about ⁇ 8 million to about 200 million cells pert kilogram body weight, more preferably about 16 million to about 100 million cells per kilogram body weight, and more preferably about 32 million to about 50 million cells per kilogram body weight.
  • the therapeutic leukocytes are administered to the subject systemically, more preferably via the intravenous route.
  • the therapeutic leukocytes may be administered to the subject according to any of various other routes, including, but not limited to the parenteral, intraperitoneal, intramuscular, subcutaneous, oral, transnasal and rectal routes.
  • the therapeutic leukocytes are administered to the subject suspended in a suitable physiological buffer, such as saline solution, PBS, HBSS, and the like.
  • a suitable physiological buffer such as saline solution, PBS, HBSS, and the like.
  • a disease of the present invention (systemic lupus erythematosus), was effectively treated in a mouse (average weight 0.025 kilograms) by intravenous administration of 5 doses of one million therapeutic lymphocytes at weekly intervals, which corresponds to the aforementioned preferred total and unit doses of 200 million and 40 million cells per kilogram body weight, respectively.
  • disease treatment may be advantageously effected according to the teachings of the present invention, in conjunction with standard prior art therapies, and/or by co-administration of an immunosuppressive molecule, such as IL-10 or TGF-beta.
  • an immunosuppressive molecule such as IL-10 or TGF-beta.
  • disease status will preferably be closely monitored so as to optimize and suitably modify the treatment.
  • levels of any of various pro-inflammatory cytokines, chemokines or other molecules may be monitored in the patient to facilitate monitoring of disease treatment.
  • tissue levels of relevant autoantibodies may be measured for monitoring disease treatment.
  • systemic autoimmune disease such as systemic lupus erythematosus
  • autoantibodies include those specific for double-stranded DNA, and those specific for phospholipids.
  • One of ordinary skill in the art such as a physician, preferably a specialist in the disease to be treated, will possess the necessary expertise for applying the teachings of the present invention so as to effectively treat a disease of the present invention in a human subject.
  • the present inventors have devised a novel disease treatment device which can harvest blood from the subject, generate desired therapeutic leukocytes from the harvested blood, and re-infuse the therapeutic leukocytes to the subject.
  • a disease treatment device an example of which is shown in FIG. 11 .
  • the disease-treatment device 40 comprises a pump 42 for pumping blood from a subject into the device and returning blood to the subject from the device; a leukocytes separator 44 in communication with the pump for separating circulating leukocytes from whole blood; and the apoptosis-inducing device 10 of the present invention in communication with the leukocytes separator for inducing apoptosis of the leukocytes, and further in communication with the pump for administering the apoptotic leukocytes to the subject.
  • the disease-treatment device of the present invention is configured essentially as a prior art blood cell apheresis device capable of harvesting blood from a subject, isolating blood cells, subjecting the isolated cells to a given treatment, and re-infusing the treated cells back into the subject.
  • Such prior art devices are widely used, for example, for practicing CD34+ cell leukapheresis, or leukocyte photopheresis.
  • the disease-treatment device of the present invention comprises the novel and inventive feature of including the apoptosis-inducing device of the present invention for inducing apoptosis, in accordance with the method of the present invention, of separated leukocytes prior to their re-infusion into the subject.
  • leukocyte apheresis devices such as the disease-treatment device of the present invention, and their use, is provided in the literature of the art (refer, for example, to: Burgstaler E A. et al., 2004; Hematopoietic progenitor cell large volume leukapheresis (LVL) on the Fenwal Amicus blood separator. J Clin Apheresis. 19:103-11; Schwella N. et al., 2003. Comparison of two leukapheresis programs for computerized collection of blood progenitor cells on a new cell separator. Transfusion. 43(1):58-64; Kohgo Y. et al, 2002.
  • LDL Hematopoietic progenitor cell large volume leukapheresis
  • Leukocyte apheresis using a centrifugal cell separator in refractory ulcerative colitis a multicenter open label trial.
  • Present status of apheresis technologies Part 1. Membrane plasma separator.
  • lymphocyte apheresis devices and techniques are provided in the literature of the art, (refer, for example, to: Zic J A., 2003. The treatment of cutaneous T-cell lymphoma with photopheresis. Dermatol Ther. 0.16:337-46; Foss F M. et al., 2002. Extracorporeal photopheresis in chronic graft-versus-host disease. Bone Marrow Transplant. 29:719-25; Oliven A, Shechter Y. 2001. Extracorporeal photopheresis: a review. Blood Rev. 15:103-8; Rook A H. et al. 1999. Photopheresis: clinical applications and mechanism of action. J Investig Dermatol Symp Proc. 4:85-90).
  • the disease-treatment device of the present invention presents various advantages over prior art apheresis devices used for disease treatment.
  • the device particularly enables practicing of photopheresis for treatment of diseases characterized by pathological immune responses with greater safety and effectiveness relative to the prior art since it avoids generation of pro-inflammatory leukocyte necrosis inherent to prior art devices, by virtue of enabling non-pro-inflammatory leukocyte apoptosis, such as monocyte and neutrophil apoptosis.
  • Embodiments of the present invention can be used to treat any of various diseases characterized by a pathological immune response.
  • the disease is an autoimmune disease or a transplantation-related disease.
  • the autoimmune disease is a systemic autoimmune disease and/or an antibody-mediated autoimmune disease.
  • the autoimmune disease is systemic lupus erythematosus (SLE).
  • the transplantation-related disease is graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • the disease characterized by a pathological immune response may be any of various inflammatory/inflammation-associated diseases.
  • the present invention can be used to treat a disease which is characterized by a pathological immune response in any of various anatomical compartments of the body.
  • antibody-mediated autoimmune diseases include but are not limited to rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July; 15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al. Immunol Res 1998;17 (1-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al.
  • vasculitises necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomeruloniephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel L H. Ann Med Interne (Paris). 2000 May; 151 (3):178); antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4):171); heart failure, agonist-like beta-adrenoceptor antibodies in heart failure (Wallukat G.
  • organ/tissue specific autoimmune diseases comprise cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
  • autoimmune cardiovascular diseases comprise atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135), myocardial infarction (Vaarala G. Lupus. 1998; 7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al., Wien Klin Klin Klin Klinschr 2000 Aug. 25; 112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., Semin Thromb Hemost.
  • autoimmune rheumatoid diseases comprise rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July; 15 (3):791; Tisch R, McDevitt H O. Proc Natl Acad Sci units S A 1994 Jan. 18; 91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189).
  • autoimmune glandular diseases comprise pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome diseases comprise autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth G S. Ann. Rev. Immunol. 8:647 Zimmet P. Diabetes Res Clin Pract 1996 October; 34 Suppl:S125), autoimmune thyroid diseases, Graves′ disease (Orgiazzi J.
  • autoimmune gastrointestinal diseases comprise chronic inflammatory intestinal diseases (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 January; 23 (1):16), celiac disease (Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122), colitis, ileitis and Crohn's disease.
  • autoimmune cutaneous diseases comprise autoimmune bullous skin diseases, such as, but not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus, discoid lupus erythematosus.
  • autoimmune hepatic diseases comprise hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 March; 54 (3):382), primary biliary cirrhosis (Jones D E. Clin Sci (Colch) 1996 November; 91 (5):55-1; Strassburg C P. et al., Eur J Gastroenterol Hepatol. 1999 June; 11 (6):595) and autoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2):326).
  • autoimmune neurological diseases comprise multiple sclerosis (Cross A H. et al., J Neuroimmunol 2001 Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997; 49:77), myasthenia gravis (Infante A J. And Kraig E, Int Rev Immunol 1999; 18 (1-2):83; Oshima M. et al, Eur J Immunol 1990 December; 20 (12):2563), neuropathies, motor neuropathies (Kornberg A J. J. Clin Neurosci.
  • autoimmune muscular diseases comprise myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 September; 123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 June; 53 (5-6):234).
  • autoimmune nephric diseases comprise nephritis and autoimmune interstitial nephritis (Kelly C J. J Am Soc Nephrol 1990 August; 1 (2): 1-40).
  • autoimmune diseases related to reproduction comprise repeated fetal loss (Tincani A. et al., Lupus 1998; 7:Suppl 2:S-107-9).
  • autoimmune connective tissue diseases comprise ear diseases, autoimmune ear diseases (Yoo T J. et al., Cell Immunol 1994 August; 157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec. 29; 830:266).
  • systemic autoimmune diseases comprise systemic lupus erythematosus (Erikson J. et al., Immunol: Res 1998; 17 (1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagin Lab Immunol. 1999 March; 6 (2):156), Chan O T. et al., Immunol Rev 1999 June; 169:107).
  • transplantation-related diseases include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft-versus-host disease (GVHD).
  • inflamatory inflammation-associated diseases include, but are not limited to, restenosis following percutaneous transluminal coronary angioplasty (PTCA), restenosis following PTCA with stent implantation, myocardial infarction, inflammation associated with mechanical injury, neurodegenerative diseases, ulcers, prosthetic implants, menstruation, septic shock, anaphylactic shock, toxic shock syndrome, cachexia, gangrene, musculo-skeletal inflammation, idiopathic inflammation.
  • the devices and methods of the present invention can be used to treat a broad range of diseases associated with pathological immune responses, such as autoimmune diseases, transplantation-related diseases and inflammatory/inflammation-related diseases, with improved safety and effectiveness relative to prior art methods which involve administration of harmful immunosuppressive drugs, and/or which inherently and unknowingly involve counterproductive and harmful administration of pro-inflammatory mediators, as is presently taught for the first time in the art.
  • diseases associated with pathological immune responses such as autoimmune diseases, transplantation-related diseases and inflammatory/inflammation-related diseases
  • Autoimmune diseases such as systemic lupus erythematosus (SLE)
  • SLE systemic lupus erythematosus
  • An optimal strategy for treating such diseases would be to present targeted antigens to the immune system of an individual afflicted with such a disease in, such a way as to induce tolerances to such antigens by the immune system of the individual.
  • An optimal way to achieve this goal would be to employ autologous apoptotic cells, which would obviate or minimize the necessity for administration of toxic immunosuppressive agents, the standard means of treatment in the art.
  • MRL/MpJ-Fas Ipr and C3H-SnJ mice were obtained from Jackson Laboratories, Bar Harbor, Me. Thymocytes and splenocytes were prepared from 4 to 8 week-old mice according to standard methodology. Apoptosis of thymocytes or splenocytes was induced by either serum deprivation, 1 micromolar dexamethasone, or gamma-irradiation-(66 rad). Apoptosis was confirmed via flow cytometric analysis of annexin-FITC staining, DNA fragmentation and propidium iodide staining of fragmented DNA.
  • MRL/MpJ-Fas Ipr and C3H-SnJ mice obtained from Jackson Laboratories, Bar Harbor, Me., were administered a total of 5 ⁇ 10 6 syngeneic sex- and age-matched apoptotic cells per mouse, as 5 weekly injections of 1 ⁇ 10 6 cells per mouse.
  • the route of administration was intravenous injection into the tail vein of cells suspended in a volume of 200 microliters.
  • Autoimmune response anti-self DNA antibody ELISA: Serum samples were obtained immediately prior to treatment and at two-week intervals following treatment. The immune response was evaluated by quantifying serum antibodies specific for single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) by enzyme-linked immunosorbent assay (ELISA) of 100-fold diluted serum.
  • ssDNA single-stranded DNA
  • dsDNA double-stranded DNA
  • Pathological evaluation Mice were examined every day for pathological signs of disease and once a month for hematuria or proteinurea. After four months the mice were sacrificed and their were kidneys examined histologically and via immunofluorescence.
  • MRL/MpJ-Fas Ipr mice develop SLE-like disease due to mutation in Fas, a receptor that mediates apoptosis and activation of induced cell death of the immune system. Since in SLE patients, as well as in MRL/MpJ-Fas Ipr mice, the development of autoantibodies and kidney disease are the most specific pathophysiological parameters, those parameters were evaluated in MRL/MpJ-Fas Ipr mice following administration of apoptotic cells.
  • mice Two groups of age- and sex-matched MRL/MpJ-Fas Ipr mice were compared.
  • 1 ⁇ 10 6 syngeneic apoptotic cells were injected intravenously into each of five mice, five times at weekly intervals, for a total dose of 5 ⁇ 10 6 cells per mouse.
  • 200 microliters of saline carrier alone was injected.
  • IgG anti-ssDNA levels were then measured via ELISA at two-week intervals and were found to be comparable in both groups prior to treatment, with a mean O.D. value of 0.096 plus/minus 0.018 ( FIG. 1 ).
  • mice administered with vehicle alone displayed, as expected in mice which develop lupus-like disease, increased anti-ssDNA antibody levels, as evidenced by an ELISA O.D. value of 0.308 plus/minus 0.029 (p ⁇ 0.0000, student t-test).
  • mice injected with 1 ⁇ 10 6 syngeneic apoptotic cells unexpectedly had significantly reduced levels of autoantibodies, with an ELISA O.D. value obtained of 0.193 plus/minus 0.017 (p ⁇ 0 . 0000 student t-test).
  • mice injected with saline were 0.198 plus/minus 0.017, 0.205 plus/minus 0.02, 00.300 plus/minus 0.033 and 0.378 plus/minus 0.037; and for mice treated with apoptotic cells were 0.108(+0.03), 0.170(+0.07), 0.186(+0.04) and 0.203(+0.8).
  • mice treated with apoptotic cells were 0.108(+0.03), 0.170(+0.07), 0.186(+0.04) and 0.203(+0.8).
  • anti-ssDNA IgG levels were unexpectedly found to significantly decrease following injection of apoptotic cells, with ELISA O.D.
  • FIG. 1 As shown in FIG. 1 , at 16 weeks of age, i.e. 10 weeks post-treatment, a surprising marked decrease in anti-ssDNA antibody titers was noted in all mice injected with the apoptotic cells.
  • anti-dsDNA antibody titers were measured immediately prior to treatment, and 6-8 weeks post-treatment, when the mice were sacrificed. As shown in FIG. 2 , anti-dsDNA antibody titers were surprisingly found to be significantly reduced (p ⁇ 0.00) in mice injected with apoptotic cells.
  • An average O.D. value of 0.599 plus/minus 0.026 was obtained in an anti-dsDNA antibody ELISA of serum from mice injected with saline, and an average O.D. value of 0.358 plus/minus 0.038 was obtained in mice injected with the apoptotic cells.
  • mice injected with saline displayed significant elevations in proteinuria and hematuria and concomitant glomerular disease, as shown in Table 1.
  • mice injected with the apoptotic cells overall unexpectedly displayed significantly decreased proteinuria and hematuria and concomitant glomerular disease, consistent with the serological response. Strikingly, in two out of five mice treated with apoptotic cells, no deterioration or very slight deterioration was noticed.
  • Immune/hematological diseases such as graft versus host disease (GVHD)
  • GVHD graft versus host disease
  • the optimal strategy for treating such diseases involves performing apheresis procedures.
  • apheresis procedures involve removing blood from an individual, separating the blood into fractions and performing therapeutic treatment of specific fractions, removing undesirable pathological fractions and reinfusing the remainder to the individual, or harvesting desired may be associated with undesirable side-effects and/or suboptimal effectiveness.
  • a potentially optimal strategy for, performing apheresis involves identifying harmful effects of apheresis procedures on blood components so as to enable design of optimal methods and devices for performing apheresis. While reducing the present invention to practice, as described below the inductions of necrosis of monocytes, and the concomitant secretion of harmful pro-inflammatory mediators thereby resulting from their ex-vivo suspension, as typically occurs during apheresis procedures was unexpectedly uncovered, as opposed to serum deprivation and substrate-adherent conditions which were surprisingly found to induce apoptosis of monocytes, in the absence of the aforementioned secretion of pro-inflammatory mediators.
  • Human mononuclear cells were isolated from heparinized peripheral blood by density gradient centrifugation. The isolated mononuclear cells were separated into monocyte, B-cell and T-cell populations by positively selecting monocytes as the CD14+ fraction by magnetic bead separation (Miltenyi Biotec., Auburn, Calif., USA), positively selecting B-cells as the CD22+ fraction, and negatively selecting T-cells as the CD14-CD22 ⁇ fraction. Purity was greater than 95 percent for monocytes, greater than 95 percent for B-cells and greater than 88 percent for T-cells.
  • Polymorphonuclear cells were separated by density gradient centrifugation of the upper fraction obtained following incubation of peripheral blood with Plasmasteril (GmbH, Bad Homburg; Germany). When necessary, red blood cells in the pellets were hemolysed under, hypoosmotic conditions. Anti-CD15 magnetic beads were employed to purify neutrophils to greater than 95 percent purity. Alternately, monocyte isolation was performed concomitantly with apoptosis induction by adherence, as described below.
  • Cell death induction Leukocyte death was induced by serum deprivation or suspension, and necrosis of leukocytes was induced.
  • monocytes were incubated at 37 degrees centigrade in serum-free RPMI culture medium in polypropylene tubes.
  • Necrosis was induced hyperthermally by incubation at 56 degrees centigrade for 20 minutes, and confirmed by greater than 95 percent trypan blue positive cells and swollen cells detected via flow cytometry forward-scatter.
  • Monocyte apoptosis was induced via substrate-adherence+serum withdrawal by either of two methods.
  • monocytes isolated using anti-CD 14 conjugated magnetic beads (Miltenyi Biotech Bergisch Gladbach, Germany) were incubated in serum-free RPMI at a concentration of 7.5 million to 20 million cells per milliliter in 35 mm diameter tissue culture-treated Petri dishes (Corning, USA, Cat. No. 430165).
  • isolated PBMCs were incubated in serum-free RPMI at a concentration of 15 million to 30 million cells per milliliter in 35 mm diameter tissue culture-treated Petri dishes (Corning, USA, Cat. No. 430165), and after 40 minutes, non-adherent-cells were washed away, leaving behind the adherent, apoptotic monocytes.
  • Cell death inhibition assays In some of the experiments the cells were pretreated or co-treated (as indicated) with different reagents to achieve cell death inhibition.
  • anti-Fas inhibitory mAb ZB4 MBL, Nagoya, Japan
  • anti-Fas mAb DM542A Acris Antibodies, Hiddenhausen, Germany
  • Caspase-1 was inhibited using caspase-1 (ICE) fink inhibitor Z-WEHD (R & D Systems).
  • proteasome inhibition cells were exposed for 45 minutes to 50 micromolar of the proteasome inhibitor MG132 (Calbiochem, San Diego, Calif., USA).
  • P38 and JNK were inhibited using 10 micromolar SB203580 (Calbiochem, Darmstadt, Germany) or 20 millimolar L-JNKI1 (Alexis, San Diego, Calif., USA), respectively.
  • 10 micromolar SB203580 Calbiochem, Darmstadt, Germany
  • 20 millimolar L-JNKI1 Alexis, San Diego, Calif., USA
  • For transcription inhibition 5 micrograms/ml Actinomycin D was used and for translation inhibition, 15 micrograms/ml cycloheximide (Sigma, St. Louis, Mo., USA) was used.
  • Monocyte activation was induced with 500 micrograms/ml, 1 mg/ml Zymosan, or 1 microgram/mil of LPS (Sigma, St. Louis, Mo., USA).
  • GEArray gene expression array systems hGEA9912090, hGEA9913030 and hGEA9913040 (Super Array, Bethesda, Md., USA) were used. Each array consists of 56 coordinates containing specific cDNA fragments spotted in duplicates as well as control sequences [PUC] 18 as negative control beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as positive control].
  • cDNA probes were synthesized from total RNA samples using the manufacturer's primer mix as a reverse transcriptase primer. The cDNA probes were hybridized to gene-specific cDNA fragments spotted on the membranes. The relative expression level of the genes was adjusted based on intensity of hybridization signals to the housekeeping genes beta-actin and GAPDH, then gene expression was quantified by scanning densitometry Each experiment was performed at least three times to ensure reproducibility of results.
  • Cytokine/chemokine analysis concentrations of the cytokines/chemokines IL 4, IL-6, IL-8, IFN-gamma, TNF-alpha, TGF-beta, and MIP-1-alpha were determined via ELISA immunoassay (R&D systems, Minneapolis, Minn., USA) according to the instructions provided by the manufacturer.
  • CD14+ monocytes were isolated and subjected to either substrate-adherence+serum deprivation, or suspension+serum adherence. Surprisingly, serum deprivation resulted in apoptosis of the cells ( FIG. 3 ) with no decline in cell numbers for the first 10 hours, whereas suspension+serum withdrawal resulted in very rapid death with 50 percent reduction in cell numbers reached in 4 hours ( FIG. 4 ). Very few suspended monocytes did not stain positive for propidium iodide, and from the start of suspension most of the cells were found to be positive for both annexin and propidium iodide in constant proportion ( FIG.
  • apoptotic monocytes or monocytes subjected to hyperthermia-induced necrosis secrete pro-inflammatory cytokines Comparison of IL-1-beta secretion among apoptotic monocytes, viable monocytes, and monocytes rendered necrotic via suspension, showed that secretion is specific to cells undergoing suspension-induced death ( FIG. 7 a ).
  • monocytes undergoing apoptosis were exposed to the pan-caspase inhibitor Zvad/fmk.
  • IL-1-beta is the key initiator of the innate immunity acute inflammatory response [21, 22].
  • LPS lipopolysaccharide
  • IL-1-beta is synthesized in human monocyte-lineage cells as the biologically inactive 31 kDa precursor pro-IL-1-beta.
  • IL-1-beta is not secreted through the classical endoplasmic reticulum-Golgi pathway [23] due to a lack in the N-terminal amino acid leader sequence that would allow translation at the endoplasmic reticulum associated ribosomes and subsequent packaging into secretory vesicles.
  • IL-1-beta is also not stored in or released from exocytotic granules [24].
  • pro-IL-1-beta In order to be released as biologically active 17 kDa IL-1-beta, pro-IL-1-beta must be further proteolytically cleaved by caspase-1, which undergoes activation from its pro-caspase zymogenic form.
  • Activation of P2X7 receptors by extra cellular ATP following NFkappaB activation causes phosphatidylserine (PS) flip in the plasma membrane and loss of membrane asymmetry with respect to its positioning.
  • PS phosphatidylserine
  • Readily releasable phosphatidylserine-exposing micro-vesicles containing 17 kDa IL-1-beta are then pinched off from the cell within a few seconds [25].
  • Assays were performed to verify that IL-1-beta secretion does not result from monocyte activation, and follows the immediate pattern described above upon activation. It was shown that IL-1-beta secretion was not intermediate, ( FIG. 6 a ) and was dependent on de-novo mRNA synthesis ( FIG. 5 b ). Assays were then performed to verify whether it was caspase-1-dependent, and, as shown in FIG. 7 b , specific inhibition of caspase-1 did not influence IL-1-beta secretion.
  • Monocytes were recently shown to exhibit pro-inflammatory signaling following Fas-induced apoptosis [28].
  • human monocytes displayed Fas-dependent IL-8 and TNF-alpha secretion, which was associated with NFkappaB activation and shown to occur even in the absence of apoptosis [29].
  • NFkappaB activation was not detected in monocytes subjected to suspension-induced death ( FIGS. 8 a - c ).
  • Fas-mediated signaling for pro-inflammatory cytokines/chemokines
  • monocytes undergoing apoptosis were exposed to two different Fas inhibiting antibodies.
  • FIG. 9 a using two different inhibitory antibodies for Fas mediated apoptosis did not significantly inhibit suspension-induced monocyte death.
  • both inhibitory antibodies did not decrease IL-1-beta secretion and even caused elevation in IL-1-beta levels (data not shown).
  • pro-inflammatory IL-1-beta, IL-8, and MIP-1-alpha were all secreted at significant levels and in a transcriptional- and translational-dependent pattern in monocytes subjected to suspension-induced death.
  • the cells showed a necrotic pattern with rapid lysis and their death was neither caspase- nor Fas-dependent.
  • Apoptotic cells have been shown to signal neighboring cells in a variety of ways.
  • Pro-phagocytic signals on apoptotic cells serve as markers for phagocytes to specifically recognize the apoptotic cells and subsequently ingest them. Such signals can appear on the membrane of apoptotic cells.
  • Direct signals include alterations in cell surface phospholipid composition [32], changes in cell surface glycoproteins, or in surface charge [33].
  • certain serum proteins can opsonize an apoptotic cell surface, and signal to phagocytes to engulf the opsonized apoptotic cells [34, 35].
  • viable cells express phagocytosis-inhibitory signals by restriction of phosphatidylserine to the inner leaflet of the plasma membrane or CD31 expression [36].
  • Apoptotic cells can also secrete molecules which are important for recruitment of phagocytic cells, phagocytosis, and immune responses in the immediate milieu. Examples of mediators of immune suppression and phagocyte recruitment include TGF-beta [37] and phosphoisocholine [38]. Most of these mechanisms have suggested that there occurs efficient-identification and clearance of apoptotic cells, in processes leading to non-inflammatory and non-autoimmune consequences [11, 12].
  • Monocytes were shown here to be capable of generating pro-inflammatory cytokines/chemokines when subjected to suspension-induced controlled necrosis. As such monocytes may have a unique and crucial role in host defense, in autoimmunity, and in the generation of inflammation. In monocytes, pro-inflammatory cytokines/chemokines may induce cross-priming, whereas anti-inflammatory cytokines may induce cross-tolerization.
  • FasL Fas ligand
  • Fas was proposed to mediate pro-inflammatory cytokines such as IL-1-beta [39] and recently it has been suggested that, following anti-Fas (CH11)-induced apoptosis human monocytes produced Fas-dependent IL-8 and TNF-alpha secretion; which was associated with NFkappaB activation, and was shown to occur in macrophages even in the absence of apoptosis [29].
  • the presently disclosed experimental results reveal for the first time a novel non-Fas-dependent, non-caspase dependent pattern of pro-inflammatory cytokine/chemokine secretion that is associated with MAPK activation in monocytes subjected to suspension-induced controlled necrosis.
  • MAPKs are divided into three major groups: ERKs, JNKs/stress-activated protein kinases, and p38, based on their degree of homology, biological activities, and phosphorylation motifs.
  • JNK may contribute to death receptor transcription-dependent apoptotic signaling via c-Jun/AP-1, leading to transcriptional: activation of FasL.
  • monocytes secrete pro-inflammatory cytokines either in Fas-dependent or p38-dependent patterns.
  • monocytes are unique among leukocytes in their p38-dependent cytokine/chemokine secretion, and as such sustained activation of p38 may determine immune response in homeostatis, infection, inflammatory, and autoimmunity.
  • IL-1-beta secretion involves a signaling cascade that is completely distinct from the cascade seen upon monocyte activation or Fas signaling, is: associated with p38 phosphorylation and is completely abrogated upon exposure of monocytes to p38 inhibitor.
  • This distinct cascade may, on the one hand, help cross-priming upon infection, but on the other hand it may expose the body to persistent inflammatory and/or autoimmune response triggered by self-antigens that are derived from apoptotic monocytes in the context of pro-inflammatory cytokines and chemokines.
  • self-antigens that are derived from apoptotic monocytes in the context of pro-inflammatory cytokines and chemokines.
  • Apoptotic lymphocytes have an immunosuppressive, tolerizing, and anti-inflammatory effect provided they are isolated in the right way and therapeutically in the right conditions and if mixed with other cells, only in controlled way (which does not occur spontaneously in leukocytes from the blood). Described below are methods of suitably obtaining and administering apoptotic lymphocytes for treatment of various disease conditions.
  • lymphocytes from PBMCs using magnetic beads conjugated to ligands of lymphocyte surface markers, or by subtraction of adherent lymphocytes.
  • apoptotic lymphocytes via one of the following routes: parenterally, intravenously, intramuscularly, subcutaneously, intra-dermally, and orally.
  • the cell dosages described below are suitable for a 70 kg patient and may be adjusted according to body weight.
  • Prophylactic treatment Administer 10 million to 5 billion cells 2-24 hours prior to, and 24 hours following, transplantation. Administer 10 million to 5 billion cells every 2 weeks, as needed.
  • Treatment for overt GVHD Administer 10 million to 5 billion cells every 2 weeks, as needed.
  • Treatment of active disease Administer 10 million to 5 billion cells every 2 weeks, as needed.
  • Treatable autoimmune diseases include rheumatoid arthritis, idiopathic polyarthritis, multiple sclerosis, inflammatory bowel disease, scleroderma, Sjogren's syndrome, polymyositis or dermatomyositis, systemic or localized vasculitis, celiac disease, Guillain-Barre syndrome, myasthenia gravis, diabetes mellitus type I, antiphospholipid syndrome, thyroiditis. Grave's disease, and psoriasis. Can be used for treating active disease or preventing flares. Administer 10 million to 5 billion cells every 2-4 weeks, as needed.
  • FMF Familial Mediterranean Fever
  • FMF Familial Mediterranean Fever
  • Apoptotic monocytes have an immunosuppressive, tolerizing, and anti-inflammatory effect provided they are isolated and therapeutically administered in the right way. Otherwise they may undergo pro-inflammatory necrosis. Described below are methods of suitably obtaining and administering apoptotic monocytes for treatment of various disease conditions.
  • apoptotic lymphocytes via one of the following routes: parenterally, intravenously, intramuscularly, subcutaneously, intra-dermally, and orally.
  • the cell dosages described below are suitable for a 70 kg patient and may be adjusted according to body weight.
  • Prophylactic treatment Administer 10 million to 1 billion cells 2-24 hours prior to, and 24 hours following, transplantation. Administer 10 million to 1 billion cells every 2 weeks, as needed.
  • Treatment for overt GVHD Administer 10 million to 1 billion cells every 2 weeks, as needed.
  • Treatment of active disease Administer 10 million to billion cells every 2 weeks, as needed.
  • Treatable autoimmune diseases include rheumatoid arthritis, idiopathic polyarthritis, multiple sclerosis, inflammatory bowel disease scleroderma, Sjogren's syndrome, polymyositis or dermatomyositis, systemic or localized vasculitis, celiac disease, Guillain-Barre syndrome, myasthenia gravis, diabetes mellitus type I, antiphospholipid syndrome, thyroiditis. Grave's disease, and psoriasis. Can be used for treating active disease or preventing flares. Administer 10 million to 1 billion cells every 2-4 weeks, as needed.
  • Apoptotic neutrophils have an immunosuppressive, tolerizing, and anti-inflammatory effect provided they are isolated in the right way and therapeutically administered in the right conditions and if mixed with other cells, only in controlled way (which does not occur spontaneously in leukocytes from the blood).
  • Neutrophils contain proteases and other contents that may inhibit the anti-inflammatory, immunosuppressant effect if not administered correctly. Described below, are methods of suitably obtaining and administering apoptotic neutrophils for treatment of various disease conditions.
  • apoptotic neutrophils via one of the following routes: parenterally, intravenously, intramuscularly, subcutaneously, intra-dermally, and orally.
  • the cell dosages described below are suitable for a 70 kg patient and may be adjusted according to body weight.
  • Prophylactic treatment Administer 10 million to 5 billion cells 2-24 hours prior to, and 24 hours following, transplantation. Administer 10 million to 5 billion cells every 2 weeks, as needed.
  • Treatment for overt GVHD Administer 10 million to 5 billion cells every 2 weeks, as needed.
  • Treatment of active disease Administer 10 million to 5 billion cells every 2 weeks, as needed.
  • Treatable autoimmune diseases include rheumatoid arthritis, idiopathic polyarthritis, multiple sclerosis, inflammatory bowel disease, scleroderma, Sjogren's syndrome, polymyositis or dermatomyositis, systemic or localized vasculitis, celiac disease, Guillain-Barre syndrome, myasthenia gravis, diabetes mellitus type. 1, antiphospholipid syndrome, thyroiditis. Grave's disease, and psoriasis. Can be used for treating active disease or preventing flares. Administer 10 million to 5 billion cells every 2-4 weeks, as needed.
  • FMF Familial Mediterranean Fever
  • FMF Familial Mediterranean Fever

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US11/121,048 US20050202098A1 (en) 2001-01-31 2005-05-04 Disease therapy using dying or dead cells
EP10183710A EP2283847A3 (fr) 2005-05-04 2006-05-04 Traitement de maladie au moyen de cellules mourantes ou mortes
JP2008509575A JP2008540400A (ja) 2005-05-04 2006-05-04 瀕死細胞または死細胞を使用する疾患治療
EP06728323.4A EP1879601B1 (fr) 2005-05-04 2006-05-04 Traitement de maladie au moyen de cellules mourantes ou mortes
PCT/IL2006/000527 WO2006117786A1 (fr) 2005-05-04 2006-05-04 Traitement de maladie au moyen de cellules mourantes ou mortes
CA002606803A CA2606803A1 (fr) 2005-05-04 2006-05-04 Traitement de maladie au moyen de cellules mourantes ou mortes
IL187122A IL187122A (en) 2005-05-04 2007-11-01 Use of dying or dead cells for the preparation of medicaments for disease therapy
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US20040244806A1 (en) * 2003-06-09 2004-12-09 Ferree Bret A. Treating disc herniation and other conditions with leukocytes
WO2006117786A1 (fr) * 2005-05-04 2006-11-09 Tolarex Ltd. Traitement de maladie au moyen de cellules mourantes ou mortes
WO2010031006A1 (fr) * 2008-09-12 2010-03-18 Cryopraxis Criobiologia Ltda. Thérapie cellulaire de tissus ischémiques
US20150275175A1 (en) * 2012-12-06 2015-10-01 Enlivex Therapeutics Ltd. Therapeutic apoptotic cell preparations, method for producing same and uses thereof
WO2016022972A1 (fr) * 2014-08-08 2016-02-11 Alfred E. Mann Institute For Biomedical Engineering At The University Of Southern California Corps apoptotiques
US20170360836A1 (en) * 2015-02-18 2017-12-21 Enlivex Therapeutics Ltd. Combination immune therapy and cytokine control therapy for cancer treatment
WO2021013214A1 (fr) * 2019-07-23 2021-01-28 四川大学 Utilisation de granulocytes neutrophiles dans la préparation de médicaments pour traiter et/ou prévenir l'hépatite auto-immune
AU2016221305B2 (en) * 2015-02-18 2021-05-27 Enlivex Therapeutics Rdo Ltd Combination immune therapy and cytokine control therapy for cancer treatment
CN113423411A (zh) * 2018-10-19 2021-09-21 明尼苏达大学董事会 用碳二亚胺处理的耐受性疫苗诱导移植物耐受
US11304976B2 (en) 2015-02-18 2022-04-19 Enlivex Therapeutics Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US11318163B2 (en) 2015-02-18 2022-05-03 Enlivex Therapeutics Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US11497767B2 (en) 2015-02-18 2022-11-15 Enlivex Therapeutics R&D Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US11596652B2 (en) 2015-02-18 2023-03-07 Enlivex Therapeutics R&D Ltd Early apoptotic cells for use in treating sepsis
US11730761B2 (en) 2016-02-18 2023-08-22 Enlivex Therapeutics Rdo Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US11883429B2 (en) 2015-04-21 2024-01-30 Enlivex Therapeutics Rdo Ltd Therapeutic pooled blood apoptotic cell preparations and uses thereof

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ES2657675T3 (es) 2013-01-07 2018-03-06 Institut National De La Santé Et De La Recherche Médicale (Inserm) Terapia de enfermedad mediante el uso de una preparación farmacéutica tolerogénica
EP4197547A1 (fr) * 2021-12-20 2023-06-21 Aposcience AG Composition pour le traitement ou la prévention de la vasculite et des maladies associées à la vasculite

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US20040244806A1 (en) * 2003-06-09 2004-12-09 Ferree Bret A. Treating disc herniation and other conditions with leukocytes
US9567568B2 (en) 2005-05-04 2017-02-14 Enlivex Therapeutics Ltd. Method of preparing apoptotic monocytes
WO2006117786A1 (fr) * 2005-05-04 2006-11-09 Tolarex Ltd. Traitement de maladie au moyen de cellules mourantes ou mortes
EP2283847A2 (fr) * 2005-05-04 2011-02-16 Tolarex Ltd. Traitement de maladie au moyen de cellules mourantes ou mortes
EP2283847A3 (fr) * 2005-05-04 2011-08-10 Tolarex Ltd. Traitement de maladie au moyen de cellules mourantes ou mortes
WO2010031006A1 (fr) * 2008-09-12 2010-03-18 Cryopraxis Criobiologia Ltda. Thérapie cellulaire de tissus ischémiques
US8784802B2 (en) 2008-09-12 2014-07-22 Cryopraxis Criobiologia Ltda. Ischemic tissue cell therapy
CN107595889A (zh) * 2008-09-12 2018-01-19 克里奥普拉斯低温生物有限公司 缺血性组织的细胞疗法
US10077426B2 (en) * 2012-12-06 2018-09-18 Enlivex Therapeutics Ltd Therapeutic apoptotic cell preparations, method for producing same and uses thereof
US10927343B2 (en) 2012-12-06 2021-02-23 Enlivex Therapeutics Ltd Therapeutic apoptotic cell preparations, method for producing same and uses thereof
CN105102612A (zh) * 2012-12-06 2015-11-25 恩立夫克治疗有限责任公司 治疗性凋亡细胞制剂、其制备方法以及其用途
EP2929015A4 (fr) * 2012-12-06 2018-03-21 Enlivex Therapeutics Ltd. Préparations de cellules apoptotiques thérapeutiques, méthode de production et utilisations associées
US20150275175A1 (en) * 2012-12-06 2015-10-01 Enlivex Therapeutics Ltd. Therapeutic apoptotic cell preparations, method for producing same and uses thereof
AU2013353573B2 (en) * 2012-12-06 2019-01-17 Enlivex Therapeutics R&D Ltd Therapeutic apoptotic cell preparations, method for producing same and uses thereof
CN106659740A (zh) * 2014-08-08 2017-05-10 南加州大学阿尔弗雷德·E·曼生物医学工程研究所 凋亡小体
WO2016022972A1 (fr) * 2014-08-08 2016-02-11 Alfred E. Mann Institute For Biomedical Engineering At The University Of Southern California Corps apoptotiques
US10646518B2 (en) * 2014-08-08 2020-05-12 Alfred E. Mann Institute For Biomedical Engineering At The University Of Southern California Apoptotic bodies
US11000548B2 (en) * 2015-02-18 2021-05-11 Enlivex Therapeutics Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US20170360836A1 (en) * 2015-02-18 2017-12-21 Enlivex Therapeutics Ltd. Combination immune therapy and cytokine control therapy for cancer treatment
AU2016221305B2 (en) * 2015-02-18 2021-05-27 Enlivex Therapeutics Rdo Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US11304976B2 (en) 2015-02-18 2022-04-19 Enlivex Therapeutics Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US11318163B2 (en) 2015-02-18 2022-05-03 Enlivex Therapeutics Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US11497767B2 (en) 2015-02-18 2022-11-15 Enlivex Therapeutics R&D Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US11512289B2 (en) 2015-02-18 2022-11-29 Enlivex Therapeutics Rdo Ltd Combination immune therapy and cytokine control therapy for cancer treatment
US11596652B2 (en) 2015-02-18 2023-03-07 Enlivex Therapeutics R&D Ltd Early apoptotic cells for use in treating sepsis
US11717539B2 (en) 2015-02-18 2023-08-08 Enlivex Therapeutics RDO Ltd. Combination immune therapy and cytokine control therapy for cancer treatment
US11883429B2 (en) 2015-04-21 2024-01-30 Enlivex Therapeutics Rdo Ltd Therapeutic pooled blood apoptotic cell preparations and uses thereof
US11730761B2 (en) 2016-02-18 2023-08-22 Enlivex Therapeutics Rdo Ltd Combination immune therapy and cytokine control therapy for cancer treatment
CN113423411A (zh) * 2018-10-19 2021-09-21 明尼苏达大学董事会 用碳二亚胺处理的耐受性疫苗诱导移植物耐受
WO2021013214A1 (fr) * 2019-07-23 2021-01-28 四川大学 Utilisation de granulocytes neutrophiles dans la préparation de médicaments pour traiter et/ou prévenir l'hépatite auto-immune

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