EP1005531A2 - Verfahren zur verhandlung für leukozyten, leukozyten enthaltende zusammensetzungen und deren verwendungen - Google Patents

Verfahren zur verhandlung für leukozyten, leukozyten enthaltende zusammensetzungen und deren verwendungen

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Publication number
EP1005531A2
EP1005531A2 EP98936943A EP98936943A EP1005531A2 EP 1005531 A2 EP1005531 A2 EP 1005531A2 EP 98936943 A EP98936943 A EP 98936943A EP 98936943 A EP98936943 A EP 98936943A EP 1005531 A2 EP1005531 A2 EP 1005531A2
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EP
European Patent Office
Prior art keywords
cell
population
cells
leukocytes
leukocyte
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EP98936943A
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English (en)
French (fr)
Inventor
William M. Greenman
Joshua A. Grass
Sohel Talib
Adonis Stassinopoulos
Derek J. Hei
John E. Hearst
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Cerus Corp
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Cerus Corp
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Publication of EP1005531A2 publication Critical patent/EP1005531A2/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide

Definitions

  • the present invention relates to cell compositions that are incapable of proliferation but retain function, and the methods of preparation and use of such compositions. More specifically, the present invention relates to methods for preparing proliferation inhibited leukocytes for use in transfusion.
  • BMT Bone marrow transplantations
  • Leukemias are malignant neoplasms of hematopoietic tissues. These neoplasms are categorized into two predominant forms: chronic and acute. While acute leukemias are characterized by undifferentiated cell populations (e.g. Acute Lymphocytic Leukemia or "ALL” and Acute Myelogenous Leukemia or "AML”), chronic Leukemias usually present a more mature morphology (e.g., Chronic Myelocytic Leukemia or "CML” and Chronic Lymphocytic Leukemia or "CLL”). In the United States in 1995, there were approximately 25,700 new cases of leukemia, of which about 4,500 were CML. In 1996, approximately 7,000 new cases of CML were diagnosed in the U.S.
  • ALL Acute Lymphocytic Leukemia
  • AML Acute Myelogenous Leukemia
  • CML Chronic Myelocytic Leukemia
  • CLL Chronic Lymphocytic Leukemia
  • leukemia Society Leukemia Society Web Site, 1997.
  • Allo BMT Allogeneic bone marrow transplantation
  • a leukemia patient is first given a myeloablative dose of chemoradiotherapy. This phase of treatment is designed to eradicate all leukemia cells and their progenitors.
  • the functionally inoperative bone marrow is subsequently replaced with allogeneic disease- free stem cells.
  • this transplanted marrow facilitates the restoration of normal polyclonal hematopoietic proliferation.
  • the improvement in disease control also relies on an immune mediated response of the graft against the leukemia cells, referred to as the graft- versus-leukemia (GVL) effect.
  • VTL graft- versus-leukemia
  • Bone marrow is not the exclusive source of hematopoietic progenitors. Peripheral blood and cord blood may also be used as a source of stem cells. See McCullough, The
  • Trophic factors may be administered systemically to increase the proliferation of peripheral blood stem cells thereby creating alternatives to bone marrow for the harvest of hematopoietic progenitors.
  • Trophic factors such as granulocyte-colony-stimulating factor
  • the major obstacles to successful BMT are Graft- versus-Host
  • GVHD GVHD
  • infections and relapse of the underlying disease GVHD and infections are responsible for 10-30% of morbidity and mortality in the first 100 days following transplantation (Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994). If the BMT fails, a "relapse" can result from the unchecked proliferation of residual tumor cells. See Slavin et al, "Immunotherapy of Minimal Residual Disease by Immunocompetent Lymphocytes and their Activation Cytokines," Cancer Investigation, 10:221 (1992). The medical treatment options in the past have been quite limited in such situations. For example, second bone marrow transplants and/or the extended use of cytotoxic drugs are associated with poor survival.
  • DLIs are particularly effective for the treatment of CML, but are also used for the treatment of AML, ALL and CLL. DLIs have also been used for the treatment of myelodysplasia (MDS), non-Hodgkin's lymphoma,
  • GVL effect Barrett, J. et al, Cur. Op. One. 8:89-95, 1996.
  • the mechanism for the GVL effect is not as well understood. It is effected by
  • T-cells derived from the donor leads to higher incidences of leukemia relapse.
  • An immune response is necessary for the effect to occur which has been experimentally demonstrated by the higher rates of relapse following syngeneic BMTs as compared to allogeneic BMTs (Duell, T., Ann. Int. Med. 126:184-192, 1997).
  • the high homogeneity of the population has resulted in ineffective DLI therapy since the random donors and hosts are not sufficiently allogeneic (Takahashi, K. et al, Lancet. 343:700-702, 1994).
  • the donor T-cells are transfected with a "suicide gene", the herpes virus thymidine kinase (HSV-tk) gene which confers sensitivity to the drug gancyclovir. If GVHD symptoms are observed, the patient is given gancyclovir to eliminate the T-cells in vivo, until the symptoms regress
  • Another "suicide gene” approach utilizes the FAS gene encoding an apoptosis signaling protein (Ariad Pharmaceuticals).
  • a dimerizing agent such as FK1012 is used to induce FAS production which kills the donor cells that express the gene.
  • GVHD GVHD symptoms are used as the signal to begin treatment with the drug. Therefore, GVHD is allowed to start and must be shut down.
  • the drug used to eliminate the T-cells must be given systemically, thereby exposing the entire body to drug for the time necessary to bring GVHD under control.
  • Gancyclovir has toxic side effects and the HSV-tk protein is immunogenic. Additionally, because the patients need to be protected from the severe forms of GVHD, immunosuppressive prophylaxis remains an absolute requirement. Also, in some instances of chronic GVHD, the administration of gancyclovir was proven not to be fully effective (Bonini C. et al. Science 276:1719-1724,
  • the disadvantages include the difficulty in the synthesis and the insolubility of the dimerizing agents that induce FAS production.
  • the binding of the dimerizing agents to FKBP protein in the host may limit the available drug.
  • the currently accepted method for transfusion associated GVHD prevention is ⁇ -radiation treatment (2500 cGy).
  • the clinical dose of ⁇ -irradiation (2500cGy) used for prevention of TA-GVHD results in a 10 5 to 10 6 fold reduction in viable T-cells.
  • ⁇ - and ionizing radiation in general are not specific for nucleic acids. As a result of their high energy, ⁇ -radiation and UVC also attack proteins and other cellular components, causing significant non-specific damage (Deeg, H.J. et al, Blood Cells 18:151-162, 1992).
  • UVB has been used to abolish rat lymphocyte proliferation while preserving BM progenitor cell and primitive hematopoietic stem cell viability for engraftment.
  • CTL activity was reduced but was still present (Gowing, H. et al, Blood 87:1635-1643, 1996).
  • UVB irradiation also leads to significant cell damage, especially if lamps with a broad emission spectrum are used (Gowing, H. et al, Blood 87:1635-1643, 1996; Pamphilon, A. A. et al, Blood 77:2072-2078, 1991).
  • UVB is not selective for nucleic acids only, but modifies any chemical group which absorbs between 280 and 320 nm.
  • UVA irradiation in the presence of the photosensitizer 8-methoxy psoralen (8-MOP), used in the treatment of human leukocytes was shown to inhibit both the stimulating ability and proliferation of peripheral blood leukocytes, in mixed lymphocyte culture reaction in vitro (Kraemer, K.H. et al, J. Inv. Derm. 77:235-239, 1981; Kraemer, K.H. et al, J. Inv. Derm. 76:80-87, 1981).
  • UVA plus psoralen treatment has resulted in the inhibition of surface antigen expression, cytokine synthesis (IL-1, IL-6, IL-8 and TNF) and transcription of cytokine mRNA (Gruner, S. et al, Tiss. Ant. 27:147-154, 1986; Neuner, P. et al, Photochem. Photobiol.
  • the present invention provides an effective method for treating leukocytes from an allogeneic donor to produce leukocytes that are unable to proliferate but maintain viability and immune function effective to promote destruction of a diseased cell or pathogen. Since the treated leukocytes are incapable of proliferation, they are unable to induce GVHD when introduced into an allogeneic host (in a syngeneic or autologous transfusion, GVHD is not an issue) and are therefore ideal for use in DLI for various clinical indications and especially for the treatment of cancer and to provide immune function to an immunocompromised mammal.
  • An isolated cell population comprising a population of leukocytes wherein a portion of the leukocyte population is non-proliferating, such that the cell population is incapable of eliciting graft-versus-host disease (GVHD) in an allogeneic host; and a portion of the leukocyte population retains immunological activity, including the ability to promote destruction of a diseased cell or a pathogen.
  • GVHD graft-versus-host disease
  • at least 90% of the leukocytes in the population are non-proliferating.
  • the population of leukocytes of the preceding embodiments is further characterized as 1) preferably comprising T cells, NK cells and antigen presenting cells; 2) capable, upon appropriate stimulation, of synthesizing and/or secreting cytokines such as IL-2, IFN- ⁇ , IL-10 and GM-CSF; 3) capable of expressing surface markers characteristic of T-cell activation and immune function, such as CD4, CD 8, CD 16 and CD56, among others; and 4) exhibiting an increased ratio of killer function to proliferative function, compared to untreated leukocyte populations.
  • the leukocyte population of the invention is, on the whole, incapable of proliferation but retains immunological activity. This leukocyte population will be capable of the following activities, among others: 1. Promoting destruction of a diseased cell, an infected cell, or a pathogen.
  • the second population of cells can be a suspension of cells or an organized collection of cells, such as a patch of tissue or an organ.
  • Such cells can include, for instance, hematopoietic cells, myeloid cells, leukocytes, bone marrow cells, islet cells, hepatic cells, neuronal cells, myocardial cells, mesenchymal cells and endothelial cells.
  • the population of leukocytes is effective to promote destruction of a cancerous cell.
  • the cancerous cell is selected from the group consisting of Chronic Myelogenous Leukemia (CML) cell, Chronic myelomonocytic Leukemia (CmML) cell, Chronic Lymphocytic Leukemia (CLL) cell, Acute Myelogenous
  • the cancerous cell is a Chronic Myelogenous Leukemia (CML) cell or a multiple myeloma cell.
  • the population of leukocytes is effective to promote destruction of a diseased cell which is an infected cell. Infected cells include cells infected with a virus.
  • the virus infecting the cell is selected from the group consisting of cytomegalo virus (CMV), Epstein Barr virus (EBV), Adenovirus (Ad) and Kaposi's Sarcoma associated Herpes virus.
  • CMV cytomegalo virus
  • EBV Epstein Barr virus
  • Ad Adenovirus
  • Kaposi's Sarcoma associated Herpes virus a virus infecting the cell.
  • the leukocyte population is effective to promote destruction of a pathogen.
  • Pathogens include bacteria, fungi and parasites.
  • the leukocyte population is effective to facilitate engraftment by a second population of cells.
  • the cells used for engraftment can be, for example, hematopoietic cells, myeloid cells, leukocytes, bone marrow cells, islet cells, hepatic cells, neuronal cells, myocardial cells, mesenchymal cells and endothelial cells.
  • the second population of engrafted cells can comprise a solid organ or portion thereof.
  • Subsets of the leukocyte population described above, selected by various methods, are also provided by the invention.
  • the subsets include lymphocytes and T-lymphocytes; and subsets can be obtained by both positive and negative selection based on, for example, surface marker expression.
  • Preferred surface markers include CD8, CD4, CD 16 and CD56.
  • Methods for selection of subsets of leukocyte populations from whole blood include leukophoresis and red cell removal.
  • the invention provides a variety of therapeutic methods utilizing cell populations as described above, including donor leukocyte infusion, leukocyte add-back, immune reconstitution, adoptive immunotherapy, treatment of mixed chimerism, and methods for enhancing engraftment of a second population of transplanted cells.
  • the cell populations of the invention can be introduced into the host prior to, at the same time as, or subsequent to transplantation of the second population of cells.
  • Also provided by the invention is a method for preparing a treated leukocyte population wherein the leukocyte population as a whole is non-proliferating and incapable of eliciting graft-versus-host disease (GVHD) in an allogeneic host, the method comprising the steps of: i) providing a sample comprising a population of leukocytes; and ii) combining the sample with a compound capable of forming a covalent bond with a nucleic acid, in an amount such that the compound forms about 1 to 10 4 adducts per 10 8 base pairs of genomic DNA of the leukocytes, thereby inhibiting proliferation but maintaining immunological activity, including the ability of the leukocyte population to promote destruction of a diseased cell or a pathogen.
  • GVHD graft-versus-host disease
  • the compound is present in an amount effective to form from about 5 to 10 3 adducts per 10 8 base pairs of genomic DNA of the leukocytes.
  • the method results in proliferation being inhibited in at least 90% of the T cells within the treated leukocyte population.
  • the compound capable of forming a covalent bond with a nucleic acid further comprises a nucleic acid binding moiety capable of binding non-covalently with a nucleic acid.
  • the compound comprises: a nucleic acid-binding moiety; a moiety capable of reacting to form a covalent bond with nucleic acid; and a frangible linker covalently linking the nucleic acid-binding moiety and the effector moiety.
  • the nucleic acid-binding moiety is an aromatic intercalator and the effector moiety is a mustard.
  • the compound capable of forming a covalent bond with a nucleic acid comprises a photoactivatable moiety, which upon electromagnetic stimulation, forms a covalent bond with a nucleic acid.
  • the method will further comprise the step of exposing the sample of leukocytes that have been combined with the compound to light, to photoactivate the photoactivatable moiety, thereby resulting in the photoactivatable moiety forming a covalent bond with leukocyte genomic DNA.
  • the compound having a photoactivatable moiety is selected from the group consisting of furocoumarins, actinomycins, anthracyclinones, anthramycins, benzodipyrones, fluorenes, fluorenones, monostral fats blue, norphillin A, organic dyes; phenanthridines, phenazathionium salts, phenazines, phenothiazines, phenylazides, quinolines and thiaxanthenones acridines and ellipticenes.
  • a preferred furocoumarin is a psoralen.
  • Preferred psoralens include PAP, 8- methoxy psoralen (8-MOP), 4'-aminomethyl 4, 5', 8-trimethylpsoralen (AMT), 5-methoxy psoralen, and trioxalen 4, 5' 8-trimethylpsoralen.
  • the psoralen is preferably present at a concentration in the range of 10 "4 to 150 ⁇ M and the sample of leukocytes is exposed to ultraviolet light having a wavelength in the range of 200 to 450 nm, preferably between 320 and 400 nm.
  • the ultraviolet light is provided at a dosage of between 10 " to 100 J/cm .
  • the sample of leukocytes will be exposed to the ultraviolet light for a period of 1 second to 60 minutes.
  • the method for preparing a treated leukocyte population employs the psoralen referred to herein as S-59, having the formula:
  • the sample of leukocytes combined with S-59 is exposed to ultraviolet light having a wavelength in the range of 200 to 450 nm, preferably between 320 and 400 nm.
  • the sample of leukocytes combined with S-59 is preferably exposed to the ultraviolet light at a dosage of between 10 "3 to 100 J/cm 2 , more preferably, 3 J/cm 2 .
  • the sample of leukocytes is preferably exposed to the ultraviolet light for a period of between 1 second to 60 minutes, more preferably, for 1 minute.
  • the sample of leukocytes will preferably be provided at a cell density of 10 to 10 9 cells per mL, more preferably between 10 2 and 10 8 cells pre mL, most preferably at 2 x 10 6 cells per mL.
  • a leukocyte population produced according to the preceding methods is provided, including a leukocyte population specifically treated with S-59.
  • Yet another aspect of the invention is a method of promoting destruction of a diseased cell or a pathogen, comprising mixing a leukocyte population produced by the aforementioned methods with a population of allogeneic cells containing the diseased cell or pathogen.
  • the method can be performed in vitro or in vivo.
  • the leukocyte population is mixed with the population of allogeneic cells of a mammalian host in vivo by donor leukocyte infusion into said host.
  • the donor leukocyte infusion is administered to a mammalian host suffering from relapse from leukemia or multiple myeloma post BMT.
  • the diseased cell is a cancerous cell.
  • Cancerous cells from the following cancers are encompassed: Chronic Myelogenous Leukemia (CML) cell,
  • CmML Chronic myelomonocytic Leukemia
  • CLL Chronic Lymphocytic Leukemia
  • AML Acute Myelogenous Leukemia
  • ALL Acute Lymphoblastic Leukemia
  • MM multiple myeloma
  • Hodgkin's lymphoma cell Hodgkin's lymphoma cell and non-Hodgkin's lymphoma cell.
  • the cancerous cell is a Chronic Myelogenous Leukemia cell or a multiple myeloma cell.
  • cancerous cell is a cancerous cell selected from the group consisting of breast cancerous cell, lung cancerous cell, ovarian cancerous cell, testicular cancerous cell, prostate cancerous cell, colon cancerous cell, melanoma cell, renal carcinoma cell, neuroblastoma cell, head tumor cell and neck tumor cell.
  • the diseased cell is an infected cell.
  • Preferred infected cells encompass cells infected with a virus such as cytomegalovirus (CMV), Epstein Barr virus (EBV), Adenovirus (Ad) or Kaposi's Sarcoma associated Herpes virus.
  • the leukocyte population has been stimulated with one or more epitopes of an antigen specific to the diseased cell or pathogen, to expand the number of cytotoxic T cells specific to the antigen.
  • Stimulation or antigen-specific T cell expansion can be performed in vivo, ex vivo, or in vitro. In vivo stimulation is performed by vaccination of the leukocyte donor with an antigen specific to the diseased cell or pathogen prior to isolation of the leukocyte population from the donor.
  • the leukocyte population is preferably stimulated with a bcr-abl antigen of the CML cell.
  • the leukocyte donor can be vaccinated with the idiotype antigen of the patient's myeloma cell.
  • the leukocyte population has been stimulated with a mitogen.
  • Stimulation is preferably conducted in vitro with a mitogenic composition such as a phorbol myristate acetate (PMA) plus ionomycin, or a phytohemagglutinin.
  • PMA phorbol myristate acetate
  • Figure 2 shows 3 H-thymidine incorporation by effector cells treated with S-59 at the various drug dosages, in a MLR assay (see Example 2).
  • Figure 3 shows the level of IL-2 production in a MLR by effector cells photochemically treated with S-59 at the various drug dosages (see Example 2).
  • Figure 4 shows the level of IFN- ⁇ production in a MLR by effector cells photochemically treated with S-59 at the various drug dosages (see Example 2).
  • Figure 5 shows the levels of IL-8 generated after PCT of leukocytes in PC with different concentrations of S-59 and 0.5 J/cm UVA, as a function of time after PCT (see Example 2)
  • Figure 6 shows the levels of CD69 marker expression after PCT with different doses of AMT and 1 J/cm 2 UVA, as a function of time after treatment (see Example 3)
  • Figure 7 shows psoralen-DNA adduct formation following photochemical treatment with various psoralens in PC.
  • S-59 squares
  • AMT triangle
  • 8-MOP circles
  • 1.9 J/cm 2 UVA see Example 3
  • Figure 8 shows the effects of S-59 + UVA treatment on proliferation of treated cells, measured by H thymidine incorporation in a MLR assay.
  • NR denotes untreated cells
  • UV denotes cells irradiated with UVA in the absence of S-59.
  • Figure 9 shows the effects of S-59 + UVA treatment on proliferation of treated cells, measured after activation of treated cells with anti-CD3 antibody. See Example 14 for details.
  • Figure 10A shows production of IL-2 by human PBMC that have been photochemically treated with S-59 + UVA and stimulated in a MLR. Measurements are by sandwich ELISA. See Example 14 for details.
  • Figure 10B shows production of IFN- ⁇ by human PBMC that have been photochemically treated with S-59 + UVA and stimulated in a MLR. Measurements are by sandwich ELISA.
  • Figure 11 A shows CD69 expression in treated and control cells measured at different times (hours) after activation by anti-CD3.
  • Figure 11B shows CD25 expression in treated and control cells measured at different times (hours) after activation by anti-CD3.
  • Figure 12 shows CD40L expression in treated and control untreated cells measured at different times (hours) after activation by anti-CD3.
  • Figure 13 shows cytotoxic T-cell activity of photochemically-treated, activated leukocytes, measured by 51 Cr release from target cells that were co-incubated with the treated leukocytes.
  • E:T represents effector cell to target cell ratio.
  • Figure 14 shows measurements of average body weight in irradiated mice which received a MHC-mismatched bone marrow transplant along with S-59 + UVA-treated splenocytes, and were then challenged with leukemia cells three days after transplant. Splenocytes were exposed to UVA in the presence of 0.01 ⁇ M S-59 for different amounts of time, as indicated in the figure.
  • Figure 15 shows analysis of GVL activity in leukemia-challenged mice, which received bone marrow transplants with or without infusion of treated leukocytes. GVL activity is demonstrated by percent leukemia-free survival (survival ratios given in parentheses).
  • Figure 16 shows analysis of proliferative ability of S-303-treated responder cells, measured by H-thymidine incorporation between 6 and 7 days after co-culture with gamma-inactivated allogeneic stimulator cells in a MLR.
  • Figure 17 shows levels of IFN- ⁇ in MLR supernatants in which the responder cells were treated with different concentrations of S -303 prior to co-culture.
  • BMT bone marrow transplantation
  • DLI donor leukocyte infusion
  • LDA limiting dilution analysis
  • MLR mixed lymphocyte reaction
  • PA photochemically arrested
  • PCT photochemical treatment
  • PI-DLI Photochemically inactivated donor leukocyte infusions
  • PUVA Psoralen and ultraviolet A radiation.
  • PAP 4'- and 5 '-primary amino-substituted psoralen
  • S-59 a primary amino-substituted psoralen having the following formula:
  • PBSC peripheral blood stem cell
  • PC platelet concentrate
  • TA-GVHD transfusion associated graft-versus-host -disease
  • PHA phytohemagglutinin
  • Allogeneic refers to the relationship that exists between genetically nonidentical members of the same species, i.e., the members are not syngeneic.
  • “Host” is used interchangeably with “recipient” and refers to a mammalian recipient of allogeneic donor leukocytes. Generally, the host will be a human but other mammals such as mice, dogs, cats, monkeys, horses, etc. are encompassed.
  • a “leukocyte” refers to any white blood cell including the lineage progenitor cells.
  • Antigen presenting cells include macrophages, and dendritic cells which are present in low numbers in peripheral blood, as well as their respective precursor cells. Leukocytes are present in circulating blood and in bone marrow as well as in the myelopoietic, lymphoid and reticular sites of the reticuloendothelial system.
  • leukocyte population refers to a population of leukocytes that contains more than one leukocyte cell type and will include at least T cells, antigen presenting cells and NK cells.
  • Donor leukocytes refer to leukocytes that are not endogenous to the host animal but are derived from an allogeneic donor.
  • the leukocyte population and donor leukocytes can be from a mammal including rodents, rabbits, dogs, etc.
  • purified is meant that the leukocyte population has been isolated from the body and processed to be substantially free from non-leukocyte cell types and preferably from other contaminants such as cellular debris that are present in the source of the leukocytes, the source generally being peripheral blood, bone marrow or even splenocytes.
  • the leukocyte population is purified such that it contains less than 13% red blood cells (RBC), more preferably less than 5% , even more preferably, less than 1%. In a most preferred embodiment, the hematocrit level is less than 0.5%.
  • Treated leukocytes refers to leukocytes that have been exposed to or contacted with a compound capable of forming one or more covalent bonds with nucleic acid.
  • the leukocytes can be "treated” by PCT or with an alkylator compound.
  • Photochemically inactivated or “photochemically arrested” (PA) or “PCT-arrested” leukocytes refers to PCT leukocytes that are incapable of proliferation even upon stimulation, and does not imply that the leukocytes are inactivated in protein expression and immune function.
  • a population of leukocytes of the invention contains but is not limited to cells that are "non-proliferating". Cells are "non-proliferating” if they are arrested in DNA replication and are thus, unable to divide to generate new cells even upon stimulation with reagents such as mitogens, cytokines, antigens, antibodies or other proliferation stimuli. It is not essential that the methods of the present invention result in replication arrest of every leukocyte in the preparation. For the purpose of this invention, it is adequate that the leukocyte population is arrested in proliferation in at least 90%, more preferably in at least 95%) of the leukocytes within the purified population, provided that the small fraction of leukocytes capable of proliferation is insufficient to cause GVHD in an allogeneic host.
  • 99% or more of the leukocytes within the population are non-proliferating.
  • Proliferation activity can be assayed, e.g., by limiting dilution assay (LDA), or by H-thymidine incorporation separately, or as part of a MLR assay as described in the Examples below.
  • LDA limiting dilution assay
  • T-cells in the population are proliferation inhibited to the limit of detection by LDA. The results of these assays have been shown to be predictive of the ability of the treated leukocytes to proliferate in vivo.
  • GVHD vascular endothelial growth factor-induced hypertension
  • the occurrence of GVHD is attributed to minor histocompatibility antigens.
  • the donor T-cells are able to proliferate unchallenged in that environment and attack host tissues, causing the pathological symptoms.
  • antigen-presenting-cells of the host interact with the T-cells of the donor in the context of the major histocompatibility complexes (MHC) I and II and induce their activation against cells bearing host-specific (minor) antigens. This leads to clonal proliferation of the activated
  • T-cells which attack the host tissues and release cytokines.
  • Cytokines secreted by the T-cells also activate a variety of other effector cells of the host which adds to the tissue damage through the additional generation of cytokines (cytokine storm).
  • cytokine storm e.g., Burakoff, S.J. et al, Graft- Versus-Host Disease Immunology, Pathophysiology, and Treatment, in Brinkhous KMS, S.A. (ed): Hematology, vol. 12 (ed 1st). New York, Marcell Dekker, Inc., 1990, p 725. Burakoff, S.J.
  • GVHD When GVHD is diagnosed, the patient is usually given a high dose of immunosuppressive drugs to suppress the GVHD.
  • immunosuppressive drugs also render the graft leukocytes ineffective in GVL and the patient may relapse into leukemia.
  • GVHD The capability of a leukocyte population to elicit GVHD can be determined in vivo, e.g., as described in Example 4 below, or in vitro by limiting dilution assay (LDA), or by
  • the leukocyte population is judged incapable of eliciting GVHD if the number of clonable T cells in the population, as assayed by LDA, is within about 10 "3 to 10 "4 of that present in the untreated leukocyte control population (positive control for GVHD and proliferation).
  • lack of clinical symptoms of GVHD (symptoms described in Example 4) in an allogeneic recipient of the leukocyte population indicates incapability of the population to elicit GVHD
  • a leukocyte population is considered "effective to promote destruction of a diseased cell or pathogen" if it includes leukocytes that are able to participate, directly or indirectly, in immune responses that are effective to kill or clear the targeted diseased cell or pathogen from the body, or to limit the proliferation of the diseased cell or pathogen.
  • the diseased cell or pathogen can be destroyed via any mechanism. It is not the intention of the invention to have the methods of treating leukocytes be limited by particular mechanisms by which the treated leukocytes can mediate destruction.
  • the diseased cell may be destroyed by cytolysis by T or NK cells, but can also be killed by other mechanisms such as by being induced to undergo apoptosis.
  • the diseased cell or pathogen can also be destroyed by antibody-dependent cell-mediated cytotoxicity (ADCC), by phagocytosis by macrophages, or be cleared by the reticuloendothelial system via any immune mechanism naturally existing in a mammal.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • a pathogen that resides inside a host cell can be destroyed indirectly by killing the host cell upon which it depends for its survival.
  • the "effective" treated donor leukocyte can be an effector cell such as a cytotoxic T cell (CTL), a NK cell, or a macrophage that directly kills a target diseased cell or pathogen, or that induces the diseased cell to undergo apoptosis.
  • CTL cytotoxic T cell
  • NK cell a NK cell
  • macrophage that directly kills a target diseased cell or pathogen, or that induces the diseased cell to undergo apoptosis.
  • the treated donor leukocyte can mediate destruction by stimulating other leukocytes, e.g., the leukocytes from a transplanted bone marrow, a previous DLI, or the host's own leukocytes, to perform the killing or clearance.
  • Stimulation of other leukocytes can be by surface antigen expression, cytokine secretion or any appropriate mechanism. Cytolysis can occur via MHC -restricted and/or non-MHC restricted (such as by NK cells) mechanisms.
  • the effectiveness of a leukocyte population to promote destruction of a diseased cell or pathogen can be measured by various assays, such as by MLR or by 51 Cr release assay as described below in the Examples. Cytolytic effectiveness is demonstrated, e.g., by the ability to mediate killing of a leukemic cell in a 51 Cr release assay.
  • a leukocyte population is considered effective to promote destruction of a diseased cell if, in an appropriate assay (e.g., the 51 Cr release assay), the population exhibits cytolytic activity at a level of at least about 20% above that of the negative control population.
  • an appropriate assay e.g., the 51 Cr release assay
  • the types of diseased cells and pathogens targeted for killing are provided below.
  • Cell viability is defined herein as survival in circulation i.e., in the blood stream and other tissues.
  • the leukocyte population includes leukocytes able to secrete at least interleukin-2 (IL-2) and interferon- ⁇ (IFN- ⁇ ) under stimulation.
  • IL-2 interleukin-2
  • IFN- ⁇ interferon- ⁇
  • cytokines are relevant because of their established role in T-cell activation and GVHD/GVL.
  • Other relevant cytokines include granulocyte-macrophage colony- stimulating factor (GM-CSF) and interleukin- 10 (IL-10). Secretion of cytokines can be readily assayed e.g., by using cytokine assay kits (e.g. kits from R&D Systems) as described in the Examples section below.
  • the surface antigens (also referred to as surface markers) expressed on the leukocytes in the purified leukocyte population will depend on the specific cell type.
  • the population will include leukocytes expressing the following surface antigens which are known to be involved in interactions associated with T-cell and NK cell activation and immune function: CD2, CD28, CTLA4, CD40 ligand (gp39), CD18, CD25, CD69 (lymphocyte activation marker) and CD16/CD56, antigens.
  • the leukocytes will also express other surface markers including MHC Class I and Class II,
  • CD8 CD4, CD3/TcR (T cell receptor), adhesion molecules such as CD54 (ICAM -1), LFA-1 and VLA-4, and other co-stimulatory molecules.
  • the expression of surface antigen can be detected and measured by various assays such as by staining cells with antibodies specific to the particular antigen and detecting the antibodies which have been labeled directly or indirectly, by standard FACS-SCAN analysis. Other methods known in the art include immunoprecipitation of surface antigens and immunoblotting.
  • Diseased cell refers to a cancerous cell, an infected cell, or a cell in any type of pathological state, which may be present in vivo or in vitro.
  • the cancerous or malignant cell can be from any type of cancer, of any tissue or cell type origin.
  • Such malignant or cancerous cells include but are not limited to cells of the following malignancies: Leukemia including Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic leukemia (CLL), Acute Myelogenous Leukemia (AML), and Acute Lymphoblastic Leukemia (ALL); Multiple myeloma (MM); Non-Hodgkin lymphoma and Hodgkin's disease (lymphoma); solid tumors, including breast, lung, ovarian and testicular cancers, prostate cancer, colon cancer, melanoma, renal carcinoma cell, neuroblastoma, and head and neck tumors.
  • Infected cell encompasses cells infected by any of the following: a virus, a bacterium, a fungus, a parasite or any other pathogenic microorganism.
  • Pathogen is defined as any agent containing nucleic acid and capable of causing disease in a human, other mammals, or vertebrates.
  • pathogens are bacteria, viruses, protozoa, fungi, yeasts, molds, and mycoplasmas which cause disease in humans, other mammals, or vertebrates.
  • the genetic material of the pathogen may be DNA or RNA, and the genetic material may be present as single-stranded or double-stranded nucleic acid.
  • the pathogen can be outside a cell or residing inside a cell as exemplified by HIV in a macrophage.
  • Light dose or dosage or “PCT dose”, as measured in units of J/cm 2 refers to the total light energy per unit area that a sample of leukocytes receives during PCT. Light dosage can be altered by varying the intensity of light and time of exposure of the cell sample to the light.
  • Intensity of light as measured in units of mW/cm 2 refers to the energy of light received per unit of the sample, per second.
  • a “photoactivatable moiety” is defined herein as a moiety which undergoes a chemical change in response to electromagnetic radiation.
  • the photoactivated moiety forms a covalent bond with a nucleic acid to form a compound: nucleic acid complex, referred to herein as an "adduct".
  • Aromatic intercalator refers to a compound or region thereof having aromatic ring structure, that can intercalate nucleic acids.
  • Aromatic intercalators include but are not limited to, anthracene, acridine, napthalene, napthoic acid, etc.
  • An "isolated population of cells,” as used herein, comprises a population of cells existing outside of the organism in which the cells normally reside.
  • Immunological activity refers to functions expressed by cells of the immune system, such as T-cells, B-cells, NK cells, antigen-presenting cells (APCs) and others, such functions including, but not limited to, synthesis and secretion of cytokines, cell-mediated cytotoxicity, synthesis and secretion of antibodies, processing and presentation of antigens and antigen fragments, and expression of surface markers characteristic of immune cells.
  • T-cells T-cells, B-cells, NK cells, antigen-presenting cells (APCs) and others, such functions including, but not limited to, synthesis and secretion of cytokines, cell-mediated cytotoxicity, synthesis and secretion of antibodies, processing and presentation of antigens and antigen fragments, and expression of surface markers characteristic of immune cells.
  • APCs antigen-presenting cells
  • the present invention provides a method of treating a population of isolated leukocytes so as to render a portion of the population non-proliferating to the extent that the population causes minimal GVHD while still retaining sufficient immune function effective to promote destruction of a diseased cell or pathogen.
  • minimal GVHD is meant that the extent of the GVHD is insufficient to result in mortality of the mammal or to abolish the ability of the leukocyte population to promote destruction of a diseased cell or pathogen and/or to mediate GVL effect.
  • GVHD is clearly associated with the clonal expansion of the donor T-cells and, in principle, any treatment which removes, inactivates or kills leukocytes would be effective against it.
  • GVL is associated with an immune response of the donor leukocytes and may be the result of cytolytic action, presentation of antigens, synthesis of specific cytokines or a combination of the above. This necessitates some of the functions of the donor leukocytes to be intact for a GVL effect to be observed.
  • the inactivation of donor leukocyte function in efforts to avoid GVHD also renders the donor BM or leukocytes ineffective in providing necessary immune functions to an immunocompromised or BMT host.
  • the proliferating ability of leukocytes must be selectively removed, while retaining some level of leukocyte function.
  • the invention provides populations of leukocytes with these characteristics.
  • the isolated population of leukocytes of the present invention provide various advantages over those previously described and used.
  • One significant benefit is that eliminating the onset of GVHD will allow the safe infusion of donor leukocytes into patients.
  • the host who is lacking an immune system or has a chimeric immune system, can receive either a larger dose of treated donor leukocytes and/or multiple DLIs, thus enhancing the efficacy of treatment of the disease.
  • the threat of GVHD limited the number of leukocytes infused.
  • the population of non-proliferating leukocytes is effective to provide the host with sufficient immune defense to combat cancer and infections.
  • leukocytes are useful for treating relapsing hematological malignancies such as chronic myelogenous, acute myelogenous, acute lympholytic, multiple myeloma and other forms of leukemia and myeloma, as part of post-BMT treatment or as an alternative to BMT.
  • a GVL effect from the DLI is useful not only to remove minimal residual disease but can also be applied to the treatment of a higher malignant cell load, especially since absence of leukocyte expansion allows for the safe infusion of greater numbers of cells.
  • the treated leukocyte populations of the invention are also useful for the treatment of certain solid tumors (e.g.
  • the method of treating leukocytes of the invention involves the following.
  • Leukocytes can be obtained from bone marrow, cord blood or whole blood.
  • the most convenient source of leukocytes is peripheral blood. Methods of isolating leukocytes from other sources are described in the scientific literature.
  • Whole blood is processed to purify and enrich for leukocytes by, for example, red cell removal or leukophoresis.
  • the resultant purified population of leukocytes is about 99% free of red blood cells which are of different size and density compared to leukocytes and are readily removed by these processes.
  • the population of leukocytes is then mixed with a compound, and treated under conditions that are effective to arrest proliferation of the leukocytes without compromising cell viability and integrity.
  • compound will refer to a compound capable of forming a covalent bond with a nucleic acid. Covalent bond formation may form about from 1 to 10 4 adducts, preferably from 5 to 10 adducts, most preferably 10 adducts per 10 base pairs of leukocyte genomic DNA. It is important that the treatment conditions minimize nonspecific damage to proteins and other cellular components and avoid membrane damage. Membrane integrity of the majority of treated leukocytes is necessary for a GVL effect to take place. These treatment conditions will also be effective to render the treated leukocytes incapable of eliciting
  • GVHD if introduced into an allogeneic host, or non-responsive in an MLR assay in vitro.
  • the treated leukocytes should maintain protein expression effective to accomplish immune function, specifically to promote destruction of a diseased cell or pathogen either in vivo or in vitro.
  • the compounds may include a moiety capable of forming a covalent bond with a nucleic acid which is photoactivatable or chemically reactive.
  • the compound inhibits proliferation of cells by forming covalent bonds with the genomic DNA, thus interfering with the function of DNA polymerases and rendering the leukocytes non-proliferating.
  • the number of covalent adducts that the compound forms with leukocyte nucleic acid can be modulated so that the compound inhibits proliferation but maintains immune functions effective to promote destruction of a diseased cell or pathogen.
  • the effect can be modulated by adjusting the concentration of the compound and the length of time the leukocytes are contacted with the compound before removing unbound compound. The concentration required will depend on the characteristics of the particular compound, such as its solubility in aqueous solution and the DNA binding constant.
  • the compound will typically be used at concentrations effective to generate about 1 to 10 4 adducts of the covalent binding compound per 10 8 base pairs of leukocyte genomic DNA, preferably about 5 to 10 3 adducts, even more preferably, about 10 2 to 10 3 adducts. Ideally, the conditions for treating the leukocyte population will generate about 10 adducts per 10 base pairs of genomic DNA.
  • concentration of compound effective to achieve the leukocyte compositions of the present invention is the preferred concentration.
  • the PCT effect can be modulated by adjusting the concentration of the compound, the wavelength of light, and the length of time of exposure to that light.
  • concentration of the compound the concentration of the compound, the wavelength of light, and the length of time of exposure to that light.
  • the conditions for PCT using psoralen and other photoactivatable compounds are described in detail below.
  • Treatment conditions will be targeted to generate about 1 to 10 adducts of the covalent binding compound, per 10 base pairs of leukocyte genomic DNA, preferably about 5 to 10 4 adducts, even more preferably, about 10 to 10 adducts. Ideally, the treatment conditions will generate about 10 adducts per 10 base pairs of genomic DNA.
  • the number of compound-DNA adducts resulting from treatment can be measured e.g., by using radiolabeled DNA binding compound as described in the Examples below.
  • Another type of compound that can be used in the treatment of leukocytes is a small molecule that acts as an inhibitor of DNA replication.
  • Such small molecule replication inhibitors can optionally comprise a linker (frangible or otherwise) and an effector, as described above. Any of the steps of DNA replication can serve as a target for inhibition, including formation of an origin recognition complex (ORC), recruitment of the
  • the compounds of the present invention may be introduced into a suspension of leukocytes (leukocyte sample) in several forms.
  • the compounds may be introduced as an aqueous solution in water, saline, a synthetic media such as "SterilyteTM 3.0", or in the solution that the leukocytes are suspended in.
  • Synthetic media is herein defined as an aqueous synthetic blood or blood product storage media.
  • the compounds can further be provided as dry formulations, with or without adjuvants. The cell suspension is then agitated to mix in the compound.
  • the leukocytes to be treated with compound are resuspended in physiologically balanced solution such as plasma, synthetic media or a combination thereof.
  • physiologically balanced solution such as plasma, synthetic media or a combination thereof.
  • the leukocytes can be provided at a volume of from 200 mL to 1 liter.
  • the leukocytes for treatment will preferably be contained in a reaction vessel such as a blood bag. Blood bags are known in the art.
  • the effect of treatment with the compound on the viability and function of the leukocyte population can be monitored by in vitro as well as in vivo assays to determine the optimum treatment conditions that minimize proliferation and GVHD activity while maximizing cytotoxic function and/or the GVL effect.
  • Optimum conditions will vary with the properties of the compound used and will be validated for the disease or pathogen.
  • the most preferred PCT condition will comprise the lowest concentration of compound and the lowest light dosage found sufficient for providing a population of leukocytes that is arrested in proliferation but effective to promote destruction of a diseased cell or pathogen.
  • the population of treated leukocytes will be characterized as follows. The cell viability of the population of treated leukocytes will be assessed.
  • Cell viability can be measured in vivo, for example, by PCR analysis to detect sequences specific to donor leukocytes, as described in the Examples section below.
  • the population of leukocytes will have a cell viability of at least 3 weeks. Fast clearance of leukocytes upon treatment may affect their ability to induce an antileukemic response.
  • Membrane integrity of the treated leukocytes is also necessary for a GVL effect to take place.
  • Membrane integrity can be determined by trypan blue or propidium iodide dye exclusion.
  • the PCT procedure, if used, is optimized for the light dose and/or the concentration of the compound which will maximize the number of leukocytes with intact membranes.
  • Proliferation activity can be measured, e.g., by Limiting Dilution Assay (LDA) or Mixed Lymphocyte Reaction (MLR) Assay, as described in the Examples below.
  • LDA Limiting Dilution Assay
  • MLR Mixed Lymphocyte Reaction
  • the activity of the leukocytes in a Mixed Lymphocyte Reaction (MLR) assay will be predictive of the ability of the treated leukocytes to mediate GVHD in vivo.
  • GVHD can be measured by monitoring GVHD symptoms and/or morbidity and mortality induced by GVHD in MHC matched animals transfused with the treated leukocytes.
  • Leukocyte activity can be determined by monitoring cytokine synthesis, expression of surface antigenic markers and ability to lyse target cells. Expression of surface antigenic markers is determined through the use of the appropriate antigen-specific antibodies and standard FACS-SCAN analysis. The presence of antigenic markers, CD2,
  • CD28, CTLA4, CD40 ligand (gp39), CD 18, CD25, CD69 (lymphocyte activation marker) and CD16/CD56 which are known to be involved in interactions associated with T-cell and NK cell activation and immune function, will be determined as a function of concentration of the covalent bonding compound and, if used, the PCT light dose. Stability of surface molecules under the PCT conditions is not expected to be influenced given the mild nature of PCT on proteins. Treatment conditions, for example, PCT, will be chosen such that they do not adversely affect the expression of the surface molecules or reduce cytokine production below desired levels (preferred levels disclosed above).
  • Cytolytic activity indicative of GVL activity can be measured, e.g., by lysis of human or mouse leukemia cell lines in a 51 Cr release assay.
  • the anti-leukemia effect can be assayed in vitro as described in Choudhury et al. Blood 89:1133-1142, 1997, or in vivo as described in Johnson et al. Blood 85:3302-3312, 1995.
  • Activity against solid tumors can be tested, e.g., using murine models of solid tumors.
  • the ability to mediate lysis of an infected cell or a pathogen can be assayed using infected animals in an animal model.
  • GVHD and GVL capabilities can also be measured in vivo as described in Examples 4 and 5 below.
  • the concentrations of compound determined to be effective will be tested on a representative number of patients and the most effective concentration of compound that results in alleviation of the patient's disease will be used. Conditions which work for the majority of patients will be most preferred.
  • the patients will generally be post BM transplant patients.
  • HLA-A and -B are HLA I genes and HLA-DR is a HLA II gene. Each of these genes exist in multiple different alleleic forms. Since each individual inherits 2 copies of chromosome 6 (containing HLA genes), the individual can express up to 6 different HLA-A, -B and -DR proteins (products of 2 different alleles at each locus).
  • the allogeneic donor is identical genotypically in 3 or more of the 6 HLA loci of HLA-A, B, and DR.
  • the donor and host are matched in 3 of the 6 HLA loci. Ideally, the donor and recipient are identical for HLA-A, B and DRB 1. Unrelated HLA-matched donors can be searched through the U.S. National Marrow Donor Program (NMPD). Currently, for patients up to the age of 55 years who receive unmodified non-T cell depleted graft, the patient's and donor's HLA-A, B and DRB1 genes have to be identical. A patient who is 36 years of age or younger may be transplanted from a donor who differs by no more than one minor antigen mismatch for HLA-A, -B or -DR.
  • NMPD U.S. National Marrow Donor Program
  • HLA-A or B minor mismatch is defined as 2 antigens that belong to the same cross reactive group.
  • An HLA-DR minor mismatch is defined as two haplotypes that express the same DR specificity but differ for DRB1 alleles.
  • the starting material for the leukocyte populations of the present invention can be provided, for example, by regional blood processing service centers similar to those currently in existence for the processing of peripheral blood stem cells, as well as other sources of hematopoietic stem cells, such as bone marrow.
  • Leukocytes from the donor can be apheresed (apheresis can be done at a variety of facilities including the local blood center or hospital blood bank) and treated at any appropriate facility equipped to perform the treatment. Alternatively, apheresed leukocytes can be shipped overnight to a Good
  • the treated leukocytes for DLI can then be sent by same day delivery to the patient's hospital for administration to the patient.
  • the treated leukocytes can be cryopreserved using methods known in the art such as by control rate freezing in 10%) DMSO and storage in liquid nitrogen (see Russel et al. Bone Marrow Transpl. 19:861-866,
  • the frozen leukocytes will be thawed and washed to remove the DMSO.
  • One conventional method for the preparation of purified leukocytes involves isolation from whole blood by density gradient centrifugation. This typically involves the isolation of T cell-enriched white blood cells by centrifugation through a Ficoll gradient.
  • the present invention contemplates processing blood as done by a leukophoresis machine (e.g., COBE Spectra Apheresis System, COBE BCT, Inc. Lakewood, CO) according to the manufacturer's directions.
  • a leukophoresis machine e.g., COBE Spectra Apheresis System, COBE BCT, Inc. Lakewood, CO
  • an apheresis machine that provides a leukocyte preparation with the lowest percentage hematocrit is used.
  • the leukocyte preparation can then be washed and diluted with the appropriate solution (plasma, infusion grade solution, etc. as described above) to achieve a final hematocrit of less than 0.5%.
  • Percoll separation can be applied as a further purification step if the above procedure was insufficient to reduce the hematocrit levels.
  • red cell removal methods are well-known to those of skill in the art.
  • selected cell subsets can be isolated from the leukocyte preparation, e.g., by using fluorescence activated cell sorting (FACS) using positive and/or negative selection with the appropriate antibodies that identify specific cell surface markers.
  • FACS fluorescence activated cell sorting
  • surface markers include, but are not limited to, CD4, CD8, CD16 and CD56.
  • Purified leukocyte populations are suspended in the appropriate solution as disclosed above.
  • the purified leukocyte populations can be treated immediately or cryopreserved for later treatment.
  • the purified, mixed leukocyte preparation is resuspended in an isotonic solution such as plasma, synthetic media, or a combination of the two.
  • the purified leukocyte population will generally be prepared at a cell density of 10 to 10 9 cells per ml, preferably 10 4 to 10 7 cells/ml, ideally 2 x 10 6 cells/ml.
  • the invention contemplates various scenarios for the preparation and administration or use of the treated leukocytes.
  • the peripheral blood or alternative leukocyte source including bone marrow or cord blood
  • the treated leukocyte populations can subsequently be used to infuse a patient.
  • compounds capable of forming a covalent bond with a nucleic acid are suitable for the purposes of achieving the leukocyte population of the present invention.
  • These compounds preferably include a moiety which binds non-covalently with a nucleic acid, as well as the same or different moiety which is capable of reacting to form a covalent bond with a nucleic acid.
  • the compound will preferably have the following properties: i) high binding affinity for nucleic acid; ii) solubility in aqueous solution; and iii) ability to penetrate the leukocyte membrane.
  • Moieties capable of forming a covalent bond with nucleic acid include chemically reactive moieties, which do not require an outside stimulation in order to be activated and react with a nucleic acid, and photoactivatable moieties, which react with nucleic acids only after stimulus in the form of electromagnetic radiation.
  • alkylator compound refers to a compound including at least one chemically reactive moiety capable of reacting to form a covalent bond with a nucleic acid, in the absence of an external stimulus, such as light stimulation.
  • the compound capable of forming a covalent bond with a nucleic acid further may be photoactivatable.
  • the "photoactivatable compound”, as defined herein, includes a moiety which is capable of forming a covalent bond with a nucleic acid upon photoactivation by stimulus of light of a certain wavelength.
  • the compound includes both a moiety capable of reacting to form a covalent bond with a nucleic acid, and a moiety capable of binding non-covalently with a nucleic acid.
  • the moiety which is capable of binding with a nucleic acid also may serve as the moiety which is capable of covalently reacting with the nucleic acid, and may be, for example, photoactivatable.
  • the alkylator compound comprises: a nucleic acid binding moiety; a moiety capable of reacting to form a covalent bond with nucleic acid ("referred to herein as an effector moiety"); and a frangible linker covalently linking the nucleic acid moiety and the effector moiety.
  • these compounds in aqueous solution, at appropriate pH values, have a period of activity during which they can bind to and react with nucleic acid. After this period, the compounds break down to products which are no longer able to bind well to nor react with nucleic acid.
  • alkylator compounds can be broadly described as an anchor, covalently bonded to a frangible linker, which is covalently bonded to an effector.
  • Anchor also referred to as “nucleic acid binding moiety” is defined as a moiety which binds non-covalently to a nucleic acid biopolymer (DNA, RNA, or synthetic analogues thereof).
  • Effector or “Effector moiety” is defined as a moiety which reacts with nucleic acid by a mechanism which forms a covalent bond with the nucleic acid.
  • “Frangible linker” is defined as a moiety which serves to covalently link the anchor and effector, and which will degrade under certain conditions so that the anchor and effector are no longer linked covalently.
  • the anchor-frangible linker-effector arrangement enables the compounds to bind specifically to nucleic acid (due to the anchor's binding ability). This brings the effector into proximity for reaction with the nucleic acid.
  • the nucleic acid binding moiety is selected from the group consisting of aromatic intercalators, acridines, acridine derivatives, ellipticenes, 2- polyamines, groove binders, and hydrophobic or shape selective binders.
  • the frangible linker comprises a functional unit selected from the group consisting of forward esters, reverse esters, forward thioesters, reverse thioesters, forward and reverse thionoesters, forward and reverse dithioic acids, sulfates, forward and reverse sulfonates, phosphates, and forward and reverse phosphonate groups.
  • the effector moiety preferably comprises a functional group selected from the group consisting of mustard groups, mustard group equivalents, epoxides, aldehydes, and formaldehyde synthons.
  • the effector portion of the compound reacts with nucleic acid present with which it comes into contact. Effector moieties which do not react with nucleic acid are gradually hydrolyzed by the solvent. Hydrolysis of the frangible linker occurs concurrently with the effector-nucleic acid reaction and effector hydrolysis. It is desirable that the frangible linker break down at a rate slow enough to permit inactivation of leukocyte proliferation; that is, the rate of breakdown of the frangible linker is slower than the rate at which the compound reacts with leukocyte nucleic acid.
  • the compound After a sufficient amount of time has passed, the compound has broken down into the anchor (which may also bear fragments of the frangible linker) and the effector- nucleic acid breakdown products (where fragments of the frangible linker may also remain attached to the effector), or into the anchor (which may also bear fragments of the frangible linker) and the hydrolyzed effector breakdown products (where fragments of the frangible linker may also remain attached to the effector). Additional fragments of the frangible linker may also be generated upon degradation of the compound which do not remain bonded to either the anchor or the effector.
  • the exact embodiment of the alkylator compound of the invention determines whether the anchor breakdown product or the effector breakdown product bears fragments of the frangible linker, or whether additional fragments of the frangible linker are generated which do not remain bonded to either the anchor or the effector breakdown products.
  • Preferred alkylator compounds are compounds which, upon cleavage of the frangible linker, result in breakdown products of low mutagenicity. Mutagenicity of the compounds, after hydrolysis of the effector, is due primarily to the anchor moiety, as the anchor interacts with nucleic acid and may have the potential to interfere with nucleic acid replication, even if the effector moiety has been hydrolyzed. After cleavage of the frangible linker, the anchor fragment has substantially reduced mutagenicity.
  • the compound capable of forming a covalent bond with nucleic acid is removed from the reaction mixture after a certain period of time.
  • the optimal reaction time can be determined empirically, as described infra in the Examples. Procedures and compositions for removal of compounds from the reaction mixture are provided in co-owned U.S. Patent Applications Serial Nos. 08/779,830;
  • anchor groups are available for use as the anchors, linkers, and effectors.
  • anchor groups which can be used in the alkylator compound include, but are not limited to, intercalators (including aromatic intercalators), minor groove binders, major groove binders, molecules which bind by electrostatic interactions or hydrophobic interactions, and molecules which bind by sequence specific interactions.
  • intercalators including aromatic intercalators
  • minor groove binders including aromatic intercalators
  • major groove binders molecules which bind by electrostatic interactions or hydrophobic interactions
  • sequence specific interactions molecules which bind by sequence specific interactions.
  • acridines and acridine derivatives, e.g.
  • fluorenones fluorenodiamines
  • phenazines phenanthridines, phenothiazines (e.g., chlorpromazine), phenoxazines, benzothiazoles, xanthenes and thioxanthenes, anthraquinones, anthrapyrazoles, benzothiopyranoindoles, 3,4-benzopyrene, 1-pyrenyloxirane, benzanthracenes, benzodipyrones, quinolines (e.g., chloroquine, quinine, phenylquinoline carboxamides), furocoumarins (e.g., psoralens and isopsoralens), ethidium, propidium, coralyne, and polycyclic aromatic hydrocarbons and their oxirane derivatives; distamycin, netropsin, other lexitropsins, Hoechst 33258 and other Hoechst dyes,
  • DAPI 4,6-diamidino-2-phenylindole
  • berenil and triarylmethane dyes
  • aflatoxins spermine, spermidine, and other polyamines
  • nucleic acids or analogs which bind by sequence specific interactions such as triple helix formation, D-loop formation, and direct base pairing to single stranded targets.
  • linkers which can be used in the invention are, but are not limited to, compounds which include functional groups such as ester (where the carbonyl carbon of the ester is between the anchor and the sp oxygen of the ester; this arrangement is also called “forward ester"), "reverse ester” (where the sp oxygen of the ester is between the anchor and the carbonyl carbon of the ester), thioester (where the carbonyl carbon of the thioester is between the anchor and the sulfur of the thioester, also called “forward thioester”), reverse thioester (where the sulfur of the thioester is between the anchor and the carbonyl carbon of the thioester, also called “reverse thioester”), forward and reverse
  • forward orientation is that orientation of the functional groups wherein, after hydrolysis of the functional group, the resulting acidic function is covalently linked to the anchor moiety and the resulting alcohol or thiol function is covalently linked to the effector moiety.
  • the reverse orientation is that orientation of the functional groups wherein, after hydrolysis of the functional group, the resulting acidic function is covalently linked to the effector moiety and the resulting alcohol or thiol function is covalently linked to the anchor moiety.
  • effectors which can be used in the invention are, but are not limited to, mustard groups, mustard group equivalents, epoxides, aldehydes, formaldehyde synthons, and other alkylating and cross-linking agents.
  • Mustard groups are defined as including mono or bis haloethylamine groups, and mono haloethylsulfide groups.
  • Mustard group equivalents are defined by groups that react by a mechanism similar to the mustards (that is, by forming an aziridinium intermediate, or by having or by forming an aziridine ring, which can react with a nucleophile), such as mono or bis mesylethylamine groups, mono mesylethylsulfide groups, mono or bis tosylethylamine groups, and mono tosylethylsulfide groups.
  • Formaldehyde synthons are defined as any compound that breaks down to formaldehyde in aqueous solution, including hydroxymethylamines such as hydroxymethylglycine. Examples of formaldehyde synthons are given in U.S. Pat. No. 4,337,269 and in International Patent Application WO 97/02028.
  • Alkylator compounds that can be used to prepare the leukocytes of this invention are described by the following general formulas I, II, and III.
  • General formula I is:
  • V is independently -R ⁇ -, -NH-Ri i- or -N(CH 3 )-R! i-, where -Rn- is independently
  • X is independently -Rn-;
  • E is independently selected from the group consisting of -N(R 12 ) 2 , -N(R ⁇ 2 )(R ⁇ 3 ), -S-R 12 , and
  • R 20 is -H or -CH ; and R 2 ⁇ is -R ⁇ -W-X-E, where -Rn- is independently -C ⁇ -8 alkyl-, -C 1- heteroalkyl-, -aryl-, -heteroaryl-, -C ⁇ -3 alkyl-aryl-, -C 1-3 heteroalkyl-aryl-, -C ⁇ -3 alkyl-heteroaryl-, -C] -3 heteroalkyl-heteroaryl-, -aryl-C ⁇ -3 alkyl-, -aryl-C ⁇ -3 heteroalkyl-, -heteroaryl-C 1-3 alkyl-, -heteroaryl-C] -3 heteroalkyl-, alkyl-, -C 1-3 heteroalkyl-aryl-C ⁇ -3 alkyl-, alkyl-, -C ⁇ -3 alkyl-aryl-C ⁇ -3 heteroalkyl-,
  • X is independently -Rn-;
  • E is independently selected from the group consisting of -N(R !2 ) 2 , -N(R ⁇ 2 )(Ri 3 ), -S-R 12 , and
  • R 4 , R 55 , R 3 , R4, R 5 , and R 8 is -V-W-X-E, and the remainder of R , R 55 , R 3 , R ⁇ R 5 , and R 8 are independently selected from the group consisting of -H,
  • V is independently -Rn-, -NH-Rj ⁇ - or -N(CH3)-Rn-, where -Ri 1- is independently
  • X is independently -Rn-; and E is independently selected from the group consisting of -N(R 12 ) 2 , -N(Ri2)(Ri3),
  • -R 12 is -CH 2 CH 2 -G, where each G is independently -CI, -Br, -I,
  • R 13 is independently -C1-8 alkyl, -C ⁇ -8 heteroalkyl, -aryl, -heteroaryl, -C ⁇ - alkyl-aryl, -Cj_ 3 heteroalkyl-aryl, -C ⁇ .
  • the acridine nucleus is the anchor moiety
  • the -V-W-X- group(s) comprises the frangible linker
  • the E group(s) is the effector group
  • the psoralen nucleus is the anchor moiety
  • the -V-W-X- group(s) comprises the frangible linker
  • the E group(s) is the effector group.
  • General formula II is a subset of general formula I.
  • Examples 7-12 illustrate the synthesis of these compounds to produce the leukocytes of the invention.
  • S-59 4'-(4-amino-2-oxa) butyl-4,5',8- trimethylpsoralen
  • Photoactivatable compounds suitable for use in the methods of this invention include: Furocoumarins; Actinomycins; Anthracyclinones; Anthramycins; Benzodipyrones; Fluorenes and Fluorenones; Monostral Fats Blue; Norphillin A; Organic Dyes; Phenanthridines; Phenazathionium Salts; Phenazines; Phenothiazines; Phenylazides; Quinolines and Thiaxanthenones.
  • the preferred species of photoactivatable compounds described herein is commonly referred to as the furocoumarins.
  • Psoralens are planar, aromatic organic compounds belonging to the group of furocoumarins. Psoralens are found in nature, principally in plants, including limes, cloves, celery, parsnips and figs. In humans, psoralens have been used in photochemotherapy for the management of vitiligo, psoriasis and mycosis fungoides. For a review of psoralen photochemistry, see Parsons, B.J. Photochem. Photobiol. 32:813-821, 1980. The basic structure of psoralen is shown below.
  • the present invention contemplates psoralens, [7H-furo(3,2-g)-(l)- benzopyran-7-one, or b-lactone of 6-hydroxy-5-benzofuranacrylic acid], which are linear:
  • Psoralen derivatives are derived from substitution of the linear furocoumarin at the 3, 4, 5, 8, 4', or 5' positions.
  • psoralen is already present in our diets. Psoralens are relatively inactive in the absence of long- wavelength UVA radiation. The effective half-life of UVA-activated psoralen is measured in milliseconds. Any drug remaining after radiation returns to the inactive state, thus limiting side effects.
  • 8-Methoxypsoralen (known in the literature under various named, e.g., xanthotoxin, methoxsalen, 8-MOP) is a naturally occurring psoralen with low mutagenicity in the Ames assay.
  • AMT 4'-Aminomethyl-4,5',8-trimethylpsoralen
  • 4'-primaryamino-substituted psoralens are defined as psoralen compounds which have an NH 2 group linked to the 4'-position of the psoralen by a hydrocarbon chain having a total length of 2 to 20 carbons, where 0 to 6 of those carbons are independently replaced by NH or O, and each point of replacement is separated from each other point of replacement by at least two carbons, and is separated from the psoralen by at least one carbon.
  • 5'-primaryamino-substituted psoralens also referred to herein by the acronym “5-PAP” are defined as psoralen compounds which have an NH 2 group linked to the 5'- position of the psoralen by a hydrocarbon chain having a total length of 1 to 20 carbons, where 0 to 6 of those carbons are independently replaced by NH or O, and each point of replacement is separated from each other point of replacement by at least two carbons, and is separated from the psoralen by at least one carbon.
  • 4-PAP and 5-PAP as well as their methods of synthesis are disclosed in U.S. Patents Nos. 5,585,503; 5,578,736; 5,556,993; and 5,399,719.
  • Preferred psoralens include 5'-primaryamino-substituted psoralens and 4'-primaryamino-substituted psoralens (collectively referred to herein as PAP), 8-methoxy psoralen (8-MOP), 4'-aminomethyl 4,5',8-trimethylpsoralen (AMT), 5-methoxy psoralen (5-MOP), and trioxalen 4, 5' 8-trimethylpsoralen.
  • AMT, 5-MOP, 8-MOP, and trioxalen are commercially available.
  • the most preferred psoralen is S-59 which has the formula shown above (see under Abbreviations, S-59).
  • S-59 is a synthetic psoralen that can bind reversibly to nucleic acids by intercalation. Upon illumination with UVA, intercalated S-59 forms monoadducts and interstrand crosslinks with RNA and DNA. S-59 photochemical treatment is nucleic acid specific and S-59 readily penetrates cellular and nuclear membranes. S-59 combines a higher water solubility and nucleic acid binding affinity, with a high quantum yield for the formation of nucleic acid adducts. This allows the use of lower psoralen concentrations for PCT for inhibiting proliferation in the absence of nonspecific cellular damage.
  • a cell population comprising a population of leukocytes is treated with a photoactivatable compound including a moiety capable of forming a covalent bond with a nucleic acid upon photoactivation.
  • the photoactivatable compound must be activated by light.
  • Light encompasses ultraviolet light (UVA, UVB, UVC) and visible light.
  • wavelengths used for PCT range from 200-450 nm. In a preferred embodiment, the light used for PCT is in the wavelength of 320-400 nm.
  • the cell population is photochemically treated with UV light having a wavelength in the range of 320 to 400 nm. UVA radiation alone causes minimal damage to cells.
  • the photoactivatable compound comprises a psoralen molecule.
  • PCT with the appropriate concentration of psoralen and dose of UVA light will result in proliferation inhibition with the retention of leukocyte immunogenic functions effective, for example, to promote destruction of a diseased cell, an infected cell, or a pathogen, induce an antileukemic effect, facilitate engraftment by a second population of cells, promote immune reconstitution, facilitate immunotherapy, and treat mixed chimerism.
  • Psoralen reactivity is not only specifically directed towards the nucleic acids versus the proteins in a cell, but there is also a differential binding affinity of psoralens for DNA vs RNA molecules due to the difference in nucleic acid structure that allows better intercalation in DNA (Cimino, G.D. et al, Ann. Rev. Biochem. 54:1151-1193, 1985). Additionally, using this technology, it may be possible to differentially modify transcriptionally active (vs. inactive) genes, due to their different conformation and state of histone binding which will affect psoralen binding.
  • PCT cancer-derived neurotrophic factor
  • Treatments such as PCT therefore have the ability to interfere with cellular function, primarily at the level of DNA synthesis (replication), with a lesser effect on transcription, and minimal or no effect on protein synthesis.
  • the level of interference will be determined by the concentration of the psoralen and dose of UVA light used.
  • a photoactivation device suitable for the present methods of PCT comprises the following: (a) a means for providing appropriate wavelengths of electromagnetic radiation to cause photoactivation of a photoactivatable compound; (b) means for supporting a plurality of samples in a fixed relationship with the radiation providing means during photoactivation; and (c) means for maintaining the temperature of the samples within a desired temperature range during photoactivation.
  • the photoactivation device is capable of emitting a given intensity of a spectrum of electromagnetic radiation comprising wavelengths between 200 and 450 nm, preferably between 320 nm and 400 nm. Particular wavelengths can be selected using appropriate filters within the photoactivation device.
  • a suitable photoactivation device as well as methods of photoactivating photoactivatable compounds by the use of the device are disclosed in U.S. Patent Nos. 5,184,020 and 5,503,721, both to Hearst et al. and in WO 96/39820.
  • Other photoactivation devices that may be used include General Electric type F20TI2-BLB fluorescent UVA bulbs (Alter, H. J., et al., The Lancet, 24:1446 (1988), and Type A405-TLGW/05 long wavelength ultraviolet lamp manufactured by P. W. Allen Co., London.
  • Stock solutions of psoralens are prepared in water or in the appropriate solvent as exemplified below in the Examples.
  • the stock psoralen solutions are added to suspensions of purified donor leukocyte populations to the desired final concentration. Units that were treated with 8-MOP were prepared by using PAS saturated with 8-MOP in the preparation procedure discussed above.
  • the purified donor leukocytes are illuminated with UVA light to a final dose of between 10 " to 100 J/cm in a photoactivation device. The samples are agitated during illumination. Samples of the leukocytes are taken prior to illumination as controls.
  • the PAP is added to the purified leukocyte preparation at a concentration of between 10 "4 and 150 ⁇ M, preferably between 10 "3 and 150 ⁇ M.
  • the leukocytes will generally be at a cell density of between about 10 to 10 9 cells/ml, preferably between about
  • PCT is performed on leukocytes combined with S-59 using ultraviolet light of wavelength of between 320-400 nm. Restricting the photoactivation process to wavelengths greater than 320 nm minimizes direct nucleic acid damage since there is very little absorption by nucleic acids above 313 nm.
  • the cells will preferably receive a light dose in the range of about 10 "3 to 100 J/cm 2 , more preferably 1 to
  • the light dose is at 3 J/cm 2 .
  • Illumination will generally be at a light intensity of between 1 to 50 mW/cm .
  • the time period for exposure of the leukocyte sample to UV light will be between about 1 second to 60 minutes, preferably about 3 minutes, more preferably about one minute.
  • the leukocyte samples will be assayed for effectiveness to proliferate and to participate in leukocyte activity, as described above and in the Examples.
  • cell viability cell viability
  • proliferation activity cell proliferation activity
  • cytokine secretion cell proliferation activity
  • surface antigen expression cell viability
  • the treated leukocyte populations of the present invention which are non- proliferating but retain immunological activity, have various prophylactic and therapeutic uses as described below.
  • individuals receiving DLIs containing treated leukocyte populations will include but are not limited to, transplant patients such as post BM transplant patients or patients anticipating a solid organ transplant.
  • transplant patients such as post BM transplant patients or patients anticipating a solid organ transplant.
  • the fact that the treated leukocytes do not elicit GVHD enables greater numbers of leukocytes (i.e. larger cell doses) to be administered in the DLI, thus maximizing the efficacy of treatment.
  • the treated leukocytes are useful for donor leukocyte infusion (DLI) especially to provide immunocompetence to allo-BMT patients, or immunocompromised patients such as elderly patients suffering from CLL.
  • DLI donor leukocyte infusion
  • DLI using treated leukocytes are useful to alleviate various conditions including the following: Leukemia including Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic leukemia (CLL), Chronic myelomonocytic Leukemia (CmML), Acute Myelogenous
  • AML Acute Lymphoblastic Leukemia
  • ALL Acute Lymphoblastic Leukemia
  • MM Multiple Myeloma
  • Non-Hodgkin's lymphoma and Hodgkin's lymphoma Large Cell Lymphoma (LCL); and Epstein-Barr virus induced B lymphoproliferative disorder (EBV-BLPD or EBV-induced lymphoma);
  • Solid tumors including breast, lung, ovarian, testicular, prostate and colon cancer, melanoma, renal cell carcinoma, neuroblastoma, head and neck tumors;
  • Aplastic anemia e.g. Myelodysplasia
  • Immunodeficiencies e.g. Common variable, SCID, Wiskott-Aldrich syndrome,
  • DLIs are useful in various formats for prophylactic or therapeutic purposes: (a) adoptive immunotherapy for all allogeneic transplants;
  • the BMT light protocol is a two step process which involves: i) induction of transplantation tolerance using a minimum conditioning regimen and donor stem cells; and ii) immunotherapy with DLI repeatedly. This protocol circumvents the toxicity of chemotherapy regimens.
  • the BMT light protocol is suitable in the following indications: for patients who are not normally eligible for transplants, particularly elderly patients (> 65 years old); and for CLL patients, most of whom are severely immunocompromised. BMT light regimen reduces toxicity, GVHD, and infection.
  • An exemplary BMT light protocol is described in Example 13 below.
  • DLI provided concurrently with CD34+ selected cells are useful to confer adoptive immunotherapy against infections, such as by CMV, Epstein Barr virus (EBV), Adenovirus (Ad), Kaposi's Sarcoma associated
  • PA-DLI is administered in haploidentical (haplo) transplantation.
  • haplo transplantation requires almost complete T cell depletion, leading to severe, sustained immunosuppression.
  • PA-DLI would be useful to maintain immunocompetence in the host; additionally, a GVL effect may be provided for the treatment of cancer.
  • the leukocytes of the present invention that are incompetent in GVHD but retain some immune function will be used in DLI for the alleviation of relapsing cancer or removal of minimal residual disease following allogeneic BMT, particularly in CML and acute leukemias, lymphomas and multiple myelomas.
  • the high number of donor CTLs specific to the bcr-abl protein of CML contributes to the efficacy of DLI in relapsing CML.
  • a cancer patient is determined to be suffering a "relapse' if malignant cells are detected, by means routinely used by physicians to monitor the presence of cancerous cells.
  • DLI containing the non-proliferating leukocytes is used in the treatment of leukemia patients during induction chemotherapy (initial round of chemotherapy) to avoid myeloablative regimens and to remove minimal residual disease.
  • induction chemotherapy initial round of chemotherapy
  • the toxic dose of chemotherapeutic drug given during induction could be reduced.
  • PA-DLI can also be applied in solid organ transplants (e.g., kidney, heart, skin) as a means to increase the lifetime of allografts and the chances of donor organ acceptance.
  • solid organ transplants e.g., kidney, heart, skin
  • Most of the organ rejections in the allogeneic transplant occur due to the presence of passenger leukocytes in the donor organ.
  • the DLI using leukocytes from the organ donor
  • Tolerization of the host for solid organ transplant would consist of the following: before or during organ transplant procedure, infusion of PA-DLI with or without donor stem cells with some level of myeloablative regimen to allow donor cells to survive in circulation. DLI may facilitate engraftment of donor stem cells in this setting, allowing for mixed chimerism in the host.
  • treated leukocytes can be administered to the host before, at the same time as, or after the transplant or graft. Multiple infusions of treated cell populations can be made at any of these times.
  • the non-proliferating, immune functional leukocytes are especially invaluable for DLI in patients for whom traditional allogeneic BMT would be contraindicated, because of advanced age or comorbid disease.
  • Elderly patients, (> 65 years old) presenting cancer are not prime candidates for allo BMT partly because the transplant cannot reconstitute an aged immune system very well and because of the severity of the procedure.
  • the only option available to control cancer in elderly patients is chemotherapy, the side effects of which can be intolerable for these patients.
  • the treated leukocytes of the present invention provide a much needed alternative to treating cancer in elderly patients and to providing immune defense against opportunistic infections. DLI will be useful to destroy cancerous cells without attendant GVHD.
  • the donor leukocytes In certain cases of treatment, it may be desirable to expand subpopulations of the donor leukocytes, particularly antigen-specific T cells and precursor cells, before subjecting the donor leukocyte population to treatment with the above-described compounds.
  • the leukocyte population can be stimulated with an antigen specific to the diseased cell (e.g., tumor-associated antigens) or pathogen to expand/enrich for the number of cytotoxic and helper T cells specific to the antigen.
  • the stimulation can be performed in vivo by vaccination of the donor with the appropriate antigen prior to isolation of the leukocytes from the stimulated donor; alternatively stimulation of isolated leukocytes can be carried out in vitro prior to treatment of the leukocyte population with the compound.
  • MM myeloma-specific precursor CTLs.
  • MM myeloma-specific precursor CTLs.
  • Pulsing dendritic cells with tumor-associated peptides for immunization of patients with non-Hodgkin's lymphoma is described in Hsu, FJ et al. Nat. Med. 2:52-58, 1996. Techniques of pulsing dendritic cells with peptide/antigens are reviewed in Stingl, G. et al.
  • cDNA immunization transfecting the dendritic cell with the appropriate cDNA
  • cDNA immunization is described in Pardoll, D. et al. Immunity 3:165-169, 1995.
  • donors can be vaccinated with bcr- abl peptides before isolation of donor leukocytes for treatment by the method of the invention and subsequent infusion.
  • Effective and immunogenic peptides for vaccination are described, e.g., by ten Bosch, G. et al. Blood 88:3522-3527, 1996.
  • donor leukocytes for the treatment of prostate cancer are pre-stimulated with prostate specific antigen (PSA) either in vivo or in vitro, before treatment according to the method of the invention.
  • PSA prostate specific antigen
  • Such vaccinations can be applied to boost precursor T cell populations and/or populations of T cells specific to other tumor specific antigens, viral antigens and antigens of other pathogens.
  • Leukocytes from donors vaccinated with the appropriate antigens are useful prophylactically or therapeutically, for providing an immunocompromised patient with immune defense against opportunistic infections such as cytomegalovirus (CMV), Epstein Barr virus (EBV), Kaposi's Sarcoma associated Herpes virus or adenovirus (Ad) infections.
  • CMV cytomegalovirus
  • EBV Epstein Barr virus
  • Ad Kaposi's Sarcoma associated Herpes virus
  • Ad adenovirus
  • donors can be vaccinated against CMV antigens and their leukocytes isolated and subjected to treatment according to the methods of the invention for use in DLI to treat patients presenting CMV infections as a result of immunosuppression.
  • Immunocompromised patients include patients under immunosuppressive drug treatment such as organ transplant patients (BM and other organs), HIV or EBV infected individuals, cancer patients, and individuals with immune deficiency diseases.
  • Donor leukocytes can also be stimulated in vitro (also referred to as ex vivo expansion) to expand precursor pools.
  • donor leukocytes can be stimulated by in vitro culture with patient specific MM antigen presented by dendritic cells, before treatment to block GVHD activity.
  • Ex vivo or in vitro stimulation can also be employed to expand the population of antileukemic T cells for the treatment of CML.
  • CML the classical translocation of the c-abl oncogene on chromosome 9 to the break point cluster region (bcr) on chromosome 22 (t(9;22)(q34;ql 1) results in a bcr-abl fusion gene and the expression of the bcr-abl 210 kD fusion oncoproteins.
  • the bcr-abl fusion protein is CML specific and the two main variants of the bcr-abl protein are well characterized antigens.
  • In vitro stimulation to expand T cells specific for the bcr-abl protein can be performed as described, e.g., by
  • dendritic cells can be generated in vitro from peripheral blood cells of CML patients and used as antigen-presenting cells for the ex vivo expansion of antileukemic T cells (Choudhury et al, 1997, supra).
  • donor antigen-presenting cells APCs
  • APCs can be pulsed with antigen characteristic of a diseased cell, or can be co-cultured with diseased cells (e.g., inactivated tumor cells); and these APCs can then be exposed to donor leukocytes prior to infusion of the donor leukocytes into a recipient.
  • direct contact between the donor leukocyte and a diseased cell (or a diseased cell antigen) can be used to expand the T-cell population in the donor lymphocytes.
  • the "antigen" used to expand the T cells can be a polypeptide, a lipid or a carbohydrate moiety (or any combination, e.g., a glycoprotein), and can be isolated from the diseased cell or pathogen or recombinantly produced.
  • a polypeptide-containing antigen need not constitute a full-length protein, as long as it includes at least one epitope.
  • the antigen can be contained in a vaccine composition as a full length protein or fragment thereof or as a recombinantly produced fusion protein, and may or may not be conjugated or otherwise presented with an adjuvant.
  • the vaccine can take various forms: diseased cell expressing the antigen, or cell membrane preparation thereof; or the pathogen, preferably in inactivated form. Methods of preparing vaccines are taught in the literature, see, e.g.,
  • the leukocytes will generally be administered in plasma, synthetic media or other physiologically buffered solution at a dosage of about 10 5 to 10 11 leukocytes per kg weight per infusion, in a volume of about 50 to 500 ml or more. These parameters will vary with the treatment and disease and will be decided by the physicians treating the patient.
  • the standard DLI practice is to administer leukocytes at a dose of 10 7 to 10 8 cells per kg of body weight. DLI can be administered in conjunction with chemotherapy.
  • the leukocytes of the present invention will generally be administered intravenously.
  • GVHD is usually apparent within 90 days after the BMT.
  • DLI is performed preferably within 1 week to 3 months after diagnosis of relapse.
  • DLI can be delivered before to several years after BMT, as maintenance therapy.
  • the DLI is preferably administered soon after diagnosis of the cancer.
  • DLI can be provided before, during or after chemo and irradiation regimens or other therapeutic regimens. In all indications, the appropriate timing of the DLI will be determined by the physicians of skill in the treatment of the disease.
  • GVHD GVHD is usually apparent within 90 days post infusion.
  • Clinical GVHD symptoms include skin rash, swelling and lesions in the liver, gut, lungs and joints, severe diarrhea and jaundice.
  • the level of the patient's bilirubin and liver function enzymes can be measured and a liver biopsy can also be done.
  • Patients receiving DLI can be monitored regularly for detailed clinical history, physical examination, general laboratory evaluation including complete blood count and differential, urinalysis, blood urea nitrogen creatinine, bilirubin, aspartate transaminase (AST), alanine transferase (ALT), alkaline phosphatase, Na + , K + , CI " , albumin, total protein, glucose, and radiographic examination.
  • the status of the cancer can be assessed for example, by examination of marrow aspirates and biopsy, cytogenetic examination (including immunohistochemical analysis), and molecular analysis.
  • a recipient of the present treated leukocytes is alleviated of the disease (disease refers to malignancy or infection) if there is visible or measurable improvement in the symptoms of or resulting from the disease.
  • the symptoms and methods of assessing improvement in them will vary with the disease condition but will be familiar to the clinician.
  • One useful endpoint for evaluating treatment success is
  • the methods and compositions of the invention are also useful for prevention of GVHD is situations other than DLI. These include, but are not limited to, platelet transfusions and febrile, non-hemolytic transfusion reactions resulting from cytokine accumulation during platelet storage.
  • the present methods and the treated, non-proliferating leukocytes of the present invention also have in vitro uses.
  • the PCT procedure for example, is a minimally invasive method of inhibiting DNA synthesis without affecting the biosynthesis of cells in primary cultures or cell lines which have the ability to differentiate, such as progenitor cells and stem cells.
  • treatment conditions can be used to prepare isolated stem cells, leukocytes or other cell lines in an assay to test whether a cell differentiation or development step is dependent on proliferation/DNA synthesis.
  • Such studies may identify target molecules along the differentiation or development pathway and facilitate development of drugs for either inhibiting or promoting differentiation.
  • Treated leukocytes can also be used, e.g., as stimulator cells in a one way MLR assay, to stimulate immune responses of proliferation competent allogeneic cells without the simultaneous proliferation of the stimulator cells themselves.
  • PCT and related treatments using compounds capable of forming a covalent bond with DNA, would replace the current use of irradiation or mitomycin C to achieve cytostatis of the stimulator cells.
  • compositions of treated leukocytes or other cells can be provided either alone or as part of an assay kit.
  • the assay kit can be for MLR or for the differentiation assay.
  • the treated cells will generally be provided in frozen form.
  • kits may provide reagents and instructions for preparation of leukocytes or other cells having the above-described characteristics.
  • the reagents will include one or more compounds capable of forming a covalent bond with DNA.
  • a kit to be used for PCT will include one or more photoactivatable compounds, preferably a PAP or an acridine, more preferably, S-59.
  • test compound e.g. DNA replication inhibitor or mitogen
  • Media is prepared that typically contains 1 ⁇ Ci/50 ⁇ l/well using 3 H thymidine of approximately 20 Ci/mmol specific activity.
  • This media/label is added to the cells and the cells are incubated at 37°C for approximately 4-6 hours or longer.
  • a multichannel cell harvester is used to transfer cells to paper filter discs and to wash away free (unincorporated) label. The discs are then dried, transferred to vials and immersed in scintillation fluid. The vials can then be placed in an automated counter, counting for the full tritium range, and the average counts per minute (cpm) for triplicate samples is determined.
  • 3 H-labeled PAP was used to measure the extent of DNA modification.
  • PAP was added to the desired final concentrations in the purified leukocyte samples. Aliquots of 20 mL in mini-PL2410 plastic containers were illuminated with the appropriate UV light dose. The control sample was treated with PAP alone without UVA. After illumination, leukocyte DNA was purified. The DNA content of each sample was determined by measurement of absorbance at 260 nm. The number of psoralen adducts per 1,000 base pairs (bp) was calculated from the radioactivity in the DNA samples. A standard curve correlates the amount of 3 H counts with the number of adducts.
  • PCR Polymerase chain reaction
  • DNA samples (1 ⁇ g) obtained from photochemically-treated or gamma irradiated platelet concentrates were PCR amplified for a 242 bp sequence in the HLA-DQ ⁇ locus or for a 439 bp sequence in the ⁇ -globin gene locus.
  • Control (untreated) DNA was serially diluted (1 :10) and then amplified. The treated DNA was amplified without dilution. PCR amplification was carried out to 35 cycles. This assay provided a means to correlate functional T-cell proliferation inhibition, as measured in a cell proliferation assay, with direct modification of nucleic acid.
  • Example 1 demonstrated that leukocytes can be inactivated by PCT in a dose dependent manner.
  • exemplary psoralens S-59, AMT and 8-MOP were used.
  • the PCT dose dependence varies with the properties of the psoralen used, S-59 being the most efficient tested.
  • Fig. 1 The dose related effect of photochemical treatment with S-59 (Squares), 4'-aminomethyl 4,5',8-trimethylpsoralen [AMT] (triangles), and 8-methoxypsoralen [8-MOP] (circles) on leukocytes in platelet concentrates (PC) was characterized and the results shown in Fig. 1. Platelet concentrates were prepared and treated as described in U.S. Patent No. 5,593,823, incorporated herein by reference. Leukocytes from photochemically-treated and untreated pooled random donor PC were plated in an LDA assay. Varying concentrations of the three psoralens were used with a constant light dose of 1 Joule/cm 2 UVA.
  • T-cells were inactivated to the limit of detection by LDA (indicated by arrows) with 0.05 ⁇ M S-59, 1.0 ⁇ M AMT, and 10.0 ⁇ M 8-MOP. These data clearly demonstrate that S-59 is a superior photoreagent, compared to AMT and 8-MOP, in inhibiting proliferation of T cells.
  • Peripheral blood was drawn into anticoagulant citrate dextrose (ACD) tubes from four individuals.
  • the peripheral blood mononuclear cells (PBMC's) were isolated by density gradient centrifugation over Ficoll.
  • the PBMC's from three donors were pooled and served as allostimulators.
  • the PBMC's from the remaining individual was used as the responder.
  • the stimulator and responder PBMC's were washed two times with RPMI supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin and resuspended in the supplemented media.
  • FBS fetal bovine serum
  • streptomycin 100 ⁇ g/mL streptomycin
  • the stimulator PBMC's were exposed to 2500 cGy gamma radiation from a blood bank 137 cesium irradiator.
  • the stimulator cells were then plated in 96 well round bottom plates in aliquots of 100 ⁇ L containing 1.0 x 10 5 cells.
  • the responder cells were subjected to PCT with S-59 as follows. Responder cells were resuspended in 30 mL of the above cell culture media and aqueous S-59 stock solution (dissolved in water) was added to the cell suspension to the final concentrations specified in Figure 2, Figure 3 or Figure 4. The concentration of S-59 stock solution and volume used varied depending on the final S-59 concentration desired. The 30 mL cell solution containing S-59 was then transferred to a small PL2410 bag and illuminated with
  • UVA light at a dosage of 3 J/cm .
  • the light dosage was 3.0 J/cm .
  • the cells were then centrifuged, resuspended in cell culture media and plated in aliquots of 100 ⁇ L containing 1.0 x 10 5 cells and mixed with the stimulator cells for a final volume of 200 ⁇ L per well.
  • Example 3 demonstrated that leukocyte activity, as measured by cytokine synthesis and surface marker expression (exemplified by CD69 lymphocyte activation marker expression) can be modulated by the concentration of the psoralen and the light dose used for the PCT.
  • the data provide evidence that UVA light doses and concentrations of photoactivatable compounds can be titered to obtain appropriate ranges effective both to block proliferation of T cells in the leukocyte population, and to maintain leukocyte activity, as demonstrated by cytokine synthesis and surface marker expression.
  • the induction of the CD69 expression by leukocytes upon their activation by PMA and calcium ionophore was measured with the use of a fluorescent antibody to CD69 and FACS-Scan analysis (see Figure 6).
  • the expression of CD69 was measured as a function of time after photochemical treatment with different concentrations of AMT and 1 J/cm 2 UVA.
  • the effect of PCT on the induction of CD69 expression was also found to be psoralen dose dependent, with expression of CD69 showing greater inhibition at increasing concentrations of AMT. At the low doses anticipated for S-59 in this study, induction will remain intact.
  • Example 4 demonstrated that the effect of PCT on the properties of the treated leukocytes correlates with leukocyte DNA modification and inhibition of polymerase activity.
  • Psoralen adducts on leukocyte genomic DNA were measured after photochemical treatment in pooled random donor PC by using H radiolabeled S-59, AMT, and 8-MOP plus 1.9 J/cm 2 UVA and scintillation counting of the isolated DNA of treated leukocytes ( Figure 7). Photochemical treatment with 150 ⁇ M S-59, AMT, and 8-MOP induced 12.0, 6.0, and 0.7 adducts/1000 bp of DNA respectively.
  • the following murine transfusion model is a well characterized model that simulates the human clinical syndrome of TA-GVHD (Fast, LD et al. Blood 82: 292, 1993). Based on the promising in vitro results, the effect of S-59 photochemical treatment on the inhibition of TA-GVHD was evaluated, using this in vivo model, by transfusing splenocytes from a homozygous parent (strain A, H-2 a ) into immunocompetent heterozygous Fj hybrid recipients (strain B6AF 1? H-2 a/b ).
  • Each recipient (F was transfused with approximately 10 8 donor (A or Fj) splenocytes via the lateral tail vein.
  • Splenocytes obtained from density gradient centrifugation were used as the source of viable T cells.
  • Three transfusion groups were utilized: (1) handling control group (F ⁇ -»F
  • Recipients were monitored for biological evidence of TA-GVHD two weeks post- transfusion.
  • Spleens of recipients were analyzed for donor T-cell engraftment using two- color, fluorescence-activated flow cytometry.
  • splenic T-cells were stained with a pan-T-cell, anti-CD3 antibody and also with an anti-H-2 b antibody.
  • Donor A T-cells were detected by the absence of reaction with the anti H-2 b antibody.
  • recipients were labeled with the anti-H-2 b antibody.
  • cytotoxic lymphocytes CTLs
  • H-2 b 51 Cr-labeled EL-4 cells
  • the cytotoxic activity of CTLs was determined by the target cell lysis and release of 51 Cr into the culture medium. The results indicated that while recipients in the control F
  • TA-GVHD was characterized by splenomegaly, donor T-cell (H-2 a ) engraftment (28.6 ⁇ 11.3%) and the presence of CTLs (35 ⁇ 18.7% 51 Cr lysis) in recipient spleens (Table 1).
  • Splenomegaly was evaluated two weeks post-transfusion by measuring the spleen: body weight ratio. Development of immunodeficiency was detected by evaluation of thymic cellularity 3 weeks post-transfusion. Thymic hypoplasia is a reliable index of acquired immune deficiency associated with TA-GVHD. Recipients were also monitored for clinical symptoms of GVHD which included body weight, posture, activity, skin integrity, fur texture, white blood cell count, red blood cell count, and platelet count.
  • Tissue sections were prepared and examined for histologic abnormality after blind coding.
  • the liver was evaluated for the presence of lymphoid infiltrates with bile duct destruction and vascular endothelial inflammation and infiltration.
  • Splenic histology was assessed for preservation or destruction of lymphoid follicles.
  • Skin sections were evaluated for lymphoid infiltrates in sub-dermal areas and appendages.
  • Bone marrow sections were evaluated for cellularity and maturation within each major lineage: myeloid, erythroid, and megakaryocytic.
  • A- F ⁇ mice developed histologic evidence of GVHD in liver, spleen, bone marrow, and oral mucosa in a blinded study.
  • PCT- A photochemically treated donor cells
  • Fj syngeneic cells
  • Example 6 This example describes the approach for determining the PCT conditions suitable to suppress GVHD while preserving the GVL effect of DLIs for application, e.g., in the treatment of relapsing leukemia patients post-allogeneic BMT.
  • the experimental objectives are summarized below.
  • Stock solutions of psoralen are prepared as exemplified below.
  • a 15 mM stock solution of AMT was prepared by dissolving 50 mg of AMT powder in 10 mL of distilled water. The solution was mixed vigorously and filtered through a 0.2 ⁇ m syringe filter.
  • the concentration of AMT in the filtered solution was determined by measuring the absorbance of the solution at 250 nm using a Shimadzu UVI60U spectrophotometer.
  • AMT concentration was calculated using a value of 25000 M " 'cm " for the extinction coefficient.
  • a stock solution of S-59 psoralen was prepared by dissolving a powder of S-59 in distilled water. The solution was mixed vigorously and filtered through a 0.2 ⁇ m syringe filter. The concentration of S-59 in the filtered solution was determined by measuring the absorbance of the solution at 250 nm using a Shimadzu UV160U spectrophotometer. The
  • S-59 concentration was calculated using a value of 25400 M “ 'cm " for the extinction coefficient.
  • the stock psoralen solutions are added to suspensions of purified donor leukocyte populations to the desired final concentration.
  • Units that were treated with 8 -MOP were prepared by using PAS saturated with 8-MOP in the preparation procedure discussed above.
  • the purified donor leukocytes are illuminated with UVA light to a final dose of between 10 " to 100 J/cm in a photoactivation device.
  • the units were agitated at 70 cycles/min during illumination. Samples of the leukocytes are taken prior to illumination for use as controls.
  • the S-59 is added to the purified leukocyte preparation at a concentration of
  • the leukocytes will be provided at a cell density of 10 to 10 cells/mL, preferably at 10 7 cells/mL, more preferably at 2 x 10 6 cells/mL.
  • the sample of a leukocyte population mixed with S-59 is exposed to ultraviolet light of wavelength of between 200 and 450 nm, preferably 320-400 nm.
  • the cells will preferably receive a light dose in the range of about 10 "3 to 100 J/cm 2 . In a preferred embodiment, the light dose is at 3 J/cm .
  • the time period for UV light exposure will be about 1 second to 60 minutes, preferably about 1 minute.
  • the leukocyte samples will be assayed for the ability to proliferate and to participate in leukocyte activity, as described above and in the Examples. The following will be measured: cell viability and integrity; proliferation activity; surface antigen expression; cytokine secretion; GVHD and GVL activity.
  • the following assays can be applied to both alkylator compound-treated or photochemical treated leukocytes.
  • This assay directly measures the number of clonable T cells, which correlates directly in vivo with the incidence and severity of GVHD (Kernan, N.A. et al, Blood 68:770-773, 1986). LDA was used by the FDA to set the current guideline for gamma irradiation of blood products. This assay is conducted according to the procedure of
  • human leukocytes are assayed for the number of proliferating cells after PCT treatment with different concentrations of drug and a constant dose of light.
  • Lethally ⁇ -irradiated pooled allostimulator cells are used to stimulate growth.
  • Controls for proliferation include untreated leukocytes (as positive control for proliferation) and pre-PCT leukocytes that have been lethally ⁇ -irradiated
  • a correlation between the dose used and the number of cells that are proliferation-inhibited is determined for human leukocytes.
  • the dose response obtained from this experiment is expected to be similar to the one obtained for the leukocytes in platelet concentrates.
  • LDA was performed as follows. Leukocytes were isolated from peripheral blood by leukophoresis. Viable T-cells were stimulated to proliferate in wells of microtiter plates containing RPMI medium supplemented with fetal bovine serum (FBS), phytohemagglutinin (PHA), recombinant interleukin-2 (rIL-2) and T-cell growth factor (TCGF). Each well also contained 10 5 allostimulator cells prepared from a pool of FBS, phytohemagglutinin (PHA), recombinant interleukin-2 (rIL-2) and T-cell growth factor (TCGF). Each well also contained 10 5 allostimulator cells prepared from a pool of FBS, phytohemagglutinin (PHA), recombinant interleukin-2 (rIL-2) and T-cell growth factor (TCGF). Each well also contained 10 5 allostimulator cells prepared from a pool of FBS, phytohemagglutinin (PHA), recombinant interle
  • PBMCs from ten individuals and irradiated with 5000 cGy of gamma irradiation.
  • Leukocytes from each untreated control sample were diluted in two independent series. Aliquots of 100 ⁇ L containing in one series 10 5 , 10 4 , 10 3 , 10 2 , 10 1 and 10° cells or in a second series 300, 100, 33, 11 and 4 cells were plated per well. Each dilution was plated in ten replicates. An 11 mL aliquot of the photochemically treated sample containing 1.1 x 10 7 total leukocytes was cultured to detect viable T-cells. Aliquots of
  • microtiter plates were incubated in a CO 2 incubator at 37°C for 3 weeks. After
  • each well was fed with additional TCGF, FBS, rIL-2 and PHA. At the end of the 3 -week incubation period, each well was scored for the presence of T-cell clones. Wells with one or more T-cell clones were scored positive.
  • T-cell reduction factor fcontroi/ftreated, where f CO ntroi and f tre ated are T-cell frequencies for the control and treated samples, respectively.
  • Control aliquots were either not treated or were treated with UVA only or S-59 only. One aliquot was irradiated with the clinical dose of 2500 cGy of gamma. To the remaining aliquots, S-59 was added to final concentrations ranging from 10 "4 ⁇ M to 150 ⁇ M. They were then illuminated at a UV light dose ranging from 10 '3 to 100 Joules/cm .
  • This assay also known as the MLC test is conducted according to the procedure of Kraemer, K.H. et al, J. Inv. Derm. 77:235-239, 1981 and Kraemer, K.H. et al, J. Inv. Derm. 76:80-87, 1981.
  • Kraemer K.H. et al, J. Inv. Derm. 77:235-239, 1981
  • Kraemer K.H. et al, J. Inv. Derm. 76:80-87, 1981.
  • the lymphocytes of 2 HLA-disparate individuals When the lymphocytes of 2 HLA-disparate individuals are combined in tissue culture, the cells enlarge, synthesize DNA, and proliferate, whereas HLA-identical cells remain quiescent.
  • the DNA synthesis induced is determined through the incorporation of 3 H-thymidine into the nucleic acid inside the proliferating cells. Cells are then harvested, washed free of unbound radioactivity and counted in a beta counter for internalized radioactivity.
  • the leukocytes are assayed for their responder and stimulator functions in one-way MLR.
  • the standard, appropriate positive and negative controls are included.
  • the responder function is assayed by incubating responder cells with lethally ⁇ -irradiated pooled human leukocytes (stimulator cells).
  • the stimulator function of the treated leukocytes (treated by PCT or other alkylator compounds) is determined, after they have been lethally ⁇ -irradiated to block thymidine uptake, by incubating with normal pooled human leukocytes.
  • the results obtained as a function of treatment dose are compared to a two-way MLR.
  • the MLR will provide a subset of the information provided by LDA, it is able to provide information on the ability of treated leukocytes to stimulate other cells.
  • Stimulation by treated leukocytes in an MLR can be used as a model for the induction of an immune response against leukemia, activated by the DLI in a BMT transfused patient.
  • the immune response against leukemia may be launched by the host or donor cells present in the BMT.
  • Example 14 for characterization of treated human lymphocytes with respect to proliferation, surface marker expression, cytokine synthesis and cytotoxic activity.
  • the cytolytic ability is investigated against a series of human and mouse leukemia cell lines (available from ATCC). The experiment is conducted according to the conditions of Jiang, Y.Z. et al, Bone Marrow Transplant. 8:233-238, 1991. Leukemia cells are labeled under standard conditions with 51 Cr as described in Coligan, J.E. et al, Current Protocols in Immunology: Current Protocols. John Wiley & Sons, Inc., New York, 1991. Any lysis achieved will be measured by the release of 51 Cr radioactivity in the supernatant and compared to controls for background and total lysis. The effect of the treatment dose on the cytolytic ability of treated T-cells is monitored.
  • cytotoxic activity can be determined by measuring cytokine production, e.g., IFN- ⁇ , IL-2 or GM-CSF production.
  • cytokine measurements are conducted through the use of commercially available Elisa kits (R&D Systems) in culture supernatants following co cultivation of effector cells with stimulator cells. Assays are performed according to the instructions provided by the Elisa manufacturer. Results are evaluated by comparing absorbance measurements for each sample to a standard curve generated by cytokine standards supplied with each assay kit. Cytokine measurements are performed on treated leukocytes as a function of the compound concentration and, in the case of PCT, of the light dose, as well as the time interval between treatment and measurement. IL-1 , IL-2,
  • IL-4, IL-10, IFN- ⁇ cytokines are measured because of their established role in T-cell activation and GVHD/GVL.
  • Measurement of cytokine synthesis by treated leukocytes is important in determining the capability of the cells to function as inducers of an immune response through their production; it also serves as a measure of biochemical function and protein synthesis.
  • mice are a well established model for studying graft vs host disease and graft versus leukemia effects. In order to differentially assess the effect of treated leukocytes on
  • mouse splenocytes are subjected to treatment before transfusion to test the inhibition of GVHD by the treatment, as measured by mortality, and also to provide the lowest dose to inhibit the onset of GVHD.
  • Splenocytes are the accepted substitute for blood leukocytes in mice since the volume of blood in mice is too small for such experiments.
  • the recipients are infused with, for example, PA allogeneic splenocytes and then challenged 7 days later with AKR leukemia cells.
  • AKR leukemia cells Infusion of the mice with 5 X 10 4 AKR leukemia cells after recovery from BMT (28 days) is known to result in leukemia and 95% mortality 25 days after the leukemia challenge (Johnson, B.D. et al, Transplantation 54:104-112, 1992).
  • infusion with allogeneic splenocytes 21 days after BMT has been shown to induce a GVL effect upon challenge with AKR cells 7 days later. This course of action results in 70% survival after 70 days.
  • treatment of the splenocytes before transfusion will allow the detection of induction of GVL in the absence of GVHD. This is measured by the survival rate of treated splenocyte-infused BMT recipients, which were challenged with AKR cells.
  • the effect of DLI using treated leukocytes on the survival of a leukemic challenge is tested as a function of the treatment dose and compared with a control group where infusion of untreated splenocytes is performed.
  • the surviving mice are tested for chimerism and leukemic load through the use of a reported PCR assay as described in Johnson, B.D. et al, Bone Marrow Transplant. 11 :329-336, 1993).
  • a leukemic cause of death can be diagnosed by either white cell count or PCR analysis of the blood immediately before death, since leukemia will cause an expansion of the AKR leukemic cells. Primers specific to AKR leukemia cell specific markers will be used for the PCR analysis.
  • GVHD Death from GVHD is diagnosed by low white cell count and spleen atrophy, as well as through characteristic clinical symptoms (fur and posture changes, sticky feces etc.).
  • the efficacy of PA-DLI against minimal residual disease or higher tumor load is tested by injecting smaller or larger numbers of AKR leukemia cells.
  • Treatment of leukocytes according to the invention is deemed to be efficient to induce a GVL effect if, under conditions which do not cause GVHD, it induces a statistically significant increase in the survival of the mice treated with the treated-cell DLI, after a leukemic challenge is administered.
  • the genetic type of the surviving animals should also be completely chimeric as this has been associated in humans with a higher incidence of a disease-free long-term survival. Increase of survival will be easy to demonstrate within 100 days after the original BMT.
  • Splenocytes will be subjected to treatment under conditions described above for donor leukocytes.
  • Table 2 indicates the designation of compound number used for the various compounds.
  • Step A ⁇ -Alanine, N-(tert-butoxycarbonyl), 2-[bis(2-hydroxyethyl)amino]ethyl ester
  • Step B ⁇ -Alanine, N-(tert-butoxycarbonyl), 2-[bis(2-tert- butyldimethylsilyloxyethyl)amino] ethyl ester
  • reaction mixture was allowed to warm to 23 °C and stirred for 2 h followed by removal of the resultant white precipitate (imidazole » HCl) by vacuum filtration.
  • the acetonitrile was removed in vacuo from the filtrate and the remaining material was partitioned between saturated brine (600 mL) and EtOAc (3 x 200 mL). The combined organic layers were dried over Na2SO4.
  • Step C ⁇ -Alanine, 2-[bis(2-tert-butyldimethylsilyloxyethyl)amino] ethyl ester
  • N-(tert-butoxycarbonyl) 2-[bis(2-tert- butyldimethylsilyloxyethyl)amino]ethyl ester from step B
  • 2-[bis(2-tert- butyldimethylsilyloxyethyl)amino]ethyl ester from step B (3.01 g, 5.48 mmol) was added neat trifluoroacetic acid (5 mL) resulting in the evolution of CO2 gas.
  • the reaction mixture was stirred for 5 min. and the trifluoroacetic acid was removed in vacuo.
  • Step D ⁇ -Alanine, N-(2-carbomethoxyacridin-9-yl), 2-[bis(2-hydroxyethyl)amino]ethyl ester
  • the ⁇ -Alanine, 2-[bis(2-tert-butyldimethylsilyloxyethyl)amino]ethyl ester (736 mg, 1.64 mmol) was reacted with methyl 9-methoxyacridine-2-carboxylate (669 mg, 2.50 mmol) by stirring in 10 mL of CHCI 3 for 12.5 h at room temperature.
  • Example 7 The compounds synthesized in Example 7 can also be prepared by the following method: Synthesis of ⁇ -Alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride (Compound V): Method II
  • Step A ⁇ -Alanine, N-(acridin-9-yl), methyl ester hydrochloride 9-Chloroacridine (11.7 g, Organic Synthesis, Coll. Vol III, pg. 57), ⁇ -alanine methyl ester hydrochloride (9.9 g) and sodium methoxide (3.26 g) were combined and 60 mL of methanol was added. The mixture was stirred with a magnetic stirrer and refluxed for 5.5 h . Heat was removed and the suspension was filtered while warm ( ⁇ 35 °C). The solid salts were rinsed with about 10 mL of additional methanol and the combined dark green filtrate was concentrated to give 21 g of a moist greenish-yellow solid.
  • Step B ⁇ -Alanine, N-(acridin-9-yl), 2-[bis(2-hydroxyethyl)amino]ethyl ester dihydrochloride
  • the ⁇ -Alanine, N-(acridin-9-yl), methyl ester hydrochloride, from Step A, (5.00 g) was partitioned between toluene (750 mL), saturated aqueous Na2CO3 (200 mL) and H2O (50 mL).
  • the aqueous layer was extracted again with toluene (3 x 250 mL) and the organic layers were combined and washed with saturated aqueous Na2CO3 (50 mL).
  • the volume of toluene was reduced to about 100 mL by rotary evaporation.
  • Triethanolamine (30 mL) was then added to form a partially immiscible system.
  • the acid solution was made basic with powdered K2CO3( s ) in the presence of CH2CI2 (200 mL).
  • the organic layer was separated and the aqueous layer was extracted again with CH2CI2 (5 x 100 mL).
  • the combined organic layers were washed with brine (50 mL), dried with anhydrous Na2SO4(s), and stripped to give crude diol free amine (5.02 g), a sticky yellow gum.
  • This material was identical by NMR to that prepared in Example 1 by an alternate procedure.
  • Step C ⁇ -Alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)arnino]ethyl ester dihydrochloride
  • Example 10 0 ⁇ -alanine, N-(4-methoxy-acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester dihydrochloride,
  • ⁇ -alanine, N-(4-methoxy-acridin-9-yl), methyl ester was prepared by mixing 1.4 g (5.84 mmol) of 4,9-dimethoxyacridine, 0.89 g (6.42 mmol) of ⁇ -alanine methyl ester hydrochloride and 20 mL of methanol and then heating to reflux for 12h under N2- The reaction was then concentrated in vacuo, dissolved in CHCl3-isopropanol (50 mL, 4:1 v/v), and washed with 50% NH4OH (2 x 25 mL) and brine (1 x 25 mL).
  • the dihydrochloride salt could be isolated in crude form by concentrating the reaction in vacuo with azeotropic removal of excess thionyl chloride (2 x 5 mL toluene). HPLC analysis indicated complete consumption of the starting material and 4-methoxy acridone (Rj 22.3 min) to be the major impurity.
  • Step A ⁇ -Alanine, [N, N-bis(2-triisopropylsilyloxy)ethyl]ethyl ester
  • Step B ⁇ -Alanine, [N, N-bis(2-triisopropylsilyloxy)ethyl]-
  • the ⁇ -Alanine, [N, N-bis(2-triisopropylsilyloxy)-ethyl] ethyl ester from Step A above (5.60 g, 10.8 mmol) and lithium hydroxide (0.59 g, 14.1 mmol) were stirred in ethanol and refluxed for 3h. The solvent was removed and the crude product was partitioned between CH2CI2 and dilute NaHCO3(aq.).
  • Step C ⁇ -Alanine, [N,N-bis(2 -hydroxy ethyl)], 3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester
  • Step D ⁇ -Alanine, [N,N-bis(2-chloroethyl)], 3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester dihydrochloride, Compound XI.
  • Step A [N,N-Bis(2-hydroxyethyl)]-2-aminoethyl 4,5',8-trimethyl-4'-psoralenacetate
  • a slurry of methyl 4,5',8-trimethyl-4'-psoralenacetate (250 mg, 0.832 mmol), triethanolamine (12 mL) and 1M HCl in ether (2 mL) were stirred at 100°C for 2 h.
  • the resulting clear brown solution was allowed to cool to room temperature and partitioned between CH 2 CI2 and saturated NaHCO 3 (aq.).
  • the organic layer was rinsed several times with saturated NaHCO 3 (aq).
  • Step B [N,N-Bis(2-chloroethyl)]-2-aminoethyl 4,5',8-trimethyl-4'-psoralenacetate hydrochloride
  • the patient presenting CLL is given a nonablative preparative regimen prior to transplantation with allogeneic peripheral blood stem cells. Transplantation is followed by support with DLI as necessary to achieve a maximum graft-versus-malignancy effect.
  • the mildly toxic preparative regimen is fludarabine (FAMP)-based and is modified according to malignancy type.
  • FAMP fludarabine
  • the patient is given FAMP at 300 mg/m2/dx3 with cyclophosphamide at 30 mg/m2/dx3.
  • Richter and Large Cell Lymphoma (LCL) are treated with a regimen consisting of FAMP 30 mg/m2/dx2, cisplatinum 25 mg/m2/d/CIx4, and Ara-c 0.5 gm mg/m2/dx2.
  • GVHD prophylaxis With the exception of treatment for LCL, no GVHD prophylaxis is given.
  • the regimen is intended to generate chimerisms and allow engraftment while decreasing the toxicity of conventional induction therapy.
  • DLI can be given 1 month post-transplant to boost engraftment. Successful engraftment allows for subsequent DLI administered as necessary to enhance a graft vs. leukemia effect and expedite immune recovery.
  • PBLs peripheral blood lymphocytes
  • PBLs subjected to 10 nM S-59 at 3 J/cm 2 UVA were assayed for viability by trypan blue exclusion. Survival was 75% after 2 days, 50% after three days, and 25% after 4 days. In related experiments, a dose-dependent survival of treated lymphocytes was observed.
  • Lymphocyte proliferation was measured by 3 H-thymidine incorporation into the DNA of allostimulated T-cells.
  • Peripheral blood mononuclear cells PBMCs
  • PBMCs Peripheral blood mononuclear cells isolated from three donors, pooled, ⁇ -irradiated (2500 cGy) to prevent autostimulation, and used as stimulator cells in a MLR.
  • PBMCs isolated from a fourth donor were used as responder cells.
  • Responder cells were treated with 0.05, 0.5 or 5 nM 4'-(4-amino-2-oxa) butyl-4,5',8-trimethylpsoralen ("S-59”) at 3 J/cm 2 UVA and co-cultured with stimulator cells. Control, untreated responder cells were also co-cultured with the stimulator cells.
  • FIG. 8 shows that proliferation of treated leukocytes, as measured by 3 H-thymidine uptake in a MLR, was eliminated in a dose-dependent manner between 0.1 nM and 10 nM S-59, with complete inhibition of proliferation at 10 nM S-59.
  • Proliferative ability of treated leukocytes was also analyzed by an alternative method, in which treated cells were activated by surface-bound anti-CD3 antibody.
  • PBMCs were either untreated, treated with 0.0001, 0.001 or 0.01 ⁇ M S-59 at a UVA dose of 3 J/cm 2 , or irradiated in the absence of S-59.
  • Cells were then incubated at 37°C in 5% CO 2 in plates to which anti-CD3 antibody had been attached, to induce polyclonal activation of T-cells, and H-thymidine incorporation was measured after 1 , 2 and 3 days of culture.
  • the results, ( Figure 9) show a dose-dependent reduction in proliferative capacity under all treatment conditions, and complete inhibition of proliferation at 10 nM
  • Human lymphocytes were cultured in a MLR as described in the previous section on analysis of proliferation. Responder cells were treated with different concentrations of
  • FIG. 10A Analysis of IL-2 production is shown in Figure 10A.
  • Secretion of IL-2 by untreated lymphocytes and by lymphocytes treated with lower concentrations of S-59 (i.e., 0.05 and 0.5 nM) peaked at 2 days in culture, and then declined due to consumption of IL-2 by proliferating lymphocytes in the population.
  • S-59 i.e., 0.05 and 0.5 nM
  • lymphocytes treated with 5 nM S-59 IL-2 was not consumed by the non-proliferating leukocytes, and remained present in the medium, at fairly high levels, at days 4, 6 and 7.
  • Lymphocytes were obtained by Ficoll gradient centrifugation, and were activated through in vitro stimulation by surface-bound anti-CD3 antibody. Surface antigen expression by activated, treated lymphocytes was measured by fluorescence-activated cell sorting
  • FACS Fluorescence Activated Cell Sorting
  • CD69 is an early marker of lymphocyte activation.
  • Figure 11A shows that cells treated with either 1 nM or 10 nM S-59 + 3 J/cm 2 UVA have surface CD69 levels similar to, or slightly higher than, those of untreated cells, for up to 48 hours post-treatment. Thus, although treated cells are unable to proliferate, signalling pathways related to activation remain functional.
  • CD 25 is the IL-2 receptor, and its expression is detected somewhat later (after activation) than that of CD69.
  • Figure 1 IB shows that, in cells treated with either 1 nM or
  • the CD40 ligand (CD40L) on T-cells recognizes the CD40 molecule expressed on the surface of B-cells as part of the T-cell activation process. Inhibition of CD40L expression on T-cells can induce cell unresponsiveness or anergy. Therefore, unperturbed expression of CD40L on T-cells after treatment is an indication of normal T-cell activation.
  • the effect of PCT on expression of CD40L was examined following activation of treated cells with anti-CD3 antibody ( Figure 12). The results of this analysis indicate that, for treatment conditions under which subsequent proliferative ability is abolished, CD40L expression by treated cells closely parallels that observed in untreated cells.
  • cytotoxic T-cells against gamma irradiation-inactivated allogeneic "stimulator" cells in a MLR. These cytotoxic T-cells were then tested for their ability to lyse 51 Cr-labeled target cells that were obtained from the same donor used to provide the inactivated stimulator cells.
  • PBMCs Peripheral blood (100 ml) was removed from two donors, one designated the "stimulator” and the other, the “responder.”
  • PBMCs were isolated from both populations by Ficoll gradient centrifugation.
  • Responder PBMCs were depleted of monocytes by placing them in a tissue culture flask, at a cell density of 5x10 6 cells/ml, for one hour.
  • Stimulator PBMCs were ⁇ -irradiated (2500cGy) to block cell division. Both the monocyte-depleted responders and the irradiated stimulators were separately resuspended in RPMI complete medium to a concentration of lxl 0 6 cells/ml. Equal volumes of responder and stimulator cell suspensions were then combined for MLR, and incubated at 37°C for 7 days in a 5% CO 2 incubator.
  • Peripheral blood 50 ml was drawn from the same donor that provided the stimulator cells for MLR.
  • PBMCs were isolated as above and resuspended at a concentration of 1 x 10 6 cells/ml in RPMI medium, then stimulated with 2 ⁇ g/ml PHA-M (phytohemagglutinin-M) and 10 Units/ml of recombinant IL-2 for 7 days at 37°C.
  • PHA-M phytohemagglutinin-M
  • Target cells were incubated in the presence of 200 ⁇ Ci Na 2 51 CrO 4 at 37°C for two hours, then washed three times to remove unincorporated label.
  • Responder cells (monocyte-depleted, as described above) were divided into four equal portions, each of which was resuspended in 30 ml of PBS containing 1% bovine serum albumin. S-59 was added to three of the portions to a final concentration of 0.1, 0.01 or 0.001 ⁇ M; the remaining portion served as an untreated control. Samples containing S-59 were transferred into 30 ml 2410 blood bags and each was illuminated with 3 J/cm UVA on a Baxter/Fenwall UVA illumination device.
  • Cytotoxic T-lymphocyte (CTL) activity of the treated responder cell population was assayed as follows.
  • the treated cells or control untreated cells
  • the treated cells were resuspended to a final concentration of 5 x 10 6 cells/ml in a final volume of 1 ml, and serial two-fold dilutions were made to obtain final concentrations of 2.5 x 10 6 , 1.25 x 10 6 and 0.625 x 10 6 cells/ml.
  • a 100 ⁇ l sample of the resuspended treated responder cells and of each treated responder cell dilution ("effector cells”) was added to each of three wells of a round- bottom 96-well microtiter plate.
  • a 100 ⁇ l sample of labeled target cells (prepared as described above), containing 5 x 10 3 cells, was then added to each well of effector cells, to give effector cel target cell ratios of 100:1, 50:1, 25:1 and 12.5:1, and the mixtures were incubated at 37°C for 4 hours. After the incubation, the plate was centrifuged for 5 min at 250 x g, and 100 ⁇ l of supernatant was removed from each well. The amount of 51 Cr in the supernatants was quantitated on a gamma counter. Average 5I Cr cpm, and standard deviation, for each triplicate set of wells was calculated. Controls
  • Unlabeled target cells were diluted and plated with labeled target cells, at the same ratios as the effector cells, to measure spontaneous release of 51 Cr. Labeled target cells were also incubated with RPMI and 1% Triton X-100 (3 wells each) to measure maximal release of 51 Cr. Average 5 l Cr cpm, and standard deviation, for each triplicate set of wells was calculated.
  • Figure 13 shows data for release of 51 Cr at different effector :target (or unlabeled target: labeled target) ratios for treated and untreated leukocytes.
  • Donor splenocytes were passed through a fine mesh screen to obtain single-cell suspensions of unseparated splenocytes and were then treated with 0.01 ⁇ M S-59 and varying doses of UVA (0.5 min, 1 min, 2 min and 8 min).
  • mice 1.2 + ) were injected into AKR, H-2k, Thy 1.1 + hosts that had been subjected to 1100 R of total body irradiation.
  • Each group of mice (6 animals per group) was challenged with 250 AKR-M2 leukemia cells 3 days after transplant. Transplanted animals were observed for clinical symptoms of GVHD, leukemia relapse and death. In addition, body weight of the transplanted animals was recorded every 3-4 days after transplant.
  • lymphocytes treated with 1 min of UVA + 0.01 ⁇ M S-59 were effective in preserving their GVL activity (3 of 3 survivors at 70 days post-leukemia challenge) with no signs of GVHD. See Table 3 and Figure 15. Body weight of animals treated with 1 min of UVA + 0.01 ⁇ M S-59 ( Figure
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs isolated from a different donor were used as responder cells.
  • Responder cells at a concentration of 2xl0 6 cells/ml were treated with 0.1, 0.2, 0.3, 0.4, or 0.5 ⁇ M ⁇ -Alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester (S-303) at room temperature for approximately 15 min and washed twice with PBS containing 1% bovine serum albumin.
  • S-303 -treated and untreated responder cells were co-cultured with the gamma-irradiated stimulator cells at a 1 :1 ratio.
  • Lymphocyte proliferation was measured by addition of 3 H-thymidine to the co- culture six days after the start of co-culture, harvesting cells on day 7, and measuring incorporation of H cpm in the cells. Cytokine production was analyzed by sandwich ELISA of culture supernatants taken 24 and 48 hours after the start of co-culture.
  • IFN- ⁇ Gamma interferon

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EP98936943A 1997-07-21 1998-07-21 Verfahren zur verhandlung für leukozyten, leukozyten enthaltende zusammensetzungen und deren verwendungen Withdrawn EP1005531A2 (de)

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AU2002218019A1 (en) * 2000-11-03 2002-05-15 Nexell Therapeutics Inc. Methods for depleting and isolating alloreactive and antigen-reactive t cells from hematopoietic donor cells
DE10112851C1 (de) 2001-03-16 2002-10-10 Gsf Forschungszentrum Umwelt Semi-allogene Antitumor-Vakzine mit HLA-haplo-identischen Antigen-präsentierenden Zellen
WO2004110481A2 (en) 2003-02-06 2004-12-23 Cerus Corporation Listeria attenuated for entry into non-phagocytic cells, vaccines comprising the listeria, and methods of use thereof
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US7695725B2 (en) 2003-02-06 2010-04-13 Aduro Biotech Modified free-living microbes, vaccine compositions and methods of use thereof
CA2743669C (en) 2008-11-24 2018-10-16 Helmholtz Zentrum Muenchen Deutsches Forschungszentrum Fuer Gesundheit Und Umwelt (Gmbh) High affinity t cell receptor and use thereof
DE102011085695A1 (de) * 2011-11-03 2013-05-08 Jörg Pohl Einmalig dosierte Oxazaphosphorine zur Therapie von Krankheiten
US20190224494A1 (en) * 2012-03-20 2019-07-25 Fenwal, Inc. Apparatus and method for batch photoactivation of mononuclear cells with cryopreservation
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US10688166B2 (en) 2014-11-03 2020-06-23 Cerus Corporation Compositions and methods for improved car-T cell therapies
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