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WO2002036750A2 - Cellules t a reconnaissance specifique des antigenes mineurs d'histocompatibilite et leurs utilisations dans l'elimination des cellules visees - Google Patents

Cellules t a reconnaissance specifique des antigenes mineurs d'histocompatibilite et leurs utilisations dans l'elimination des cellules visees Download PDF

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Publication number
WO2002036750A2
WO2002036750A2 PCT/CA2001/001477 CA0101477W WO0236750A2 WO 2002036750 A2 WO2002036750 A2 WO 2002036750A2 CA 0101477 W CA0101477 W CA 0101477W WO 0236750 A2 WO0236750 A2 WO 0236750A2
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cells
recipient
miha
donor
immunodominant
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PCT/CA2001/001477
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WO2002036750A3 (fr
Inventor
Claude Perreault
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Compatigene Inc
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Priority to AU2002213694A priority Critical patent/AU2002213694A1/en
Publication of WO2002036750A2 publication Critical patent/WO2002036750A2/fr
Publication of WO2002036750A3 publication Critical patent/WO2002036750A3/fr

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    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the present invention is concerned with the elimination of undesirable target cells such as cancerous cells, viral infected cells and abnormal cells of a recipient. More particularly, the invention is concerned with the isolation, selection and use of T-cells from a donor for the treatment of hematopoietic cancers.
  • Adoptive immunotherapy is a main approach that is currently being investigated in the field of cancer immunotherapy.
  • Adoptive immunotherapy is based on the use of T-cells specifically recognizing minor histocompatibility antigens (MiHAs).
  • MiHAs minor histocompatibility antigens
  • AHCT allogeneic hematopoietic cell transplant
  • VTL graft-versus-leukemia
  • GVL graft- versus-host-disease
  • the potentially fatal condition of GVHD has also greatly limited the use of MiHA-based adoptive immunotherapy to hematopoietic cancer treatments only, although this approach could, in theory, be used for treating other types of cancers and other diseases such as chronic viral infections and abnormal cells proliferation.
  • T-cells that could be used in adoptive immunotherapy methods without causing the recipient a graft-versus-host disease (GVDH) reaction. Also needed are T-cells that would allow to eradicate not only hematopoietic cancerous cells of a recipient, but also other types of cancerous cells, viral infected cells and abnormal cells. There is also a need for methods for selecting such T-cells and for therapeutic physiological solutions comprising the same. There is further a need for adoptive immunotherapy methods wherein ubiquitous minor histocompatibility antigens (MiHAs) are targeted, i.e. minor histocompatibility antigens that are expressed not only by target cells to be eliminated but also expressed by non-target cells. There is more particularly a need for methods for treating an hematopoietic cancerous recipient without causing him a graft-versus-host disease (GVHD) reaction.
  • MiHAs ubiquitous minor histocompatibility antigens
  • the present invention fulfils these needs, since it demonstrates for the first time that MiHA based immunotherapy can cure leukemia without causing GVHD.
  • the invention fulfils also other needs which will be apparent to those skilled in the art upon reading the following specification.
  • T-cells T-cells, method for selecting the same, therapeutic physiological solutions and methods of treatment, are provided that are effective for eliminating target cells in a patient without causing him a GVDH reaction.
  • the invention is more particularly concerned with the treatment of hematopoietic cancers.
  • the invention is directed to a population of T-cells that have been isolated from a donor and that have been selected for specifically recognizing an immunodominant minor histocompatibility antigen (MiHA) expressed by target cells of a recipient but not expressed by any of the donor's cells.
  • MiHA immunodominant minor histocompatibility antigen
  • the T-cells from the population of T-cells are capable of being primed against the immunodominant MiHA such that, when transferred into a recipient, the primed T-cells eliminate the recipient's target cells without causing him a graft-versus-host disease (GVHD) reaction.
  • the T-cells population comprises a plurality of CD8+ T-cells that have been isolated from a hematopoietic cancer-free donor and that have been selected for specifically recognizing a ubiquitous and immunodominant minor histocompatibility antigen (MiHA) expressed by hematopoietic cancerous cells of a recipient, but not expressed by any of the donor's cells.
  • MiHA ubiquitous and immunodominant minor histocompatibility antigen
  • the invention is directed to the use of population(s) of the above-mentioned T-cells for the elimination of target cells, such as hematopoietic cancerous cells in a recipient and to therapeutic physiological solutions comprising such population(s) of T-cells.
  • the invention is directed to methods for selecting T-cells from a donor which are capable of being transferred into a compatible recipient without causing him a graft-versus-host disease (GVHD) reaction.
  • GVHD graft-versus-host disease
  • the invention is directed to methods for treating an hematopoietic cancer wherein CD8+ T-cells from a hematopoietic cancer-free donor are isolated and transferred into a compatible hematopoietic cancerous recipient.
  • Figures 1A and 1B are graphs showing survival of primed (Fig. 1A) and unprimed (Fig. 1B) mice following injection of 10 5 EL4 cells i.v. Priming was performed by i. p. injection of 2 x 10 7 cells (either normal splenocytes or EL4 cells) on day -14, or of 300 ⁇ g B6 do 1 peptide (AAPDNRETF; sequence published in Perreault et al., J. Clin. Invest, 98:622, 1996) in incomplete Freund's adjuvant s.c. on day -21 and -14. EL4 cells used for priming were irradiated (10 4 cGy). Ten to fifteen mice per group.
  • Figure 2 is a graph illustrating the assessment of B6 dom1 -specific CD8 + T-cells by staining with B6 do 1 /D tetramers. Numbers indicate the percentage of CD8 + T-cells that were tetramer + .
  • B6 om1 -specific cell line shows that B6 dom /D b tetramers were highly specific and sensitive reagents.
  • the mean percentage (+ SD) of tetramer" CD8 + T-cells was 0.9 ⁇ 0.2 for B10.H7 b mice primed with B6 dom1 peptide, 2.4 ⁇ 0.4 for B10.H7 b mice primed with B10 cells, and 1.3 ⁇ 0.3 for C3H.SW mice primed with B6 dom1 peptide. Priming was performed as described in Figure 1.
  • Figure 3 depicts in graphs that B6 dom1 is expressed on both hematopoietic and non hematopoietic cells.
  • Peptides were extracted from the liver of B6.PL, B10.H7 b , B6.PL- C3H.SW and C3H.SW ⁇ B6.PL mice. Extracts containing 5 mg proteins were fractionated by HPLC, and titrated amounts of HPLC fraction 15, in which B6 dom1 can be found, were incubated with 51 Cr-labeled C3H.SW Con A blast targets. Specific cytotoxicity is shown as means of triplicate cultures.
  • FIG 4 is a graph showing that adoptive transfer of B6 dom1 -primed T-cells can eradicate EL4 cells.
  • Irradiated mice 1000 cGy
  • Donors were primed with EL4 or B10 cells on day -14 as described in Figure 1.
  • the type of cells used for priming is shown in parentheses.
  • B10.H7 b (B10)-»B10 refers to transfer of cells from B10.H7 b donors primed against B10 cells into irradiated B10 hosts.
  • mice received 10 5 EL4 cells i. v. Ten mice per group.
  • the present invention relates to the elimination of target cells in vertebrates, and more particularly the eradication of hematopoietic cancer cells, by selective transfer of T-cells specific for selected minor histocompatibility antigens (MiHAs).
  • the invention provides T-cells with the potent activity of eliminating target cells when injected into a recipient, without being toxic, i.e., without causing the recipient a graft-versus-host disease (GVHD) reaction.
  • GVHD graft-versus-host disease
  • the invention is based on: 1) the priming of T-cells specifically reacting against a selected ubiquitous MiHA, viz.
  • MiHA that is expressed by target cells and by non-target cells of a recipient; and also 2) the selection of a 100% purified population of T-cells that specifically react against a minor histocompatibility antigen which is ubiquitously expressed by the recipient's cells or selectively expressed by specific recipient's target cells only.
  • MiHA-specific T-cell responses are of remarkable potency and represent the most conclusive documentation that the immune system can cure cancer in human. It was shown recently that anti-MiHAs T-cell responses are so effective that leukemic patients can be cured by AHCT without the need for myeloablative chemo/radiotherapy.
  • the present invention encompasses the elimination of all these types of cells ⁇
  • the present invention encompasses the eradication of target cells in members of the class Vertebrates into which it would be preferable to eliminate such cells.
  • the vertebrate is a mammalian subject including, without limitation, human and nonhuman primates, farm animals, domestics animals, laboratory animals.
  • the invention is directed to a population of T-cells comprising a plurality of T-cells isolated from a donor and selected for specifically recognizing an immunodominant minor histocompatibility antigen (MiHA) expressed by target cells of a recipient but not expressed by any of the donor's cells.
  • MiHA immunodominant minor histocompatibility antigen
  • At least some of the T-cells from the T-cells population are capable of being primed against the immunodominant MiHA such that, when transferred into a recipient, the primed T-cells eliminate the recipient's target cells without causing him a graft-versus-host disease (GVHD) reaction.
  • GVHD graft-versus-host disease
  • the population of T-cells comprises CD4+ and/or CD8+ T-cells.
  • CD4+ T-cells can recognize immunodominant MiHAs.
  • the T-cells are CD8+ cells.
  • the T-cells may be isolated from a compatible donor using methods well known in the art. Theoretically, the T-cells could also come from a plurality of donors provided that these T-cells recognize specifically the selected immunodominant MiHA(s). According to the invention, selection, priming, and adoptive transfer of such T-cells targeted to immunodominant MiHAs could be very useful to eliminate various types of target cells such as cancerous cells, abnormal cells and virus infected cells.
  • the population of T-cells comprises a plurality of CD8+ T-cells isolated from a hematopoietic cancer-free donor and selected for specifically recognizing a ubiquitous and immunodominant minor histocompatibility antigen (MiHA) expressed by hematopoietic cancerous cells of a recipient, but not expressed by any of the donor's cells.
  • MiHA ubiquitous and immunodominant minor histocompatibility antigen
  • “immunodominant MiHA” refers to an immunogenic amino acid sequence of a selected MiHA (a peptidic portion of the MiHA or the whole protein) that would "prime” the T-cells and cause an effective immune response against the target cells.
  • the MiHAs and fragments thereof useful in the present invention may be purified from living organisms or obtained by synthetic means, i.e. chemical synthesis of the polypeptide from component amino acids by methods known to those of ordinary skill in the art.
  • the polypeptide may be obtained by its production in prokaryotic or eukaryotic host cells using techniques known in the art.
  • Immunogenic analogs of MiHAs resulting from amino acid substitution, deletion addition and/or chemical modifications of the MiHA polypeptide are suitable according to the present invention, if these analogs do not materially reduce the immunogenicity of the MiHAs and that they permit to prime efficaciously the donor's T-cells.
  • the invention encompasses immunodominant MiHAs which are specifically expressed by various cells or tissues and also ubiquitous MiHAs expressed by most or all the recipient's cells.
  • some MiHAs are specifically expressed by cancerous cells, viral infected cells, and abnormal cell at a specific phase of the cell cycle or at a specific differentiation stage.
  • some immunodominant MiHAs which are specifically expressed by hematopoietic cells have already been identified.
  • International PCT patent applications published under Nos. WO 97/05169, WO 99/05173 and WO 99/05174 and also U.S. patent No. 5,770,201 disclosed such selectively expressed MiHAs.
  • the ubiquitous MiHA is expressed by the target cells of the recipient at a level of at least about 100 copies per cell.
  • an available approach to assess MiHA expression on cells is the titration of peptide extracts in cytotoxic assays. Shirle et al. ⁇ Europeen Jour. Immunol., 30:2216, 2000) describe another approach that uses mass spectrometry on peptides extracted from tissues or cells suspensions.
  • the present invention encompasses the use of suitable combination(s) of more than one MiHAs.
  • the T-cells from the population of T-cells have been genetically modified for producing at least one lymphokine.
  • the lymphokine is selected from the group consisting of IL- 2, IL-4, IL-6, IL-7 and IL-15. More preferably, the T-cells are genetically modified so as to produce IL-15. Methods for genetically modifying eucaryote cells such as T-cells are well known in the art.
  • the invention is directed to a therapeutic physiological solution for eliminating target cells of a recipient, and comprising: a population of
  • T-cells as defined hereinbefore, and a physiologically acceptable diluent.
  • the therapeutic physiological solution is used for eliminating hematopoietic cancerous cells of a hematopoietic cancerous recipient.
  • Such anti- cancerous solution comprises a physiologically acceptable diluent and CD8+ T-cells isolated from a compatible hematopoietic cancer-free donor as defined hereinbefore.
  • Physiologically acceptable diluents include saline and aqueous buffer solutions.
  • the therapeutic physiological solution may further comprise therapeutically active agents such as anti-inflammatory agents, compounds modulating immunity, growth factors, nucleic acids (DNA, anti-sense, RNA), antibodies, etc. It may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, colorants, salts, buffers, coating agents or antioxidants.
  • therapeutic physiological solution of the invention may be administered by any suitable route and the amount to be administered is that amount necessary for inducing an effective immune response towards the selected MiHA and eliminating the target cells of the recipient.
  • Suitable dosages will vary, depending upon factors such as the type and amount of T-cells in the solution, the type of disease to be treated, the nature of the selected MiHA(s), the route of administration and the age and weight of the individual to be treated. Dosage regimens could be adjusted to provide the optimum therapeutic response.
  • the therapeutic physiological solution of the invention would be prepared using methods well known in the art, preferably in the form of a sterile injectable aqueous solution.
  • the invention is directed to a method for selecting T-cells from a donor which are capable of being transferred into a compatible recipient. This method is very useful since the selected T-cells are capable of being transferred into the recipient without causing a graft-versus-host-disease (GVHD) reaction.
  • GVHD graft-versus-host-disease
  • the method comprises the steps of: a) selecting an immunodominant minor histocompatibility antigen (MiHA) expressed by target cells of the recipient but not expressed by any of the donor's cells, wherein the immunodominant MiHA is expressed by the recipient's target cells at a level of at least about 100 copies per cell; b) isolating T-cells from the donor; and c) • positively selecting, from the T-cells isolated at step b), T-cells that specifically recognize the immunodominant MiHA of step a); and/or
  • MiHA immunodominant minor histocompatibility antigen
  • step c) • eliminating, from the T-cells isolated at step b), T-cells reacting against any immunodominant minor histocompatibility antigen(s) other than the immunodominant MiHA of step a); whereby the T-cells selected/not eliminated at step c) are capable of being transferred into a compatible recipient without causing him a graft-versus-host disease (GVHD) reaction. More preferably, step c) is performed so that 100% of the T-cells selected/not eliminated specifically recognize said immunodominant MiHA.
  • GVHD graft-versus-host disease
  • the T-cells are selected from CD4+ and CD8+ T-cells and the recipient's target cells are selected from the group consisting of cancerous cells, viral infected cells, and abnormal cells at a specific phase of the cell cycle or at a specific differentiation stage.
  • the selection method is used for selecting CD8+ T-cells from a hematopoietic cancer-free donor which are capable of eliminating hematopoietic cancerous cells when transferred into a compatible hematopoietic cancerous recipient without causing him a graft-versus-host disease (GVHD) reaction.
  • GVHD graft-versus-host disease
  • This preferred method comprises the steps of: a) selecting a ubiquitous and immunodominant minor histocompatibility antigen (MiHA) expressed by hematopoietic cells of the recipient but not expressed by any of the cells of the donor; b) isolating CD8+ T-cells from the donor; c) • positively selecting, from the T-cells isolated at step b), the CD8+ T-cells that specifically recognize the MiHA of step a); and/or
  • MiHA ubiquitous and immunodominant minor histocompatibility antigen
  • the CD8+ T-cells primed at step d) are capable of being transferred into a compatible hematopoietic cancerous recipient for eliminating hematopoietic cancerous cells of said recipient without causing him a graft-versus-host disease (GVHD) reaction.
  • GVHD graft-versus-host disease
  • the selection method may further comprise the step of priming in vitro the T-cells selected/not eliminated at step c) against the selected MiHA such that, when transferred into the recipient, the primed T-cells eliminate the target cells of the recipient.
  • the selection method may also further comprise the step of genetically modifying the T-cells isolated from said donor such that they produce at least one lymphokine selected from the group consisting of IL-2, IL-4, IL-6, IL-7 and IL-15.
  • the invention is directed to an improved method for eliminating, in a recipient, target cells expressing a selected immunodominant minor histocompatibility antigen (MiHA), without causing the recipient a graft-versus-host-disease reaction; the known method comprising the steps of:
  • MiHA immunodominant minor histocompatibility antigen
  • the invention is directed to an improved method for treating an hematopoietic cancer wherein CD8+ T-cells from a hematopoietic cancer-free donor are isolated and transferred into a compatible hematopoietic cancerous recipient.
  • the known method is improved in that the CD8+ T-cells from the donor are primed against a ubiquitous minor histocompatibility antigen (MiHA) expressed by all the recipient's cells (including hematopoietic cancerous cells), but not expressed by any of the donor's cells.
  • the invention is directed to a method for treating an hematopoietic cancer comprising the steps of:
  • hematopoietic stem-cells transplant from a hematopoietic cancer-free donor to a compatible hematopoietic cancerous recipient; - selecting a ubiquitous and immunodominant minor histocompatibility antigen (MiHA) expressed by hematopoietic cancerous cells of the recipient but not expressed by hematopoietic cells of the donor;
  • MiHA ubiquitous and immunodominant minor histocompatibility antigen
  • CD8+ T-cells from the donor and injecting these isolated CD8+ T-cells into the recipient; and - priming the donor's CD8+ T-cells against the selected ubiquitous immunodominant MiHA such that these primed CD8+ T-cells eliminate the hematopoietic cancerous cells of the recipient.
  • the invention is directed to an improved method for treating an hematopoietic cancer comprising the steps of: - isolating CD8+ T-cells from a hematopoietic cancer-free donor;
  • the invention is directed to a method for treating an hematopoietic cancer comprising the steps of: - proceeding to a hematopoietic stem-cells transplant from a hematopoietic cancer-free donor to a compatible hematopoietic cancerous recipient;
  • MiHA minor histocompatibility antigen
  • CD8+ T-cells from the donor; - injecting into the recipient a population of isolated CD8+ T-cells wherein 100% of the CD8+ T-cells specifically recognize the selected MiHA; and - priming the donor's CD8+ T-cells against the selected MiHA such that these primed lymphocytes eliminate the hematopoietic cancerous cells of the recipient.
  • the CD8+ T-cells are generally transferred following a hematopoietic stem- cells transplantation from an hematopoietic cancer-free donor to a compatible hematopoietic cancerous recipient.
  • Such transplantation is performed according to regular methods well known in the art of hematopoietic stem-cells purification and transplantation.
  • compatibility between a donor and a recipient depends on the number of HLA genes they shared, and that in order to be compatible, the donor and the recipient must have at least some HLA genes in common.
  • the CD8+ T-cells to be used are isolated from the donor and injected into the recipient using well known methods. Theoretically, the T-cells could, if necessary, be isolated from more than one compatible donor as explained previously.
  • the CD8+ T-cells are also preferably "primed" against a selected minor histocompatibility antigen (MiHA) in order to elicit an immune response towards the selected MiHA and lead to the elimination of the hematopoietic cancerous cells expressing the selected MiHA.
  • Priming of CD8+ T-cells is performed by exposing the CD8+ T-cells of the donor to an immunogenic amino acid sequence of the selected MiHA (a peptidic portion of the MiHA or the whole protein) using methods within the skill of the art.
  • the priming be performed in vitro prior to transfering the CD8+ T-cells into the recipient.
  • In vivo priming can be done by injecting into the donor/recipient the whole MiHA or a peptidic portion thereof, preferably in combination with an adjuvant.
  • T-cells transferred from the donor into the recipient specifically recognize the selected MiHA (ubiquitous or specific to the target cells).
  • Example 1 hereinbelow demonstrates that, for reducing to a minimum any risk of GVHD, one must take specific measures to deplete T-cells reactive to recipient antigens other than the target MiHA in all donor-derived hematopoietic cell preparations injected to the recipient before or at the time of immunotherapy. Therefore, in a preferred embodiment of the invention, all T-cells reacting against minor histocompatibility antigen(s) other than the selected MiHA are eliminated prior to their transfer into the recipient.
  • the methods of the invention preferably further comprise at least one step selected from the steps of: a) positively selecting isolated CD8+ T-cells that specifically recognize the selected MiHA and transferring solely into the recipient these positively selected CD8+ T-cells; and b) eliminating CD8+ T-cells reacting against immunodominant minor histocompatibility antigen(s) other than the recipient's selected MiHA prior to transferring the donor's CD8+ T-cells into the recipient.
  • the selection/elimination steps can be done using methods well known in the art such as flow cytometry and in vitro expansion of CD8+ T-cells specifically recognizing the selected MiHA.
  • the CD8+ T-cells selected/not eliminated are preferably injected to a recipient in need of treatment for hematopoietic cancers and therefore, the steps of selecting the MiHA, selecting/eliminating the CD8+ T-cells and genetically modifying the CD8+ T-cells can be done as described hereinbefore in the aspect concerning the population T-cells.
  • the methods for treating hematopoietic cancers further comprise the step of preventing activation induced cell death (AICD) of the CD8+ T-cells transferred into the recipient.
  • AICD activation induced cell death
  • the invention provides T-cells specifically recognizing minor histocompatibility antigen(s), methods for selecting these cells and uses thereof for eliminating target cells and more particularly to eliminate hematopoietic cancers cells.
  • B6 do 1 (H7 a ) is expressed at extremely high levels (1 ,000 copies/cell) on hematopoietic cells. Initially, based on previously published work, it was not believed that B6 do 1 represented a good target for immunotherapy of hematopoietic malignancies since this MiHA peptide was present in practically all tissues and organs. Considering the wide tissue distribution of B6 dom1 , the Applicant was expecting that transfer of B6 om1 -reactive T-cells would cause severe GVHD. This assumption was strengthened when it was observed that several mice injected with T-cells from C3H.SW donors primed with B6 dom1 peptide preparations presented mild signs of cutaneous GVHD.
  • mice The following strains of mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in the Guy ⁇ Bernier Research Center: B6.PL-777y- a /Cy
  • H7 b 47N
  • Sn B10.H7
  • LP/J LP/J. Mice were used between 6 and 16 weeks of age and were maintained in specific pathogen-free conditions according to the standards of the Canadian Committee for Animal Protection.
  • EL4 thymoma cell line (of C57BIJ6J origin) was obtained from the American Type Culture Collection (Rockville, MD). The B6 do 1 - specific T-cell lines have been previously described (Eden PA et al. J. Immunol. 162:4502, 1999).
  • mice were transplanted as described previously (Fontaine P et al, Immunogenetics 34:222, 1991). In most experiments, recipient mice received 1000 cGy total body irradiation from a 60 Co source at a dose rate of 128 cGy/min on day 0, the day of transplant. The dose of irradiation was increased to 1200 cGy for production of chimeras used in experiments depicted in Figure 3. Bone marrow cells were obtained from the tibiae and femurs of donor mice.
  • Bone marrow cells (10 7 ) mixed with spleen cells (5 x 10 7 ) were given as a single intravenous injection, via the tail vein, in a volume of 0.5 ml of serum-free RPMI-1640 medium. Transplanted mice were observed twice a week for skin signs of GVHD (hair loss, dermatitis), and the day of death was recorded.
  • B6 do 1 peptide was extracted from tissue homogenates in 0.1% trifluoroacetic acid, in the presence of protease inhibitors (25 mM iodoacetamide, 1mM aprofinine, 1mM PMSF) as described previously (Eden PA et al. J. Immunol. 162:4502, 1999). After a pre- purification on a C18 SEP-PAKTM column (Waters, Milford, MA) extracts containing 5 mg proteins were fractionated on an HPLC system using a LUNATM C18 column (5 ⁇ m, 4.6 x 250 mm, Torrance, CA).
  • protease inhibitors 25 mM iodoacetamide, 1mM aprofinine, 1mM PMSF
  • Solvents used were 99.9% water/0.1% trifluoro-acetic acid (solvent A) and 99.9% acetonitrile/0.1% trifluoro-acetic acid (solvent B).
  • the gradient consisted of the following linear step intervals: 0% B (0-5 minutes), up to 20% B at 10 minutes, up to 55% B at 55 minutes, plateau at 55% B from 55 to 60 minutes, and up to 100% B at 70 minutes.
  • Flow rate was 1 ml/minute, and 1 ml fractions were collected and lyophilized.
  • MHC class l/peptide tetramers MHC class I (H2D b )/peptide (B6 dom1 ) tetramers were produced as previously described (Altman JD et al., Science 274:94, 1996; and Gallimore A et al., J. Exp. Med. 187:1383, 1998). Recombinant ⁇ 2 - microglobulin and the extracellular domain of the MHC class I heavy chain containing the BirA recognition sequence in frame at its C terminus were overexpressed in Escherichia coli as insoluble aggregates that formed inclusion bodies.
  • H2D b complexes were refolded around peptide by dilution of denaturing buffer. Monomeric complexes were recovered by anion exchange chromatography over a MONO Q HRTM column (Pharmacia, Upsala, Sweden). H2D b /peptide complexes were biotinylated using BirA enzyme (Avidity, Denver, CO) as described. Tetramers were generated by mixing the biotinylated monomeric complexes with NEUTRAVIDIN-PETM (Molecular Probes, Eugene, OR) at a 6:1 molar ratio.
  • biotinylated tetramers were purified by gel filtration over a SUPERDEX 200 HRTM column (Pharmacia). Purified tetramers were stored at 1 mg/ml at 4°C and were frequently tested on the B6 dom1 -specific T-cell line to document maintenance of staining capacity and signal intensity. Cell staining and flow cytometry. Evaluation of chimerism was performed with PE- conjugated anti-Thy-1.2 (clone 30-H12) and FITC-conjugated anti-Thy-1.1 (clone OX-7) antibodies from Pharmingen (Mississauga, Canada).
  • tetramer + splenocytes For estimation of tetramer + splenocytes, cell suspensions (100 ⁇ l) were stained with anti-CD8 (APC) antibody (clone 53-6.7; Pharmingen) in PBS/BSA 0.1% for 25 minutes at 4°C, washed, and then incubated with 1 ⁇ g PE-labeled tetramers at 37°C for 40 minutes. TCR expression was monitored for antigen specific T-cell lines with anti- TCR ⁇ (FITC) antibody (clone H57-597; Pharmingen). Cells were analyzed on a FACSCALIBURTM (Becton Dickinson, Mountain View, CA) using CELLQUESTTM software.
  • APC anti-CD8
  • FITC anti- TCR ⁇
  • B6 dom1 (H7 a ) confers resistance to EL4 thvmoma cells.
  • the Applicant first addressed the question as to whether an immune response targeted to the immunodominant B6 dom1 /H7 a MiHA would confer resistance to (B6-derived) EL4 leukemic cells. It is known that B6 dom1 is absent on cells from B10.H7 b , C3H/HeJ and C3H.SW mice, but is present on B6, B10, EL4 and LP cells. It is also known that EL4 cells do not express MHC class II antigens, but express MHC class I antigens as well as six tumor specific MHC class l-associated CTL epitopes. As shown in Figure 1A, B6 mice injected with 10 5 EL4 cells died within 40 days. Thus, TAAs found on EL4 cells do not elicit protective immune response in unprimed animals.
  • B10 mice differ from B6 mice (and EL4 cells) at the H9 locus which encodes a non-dominant MiHA; B10 have the H9 a allele while B6 have a "non a" allele ⁇ H9 'a ).
  • host vs EL4 disparity at the H9 locus had no effect since survival of B10 recipients was not prolonged relative to that of B6 hosts.
  • survival of recipients with disparity either for the H7 locus (which encodes the immunodominant B6 do 1 MiHA) or the full MHC (C3H/HeJ) was 30% and 100%, respectively.
  • Figure 1 B shows results wherein the susceptibility to EL4 cells in mice primed against specific antigens was assessed.
  • the Applicant transferred 10 7 bone marrow cells (as a source of hematopoietic progenitors) and 5 x 10 7 splenocytes (as a source of T-cells) from either primed (against B6 dom1 ) or unprimed donors, and assessed survival and skin signs of GVHD in irradiated B10 recipients.
  • Two types of donors were used: B10.H7 b mice which differ from B10 hosts at the H7 locus, and C3H.SW mice which differ from B10 for numerous MiHAs including H7.
  • B6 do 1 peptide (AAPDNRETF) in incomplete Freund's adjuvant s.c. day -21 and -14, or 2 x 10 7 splenocytes i. p. on day -14.
  • C3H.SW- and B10.H7 b -derived T-cells only the former contain T-cell precursors reactive to MiHAs other than B6 dom1 on B10 cells.
  • a logical inference is that the occurrence of a mild form of GVHD in B10 recipients of T-cells from B6 do 1 - primed C3H.SW donors is best explained by epitope spreading, a known process whereby epitopes distinct from and non-cross-reactive with an inducing epitope become targets of an evolving immune response.
  • destruction of host hematopoietic cells by B6 dom1 -primed T-cells has no deleterious consequence per se (c.f. B10.H7 b donors).
  • the ensuing inflammatory reaction provides an ideal milieu to prime T-cells specific for other host MiHAs (provided they are present in the injected inoculum, c.f. C3H.SW donors), and initiate GVHD.
  • B6 dom1 is present on non- hematopoietic cells.
  • An alternative explanation would be that B6 dom1 recovered from non lymphoid organs derives from resident hematopoietic cells (lymphocytes, macrophages, dendritic cells). To discriminate between these two possibilities, the Applicant created hematopoietic chimeras that expressed B6 dom1 either on hematopoietic cells or non-hematopoietic cells.
  • mice irradiated (1200 cGy) C3H.SW (Thy1.2, B6 do 1 neg) mice were transplanted with B6.PL (Thy1.1 , B6 dom pos) bone marrow + spleen cells (B6.PL ⁇ C3H.SW), and vice versa (C3H.SW ⁇ B6.PL).
  • B6.PL Thy1.1 , B6 dom pos
  • C3H.SW ⁇ B6.PL C3H.SW ⁇ B6.PL
  • mice were complete hematopoietic chimeras as determined by flow cytometry analysis of spleen cells with anti-Thy1.1 and anti-Thy1.2 antibodies ( ⁇ 1% host cells; data not shown).
  • B6 dom1 was recovered from the liver of B6 but not from B10.H7 b mice. This indicates that the B6 dom1 -specific T-cel! line does not crossreact with other peptides from the C57BL background ( Figure 3). Notably, ⁇ d om i was p resen t j n s i m ilar amounts in liver extracts from B6.PL-»C3H.SW and C3H.SW->B6.PL mice.
  • B6 dom1 is present in both hematopoietic and non- hematopoietic cells. Similar results were obtained when peptides were extracted from the lung (data not shown). Titration of peptide extracts in cytotoxic assays, the standard available approach to assess MiHA expression, provides only gross semiquantitative estimates.
  • Recipients were either B10.H7 b or B10 mice; in the former group, B6 dom1 was present only on injected EL4 cells, whereas in the latter, it was also present on host cells. Mice were observed for skin signs of GVHD, and autopsy was performed on mice dying before day 100 to ascertain whether their demise was due to GVHD (lymphoid atrophy) or leukemia (hepatosplenomegaly and enlarged lymph nodes).
  • Figure 4 shows that, as expected, mice that received T-cells from B10 donors (i. e., containing no anti-B6 dom1 T-cells) were not protected and died of EL4 leukemia between day 23 and 43.
  • Differential susceptibility to MiHA-reactive T-cells is presumably related to the fact that the amount of MiHA peptide on the cell surface is determined by the level of expression of MHC molecules which is lower on non-hematopoietic than hematopoietic cells.
  • This idea is consistent with Applicant's estimate of B6 dom1 expression being one order of magnitude lower on hepatocytes relative to K ⁇ pffer cells. Accordingly, the Applicant infers that presence of an immunodominant MiHA on non-hematopoietic cells does not preclude its selection as an immunotherapy target provided its expression level is not above 10% than that of hematopoietic cells.
  • B10.H7 b mice showed 100% resistance to a lethal inoculum of EL4 cells (Fig. 1), and eradicated leukemic cells following adoptive transfer in 100% of syngeneic hosts (Fig. 3).
  • the cure rate was decreased to 60% when these T-cells were injected into B10 hosts i.e., when the target MiHA was expressed not only on leukemic cells but also on host cells. This shows that the fate of adoptively transferred T-cells, and thus the efficacy of the GVL effect, is influenced by the target antigen load encountered in the recipient. It has been shown in numerous models that high antigen load causes AICD of antigen-reactive T-cells.
  • the Applicant therefore proposes that in vitro transfection into donor-derived T-cells of cDNA coding for cytokines of the IL-2 family and/or for IL-6 should be a most appropriate strategy to enhance the survival and GVL efficacy of adoptively transferred T-cells targeted to MiHA with a wide tissue distribution.

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Abstract

L'invention concerne des cellules T qui reconnaissent spécifiquement les antigènes mineurs d'histocompatibilité, ainsi que des méthodes de sélection de ces cellules T et leurs utilisations pour éliminer les cellules visées, plus particulièrement pour éliminer les cellules cancéreuses hématopoïétiques. L'invention repose sur: 1) l'amorçage de cellules T réagissant spécifiquement contre un antigène mineur d'histocompatibilité (MiHA) immunodominant ubiquiste sélectionné, exprimé par les cellules visées et par les cellules non visées; et 2) la sélection d'une population de cellules T 100 % purifiée qui réagit spécifiquement contre un antigène mineur d'histocompatibilité immunodominant exprimé de façon ubiquiste par les cellules du receveur, ou sélectivement exprimé par des cellules visées spécifiques du receveur uniquement. Le principal avantage de l'invention réside dans le fait que les cellules T utilisées dans l'invention peuvent être transférées d'un donneur à un receveur compatible sans entraîner chez ce dernier une réaction de rejet de greffe (GVHD).
PCT/CA2001/001477 2000-11-02 2001-10-19 Cellules t a reconnaissance specifique des antigenes mineurs d'histocompatibilite et leurs utilisations dans l'elimination des cellules visees WO2002036750A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003054008A3 (fr) * 2001-12-20 2004-01-15 Compatigene Inc Proteine mammalienne simp, sequence genetique et leurs utilisations dans la therapie anticancereuse
WO2016127249A1 (fr) * 2015-02-09 2016-08-18 Université de Montréal Nouveaux antigènes mineurs d'histocompatibilité et leurs utilisations
WO2018152633A1 (fr) * 2017-02-22 2018-08-30 Université de Montréal Nouveaux antigènes mineurs d'histocompatibilité et leurs utilisations

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1020519A1 (fr) * 1999-01-15 2000-07-19 Introgene B.V. Antigènes d'histocompatibilité mineurs et leur utilisation pour le diagnostic et le traitement des tumeurs

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003054008A3 (fr) * 2001-12-20 2004-01-15 Compatigene Inc Proteine mammalienne simp, sequence genetique et leurs utilisations dans la therapie anticancereuse
WO2016127249A1 (fr) * 2015-02-09 2016-08-18 Université de Montréal Nouveaux antigènes mineurs d'histocompatibilité et leurs utilisations
US10414813B2 (en) 2015-02-09 2019-09-17 Université de Montréal Minor histocompatibility antigens and uses thereof
WO2018152633A1 (fr) * 2017-02-22 2018-08-30 Université de Montréal Nouveaux antigènes mineurs d'histocompatibilité et leurs utilisations

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