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  • C12N5/0602—Vertebrate cells
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  • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/46—Cellular immunotherapy
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  • A61K39/464838—Viral antigens
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• • A—HUMAN NECESSITIES
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/10—Anthelmintics
  • A61P33/12—Schistosomicides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells
  • A61K2039/5158—Antigen-pulsed cells, e.g. T-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
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • 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
• • 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
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Definitions

• the invention relates to obtaining ex vivo specific CD4 T lymphocytes of the TH1 type.
• lymphocytes relates more particularly to such lymphocytes and a method for obtaining them by ex vivo induction of specific CD4 T lymphocyte lines of THl type for an immunoprophylactic or therapeutic purpose with regard to infections caused by an infectious agent such as 'a virus, bacteria or parasite.
• HIV Human Immunodeficiency Virus
• HIV infection is characterized by chronic overactivity of the immune system which contrasts with a deficiency in many of the cells of the immune system.
• vaccination against HIV should make it possible to transform chronic infection into acute infection, this by amplifying and accelerating the natural immune response.
• vaccine research on HIV has mainly focused on two axes: on the one hand, the induction of a humoral response mediated by antibodies neutralizing the virus and, on the other hand, the induction of cell-mediated immunity.
• neutralization experiments have made it possible to observe positive results in terms of protection, the exclusive use of neutralizing antibodies for vaccination purposes appears to be of little application due to important limitations such as the restriction to minority viral isolates as well that a very high required antibody concentration.
• CD8 T lymphocytes cytotoxic T lymphocytes (Cytotoxic T Lymphocytes or CTL)] reveal a correlation between the appearance of this activity and control of viral infection. Cytotoxic T lymphocytes directed against sequences derived from viral proteins appear very rapidly during the im response triggered by viral infection. In general, they play an important role in eliminating cells infected with the virus in acute infections and in blocking viral replication during persistent infections.
• T helper T helper
• CD4 T cells T helper cells which express the CD4 marker
• a response of the THl type (IFN- ⁇ , IL-2) or of the TH2 type (IL-4) does not have the same meaning because it is feared that in the second case, it is for example more harmful than useful for vaccination, and all of the work carried out to date clearly demonstrate the importance of the IFN- ⁇ -producing TH1 cells in protective anti-HIV immunity. But the orientation towards one type or the other of response is very difficult to control after injection of an antigen to an individual, in function of the genetic polymorphism expressed by the latter.
• Patent application WO 94/02156 (PCT / US93 / 06653) filed on July 15, 1993 and which claims priority from July 16, 1992, relates to methods using dendritic cells to activate T cells. These methods are based on knowledge which were available at that time with regard to the cellular components of the immune response, in particular anti-HIV.
• the authors propose a protocol for purifying dendritic cells for cell therapy purposes. These dendritic cells are directly drawn by an antigen, to activate and expand specific T cells (CD4 and CD8). This protocol requires several isolation steps to obtain a purity of 80 to 90% of the dendritic cells.
• CD8 T cells in the late stages of the disease, by transfer of CD8 T cells from healthy HLA compatible subjects, - in the early stages of the disease, by transfer of autologous CD8 T cells (after ex vivo reactivation) or healthy HLA compatible subjects.
• patent application WO 94/02156 in the particular case of HIV infection, constitutes an approach for obtaining a CD8 response.
• it does not expose the means making it possible to induce a specific CD4 TH1 response (more particularly generating a production of IFN- ⁇ ), allowing protection against infectious disorders, regardless of the HLA genotype of the donor.
• the methods and protocols described or provided for in this application are tedious or long (purification of dendritic cells in several stages, expansion over 6 to 8 weeks of cultures of CD4 or CD8 cells).
• the present invention overcomes these shortcomings and drawbacks.
• a line of specific CD4 T lymphocytes of the TH1 type effectively inducing cytotoxic T lymphocytes, that is to say an effective CD8 response, against an infection caused by an infectious agent.
• an infectious agent such as a virus, bacteria or parasite.
• the subject of the invention is a method making it possible to induce ex vivo, in a standardized manner, such a line.
• the subject of the invention is also a method of ex vivo induction of a line of specific CD4 T lymphocytes of TH1 type as defined above, which method essentially comprises the steps consisting in: a) taking from a donor a biological sample containing T lymphocytes; b) isolating the CD4 T lymphocytes from the sample taken in step a); in parallel c) obtain dendritic cells isolated from the same sample or from another sample from from the same donor; d) subjecting the CD4 T lymphocytes isolated in step b) to an immunological reaction or in vitro immunization with a peptide of a protein of the infectious agent having at least one T epitope, preferably a T epitope and a B epitope , in the presence of the dendritic cells of step c); e) performing at least one restimulation, preferably from one to three restimulations, under the same conditions as the immunization, optionally replacing the dendritic cells with B cells from the same donor
• peptide of a protein of the infectious agent is understood here to mean a peptide whose sequence is identical to that of the corresponding fraction of said protein or is modified by contribution of a lipid part or by induction of a controlled degeneration chemically, to an extent such that its function is maintained, or a mixture of at least two peptides as defined above.
• CD4 + T lymphocytes isolated in a previous step, are subjected directly to an immunological reaction or immunization in vitro with a peptide of a protein of the infectious agent to be combated, in the presence of dendritic cells previously isolated.
• the process can be implemented on any human or animal biological sample containing T lymphocytes. It presents, a priori, a particular interest in its applications to human beings.
• the biological sample used is preferably blood and, for applications of the immunoprophylactic or therapeutic type, it is autologous blood.
• the ability of this method to induce specific CD4 T lymphocyte lines of TH1 type necessary for the generation of CTL has, as shown in the examples described below, been verified on various pathogens.
• the specific CD1 T lymphocyte lines of TH1 type necessary for the generation of CTL can be reinjected into anti-HIV cell therapy protocols.
• the patients treated can in particular be seropositive patients in asymptomatic phase with CD4 T cell level still intact or patients undergoing cell renewal under high-efficiency antiviral therapy (Highly Active AntiRetroviral Therapy (HAART).
• HAART Highly Active AntiRetroviral Therapy
• the specific CD4 T lymphocyte lines of THl type obtained ex vivo according to the invention can therefore be used in addition to a HAART, for example triple therapy, for restoring THl potential. It goes without saying that for infections caused by other infectious agents, the pro usage tocol may be different.
• the lines can in particular, in certain cases, be used alone, that is to say not serve as a complement to therapy, for example when it is a question of carrying out the vaccination of a healthy subject.
• the specific CD4 T lymphocytes of the TH1 type according to the invention are autologous cells whose transfer is capable of inducing an effective CD8 response in vivo, without supply of CD8 cells.
• Example 1 Preparation of a Line of Specific CD4 T Lymphocytes of Type TH1, Anti-HIV and Results
• the inducer is the peptide
• Nef Native Factor
• the Nef gene is located 3 'to the HIV Env gene.
• the sequence of the Nef protein is described in:
• the peptide 56-68 sequence of the HIV Nef protein is
• This peptide contains both a T epitope and a B epitope.
• the biological medium used is peripheral human blood.
• the samples (100 to 150 ml) listed in the following table came from different donors typed for molecules of class II of the major complex histocompatibility (CMH), in particular HLA-DR.
• CMH major complex histocompatibility
• CD14 + blood monocytes are isolated by positive selection (Macs system, Milteny Biotech, Germany). The dendritic cells are then obtained at a purity of at least 80 to 90%, in a single step, by in vi tro differentiation of these CD14 + cells placed in the presence of IL-4 (1000 U / ml) and GM- CSF (800 U / ml) for five days in complete medium at 10% fetal calf serum (SVF). The CD14 "cells are for their part stored in complete medium at 10% human serum of group AB + for also five days.
• the CD4 + T lymphocytes are isolated by depletion or negative selection (Macs system, Milteny Biotech) from the CD14 ⁇ cells obtained previously.
• the CD4 + T lymphocytes (1.10 6 / ml) are then immunized in vitro by peptide 56-68 of the protein Nef (50 ⁇ g / ml) in the presence of the dendritic cells (DC) obtained in parallel (1.10 6 / ml).
• peptide 56-68 of the protein Nef 50 ⁇ g / ml
• DC dendritic cells
• the inventors having been able to show that freezing does not affect the functionality of the dendritic cells, the remaining dendritic cells are frozen and used for the first restimulation by the peptide Nef 56-68 which takes place fifteen days after the immunization.
• the dendriditic cells are generally replaced, in particular beyond the second restimulation, by donor B cells, isolated from the same sample by depletion of CD4 ⁇ cells into CD8 + cells (Macs system, Milteny Biotech).
• donor B cells isolated from the same sample by depletion of CD4 ⁇ cells into CD8 + cells (Macs system, Milteny Biotech).
• frozen dendritic cells can be used in all restimulations, as long as there are sufficient amounts.
• Diagram 1 which follows summarizes the various stages of obtaining, from whole blood, the various cells used: dendritic cells, CD4 + T lymphocytes and B cells.
• CD4 + Cell Dendritics Cell, CD4 + Cell.
• CD4 + cells + dendritic cells + peptide Culture 15 days
• IFN- ⁇ , IL-2, IL-4 and IL-5 was sought in culture supernatants of CD4 + T cells 24 h and 48 h after in vitro immunization ( I) and after successive restimulations (1S, 2S and
• CD4 + T cells very quickly (within 10 days) are generated having a phenotype of effector memory cells producing IFN- ⁇ after restimulation with peptide 56-68 from Nef.
• This phenotype is given on the basis of a decrease in markers CD45RA (characterizing naive T cells) and CD62Ligand (T cell homing receptor) and an increase in marker CD45RO (characterizing memory T cells) which reaches 36% at D10.
• Sm 28 GST 190-211 The procedure is as in Example 1 using peptide 190-211 of Sm 28 GST, Glutathione S Transferase of 28 kDa from the parasite Schi s tosoma mans oni, called Sm 28 GST 190-211 whose sequence is:
• Example 3 The procedure is as in Example 1 using the peptide 830-846 of the tetanus toxin. This peptide is called TT 830-846. Its sequence is:
• the CD4 T lymphocytes After immunization in vi tro and a restimulation, the CD4 T lymphocytes have a THl type orientation (with significant productions of IFN- ⁇ and / or IL-2 but never of IL-4 or IL-5 ) of the response obtained, regardless of the DR genotype of the sample.
• Example 2 The procedure is as in Example 1 using peptide 307-319 of 1 hemaglutinin of the Influenza virus. This peptide is called HA 307-319. Its sequence is:
• the CD4 T lymphocytes After immunization in vi tro and a restimulation, the CD4 T lymphocytes have a THl type orientation (with significant productions of IFN- ⁇ and / or IL-2 but never of IL-4 or L-5 ) of the response obtained, regardless of the DR genotype of the sample.
• Examples 2 to 4 show that the method which has been described in detail with regard to HIV can be generalized to infections due to other viruses, bacteria and parasites. The inventors have demonstrated that despite this generalization of the method, the T repertoire (determined on the basis of the expression of the Vbeta regions of the T receptor) expressed by the induced CD4 T cell was specific for the peptide used for in vitro immunization.
• the T clones derived after in vitro immunization and a restimulation by the peptide 56-68 of the protein Nef all exhibit a TH1 response profile and a preferential expression of V ⁇ 6.1.
• the present invention makes it possible to generate very quickly CD4 TH1 cells, specific, producing IFN- ⁇ , having a phenotype of memory cells and this whatever the HLA genotype of the donor.
• these cells can be used in cell therapy protocols, by adoptive transfer within 10 days of ex vivo immunization, in all pathologies where this type of cell has been described as protective and in particular in infection. by HIV (in particular by the generation of cells specific for peptide 56-68 of the protein Nef).

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• 1999
  • 1999-12-30 FR FR9916716A patent/FR2803307A1/fr active Pending
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