WO2021101466A1 - A method for producing multivirus specific t cells - Google Patents
A method for producing multivirus specific t cells Download PDFInfo
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- WO2021101466A1 WO2021101466A1 PCT/TR2019/051071 TR2019051071W WO2021101466A1 WO 2021101466 A1 WO2021101466 A1 WO 2021101466A1 TR 2019051071 W TR2019051071 W TR 2019051071W WO 2021101466 A1 WO2021101466 A1 WO 2021101466A1
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- cells
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- multivirus
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Classifications
-
- 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
- A61K39/12—Viral antigens
-
- 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
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- 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
- A61K39/464838—Viral antigens
-
- 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
- C12N5/0636—T lymphocytes
-
- 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
- A61K2039/70—Multivalent vaccine
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
- C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
- C12N2710/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
- C12N2710/22034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a method for production and expansion of particularly multivirus specific T cells which are convenient for use in antiviral treatment products and which are effective against donor-derived and multiple viruses.
- CMV cytomegalovirus
- AdV Epstein- Barr virus
- BKV BK virus
- Pharmacological agents that are used in the treatment of these infections have several side effects, especially toxicity. It is known that agents such as ganciclovir, valganciclovir, foscarnet, cidofovir, acyclovir cause side effects such as myelosuppression, leucopenia, neutropenia, nephrotoxicity, liquid-electrolyte imbalance. On the other hand, cidofovir and ribavirin agents used in AdV treatment may cause side effects in the central nervous system and toxicity; leflunomide and cidofovir agents used in BKV treatment may cause liver toxicity.
- T cells are combined with the antigen-presenting cells (APCs), which are transduced with a viral vector coding the relevant antigens or with plasmids.
- APCs antigen-presenting cells
- Antigen-presenting cells are used to stimulate T cells until the cells with enough specificity and number are reproduced.
- APCs antigen-presenting cells
- US Patent No. 6610542B1 discloses a method based on an ex-vivo expansion of CD4, CD8 and DP T cells obtained from HIV infected patients and on inhibition of the HIV gene. It is noted that the invention can be used in cell banks and organizations for restructuring immunity, to increase therapeutic effectiveness.
- virus-specific T cells T cells are incubated with multimers, which mimicking the MHC (major histocompatibility complex) bonding peptide of an APC. T cells that bonding the multimer are then isolated by using magnetic beads. Also a high amount of blood is needed to apply this method. Moreover, as the produced virus-specific T cells (VST) will be monospecific, it will not be possible to apply these to different patients.
- VST virus-specific T cells
- TR2018/05417 discloses a complex comprising an antibody- based part bonding a target antigen specifically and a virus-based peptide bonding an MHC class I protein complex. It is described that, by this complex, the virus-specific cytotoxic T cells (T memory cells or T effector cells) circulating in an individual can be directed to the said cells, by imitating an acute viral infection with MHC class I complexes of the cells expressing the target antigen to which the antibody-based part of the covalent complex specifically bonds.
- the invention provides this complex and the methods for producing and using this complex.
- T cells are activated by using necessary peptides. After the T cells are stimulated, antibodies bond to T cells secreting IFN-y by the magnetic selection, to allow their isolation. There is a need for leukapheresis in this method and the produced VSTs are monospecific like in the method of multimeric selection.
- Rapid cytotoxic T lymphocyte production is another method applied in the art.
- APC antigen-presenting cells
- Mononuclear cells are fused with peptides containing the desired viral antigens.
- APCs then promote the proliferation of T cells. Since CD45RA depletion is not performed in this method, there is a risk that naive T cells cause Graft-versus-Host disease (GVHD) in the recipient.
- T cell subgroups T S CM, T C M, T E F
- TEF effector T cell
- T S CM stem cell memory T cell
- T C M central memory T cell
- HLA human leukocyte antigen
- the present invention relates to a method for producing multivirus specific T cells, which meets the abovementioned requirements, removes all disadvantages and brings some additional advantages.
- the primary object of the invention is to develop a method for producing multivirus specific T cells that provide effective treatment against multiple viruses from donor T lymphocytes having immunity against target viruses.
- Another object of the invention is to develop a method for producing multivirus specific T cells that enable the treatment of the patient without developing resistance, without causing toxicity.
- a further object of the invention is to develop a method that provides depletion of CD45RA, which causing GVHD, and which paves the way for achieving a treatment product that is convenient for worldwide use.
- a further object of the invention is to develop a method which paves the way for a treatment product convenient for use on more than 80% of country populations, based on information obtained from HLA pools of different countries to form the necessary reaction being recognized by the viral antigen donor cells expressed by human leukocyte antigens (HLA).
- HLA human leukocyte antigens
- Yet another object of the invention is to develop a method providing stem cell memory, central memory and effector cells (T S CM, T C M, T E F) proliferation that shows longer and more effective activity against viral pathogens when compared to other T lymphocytes.
- the invention to fulfill the abovementioned objects is a method for producing multivirus specific T cells convenient for cellular therapy purposes against multiple viruses worldwide; comprising the steps of i. Making donor selections using HLA tissue information data and apheresis of blood drawn from the donor ii. Separating naive T cells (1) from apheresis allograft (2) by selectively labeling with magnetic CD45RA beads (3) iii.
- Figure 1 is a schematic view of steps (ii) to (iii) according to an embodiment of the inventive method.
- Figure 2 is a schematic view of steps (iv) to (vii) according to an embodiment of the inventive method.
- the invention is a method of production of multivirus-specific T cells convenient for use of worldwide for cellular therapy against multiple viruses; comprising the steps of i. Making donor selections using HLA tissue information data and apheresis of blood drawn from the donor ii. Separating naive T cells (1) from apheresis allograft (2) by selectively labeling with magnetic CD45RA beads (3) iii. Realizing CD45RA + depletion with the help of a magnetic column (4) and collecting naive T cells (5) under the magnetic column (4) iv. Stimulating separated mononuclear T cells (5) with a viral peptide pool (6) at least once v.
- the HLA mentioned in process step (i) is a human leukocyte antigen that forms the tissue type of the individual.
- HLA typing is used for the compatibility of patients and donors in marrow or cord blood transplants. If there is no HLA compliance during the transplant, the recipient perceives the externally given cells as foreign matter and the transplant fails from the beginning. HLA is important; because close compatibility increases the chance of transplant success, contributes to engraftment and reduces the risks associated with Graft- versus-Host Disease (GVHD).
- GVHD Graft- versus-Host Disease
- step (i) HLA pool information from different countries is examined to obtain a world-wide final product and more than one donor selection is made accordingly. At least two HLA alleles are sought for selection. In the most preferred embodiment of the invention; the compatibility of at least two HLA alleles of donors is at least 80%.
- naive T cells (1) It is known in the literature that alloreactivity is derived mainly from naive T cells (1). Therefore, in the invention, processing steps are selectively performed on naive T cells (1).
- the selective depletion of naive T cells (1) from allografts (2) protects the memory T cell response to pathogens. Therefore, in the preferred embodiment of the invention; in process step (ii), the naive T cells (1) are selectively separated from the apheresis allograft (2) from the blood obtained from the donor. Magnetic CD45RA beads (3) are used for this purpose.
- the cell suspension containing the naive T cells (1) marked with CD45RA beads (3) is passed through the magnet column (4) so that the CD45RA beads (3) remain in the magnet column (4) and CD45RA depleted mononuclear T cells (5) are collected under the magnet column (4).
- T cells collected here; T S CM, T C M, and T E F are composed of T cells.
- This step is a very important part of the invention as it eliminates the risk of compliance problems and GVHD formation in the patient the final product is transplanted.
- Graft-versus-host disease is a complex clinical syndrome resulting from severe immunological reaction mediated by healthy T lymphocytes taken from the donor and delivered to the patient with stem cells.
- the viral peptide pool (6) mentioned in process step (iv) comprises AdV, EBV, CMV, BKV peptides. These peptides are selected from the most common peptides that cause viral infection, thereby targeting to obtain a final product appealing to multiple patients.
- said viral peptide pool (6) comprises AdV5 Hexon, CMV pp65, EBV LMP2A, EBV EBNA-1 , BKV VP1 peptides.
- step (iv) is carried out on day 0 of the process.
- the specifically selected cytokine feed (7) containing IL-2 and IL-7 in process step (v) is intended to make the populations of T S CM, T C M cells survive. This step is carried out 3 times, preferably on days 0, 3 and 5 of the process; thus, virus-specific T cell activation (8) is provided.
- the viral peptide pool referred to in process step (vi) comprises AdV, EBV, CMV, BKV peptides, like process step (6).
- This step makes it possible to obtain multivirus-specific T cells (12).
- said viral peptide pool (6) comprises AdV5 Hexon, CMV pp65, EBV LMP2A, EBV EBNA-1 , BKV VP1 peptides.
- step (vi) is carried out on day 6 of the process.
- the cytokine feed (10) containing specifically selected IL-2, IL-7, and IL-15 in process step (vii) is intended to produce a cell population comprising T S CM, T C M cell populations as well as T E F cells.
- the dose of IL-2 is kept higher than the dose of IL-2 in process step (v).
- process step (vii) is carried out 3 times, on days 7, 9 and 11 of the process; thus, proliferation (11) of multivirus-specific T cells is provided.
- process step (viii) is carried out on day 13 of the process.
- VST activity and cytotoxicity experiments were performed from mononuclear cells from healthy donors.
- T2 cells B cell line
- virus-specific peptide control
- 7AAD- live T2 cell ratios were evaluated by flow cytometric analysis with CD19 labeling.
- VST activation CD25
- cytotoxicity level dead (7AAD-) CD19 + cells
- T cells With the method of the invention; without causing toxicity, it is possible to realize a product that allows the patient to be treated without developing resistance. Also, it is globally convenient as the depletion of CD45RA is provided in T cells by the method. In the method developed based on the information obtained from HLA pools, it becomes possible to obtain a treatment product convenient for use in more than 80% of the country's populations. Furthermore, compared with T lymphocytes used in the state of the art, proliferation of stem cell memory, central memory and effector T cells (T S CM, T C M, T E F), which have longer and more effective activity against viral pathogens is provided.
- T S CM, T C M, T E F central memory and effector T cells
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Abstract
The present invention relates to a method for producing multivirus specific T cells convenient for use for cellular therapy purposes against multiple viruses worldwide, comprising the steps of making donor selections using HLA tissue information data and apheresis of blood obtained from the donor; separating naive T cells (1) from apheresis allograft (2) by selectively labeling with magnetic CD45RA beads (3); performing CD45RA+ depletion with the help of a magnetic column (4) and collecting naive T cells (5) under the magnetic column (4); stimulating separated mononuclear T cells (5) with a viral peptide pool (6) at least once; feeding cytokines with IL-2 and IL-7 content (7) on different days to the T cell (8) population formed; multivirus specification of T cell (8) population by re-stimulation with viral peptide pool (6); ensuring the expansion of multivirus specific T cell (11) population by feeding cytokines (10) with IL-2, IL-7 and IL-15 content on different days; and determining the peptide-specific activity of the produced cells.
Description
A METHOD FOR PRODUCING MULTIVIRUS SPECIFIC T CELLS
Technical Field
The present invention relates to a method for production and expansion of particularly multivirus specific T cells which are convenient for use in antiviral treatment products and which are effective against donor-derived and multiple viruses.
State of the Art
In allogeneic transplants, one of the most crucial complications is viral infections. Agents used in the treatment of these infections are extremely limited and have high rates of side effects. In treatments with pharmacological agents, complete recovery cannot be achieved, apart from cytomegalovirus (CMV) infection. Moreover, the continuous use of the drug, due to low efficacy, causes the virus to develop resistance against the drug. Besides all these, there are various side effects, toxicity being in the first place. In a treatment method by which complete recovery cannot be achieved, the constant use of pharmacological agents also has high costs. Viral infections that are frequently seen following the allogeneic transplant can be listed as cytomegalovirus (CMV), adenovirus (AdV), Epstein- Barr virus (ENV) and BK virus (BKV).
Pharmacological agents that are used in the treatment of these infections have several side effects, especially toxicity. It is known that agents such as ganciclovir, valganciclovir, foscarnet, cidofovir, acyclovir cause side effects such as myelosuppression, leucopenia, neutropenia, nephrotoxicity, liquid-electrolyte imbalance. On the other hand, cidofovir and ribavirin agents used in AdV treatment may cause side effects in the central nervous system and toxicity; leflunomide and cidofovir agents used in BKV treatment may cause liver toxicity.
Due to these disadvantages brought along by the pharmacological agents used for treatment in viral infections, virus-specific T cell applications are preferred today.
One of these applications is the ex-vivo expansion method. In this method, T cells are combined with the antigen-presenting cells (APCs), which are transduced with a viral vector coding the relevant antigens or with plasmids. Antigen-presenting cells are used to stimulate T cells until the cells with enough specificity and number are reproduced. For instance, US Patent No. 6610542B1 discloses a method based on an ex-vivo expansion of CD4, CD8 and DP T cells obtained from HIV infected patients and on inhibition of the HIV gene. It is noted that the invention can be used in cell banks and organizations for restructuring immunity, to increase therapeutic effectiveness. However, placing the viral vector or the plasmid into the antigen-presenting cell requires a large scale of manipulation. Moreover, isolation of dendritic cells, which were used as antigen- presenting cells is difficult; because these cells have lost their mitosis characteristics in LH (Langerhans cell) and IDC (interdigitating dendritic cell) stages. Therefore, even though they are isolated, it is impossible to replicate them.
Another method used in the state of the art is the multimeric selection. To prepare virus-specific T cells, T cells are incubated with multimers, which mimicking the MHC (major histocompatibility complex) bonding peptide of an APC. T cells that bonding the multimer are then isolated by using magnetic beads. Also a high amount of blood is needed to apply this method. Moreover, as the produced virus-specific T cells (VST) will be monospecific, it will not be possible to apply these to different patients.
Regarding this, TR2018/05417 discloses a complex comprising an antibody- based part bonding a target antigen specifically and a virus-based peptide bonding an MHC class I protein complex. It is described that, by this complex, the virus-specific cytotoxic T cells (T memory cells or T effector cells) circulating in an individual can be directed to the said cells, by imitating an acute viral infection with MHC class I complexes of the cells expressing the target antigen to which the antibody-based part of the covalent complex specifically bonds. In the said document, the invention provides this complex and the methods for producing and using this complex.
In the gamma capture technique, which is another method, T cells are activated by using necessary peptides. After the T cells are stimulated, antibodies bond to T cells secreting IFN-y by the magnetic selection, to allow their isolation. There is a need for leukapheresis in this method and the produced VSTs are monospecific like in the method of multimeric selection.
Rapid cytotoxic T lymphocyte production is another method applied in the art. In this method, antigen-presenting cells (APC), which are present in peripheral blood mononuclear cells, are used. Mononuclear cells are fused with peptides containing the desired viral antigens. APCs then promote the proliferation of T cells. Since CD45RA depletion is not performed in this method, there is a risk that naive T cells cause Graft-versus-Host disease (GVHD) in the recipient. In this production method, T cell subgroups (TSCM, TCM, TEF) which are formed only due to the use of IL-2 are not homogeneous and most of them have short-lived TEF (effector T cell) character.
Methods in the state of the art are mostly for virus-specific T cell production and result in obtaining a low number of VST, and a high amount of donor blood is needed for the production. Also, it is impossible to use such products for more than one patient. Generally, IL-2 based proliferation techniques are used for this in the present day. However, by this technique, the generation of TSCM (stem cell memory T cell) and TCM (central memory T cell) are less , which results short- termed product effectiveness.
Besides these, there is a requirement for at least one HLA (human leukocyte antigen) compatibility for global virus-specific T lymphocytes produced worldwide, while there is no planned production and donor selection technique peculiar to country populations based on country-specific HLA tissue information data. It is an important problem that donor-derived virus-specific T lymphocytes cause GVHD (graft versus host disease) risk due to tissue reaction in the patient, and it restricts the applicability of the treatment.
Analyzing the studies in the state of the art, it is seen that there is a need for multivirus specific T cells that provide effective cellular therapy against multiple viruses, prepared by using more than one donors and convenient to use safely in more than 80% of country populations, and that provide long-term effectiveness. Also, a method for the production thereof.
Brief Description of the Invention
The present invention relates to a method for producing multivirus specific T cells, which meets the abovementioned requirements, removes all disadvantages and brings some additional advantages.
The primary object of the invention is to develop a method for producing multivirus specific T cells that provide effective treatment against multiple viruses from donor T lymphocytes having immunity against target viruses.
Another object of the invention is to develop a method for producing multivirus specific T cells that enable the treatment of the patient without developing resistance, without causing toxicity.
A further object of the invention is to develop a method that provides depletion of CD45RA, which causing GVHD, and which paves the way for achieving a treatment product that is convenient for worldwide use.
Still, a further object of the invention is to develop a method which paves the way for a treatment product convenient for use on more than 80% of country populations, based on information obtained from HLA pools of different countries to form the necessary reaction being recognized by the viral antigen donor cells expressed by human leukocyte antigens (HLA).
Yet another object of the invention is to develop a method providing stem cell memory, central memory and effector cells (TSCM, TCM, TEF) proliferation that shows longer and more effective activity against viral pathogens when compared to other T lymphocytes.
The invention to fulfill the abovementioned objects is a method for producing multivirus specific T cells convenient for cellular therapy purposes against multiple viruses worldwide; comprising the steps of i. Making donor selections using HLA tissue information data and apheresis of blood drawn from the donor ii. Separating naive T cells (1) from apheresis allograft (2) by selectively labeling with magnetic CD45RA beads (3) iii. Realizing CD45RA+ depletion with the help of a magnetic column (4) and collecting naive T cells (5) under the magnetic column (4) iv. Stimulating separated mononuclear T cells (5) with a viral peptide pool (6) at least once v. Feeding cytokines with IL-2 and IL-7 content (7) on different days to the T cell (8) population formed vi. Re-stimulating the T cell (8) population at least once with the viral peptide pool (6) to make the multi virus-specific vii. Ensuring the expansion of multivirus specific T cell (11) population by feeding cytokines (10) with IL-2, IL-7, and IL-15 content on different days viii. Determining the peptide-specific activity of the produced cells.
The structural and characteristic features and all advantages of the invention will become more apparent from the following figure and the detailed description written regarding this figure. Therefore, the evaluation should be made considering this detailed explanation.
Figures to Better Understand the Invention
Figure 1 is a schematic view of steps (ii) to (iii) according to an embodiment of the inventive method. Figure 2 is a schematic view of steps (iv) to (vii) according to an embodiment of the inventive method.
Description of References
1 . Naive T cell
2. Allograft
3. CD45RA bead
4. Magnetic column
5. Mononuclear T cell with CD45RA depletion 6. Viral peptide pool
7. Cytokine feed with IL-2 and IL-7 content
8. Virus-specific T cell activation
9. Virus-specific T cell
10. Cytokine feed with IL-2, IL-7, and IL-15 11. Multivirus specific T cell proliferation
12. Multivirus specific T cell
Detailed Description of the Invention
In this detailed description, preferred embodiments of a method to produce multivirus-specific T cells are described for a better understanding of the subject and with no limiting effect.
The invention, is a method of production of multivirus-specific T cells convenient for use of worldwide for cellular therapy against multiple viruses; comprising the steps of i. Making donor selections using HLA tissue information data and apheresis of blood drawn from the donor ii. Separating naive T cells (1) from apheresis allograft (2) by selectively labeling with magnetic CD45RA beads (3) iii. Realizing CD45RA+ depletion with the help of a magnetic column (4) and collecting naive T cells (5) under the magnetic column (4) iv. Stimulating separated mononuclear T cells (5) with a viral peptide pool (6) at least once v. Feeding cytokines with IL-2 and IL-7 content (7) on different days to the T cell (8) population formed vi. Re-stimulating the T cell (8) population at least once with the viral peptide pool (6) to make the multivirus specific
vii. Ensuring the expansion of multivirus specific T cell (11) population by feeding cytokines (10) with IL-2, IL-7, and IL-15 content on different days viii. Determining the peptide-specific activity of the produced cells.
The HLA mentioned in process step (i) is a human leukocyte antigen that forms the tissue type of the individual. HLA typing is used for the compatibility of patients and donors in marrow or cord blood transplants. If there is no HLA compliance during the transplant, the recipient perceives the externally given cells as foreign matter and the transplant fails from the beginning. HLA is important; because close compatibility increases the chance of transplant success, contributes to engraftment and reduces the risks associated with Graft- versus-Host Disease (GVHD).
In a preferred embodiment of the invention, in step (i), HLA pool information from different countries is examined to obtain a world-wide final product and more than one donor selection is made accordingly. At least two HLA alleles are sought for selection. In the most preferred embodiment of the invention; the compatibility of at least two HLA alleles of donors is at least 80%.
It is known in the literature that alloreactivity is derived mainly from naive T cells (1). Therefore, in the invention, processing steps are selectively performed on naive T cells (1). The selective depletion of naive T cells (1) from allografts (2) protects the memory T cell response to pathogens. Therefore, in the preferred embodiment of the invention; in process step (ii), the naive T cells (1) are selectively separated from the apheresis allograft (2) from the blood obtained from the donor. Magnetic CD45RA beads (3) are used for this purpose.
In a preferred embodiment of the invention; in process step (iii), the cell suspension containing the naive T cells (1) marked with CD45RA beads (3) is passed through the magnet column (4) so that the CD45RA beads (3) remain in the magnet column (4) and CD45RA depleted mononuclear T cells (5) are collected under the magnet column (4). T cells collected here; TSCM, TCM, and TEF (stem cell memory, central memory, and effector) are composed of T cells. This
step is a very important part of the invention as it eliminates the risk of compliance problems and GVHD formation in the patient the final product is transplanted. Graft-versus-host disease is a complex clinical syndrome resulting from severe immunological reaction mediated by healthy T lymphocytes taken from the donor and delivered to the patient with stem cells.
In a preferred embodiment of the invention; the viral peptide pool (6) mentioned in process step (iv) comprises AdV, EBV, CMV, BKV peptides. These peptides are selected from the most common peptides that cause viral infection, thereby targeting to obtain a final product appealing to multiple patients. According to the most preferred embodiment of the invention; said viral peptide pool (6) comprises AdV5 Hexon, CMV pp65, EBV LMP2A, EBV EBNA-1 , BKV VP1 peptides. In a preferred embodiment of the invention, step (iv) is carried out on day 0 of the process.
In a preferred embodiment of the invention; the specifically selected cytokine feed (7) containing IL-2 and IL-7 in process step (v) is intended to make the populations of TSCM, TCM cells survive. This step is carried out 3 times, preferably on days 0, 3 and 5 of the process; thus, virus-specific T cell activation (8) is provided.
In a preferred embodiment of the invention; the viral peptide pool referred to in process step (vi) comprises AdV, EBV, CMV, BKV peptides, like process step (6). This step makes it possible to obtain multivirus-specific T cells (12). According to the most preferred embodiment of the invention; said viral peptide pool (6) comprises AdV5 Hexon, CMV pp65, EBV LMP2A, EBV EBNA-1 , BKV VP1 peptides. In a preferred embodiment of the invention, step (vi) is carried out on day 6 of the process.
In a preferred embodiment of the invention; the cytokine feed (10) containing specifically selected IL-2, IL-7, and IL-15 in process step (vii) is intended to produce a cell population comprising TSCM, TCM cell populations as well as TEF cells. In this step, preferably, the dose of IL-2 is kept higher than the dose of IL-2 in process step (v). In a preferred embodiment of the invention; process step (vii)
is carried out 3 times, on days 7, 9 and 11 of the process; thus, proliferation (11) of multivirus-specific T cells is provided.
In a preferred embodiment of the invention; process step (viii) is carried out on day 13 of the process.
VST activity and cytotoxicity experiments were performed from mononuclear cells from healthy donors. T2 cells (B cell line) incubated in the presence or absence of virus-specific peptide (control) at different concentrations (3000-1000-100-10-1 nM) were washed and co-cultured with VST cells. After 24 hours, live (7AAD-) T2 cell ratios were evaluated by flow cytometric analysis with CD19 labeling. In comparative analyses, it was found that VST activation (CD25) and cytotoxicity level (dead (7AAD-) CD19 + cells) were increased at the increased concentration level of virus peptides. The proportional differences in the activity of virus-specific peptides on VST indicate that VST should be produced from different donors and that using pools is beneficial.
With the method of the invention; without causing toxicity, it is possible to realize a product that allows the patient to be treated without developing resistance. Also, it is globally convenient as the depletion of CD45RA is provided in T cells by the method. In the method developed based on the information obtained from HLA pools, it becomes possible to obtain a treatment product convenient for use in more than 80% of the country's populations. Furthermore, compared with T lymphocytes used in the state of the art, proliferation of stem cell memory, central memory and effector T cells (TSCM, TCM, TEF), which have longer and more effective activity against viral pathogens is provided.
Claims
1. A method for producing multivirus specific T cells convenient to use for cellular therapy purposes against multiple viruses worldwide; characterized in comprising process steps of; i. Making donor selections using HLA tissue information data and apheresis of blood obtained from the donor, ii. Separating naive T cells (1) from apheresis allograft (2) by selectively labeling with magnetic CD45RA beads (3), iii. Performing CD45RA+ depletion with the help of a magnetic column (4) and collecting naive T cells (5) under the magnetic column (4), iv. Stimulating separated mononuclear T cells (5) with a viral peptide pool (6) at least once, v. Feeding cytokines with IL-2 and IL-7 content (7) on different days to the T cell (8) population formed, vi. Re-stimulating the T cell (8) population at least once with the viral peptide pool (6) to make the multivirus specific, vii. Ensuring the expansion of multivirus specific T cell (11) population by feeding cytokines (10) with IL-2, IL-7, and IL-15 content on different days, viii. Determining the peptide-specific activity of the produced cells.
2. The method according to Claim 1 , characterized in that compatibility of at least two HLA alleles is sought in the selection of donors mentioned in the process step (i).
3. The method according to Claim 2, characterized in that compatibility of at least two HLA alleles of donors is at least 80%.
4. The method according to Claim 1 , characterized in that the mononuclear T cells (5) mentioned in the process step (iii) comprise TSCM, TCM and TEF cells.
5. The method according to Claim 1 , characterized in that the viral peptide pool (6) mentioned in process step (iv) comprises AdV, EBV, CMV, BKV peptides.
6. The method according to Claim 5, characterized in that the viral peptide pool (6) mentioned in the process step (iv) comprises AdV5 Hexon, CMV pp65, EBV LMP2A, EBV EBNA-1 , BKV VP1 peptides.
7. The method according to any one of the Claims 1 , 5 and 6, characterized in that the process step (iv) is carried out on day 0 of the process.
8. The method according to Claim 1 , characterized in that cytokine feed (7) with IL-2 and IL-7 content mentioned in the process step (v) is carried out 3 times, on days 0, 3 and 5 of the process.
9. The method according to Claim 1 , characterized in that the viral peptide pool mentioned in the process step (vi) comprises AdV, EBV, CMV, BKV.
10. The method according to Claim 10, characterized in that the viral peptide pool (6) mentioned in the process step (vi) comprises AdV5 Hexon, CMV pp65, EBV LMP2A, EBV EBNA-1 , BKV VP1 peptides.
11. The method according to any one of Claims 1 , 9 and 10, characterized in that the process step (vi) is carried out on day 6 of the process.
12. The method according to Claim 1 , characterized in that the cytokine feed (10) with IL-2, IL-7, and IL-15 content is carried out 3 times, on days 7, 9 and 11 of the process.
13. The method according to Claim 1 , characterized in that the process step (viii) is carried out on day 13 of the process.
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| EP19928301.1A EP4061386A4 (en) | 2019-11-19 | 2019-12-13 | A method for producing multivirus specific t cells |
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| TR201918044 | 2019-11-19 | ||
| TR2019/18044 | 2019-11-19 |
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| ES2928851T3 (en) * | 2012-02-09 | 2022-11-23 | Baylor College Medicine | Pep mixes to generate multiviral CTLs with broad specificity |
| AU2017326173B2 (en) * | 2016-09-16 | 2022-08-18 | Baylor College Of Medicine | Platform for activation and expansion of virus-specific T-cells |
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Non-Patent Citations (5)
| Title |
|---|
| FAIST BENJAMIN, SCHLOTT FABIAN, STEMBERGER CHRISTIAN, DENNEHY KEVIN M., KRACKHARDT ANGELA, VERBEEK MAREIKE, GRIGOLEIT GÖTZ U., SCH: "Targeted in-vitro-stimulation reveals highly proliferative multi-virus- specific human central memory T cells as candidates for prophylactic T cell therapy", PLOS ONE, vol. 14, no. 9, 30 September 2019 (2019-09-30), pages e0223258, XP055830387, DOI: 10.1371/joumal.pone.0223258 * |
| LITVINOVA L.S. ET AL.: "The influence of immunoregulatory cytokines IL -2, IL -7, and IL -15 upon activation, proliferation, and apoptosis of immune memory T-cells in vitro", CELL TISS. BIOL., vol. 7, 11 December 2013 (2013-12-11), pages 539 - 544, XP035366789, DOI: 10.1134/S1990519X13060072 * |
| QIAN C, WANG Y, REPPEL L, D'AVENI M, CAMPIDELLI A, DECOT V, BENSOUSSAN D: "Viral-specific T- cell transfer from HSCT donor for the treatment of viral infections or diseases after HSCT", BONE MARROW TRANSPLANT, vol. 53, no. 2, 23 October 2017 (2017-10-23), pages 114 - 122, XP055830388, DOI: 10.1038/bmt.2017.232 * |
| See also references of EP4061386A4 * |
| TESCHNER D. ET AL.: "Depletion of naive T cells using clinical grade magnetic CD 45RA beads: a new approach for GVHD prophylaxis", BONE MARROW TRANSPLANTATION, vol. 49, 12 August 2013 (2013-08-12), pages 138 - 144, XP055358248, DOI: 10.1038/bmt.2013.114 * |
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| EP4061386A4 (en) | 2024-01-10 |
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