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EP4061385A1 - Procédé de reconception et d'expansion de cellules nk92 destinées à être utilisées en immunothérapie - Google Patents

Procédé de reconception et d'expansion de cellules nk92 destinées à être utilisées en immunothérapie

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Publication number
EP4061385A1
EP4061385A1 EP19926735.2A EP19926735A EP4061385A1 EP 4061385 A1 EP4061385 A1 EP 4061385A1 EP 19926735 A EP19926735 A EP 19926735A EP 4061385 A1 EP4061385 A1 EP 4061385A1
Authority
EP
European Patent Office
Prior art keywords
cells
car
assassin
egfrt
expansion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP19926735.2A
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German (de)
English (en)
Other versions
EP4061385A4 (fr
Inventor
Ercüment OVALI
Derya DILEK KANÇAGI
Cihan TASTAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Acibadem Labmed Saglik Hizmetleri AS
Acibadem Mehmet Ali Aydinlar Ueniversitesi
Original Assignee
Acibadem Labmed Saglik Hizmetleri AS
Acibadem Mehmet Ali Aydinlar Ueniversitesi
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Application filed by Acibadem Labmed Saglik Hizmetleri AS, Acibadem Mehmet Ali Aydinlar Ueniversitesi filed Critical Acibadem Labmed Saglik Hizmetleri AS
Publication of EP4061385A1 publication Critical patent/EP4061385A1/fr
Publication of EP4061385A4 publication Critical patent/EP4061385A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
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    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
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    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
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    • A61K40/4204—Epidermal growth factor receptors [EGFR]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
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    • 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
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • 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
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
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    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention is related to a method for the production and expansion of the modified NK92 cells for use in cancer immunotherapy.
  • the invention particularly relates the production of ASSASSIN (CD16+IL-12 or IL-18+ NK92) cells, which carry innate immune simulator CD16 receptor and secrete IL-12 or IL- 18 cytokines, and CAR-ASSASSIN (CAR+CD16+IL-12 or IL-18 +NK92) cells, which synthesize chimeric antigen receptor (CAR), to be used in the cancer immunotherapy.
  • ASSASSIN CD16+IL-12 or IL-18+ NK92
  • CAR-ASSASSIN CAR+CD16+IL-12 or IL-18 +NK92
  • Cancer is a complex disease caused by uncontrolled division and proliferation of cells, and that is under the influence of genetic and environmental conditions. Although there are more than a hundred known types of cancers and standard approaches are present for certain types of cancers, cancer is indeed a personal disease. It is not surprising that people respond differently to similar treatments because the genome of any human is not alike. With the advancement of technology, new treatment methods are being developed in addition to existing treatments. Immunotherapy is the most important and promising one.
  • the immunotherapy method supports the body resistance who targets cancer cells, increases immunity and tries to get rid of cancer cells in this manner.
  • Immunotherapy is very beneficial for the patient and comprises using some immune system cells that have cytotoxic activity against target cancer cells.
  • the agents used in the cellular immunotherapy against cancer are not effective in causing complete remission.
  • the main reason for this is that although present immune cells are stimulated byb the cancer tissue, they are not enough in specific mechanisms.
  • Natural Killer (Naturel killer, NK) cells are very popular in the cellular immunotherapy depending on its anti-tumor feature.
  • the NK cells represent approximately 10- 15% of the lymphocytes in the peripheral blood and kill the targeted cells including the virus-infected cells and many malign cells concerning the antigen specificity and without prior immune sensitization. Killing the targeted cells is performed by means of inducing the cell lysis.
  • the studies show that allogeneic NK cell lines including NK92 fulfill the in vivo cytotoxic functions. In GMP standards, since high numbers are reached through culture, it can be used as a continuous source in the immunotherapy for many patients (Klingemann, Frontiers in Immunology, 2016).
  • Transgenic CAR-NK92 cells coding NK92 or chimeric antigen receptor (CAR) are used in clinical studies for cancer patients depending on its anti-tumor activity (NCT00900809, NCT03383978, NCT02465957, NCT02944162, NCT00990717). Also, together with the NK92 cells coding CD16 preclinical in vivo studies are started (Williams, Haematologica, 2018). For this reason, the actively cytotoxic NK92 cell line is seen as a ready for sale (of-the-shelf) and standard immunotherapy agent (Oelsner, Cytotherapy, 2017).
  • US20080247990 A1 discloses the presence of a natural killer cell, NK92, or an inhibitory killer cell immunoglobulin-like receptor (KIR) modified to express a CD16 receptor.
  • NK92 natural killer cell
  • KIR inhibitory killer cell immunoglobulin-like receptor
  • NK92 cells do not show cancer-specific properties, they should be directed to the target tumor (Raulet et al., Nature Reviews Immunology, 2006).
  • CD19 which is a target antigen diminishes on the cancer cells so that CAR-T cell effect remains insufficient and tumor relapses.
  • an improvement in the relevant technical field was required.
  • the present invention relates to a method for production of modified NK92 cells for use in cancer immunotherapy that meets the above-mentioned requirements, eliminates all disadvantages and brings some additional advantages.
  • NK92 cells do not express the FcyRIII receptor (CD16). Therefore, NK92 cells do not cause antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Interleukin-12 is a pleiotropic cytokine that plays an important role in the immune response against cancer (anti-tumor activity).
  • the present invention aims to create an effective cancer immunotherapy model with IL-12-induced innate (NK cells, monocytes and macrophages) and adaptive (cytotoxic T lymphocytes, T helper cells) immunity to be secreted by NK92 cells.
  • IL-18 Transgenic CD16 & IL-18 synthesizing NK92 cells will be produced if IL-12 secretion causes toxicity or poor anti-tumor activity. Therefore, the gene sequence of IL-18 (Uniprot, Q14116, 157 amino acid) with CD16 is encoded in the lentivirus. IL-18-secreting CAR- modified T cells showed superior antitumor activities in various tumor models (Cell Rep. 2017 Dec 12; 21 (11): 3205-3219). The effect of IL-18, which belongs to the cytokine family of IL-1 and is produced mainly by macrophages, DCs, epithelial cells, fibroblasts, and microglia cells, is pleiotropic with both pro-inflammatory and immune regulatory functions. IL-18 was initially identified as a stimulating factor due to its IFNy-stimulating effects on natural killer (NK) cells, monocytes, dendritic cells (DC), B cells as well as T cells.
  • NK natural killer
  • DC dendritic cells
  • TAA cancer-specific tumor-associated antigen
  • CAR chimeric antigen receptor
  • the invention provides the first use of an off-the-shelf modified NK92 cell line that secretes IL-12 or IL-18 cytokines and can be simultaneously targeted to various tumor- specific antigens through CD16 receptor, in the context of bridging therapy (to shrink the tumor before the main treatment). It is expected that the IL-12 or IL-18 induced host immune system will continue to fight with the tumor even after the irradiated ASSASSIN (CD16+ IL-12 or IL-18+ NK92) or CAR-ASSASSIN (CAR + CD16 + IL-12 or IL-18+ NK92) cells are destroyed.
  • ASSASSIN CD16+ IL-12 or IL-18+ NK92
  • CAR-ASSASSIN CAR + CD16 + IL-12 or IL-18+ NK92
  • the invention provides a method for the production of ASSASSIN (CD16+ IL-12 or IL-18+ NK92) and CAR-ASSASSIN (CAR+ CD16+ IL-12 or IL-18+ NK92) cells that express the innate immunostimulatory CD16 receptor and synthesize IL-12 or IL-18 cytokines along with chimeric antigen receptor (CAR).
  • ASSASSIN CD16+ IL-12 or IL-18+ NK92
  • CAR-ASSASSIN CAR+ CD16+ IL-12 or IL-18+ NK92
  • NK cells do not resemble T and B cells. There is no thymic maturation, and the presence of NK cells was demonstrated in animals upon thymus removal. These cells do not contain Ig, TCR and CD3 molecules. In contrast, they contain the FcRIII receptor, CD16, which shows a low affinity for the Fc fragment of the CD2 molecule and IgG. Monoclonal antibodies used against this CD16 receptor are important for the identification of NK cells.
  • NK92, CD16+ NK92 or transgenic CAR-NK92 cell therapies have been tested in many preclinical and/or clinical studies as bridging therapy to the transplant to prolong survival of patients who were unresponsive to standard methods. Since the cells were irradiated during this treatment, their efficacy was observed for a short time. To increase cytotoxic efficiency and specificity in the treatment against multiple types of cancer (for universal targeting), CD16-expressing NK92 cells were generated. Thus, antibody-dependent cellular cytotoxicity (ADCC) through CD16 and tumor antigen-specific monoclonal antibodies will be induced to increase anti-tumor activity.
  • ADCC antibody-dependent cellular cytotoxicity
  • ASSASSIN CD16+ IL-12 or IL-18+ NK92
  • CAR-ASSASSIN CAR+ CD16+ IL-12 or IL-18+ NK92
  • IL-12 or IL-18 cytokines are expected to trigger the patient's innate and adaptive immune system against cancer.
  • the activated host immune system will continue to fight with the tumor even after the destruction of the irradiated ASSASSIN or CAR-ASSASSIN cells.
  • an off-the-shelf modified NK92 cell line secreting IL-12 or IL-18 cytokines that can be simultaneously targeted to multiple tumor-specific antigens through CD16 along with CAR will be used for the first time in bridging therapy in cancer treatments.
  • the modified NK-92 cells or cell lines comprises IL-12 or IL-18, CD16, CAR.
  • IL-12 Interleukin-12
  • IL-18 enables increasing the anti-tumor activity by stimulating the innate and the adaptive immune cells of the patient.
  • Fc ⁇ lll receptor CD16
  • TAA tumor associated antigen
  • CAR provides cytotoxic effect to the target tumor by means of the chimeric antigen receptor specific to the TAA.
  • EGFRt truncated form of Epidermal Growth Factor Receptor
  • CAR-NK92 cells are directed to the programmed cell death with the EGFR-specific monoclonal antibody when required.
  • NK and NK92 cells During usage of NK and NK92 cells in a method for cancer immunotherapy, the realized biological processes are shown in the following in a comparative manner.
  • the production method of the modified NK92 cells for use in cancer immunotherapy consists of the following process steps; a) Lentiviral CD16-IL-12 or IL-18 gene design b) CD16-IL-12 or IL-18 coding lentivirus production c) Expansion of the NK92 cells transduced with CD16-IL-12 or IL-18 Lentiviruses d) Marking and isolation of the expanded NK92 cells expressing CD16 e) Expansion of CD16+ IL-12 or IL-18+ NK92 (ASSASSIN) cells f) Determination of IL-12 or IL-18 secretion from CD16+NK92 (ASSASSIN) cells g) TAA specific chimeric antigen receptor (CAR) & EGFRt gene design h) CAR-EGFRt lentivirus production i) Gene transfer with the 2 nd transduction agent coding CAR-EGFRt and expansion of CD16+ IL-12 or IL-18+ NK92 cells j
  • ASSASSIN CD16+ IL-12 or IL-18+ NK92
  • CAR-ASSASSIN CAR+ CD16+ IL-12 or IL-18+ NK92
  • CD16 - Fc ⁇ RIIIa receptor sequence (P08637, UniProtKB; 1-254 amino acids) which will stimulate the antibody dependent cellular cytotoxicity (ADCC)
  • CD16-IL-12 lentivirus production is enabled following transfection on HEK293 cells.
  • the positive selection and isolation of CD16+ NK92 cells is performed with Streptavidin-bead through the magnetic column.
  • CD16+IL-12+NK92 ASSASSIN
  • the cells are targeted to the tumor by means of CD16 after the transfusion of tumor associated antigen (TAA) specific monoclonal antibodies (for example, by means of providing tumor specificity by using CD20 antibody /Mabthera, the antibody dependent cytotoxicity would be triggered).
  • TAA tumor associated antigen
  • ADCC antibody dependent cellular cytotoxicity
  • the CD16+NK92 cells at the same time synthesize the IL-12a and IL-12b subunit proteins.
  • the produced IL-12a and IL-12b proteins would form a heterodimer structure and provides formation of IL-12.
  • the activated immune cells start to exhibit anti-tumor effects by means of migrating towards the tumor cells.
  • CD16-IL-12+ NK92 cells Transduction of CD16-IL-12+ NK92 cells; following chimeric antigen receptor (CAR) & EGFRt gene design and lentivirus production (10):
  • CD8a protein signal peptide sequence P01732, UniProtKB; 1-21 amino acids
  • CAR chimeric antigen receptor’s
  • - ScFv antibody sequence for example, aCD19, aMUC1 , aGD2, aBCMA, scFv clone
  • TAA for example, CD19, MUC1 , GD2, BCMA
  • Immunoglobin lgG4 CH3/lgD hinge line which would enable bonding CAR protein to cell surface - CD8a transmembrane sequence (P01732, UniProtKB; 183-203. Amino acids) or NK/NK92 cell activator and NKG2D transmembrane sequence (P26718, UniProtKB; 52-72. Amino acids) creating costimulated factor effect or CD28/41 BB transmembrane sequence
  • CD3 zeta (CD247) line production (P20963, UniProtKB; 52-164 amino acids) which generates activation signal of CAR protein
  • CAR-EGFRt lentivirus production is enabled following the transfection on HEK293 cells by using CAR-EGFRt encoding Lentiviral plasmid DNA and virus packaging plasmids (VSVG & psPAX2).
  • CD16-IL-12+ NK92 cells expressing TAA-specific CAR detects the TAA presenting cancer cell. It shows cytotoxicity feature against tumor by means of activation.
  • CAR-ASSASSIN CAR+ CD16-IL-12+ NK92 cells are used with TAA- specific monoclonal antibodies, ADCC is triggered. At the same time, cytotoxic effect is further increased by stimulation of the CAR receptor. Thus, the target cancer cell is subject to apoptosis together with the bispecific orientation. Marking and isolating the cells expressing EGFRt following transduction of CD16- IL-12+NK92 cells with CAR-EGFRt lentivirus (12):
  • the flow cytometry analysis is performed with a fluorochrome conjugated anti- EGFR (for example A488-anti-EGFR) to label CD16-IL-12+ NK92 cells expressing CAR.
  • a fluorochrome conjugated anti- EGFR for example A488-anti-EGFR
  • isolation is performed with Streptavidin-bead following labeling with Biotin conjugated anti-EGFR antibodies.
  • the programmed cell death (as a suicide gene for security control) is enabled by means of EGFR-specific monoclonal antibody in order to remove the transfused proliferating CAR+CD16-IL-12+NK92 cells to the patients when necessary.
  • the freezing process following expansion of ASSASSIN (CD16+IL-12+ or IL-18+ NK92) and CAR-ASSASSIN (CAR+ CD16+IL-12+ or IL-18+ NK92) cells is performed at -196°C.

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Abstract

L'invention concerne un procédé de production et d'expansion des cellules NK92 modifiées destinées à être utilisées en immunothérapie anticancéreuse. L'invention couvre en particulier la production d'ASSASSIN (CD16 + IL-12 ou IL-18 + NK92) qui exprime le récepteur CD16 du simulateur immunitaire inné conjointement avec la sécrétion de cytokines IL-12 ou IL-18, et de cellules CAR-ASSASSIN (CAR + CD16 + IL-12 ou IL-18 + NK92), qui synthétisent un récepteur antigénique chimérique (CAR) sur leur surface.
EP19926735.2A 2019-11-19 2019-12-18 Procédé de reconception et d'expansion de cellules nk92 destinées à être utilisées en immunothérapie Withdrawn EP4061385A4 (fr)

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AU2021246671A1 (en) * 2020-03-28 2022-10-13 Board Of Regents, The University Of Texas System Cell immunotherapy for the treatment of cancer
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WO2019141270A1 (fr) * 2018-01-19 2019-07-25 科济生物医药（上海）有限公司 Expression régulée de l'il12 par le récepteur synnotch
US11813292B2 (en) * 2018-03-12 2023-11-14 Immunity Bio, Inc. Use of CD33CAR modified high affinity NK cells (t-haNK) to reduce myeloid-derived suppressor cells suppressor activity (or reduce negative impact on NK cell activity)
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