EP4061385A1 - A method for redesign and expansion of nk92 cells for use in immunotherapy - Google Patents
A method for redesign and expansion of nk92 cells for use in immunotherapyInfo
- 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.)
- Pending
Links
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- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2318—Interleukin-18 (IL-18)
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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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
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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
- C12N2510/00—Genetically modified cells
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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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- 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
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 covers the production of ASSASSIN (CD16+ IL-12 or IL-18+ NK92), which express innate immune simulator CD16 receptor along with secretion of IL-12 or IL-18 cytokines, and CAR- ASSASSIN (CAR+ CD16+ IL-12 or IL-18+ NK92) cells, which synthesizes chimeric antigen receptor (CAR) on their surface.
Description
A METHOD FOR REDESIGN AND EXPANSION OF NK92 CELLS FOR USE IN
IMMUNOTHERAPY
Technical Field
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.
State of the Art
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.
Each day millions of cancer cells are born in our body. There are cells in the immune system of the human being to defeat the cancer cells. Immunity, in other words, the immune system kills the cancer cells as soon as they detect these cells. The immunotherapy method supports the body resistance who targets cancer cells, increases immunity and tries to get rid of cancer cells in this manner.
Today immunotherapy also develops rapidly. 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. In general, 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.
However, there are still some problems with the studies. Some of the problems encountered are;
- NK92 or transgenic CAR-NK92 cell therapies have been assessed in many clinical studies to prolong the survival of patients who are unresponsive to standard treatment as a bridging therapy to the transplant (Klingemann, Frontiers in Immunology, 2016; Oelsner, Cytotherapy, 2017).
- Since NK92 cells do not show cancer-specific properties, they should be directed to the target tumor (Raulet et al., Nature Reviews Immunology, 2006). Particularly, the most important problem in CD19 CAR-T cell therapies is that CD19 which is a target antigen diminishes on the cancer cells so that CAR-T cell effect remains insufficient and tumor relapses.
As a result, due to the negativities described above and the inadequacy of the existing solutions on the subject, an improvement in the relevant technical field was required.
Brief Description of the Invention
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). To achieve this effect for anti tumor purposes, we designed the lentiviral vector to express the CD16 FcyRIII receptor on NK92 cells. Thus, the monoclonal antibodies (mAb) to be used are intended for cancer cell death by stimulation of ADCC specific to the cancer type.
Interleukin-12 (IL-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.
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.
Bi-specific (double-hit) cytotoxic effect is contemplated by the expression of the cancer- specific tumor-associated antigen (TAA, eg CD19, CD20, MUC-1 , etc.) specific chimeric antigen receptor (CAR) on the modified NK92 cells which express CD16, too. Thus, it is aimed to overcome the loss of anti-tumor efficacy caused by the loss of the TAA antigens, which is the most important problem in classical CAR-T cell therapies.
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.
To achieve the above objects, 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).
The structural and characteristic features and all advantages of the invention will be more clearly understood and should be evaluated concerning the following figures and the detailed descriptions.
Detailed Description of the Invention
In this detailed description, the method of producing the modified NK92 cells for use in cancer immunotherapy according to the invention is explained for a better understanding of the subject without any limiting side.
In terms of surface phenotype, 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. On the other hand, expression of the TAA-specific CAR receptor in CAR-ASSASSIN (CAR+ CD16+ IL-12 or IL-18+ NK92) cells together with use of the monoclonal antibodies is designed to generate a bispecific (double-hit / double-hit) cytotoxic effect. As a result, it is aimed to overcome the decline of the anti-tumor efficacy due to the loss of the target antigens, which is the most important problem in classical CAR-T cell therapies. Also, ASSASSIN (CD16+ IL-12 or IL-18+ NK92) or CAR-ASSASSIN (CAR+ CD16+ IL-12 or IL-18+ NK92) cells that secrete IL-12 or IL-18 cytokines are expected to trigger the patient's innate and adaptive immune system against cancer. Thus, the activated host immune system will continue to fight with the tumor even after the destruction of the irradiated ASSASSIN or CAR-ASSASSIN cells. As a result, 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.
According to an embodiment of the invention, the modified NK-92 cells or cell lines comprises IL-12 or IL-18, CD16, CAR.
IL-12 (Interleukin-12) or IL-18 enables increasing the anti-tumor activity by stimulating the innate and the adaptive immune cells of the patient.
Fcγlll receptor (CD16) generates ADCC response with the tumor associated antigen (TAA) specific monoclonal antibodies.
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) is used in marking and selection of the generated CAR-NK92 cells; also, CAR-NK92 cells are directed to the programmed cell death with the EGFR-specific monoclonal antibody when required.
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.
According to an embodiment of the invention, 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 2nd transduction agent coding CAR-EGFRt and expansion of CD16+ IL-12 or IL-18+ NK92 cells j) Marking and isolation of the expanded CAR-ASSASSIN (CD16+IL-12 or IL- 18+ NK92) cells synthesizing CAR-EGFRt k) Freezing ASSASSIN (CD16+IL-12 or IL-18+ NK92) and CAR-ASSASSIN (CAR+ CD16+ IL-12 or IL-18+ NK92) cells following the expansion process.
According to an example embodiment of the invention, during the production of ASSASSIN (CD16+ IL-12 or IL-18+ NK92), which express innate immune simulator CD16 receptor along with secretion of IL-12 or IL-18 cytokines, and CAR-ASSASSIN (CAR+ CD16+ IL-12 or IL-18+ NK92) cells, which synthesizes chimeric antigen receptor (CAR) on their surface, the schemes of the realized biological reaction processes are shown herein below and in the following these processes are described in detail:
Lentiviral CD16-IL-12 gene design and lentivirus production (1):
EF-1 a promoter CD16-P2A-IL12A-T2A-IL12B vector design
- FcγRIIIa receptor (CD16) sequence (P08637, UniProtKB; 1-254 amino acids) which will stimulate the antibody dependent cellular cytotoxicity (ADCC)
- Self-cleavage (self-interceptable, SIN) peptide P2A sequence (porcine teschovirus-1 single 2A sequence)
- IL-12 subunit A sequence (P29459, UniProtKB; 1-219 amino acids) which would generate IL-12 heterodimer showing anti-tumor activity
- Self-cleavage (self-interceptable) peptide T2A (Thoseaasigna virus 2A) sequence
- Subunit B sequence (P29460, UniProtKB; 1-328 amino acids) which would generate IL-12 heterodimer showing anti-tumor activity
- By using generated CD16-IL-12 coding lentiviral vector and virus packaging plasmids (VSVG & psPAX2), CD16-IL-12 lentivirus production is enabled following transfection on HEK293 cells.
Marking and isolating NK92 cells expressing CD16 following transduction of NK92 cells (2):
- Marking the NK92 cells expressing CD16 with anti-CD16 monoclonal antibodies conjugated with fluorochrome (for example A488 or PE) and determining the proportion by means of the flow cytometry,
- Following marking of the NK92 cells expressing CD16 with the biotin conjugated anti-CD16 monoclonal antibodies, the positive selection and isolation of CD16+ NK92 cells is performed with Streptavidin-bead through the magnetic column.
The expansion step of CD16+IL-12+NK92 (ASSASSIN) cells is performed in GMP culture.
Targeting the CD16+ NK92 cells to the tumor with the TAA-specific monoclonal antibodies (3):
- Following the transplantation of CD16+NK92 cells to the patient, 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).
Occurrence of the antibody dependent cellular cytotoxicity (ADCC) effect (4):
- The activation of the CD16+NK92 cells that are targeted to the tumor with TAA- specific monoclonal antibodies would lead to the reveal of the antibody dependent cytotoxic (ADCC) effect.
Perforin and granzyme mediated tumor cell apoptosis (5):
- Perforin and granzyme proteins are secreted for killing the cancer cells from the CD16+ NK92 cells with the ADCC effect. Thus, the tumor cells are targeted to apoptosis.
IL-12 synthesis in CD16+NK92 cells (6):
- 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.
Assessment of IL-12 secretion from CD16+NK92 cells (7):
- IL-12, which is produced in CD16+NK92 cells, is extracellularly secreted.
- In order to mark functional IL-12 protein and determine its concentration in vitro culture, ELISA analysis method composing IL-12 monoclonal antibodies is used.
Activation of innate& adaptive immune cells by IL-12 (8):
- IL-12, which will be secreted from CD16+NK92 cells, in the patient’s body stimulates the innate and adaptive immune system cells of the patient.
Formation of anti-tumor activity by means of the immune cells following IL-12 activation (9):
- The activated immune cells start to exhibit anti-tumor effects by means of migrating towards the tumor cells.
Transduction of CD16-IL-12+ NK92 cells; following chimeric antigen receptor (CAR) & EGFRt gene design and lentivirus production (10):
EF-1a promoter TAA scFV- CD8 hinge- CD8 TM- CD28 CD- CD3- P2A- EGFRt vector design
- TAA-specific CAR-EGFRt gene coding Lentiviral vector design and lentivirus production
- CD8a protein signal peptide sequence (P01732, UniProtKB; 1-21 amino acids) which will provide chimeric antigen receptor’s (CAR) expression on NK92 cell surface
- ScFv antibody sequence (for example, aCD19, aMUC1 , aGD2, aBCMA, scFv clone) which will detect TAA (for example, CD19, MUC1 , GD2, BCMA) expressed by the cancer types
- CD8a hinge sequence (P01732, UniProtKB; 138-182. Amino acids) or
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
- 2B4 (CD244) intracellular domain sequence (Q9BZW8, UniProtKB; 251-370. Amino acids) or 41 BB (CD137) intracellular domain sequence (Q07011 , UniProtKB; 214-255. Amino acids) or CD28 intracellular domain sequence (P10747, UniProtKB; 180-220. Amino acids) or OX-40 (CD134) intracellular domain sequence (P43489, UniProtKB; 236-277. Amino acids) which would provide NK92 cells to exhibit cytotoxic effect as a result of CAR activation.
- CD3 zeta (CD247) line production (P20963, UniProtKB; 52-164 amino acids) which generates activation signal of CAR protein
- P2A (porcine teschovirus-1 single 2A) sequence which bonds EGFRt protein line to each other to be produced on NK92 cell surface equal to the CAR protein and is subject to autonomous cleavage
- EGFRt protein sequence (P00533, UniProtKB; 18-594. Amino acids) subject to which will allow isolation of CAR+ EGFRt-i- NK92 cells
- 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).
- Transduction of CAR-EGFRt encoding lentiviruses to CD16-IL-12+ NK92 cells is performed.
Increasing TAA specific cytotoxicity following CAR expression in CD16-IL-12+NK92 cells (11):
- 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. When 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.
- For the isolation of CAR+CD16-IL-12+NK92 cells, 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.
According to an embodiment of the invention, 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.
Claims
1. A production method of the modified NK92 cells for use in cancer immunotherapy is characterized in comprising process steps of; a) Lentiviral CD16-IL-12 or IL-18 gene design b) CD16-IL-12 or IL-18 coding lentivirus production c) Expansion of NK92 cells transduced with CD16-IL-12 or IL-18 Lentiviruses d) Marking and isolation of the proliferated NK92 cells expressing CD16 e) Expansion of CD16+ IL-12 or IL-18+ NK92 (ASSASSIN) cells f) IL-12 or IL-18 secretion and determination from CD16+NK92 (ASSASSIN) cells g) TAA specific chimeric antigen receptor (CAR) & EGFRt gene design h) CAR-EGFRt lentivirus production i) Expansion of ASSASSIN (CD16+ IL-12 or IL-18+ NK92) cells transduced with the 2nd lentivirus encoding CAR-EGFRt j) Isolation of the expanded labeled CAR-ASSASSIN (CD16+IL-12 or IL-18 +NK92) cells expressing EGFRt. k) Freezing ASSASSIN (CD16+ IL-12 or IL-18+ NK92) and CAR-ASSASSIN (CAR+ CD16+ IL-12 or IL-18+ NK92) cells following the expansion process.
2. The method according to claim 1 , characterized in that; Biotin conjugated anti- CD16 monoclonal antibodies and Streptavidin-bead are used for the isolation of CD16+ NK92 cells included in step d).
3. The method according to claim 1 , characterized in that; labeling CD16+ NK92 included in step d) is made with A488 or PE fluorochrome conjugated anti-CD16 monoclonal antibody.
4. The method according to claim 1 , characterized in that; determining secretion of IL-12 or IL-18 cytokines included in step f) ELISA analysis method including IL-12 or IL-18 monoclonal antibodies.
5. The method according to claim 1 , characterized in that; determining CAR-EGFRt expressing CD16+ IL-12 or IL-18 + NK92 cells included in step j) is performed with A488 fluorochrome conjugated anti-EGFR.
6. The method according to claim 1 , characterized in that; isolation of CAR+ CD16+ IL-12 or IL-18+ NK92 cells included in step j) is performed by using Biotin conjugated anti-EGFR antibodies following marking with Streptavidin-bead.
7. The method according to claim 1 , characterized in that; the freezing process included in step k) is performed at -196°C temperature.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TR201918028 | 2019-11-19 | ||
| PCT/TR2019/051103 WO2021101467A1 (en) | 2019-11-19 | 2019-12-18 | A method for redesign and expansion of nk92 cells for use in immunotherapy |
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| Publication Number | Publication Date |
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| EP4061385A1 true EP4061385A1 (en) | 2022-09-28 |
| EP4061385A4 EP4061385A4 (en) | 2024-08-14 |
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| EP19926735.2A Pending EP4061385A4 (en) | 2019-11-19 | 2019-12-18 | A method for redesign and expansion of nk92 cells for use in immunotherapy |
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| WO (1) | WO2021101467A1 (en) |
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| EP2801583B1 (en) * | 2004-07-10 | 2018-04-25 | Fox Chase Cancer Center | Genetically modified human natural killer cell lines |
| CN107709552B (en) * | 2015-06-10 | 2022-05-13 | 南克维斯特公司 | Modified NK-92cells for the treatment of cancer |
| EP3741861A4 (en) * | 2018-01-19 | 2021-12-08 | Cafa Therapeutics Limited | Synnotch receptor-regulated expression of il12 |
| WO2019177986A1 (en) * | 2018-03-12 | 2019-09-19 | Nantkwest, 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) |
| CN113164519A (en) * | 2018-08-01 | 2021-07-23 | 南克维斯特公司 | Genetically modified tetra-cistronic system comprising a homing receptor or cytokine and a chimeric antigen receptor for immunotherapy |
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| EP4061385A4 (en) | 2024-08-14 |
| WO2021101467A1 (en) | 2021-05-27 |
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