Development of an IL-2 independent NK cell line … IL-2 sequesters the cytokine inside the cell,...
1
Disclosure Statement: KSC: Scientific advisor for and recipient of research support from Conkwest, Inc.; holds a patent on CD16- bearing NK-92 cells Results Introduction - NK cells mediated Antibody Dependent Cell- mediated Cytotoxicity (ADCC) is considered a major mechanism of action of several IgG monoclonal antibodies (mAb) used in cancer therapy 1 , - Efficacy of these mAbs is limited by the fact that only 12% of the normal human population is homozygous for expression of the high affinity variant of FcγRIIIA (CD16-158V) that provides optimal ADCC 2,3 , and overall NK cell function may be compromised in many patients, - The combination of mAbs with NK-92 modified to express CD16-158V has superior antitumor effect compared to mAb infusions alone, as confirmed in SCID mouse/human xenotransplant models, - Adoptive cell therapy using the NK-92 cell line has been shown to be safe in phase I clinical trials, - NK-92 cells require IL-2 to maintain optimal cytotoxic activity but infusion of IL-2 can cause significant adverse effects in patients, - The addition of an intracellular retention sequence to IL-2 sequesters the cytokine inside the cell, yet maintains autocrine growth while restricting secretion. Conclusions - ERIL2 allows IL-2 independent growth of NK-92 through autocrine mechanism, - Plasmid-based transduction allows for stable long- term expression of CD16, - NK-92.CD16-ERIL2 (haNK TM ) cells mediate potent ADCC, which is minimally affected by irradiation, - NK-92 cells can be frozen with minimal loss of viability and no effect on cytotoxicity after a rest period of 24h, - CD16 expression in plasmid-transfected cells is unchanged by freezing, - ERIL2 makes NK-92 easier and more cost effective to grow in large scale cultures, but avoids secretion of IL-2 in patients, - A master cell bank is being established for use in a phase I/II trial with haNK TM cells in combination with a clinical grade mAb. Material & Methods Cells and media: K562, DoHH2, SKOV-3 and MM1.R cells were cultured in RPMI 1640 medium + 10% FBS. NK-92 cells were grown in X-Vivo TM 10 medium + 5% Human Serum, supplemented or not with human recombinant IL-2 at 500 IU/ml. NK-92 cells were subjected to an irradiation dose of 1000 cGy. NK-92 cells were frozen to -80 o C using a CoolCell® device (Biocision) then transferred to a LN 2 cryotank. Electroporation: The pNeukv1 plasmid was synthesized de novo and electroporated into NK-92 cells using a Neon TM electroporator. Cytotoxicity and ADCC assays: For cytotoxicity assay, cells were mixed in various effector-to- target (E:T) ratios and co-incubated for 4h at 37 o C. For ADCC, target cells were preincubated with human monoclonal antibodies at various concentrations prior to the co-incubation with effectors at a fixed E:T ratio. The killing percentages were normalized to the corresponding negative controls (spontaneous death for cytotoxicity, killing in absence of mAb for ADCC). Flow cytometry: CD16 density on the cells surface was determined by flow cytometry using a Qifikit kit (Dako/Agilent). Objective Develop a universal “off-the-shelf” high affinity NK cell therapy product (haNK TM ) that augments the efficacy of ADCC-mediated monoclonal antibody treatments in all patients. Development of an IL-2 independent NK cell line expressing high-affinity Fc-receptor to augment monoclonal antibody therapy Laurent Boissel 1 , Kerry S. Campbell 2 , Ashley M. James 2 , Frances Toneguzzo 1 , Hans Klingemann 1 1 Conkwest, Inc., Cardiff-by-the-Sea, CA USA; 2 Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA USA Visit us at www.conkwest.com The generation of NK-92 cells that express the high-Fc affinity version of FcγRIIIa at high level that is stable over time, that are IL-2 independent, and that can be cryopreserved with minimal loss of viability makes the development of an “off-the-shelf” therapeutic product possible. haNK TM cells grown in continuous large scale cultures can be packaged into ready-to-use frozen aliquots and thawed upon request to be shipped to the treatment facility in a medium that allows them to recover their cytolytic function. Upon arrival, the cells are ready to be irradiated and infused into the patient. Infusion Washing, QA-testing Send to Treatment Facility Aliquot & Freeze Thaw upon request Wash Cells, QA-testing Irradiation Cell culture Figure 4 haNK TM cells: an “off-the-shelf” cellular therapy product 1. Seidel U.J.E., et al. Natural Killer Cell Mediated Antibody- Dependent Cellular Cytotoxicity in Tumor Immunotherapy with Therapeutic Antibodies. Front Immunol. 2013 4:76 2. Lehrnbecher T., et al. Variant genotypes of the low-affinity Fcgamma receptors in two control populations and a review of low- affinity Fcgamma receptor polymorphisms in control and disease populations. Blood 1999 94(12):4220 3. Musolino A., et al. Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. J. Clin. Oncol. 2008 26(11):1789 References haNK TM (high affinity NK) cells: Stable high expression of CD16-158V on NK-92 cells using pNEUKv1 NK-92 cells were electroporated with a bicistronic plasmid vector (pNEUKv1 plasmid) expressing CD16-158V and intracellular retained IL-2. The resulting NK-92.CD16-ERIL2 (haNK TM ) can grow in the absence of IL-2 in the culture medium and “self-select” over time to a high degree of purity (A). Expression of CD16 in NK-92.CD16-ERIL2 cells is stable for at least 5 months in culture (B), with an estimated 40,000 CD16 molecules per cell (C). NK-92.CD16-ERIL2 grow at a very similar rate to parental NK-92 (doubling time of ~35h; D) and do not display any change in surface markers profile compared to the parental cells (E). A. D. E. B. C. 0 5000 10000 15000 20000 25000 30000 0 30 60 90 120 150 CD16 MFI Time (Days) 0 20 40 60 80 100 0 30 60 90 120 150 % CD16+ Cells Time (Days) y = 1.966920x 1.127542 100 1000 10000 100000 1000000 100 1000 10000 100000 # Molecules/Cell CD16 MFI CD56 NKG2D NKp44 NKp46 NKG2A CXCR3 CXCR4 Isotype NK692 NK692.CD166ERIL2 NKp30 Isotype NK692 NK692.CD166ERIL2 1.E+05 1.E+06 1.E+07 1.E+08 0 2 4 6 8 10 12 Cells Number Time (Days) Robust growth without exogenous IL2 No change in receptor expression in haNK TM cells Figure 1 NK-92 + exogenous IL-2 NK-92.CD16-ERIL2 The ability of NK-92.CD16-ERIL2 to perform ADCC (A) was evaluated against the Her2/Neu positive SKOV-3 cell line (ovarian adenocarcinoma; B) and the CD20 positive DoHH2 cell line (non- Hodgkin’s lymphoma; C) in combination with Herceptin® or Rituxan®. NK-92.CD16-ERIL2 cells efficiently and specifically mediate ADCC against both target cell lines. Expression of the CD16- ERIL2 construct does not affect the NK-92 natural cytotoxicity against NK-sensitive K562 cells (D) and NK-resistant SKOV-3 cells (E). However, NK-92.CD16-ERIL2 appear to be much less effective at killing DoHH2 cells (F). A. B. C. D. E. F. -10 0 20 40 60 80 100 0 0.01 0.1 1 % ADCC [mAb] µg/ml -10 0 10 20 30 40 50 0 0.01 0.1 1 % ADCC [mAb] µg/ml 0 20 40 60 80 100 1:3.3 1.1:1 3.3:1 10:1 % Killing E:T Ratio 0 20 40 60 80 100 1:3.3 1.1:1 3.3:1 10:1 % Killing E:T Ratio 0 20 40 60 80 100 1:3.3 1.1:1 3.3:1 10:1 % Killing E:T Ratio NK-92 + Herceptin NK-92.CD16-ERIL2 + Herceptin NK-92 + Rituxan NK-92.CD16-ERIL2 + Rituxan NK-92 NK-92.CD16-ERIL2 haNK TM cells mediate efficient ADCC towards solid and liquid tumor in vitro Figure 2 DoHH2 SKOV-3 K562 DoHH2 SKOV-3 0 20 40 60 80 100 % CD16 + Cells 0 20 40 60 80 100 % Viability CD16 expression after freezing NK-92 viability after freezing NK-92 Cytotoxicity recovers after freezing Irradiation and cryopreservation on NK-92 and haNK TM cells Irradiation of NK-92 cells causes a dramatic decrease in viability between 8h and 72h post- irradiation (A). Irradiated NK-92.CD16-ERIL2 cells are able to mediate similar ADCC as non- irradiated cells against the MM1.R multiple myeloma cell line in combination with reactive IgG1 antibody (B). NK-92 can be cryopreserved at high cells density with only a small decrease in viability within the first 24h post-thawing (C). Cryopreservation does not affect levels of CD16 expression (D). The natural cytotoxicity of NK-92 against K562 cells is severely reduced right after thawing (E) but recovers to normal levels after 24h (F). 5x10 6 Pre- Freezing Post- Freezing A. E. D. C. B. F. -10 0 20 40 60 80 100 0 0.001 0.01 0.1 1 % ADCC [mAb] µg/ml 0 20 40 60 80 100 0 2 4 8 24 48 72 120 % Viability Time (Hours) 0 20 40 60 80 100 1.25:1 2.5:1 5:1 10:1 % Killing E:T Ratio 0 20 40 60 80 100 1.25:1 2.5:1 5:1 10:1 % Killing E:T Ratio 5x10 7 Non-Irradiated 24h Post-Irradiation 4h Post-Irradiation 0h 24h Unfrozen culture Frozen/Thawed Non-Irradiated Irradiated Cells/ml Figure 3 NK-92 viability after irradiation NK-92.CD16-ERIL2 ADCC after irradiation K562 K562 MM1.R
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Transcript of Development of an IL-2 independent NK cell line … IL-2 sequesters the cytokine inside the cell,...
Disclosure Statement: KSC: Scientific advisor for and recipient of research
support from Conkwest, Inc.; holds a patent on CD16-bearing NK-92 cells
Results
Introduction - NK cells mediated Antibody Dependent Cell-
mediated Cytotoxicity (ADCC) is considered a major mechanism of action of several IgG monoclonal antibodies (mAb) used in cancer therapy1,
- Efficacy of these mAbs is limited by the fact that only 12% of the normal human population is homozygous for expression of the high affinity variant of FcγRIIIA (CD16-158V) that provides optimal ADCC2,3, and overall NK cell function may be compromised in many patients,
- The combination of mAbs with NK-92 modified to express CD16-158V has superior antitumor effect compared to mAb infusions alone, as confirmed in SCID mouse/human xenotransplant models,
- Adoptive cell therapy using the NK-92 cell line has been shown to be safe in phase I clinical trials,
- NK-92 cells require IL-2 to maintain optimal cytotoxic activity but infusion of IL-2 can cause significant adverse effects in patients,
- The addition of an intracellular retention sequence to IL-2 sequesters the cytokine inside the cell, yet maintains autocrine growth while restricting secretion.
Conclusions - ERIL2 allows IL-2 independent growth of NK-92
through autocrine mechanism, - Plasmid-based transduction allows for stable long-
term expression of CD16, - NK-92.CD16-ERIL2 (haNKTM) cells mediate potent
ADCC, which is minimally affected by irradiation, - NK-92 cells can be frozen with minimal loss of
viability and no effect on cytotoxicity after a rest period of 24h,
- CD16 expression in plasmid-transfected cells is unchanged by freezing,
- ERIL2 makes NK-92 easier and more cost effective to grow in large scale cultures, but avoids secretion of IL-2 in patients,
- A master cell bank is being established for use in a phase I/II trial with haNKTM cells in combination with a clinical grade mAb.
Material & Methods Cells and media:
K562, DoHH2, SKOV-3 and MM1.R cells were cultured in RPMI 1640 medium + 10% FBS. NK-92 cells were grown in X-VivoTM
10 medium + 5% Human Serum, supplemented or not with human recombinant IL-2 at 500 IU/ml. NK-92 cells were subjected to an irradiation dose of 1000 cGy. NK-92 cells were frozen to -80oC using a CoolCell® device (Biocision) then transferred to a LN2 cryotank.
Electroporation: The pNeukv1 plasmid was synthesized de novo and electroporated into NK-92 cells using a NeonTM electroporator.
Cytotoxicity and ADCC assays: For cytotoxicity assay, cells were mixed in various effector-to-target (E:T) ratios and co-incubated for 4h at 37oC. For ADCC, target cells were preincubated with human monoclonal antibodies at various concentrations prior to the co-incubation with effectors at a fixed E:T ratio. The killing percentages were normalized to the corresponding negative controls (spontaneous death for cytotoxicity, killing in absence of mAb for ADCC).
Flow cytometry: CD16 density on the cells surface was determined by flow cytometry using a Qifikit kit (Dako/Agilent).
Objective Develop a universal “off-the-shelf” high affinity NK cell therapy product (haNKTM) that augments the efficacy of ADCC-mediated monoclonal antibody treatments in all patients.
Development of an IL-2 independent NK cell line expressing high-affinity Fc-receptor to augment monoclonal antibody therapy
Laurent Boissel1, Kerry S. Campbell2, Ashley M. James2, Frances Toneguzzo1, Hans Klingemann1
1 Conkwest, Inc., Cardiff-by-the-Sea, CA USA; 2 Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA USA
Visit us at www.conkwest.com
The generation of NK-92 cells that express the high-Fc affinity version of FcγRIIIa at high level that is stable over time, that are IL-2 independent, and that can be cryopreserved with minimal loss of viability makes the development of an “off-the-shelf” therapeutic product possible. haNKTM cells grown in continuous large scale cultures can be packaged into ready-to-use frozen aliquots and thawed upon request to be shipped to the treatment facility in a medium that allows them to recover their cytolytic function. Upon arrival, the cells are ready to be irradiated and infused into the patient.
Infusion Washing, QA-testing
Send to Treatment Facility
Aliquot & Freeze
Thaw upon request Wash Cells, QA-testing
Irradiation
Cell culture
Figure 4 haNKTM cells: an “off-the-shelf” cellular therapy product
1. Seidel U.J.E., et al. Natural Killer Cell Mediated Antibody-Dependent Cellular Cytotoxicity in Tumor Immunotherapy with Therapeutic Antibodies. Front Immunol. 2013 4:76
2. Lehrnbecher T., et al. Variant genotypes of the low-affinity Fcgamma receptors in two control populations and a review of low-affinity Fcgamma receptor polymorphisms in control and disease populations. Blood 1999 94(12):4220
3. Musolino A., et al. Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. J. Clin. Oncol. 2008 26(11):1789
References
haNKTM (high affinity NK) cells: Stable high expression of CD16-158V on NK-92 cells using pNEUKv1
~40,000 CD16 molecules/cell
NK-92 cells were electroporated with a bicistronic plasmid vector (pNEUKv1 plasmid) expressing CD16-158V and intracellular retained IL-2. The resulting NK-92.CD16-ERIL2 (haNKTM) can grow in the absence of IL-2 in the culture medium and “self-select” over time to a high degree of purity (A). Expression of CD16 in NK-92.CD16-ERIL2 cells is stable for at least 5 months in culture (B), with an estimated 40,000 CD16 molecules per cell (C). NK-92.CD16-ERIL2 grow at a very similar rate to parental NK-92 (doubling time of ~35h; D) and do not display any change in surface markers profile compared to the parental cells (E).
A.
D. E.
B. C.
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# M
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NKG2A% CXCR3% CXCR4%
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NK692.CD166ERIL2%
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Robust growth without exogenous IL-‐2 No change in receptor expression in haNKTM cells
Figure 1
NK-92 + exogenous IL-2 NK-92.CD16-ERIL2
The ability of NK-92.CD16-ERIL2 to perform ADCC (A) was evaluated against the Her2/Neu positive SKOV-3 cell line (ovarian adenocarcinoma; B) and the CD20 positive DoHH2 cell line (non-Hodgkin’s lymphoma; C) in combination with Herceptin® or Rituxan®. NK-92.CD16-ERIL2 cells efficiently and specifically mediate ADCC against both target cell lines. Expression of the CD16-ERIL2 construct does not affect the NK-92 natural cytotoxicity against NK-sensitive K562 cells (D) and NK-resistant SKOV-3 cells (E). However, NK-92.CD16-ERIL2 appear to be much less effective at killing DoHH2 cells (F).
A. B. C.
D. E. F.
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NK-92 + Herceptin NK-92.CD16-ERIL2 + Herceptin NK-92 + Rituxan NK-92.CD16-ERIL2 + Rituxan
NK-92 NK-92.CD16-ERIL2
haNKTM cells mediate efficient ADCC towards solid and liquid tumor in vitro
Figure 2
DoHH2 SKOV-3 K562
DoHH2 SKOV-3
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CD16 expression after freezing NK-92 viability after freezing
NK-92 Cytotoxicity recovers after freezing
Irradiation and cryopreservation on NK-92 and haNKTM cells
Irradiation of NK-92 cells causes a dramatic decrease in viability between 8h and 72h post-irradiation (A). Irradiated NK-92.CD16-ERIL2 cells are able to mediate similar ADCC as non-irradiated cells against the MM1.R multiple myeloma cell line in combination with reactive IgG1 antibody (B). NK-92 can be cryopreserved at high cells density with only a small decrease in viability within the first 24h post-thawing (C). Cryopreservation does not affect levels of CD16 expression (D). The natural cytotoxicity of NK-92 against K562 cells is severely reduced right after thawing (E) but recovers to normal levels after 24h (F).
5x106 Pre-Freezing
Post-Freezing
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E.
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Figure 3
NK-92 viability after irradiation NK-92.CD16-ERIL2 ADCC after irradiation
K562 K562
MM1.R
