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).

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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).

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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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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).

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NK-92 viability after irradiation NK-92.CD16-ERIL2 ADCC after irradiation

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