Controlled production of a bispecific antibody by an allogeneic genetically-modified stem cell triggers T cell activation and cytolysis in non-small cell lung carcinoma

Amy Wesa1, Yumei Xiong1, Tim Chan1, Jeff Rosenbloom2, Srinivas Rengarajan2, Paul Szymanski2, John A. Barrett3, Francois Lebel3, Farzad Haerizadeh2, Richard Einstein1

1Intrexon Corporation, Germantown, MD, 2Intrexon Corporation, San Diego, CA, 3ZIOPHARM Oncology, Inc., Boston, MA.

Intrexon Corporation

20358 Seneca Meadows Parkway, Germantown, MD 20876

Phone 301-556-9900 Fax 301-556-9901 www.dna.com

ZIOPHARM Oncology, Inc.

1 First Avenue, Parris Building #34, Navy Yard Plaza, Boston, MA 02129

Main 617-259-1970 Fax 617-241-2855 www.ziopharm.com

Abstract Bispecific antibodies and their derivatives can induce potent cytolytic activity through activation of T cells which target tumor cells. Cell linking moieties (CLM) represent engineered, bispecific antibodies consisting of two scFv. Previous studies have shown that the systemic distribution and pharmacokinetic profile of these agents has limited their utility for many target and effector combinations. We hypothesize that a ge-netically-modified, allogeneic cellular product could be developed to express such molecules leading to the inducible release at the tumor site following systemic delivery and thereby providing spatial and temporal control of the CLM distribution to increase the number of target-effector combinations. The RheoSwitch Therapeutic System® (RTS®) is a gene switch activated by a small molecule ligand that has been demonstrated clinically to control the timing and extent of gene expression. The RTS® platform has three essential elements: (1) switch com-ponents (co-activation partner and a ligand-controllable transcription factor); (2) activator ligand (veledimex); and (3) inducible promoter. Sans ligand, the switch complex is inactive and inhibits gene expression. By adding ligand, the switch components form an active complex, prompting gene expression in a dose-dependent manner. Endometrial regenerative cells (ERC) are a mesenchymal-like stem cell with a high replicative potential. We demonstrate that genetically modified ERC (GM-ERC) can be generated with high efficiency via nucleofection, lipofection or transduction with adenovirus, adeno-associated virus (AAV) or lentivirus. GM-ERC expressing a CLM construct comprised of anti-CD3-anti-EGFR bispecific antibody (CLM) under constitutive expression secretes up to 300 ng/ml of CLM as evaluated by ELISA. Addition of GM-ERC cells, supernatants from GM-ERC, or purified CLM to co-cultures of unstimulated peripheral blood mononuclear cells and EGFR+ A549 lung carcinoma cells led to T cell activation as measured by cytokine secretion (IL-2 and IFN-γ) and upregulation of CD69, CD25, and EGFR-specific cell-mediated cytotoxicity of A549 cells in vitro. CLM-expressing ERC were effective in coculture killing assays at doses as low as 1% of A549 target cells. Expression of CLM under the inducible promoter (RTS-CLM) yielded effective control of CLM secretion and killing ac-tivity, with greater than 80% cytotoxicity against A549 at 48-72 hours of cocultures with ERC-RTS-CLM, A549 cells and T cells when in the pres-ence of veledimex (ligand), but not vehicle control. The data demonstrate that GM-ERC can secrete a bispecific antibody that induces T cell mediated killing in vitro, and that this activity can be controlled using an RTS® expression vector in combination with an activator ligand. This provides evidence supporting the feasibility of the cytolytic activity of CLM-secreting GM-ERC as an allogeneic cell therapy product, a novel and promising approach for the therapy of lung carcinoma and other EGFR+ malignancies.

Hypothesis Endometrial regenerative cells (ERC) can be genetically modified to express a ligand inducible cell linking moiety that will elicit redirected T cell cytotoxicity response against an EGFR+ lung cancer target

RheoSwitch® proteins and activator ligand control timing and level of target gene expression

Efficient gene modification of ERC using viral or non-viral approaches with reporter genes (GFP or luciferase)

Figure 3. A. Dose-dependent transduction of ERC by adenovirus. Titration of adenoviral (Ad) vector with GFP reporter gene in ERC. GFP expression was measured after 48 hours by flow cytometry, average duplicate wells +/- S.D. is plotted. Fluorescent microscopy of GFP+ ERC in the 20K MOI cohort is shown in inset. B. Dose-dependent transduc-tion of ERC by adeno-associated viral (AAV2). Titration of AAV2 with GFP reporter gene on ERC. GFP expression was measured after 7 days by flow cytometry, average duplicate wells +/- S.D. is plotted. Fluorescent microscopy of GFP+ ERC in the 50K MOI cohort is shown in inset. C. Dose-dependent transduction of ERC by lentivirus (LV). Titration of LV vectors with fLUC reporter gene in the presence of polybrene was evaluated in ERC. Luminescence was measured after 8 days, the average duplicate wells +/- S.D. is plotted. D. ERC are efficiently modified by nucleofection. Transfec-tion of ERC by plasmid with GFP reporter gene with electroporation enabled identification of conditions resulting in high GFP expression and viability measured by flow cytometry after 48 hours. Initial screening of 132 different condi-tions (data not shown) led to secondary screening in the top six conditions illustrated here. Representative fluores-cence of 96-well culture as measured by Phenix imaging system is shown in inset.

αEGFR- αCD3 CLM mediates T cell activation and induces apoptotic cell death in A549 lung carcinoma cells

Figure 4. A. Titrated doses of purified CLM (from supernatants of 293T transfectants, purified by nickel column via His tag) were incubated with PBMC and A549 lung carcinoma cells (20:1 effector : target ratio) and cytotoxicity measured in 24h LDH release assay. B. Purified T cells (T) and/ or A549 cells (A) (10:1) were treated with 100 nM CLM, staurosporine (positive control) and/or OKT3 (specificity control). Cells and supernatant were pooled for analysis of caspase activation by biolumi-nescence assay to evaluate induction of apoptosis in A549 cells by redirected T cells. C. Titrated doses of purified CLM were incubated with T cells and A549 lung carcinoma cells (10:1), and CD25 and CD69 expression on T cells analyzed at 48 hours. D. After coincubation of PBMC with A549 or ERC at (10:1) in the presence of varying doses of CLM for 48 hours, superna-tants were harvested for analysis of cytokines (IL-2, IFN-g) by luminex. Results represent average +/- S.D.

Gene-modified ERC secrete functional CLM in vitro after transient transfection

Figure 5. A. Secretion of CLM as measured by electro-chemiluminescent ELISA from transiently transfected ERC. Results represent average of duplicate samples. B. Supernatants of transiently transfected ERC were incubated with A549 and PBMC, and cytotoxicity measured by LDH release assay after 24 hours. Results represent the av-erage of a minimum of quadruplicate samples wells +/- S.D.

ERC-CLM stable transfectants reduce A549 target cell confluency in a dose-dependent manner in the presence of T cells

Figure 6. A-B. 1000 A549-GFP cells seeded into each well of 384 well plate and allowed to adhere overnight. ERC [stable, constitutive CLM transfectants (A); or untransfected ERC (untx) (B)] were added into co-culture at various doses (shown as a percentage of A549 target cells, ranging from 0 to 200%) the following day with 10,000 un-stimulated PBMC. From day 1 to day 5, wells imaged using the Phenix imager. Results represent average conflu-ence analysis +/- SD of quadruplicate wells. C-D. Dilutions of supernatants of stable ERC transfectants (C) or con-trol ERC (D) were incubated with A549-GFP and T cells (10:1 ratio), and imaged from day 1 to day 5. Results rep-resent average confluence analysis +/- SD of quadruplicate wells. E. Representative images from Phenix imager on day 4 of coculture of A549-GFP+ cells with ERC and T cells.

Veledimex control of CLM secretion by ERC elicits T cell mediated killing of A549 target cells

Figure 7. Evaluation of ligand inducible ERC cytotoxicity in cocultures of RTS-CLM-ERC + A549 + T cells (1:1:20) after 48 (A) and 72 hours (B, C). CFSE-labeled A549 cells were cultured with stably transfected ERC and purified T cells with rCLM or 100 nM veledimex or vehicle (DMSO) (A, B) or titrated doses of veledi-mex (C). Harvested cocultures were incubated with PE-conjugated CD105 mAb, and a viability dye was used to measure the frequency of dead cells among CFSE+CD105PE- A549 cells. Average frequencies are shown in A and C, with representative plots shown in B. Two independent stable RTS-CLM-ERC lines are shown for C. D. Veledimex was titrated on two stable RTS-CLM ERC lines and CLM secretion was measured by electrochemiluminescent ELISA in 48 hour supernatants.

Conclusions

Model for cell linking moiety mediated anti-tumor activity via an allogeneic Embedded Cellular Bioreactor

Figure 1. Genetically modified-ERC(GM-ERC) stem cells expressing CLM under RheoSwitch® system control after infu-sion into the subject are hypothesized to permit redirected killing of EGFR+ tumor targets under spatial and temporal control to increase the therapeutic index

Figure 2. The RheoSwitch Therapeutic System® (RTS®) contains three basic components: (1) an inducible promoter; (2) a ligand-inducible transcription factor and a co-activation partner; (3) RheoSwitch® activator ligand (AL), veledi-mex. In the absence of ligand, the switch protein complex provides an “off” signal which limits gene transcription. In con-trast, in the presence of ligand, the complex changes conformation and provides a dose-dependent “on” signal for target gene expression. In vivo, the orally administered AL turns on gene expression within 24 hours, and upon with-drawal of the AL, gene expression returns to baseline levels within about 24 hours.

RXR Gal4

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T cell activation marker upregulation Cytokine production by rCLM

Ligand-regulated CLM secretion from two ERC-RTS-CLM lines

Apoptosis induction by rCLM B.