"You can see control of both CAR expression as well as membrane-bound IL15 production through RheoSw | ZIOP Message Board Posts

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Msg  76584 of 113342  at  3/21/2017 6:04:00 PM  by

Rob Cos

The following message was updated on 3/21/2017 7:03:10 PM.

 In response to msg 76302 by  Steve1d
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Strong Buy

"You can see control of both CAR expression as well as membrane-bound IL15 production through RheoSwitch platform utilizing the Sleeping Beauty system. This unique capability through our gene switch enables an unrivaled control approaching CAR-T cell "


First let me repeat in blue this msg 76302 on ZIOP IL-15 advantage from Steve1d - one of the best posters on the board.... (Steve please reply to this with more thoughts - some of your posts have the most recommendations on this board for a reason) - and then expand on it

“ IL15 is a potent longevity factor for T stem cell compartment as well as increasing numbers and activity. This study demonstrating that pre- conditioning chemotherapy caused the production of IL15, expansion of CART and remissions. ZIOP membrane bound IL15 able to be regulated within the CART by velemedex will limit associated toxicity that was observed in the KITE trial and at the same time can regulate up or down the numbers and activity of the CART as required to fight the cancer optimally......all without chemotherapy. KITE gets an initial burst of IL15 activity but cannot keep it going whereas ZIOP could potentially keep IL15 on longer or turn it on again as required. This is important science that ZIOP is controlling.”

The preclinical trials were to avoid the problems of cost and control - remember from the Rosenberg publication - the viral TCR solutions will be TEN TIMES THE COST OF ZIOP NON-VIRAL without the control - also with the burden in hospital and related costs of a bone marrow transplant (per GILD CEO - see quote below)....Once the Point of Care concept - able to deliver faster treatment (now 11 days going to 2 days, then 0 days) at the point of care without flying to a major treatment center and without a huge manufacturing facility and the cost and time of creating enough lentivirus - gets a little further - the advantages over viral non-controlled high cost will become crystal clear.

"Also, not everybody in the pharma/biotech sector is really enthusiastic about the kind of bespoke CAR-T therapies, which KITE is offering. John F. Milligan, CEO of Gilead (NASDAQ:GILD), for example, was rather skeptical about the new approach.

"It's a very labor-intensive kind of business (…) Those programs are projected to be quite expensive and are more akin to a bone marrow transplant, with a lot of supportive care and hospitalization costs, and those are the sorts of things that make me nervous."

See blog for Cooper’s direct quote on point of care:


A day of reckoning will come for the viral companies within the next 18 months imo, but until then we can settle for vindication and validation with a Big Pharma large deal coming soon imo.

Remember the comment from the Feb 16 ZIOP P/R below - followed by Andy Lasts comments on the XON call right after this:

ZIOP P/R membrane-bound IL-15 comments XON call

Announced Publication Demonstrating Membrane-bound IL-15 (mbIL15) Enhanced Persistence of CD19-Specific T Cells. In November 2016, ZIOPHARM announced the publication of data demonstrating enhanced persistence of genetically modified T cells targeting leukemia through utilization of its non-viral SB system to co-express mbIL15 and a CD19-specific CAR. The article, titled "Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells," was published in the Proceedings of the National Academy of Sciences and is available online here.

Using the SB system, researchers generated genetically modified T cells that preserved stem-cell memory (TSCM) cells by co-expressing the CAR with mbIL15. Engineered T cells were effective in treating established CD19+ leukemia in mice by facilitating the long-term persistence of TSCM cells sustained by signaling through recombinant IL-15. These findings provide for a translational pipeline of immunotherapies with improved potential by combining mbIL15 and T cells with diverse specificities.

The Company previously announced the publication of data highlighting the benefits of using the non-viral SB system to genetically modify T cells to express a CAR for use against CD19-expressing leukemias and lymphomas. The article, titled "Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells," was published in the Journal of Clinical Investigation (doi:10.1172/JCI86721), and is available online here.

Andy Last comments XON call

Slide 15 provide some detail around new leading edge work in developing non-viral approaches to T-cell immunotherapy including CAR-T and TCR. The top part of the slide shows what we believe to be the largest multi-gene program in a T-cell delivered through a single non-viral vector. The genetic software package in this case is roughly 12 KB and includes the RheoSwitch system, the expression of CAR and the control manufacture of membrane-bound cytokine IL15.

As you can see here, the addition of veledimex leads to dramatic induction of IL15 expression and a complete loss of that expression when veledimex is removed, this achievement was made possible by continued development of AttSite Recombinases platform and paves the way for delivering even more powerful and flexible multi-gene systems. An important point here is that this furthers our capability and industrializing CAR-T and TCR therapies, and enables us and our partners to leapfrog competitors in this fast moving field.

In the bottom half of the slide, you can see control of both CAR expression as well as membrane-bound IL15 production through RheoSwitch platform utilizing the Sleeping Beauty system. This unique capability through our gene switch enables an unrivaled control approaching CAR-T cell therapy. We intend to move this distinctive approach from bench to bedside in the near future.


Now on the neoantigen targeting side - remember Cooper comments

Treating liquid tumors with chimeric antigen receptors has yielded extraordinary results with genetically engineered T cells and the next stage in the evolution of this immunotherapy is the expression of T-cell receptors to target solid tumors,"said Laurence Cooper, MD, PhD, Chief Executive Officer of ZIOPHARM. "Through use of the scalable non-viral Sleeping Beauty platform to express an array of TCRs that recognize neoantigens within each patient's tumor, we can customize T-cell therapies and enhance their function through cytokines and switches."

The SB transposon-transposase is a unique system for introducing genes encoding chimeric antigen receptors (CARs) and TCRs into lymphocytes. This non-viral platform may play an important role in immunotherapy and has several potential advantages over viral-based delivery systems including lowering the cost of genetic modification and the generation of T cells with minimal ex vivo processing supporting the personalization of T-cell therapy.

Dr. Cooper added, "The Sleeping Beauty system is the most advanced non-viral cell engineering platform in clinical development and we look forward to working with Dr. Rosenberg and the NCI to explore its potential to express neoantigen-specific TCRs to develop individualized immunotherapies for patients with cancer.


Remember the 1/10th cost comment from Rosenberg in his scientific publication in Molecular theory...

Molecular Therapy: (Dr Cooper and Dr Rosenberg) Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System


The α/β TCR that confers reactivity against aneoantigencan then be cloned into an expression vector, for example an integrating virus or the Sleeping Beauty (SB) transposon/transposase system. T cell subsets isolated from the peripheral blood of the patient can finally be modified with this expression vector, expanded in vitro to numbers sufficient for treatment, and re-infused back into the patient. TL, transmitted light; TN, T naive; TSCM, T stem cell memory; TCM, T central memory; SB11, SB transposase 11.

As an alternative to retroviral and lentiviral vectors, nonviral gene-transfer methods can potentially be used to genetically introduce antigen receptors. Because nonviral integration systems use oligonu­cleotides and recombinant proteins, they can be considerably cheaper to manufacture and easier to implement for single-use applications com­pared with viral vectors. By some estimates, production of nonviral rea­gents may cost one-tenth that of GMP-grade virus118. Presently, use of the Sleeping Beauty (SB) transposon/transposase system has advanced farthest in clinical development22.

Per the Steven Rosenberg Nature Medicine article SB is the farthest into clinic non-viral....

Nature Medicine (Dr Steven Rosenberg) - Prospects for gene-engineered T cell immunotherapy for solid cancers


The Sleeping Beauty transposon-transposase is a unique non-viral system for introducing genes encoding CARs and T-cell receptors (TCRs) into lymphocytes and is exclusively licensed by Intrexon Corporation (NYSE:XON) through The University of Texas MD Anderson Cancer Center and accessed as part of ZIOPHARM's collaboration. This non-viral approach may play an important role in immunotherapy and has several potential advantages over viral delivery systems, including:

  • Lower cost of generating genetically modified T cells
  • Generate T cells with minimal ex vivo processing
  • Conduit to targeting solid tumor neo-antigens using TCRs
  • Pathway to overcome regulatory hurdles

And remember Dr Rosenberg's direct quote

"The Sleeping Beauty transposon-transposase system represents a unique non-viral system for introducing encoding T-cell receptors and chimeric antigen receptors into lymphocytes that can be of great value in the development of personalized immunotherapies for patients with cancer"

Dr Steven A. Rosenberg MD Ph.D.

The slow pace has nothing to do with financing - ZIOP has many options and is not out of money and won't be - it’s to take the time to do the trials correct and avoid the pitfalls the viral companies will face.... remember Kirk's comments...

"So while we've seen several companies plow ahead running basically with a very high set of costs and therapies that really have to be administered in, you know, very top-flight research institutes in order to manage the side effects that are associated with the on-target toxicity and so forth, we just didn't consider that the motif as practiced by the first-generation of companies in this field is really workable, or to kind of put it in drug development parlance, is it a drug? Our conclusion is no, it's not.

So we weren't interested in joining that race because it's -- we think it's a race that ultimately leads to nowhere, except for the fact that obviously three or four companies are going to have a successful CD19 CAR T. But beyond that, I'm not really sure what they're racing for or toward. What we realized was that there were significant technical hurdles that needed to be overcome. We focused on those hurdles. We highlighted how some of our technology could help us get over those hurdles. And based on all that, I will say we're very, very pleased….in summary, let me just say, please remember that our strategy here is not to race to the clinic as fast as we can go with a me-too CD-19 CAR T or TCR. Our strategy here is to leapfrog the others technically. We think we've shown a lot of really encouraging data that lead us to believe that we'll be able to do that."

Mr Randall Kirk

And the Griffin comments

The difference between the two approaches can be measured in time and money, and the result weighs heavily in favor of Sleeping Beauty because it directly inserts one or two copies of the TCR gene into a cell’s DNA. Use of a viral vector for this purpose is fine if the goal is to create a single, large batch of T cells for many individuals. Yet, the technology is not suitable for many small batches because of the time and cost involved in creating each viral vector. And that is where the problem is – TCR therapies must not only target the appropriate antigen(s), but also express human leukocyte antigens (HLA) that are compatible with the patient’s.


Also reminder these excerpt comments from Markey recent on ZIOPs - I have include the link to full report so you can see all the key graphics that won't paste - this also provides the disclosures some are obsessed about to the point where they will not discuss the commentary and science. Markey does by far the best job of providing detail behind the ZIOP and MDA science and I HIGHLY recommend viewers read his commentary in detail. Markey clearly has the closest relationship to Cooper and Kirk of all the analysts IMO. He also has access to former ZIOP analyst Chrystyna Bedrij who brought ZIOP and XON together and who I believe spends the majority of her time working with Kirk and those two companies. Sure this leads to the disclosures included in the link above - but those of us who have met Markey in person also understand all of the above provides him with additional detail of the science that is important. I am thankful for his detailed worked and discussion below...

Griffin Securities-Exceptional Therapies in the Clinical Queue-Ziopharm will break new ground with seven clinical studies in 2017 - Raising Target to $23

For all charts and graphs and Griffin disclosures and other info see full report here


Ziopharm February 6, 2017 Griffin Securities Equity Research

12 mos. Target Price $23.00

Ziopharm BUY

Target Price Change : Biotechnology

Exceptional Therapies in the Clinical Queue

Ziopharm will break new ground with seven clinical studies

in 2017. This report discusses the trials, their clinical/commercial

significance, and the Company’s valuation relative to its peers.

A Paradigm Shift with Point-of-Care T Cells: Ziopharm

has cut the preparation time of CAR T cells to two days

with its Sleeping Beauty technology. A Phase 1 trial is set

to begin to test a third-generation CAR T cell that targets

CD19 and expresses membrane-bound interleukin-15 (IL-15).

These cells are younger than T cells that are typically expanded

ex vivo and they include memory T stem cells. Favorable results

from the study could put the therapy on a path to a 2019 launch.

The Versatile IL-12 Gene Therapy: Preparations are under

way for a pivotal trial of Ziopharm’s IL-12 gene therapy for

glioblastoma. An “end of Phase 2” meeting was held with the

FDA to discuss Phase 1 trial results and set a path forward. The

data point to a median overall survival benefit of more than 12

months, which is 2 to 3 fold longer than expected for patients

with advanced disease. We believe the pivotal trial will support

commercialization in 2019.

Two more studies of the IL-12 gene therapy will commence.

One is a dose escalation trial that will test it in combination with

a checkpoint inhibitor in adults with glioblastoma. The other will

enroll children with brain tumors for whom today’s treatments

offer little benefit. The studies may yield some data late this year

and improve the therapy’s efficacy and/or broaden its use.

A Choice of Two Therapies for AML: Based on their

constitution, patients will receive one of two immunotherapies in

the 2019 clinical trials. CD33-CAR T cells may offer a cure for

patients eligible for a hematopoietic stem cell transplant while

allogeneic NK cells may yield a survival benefit for the infirm.

Neoantigens & Personalized Cancer Care: The age of

personalized immunotherapy will dawn with a trial targeting

solid tumor neoantigens. This major advance will test T cells

modified via the Sleeping Beauty technology to express TCRs

against a neoantigen identified in each patient’s tumor. This

contrasts with an approach targeting cancer antigens that are

characteristic of certain malignancies. The initial study, which

will likely commence later this year, will be conducted under the

CRADA with Dr. Rosenberg of the NIH.

We are maintaining our BUY rating on ZIOP shares and

raising our price target from $21 to $23.


We’ve performed a comparative analysis in setting our price target for this report. The results indicate that Ziopharm is undervalued relative to its peers today and that its stock offers superior appreciation potential over the next 12months based simply on execution of the clinical trials that are scheduled to commence this year. The R&D pipeline, shown in Figure 1, illustrates the many products that will be involved in human testing and the breadth of the underlying technologies. Ziopharm’s R&D pipeline incorporates four basic technologies:

  1. control of gene expression via the RheoSwitch® (e.g., interleukin-12 gene, identified as Ad-RTS-hIL-12),
  2. (ii) chimeric antigen receptors (CAR) on T cells produced at the point of care,
  3. (iii) natural killer cells (NK cells), and
  4. (iv) synthetic T cell receptors (TCR).

In addition, the Company has two distinct methods of preventing/treating graftversus-host disease (GvHD) in preclinical development, involving in one case T cells that express interleukin-2 under RheoSwitch control and in the other, a common food bacterium that delivers the same cytokine to immune cells lining the gastrointestinal tract.

Figure 1. The Ziopharm R&D Pipeline

Overall, seven clinical studies are scheduled to commence this year, including a pivotal trial of its IL-12 gene therapy for glioblastoma (GBM). Four should start in the first half, including two investigating CD33-CAR T cells and off-the-shelf NK cells for acute myeloid leukemia (AML). The other two studies will involve patients with tumors of the central nervous system. The pediatric study will employ only the IL-12 gene therapy, while the GBM study will use it in combination with a checkpoint inhibitor targeting the PD-1 pathway. A trial of a 3rd generation CD19-CAR T cell that expresses IL-15 bound to its receptor is planned for the second half at MD Anderson (MDACC), and the first TCR therapy targeting neoantigens will commence at the National Cancer Institute (NCI) via the CRADA with Dr. Steven Rosenberg.

While our valuation analysis is based on the number of distinct products and indications being evaluated, we have not included several key “assets,” even though they distinguish Ziopharm in the field of immunotherapies:

  1. The Company is run by an expert, Dr. Laurence Cooper, who renowned for his track record of innovation in the field.
  2. Ziopharm has exclusive rights to the Sleeping Beauty transposon/transposase technology for modifying cells through the insertion of therapeutic genes. This technology is the keystone in production of immunotherapies at the point of care that will be tested clinically this year.
  3. A collaboration with Intrexon Corporation is providing expertise in synthetic biology and exclusive access to the RheoSwitch Therapeutic System® that enables thermostat-like control over gene expression in vivo via an oral activator called veledimex. And
  4. the Company has secured patents underpinning competitive advantages, such as membrane-bound IL-15 for the production of cells at the point of care and elimination of HLA-A antigen for broad applicability of allogeneic T cells.

Ziopharm has sufficient cash on hand to finance operations into the first quarter of 2018, including the seven clinical trials that are scheduled for this year. We note that the trials labeled with “MDACC” in Figure 1 will be paid through the Company’s 2015 collaboration agreement with MD Anderson, which stipulates annual payments of $15- $20 million to the cancer center. The trial labeled “NCI” is being financed via the National Cancer Institute under the recently signed CRADA with another immunotherapy expert, Dr. Steven Rosenberg. Accordingly, the only new trials that will be funded by Ziopharm are the three testing the IL-12 gene therapy. However, we would not be surprised if Ziopharm outlicenses that drug to a large pharmaceutical corporation, since the Phase 1 study yielded

clear safety and efficacy data, including a large median overall survival benefit. Our discussions with Dr. Cooper recently led us to believe there are several companies are interested in partnering. Our valuation analysis, which compares the clinical programs of Ziopharm against those of other companies, determined that ZIOP shares are undervalued today and that they offer superior appreciation potential over the next 12 months. We are maintaining our BUY rating and have raised our price target from $21 to $23.


Here, we provide background information about the clinical trials that Ziopharm will initiate this year and we discuss the design of a few trials to the extent possible. (Details of the trials have not been made public yet, but all will be available on the Clinicaltrials.gov website. The Phase 1 trials will be small, probably with fewer than 20 patients each, and dedicated to identifying safety issues and seeking some evidence of efficacy.) We begin with the single most important valuation-driving study, the pivotal trial of IL-12 gene therapy for GBM, and then progress down the list as presented in the R&D Pipeline illustration.


As perhaps the most potent stimulator of the immune system, IL-12 has garnered considerable attention by scientists worldwide for combating cancer and other diseases. But its potency is also its limitation, because it can cause severe adverse events including cytokine storm, neurotoxicity, and death. Using Intrexon’s RheoSwitch to control expression, Ziopharm has been able to identify an activator dose that generates sufficient IL-12 from its gene therapy to markedly improve survival of patients with advanced disease and minimize the number and severity of adverse events.

The Pivotal IL-12 Gene Therapy Study

Ziopharm recently met with the FDA to discuss the results of its initial clinical trial of the IL-12 gene therapy. The agency deemed it to be an “end of Phase 2” meeting even though it has been designated to be a Phase 1 trial. This distinction is important to understanding the status of the therapy’s development and its contribution to the valuation of ZIOP shares.

Two Potential Trial Designs: The Company has not finalized its plans for the pivotal study yet, since it will meet with the FDA (and possibly the EMA) to discuss the alternatives. One of the two designs that are considered possible is a single-arm study in which the overall survival benefit of the gene therapy would be compared against historical data. This alternative may be acceptable since today’s best care for patients with advanced glioblastoma (stage III and IV) provides little/no benefit. An obvious advantage of using this type of trial is that it would require fewer patients, most likely in the range of 50 – 100. The other design under consideration is a standard two-arm trial in which 100 – 200 patients, probably enrolled in the United States and Europe, would be randomized between physician’s choice of care and the IL-12 gene therapy plus physician’s choice of care. Regardless of which alternative is selected, the patients will have to be candidates for surgery upon enrollment. After the initial surgery + IL-12 treatment (1 x 1012 viral particles and 14 days on 20mg/day veledimex), the patients may be treated surgically again but they would not be permitted to receive a second cycle of veledimex.

Foundational Work: The Phase 1 trial identified the 20 mg/day dose of veledimex as both efficacious and well tolerated. The most recent update compared the median overall survival of glioblastoma patients who received the IL-12 gene therapy against others who received other treatments at similar stages of development in clinical trials. We believe the comparison (see Figure 2) is telling – as of January 6th, the IL-12 patients had an interim median overall survival of 12.7 months, versus approximately 9 months achieved with the next best drug, temozolomide.1

Figure 2. A Comparison of Median Overall Survival Rates1

The significance of the pivotal study extends beyond gaining regulatory approval of the IL-12 gene therapy for glioblastoma. As discussed below, the Company will soon test the treatment for pediatric brain tumors. But more important, this gene therapy may ultimately be used in combination with other immunotherapies or it may be a stepping stone to a cell therapy that expresses IL-12 under a conditional control, such as binding to a tumor antigen. IL-12 administered directly into individual tumors has initiated an immune response.2,3 But then, Dr. Steven Rosenberg’s lab and others have shown that immune cells modified to express IL -12 are more effective by creating a local inflammatory environment, increasing tumor infiltration by lymphocytes, and restoring the ability of tumor associated myeloid cells to present tumor antigen to adoptive immune cells without causing toxicities typically seen with systemic IL-12 administration.4,5,6,7

IL-12 for Pediatric Brain Tumors

This trial reflects Dr. Cooper’s genuine desire to help individuals diagnosed with cancer. Today, very little can be done for youngsters who develop brain tumors at an early age. Indeed, some cannot be treated surgically, especially in the pontine region of the brain stem (see Figure 3). IL-12 offers a new approach to this disease by stimulating the patient’s immune system to mount an attack against the malignancy. Success in developing this therapy would qualify Ziopharm for a highly valued Priority Review Voucher via the FDA’s Rare Pediatric Disease


1 Cooper, LJ. Presentation at the JP Morgan Healthcare Conference on January 11, 2017.

2 Rook, AH, et al. Interleukin-12 therapy of cutaneous T-cell lymphoma induces lesion regression and cytotoxic T-cell responses. Blood (1999);

94(3): 902.

3 Van Herpen, CML,, et al. Intratumoral recombinant human interleukin-12 administration in head and neck squamous cell carcinoma patients

modifies locoregional lymph node architecture and induces natural killer cell infiltration in the primary tumor. Clin Cancer Res (2005); 11(5):


4 Wagner, HJ, et al. A strategy for treatment of Epstein-Barr virus-positive Hodgkin’s disease by targeting interleukin 12 to the tumor

environment using tumor antigen-specific T cells. Cancer Gene Ther (2004); 11(2): 81.

5 Kerkar, SP, et al. Tumor-specific CD8+ T cells expressing interleukin-12 eradicate established cancers in lymphodepleted hosts. Cancer Res

(2010); 70(17): 6725.

6 Zhang, L, et al. Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther (2011);

19(4): 759.

7 Kerkar, SP, et al. Collapse of the tumor stroma is triggered by IL-12 induction of Fas. Mol Ther (2013); 21(7): 1369.

Figure 3. The Human Brain (see link for graphic)

Trial Design: The Phase 1 study will initially enroll children with tumors in the cerebrum where higher thought

processes take place. The initial goal is to evaluate the safety of different doses of the gene’s activator ligand and

seek evidence of efficacy. Once a suitable dose is identified, children with tumors in the pons will also be enrolled. The reason for this specific approach is based on the crucial role that the pons plays in controlling many bodily functions and in serving as a conduit for nerve fibers between the higher brain and the spinal cord.

Preparation: The Phase 1 study in adult glioblastoma patients has helped to define a range of veledimex dosage strengths (no more than 20 mg/day) to test in this pediatric study. It has also provided physicians with experience in identifying the onset of an adverse reaction that should be addressed quickly. In addition, MD Anderson surgeons have already demonstrated an ability to place a shunt into the fourth ventricle in a study for delivering natural killer (NK) cells to treat pontine tumors.8 (Note: this is one of many trials the cancer center has conducted that may lay the groundwork for a Ziopharm study and is not recognized as Company-related.)

A Combination of IL-12 and a Checkpoint Inhibitor for GBM

This trial is intended to investigate clinically a combination of IL-12 and heckpoint inhibitor that showed a synergistic effect in preclinical testing.9 In that experiment, mice were treated with one of five alternative therapies implanted with glioma cells five days prior to the initiation of a treatment with the IL-12 gene therapy (5 x 109 viral particles + veledimex, 30 mg/m2/day for 14 days) + PD-1 inhibitor (7.5 or 15 mg/m2, 4x/day for 5 days). The combination of IL-12 therapy and the higher dose of the PD-1 inhibitor enabled all of the animals to survive for 90 days, while the gene therapy alone or with the lower PD-1 inhibitor dose were less effective with survival rates of approximately 60% and 80% respectively. The two doses of PD-1 inhibitor alone were less effective still, with survival rates in the 30% - 40% range at 90 days.

Figure 4. Synergistic Effect of IL-12 & a PD-1 Inhibitor9

8 See NCT 02271711 at www.clinicaltrials.gov

9 Barrett, JA, et al. Localized regulated expression of IL-12 as a gene therapy concomitant with blockade of PD-1 for treatment of glioma.

Presented at the American Society of Gene & Cell Therapy meeting on May 6, 2016.

4th Ventricle

Thus, the preclinical test provided a good foundation for a clinical investigation of a combination of the IL-12 gene therapy and a PD-1 inhibitor. The planned study, which will likely resemble the Phase 1 trial of the gene therapy, is intended to offer glioblastoma patients better care and possibly expand the combination’s use to other cancers.


Ziopharm’s news releases in 2016 mentioned some of the most exciting developments in CAR T cells. Two developments were particularly noteworthy and remain so, since they underpin the CAR T cell trials of this year.

Point-of-Care CAR T cells

Ziopharm has applied a recently unveiled approach to improving the quality and reducing the production time of CAR T cells. Specifically, the Company used the Sleeping Beauty system to create CD19-CAR T cells that also express IL-15 tethered to its receptor on the surface of T cells. The proprietary IL-15/receptor complex, which has also been called membrane bound IL-15 (mbIL-15), is a construct that Dr. Cooper developed long before joining Ziopharm.10 (This is known because its effect on T cells was investigated over a period of two years in cell culture starting in 2014.) Those experiments determined that the modified T cells divided normally, maintained normal phenotypes, and included a subpopulation of memory T stem cells. The discovery of memory T stem cells was a remarkable finding because those cells have the ability to recognize a targeted antigen (e.g., a cancer biomarker such as CD19), expand into cytotoxic T cells to destroy the malignant cells, and then remain on guard should the cancer return. This modification to Ziopharm’s second generation CD19-CAR T cells underpins its plan to create CAR T cells in only two days at the point-of-care.

We believe there are three reasons that the creation of genetically modified T cells at the point of care will be a major competitive advantage for Ziopharm:

1 Cell therapies created at the point of care with minimal time ex vivo have a healthier, more normal mix of T cells. Indeed, the Company demonstrated this in an experiment discussed at the 2016 American Society of Hematology meeting.11 As shown in Figure 5, expression of mbIL-15 drastically reduced the time required in culture to prepare CAR T cells

2. CAR T cells with mbIL-15 have potent anti-tumor activity. As shown in Figure 6, leukemic mice treated with point-of-care modified CAR T cells expressing mbIL-15 survived significantly longer than mice treated with the CAR T cells lacking mb-IL-15 (POC-CAR T cells) and two control groups, tumor only and T cells lacking a CAR (POC-CARneg T cells). Hence, the short ex vivo cell expansion with point-of-care production yields fewer, but more robust T cells.

10 Hurton, LV, et al. Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Proc Natl

Acad Sci USA (2016); 113(48): e7788.

11 Hurton, LV, et al. Very rapid production of CAR+ T cells upon non-viral gene transfer using the Sleeping Beauty system. Poster #2807

presented at the 2016 American Society of Hematology meeting, December 4, 2016

Figure 5. Point-of-care production yields better-quality cells.10

Ziopharm examined the effect of time in cell culture on T cell

subpopulations. All of the cells were modified with the Sleeping

Beauty DNA transposon & transpose to express a CAR and then

grown in culture for 28, 14, or 2 days. The cells to be grown for

only 2 days included a gene for mbIL-15, while cells grown for 14

and 28 days were stimulated according to protocol by antigen

presenting cells (K5652 AaPC). An analysis showed the longer the

cells were grown ex vivo, the more homogeneous was the final

population. In other words, the 2-day point-of-care production

yielded a richer, more normal mix of cells that included three

basic T cells: naïve (TN), effector (TEff), and memory, including

three types of memory T cells: memory stem (TSCM), central

memory (TCM), and effector memory (TEM)].

Figure 6. Increased Survival with mbIL-15 CAR T Cell Therapy

3. Membrane-bound IL-15 supports memory T stem cells, as well as two other types of memory T cells (see Figure 5). This is not inconsequential. Memory T stem cells have a special place in the immune system standing guard against disease or infection long after the initial offensive agent has been eradicated. When

they encounter a target they divide, become effector T cells, and eradicate the threat. Thus, they protect against disease recurrence.

4. Point-of-care production comes with competitive advantages. Preparation of CAR T cells expressing mbIL-15 at the point of care will eliminate shipping costs and other logistics-related expenses, and greatly reduce cell culture costs. In addition, the culture system required for point-of-care production will be small

and relatively inexpensive compared with the cost of a centralized manufacturing plant. As a result, Ziopharm will likely have a pricing advantage.

This year’s trial of Ziopharm’s 3rd generation CD19-CAR T cells, which express mbIL-15, will be the first to test clinically the point of care production method and the most advanced CAR T cells. Given the extensive preclinical

testing performed to assure safety and demonstrate anti-tumor activity, the Phase 1 trial will likely yield results that lay the foundation for a pivotal study and commercialization in 2019. Moreover, we believe the results from the Phase 1 study will lead to a partnering agreement.

CD33-CAR T cells for AML

AML Therapeutic Strategy: Ziopharm’s AML program consists of two therapies, the CD33-CAR T cells and off-the-shelf NK cells, which are discussed in the next section of this report. The two offer promise against an insidious disease that cuts short the lives of 60% of younger patients and 90% of the elderly. The difference in overall survival rates is related to unfavorable biological features associated with the elderly’s disease and an inability to treat them with consolidation therapy, notably allogeneic stem cell transplantation, due to co-morbidities. The CD33-CAR T cells are intended for individuals who are sufficiently healthy to undergo hematopoietic stem cell transplantation, since CD33 is a biomarker found on both normal and the vast majority of malignant myeloid cells.12 Treatment with the CAR T cells therefore will be followed by the stem cell transplantation to restore the normal myeloid cell populations.

Preclinical Research: The Company has demonstrated the benefit of its CD33-CAR T cells in immunocompromised mice that were administered a commonly studied human AML tumor cell line expressing a fluorescent marker.13 The malignant cells were allowed to engraft for eight days prior to administration of T cells that expressed a CD33 CAR and a kill switch, consisting of a segment of the human epidermal growth factor receptor that initiated cell death upon exposure to the clinically available drug cetuximab (marketed under the brand name Erbitux® by Eli Lilly in North America and by Merck KGaA elsewhere). The CD33-CAR T cells engrafted and expanded in vivo between days 8 and 32, as shown in Figure 7, and the cells could be eliminated with exposure to cetuximab, which confirmed the effectiveness of the kill switch.

12 Ehninger, A, et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. Blood Cancer J (2014); 4:


13 Song, D, et al. Chimeric antigen receptor-modified T cells for the treatment of acute myeloid leukemia expressing CD33.

Figure 7. Expansion& Persistence of CD33-CAR T cells In Vivo13

The CD33-CAR T cells almost completely eliminated the AML tumor about 7 days after administration (see left panel in Figure 8), resulting in a survival benefit (see right panel in Figure 8) when compared with three control

groups that received saline, untransduced T cells, or CD19-CAR T cells. (CD19 is not expressed on AML cells.)

Figure 8. CD33-CAR T Cells Are Effective Against AML13

The clinical program for the CD33-CAR T cells has not been finalized, but it seems likely that the trial, like the other Phase 1 studies planned for this year, will be relatively small and probably focused on a few doses of the CAR T cells, assessing safety and seeking evidence of efficacy. However, we note that there have been a number of studies of CD33 antibodies that provide evidence of clinical activity against AML and a small number of investigations involving CD33-CAR T cells.14,15,16,17


Ziopharm has two therapies under development utilizing NK cells. One has not been identified as it is at a preclinical stage, while the other is the second therapy for AML, which will enter clinical testing soon. These cells are an attractive alternative to T cells, in part because they kill independently of a specific antigen, but respond to activating and inhibitory signals on potential target cells. Then, too, they do not need genetic modification to avoid graft-versus-host disease that often comes with T cells. (Evidence indicates that off-the-shelf NK cells present a very

low risk of causing graft-versus-host disease.) Yet, allogeneic NK cells can enter an activated state and initiate a 14 Laszlo, GS, et al. The past and future of CD33 as a therapeutic target in acute myeloid leukemia. Blood Rev (2014); 28(4): 143.

15 Harrington, KH, et al. The broad anti-AML activity of the CD33/CD3 BiTE antibody construct, AMG330, is impacted by disease stage and risk. PLoS ONE (2015); 10(8): e0135945.

16 Want, QS, et al. Treatment of CD33-directed chimeric antigen receptor-modified T cells in one patient with relapsed and refractory acute

myeloid leukemia. Mol Ther (2015); 23(1): 184.

17 Cartellieri, M, et al. Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts. Blood Cancer J

(2016); 6(8): 458.

graft-versus-leukemia response against a recipient’s malignant cells, since they may not be subject to the signals (e.g., HLA antigens) that inhibit the recipient’s own NK cells.

NK cells for AML

AML is an ideal candidate for an NK cell therapy, as allogeneic cells require no artificial modification to direct them to target a recipient’s malignant cells. And because they do not target a single biomarker by their very nature, they will likely mount an attack against the various abnormalities associated with multiple myeloma and AML. Use of allogeneic NK cells may also prove particularly beneficial for AML patients who have a poor prognosis today since their NK cells have become defective.18

cGMP Production of NK Cells: Dr. Cooper’s lab at MD Anderson developed a GMP-compliant method for expanding umbilical cord NK cells in culture long before he joined Ziopharm.19,20 Briefly, the procedure, which is illustrated in Figure 9, involves harvesting umbilical cord blood mononuclear cells (not peripheral blood as depicted), expansion of the cells in the presence of antigen-presenting cells expressing IL-21 on their surface (K562 m-IL21 aAPC), depletion of T cells to prevent the potential of graft-versus-host disease, and preservation in

aliquots. This two-week process yields 1.9 x 107 NK cells/kg for an average 70 kg adult from 1/10th of a cord blood unit. Just as important, the NK cells expanded and preserved according to this protocol exhibit strong cytotoxic activityin vitro and in vivo. Ziopharm will employ cord blood rather than peripheral blood because of a lower risk of graft-versus-host disease and the ready availability of more than 600,000 banked units worldwide.

Figure 9. Preparation of NK Cells21

18 Khaznadar, Z, et al. Defective NK cells in acute myeloid leukemia patients at diagnosis are associated with blast transcriptional signatures of

immune evasion. J Immunol (2015); 195(6): 2580.

19 Denman, CJ, et al. Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells. PLoS ONE (2012); 7(1):


20 Shah, N, et al. Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with

anti-myeloma activity. PLoS ONE (2013); 8(10): e76781.

21 Cooper, L. Therapeutic implications of preparing and administering innate immune cells. Presented at the Innate Killer Summit on May 16,


AML Trial of NK Cells: The study of NK cells for AML will complement the CD33-CAR T cell trial in that it will enroll patients who are not candidates for a hematopoietic stem cell transplant, while the latter will treat patients who will receive a transplant. The research that contributed to the development of the cGMP production process suggested that NK cells might be best used in combination with alkylating or immunomodulatory agents. As a result, we would not be surprised if such a combination is employed in this study.


The TCR clinical trial that is planned for this year will be performed through a collaboration with Dr. Rosenberg of the National Cancer Institute. The type(s) of solid tumors that will be involved in the study have not been identified. However, we believe the trial will follow the work that has been done already in two areas, the identification of TCRs already expressed on the patient’s T cells and employing the Sleeping Beauty system to express the neoantigen-specific TCRs on therapeutic autologous T cells.22 Two techniques developed by Drs. Rosenberg and Cooper have facilitated the identification of TCRs. One involves using the expression of the PD-1 checkpoint on cytotoxic T cells (i.e., CD8+ T cells) to rapidly identify T cells targeting neoantigens and the other is a highthroughput

system for determining which TCRs have the optimal affinity for the selected neoantigen(s).23,24

Accordingly, we believe Ziopharm is well positioned to advance the field of personalized immunotherapies for solid tumors.

22 Deniger, DC, et al. Stable, non-viral expression of mutated tumor neoantigen-specific T-cell receptors using the Sleeping Beauty

transposon/transposase system. Mol Ther (2016); 24(6): 1078.

23 Gross, A, et al. Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients. Nat Med (2016);

22(4): 433.

24 Zong, S, et al. Personalization of T-cell therapy using a high-throughput platform to identify tumor-specific T-cell receptors. Poster 3359

presented at the American Society of Hematology on December 4, 2016.


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76713 Re: "You can see control of both CAR expression as well as membrane-bound IL15 production through RheoSwitch platform utilizing the Sleeping Beauty system. This unique capability through our gene switch enables an unrivaled control approaching CAR-T cell Steve1d 96 3/22/2017 4:34:04 PM

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