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April 2018 - Volume 13, No.2

Cliona Rooney, PhD Professor, Departments of Molecular

ology & Microbiology, Pediatrics and Pathology Center for Cell & Gene Therapy

Baylor College of Medicine Houston, Texas 77030

Q. Please describe your clinical and research interests. A: Clinical Research Interests: I have a longstanding interest using immuno­therapy with anƟ gen-specific T cells to treat viral disease in immunosuppressed individuals, and as therapy for malignant diseases. My iniƟal studies focused on lymphomas resulƟng from Epstein Barr virus infecƟ on post-hematopoieƟc transplantaƟon, where we were able to show that treatment with EBV- specific T cells was safe and highly effecƟve. We then extended this approach to the EBV-associated cancers Hodgkin’s disease (HD) and nasopharyngeal cancer (NPC) in immunocompetent individuals. In HD we were able to demonstrate that the T cells could expand, traffic to the tumor sites and induce clinical responses. In NPC two paƟents with advanced disease had complete sustained remissions, and two others had parƟal responses. This approach is now under study in clinical trials supported by biotechnology companies. I am currently working on geneƟ c modificaƟon of the T cells to overcome tumor evasion mechanisms. As a component of this approach, our group has been involved in the development of a suicide gene to destroy therapeuƟc T cells that may be producing adverse side effects. The gene is acƟvated by the administraƟon of a dimerizing drug to the paƟent, and results in rapid destrucƟon of only those T cells that have been geneƟ cally modified.

Q. Can you describe the projects in which PACT supported your work? A: CAGT previously received PACT support for a number of projects that involved generaƟ ng anƟ gen-specific T cells for clinical use. This allowed the iniƟaƟon of Phase 1 trials that produced data that were used to change and accelerate manufacturing procedures, provide informaƟon on tumor evasion strategies and devise strategies for increasing effector cell efficacy. My current project reflects the new translaƟonal focus of PACT. The use of retroviral vectors to geneƟ cally modify T cells has always been a concern. The vectors are expensive to manufacture and test. This can be avoided by the use of the Piggy Bac transposon, which is a mobile geneƟc element that transposes between vectors and chromosome via a cut and paste mechanism. The cells are geneƟ cally modified with transposon and transposase plasmids, using electroporaƟon. The system is effecƟve but can be highly toxic. The aim of the project is to (i) increase transgene expression by lowering DNA transfecƟon-induced cell death; (ii) increase the expansion of transgene expressing-cells; (iii) improve understanding of the reasons for DNA transfecƟon-induced T cell toxicity, and (iv) use the data to develop a GMP-compliant strategy that is effecƟve for both virus-specific T cells (VSTs) and CD3 and CD28 anƟbody sƟmulated T cells (ATCs).

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Contact PACT Please contact the PACT CoordinaƟng Center if you have any quesƟons regarding the PACT program.

Visit our website at: www.pactgroup.net

Contact Debbie Wood or Lani Ibenana

Address The Emmes CorporaƟon 401 North Washington Street Suite 700 Rockville, MD 20850 Telephone: (301) 251-1161 Email: pacƟ nfo@pactgroup.net

In this issue: PI Highlight

Harnessing the AnƟ tumor PotenƟal of Natural Killer Cells

EducaƟ onal Updates

Don't Miss PACT

Want to apply to PACT?

Products & Services

General Facility SOPs

PACT Cell Processing FaciliƟes

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Harnessing the Ant/tumor Potential o,Natural

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Shelly M. Williams and David H. McKenna, Jr.

The University of Minnesota Molecular and Cellular Therapeutics

Natural killer (N K) cells are large granular lymphocytes of the innate immune system with

effector and immunoregulatory functions. Circulating NK cells surveil for virally-infect ed and

transformed cells and exert cytotoxic activity in the absence of prior antigenic sensitization. Unlike T lymphocytes, NK cells do not require antigen

presentation by major histocompatibility complex class I (MHC-I). In fact, NK cells are "educated" to recognize

self and remain quiescent by engaging MHC-I while simultaneously becoming activated to lyse target cells

Figure 1. Comparative manufacturing of clinical-scale, that lack or downregulate this expression (1). They are distinguished by surface expression of CD56 and lack o f surface

CD3 expression .

Exploitation of NK cells for their antitumor potential has led to human clinical trials in adoptive immunotherapy for solid and

hematologic malignancies. Allogeneic haploidentical NK cells have the ability to overcome the self-tolerance seen with autologous NK cells and can produce a graft-versus-tumor effect without apparent graft-versus-host disease (2,3). Thus far, the greatest benefit has been shown in patients with acute myelogenous leukemia, including as salvage therapy for relapsed or refractory disease, consolidation therapy following hematopoietic cell transplant, and NK donor lymphocyte infusions for relapse or graft rejection (4-9).

We recently reported our 15-year single institutional experience in clinical-scale production of NK cells under cGMP conditions for use in early phase human trials (10). Briefly, non-mobilized peripheral blood mononuclear cells were collected by apheresis and incubated with immunomagnetic microbeads (anti-CD 3, anti-CD56, or anti-CD19) prior to loading on a column in an automated cell selection system. The enriched NK cell products were incubated overnight in cytokine (IL-2 or IL-15) to promote cytotoxic activation and administered to patients following successful lot release testing. See figures 1 and 2 for comparative manufacturing processes and product characterization.

Patients receiving our initial T (CD3+) cell-depleted NK cell products achieved clinical remissions; however, complications related to B cell contamination caused us to move to a higher level of NK cell purification using sequential CD3 depletion and CD56 enrichment (4). Unfortunately, this 2-step process led to significant cell loss and poor NK cell recovery. Despite these shortcomings, we continue to explore the efficacy of the more highly purified NK cell product with an open prospective randomized controlled trial comparing CD3/CD19 depleted to CD3 depleted/CD56 enriched haploidentical NK cell therapy (clinicaltrials.gov, NCT03152526). Presently our primary method of simultaneous CD3/CD19 depletion effectively minimizes T cell and B cell contamination in a single manipulation without compromise to NK cell recovery, and clinical remissions have been achieved (5). For a more in-depth review, please refer to the full study in Transfusion (10).

References

1. Farag SS, Fehniger TA, Ruggeri L, et al. Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood (2002); 100(6): 1935-1947.

2. Ruggeri L, Capanni M , Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science (2002); 295(5562): 2097-2100.3-4301.

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Figure 2. ComparaƟve flow cytometry characterizaƟon of the unmanipulated apheresis mononuclear cells and the NK cell products from each manufacturing process10.

References (continued) 3. Olson JA, Leveson-Gower DB, Gill S, et al.

NK cells mediate reducƟon of GVHD by inhibiƟ ng acƟ vated, alloreacƟve T cells while retaining GVT effects. Blood (2010); 115(21): 4293-4301.

4. Miller JS, Soignier Y, Panoskaltsis-Mortari A, et al. Successful adopƟve transfer and in vivo expansion of human haploidenƟcal NK cells in paƟents with cancer. Blood (2005); 105(8): 3051-3057.

5. Bachanova V, Cooley S, Defor TE, et al. Clear ance of acute myeloid leukemia by haploiden­Ɵcal natural killer cells is improved using IL-2 diphtheria toxin fusion protein. Blood (2014); 123(25): 3855-3863.

6. Shaffer BC, Le Luduec JB, Forlenza C, et al. Phase II Study of HaploidenƟcal Natural Killer Cell Infusion for Treatment of Relapsed or Persistent Myeloid Malignancies Following Allogeneic HematopoieƟ c Cell TransplantaƟon. Biol Blood Marrow Transplant (2016); 22(4): 705-709.

7. CurƟ A, Ruggeri L, Parisi S, et al. Larger Size of Donor AlloreacƟve NK Cell Repertoire Correlates with BeƩer Response to NK Cell Immu­notherapy in Elderly Acute Myeloid Leukemia PaƟents. Clin Cancer Res (2016); 22(8): 1914-1921.

8. Rubnitz JE, Inaba H, Ribeiro RC, et al. NKAML: a pilot study to determine the safety and feasibility of haploidenƟcal natural killer cell transplantaƟon in childhood acute myeloid leukemia. J Clin Oncol (2010); 28(6): 955-959.

9. Passweg JR, Tichelli A, Meyer-Monard S, et al. Purified donor NK-lympho cyte infusion to consolidate engraŌ ment aŌ er haploidenƟcal stem cell transplantaƟon. Leukemia (2004); 18(11): 1835-1838.

10. Williams SM, Sumstad D, Kadidlo D, et al. Clinical-scale producƟon of cGMP compliant CD3/CD19 cell-depleted NK cells in the evoluƟon of NK cell immunotherapy at a single insƟtuƟon. Transfusion (2018), accepted for publicaƟon.

PACT is excited to announce a web seminar on the development of GMP cell manufacturing of cardiac stem cells.

The web seminar will be on June 19, 2018, 12:00-1:00 PM EST, with speakers from the University of Miami:

• Joshua Hare, MD • Norma Sue Kenyon, PhD • Aisha Khan, MSc, MBA

Visit www.pactgroup.net for further updates on this web seminar.

Visit the PACT Group exhibit booth at the InternaƟonal Society for Cellular Therapy Annual MeeƟng (ISCT) on May 2-5, 2018 in Montréal, Canada.

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Center for Cell and Gene TherapyBaylor College of MedicinePI: Adrian Gee, MI Biol, PhD

Contract Number: HHSN268201600015I

Interdisciplinary Stem Cell InsƟ tute Cellular Manufacturing Program,

University of Miami, Miller School of MedicinePI: Joshua Hare, MD Contract Number:

HHSN268201600012I

University of Minnesota, Molecularand Cellular TherapeuƟ cs Facility

PI: David McKenna, MD Contract Number:

HHSN268201600014I

City of Hope, Center for Biomedicine and GeneƟcs

PI: Joseph Gold, PhDContract Number:

HHSN268201600011I

MoffiƩ Cancer Center PI: Linda Kelley, PhDContract Number:

HHSN268201600013I

PACT CoordinaƟ ng Center:The Emmes CorporaƟon

Contract Number: HHSN268201600020C

PACT is accepƟ ng applicaƟons!

If you are interested in applying for PACT services, please review the materials available on the PACT website www.pactgroup.net under the Apply to PACT tab including informaƟ on regarding the PACT scope and evaluaƟ on criteria used for evaluaƟng PACT applicaƟons.

If you think your service request meets the PACT scope criteria, you may register and apply through the online ApplicaƟon System at any Ɵme. You can also contact the CoordinaƟng Center at pactgroup@ emmes.com if you need assistance with your applicaƟon or have any addiƟonal quesƟons.

PLEASE NOTE that these SOPs are for INFORMATIONAL PURPOSES ONLY and therefore require validaƟon by your own facility. To see a full list of SOPs available for request go to the PACT website and look under the Resource Center tab.

SOP Categories available for request: Cleaning Procedures

DeviaƟ on Management Environmental Monitoring Personnel Training Quality Assurance / Quality Control Quality Management SOP: Development and Management ValidaƟ on Process

This project has been funded in whole or in part with Federal funds from the NaƟonal Heart, Lung, and Blood InsƟ tute, NaƟ onal InsƟtutes of Health, Department of Health and Human Services.

Progenitor CellsC-kit+ cells, Endothelial cells, HSCs, MSCs, iPSCs

Lymphocytes Virus-specific T-cells, Tumor-infi ltraƟng lymphocytes (TILs),

EBV-transformed B cell lines

GeneƟ cally Modifi ed Cells CAR-T-cells, Cytotoxic T lymphocytes, fi broblasts

Services Cell culture isolaƟon, expansion, and cryopreservaƟon, cell depleƟ on/cell

enrichment, cell manufacturing for animal model studies

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