1. Natural Killer Cells: Their role in Cancer Immunotherapy, And the Significance of the Molding of the KIR Repertoire Jerome R. Trembley Senior Seminar February 19th, 2015

2. Topical Objectives  Introduction to work at MD Anderson  What are Natural Killer Cells  NK Cell Immunotherapy  NK Cells and the Killer Immunoglobulin- Like Receptor  Molding of the KIR Repertoire  Future Work…

3. What did I do? Also Shadowed Dr. Lee weekly during clinic periods Able to observe the majority of his patients, including a Bone Marrow Extraction/Transplant Img Source: Self

4. What did I do?  Conducted bench-top research at MD Anderson Cancer Center, Houston Texas, from May 19 th, to August 14 th, 2013.  14, 3 week long experimental batteries performed.  Abstract submitted, and accepted at ASH – Presented on Dec 14 th, 2013.

5. What are NK Cells – What are they?  Part of the Innate Immune System Lymphocyte.  GVL, with minimal GVHD. (Lee, 2015)  Selective Two methods of cytotoxicity (Cheng., et.al. 2013) ○ Granzymes ○ Perforin  Highly Cytotoxic to Cancer Enhanced efficacy on a range of cancers. (Cheng et.al., 2013)

6. NK Cell Utilization – Why do we need NK Cells? Img Source: Cancer Research UK, 2014

7. NK Cell Utilization – Why do we need NK Cells? Img Source: Cancer Research UK, 2014

8. What are NK Cells? – NK Cell suppression in compromised systems  NK Cells are needed to maintain innate immune defense.(Lion et.al., 2012)  NK Cells are severely compromised via current immunotherapy methods. (Lion et.al., 2012)  When compromised, cancer cells “escape” detection. (Lion et.al., 2012) Sources: Lion et.al., 2012

9. What are NK Cells? – NK Cell suppression in compromised systems  NK Cells are needed to maintain innate immune defense.(Lion et.al., 2012)  NK Cells are severely compromised via current immunotherapy methods. (Lion et.al., 2012)  When compromised, cancer cells “escape” detection. (Lion et.al., 2012) Sources: Lion et.al., 2012

10. What are NK Cells? – NK Cell suppression in compromised systems Img Source: Lion et.al., 2012

11. Img Source: Glycostem, 2014 What are NK Cells? – GVHD vs. GVL “NK cells have shown to mediate graft-versus-leukemia immunity towards recipient tumor cells without attacking normal tissues, leading to graft-versus-host disease.” - Glycostem, 2014

12. What are NK Cells? – Why Are they Effective? Video Source: Somanchi, et.al., 2011

13. What are NK Cells? – Why Are they Effective? Img Source: Cheent, K., & Khakoo, S., 2009

14. What are NK Cells? – The Killerimmunoglobulin-Like Receptor Passport “780-C1” Passport “780-C2” Passport “Bw4” KIR-2DL2/3 Utilizes same Passport, Same Crosslinked Antibody Img Source: Lanier, 2005

15. Point of Study “NK Cell-based immunotherapy holds great promise for treatment... Only moderate clinical success so far… “Progress in the field of understanding NK cell biology and function is therefore needed to assist in developing novel approaches to effectively manipulate NK cells for the ultimate benefit of treating cancer patients.” – Cheng et.al., 2013

16. Hypothesis 1 – The desired KIR Repertoire could be molded through inhibition of undesirable KIR populations through the crosslinking of relevant Anti-KIR antibodies during expansion. 2- The KIR Repertoire molding would have no effect on expanded NK Cell cytotoxicity

17. Experiment – Methodology Early Difficulties 1.Initial outlook was poor 1.Attempts to stimulate growth via antibodies proved ineffective. 2.Determined inhibition protocol in early July. 3.Was able to isolate most efficient means by July 30 th. 1.Most efficient means determined by stimulation on Day 3 by loading and stimulating with K562.

18. Experiment – Methodology Ideal Method 1.Initiate Experiment In accordance with Expansion Protocol. A.Extract Cells from Buffy Coat B.Determine Phenotype (KIR Receptors) of NK Cells Done using antibody identifiers. C.Culture in accordance with Expansion Protocol FARG Solution (Fetal Bovine Serum + Culturing Media) 2.D=3, Load NK Cells with Anti-KIR aB. 3.Crosslink with Goat Anti-Mouse aB. 4.Suspend cells with 1:1 ratio of K562 Cells in accordance with Expansion Protocol

19. Results Population results for Unloaded vs. KIR- Crosslinked cell lines. KIR2DL1- 70.4% ±19.3% KIR3DL1- 53.5% ±16.3% KIR2DL2/3– 56% ±17.5% Gfx Source: Self

20. Results Gfx Source: Self Anti KIRPercent ReductionMargin of Error (±%) 2DL170.419.3 3DL156.017.5 2DL2/353.516.3

21. Results Flow Cytometry Data: NK Cell subpopulations Vs. Isotype control. Crosslinking with individual CD158e (KIR3DL1) and Bulk KIR populations. Marked molding of KIR Repertoire is noted in undesired populations. Gfx Source: Self

22. Results Degranulation response demonstrated efficacy against tumor cells expressing relevant HLA Ligand for targeted KIR Additionally, Bulk NK Cell cytotoxicity against HLA- Negative targets remained robust. Gfx Source: Dr. Vladimir Senyukov, unpublished

23. Hypothesis Confirmed  A robust reduction of targeted KIR-Positive populations was achieved.  Cytotoxicity of bulk NK cell population remained intact

24. Implications of Work Increased Anti-tumor affect from utilization of KIR Mismatch Easy to use Immunotherapy Cheap Easily understood Easily replicable for clinical grade application Most Importantly: It has the potential to enhance the efficacy of NK Cell Immunotherapy!

25. Future Work  Further Experimentation  GVHD vs GVL – A problem…

26. Img Source: Lee, 2015 Issues with NK Cell Immunotherapy- GVHD vs. GVL – Possible Connection? 1.Direct effect via lysis or indirect activation of adaptive immunity 2.Cytokine-mediated upregulation of HLA for T-Cell recognition 3.Additional stimulation of Cytotoxic T-Cells 4.Activation of Helper T- Cells 5.Maturation of dendritic cells for antigen presentation

27. Future Work  Further Experimentation  GVHD vs GVL – A problem…  Signaling Pathway – STAT3 Confirmed! “…data show that NKG2D expression in NK cells is regulated at the transcriptional level by STAT3.” -Zhu, et.al., 2014

28. Acknowledgements  Dr. Dean Lee, MD, PhD Associate Professor – MD Anderson, Pediatrics.  Dr. Vladimir Senyukov, PhD Research Scientist, MD Anderson, Pediatrics.  St. Baldrick’s Foundation  MD Anderson Cancer Center  Fresno Pacific University – Biology Department

29. References  Cancer Research UK. Childhood Cancers by Cancer Site: 1996-2005. (2014, March 11). Retrieved February 15, 2015, from http://www.cancerresearchuk.org/cancer- info/cancerstats/childhoodcancer/mortality/childhood-cancer-mortality-statistics  Cheent, K., & Khakoo, S. (2009). Natural killer cells: Integrating diversity with function. Immunology, 126(4), 449-457. doi: 10.1111/j.1365-2567.2009.03045.x  Cheng, M., Chen, Y., Xiao, W., Sun, R., & Tian, Z. (2013). NK cell-based immunotherapy for malignant diseases. Celllular & Molecular Immunology, 10, 230-252. doi: 10.1038/cmi.2013.10  Glycostem. Allogeneic NK cells for immunotherapy. (2014). Retrieved February 16, 2015, from http://glycostem.com/technology/nk-cell-immunotherapy  Lanier, L. (2005). NK Cell Recognition. Annual Review of Immunology, (23), 225-274. doi: 10.1146/annurev.immunol.23.021704.115526  Lee, D. (2015). The off-target effects of nonspecific NK cells. Blood, 125(5), 774-775.  Lion, E., Willemen, Y., Berneman, Z., Van-Tendeloo, V., & Smits, E. (2012). Natural killer cell immune escape in acute myeloid leukemia. Leukemia, (2012 Review), 1-8. doi: 10.1038/leu.2012.87  Somanchi, S., Senyukov, V., Denman, C., & Lee, D. (2011). Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells. Journal of Visualized Experiments, 48  Zhu, S., Phatarpekar, P., Denman, C., Senyukov, V., Somanchi, S., Nguyen-Jackson, H., & Lee, D. (2014). Transcription of the activating receptor NKG2D in natural killer cells is regulated by STAT3 tyrosine phosphorylation. Blood,124(3). doi: 10.1182/blood-2013-05- 499707