Natural Killer Cells Their role in Cancer Immunotherapy

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

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…

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

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.

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)

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

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

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

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

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

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 Img Source: Glycostem, 2014

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

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

What are NK Cells? – The Killerimmunoglobulin-Like Receptor Passport “ 780 -C 1” Passport “ 780 -C 2” Passport “Bw 4” KIR-2 DL 2/3 Utilizes same Passport, Same Crosslinked Antibody Img Source: Lanier, 2005

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

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

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 K 562.

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 a. B. 3. Crosslink with Goat Anti-Mouse a. B. 4. Suspend cells with 1: 1 ratio of K 562 Cells in accordance with Expansion Protocol

Results Population results for Unloaded vs. KIRCrosslinked cell lines. KIR 2 DL 1± 19. 3% 70. 4% KIR 3 DL 1± 16. 3% 53. 5% KIR 2 DL 2/3– 56% ± 17. 5% Gfx Source: Self

Results Anti KIR Percent Reduction Margin of Error (±%) 2 DL 1 70. 4 19. 3 3 DL 1 56. 0 17. 5 2 DL 2/3 53. 5 16. 3 Gfx Source: Self

Results Flow Cytometry Data: NK Cell subpopulations Vs. Isotype control. Crosslinking with individual CD 158 e (KIR 3 DL 1) and Bulk KIR populations. Marked molding of KIR Repertoire is noted in undesired populations. Gfx Source: Self

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 Vladimir Senyukov, unpublished

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

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!

Future Work � Further Experimentation � GVHD vs GVL – A problem…

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 TCells 5. Maturation of dendritic cells for antigen presentation Img Source: Lee, 2015

Future Work � Further Experimentation � GVHD vs GVL – A problem… � Signaling Pathway – STAT 3 Confirmed! “…data show that NKG 2 D expression in NK cells is regulated at the transcriptional level by STAT 3. ” -Zhu, et. al. , 2014

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

References � � � � � Cancer Research UK. Childhood Cancers by Cancer Site: 1996 -2005. (2014, March 11). Retrieved February 15, 2015, from http: //www. cancerresearchuk. org/cancerinfo/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 NKG 2 D in natural killer cells is regulated by STAT 3 tyrosine phosphorylation. Blood, 124(3). doi: 10. 1182/blood-2013 -05499707