STRUCTURAL STUDIES OF A NEW CLASS OF IMMUNE CHECKPOINT INHIBITOR FOR CANCER IMMUNOTHERAPY By NINA HARPELL A senior thesis submitted in partial fulfillment of the requirements for the degree of BACHELOR OF SCIENCE UNIVERSITY OF CALIFORNIA SANTA CRUZ Chemistry and Biochemistry APRIL 2020 ACKNOWLEDGMENT I would like to thank my advisor, Dr. Rebecca DuBois, for providing me with the opportunity to begin this project as well as encourage my pursuit of research beyond an undergraduate degree. I would also like to thank the additional members of the Dubois Lab: Natasha George, Ana Nunez Castrejon, Lena Meyer, John Dzimianski, Jordan Ford, and Kevin Delgado-Cunningham. Additional thanks go to Nicholas Lorig-Roach, for his mentorship and support in my research and education. ii Abstract Natural Killer (NK) cells are a key component of the innate immune system that play a role in eliminating both tumors and virus-infected cells. NK cells express a deactivating receptor, KIR2DL3, which inhibits their cytotoxic function when bound by its ligand, class I MHC. Lirilumab, an Anti-KIR antibody (mAb) in clinical trials, prevents deactivation of the NK cell by binding to the KIR2DL3 receptor. However, the structural basis of lirilumabâĂŹs disruption of KIR2DL3 activity is unknown. To that end, the first aim of this project is to use X-ray crystallography to determine the structure of the lirilumab-KIR2DL3 complex to reveal how the antibody blocks MHC binding to the KIR receptor. A series of expression constructs for the KIR2DL3 receptor with different tag configurations for affinity chromatography were cloned then expressed in HEK293 and CHO cells. Biolayer interferometry assays demonstrate binding of an expressed and purified KIR2DL3 receptor with a control mAb and will also be used to determine Lirilumab’s affinity. Identification of stable expression constructs, as well as structural epitopes of the KIR-mAb construct, will implement a foundation for the study of the Lirilumab mAb as a cancer therapeutic. TABLE OF CONTENTS Page ACKNOWLEDGMENT ................................ ii ABSTRACT ....................................... iii LIST OF FIGURES ................................... vi CHAPTER 1 Introduction .................................... 1 2 Background .................................... 3 2.1 Cancer and Immunotherapy ......................... 3 2.1.1 Immunotherapy for Cancer...................... 3 2.1.2 Immune Checkpoints ......................... 4 2.1.3 Immune Checkpoint Inhibitors.................... 4 2.2 Characterizing Different Antibodies and Their Function.......... 5 2.3 Natural Killer Cells.............................. 6 2.4 NK Cells KIR2DL3 Receptor and Function................. 7 2.5 Antibodies Bound to KIR2DL3 are Immune Checkpoint Inhibitors.... 7 2.6 Prior Work................................... 9 3 Work Completed ................................. 12 3.1 Cloning the KIR2DL3 Receptor ....................... 12 3.2 Protein Expression of KIR2DL3 in HEK293 Cells ............. 14 3.3 Protein Expression of KIR2DL3 in CHO Cells ............... 18 3.4 Cloning the TRL8605 mAb.......................... 19 3.5 Protein Expression of TRL8605 in HEK293 and CHO Cells . 20 3.6 Binding studies of TRL8605 mAb and KIR2DL3 Receptor . 21 3.7 Cloning the Lirilumab mAb ......................... 25 iv 3.8 KIR2DL3 Protein Expression in E.Coli Cells................ 25 4 Future Work ................................... 28 4.1 Lirilumab Protein Expression in CHO Cells................. 28 4.2 Binding Studies of Lirilumab mAb and KIR2DL3 receptor . 29 4.3 Crystallization of anti-KIR mAb bound to KIR2DL3 Receptor . 29 5 Conclusion ..................................... 30 REFERENCES ...................................... 34 APPENDIX A Materials ...................................... 36 B Detailed Methods ................................ 39 B.1 Polymerase Chain Reaction (PCR) ..................... 39 B.2 Restriction Digestion ............................. 39 B.3 Transformation into E. coli ......................... 40 B.4 SDS-PAGE, Western Blot .......................... 40 B.5 Small-scale Protein Purification (His-tag purification via affinity chromatog- raphy) ..................................... 40 B.6 Transfection of HEK293 Cells ........................ 41 B.7 Transfection of CHO cells .......................... 42 C Sequences ..................................... 44 C.1 Protein Sequence: TRL8605 Heavy Chain Variable Region . 44 C.2 Protein Sequence: TRL8605 Light Chain Variable Region . 44 C.3 Protein Sequence: Lirilumab SCFV..................... 44 C.4 Protein Sequence: KIR2DL3 Receptor.................... 45 v LIST OF FIGURES 2.1 IgG1, 2, 3, and 4 antibodies........................... 6 2.2 Antibody Bound to Target Cell and FC Receptor of NK Cell......... 7 2.3 NK Cell KIR Deactivating Receptor and Tumor Cell HLA Antigen (MHC). 8 2.4 NK Cell Activation through Anti-KIR mAb blockade............. 8 2.5 Tumor volume as a function of mAb administered................ 10 2.6 Binding affinity of TRL mAbs for KIR2DL3 receptor on NK cells measured on ELISA...................................... 11 3.1 KIR2DL3-cmyc-6His SDS-PAGE and Western Blot. ............. 17 3.2 SDS-Page of commercial His-tag KIR2DL3 Receptor.............. 17 3.3 The 10-hisKIR2DL3 receptor protein on SDS-Page gel............. 18 3.4 SDS-Page gel of KIR2DL3 his-tagged receptor protein. ............ 19 3.5 SDS-Page gel for TRL8605 mAb produced in HEK293 and CHO cells. 21 3.6 BLI assay scheme from left to right........................ 22 3.7 BLI assay of TRL8605 mAb and KIR2DL3 receptor produced in HEK293F cells. ....................................... 23 3.8 BLI assay of the TRL8605 (CHO) mAb and KIR2DL3 receptor (CHO). 24 3.9 BLI assay of KIR2DL3 receptor and TRL8605 mAb and control mAb. 24 vi 3.10 PCR products on agarose gel of Lirilumab light chain and heavy chain variable gene fragments................................... 26 3.11 Western Blot of KIR2DL3 receptor protein pellet and lysate prior to purification. 27 vii Chapter One Introduction Behind heart disease, cancer is the second most common cause of death in the United States. Treatments, such as chemotherapy and radiation therapy have been developed to combat cancer by targeting rapidly dividing cancer cells. However, healthy cells that divide at similar rates are often damaged or killed. Because these treatments do not target specific cancer cells, damaging healthy cells poses dangerous risks for the patient to develop toxic side effects or death. Even when these traditional methods appear to work and shrink tumor volumes, relapse and regrowth of tumors after treatment ends is a daunting problem. Cancer immunotherapy is a new and promising avenue for treatment that uses a patient’s own immune system to recognize and target cancer cells. Innovative therapies such as CAR T-Cell therapy have introduced new methodologies of engineering an individual’s T-cells for targeted tumor detection and cytotoxic function [3]. Other immunotherapies are utilizing engineered mAbs to serve as checkpoint inhibitors which retain activation of T-Cells and Natural Killer Cells in the presence of tumor cells. Ipilimumab and Avelumab are both FDA approved immune checkpoint inhibitors for the treatment of skin and lung cancer [17]. Lirilumab, a monoclonal antibody (mAb) in development by Innate Pharma and Brystol- Meyers Squibb, is currently in clinical trials for a variety of cancer indications, both alone and in combination with other therapeutics [18]. Lirilumab is an immune checkpoint inhibitor that binds to the KIR2DL3 receptor on Natural Killer (NK) cells, activating them and as- 1 sisting their killing of tumor cells. While the Lirilumab mAb is known to bind KIR2DL3 and block ligand interaction, there is no structural information on the Lirilumab-KIR complex, which could reveal how this antibody is able to specifically target inhibitory KIRs without affecting the homologous activating KIRs (or some sentence like this). Another company, Trellis Biosciences, has also developed a mAb, TRL8605, similar to lirilumab with higher binding affinity for the KIR receptor [19]. The structural basis for any similarities or differences between these therapeutic mAbs is unknown.Therefore, the first goal of this project is to compare binding affinity of the TRL8605 mAb with lirilumab to verify Trellis’ claim. The second goal is to use X-Ray crystallography to determine the structures of the Anti-KIR antibodies bound to the KIR receptor. Characterizing structural components will provide better understanding of which struc- tural epitopes are active in the complex binding site. Furthermore, understanding the mecha- nism these anti-KIR mAbs use to block interaction between the NK cell and MHC of tumor cells would provide insight into developing a mAb that can effectively target cancer cells without damaging healthy cells. 2 Chapter Two Background 2.1 Cancer and Immunotherapy Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body [2]. In 2015, about 90.5 million people had cancer and about 14.1 million new cases occur a year [1]. Cancer accounts for 8.8 million (roughly 15.5%) deaths each year. The most common types of cancer such as breast cancer, lung cancer, and colon cancer which are initially treated with surgery [2]. Treatment options depend on both the cancer and the patient as well as available treatment options [2]. However, most available treatment options cannot specifically target individual cancer cells.