Functional Characterization of Potential New Agents for Cancer Immunotherapy The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:39010116 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Functional Characterization of Potential New Agents for Cancer Immunotherapy Xiaoxu Wang A Thesis Submitted to the Faculty of The Harvard Medical School in Partial Fulfillment of the Requirements for the Degree of Master of Medical Sciences in Immunology Harvard University Boston, Massachusetts. May, 2018 Thesis Advisor: Dr. Gordon Freeman Xiaoxu Wang Functional Characterization of Potential New Agents for Cancer Immunotherapy Abstract PD-1/PD-L1 checkpoint blockade has demonstrated promise in a variety of malignancies. The blockade of T cell inhibitory molecules PD-1 and PD-L1 can rejuvenate T cells and improve tumor cell killing. However, the overall response rate of PD-1/PD-L1 immunotherapy is only about 20-30%. One promising method to improve cancer immunotherapy is combinatory immunotherapies that target new receptors/ligands regulating T cell activity, so the functional characterization of these new receptors/ligands is urgent. Due to the functional importance of the B7 superfamily in T cell activation, I studied T cell regulating functions of two poorly characterized B7 superfamily members, IgC domain-truncated CD86 and butyrophilin-like 2 (BTNL2). To study their function in a natural cell-cell interaction scenario, the murine IgC domain-truncated CD86 or murine BTNL2 was cloned and transfected into Chinese Hamster Ovary Cells that already expressed MHC Class II IAd. After co-culturing the transfectants, OVA peptide 323-339 and transgenic T cells from DO11.10 mice, I tested T cell proliferation and cytokine expression. The data suggested cell-membrane bound BTNL2 will moderately stimulate T cells and increase IFN-γ secretion significantly. Similarly, stimulatory results were also demonstrated with mBTNL2 hIgG1 fusion protein and recombinant mBTNL2 protein. The stimulatory effect of BTNL2 on T cell activation suggested that BTNL2 could be targeted as a T cell activity modulator or be a potential checkpoint ligand to be lentiviral delivered to dendritic cells as a therapy. My study indicated additional B7 proteins may be another potential target for combinatory cancer immunotherapy with PD-1/PD-L1 blockade. ii Table of Contents 1. Chapter 1: Background .........................................................................................................................................1 1.1. Checkpoint Blockade.................................................................................................................................1 1.2 ICB Response and Resistance Mechanisms..............................................................................................2 1.3 New Co-stimulatory Molecules, BTNL2 and Modified CD86.................................................................6 2. Chapter 2: Materials and Methods.....................................................................................................................11 2.1. Pre-activation of T cells............................................................................................................................11 2.2. T cell Proliferation with CHO Cells.........................................................................................................12 2.3. T cell Proliferation with Plate-Bound-Antibody....................................................................................12 2.4. Cytokine Analysis......................................................................................................................................13 2.5. Truncation Design of CD86-IgV.............................................................................................................13 2.6. Primer Design, Gene In-fusion and Vector Insertion............................................................................13 2.7. Gene Cloning............................................................................................................................................14 2.8. Cell Transfection......................................................................................................................................15 2.9. Transient Transfection.............................................................................................................................15 2.10. Cell Sorting and Subcloning...................................................................................................................15 2.11. Flow Cytometry......................................................................................................................................16 3. Chapter 3: Results ..............................................................................................................................................18 3.1. mBTNL2 Expression and Subclone Selection.........................................................................................18 3.2. T cell Stimulation with CHO Cells..........................................................................................................19 3.3. T Cell Stimulation with Plate-Bound-Antibody...................................................................................24 3.4. Initial Generation of mCD86 Constructs without the IgC Domain......................................................26 3.5. Generation and Detection of Modified mCD86 without IgC..............................................................28 4. Chapter 4: Discussion........................................................................................................................................32 4.1. Limitation and Perspectives...................................................................................................................32 4.2. Perspective for Cancer Immunotherapy................................................................................................36 5. Bibliography .......................................................................................................................................................38 iii Figures Figure 1. Hypothesis for different biological activity of IgC truncated CD86: Ability to engage cell surface CD28 but not CTLA4. In cell-cell interactions, the full length CD86, which has both IgC and IgV domain, could reach both CD28 and CTLA4. However, the IgC truncated CD86 could only reach CD28 but not CTLA4 and then results in a stronger T cell stimulation. Figure 2. Cell sorting of initial transfected population and phenotyping of mBTNL2 subclones. (A) Cell sorting of CHO-IAd+mBTNL2 cells. Double staining of mouse-IAd and mBTNL2. (B) Three mBTNL2 subclones had the indicated expression. Figure 3. T cell proliferation in the presence of mBTNL2. (A) Time schedule of naïve CD4+ T cell proliferation assay (B) Naive CD4+ T cell proliferation on day 2 and (C) Day 3. Figure 4. Naive CD4+ T cell cytokine expression analysis by Luminex. Naïve CD4+ T cell cytokine expression analysis when OVA was 1µg/mL on (A) Day 2 or (B) Day 3. Figure 5. T cell proliferation in the presence of mBTNL2. (A) Time schedule of pre-activated CD4+ T cell proliferation assay (B) Naive CD4+ T cell proliferation on day 2 Figure 6. Pre-activated CD4+ T cell cytokine expression analysis on Day 2 by Luminex. Pre- activated CD4+ T cell cytokine expression analysis when OVA was 1µg/mL. Figure 7. Naïve CD4+ T cell proliferation when co-cultured with soluble mBTNL2 hIgG1 fusion protein. Naïve CD4+ T cells were co-cultured with mBTNL2-hIgG1, 1.25µg/mL anti-CD3 and 2.5 µg/mL anti-CD28 antibody. T cell proliferation was analyzed on (A) Day 2 and (B) Day 3, separately Figure 8. Naïve CD4+ T cell proliferation when co-cultured with soluble recombinant mBTNL2. Naïve CD4+ T cell were co-cultured with rmBTNL2-His, 1.25µg/mL anti-CD3 and 5 µg/mL anti- CD28 antibody. T cell proliferation was analyzed on Day 2 Figure 9. Design of “mCD86 1st ATG, IgV+Stalk+TM+Cyto” construct. The primer “mCD86 IgV reverse” was compared with “mCD86 stalk+TM+Cyto, Forward”. The highlighted sequence was the 15-bp overlap for fusion. Figure 10. Schematic diagram of generating IgC domain truncated CD86. (A) Process of truncating IgC domain. (B) Structures of natural CD86 and modified CD86 generated in this project. Figure 11. Expression of IgC truncated mCD86. (A) Double staining of mouse-IAd and IgC truncated mCD86. (B) Expression detection of IgC truncated mCD86 or full-length m CD86 with three commercial conjugated antibodies from different clones and mCD28 or mCTLA4 fusion protein iv Tables Table 1. Primers for mCD86 cloning Table 2. Constructs of IgC truncated mCD86 v Acknowledgements I would like to give my huge and sincere thanks to my thesis advisor Dr. Gordon Freeman of the Dana-Farber Cancer Institute. Dr. Freeman is so supportive that he always teaches me experimental techniques with patience by himself. He teaches me backgrounds and knowledge of each experiment. He guides me to create ideas and design experiment to approach our thoughts. He never leaves me alone when I have troubles upon my work and gave me a lot of confidence over the