Tumor Immunity and Autoimmunity Following Cancer Immunotherapy Heidi Margareta Harjunpaa MSc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 The School of Medicine, The University of Queensland QIMR Berghofer Medical Research Institute Abstract This thesis examines a number of topical studies in the field of tumor immunology focusing on two immune checkpoint receptors, CD96 and T cell immunoglobulin and ITIM domain (TIGIT). Both receptors belong to group of cell surface receptors that bind nectin and nectin-like ligands and they have recently emerged as attractive targets in cancer immunotherapy. Here, their role in anti-tumor responses and immune homeostasis are studied. Immune checkpoint blockade (ICB) targeting PD-1, PD-L1 and CTLA-4 has demonstrated unprecedented efficacy against wide variety of malignancies compared to previous cancer therapy approaches. In addition, anti-tumor efficacy was enhanced by simultaneous co-blockade of PD-1 and CTLA-4 supporting combinatorial targeting approaches in cancer patients. However, this combination therapy was also associated with an increased spectrum of treatment-related toxicities termed immune-related adverse events (irAEs). Furthermore, there is still a significant number of patients that do not respond to the currently available therapy targeting immune checkpoint receptors indicating a need to explore new therapeutic targets with greater therapeutic windows (high anti- tumor efficacy versus low level of irAEs). In Chapter 3, we utilized two mouse strains deficient for PD-1 and CD96 or TIGIT and CD96 to evaluate how targeting these receptors in combination affects immune homeostasis and thus the possible development of immune related toxicities following co- targeting of these receptors. Encouragingly, neither strain of mice displayed exacerbated changes in their immune homeostasis or enhanced immune-based disease development compared to mice deficient for PD-1, CD96 or TIGIT alone even when aged for 22 months. This suggested that co- targeting these receptors might not induce serious immune-related toxicities. In addition, in Chapter 4, we utilized the same mouse strains to evaluate how targeting CD96 in combination with PD-1 or TIGIT affects the growth of subcutaneous tumors. Interestingly, mice deficient for PD-1 and CD96 showed superior immune resistance to tumors where both CD8+ T cells and NK cells have been reported to be the main cell types controlling tumor growth. The tumor resistance of Pdcd1-/-CD96-/- mice required CD8+ T cells, NK cells and IFNγ and seemed to be due to increased CD8+ T cell to Treg or CD11b+GR-1hi myeloid cell ratios in the tumor microenvironment. These results suggested that co-targeting PD-1 and CD96 enhanced anti-tumor response over targeting PD-1 alone in certain tumors, without causing serious immune-related toxicities and thus we are encouraged to develop PD-1/CD96 co-blockade in a clinical setting. Multiple myeloma (MM) is a common haematological cancer that is caused by the proliferation of malignant plasma cells in the bone marrow (BM) which secrete circulating monoclonal antibodies (M-proteins). Despite the use of transplantation and discovery of novel MM therapies such as 1 proteasome inhibitors and immunomodulatory drugs (IMiDs), MM is still largely incurable and thus there is a vital demand for more effective treatments. Immunotherapy, including ICB, is currently being explored in the treatment of MM. However, PD-1 blockade failed to induce response as a single agent indicating that other immune checkpoint pathways might be dominating in the MM tumor microenvironment. In Chapter 5, we utilized two mouse models of MM to assess the role of TIGIT as a possible target in MM therapy. We observed that TIGIT expression on CD8+ T cells increased with MM development and correlated with tumor burden. Interestingly, deficiency or blockade of TIGIT significantly decreased tumor burden and prolonged survival of mice challenged with MM. In addition, TIGIT was also highly expressed on BM CD8+ T cells isolated from MM patients and was associated with decreased effector cell functions. Furthermore, human anti-TIGIT mAbs significantly increased cytokine production and degranulation of MM patient CD8+ T cells. These results suggested that the TIGIT pathway has a predominant inhibitory role in the MM tumor microenvironment. Importantly, our results demonstrated for the first time that TIGIT blockade enhanced anti-myeloma immune responses in mouse models of MM and improved MM patients´ CD8+ T cell effector functions, thus providing a strong rationale for developing anti-TIGIT mAbs for the treatment of human MM. Overall, the findings presented in this thesis contribute by improving our understanding of tumor immunology and the development of ICB as a novel cancer therapy. 2 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. Heidi Harjunpää The School of Medicine, The University of Queensland QIMR Berghofer Medical Research Institute 3 Publications included in this thesis 1. Heidi Harjunpää*, Stephen Blake*, Elizabeth Ahern, Stacey Allen, Jing Liu, Juming Yan, Viviana Lutzky, Kazuyoshi Takeda, Amy Roman Aguilera, Camille Guillerey, Deepak Mittal, Xian Yang Li, William C. Dougall, Mark J. Smyth and Michele W. L. Teng. Deficiency of host CD96 and PD-1 or TIGIT enhances tumor immunity without significantly compromising immune homeostasis. OncoImmunology, 2018 Mar 26;7(7):e1445949. doi: 10.1080/2162402X.2018. 1445949. *These authors contributed equally to the work. -incorporated in Chapters 3 and 4. 2. Camille Guillerey*, Heidi Harjunpää*, Nadège Carrié, Sarah Kassem, Tricia Teo, Kim Miles, Sophie Krumeich, Marianne Weulersse, Marine Cuisinier, Kimberley Stannard, Yuan Yu, Simone A. Minnie, Geoffrey R. Hill, William C. Dougall, Hervé Avet-Loiseau, Michele W. L. Teng, Kyohei Nakamura, Ludovic Martinet and Mark J. Smyth. TIGIT immune checkpoint blockade restores CD8+ T cell immunity against multiple myeloma. Blood, 2018 Jul 9. pii: blood-2018-01-825265. doi: 10.1182/blood-2018-01-825265. *These authors contributed equally to the work. -incorporated in Chapter 5. Submitted manuscripts included in this thesis No manuscripts submitted for publication. Other publications during candidature 1. Jing Liu, Stephen J. Blake, Heidi Harjunpää, Kirsten A. Fairfax, Michelle C.R. Yong, Stacey Allen, Holbrook E. Kohrt, Kazuyoshi Takeda, Mark J. Smyth and Michele W.L. Teng. Assessing immune-related adverse events of efficacious combination immunotherapies in preclinical models of cancer. Cancer Research, 2016 Sep 15;76(18):5288-301. doi: 10.1158/0008-5472.CAN-16-0194. 2. Jing Liu, Stephen J. Blake, Michelle C.R. Yong, Heidi Harjunpää, Shin Foong Ngiow, Kazuyoshi Takeda, Arabella Young, Jake S. O’Donnel, Stacey Allen, Mark J. Smyth and Michele W.L. Teng. Improved efficacy of neoadjuvant compared to adjuvant immunotherapy to eradicate metastatic disease. Cancer Discovery, 2016; 6(12), 1382-1399. 4 3. Elizabeth Ahern, Heidi Harjunpää, Deborah Barkauskas, Stacey Allen, Kazuyoshi Takeda, Hideo Yagita, David Wyld, William C. Dougall, Michele W.L. Teng and Mark J. Smyth. Co- administration of RANKL and CTLA4 antibodies enhances lymphocyte-mediated antitumor immunity in mice. Clinical Cancer Research, 2017, Oct 1;23(19):5789-5801. doi: 10.1158/1078- 0432.CCR-17-0606. 4. Elizabeth Ahern, Heidi Harjunpää, Jake S. O’Donnell, Stacey Allen, William C. Dougall, Michele W.L. Teng and Mark J. Smyth. RANKL blockade improves efficacy of PD1-PD-L1 blockade or dual PD1-PD-L1 and CTLA4 blockade in mouse models of cancer. OncoImmunology, 2018 Feb 14;7(6):e1431088. doi: 10.1080/2162402X.2018.1431088. 5. Heidi Harjunpää*, Stephen Blake*, Elizabeth Ahern, Stacey Allen, Jing Liu, Juming Yan, Viviana Lutzky, Kazuyoshi Takeda, Amy Roman Aguilera, Camille Guillerey, Deepak Mittal, Xian Yang Li, William C. Dougall, Mark J. Smyth and Michele W. L. Teng. Deficiency of host CD96 and PD-1 or TIGIT enhances tumor immunity without significantly compromising immune homeostasis. OncoImmunology, 2018 Mar 26;7(7):e1445949. doi: 10.1080/2162402X.2018. 1445949. *These authors contributed equally to the work. 6. Camille Guillerey*, Heidi Harjunpää*, Nadège Carrié, Sarah Kassem, Tricia Teo, Kim Miles, Sophie Krumeich, Marianne Weulersse, Marine Cuisinier, Kimberley Stannard, Yuan Yu, Simone A. Minnie, Geoffrey R. Hill, William C. Dougall, Hervé Avet-Loiseau, Michele W. L. Teng, Kyohei Nakamura, Ludovic Martinet and Mark J. Smyth. TIGIT immune checkpoint blockade restores CD8+ T cell immunity against multiple myeloma. Blood, 2018 Jul 9. pii: blood-2018-01-825265. doi: 10.1182/blood-2018-01-825265. *These authors contributed equally to the work.