Looking deeper into the science of Immuno-Oncology

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1 Looking deeper into the science of Immuno-Oncology Using the body s natural immune response to fight cancer Bristol-Myers Squibb At the Forefront of Immuno-Oncology

2 About These slides help explain key concepts about the rapidly eoling field of Immuno-Oncology (I-O). The information is separated into 5 topics that are color-coded for clarity. Topic 1. ESSENTIAL PRINCIPLES OF IMMUNOLOGY Topic 2. REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER Topic 3. DISCOVERING THE POSSIBILITIES OF I-O BIOMARKERS Topic 4. EVOLVING CLINICAL EXPECTATIONS IN I-O Topic 5. REALIZING THE POTENTIAL OF I-O RESEARCH

3 Topics coered Topic 1: Topic 2: Topic 3: Topic 4: Topic 5: ESSENTIAL PRINCIPLES OF IMMUNOLOGY REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER DISCOVERING THE POSSIBILITIES OF I-O BIOMARKERS EVOLVING CLINICAL EXPECTATIONS IN I-O REALIZING THE POTENTIAL OF I-O RESEARCH Differentiating self from nonself is a hallmark of the immune response Innate and adaptie immunity are complementary responses Innate immunity is rapid and antigen-independent APCs act as primary messengers between innate and adaptie immunity Adaptie immunity is durable and antigen-dependent T cells migrate throughout the body in search of antigens Select cells of the immune system Introduction to the tumor microenironment and the immune response Antitumor actiity of the innate and adaptie immune responses Key stages of the antitumor immune response Tumor cells can eade and suppress immune actiity Empowering the immune system to reestablish the antitumor response Select pathways that modulate NK-cell actiity Select pathways that modulate effector T-cell actiity Select pathways that modulate non-effector cell actiity Immune pathways combine to refine response Biomarkers in I-O research Biomarkers can help guide clinical decisions I-O biomarkers are a dynamic and dierse subset of biomarkers Multiple I-O biomarkers may be needed to proide a more precise representation of the tumor microenironment Immune responses hae the potential to deepen and sustain oer time Pseudo-progression may reflect deelopment of antitumor immunity Pseudo-progression may be considered until disease progression can be confirmed Endpoint considerations for I-O research Immune-mediated aderse reactions Depth of eidence for the immune response to cancer Broad potential of I-O research 3

4 Topic 1: ESSENTIAL PRINCIPLES OF IMMUNOLOGY The immune system identifies nonself inaders through both innate and adaptie immunity. 4

5 Differentiating self from nonself is a hallmark of the immune response The immune system is a network of tissues, cells, and signaling molecules that work to protect the body by recognizing and attacking foreign cells (nonself), while seeking to minimize the damage to healthy cells (self). 1,2 Antigens, small molecules or peptides capable of eliciting an immune response, are a key element in the process of distinguishing self from nonself. 1 APC MHC Inactie T cells search for nonself antigens by transiently binding to antigens presented by antigen-presenting cells (APCs) 3 Immune cells learn to oerlook self antigens from normal cells to preent autoimmunity 2 ESSENTIAL PRINCIPLES OF IMMUNOLOGY + Co-stimulatory signaling TCR Actiated T cell Nonself antigen should actiate T cells (initiates immune response) Although originating from normal cells, tumor antigens can be recognized as nonself and actiate cytotoxic T cells 1,4,5 Neoantigens are a type of tumor antigen that arise from self proteins that hae been mutated or modified, making them unique to the tumor 4,5 5

6 Innate and adaptie immunity are complementary responses The immune system identifies nonself inaders through both innate and adaptie immunity. Actiated through distinct and often complementary mechanisms, innate and adaptie immunity deploy different effector cells to attack and destroy abnormal / foreign cells such as cancer. 1 Number of immune cells Primary immune response dynamics The innate immune response is rapid, while the adaptie immune response is not as immediate but can produce a durable response through the deelopment of memory cells, including memory T cells 1,6 As the immune response continues to expand, some cytotoxic T cells mature into memory T cells that may proide long-term immune protection, een if the original stimulus is no longer present 7,8 ESSENTIAL PRINCIPLES OF IMMUNOLOGY

7 Innate immunity is rapid and antigen-independent Innate immunity, the body s first line of defense, is non-specific and independent of antigens, allowing for the rapid identification and elimination of foreign threats. 1 The primary effector cells of the innate immune response, natural killer (NK) cells, continually scan the body for abnormal cells to attack. 1,9,10 * NK cell Actiating receptor Tumor cell Dying tumor cells ESSENTIAL PRINCIPLES OF IMMUNOLOGY NK-cell recognition NK-cell actiation Direct tumor cell death NK cells express receptors that interact with actiating and inhibitory signals from normal and abnormal cells. The balance of these signals determines NK cell behaior. 11 *Numerous cell types are inoled with the innate immune response, including macrophages, neutrophils, dendritic cells, mast cells, basophils, eosinophils, natural killer (NK) cells, and lymphocytes (T cells). 1

8 APCs act as central messengers between innate and adaptie immunity Antigen-presenting cells (APCs) are innate immune cells that can act as central messengers between the innate and adaptie immune responses. 1 Tumor cell death, which can be initiated by the innate immune system, can release signaling molecules, such as DNA, adenosine triphosphate (ATP), and proteins These factors may cause APCs to initiate an adaptie immune response. 12,15 ESSENTIAL PRINCIPLES OF IMMUNOLOGY DNA or ATP released by dying tumor cells stimulates APCs to produce proinflammatory cytokines, through the inflammasome, which can support antitumor function and surial in actiated T cells inoled in the adaptie immune response Proteins released by dying tumor cells can be processed by APCs into tumor antigens. 18,19 APCs present these antigens to T cells, priming them to recognize tumor cells. 1,19

9 Adaptie immunity is durable and antigen-dependent Adaptie immunity is antigen-dependent and able to produce a durable response. 1 Cytotoxic T cells, the primary effector cells of the adaptie immune response, can be actiated by the detection of tumor antigens. 1,20 Once actiated, cytotoxic T cells proliferate, migrate to the location of the antigen, infiltrate it, and directly initiate cell death. 21 Tumor cell APC Tumor antigens Antigen MHC TCR Inactie T cell T cell Dying tumor cells Memory T cell ESSENTIAL PRINCIPLES OF IMMUNOLOGY Tumor antigen release Antigen presentation T-cell actiation Tumor cell death T-cell memory Unlike the innate immune response, adaptie immunity is not immediate, but can be sustained through a memory cell response, which includes memory T cells. 1,8

10 T cells migrate throughout the body in search of antigens To identify and eliminate tumor cells, cytotoxic and memory T cells must be able to scan peripheral tissues in search of an unique actiating antigen. 21,22 To make this possible, actiated T cells upregulate factors that enable them to recognize threats and migrate through blood essel walls, into affected tissues 23,24 T-cell migration occurs across nonlymphoid tissues, with documented trafficking to een particularly selectie tissues such as the eye and brain ESSENTIAL PRINCIPLES OF IMMUNOLOGY After the actiated cytotoxic T cell population diminishes, memory T cells remain capable of trafficking to surrounding tissues in the eent of antigen reoccurence 26

11 Select cells of the immune system Effector cells Effector cells such as cytotoxic T cells and NK cells are actiely inoled in the destruction of foreign pathogens and cancer. Natural killer (NK) cells are the primary effector cells of the innate immune response. NK cells express actiating and inhibitory receptors that interact directly with signals from other cells. NK cells do not require antigenbound MHC to identify and attack abnormal cells. 1,22 Cytotoxic T cells are the primary effector cells of the adaptie immune response. Following actiation by recognition of antigens presented by MHC class I molecules, T cells directly kill pathogens and abnormal cells that express the respectie antigen. 22,32 Non-effector cells Non-effector cells such as APCs, Tregs, TAMs, and MDSCs directly or indirectly modulate the cytotoxic effector T-cell response. These cells cannot induce tumor cell death on their own. Antigen-presenting cells (APCs) (such as dendritic cells) recognize, process, and present antigens to T cells through MHC molecules. 22,34 Regulatory T cells (Tregs) are a unique subset of T cells that modulate the actiation of other effector T cells to inhibit the immune response. 22,34 Tumor-associated macrophages (TAMs) are cells deried from the macrophage lineage that are recruited to the tumor microenironment to promote tumor cell surial by driing immunosuppression. 35,36 ESSENTIAL PRINCIPLES OF IMMUNOLOGY Memory T cells are deried from actiated cytotoxic T cells and represent a long-lied population of antigenexperienced cells that can rapidly respond upon antigen reocurrence. 1,33 Myeloid-deried suppressor cells (MDSCs) are cells deried from the myeloid lineage that function to suppress T-cell responses. 35

12 Topic 2: REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER The ability of the immune system to detect and destroy cancer is the foundation of Immuno-Oncology research. 12

13 Introduction to the tumor microenironment and the immune response The immune system is capable of recognizing and eliminating tumor cells in the tumor microenironment. Innate and adaptie immunity act as a complementary network of selfdefense against foreign threats. 1 This ability to recognize foreign threats (nonself) as distinct from normal cells (self), is an essential feature of the immune system. 2,4,37 Despite originating from normal cells, tumor cells can be recognized as nonself through production of tumor antigens. 5 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

14 Antitumor actiity of the innate and adaptie immune responses Innate immune response The first line of defense, it rapidly identifies and attacks tumor cells without antigen specificity 1,32,38,39 It recognizes actiating and inhibitory signals from target cells to distinguish self from nonself 11,40,41 Natural killer (NK) cells are the main effector cells of innate immunity 1 Adaptie immune response An antigen-specific response that can be actiated by recognition of tumor antigens (nonself) 1,32 Once actiated, it can be sustained through a durable memory response 26 Cytotoxic T cells are the main effector cells of adaptie immunity 1 The antitumor actiity of NK cells and cytotoxic T cells is regulated through a network of actiating and inhibitory signaling pathways 22,37,42 : ACTIVATING Stimulating pathways trigger immune responses INHIBITORY Pathways that counterbalance immune actiation The balance between actiating and inhibitory pathways normally enables the immune system to attack tumor cells, while sparing healthy cells. 22 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

15 Key stages of the antitumor immune response In both the innate and adaptie immune responses, immune cells hae the potential to recognize and eliminate tumor cells. There are 3 principal stages in this process: Presentation The innate immune system rapidly identifies and attacks tumor cells Tumor cell death releases tumor antigens, which can actiate the cytotoxic T cells of the adaptie immune system 38,43 Infiltration Tumor antigens and other factors attract immune cells to the tumor site, where they inade and attack 43 Elimination Actiated cytotoxic T cells recognize tumor cells as the source of the antigen and target them for elimination 43 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

16 Tumor cells can eade and suppress immune actiity The complex network of actiating and inhibitory pathways enables the antitumor immune response to detect and eliminate tumor cells at any point in tumor deelopment. 14,44 The success of these strategies determines the ability of immune cells to react to the tumor. 45 The tumor microenironment consists of different cell types that can help tumor cells eade antitumor immune actiity As tumors eole, they can influence the actiation and composition of cells within the tumor microenironment. 44,49 Depending upon their degree of immune cell infiltration, tumors are defined on a range from noninflamed to inflamed. 45 Noninflamed Tumors Impaired Impaired Impaired Stages of the antitumor response Characterized by poor presence of immune cells 45,50 Impaired ability to present tumor antigens to T cells or secrete key factors (chemokines) 50,51 Less able to direct tumor-specific T cells to the tumor and promote T cell infiltration, ultimately preenting tumor cell elimination 50,51 Presentation Infiltration Elimination Actie Actie Impaired Inflamed Tumors Characterized by presence of immune cells and can be an indicator of a pre-existing immune response 45,50 Antigen presentation and expression of chemokines allow for infiltration of actiated cytotoxic T cells 50,52-54 Howeer, tumor cells may increase their expression of inhibitory proteins to preent elimination by cytotoxic T cells 53,55,56 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

17 Empowering the immune system to reestablish the antitumor response The immune system uses a network of signaling pathways to detect and eliminate tumor cells. 37,42 Ongoing Immuno-Oncology research focuses on the following select pathways, either alone or in combination, to understand how they can be modulated to restore the body s natural ability to fight cancer. Reestablishing the fundamental stages that are impaired within tumors presentation, infiltration, and elimination is a key strategy in improing the broad potential of Immuno-Oncology. REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

18 Select pathways that modulate NK-cell actiity Current research is inestigating the following NK-cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: NK cell SLAMF7 SLAMF7 actiating NK cell KIR MHC Tumor cell NK cell SLAMF7 is an actiating receptor on the surface of NK cells and other immune cells. 56 When engaged, SLAMF7 actiates NK cells, the rapid responders of the immune system and the body s first line of defense against cancer. 39,58 Continuous actiation of NK cells through pathways like SLAMF7 may initiate the deelopment of long-term immunity. 38,59,60 KIR is an immune checkpoint receptor on the surface of NK cells that acts to stop NK cells from killing normal cells. 40 Tumor cells can use the KIR pathway to disguise themseles as normal cells and escape detection by NK cells. 61 Preclinical data suggest that blockade of inhibitory KIRs can help restore NK cell-mediated immune actiity. 62,63 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER inhibitory

19 Current research is inestigating the following actiating effector T-cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: CD137 GITR T cell Select pathways that modulate effector T-cell actiity (1/6) CD137L T cell GITRL TCR actiating MHC actiating Antigen MHC APC TCR Antigen APC CD137 is an actiating receptor on the surface of NK cells and T cells that can stimulate them to reproduce and generate antitumor actiity. 64,65 CD137 also plays a critical role on T cells in the deelopment of immune memory and the creation of a durable immune response, in animal models. 66 Preclinical data suggest that actiation of CD137 can stimulate both NK-cell and cytotoxic T-cell actiity and generate a lasting memory response. 67,68 GITR is an actiating receptor on the surface of T cells and other immune cells that helps to enhance cell reproduction and generate antitumor actiity GITR signaling can also block the suppressie abilities of regulatory T cells (Tregs), further enhancing cytotoxic T-cell function. 72 Preclinical data suggest that actiation of GITR signaling can help enhance immunity through the actiation of cytotoxic T cells and inhibition of Treg actiity. 73 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

20 Select pathways that modulate effector T-cell actiity (2/6) Current research is inestigating the following actiating effector T-cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: TCR T cell Antigen MHC T cell Antigen TCR MHC OX40 OX40L APC actiating ICOS ICOSL APC actiating ICOS is an actiating receptor expressed on the surface of actiated cytotoxic T cells, regulatory T cells (Tregs), NK cells, and other types of T cells that can lead to the actiation, proliferation, and surial of cytotoxic T cells, as well as the surial of memory T cells. Following T-cell actiation, upregulation of ICOS perpetuates T-cell proliferation and function Preclinical data suggest that ICOS signaling is necessary for the actiation, proliferation, and function of cytotoxic T cells under natie conditions as well as during the blockade of CTLA-4. 83,84 OX40 is an actiating receptor on the surface of actiated cytotoxic T cells and Tregs OX40 plays a dual role in the immune response, both actiating and amplifying T-cell responses. This dual effect helps create a tumor microenironment that is more faorable to antitumor response Preclinical data suggest that OX40 increases the number and actiity of cytotoxic T cells and curtails the immunosuppressie impact of Tregs REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

21 Tumor cell Select pathways that modulate effector T-cell actiity (3/6) Current research is inestigating the following actiating effector T-cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: Virus Target receptor Surface expression of immunomodulatory proteins stimulates immune response Immunomodulatory proteins Viral DNA actiating Stimulatory receptors Dying tumor cell T cell Cell death and subsequent release of tumor antigens and irus stimulate immune response Oncolytic iruses are naturally occurring or genetically engineered iruses that preferentially target and replicate within tumor cells, leading to tumor cell destruction and actiation of the antitumor immune response Tumor cells often hae altered molecular mechanisms faoring iral replication, which may make them uniquely susceptible to iral infection and propagation. 97 Oncolytic iruses can be either unarmed or armed. Both types of irus can actiate an antitumor immune response, in part by promoting tumor inflammation. 96,98 Preclinical data suggest that unarmed oncolytic iruses can cause the release of tumor antigens, promoting the actiation of cytotoxic T cells. 99 In addition, immunomodulatory proteins expressed by armed iruses may stimulate tumor inflammation and further enhance cytotoxic T-cell actiation REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

22 Select pathways that modulate effector T-cell actiity (4/6) Current research is inestigating the following inhibitory effector T-cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: CTLA-4 T cell CD28 CD80 CD86 inhibitory APC TCR Antigen MHC CTLA-4 is an immune checkpoint receptor on actiated T cells that plays a key role in preenting T-cell oeractiation Tumor cells use the CTLA-4 pathway to suppress initiation of an immune response, resulting in decreased T-cell actiation and ability to proliferate into memory T cells. 107,108 CTLA-4 signaling diminishes the ability of memory T cells to sustain a response, damaging a key element of durable immunity. 107 Preclinical data suggest that treatment with antibodies specific for CTLA-4 can restore an immune response through increased accumulation, function, and surial of T cells and memory T cells and depletion of regulatory T cells One recent approach aims to improe the specificity of CTLA-4 blockade by using pro-antibodies, antibodies masked with a protein that can be remoed by enzymes that are actie primarily at the tumor site REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

23 TCR Select pathways that modulate effector T-cell actiity (5/6) Current research is inestigating the following inhibitory effector T-cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: T cell PD-L1 Inactie T cell Antigen PD-1 MHC APC Tumor cell inhibitory LAG-3 inhibitory PD-L2 PD-1 is an immune checkpoint receptor on cytotoxic T cells that plays a key role in T-cell exhaustion and preention of autoimmunity Tumor-infiltrating T cells across solid tumors and hematologic malignancies display eidence of exhaustion, including upregulation of PD Preclinical data suggest that PD-1 blockade reinigorates exhausted T cells and restores their cytotoxic immune function. 119 Inhibiting both PD-1 ligands (PD-L1 and PD-L2) may be more effectie at reersing T-cell exhaustion than inhibiting PD-L1 alone. 120 LAG-3 is an immune checkpoint receptor on the surface of both actiated cytotoxic and regulatory T cells (Tregs). 121,122 When bound to the antigen-mhc complex, LAG-3 can negatiely regulate T-cell proliferation and the deelopment of lasting memory T cells. 123 Repeated exposure to tumor antigen causes an increase in the presence and actiity of LAG-3, leading to T-cell exhaustion. 124,125 Preclinical data suggest that when the PD-1 pathway is blocked, LAG-3 may be upregulated to maintain tumor growth. 126 Research is ongoing to understand how dual inhibition of LAG-3 and other checkpoint pathways may synergistically increase T-cell antitumor actiity compared with inhibition of either pathway alone. REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

24 Select pathways that modulate effector T-cell actiity (6/6) Current research is inestigating the following inhibitory effector T-cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: T cell CD155 CD226 TIGIT Tumor cell inhibitory Myeloid-deried suppressor cell Galectin-9 inhibitory CD112 T-cell exhaustion Inactie T cell TIM-3 TIGIT is an immune checkpoint receptor expressed on the surface of cytotoxic, memory, and regulatory T cells (Tregs), as well as natural killer (NK) cells. 127,128 On cytotoxic T cells and NK cells, interaction of TIGIT with either of its ligands suppresses immune actiation. 92,93 When TIGIT is expressed on Tregs, howeer, this interaction enhances their ability to suppress the immune response. 129 Preclinical data suggest that inhibition of TIGIT signaling increases the proliferation and function of cytotoxic T cells. 130,131 TIM-3 is an immune checkpoint receptor inoled in the suppression of both innate and adaptie immune cells. 132 It is expressed on the surface of a wide ariety of immune cells, including cytotoxic T cells, Tregs, NK cells, and some APCs such as DCs. 132,133 Binding of TIM-3 on cytotoxic T cells to its ligand, galectin-9, on immunosuppressie MDSCs can enhance MDSC expansion and suppressor actiity. 134 Preclinical data suggest that the blockade of TIM-3 can rescue NK-cell actiity, promote tumor antigen processing, and reinigorate exhausted T cells, restoring their proliferation and function REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

25 Dying tumor cell Dying tumor cell Select pathways that modulate non-effector cell actiity (1/5) Current research is inestigating the following actiating non-effector cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: ATP Tumor DNA APC Inactie IL-18 and NLRP3 IL-1β inflammasome NLRP3 and inflammasome components actiating APC STING Production of IFN and other cytokines actiating Actie IL-1β Antigen MHC MHC Antigen NK cell Actie IL-18 TCR TCR T cell NK cell T cell NLRP3 is a protein expressed in APCs such as DCs, monocytes, and macrophages. 140 NLRP3 is inoled in the assembly of the NLRP3 inflammasome, a protein complex that is a key mediator of innate immunity and the priming of T cells. 141,142 Preclinical data suggest that the NLRP3 inflammasome can actiate NK cells and initiate the priming of T cells, which promotes tumor inflammation and enhances antitumor function STING is an intracellular protein expressed in APCs, such as DCs, which seres as an innate immune actiator that stimulates APCs to drie cytotoxic T-cell actiity. 144,145 STING is triggered when an intracellular-sensing protein detects DNA from pathogens or dying tumor cells. 146,147 Preclinical data suggest that actiation of STING can increase priming of T cells, leading to increased T-cell actiation and an inflamed tumor microenironment REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

26 Select pathways that modulate non-effector cell actiity (2/5) Current research is inestigating the following inhibitory non-effector cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: Tumor cell Regulatory T cell CD39 Inactie T cell CD73 CCR2 CCR5 Stroma cell CCL5 CCL2 CCR2 CCR5 CCR5 inhibitory A2aR Myeloidderied suppressor cell Tumorassociated macrophage CCR2 Regulatory T cell Adenosine CCR2 and CCR5 regulate the recruitment of immunosuppressie cells through the stroma. CCR2 and CCR5 are both expressed on the surface of T cells, Tregs, monocytes, MDSCs, and TAMs Preclinical data suggest that depletion or blockade of CCR2 and CCR5, indiidually or in combination, has been shown to decrease the infiltration of MDSCs, TAMs, and Tregs to the tumor microenironment CD73 is a cell-surface enzyme on Tregs. CD73 is a critical checkpoint in the production of adenosine, which has been demonstrated to be a powerfully immunosuppressie molecule in cellular studies. 164 Tumor cells exploit this pathway by expressing CD73 and releasing adenosine into the tumor microenironment Preclinical data suggest that inhibition of CD73 actiity can stimulate T-cell actiity. 168 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER inhibitory

27 Select pathways that modulate non-effector cell actiity (3/5) Current research is inestigating the following inhibitory non-effector cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: Inactie T cell Immunosuppressie factors Tumor-associated macrophage inhibitory Regulatory T cell CTLA-4 CD80 CD86 CSF1 inhibitory CSF1R TCR Antigen MHC CD28 + MHC APC CSF1R is a receptor on the surface of macrophages and other cells of the myeloid lineage. 169 In the tumor microenironment, some macrophages eole from antitumor to protumor in their actiity. 36 Protumor, or tumor-associated macrophages (TAMs) can drie immunosuppression and support tumor growth. 36 Mouse models hae shown that tumor cells use CSF1 to target CSF1R on macrophages, stimulating the deelopment and surial of TAMs. 170 Preclinical data suggest that blockade of CSF1R can result in depletion of TAMs and improed T-cell responses. 171,172 CTLA-4 is an immune checkpoint inhibitor that, in addition to being expressed on actiated T cells, is also found on Tregs, where it is a key drier of their ability to suppress T-cell actiity and counterbalance excessie immune actiation. 22,34,173 Continuous expression of CTLA-4 on Tregs is critical for their suppressie actiity. 173,174 Preclinical data suggest that increased depletion of Tregs can improe cytotoxic T-cell actiation and antitumor actiity. One recent approach to regulate the degree of immune actiity and increase the depletion of Tregs uses a specific type of CTLA-4 antibody with a modified Fc region known as a fucosylated antibody. This fucosylated antibody can bind to Tregs, identifying them for elimination by other immune cells. 110,112,175 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

28 Select pathways that modulate non-effector cell actiity (4/5) Current research is inestigating the following inhibitory non-effector cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: Inactie T cell Tryptophan IDO1 Tumor cell Stromal cell Kynurenine inhibitory IL-8 CXCR1 Myeloid-deried suppressor cell APC CXCR2 IDO1 is an enzyme expressed in APCs. It metabolizes tryptophan, an amino acid that is essential for T-cell surial, into immunosuppressie kynurenine, which normally acts as a counterbalance to suppress T-cell function and preent oeractiation of the immune response Tumors can hijack this immunosuppressie process, and eole to increase IDO1 expression in both tumor cells and APCs. 176,177,179,180 Preclinical data suggest that inhibition of both IDO1 and an immune checkpoint pathway may synergistically improe T-cell proliferation and reduce Treg accumulation. 181 IL-8 is a chemokine produced by macrophages, monocytes, and stromal cells that promotes the recruitment of immunosuppressie MDSCs and also actiates the angiogenic response to generate new blood essels during the normal healing process Preclinical data suggest that blockade of IL-8 signaling reduces angiogenesis and the recruitment of CXCR1- and CXCR2-expressing MDSCs to the stromal barrier and tumor microenironment. 183,186,187 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER inhibitory

29 Select pathways that modulate non-effector cell actiity (5/5) Current research is inestigating the following inhibitory non-effector cell mechanisms to understand how they can be modulated to restore the body s natural ability to fight cancer: Regulatory T cell TGFR2 TGFR1 TGFβ Tumor cell inhibitory TGFR is a cytokine receptor expressed on the surface of tumor cells as well as immune cells, such as Tregs. TGFR initiates signaling of multiple processes, including suppression of immune actiity and cellular migration Preclinical data suggest that inhibiting TGFR actiity can reduce the number of immunosuppressie Tregs infiltrating the tumor, increasing the actiity of cytotoxic T cells and NK cells. 189 REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

30 Immune pathways combine to refine response Actiating and inhibitory signaling pathways combine to maintain immune balance by regulating the 3 key stages of the immune response: presentation, infiltration, and elimination. 37,42 Once an immune response is initiated, each stage can potentiate or limit the actiity of subsequent stages. 193 Modulating immune pathways in combination may enhance the immune response, as suggested by preclinical data REVEALING THE POTENTIAL OF THE IMMUNE SYSTEM IN CANCER

31 Topic 3: DISCOVERING THE POSSIBILITIES OF I-O BIOMARKERS Research in the field of Immuno-Oncology (I-O) biomarkers seeks to characterize the relationship between the immune system, the tumor and its microenironment, and the host. 31

32 Biomarkers in I-O research For each patient, the interaction of the immune system, cancer, and therapy is complex and unique. 198 Biomarkers are biologic molecules, cells, or processes found in tissues or body fluids (such as blood) that are a sign of a normal or abnormal process or disease. 199,200 A goal of I-O biomarker testing is to help enable a more personalized approach to treatment by identifying patients who are likely to respond to specific immunotherapies. 198,201,202 EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS

33 Biomarkers can help guide clinical decisions I-O biomarkers are a class of biomarker that can help ealuate an actie antitumor immune response within the body. 203 They can be prognostic, predictie, or pharmacodynamic : PROGNOSTIC BIOMARKERS Prognostic biomarkers may identify the likelihood of a clinical eent, such as disease progression, disease recurrence, or death, independent of the therapy receied. 204,205 PREDICTIVE BIOMARKERS Predictie biomarkers may identify whether indiiduals are more likely to experience a faorable or unfaorable response to treatment (eg, a mutation in the EGFR, BRAF, or KRAS genes). 204,205, PHARMACODYNAMIC BIOMARKERS Predictie biomarkers may identify whether indiiduals are more likely to experience a faorable or unfaorable response to treatment. 204,205 EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS

34 I-O biomarkers are a dynamic and dierse subset of biomarkers I-O biomarker research aims to further characterize the unique interplay between the immune system and tumor cells, in the following categories: TUMOR ANTIGENS 5,202,210,211 Tumor antigens are recognized as nonself or foreign by the host immune system and can initiate the adaptie immune response Neoantigens MSI-H/dMMR Tumor mutational burden IMMUNE SUPPRESSION 201,210,211 Cells and proteins within the tumor and its microenironment are associated with inhibition of the antitumor immune response LAG-3 MDSCs Tregs IDO1 INFLAMED TUMORS Inflamed tumors show eidence of immune-cell infiltration and actiation in the tumor microenironment PD-L1 PD-L2 TILs Inflammation gene signatures HOST ENVIRONMENT 49,213 Many factors within the host enironment, such as the microbiome, may play a role in modulating an immune response EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS *Effector T cell or NK cell. 34

35 Inestigational I-O biomarker: tumor antigens Proteins released by dying tumor cells can be processed by APCs into tumor antigens. 19,214 APCs present these antigens to T cells, priming them to recognize tumor cells. 1,19 Neoantigens: Newly formed antigens not preiously recognized by the immune system 5,215 Tumor mutational burden (TMB): The collectie number of somatic (acquired) mutations in the tumor genome 216,217 Microsatellite instability high/deficient mismatch repair (MSI-H/dMMR): Indicators of genomic stability 218,219 Seeral I-O biomarkers related to tumor antigens are currently under inestigation. EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS

36 Inestigational I-O biomarker: neoantigens Neoantigens are a class of tumor antigen that are deried from the unique mutations in tumor DNA that differentiate tumors from normal tissue. 215 Neoantigens are thus unique to the tumor and recognizable as nonself by the immune system. 5,215 They can initiate the adaptie immune response, a process known as immunologic priming. 5,211 Tumors with a high burden of neoantigens are more sensitie to immunotherapy, indicating that neoantigens may be a potential I-O biomarker. 220 As immunogenic neoantigens can be challenging to identify directly, TMB may potentially be used as a surrogate to indirectly assess neoantigen load. 201,221 EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS

37 Inestigational I-O biomarker: tumor mutational burden Tumor mutational burden (TMB) is defined as the number of somatic (acquired) mutations in the tumor genome. 216,217 The number of mutations can ary across different tumor types. 222,223 High TMB has been shown to be associated with infiltration of cytotoxic T cells into the tumor microenironment, supporting its use as a neoantigen surrogate. 221,224,225 TMB is assessed using next-generation sequencing (NGS), a method in which tumor DNA can be read and analyzed for mutations against a reference genome. 226,227 TMB is an emerging biomarker that may predict the likelihood of an immune response against cancer cells, which could help inform indiidualized treatment across tumor types. 201,228 EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS

38 Inestigational I-O biomarker: inflamed tumors Inflamed tumors show eidence of immune-cell infiltration and actiation in the tumor microenironment. 212 Programmed death ligand 1/programmed death ligand 2 (PD-L1/PD-L2): Ligands for the immune checkpoint receptor PD-1 expressed on the surface of immune cells, including cytotoxic T cells 229 Tumor-infiltrating lymphocytes (TILs): Immune cells that enter the tumor and its microenironment to mediate an antitumor immune response 230 Inflammation gene signature: Specific type of gene expression profile proiding a holistic iew of cellular function 231,232 Seeral I-O biomarkers related to inflamed tumors are currently under inestigation. EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS

39 Inestigational I-O biomarker: inflammation gene signatures Inflammation gene signatures are a specific type of gene expression profile. Gene expression profiling measures the expression of mrna across thousands of genes. 231 This can create a distinct molecular profile (or gene signature), proiding a holistic iew of cellular function. 231,232 Inflammation gene signatures ary across tumor types and may be a powerful diagnostic tool. 233,234 EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS Inflammation gene signatures are being inestigated as a potential I-O biomarker.

40 Inestigational I-O biomarker: immune suppression markers Cells and proteins within the tumor and its microenironment can suppress T-cell actiation, promote T-cell exhaustion, or actiate regulatory T cells (Tregs). 235,236 Lymphocyte-actiation gene 3 (LAG-3): Immune checkpoint receptor expressed on actiated cytotoxic T cells and Tregs 121,122 Regulatory T cells (Tregs): Cells that suppress the immune response by modulating the actiation of effector T cells 22,34 Myeloid-deried suppressor cells (MDSCs): Cells recruited to the tumor microenironment to suppress effector cell responses 35 Indoleamine 2,3-dioxygenase 1 (IDO1): Enzyme that initiates the breakdown of tryptophan into immunosuppressie kynurenine in the tumor microenironment Seeral I-O biomarkers related to immune suppression markers are currently under inestigation. EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS

41 Inestigational I-O biomarker: host enironment Many factors within the host enironment may play a role in modulating an immune response. Outside of the tumor microenironment, factors such as smoking, diet, UV exposure, infectious agents, and the gut microbiome can faorably or unfaorably affect the antitumor immune response. 49,198,222 Gut microbiome: A collection of bacteria and other organisms that actiely colonize the intestine 213 Factors within the host enironment, including the gut microbiome, are currently being inestigated as I-O biomarkers. EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS

42 Multiple I-O biomarkers may be needed to proide a more precise representation of the tumor microenironment As I-O biomarkers are dynamic and complex, the presence or absence of any single I-O biomarker may not proide a complete understanding of the dierse interactions occurring within the tumor microenironment. 198,237,238 Ealuating multiple I-O biomarkers in combination may proide a more accurate and comprehensie assessment of immune status. 198 Tumor antigens Inflamed tumors Immune suppression Host enironment Therefore, the goal of I-O biomarker deelopment is to enable a more personalized approach to treatment by identifying patients who are likely to respond to specific immunotherapies. 198,201,202 EXPLORING PREDICTORS OF RESPONSE: IMMUNE BIOMARKERS

43 Topic 4: EVOLVING CLINICAL EXPECTATIONS IN I-O Immuno-Oncology (I-O) is a fundamentally different approach to cancer treatment. With this new approach comes unique considerations and distinctie characteristics that continue to be researched. 43

44 Immune responses hae the potential to deepen and sustain oer time The immune response eoles and expands oer time by constantly recognizing and remembering tumor antigens. This ability to propagate and perpetuate suggests the intelligent nature of the immune response. 43 Immune responses are dynamic and hae the potential to improe and deepen oer time. 239 EVOLVING CLINICAL EXPECTATIONS IN I-O As the immune response continues to expand, some cytotoxic T cells mature into memory T cells that may proide long-term immune protection, een if the original stimulus is no longer present. 7,8,240

45 Pseudo-progression may reflect deelopment of antitumor immunity The nature of the antitumor immune response can create the appearance of disease progression, either as tumor growth or appearance of new lesions. 241 This is known as pseudo-progression. Pseudo-progression does not reflect tumor cell growth, but may be misclassified as disease progression. 241,242 Tumors may appear to grow or new lesions may appear when immune cells infiltrate the tumor site. 241 Due to the time required to mount an adaptie immune response, pseudo-progression may also reflect continued tumor growth until a sufficient response deelops. 241,243 Disease progression Baseline assessment First assessment Later assessment EVOLVING CLINICAL EXPECTATIONS IN I-O Pseudoprogression NK cell T cell

46 Pseudo-progression may be considered until disease progression can be confirmed While uncommon, pseudo-progression is an important consideration when ealuating response to Immuno-Oncology therapies. 243 Histologic confirmation is not always possible, but close monitoring of the following factors may help identify pseudo-progression 241,244 : Disease progression Pseudo-progression Performance status Deterioration of performance Remains stable or improes Systemic symptoms Worsen May or may not improe Symptoms of tumor enlargement Tumor burden: Present May or may not be present EVOLVING CLINICAL EXPECTATIONS IN I-O o Baseline Increase Initial increase followed by response o New lesions Appear and increase in size Appear then remain stable and/or subsequently respond Biopsy may reeal Eidence of tumor growth Eidence of immune-cell infiltration

47 Endpoint considerations for I-O research (1/2) The criteria currently used to assess potential benefit of cancer therapies are based on surgery, radiation therapy, and chemotherapy. 245 Howeer, for Immuno-Oncology, a different way to fight cancer, a more comprehensie approach to endpoint assessment may be needed to recognize potential benefit Magnitude and duration are both key measures of response Response can be assessed by both magnitude (size) and duration (time). 251 Oerall response rate (ORR) is the proportion of patients with a predefined decrease in tumor burden. 251 ORR reflects solely the magnitude of response, and is generally defined as a sum of partial and complete responses. 251 Duration of response (DOR) measures the time from initial tumor response to disease progression. 251 As our understanding of research continues to eole, the DOR may proe een more releant to potential benefit than the magnitude of tumor reduction. 252 EVOLVING CLINICAL EXPECTATIONS IN I-O Because responses range in both size and duration, these measures should be ealuated together to more accurately assess adances in Immuno-Oncology research. 251

48 Endpoint considerations for I-O research (2/2) Oerall surial (OS), progression-free surial (PFS), and response rate are among endpoints used to measure outcomes in oncology research. 251,252 OS is the gold standard to assess therapeutic benefit when possible. 252 Applying multiple measures can illustrate the full scope of clinical benefit. EVOLVING CLINICAL EXPECTATIONS IN I-O Assessment of these measures in combination can proide a broad and comprehensie picture of the difference between the inestigational arm and the control arm with respect to PFS and OS ,253,254

49 Immune-mediated aderse reactions I-O therapies that modulate immune pathways may enable the immune system to attack healthy cells along with tumor cells. The effects are known as immune-mediated aderse reactions. 255 When managing complications of immune-mediated aderse reactions, please consider: Patients, caregiers, and physicians should be educated to remain igilant throughout and after I-O treatment to minimize complications, some of which may be life-threatening 255 In addition, treatment algorithms are aailable for use by healthcare proiders to assist them in managing immune-mediated aderse reactions Recent guidelines hae been published that proide consensus recommendations for the management of immune-mediated aderse reactions. 257,258 Specific guidance for managing immune-mediated aderse reactions for an indiidual product can be found in the accompanying FDA-approed prescribing information EVOLVING CLINICAL EXPECTATIONS IN I-O As research in immune system actiation adances and more data are made aailable, understanding and appropriate management of immune-mediated aderse reactions will eole. 259

50 Topic 5: REALIZING THE POTENTIAL OF I-O RESEARCH Eidence for tumor immunogenicity across a wide range of solid tumors and hematologic malignancies proides the rationale for the breadth of Immuno-Oncology (I-O) research across tumor types. 50

51 Depth of eidence for the immune response to cancer Both solid tumors and hematologic malignancies are able to induce an immune response that can regulate their growth. This ability is known as tumor immunogenicity. 260,261 The body is able to recognize and attack cancer through the following mechanisms: PRESENTATION: Traditionally, immunogenic tumors are defined by a high rate of mutations. 262 These mutations create neoantigens that can be recognized by the immune system, actiating an antitumor immune response. 263 INFILTRATION: Tumor-infiltrating immune cells are present in the tumor microenironment Their presence demonstrates their capacity to identify and migrate to tumor cells. 277 REALIZING THE POTENTIAL OF I-O RESEARCH ELIMINATION: Early in their deelopment, some tumors display eidence of spontaneous regression. 278 This suggests that the immune system is able to recognize and eliminate some tumor cells, and supports the concept that the body s own immune system has the ability to induce an antitumor response against cancer. 278,279

52 Broad potential of I-O research There is eidence of immunogenicity across a wide range of malignancies 280 : Tumor type* Eidence for tumor immunogenicity PRESENTATION INFILTRATION ELIMINATION Presence of somatic mutations Eidence of immune-cell infiltration Bladder 262,274 O 262 O 274 Breast 276,281 O 281 O 276 Colorectal 275 O 275 O 275 Gastric/esophageal 267,282,283 O 282,283 O 267 Glioblastoma 263,265,284 O 263,284 O 265 Head & neck 268,285 O 285 O 268 Hepatocellular 272,286 O 286 O 272 Lung 262,267 O 262 O 267 Eidence of spontaneous regression Melanoma 262,267,278 O 262 O 267 O 278 Oarian 271,287 O 287 O 271 Pancreatic 275 O 275 O 275 Prostate 269,288 O 288 O 269 Renal 262,270 O 262 O 270 O 270 Non-Hodgkin lymphoma 264,289 O 289 O 264 Hodgkin lymphoma 273,290 O 290 O 273 Leukemia 291 O 291 Multiple myeloma 266,292 O 292 O 266 REALIZING THE POTENTIAL OF I-O RESEARCH *List of tumors represents common types of cancer, but is not exhaustie.

53 I-O RESEARCH IS CONSTANTLY EVOLVING Some of the ongoing research at Bristol-Myers Squibb focuses on: Building an understanding of the dynamic mechanisms that goern the immune system's response to cancer Understanding the role of immune signaling pathways, either alone or in combination, and how they can be modulated to restore the body's natural ability to fight cancer Identifying I-O biomarkers that clarify the unique interplay between the immune system and the tumor and that may help to optimize personalized medicine and improe patient outcomes Deeloping a more comprehensie approach to endpoint assessment, to better recognize the potential benefit of Immuno-Oncology research The potential of I-O research continues to expand, drien by the many patients with adanced cancer who await the offer of renewed hope and the potential of a longer life.

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