Targeting CTLA-4 Research Articles

Mechanism- and Immune Landscape-Based Ranking of Therapeutic Responsiveness of 22 Major Human Cancers to Next Generation Anti-CTLA-4 Antibodies.

Background: CTLA-4 was the first immune checkpoint targeted for cancer therapy and the first target validated by the FDA (Food and Drug Administration) after approval of the anti-CTLA-4 antibody, Ipilimumab. However, clinical response rates to anti-CTLA-4 antibodies are lower while the rates of immunotherapy-related adverse events (irAE) are higher than with anti-PD-1 antibodies. As a result, the effort to target CTLA-4 for cancer immunotherapy has stagnated. To reinvigorate CTLA-4-targeted immunotherapy, we and others have reported that rather than blocking CTLA-4 interaction with its cognate targets, CD80 and CD86, anti-CTLA-4 antibodies achieve their therapeutic responses through selective depletion of regulatory T cells in the tumor microenvironment. Accordingly, we have developed a new generation of anti-CTLA-4 antibodies with reduced irAE and enhanced antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP). A major unresolved issue is how to select appropriate cancer types for future clinical development. Methods: We generated a landscape of the immune tumor microenvironment from RNAseq and genomic data of 7279 independent cancer samples belonging to 22 cancer types from The Cancer Genomics Atlas (TCGA) database. Based primarily on genomic and RNAseq data from pre-treatment clinical samples of melanoma patients who were later identified as responders and nonresponders to the anti-CTLA-4 antibody Ipilimumab, we identified 5 ranking components of responsiveness to anti-CTLA-4, including CTLA-4 gene expression, ADCC potential, mutation burden, as well as gene enrichment and cellular composition that favor CTLA-4 responsiveness. The total ranking number was calculated by the sum of 5 independent partitioning values, each comprised of 1–3 components. Results: Our analyses predict metastatic melanoma as the most responsive cancer, as expected. Surprisingly, non-small cell lung carcinoma (NSCLC) is predicted to be highly responsive to anti-CTLA-4 antibodies. Single-cell RNAseq analysis and flow cytometry of human NSCLC-infiltrating T cells supports the potential of anti-CTLA-4 antibodies to selectively deplete intratumoral Treg. Conclusions: Our in silico and experimental analyses suggest that non-small cell lung carcinoma will likely respond to a new generation of anti-CTLA-4 monoclonal antibodies. Our approach provides an objective ranking of the sensitivity of human cancers to anti-CTLA-4 antibodies. The comprehensive ranking of major cancer types provides a roadmap for clinical development of the next generation of anti-CTLA-4 antibodies.

Dissecting the mechanisms of immune checkpoint therapy.

Immune checkpoint therapy with antibodies targeting CTLA4 or PD1/PDL1 has led to durable regression of disease and cures for a subset of patients with cancer, which has led to increased efforts to develop combination immunotherapy strategies to benefit an even greater number of patients. However, questions remain as to which combinations should be used and in which population of patients. In 2019, several studies reported on the differential impact of targeting CTLA4 and/or PD1 on T cell responses, with clear evidence that both CD4+ and CD8+ T cell responses are needed. These data highlight the complementary nature of targeting CTLA4 and PD1 to drive both CD4+ and CD8+ effector T cell responses, which should be considered in future treatment strategies. Furthermore, reverse translational studies, which provide immune response data from patients receiving immune checkpoint therapy, have revealed resistance mechanisms that indicate relevant targets for combination strategies. Taken together, future treatments will likely consist of anti-CTLA4 plus anti-PD1 with addition of individualized therapy on the basis of tumour type and site of metastatic disease. Studies of immune checkpoint therapy for cancer in 2019 uncovered critical insights into the differences between targeting CTLA4 versus PD1 and the role of particular T cell subsets in immune responses to cancer. Moreover, reverse translational studies are informing our understanding of resistance and response mechanisms in patients.

Mechanisms of Resistance to Checkpoint Blockade Therapy.

Immune checkpoint blockades (ICBs), as a major breakthrough in cancer immunotherapy, target CTLA-4 and the PD-1/PD-L1 axis and reinvigorate anti-tumor activities by disrupting co-inhibitory T-cell signaling. With unprecedented performance in clinical trials, ICBs have been approved by FDA for the treatment of malignancies such as melanoma, non-small-cell lung cancer, colorectal cancer, and hepatocellular carcinoma. However, while ICBs are revolutionizing therapeutic algorithms for cancers, the frequently observed innate, adaptive or acquired drug resistance remains an inevitable obstacle to a durable antitumor activity, thus leading to non-response or tumor relapse. Researches have shown that resistance could occur at each stage of the tumor's immune responses. From the current understanding, the molecular mechanisms for the resistance of ICB can be categorized into the following aspects: 1. Tumor-derived mechanism, 2. T cell-based mechanism, and 3. Tumor microenvironment-determined resistance. In order to overcome resistance, potential therapeutic strategies include enhancing antigen procession and presentation, reinforcing the activity and infiltration of T cells, and destroying immunosuppression microenvironment. In future, determining the driving factors behind ICB resistance by tools of precision medicine may maximize clinical benefits from ICBs. Moreover, efforts in individualized dosing, intermittent administration and/or combinatory regimens have opened new directions for overcoming ICB resistance.

Targeting tumor-intrinsic hexosamine biosynthesis sensitizes pancreatic cancer to anti-PD1 therapy.

Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA4, PD1, PDL1) has not been very successful against PDAC. The robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on the one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase 1 (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). In the current manuscript, we targeted this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-l-norleucine [DON]). Our results showed that DON decreased the self-renewal potential and metastatic ability of tumor cells. Further, treatment with DON decreased hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM and an increased infiltration of CD8+ T cells. Additionally, treatment with DON sensitized pancreatic tumors to anti-PD1 therapy, resulting in tumor regression and prolonged survival.

Abstract LB-B03: PARP and PI3K inhibitor combination therapy eradicates c-MYC-driven murine prostate cancers via cGAS/STING pathway activation within tumor-associated macrophages

Abstract While immune checkpoint blockade (ICB) targeting CTLA-4 and PD-1/PD-L1 has shown remarkable success across a wide range of malignancies, the majority of prostate cancer (PC) patients do not demonstrate clinically meaningful responses to these therapies. Therefore, there is a critical unmet need to discover combinatorial therapeutic strategies that enhance immune-responsiveness in ICB-refractory cancers, such as PC. Here, we show that DNA damage induced by PARP inhibitor (PARPi), rucaparib, in combination with PI3K inhibitor (PI3Ki), buparlisib, resulted in STING activation within myeloid suppressive cells within the TME. Strikingly, this myeloid reprogramming induced by rucaparib/buparlisib combination was accompanied by enhanced T cell infiltration and tumor regression of myc-driven murine PCs in vivo. Furthermore, the anti-tumor mechanism of the rucaparib/buparlisib combination was non-tumor cell autonomous and immune-mediated, as the effect was abrogated in immunodeficient NOD/SCID mice. Critically, rucaparib/buparlisib-mediated tumor regression in myc-driven murine models was abrogated with concomitant clodronate treatment in vivo, which depletes macrophages. To unravel the mechanism for activation of macrophage-mediate anti-cancer immunity elicited by DNA damage, we performed co-culture assays, which revealed that STING pathway activation elicited within macrophages was mediated by double-stranded (ds) DNA-containing microvesicles (MVs) released from tumor cells treated with PARP inhibitors, coupled with c-GAS de-repression induced by PI3K inhibition within macrophages. Interestingly, ex vivo exosome reconstitution studies revealed that DNAse treatment of the MVs completely abolished the STING activation response elicited within macrophages, thus demonstrating that the DNA fragments on the surface of exosomes drives STING pathway activation within macrophages. Strikingly, the combination of rucaparib/buparlisib with immune checkpoint blockade enhanced macrophage and T cell activation/infiltration, resulting in durable tumor clearance. Taken together, these results demonstrate that the DNA damage elicited by PARPi/PI3Ki can reprogram the myeloid tumor microenvironment (TME) to enhance anti-tumor immunity, resulting in effective tumor eradication in ICB-refractory cancers. Preliminary immune profiling of paired metastatic biopsies from a combination co-clinical trial in castrate-resistant prostate cancer patients, showed decreased immunosuppression and T cell activation within the TME following 4 weeks of treatment with rucaparib/nivolumab, thus supporting the preclinical data described above. Citation Format: Priyanka Duttagupta, Kiranj Chaudagar, Sweta Sharma-Saha, Kaela Bynoe, Eileen Parkes, Akash Patnaik. PARP and PI3K inhibitor combination therapy eradicates c-MYC-driven murine prostate cancers via cGAS/STING pathway activation within tumor-associated macrophages [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B03. doi:10.1158/1535-7163.TARG-19-LB-B03

Mechanisms of immune-related adverse events during the treatment of cancer with immune checkpoint inhibitors.

Immune checkpoint inhibitors are novel biologic agents to treat cancer by inhibiting the regulatory interactions that limit T cell cytotoxicity to tumours. Current agents target either CTLA-4 or the PD-1/PD-L1 axis. Because checkpoints may also regulate autoreactivity, immune checkpoint inhibitor therapy is complicated by side effects known as immune-related adverse events (irAEs). The aim of this article is to review the mechanisms of these events. irAEs can involve different tissues and include arthritis and other rheumatic manifestations. The frequency of irAEs is related to the checkpoint inhibited, with the combination of agents more toxic. Because of their severity, irAEs can limit therapy and require immunosuppressive treatment. The mechanisms leading to irAEs are likely similar to those promoting anti-tumour responses and involve expansion of the T cell repertoire; furthermore, immune checkpoint inhibitors can affect B cell responses and induce autoantibody production. Better understanding of the mechanisms of irAEs will be important to improve patient outcome as well as quality of life during treatment.

1912P - Pathogenesis of myocarditis following treatment with immune checkpoint inhibitors in a cynomolgus monkey model

BackgroundImmune checkpoint inhibitors (ICI) targeting PD1, PDL1, or CTLA4 are associated with Multi-organ immune-related adverse events (irAEs) including myocarditis. The pathogenesis and early diagnostic markers for ICI-induced myocarditis are poorly understood. We aimed to develop a preclinical model to further understand ICI associated myocarditis. MethodsChinese-origin cynomolgus monkeys were dosed intravenously with vehicle or nivolumab 20mg/kg plus ipilimumab 15mg/kg once weekly for a total of four doses. Immunophenotyping of lymphocyte subsets was performed on blood and tissue samples. Microscopic examination of formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained sections was performed on all major organs. Immunohistochemistry and RNA sequencing were performed using heart sections. ResultsICI combination resulted in loose faeces, lymphadenopathy, and mononuclear cell infiltrations of varying severity in heart, colon, kidneys, liver, salivary glands and endocrine organs. Three of 5 monkeys developed myocarditis characterized by mononuclear cell infiltration in myocardium, cardiomyocyte degeneration, and or increases in cardiac troponin-I and NT-pro-BNP. Mononuclear cell infiltration primarily comprised of T cells, with lower numbers of macrophages and occasional B cells. Morphologically, cardiac lesions in our monkey model are similar to the reported ICI myocarditis in humans. Increased proliferation of CD4+ and CD8+ T lymphocytes as well as an increase in activated T cells and central memory T cells in the blood, spleen, and lymph nodes, were observed. Transcriptomic analysis suggested increased migration and activation of T cells and increased phagocytosis and antigen presentation in the heart. ConclusionsWe have developed a monkey model characterized by multiple organ toxicities including myocarditis. This model may provide insight into the immune mechanisms and facilitate biomarker identification for ICI-associated irAEs. Legal entity responsible for the studyPfizer. FundingPfizer. DisclosureC. Ji: Full / Part-time employment: Pfizer.

Targeting interferon signaling and CTLA-4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of HPV+ oral cancer

BackgroundThe US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients.MethodsWe used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy.ResultsMonotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC).ConclusionsThese results support combining α-PD-1 therapy with induction of IFN-α/β signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.

Anti-CTLA-4 Activates Intratumoral NK Cells and Combined with IL15/IL15Rα Complexes Enhances Tumor Control.

Antibodies targeting CTLA-4 induce durable responses in some patients with melanoma and are being tested in a variety of human cancers. However, these therapies are ineffective for a majority of patients across tumor types. Further understanding the immune alterations induced by these therapies may enable the development of novel strategies to enhance tumor control and biomarkers to identify patients most likely to respond. In several murine models, including colon26, MC38, CT26, and B16 tumors cotreated with GVAX, anti-CTLA-4 efficacy depends on interactions between the Fc region of CTLA-4 antibodies and Fc receptors (FcR). Anti-CTLA-4 binding to FcRs has been linked to depletion of intratumoral T regulatory cells (Treg). In agreement with previous studies, we found that Tregs infiltrating CT26, B16-F1, and autochthonous Braf V600E Pten -/- melanoma tumors had higher expression of surface CTLA-4 (sCTLA-4) than other T-cell subsets, and anti-CTLA-4 treatment led to FcR-dependent depletion of Tregs infiltrating CT26 tumors. This Treg depletion coincided with activation and degranulation of intratumoral natural killer cells. Similarly, in non-small cell lung cancer (NSCLC) and melanoma patient-derived tumor tissue, Tregs had higher sCTLA-4 expression than other intratumoral T-cell subsets, and Tregs infiltrating NSCLC expressed more sCTLA-4 than circulating Tregs. Patients with cutaneous melanoma who benefited from ipilimumab, a mAb targeting CTLA-4, had higher intratumoral CD56 expression, compared with patients who received little to no benefit from this therapy. Furthermore, using the murine CT26 model we found that combination therapy with anti-CTLA-4 plus IL15/IL15Rα complexes enhanced tumor control compared with either monotherapy.

Myocarditis in Cynomolgus Monkeys Following Treatment with Immune Checkpoint Inhibitors.

Immune checkpoint inhibitors (ICI) targeting PD1, PDL1, or CTLA4 are associated with immune-related adverse events (irAE) in multiple organ systems including myocarditis. The pathogenesis and early diagnostic markers for ICI-induced myocarditis are poorly understood, and there is currently a lack of laboratory animal model to enhance our understanding. We aimed to develop such a model using cynomolgus monkeys. Chinese-origin cynomolgus monkeys were dosed intravenously with vehicle or nivolumab 20 mg/kg plus ipilimumab 15 mg/kg once weekly and euthanized on day 29. Multiple organ toxicities were observed in cynomolgus monkeys, and were characterized by loose feces, lymphadenopathy, and mononuclear cell infiltrations of varying severity in heart, colon, kidneys, liver, salivary glands, and endocrine organs. Increased proliferation of CD4+ and CD8+ T lymphocytes as well as an increase in activated T cells and central memory T cells in the blood, spleen, and lymph nodes, were observed. Transcriptomic analysis suggested increased migration and activation of T cells and increased phagocytosis and antigen presentation in the heart. Mononuclear cell infiltration in myocardium was comprised primarily of T cells, with lower numbers of macrophages and occasional B cells, and was associated with minimal cardiomyocyte degeneration as well as increases in cardiac troponin-I and NT-pro-BNP. Morphologically, cardiac lesions in our monkey model are similar to the reported ICI myocarditis in humans. We have developed a monkey model characterized by multiple organ toxicities including myocarditis. This model may provide insight into the immune mechanisms and facilitate biomarker identification for ICI-associated irAEs.

Characterizing responsive and refractory orthotopic mouse models of hepatocellular carcinoma in cancer immunotherapy.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has a high mortality rate due to limited treatment options. Hence, the response of HCC to different cancer immunotherapies is being intensively investigated in clinical trials. Immune checkpoint blockers (ICB) show promising results, albeit for a minority of HCC patients. Mouse models are commonly used to evaluate new therapeutic agents or regimens. However, to make clinical translation more successful, better characterized preclinical models are required. We therefore extensively investigated two immune-competent orthotopic HCC mouse models, namely transplanted Hep-55.1c and transgenic iAST, with respect to morphological, immunological and genetic traits and evaluated both models’ responsiveness to immunotherapies. Hep-55.1c tumors were characterized by rich fibrous stroma, high mutational load and pronounced immune cell infiltrates, all of which are features of immune-responsive tumors. These characteristics were less distinct in iAST tumors, though these were highly vascularized. Cell depletion revealed that CD8+ T cells from iAST mice do not affect tumor growth and are tumor tolerant. This corresponds to the failure of single and combined ICB targeting PD-1 and CTLA-4. In contrast, combining anti-PD-1 and anti-CTLA-4 showed significant antitumor efficacy in the Hep-55.1c mouse model. Collectively, our data comprehensively characterize two immune-competent HCC mouse models representing ICB responsive and refractory characteristics. Our characterization confirms these models to be suitable for preclinical investigation of novel cancer immunotherapy approaches that aim to either deepen preexisting immune responses or generate de novo immunity against the tumor.

Abstract 940: Development of ONCR-177, an miR-attenuated oncolytic HSV-1 designed to potently activate systemic antitumor immunity

Abstract ONCR-177 is a highly modified recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumor indications. Complementary safety mechanisms, such as tissue-specific miR attenuation of essential viral genes and UL37 mutation, were introduced to reduce replication, neuropathic activity, and latency in normal cells, while preserving oncolytic ability in tumor cells. In addition, ONCR-177 is armed with five transgenes: the NK and T cell activating cytokine IL-12, the chemokines CCL4 and FLT3LG (extracellular domain) to allow for expansion and recruitment of classical dendritic cells, and antagonists to the clinically validated immune checkpoint targets PD-1 and CTLA-4 to counter T cell exhaustion. Therefore, ONCR-177 is proposed to have a dual mechanism of action whereby the miR attenuation strategy allows for selective oncolysis of tumors cells and the transgenes mediate potent stimulation of systemic anti-tumor immunity. Since human IL-12 and the anti-CTLA-4 antagonist do not cross-react with the mouse targets, most nonclinical pharmacology studies were conducted with the mouse surrogate mONCR-171, which expresses within the same base vector as ONCR-177 the mouse equivalent to the ONCR-177 transgenes. Intra-tumoral administration of mONCR-171 in the oHSV-sensitive A20 BALB/c lymphoma bilateral tumor model resulted in response rates (partial and complete tumor regressions) of 100% and 80%, respectively, on the injected (ipsilateral) and distant (contralateral) tumor. mONCR-171 was also highly efficacious in the B16F10 melanoma model, an oHSV-resistant C57BL/6 based tumor model engineered to be permissive to oHSV by introduction of Nectin-1, and in the oHSV-resistant MC38 C57BL/6 colon carcinoma model. Abscopal anti-tumor activity could not be explained by propagation of the virus since viral DNA and transgenes were only detectable in the injected tumor. Rather, mONCR-171 treatment resulted in increased numbers of activated NK cells, CD8 and CD4 T cells, and classical dendritic cells. The proportion of Tregs decreased, resulting in large CD8/Treg ratios. These changes in immune contexture occurred in both the ipsilateral and contralateral tumor and were more pronounced with mONCR-171 treatment compared to the base vector without transgenes, indicating that the observed abscopal effects were due to the elicitation of systemic anti-tumor immunity mediated in part by the transgenes. These encouraging preclinical data warrant the clinical investigation of ONCR-177 in patients with metastatic cancer. Citation Format: Brian B. Haines, Agnieszka Denslow, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Prajna Behera, Peter Grzesik, Caitlin Goshert, Allison Colthart, Jennifer S. Lee, Terry Farkaly, Edward M. Kennedy, Lorena Lerner, Christophe Queva. Development of ONCR-177, an miR-attenuated oncolytic HSV-1 designed to potently activate systemic antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 940.

Abstract 4077: ATOR-1144 is a tumor-directed CTLA-4 x GITR bispecific antibody that acts by depleting Tregs and activating effector T cells and NK cells

Abstract ATOR-1144 is a human CTLA-4 x GITR bispecific IgG1 antibody generated by fusing a high affinity CTLA-4 binder, derived by FIND® optimization of the CTLA-4 binding domain of CD86, to an agonistic GITR antibody isolated from the human antibody library ALLIGATOR-GOLD®. CTLA-4 and GITR are highly expressed on Tregs in the tumor microenvironment, and GITR is also expressed on NK cells and certain tumor cells. ATOR-1144 was designed to induce a tumor-directed immune activation for treatment of solid tumors and hematological malignancies. The in vitro activity of ATOR-1144 in terms of Treg depletion and activation of T cells and NK cells was investigated using purified cells from healthy human donors. ATOR-1144 induced depletion of primary Tregs in an ADCC assay with NK cells. T-cell activation in terms of IL-2 and IFN-γ release was demonstrated in the presence of CTLA-4 and Fcγ receptor crosslinking. NK cells treated with ATOR-1144 released IFN-γ, perforin and granzyme B, and displayed enhanced cytolytic killing of tumor cells. Moreover, NK cell-mediated depletion of CTLA-4-expressing cells was significantly improved upon stimulation with ATOR-1144 compared to monospecific anti-CTLA-4, indicating that NK cells activated via GITR acquire an enhanced capacity to induce ADCC. In conclusion, ATOR-1144 is a first-in-class bispecific tumor directed antibody targeting CTLA-4 and GITR. ATOR-1144 acts through several mechanisms, including depletion of Tregs, activation of effector T cells and activation of NK cells for enhanced Treg depletion and tumor cell killing. Citation Format: Sara Fritzell, Mattias Levin, Ida Åberg, Maria Johansson, Magnus Winnerstam, Karin Enell Smith, Peter Ellmark, Christina Furebring, Per Norlén, Anne Kvarnhammar. ATOR-1144 is a tumor-directed CTLA-4 x GITR bispecific antibody that acts by depleting Tregs and activating effector T cells and NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4077.

Abstract 4588: Characterization of anti-CTLA-4 effects on tumor growth and immune microenvironment in a syngeneic CT26.WT colon carcinoma mouse model

Abstract Colorectal cancer is the third most common type of cancer and one of the leading causes of cancer related deaths. It is a group of various diseases at molecular level, which can make targeted drug development challenging. However, novel therapeutics including immunotherapies have shown their potential to be utilized in the future also when treating colorectal cancer. One promising example is ipilimumab, a monoclonal antibody targeting CTLA-4 (cytotoxic T lymphocyte antigen-4, also known as CD152). CTLA-4 is a cell surface receptor expressed on activated T and B lymphocytes. It regulates immunological tolerance and downregulates immune responses. The aim of the study was to characterize the effects of anti-CTLA-4 on tumor growth, survival and tumor immune microenvironment of mice subcutaneously inoculated with mouse colon carcinoma CT26.WT cells. CT26.WT cells were subcutaneously inoculated into female Balb/c mice (n=27). Body weights and tumor dimensions were determined two times a week during the course of the study. The mice were randomized to three groups (n=9/group) with similar tumor volume and variation and dosing was started when an average tumor size of approximately 50 mm³ was reached (day 7). Anti-CTLA-4 antibody (9D9, 10 mg/kg), an isotype control (mouse IgG2b) and vehicle (PBS) were administered (i.p.) twice a week. Tumor growth and survival were followed for 26 days after inoculation of the cancer cells. The mice were sacrificed individually when the maximum tumor volume (1500 mm³) was reached, the tumors were ulcerated or at the latest on study day 26. Tumor samples were collected for histological and immunohistochemical analysis and stained for helper T cells (CD4), cytotoxic T cells (CD8), myeloid-derived suppressor cells (MDSC; CD11b), tumor-associated macrophages (F4/80), and inflammatory monocytes and neutrophils (FCGR1/CD64) for characterization of tumor immune microenvironment. Maximum tumor volume and tumor ulceration were observed the earliest on study day 17. Anti-CTLA-4 suppressed tumor growth, and all mice in the group receiving anti-CTLA-4 were still in the study on day 26. Only one and four mice were left in the vehicle and isotype control groups, respectively. Immunohistochemical staining suggested an increase of CD4+ tumor-infiltrating lymphocytes (TILs) in anti-CTLA-4 treated mice. Furthermore, more TAMs were observed in tumors of anti-CTLA-4 treated mice, especially in the borderlines of necrotic areas. Large quantity of CD11b+ MDSCs and inflammatory cells were observed in CT26.WT tumors. CT26.WT subcutaneous model can be utilized for studying the effects of immunotherapies, and anti-CTLA-4 treatment suppressed tumor growth in this model. CT26.WT tumors represent a highly immunogenic hot tumor type with high immune cell infiltration, associated with a good efficacy of anti-CTLA-4 treatment in the model. Citation Format: Jenni H. Mäki-Jouppila, Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli. Characterization of anti-CTLA-4 effects on tumor growth and immune microenvironment in a syngeneic CT26.WT colon carcinoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4588.

Abstract 1136: Tumor uptake and biodistribution of 89Zirconium-labeled ipilimumab in patients with metastatic melanoma during ipilimumab treatment

Abstract Introduction Ipilimumab, a monoclonal antibody targeting CTLA-4, is approved for the treatment of metastatic melanoma and significantly improves overall survival. Because of the high costs and the potential serious toxicity of ipilimumab, it is of great importance to identify biomarkers that correlate with clinical activity and that can be used to select patients who will benefit from CTLA-4 blockade therapy. We hypothesize that patients who do not respond to treatment with ipilimumab have lower drug levels in tumor tissues as compared to patients with a good response to therapy. In addition, we hypothesize that immune related adverse events (irAEs) are associated with high drug levels in the affected tissue. As irAEs usually occur approximately 6-8 weeks after the first injection of ipilimumab, we hypothesize that the drug levels in potentially affected tissues will increase at the second injection. Experimental procedures To visualize in vivo localization of ipilimumab in patients diagnosed with metastatic melanoma, 37 MBq, 10 mg 89Zr-labeled ipilimumab was injected within 2 hours after their first ipilimumab dose (3 mg/kg). Whole body PET/CT scans were obtained at 2h, 72h and 144h post injection and this procedure was repeated three weeks later at the second ipilimumab cycle. Biodistribution and tumor uptake were assessed visually by a nuclear physician. Focal uptake in tumor lesions exceeding local background was determined in volumes of interest (VOI) and SUVpeak values were obtained. Biodistribution was quantified by defining vital organs (i.e. lungs, kidneys, spleen, liver) and calculating mean %ID/kg. Blood was drawn for dosimetry and immunophenotyping at several time points during the trial. Presented here are initial results of the first three patients, up to 29 patients are planned to be included. Results Biodistribution of 89Zr-labeled ipilimumab showed a pattern distinctive for 89Zr-labeled antibodies with uptake in liver and spleen, as well as prolonged circulating antibody in the bloodstream corresponding to the pharmacokinetics of ipilimumab. Visual evaluation confirmed uptake of 89Zr-labeled ipilimumab in 5/12 evaluable tumor lesions, visible at both first and second injection of ipilimumab. Tumor uptake was comparable for 72h and 144h post injection with a mean of 6.9 %ID/kg (range 3.3-10.1) and a SUVpeak of 4.4 (range 2.3-8.9). There were no significant differences in tumor uptake between first and second dose of ipilimumab (mean 7.31 and 6.54 %ID/kg respectively). Conclusions Preliminary data of this ongoing study showed that the tracer is able to visualize and quantify uptake of ipilimumab in tumors. Correlations between tumor uptake and response to treatment will be presented. Furthermore, special interest will be given to uptake in lymphoid organs and locations for irAEs. Citation Format: Iris H. Miedema, Gerben J. Zwezerijnen, Guus A. van Dongen, Daniëlle J. Vugts, Marc C. Huisman, Otto S. Hoekstra, Tanja D. de Gruijl, Hendrik M. Verheul, Catharina W. Menke, Alfons J. van den Eertwegh. Tumor uptake and biodistribution of 89Zirconium-labeled ipilimumab in patients with metastatic melanoma during ipilimumab treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1136.

Reduction of immunosuppressive tumor microenvironment in cholangiocarcinoma by ex vivo targeting immune checkpoint molecules

Cholangiocarcinoma is an aggressive hepatobiliary malignancy originating from biliary tract epithelium. Whether cholangiocarcinoma is responsive to immune checkpoint antibody therapy is unknown, and knowledge of its tumor immune microenvironment is limited. We aimed to characterize tumor-infiltrating lymphocytes (TILs) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TILs. We isolated TILs from resected tumors of patients with cholangiocarcinoma and investigated their compositions compared with their counterparts in tumor-free liver (TFL) tissues and blood, by flow cytometry and immunohistochemistry. We measured expression of immune co-stimulatory and co-inhibitory molecules on TILs, and determined whether targeting these molecules improved ex vivo functions of TILs. Proportions of cytotoxic T cells and natural killer cells were decreased, whereas regulatory T cells were increased in tumors compared with TFL. While regulatory T cells accumulated in tumors, the majority of cytotoxic and helper T cells were sequestered at tumor margins, and natural killer cells were excluded from the tumors. The co-stimulatory receptor GITR and co-inhibitory receptors PD1 and CTLA4 were over-expressed on tumor-infiltrating T cells compared with T cells in TFL and blood. Antagonistic targeting of PD1 or CTLA4 or agonistic targeting of GITR enhanced effector molecule production and T cell proliferation in ex vivo stimulation of TILs derived from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated with differences in TIL immune phenotype. Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells that over-express co-inhibitory receptors suggest that the tumor microenvironment in cholangiocarcinoma is immunosuppressive. Targeting GITR, PD1 or CTLA4 enhances effector functions of tumor-infiltrating T cells, indicating that these molecules are potential immunotherapeutic targets for patients with cholangiocarcinoma. The defense functions of immune cells are suppressed in cholangiocarcinoma tumors. Stimulating or blocking "immune checkpoint" molecules expressed on tumor-infiltrating T cells can enhance the defense functions of these cells. Therefore, these molecules may be promising targets for therapeutic stimulation of immune cells to eradicate the tumors and prevent cancer recurrence in patients with cholangiocarcinoma.

CCTG CO.26: Updated analysis and impact of plasma-detected microsatellite stability (MSS) and tumor mutation burden (TMB) in a phase II trial of durvalumab (D) plus tremelimumab (T) and best supportive care (BSC) versus BSC alone in patients (pts) with refractory metastatic colorectal carcinoma (rmCRC).

3512 Background: Targeting both PD-L1 and CTLA-4 may be synergistic immunotherapy approaches. CO.26 evaluated if dual inhibition leads to improved pt survival vs BSC alone in rmCRC. Methods: rmCRC pts were randomized 2:1 to D+T vs BSC. Treatment consisted of D (1500 mg) D1 q 28 days and T (75 mg) D1 for first 4 cycles, and supportive measures. Primary endpoint was overall survival (OS). Two-sided p < 0.10 was considered statistically significant. Cell-free (cf)DNA sequencing for MSI and TMB used GuardantOMNI panel and baseline plasma. Results: From 08/2016-06/2017, 180 pts were enrolled. Pt characteristics were balanced between arms. At median follow-up of 15.2 months (mos), median OS was 6.6 mos for D+T and 4.1 mos for BSC (p = 0.07; Hazard ratio (HR): 0.72, 90% confidence interval (CI): 0.54 – 0.97). Progression free survival (PFS) was 1.8 mos vs 1.9 mos, respectively (HR 1.01, 90% CI 0.76 – 1.34). Disease control rate (DCR) was 22.6% for D+T and 6.6% for BSC (p = 0.006). cfDNA analysis was successful in 168/169 pts (99.4%). Two pts were MSI-high. In 166 MSS pts, OS HR was 0.66 (p=0.024; 90% CI 0.49-0.89). Excluding the MSI-H cases (TMB of 74.7 and 247.1 mts/Mb), mean TMB was 20.4 ± 16.3 mts/Mb (range: 0.96 – 114.0). In MSS pts, a pre-specified cutpoint of 20 mts/Mb stratified pts into high and low TMB groups but was not predictive for OS , PFS, or DCR (interaction p-values > 0.7). Using a minimum p-value approach, pts with TMB >28 mts/Mb (21% of MSS pts) had the greatest OS benefit (HR 0.34, 90% CI 0.18-0.63) for D+T (interaction p = 0.07). High TMB was associated with a trend in worse prognosis for OS in the BSC arm using both 20 mts/Mb (HR 1.26, 90% CI 0.76-2.12) and 28 mts/Mb (HR 2.59 90% CI 1.46-4.62) cutpoints. Conclusions: D+T significantly prolonged OS in pts with rmCRC. High TMB may select a group of MSS pts who benefit from D+T. Plasma TMB appeared prognostic in the BSC arm. This is the first study showing combined PD-L1 and CTLA-4 inhibition prolongs survival in pts with MSS rmCRC. Updated results based on deaths in more than 90% of pts will be presented. Clinical trial information: NCT02870920.

Significance of systemic and local immune responses to the pathological therapeutic effect of neoadjuvant chemotherapy in breast cancer.

e12084 Background: Activation of the immune response including T lymphocytes, natural killer (NK) cells, and tumor microenvironment factors (TMEFs) is important in inducing a therapeutic response after neoadjuvant chemotherapy (NAC) in breast cancer. We examined the significance of systemic and local immune responses to the pathological therapeutic effect of NAC in breast cancer. Methods: From 2012 to 2018, 38 patients with stage II–III breast cancer received NAC with anthracyclines and taxanes followed by surgery. Therapeutic effects were evaluated according to the histopathology criteria for the assessment of therapeutic effects in breast cancer indicated by the Japanese Breast Cancer Society. Peripheral NK (pNK) cell activity was measured by chromium release assay. Levels of TMEFs were assessed by next-generation sequencing for CD4, CD8, NK, FOXP3, CTLA-4, PD-1, PD-L1, IL-2, IL-6, IL-12, IFN-γ, IL-10, TGF-β, and VEGF in FFPE sections collected from preoperative VAB samples and surgical specimens. Results: The stages, tumor subtypes, and therapeutic outcomes were as follows: II (N = 21), III (N = 17); luminal (N = 22), HER-2 positive (N = 12), TN (N = 4); G1a (N = 8), G1b (N = 13), G2a (N = 7), G2b (N = 4), G3 (i.e. complete) (N = 6). A G2 or better therapeutic effect were significantly associated with high post-NAC levels of NK, and potentially associated with higher CD4, CD8, and lower CTLA-4 after NAC. Multivariate logistic regression analysis showed that a G2 or better therapeutic effect was significantly associated with higher NK after NAC (OR = 1.07; 95% CI, 1.00–1.14; P = .0255). Disappearance of axillary lymph node metastasis was significantly associated with increased NK and pNK cell activity, as well as decreased VEGF level, and potentially associated with lower CTLA-4 after NAC. Conclusions: Increased NK cells after NAC are critical in producing a better therapeutic effect in collaboration with increased CD4 and CD8, and decreased CTLA-4 and VEGF. Systemic activation of pNK cells may improve the elimination of metastatic tumor cells in axillary lymph nodes by coordinating with release of immunosuppression derived from VEGF and activation of immune cells in the tumor microenvironment in breast cancer patients after NAC. An immune checkpoint inhibitor targeting CTLA-4 may improve NAC efficacy for breast cancer.

Current knowledge of Ipilimumab and its use in treating non-small cell lung cancer

ABSTRACTIntroduction: The systemic treatment of non-small cell lung cancer (NSCLC) has changed dramatically with the identification of actionable mutations and the use of targeted agents. Unfortunately, many tumors will acquire resistance and >75% of NSCLC cases lack for an actionable gene aberration. In this setting, immunotherapy rises as effective therapeutic where immune checkpoint inhibitors have entered or are entering the market in many neoplasms, including NSCLC. Ipilimumab is a monoclonal antibody targeting CTLA-4, promoting T-cell activation and its subsequent anti-tumoral immune effect. Ipilimumab might have a very important role in NSCLC as it does in melanoma because of its synergistic effect with PD-1/PDL-1 inhibitors.Areas covered: We summarize current results of clinical studies of ipilimumab for efficacy and safety in NSCLC and also the current knowledge about potential biomarkers for its efficacy.Expert Opinion: Combined use of PD-1/PDL-1 and anti-CTL4 inhibitors increases the efficacy against NSCLC and it is a very promising approach not only in NSCLC but also in small cell lung cancer (SCLC) for first or second-line therapy. It’s very important to identify biomarkers that can better select the population of patients that benefit the most with these checkpoint inhibitors.

The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation

BackgroundThe CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile.MethodsATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry.ResultsATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells.ConclusionsBy targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467).