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This file contains Supplementary Figures presenting additional information on the immunology and tumor biology of the patient case

This file contains supplementary information on the methods used to generate information on the immunology and tumor biology of the patient case

Cancer immunotherapy can result in durable tumor regressions in some patients. However, patients who initially respond often experience tumor progression. Here, we report mechanistic evidence of tumoral immune escape in an exemplary clinical case: a patient with metastatic melanoma who developed disease recurrence following an initial, unequivocal radiologic complete regression after T-cell–based immunotherapy. Functional cytotoxic T-cell responses, including responses to one mutant neoantigen, were amplified effectively with therapy and generated durable immunologic memory. However, these immune responses, including apparently effective surveillance of the tumor mutanome, did not prevent recurrence. Alterations of the MHC class I antigen-processing and presentation machinery (APM) in resistant cancer cells, but not antigen loss or impaired IFNγ signaling, led to impaired recognition by tumor-specific CD8+ T cells. Our results suggest that future immunotherapy combinations should take into account targeting cancer cells with intact and impaired MHC class I–related APM. Loss of target antigens or impaired IFNγ signaling does not appear to be mandatory for tumor relapse after a complete radiologic regression. Personalized studies to uncover mechanisms leading to disease recurrence within each individual patient are warranted. Cancer Res; 77(17); 4562–6. ©2017 AACR.

BackgroundIO112 is an immune modulatory cancer therapy under preclinical development to target arginase-1-expressing tumor cells and immune inhibitory myeloid cells, such as myeloid derived suppressor cells (MDSCs), and tumor associated macrophages (TAMs). Arginase-1 acts as a metabolic immune regulator at the tumor site by reducing availability of L-arginine to the infiltrating immune cells thus reducing T cell functionality and proliferation. Previously, we demonstrated that IO112 triggers activation of spontaneous CD4+ and CD8+ T-cell responses against arginase-1, found in both cancer patients and healthy individuals.1 These T cells are present in the memory T cell compartment, and are activated in arginase-1 inducing conditions, such as presence of TH2 cytokines IL-4 or IL-13 in vitro.2 3 In this study we aimed to explore the role of arginase-1-specific T cells as immune modulators in immune homeostasis and tumor microenvironment for the development of IO112 immunomodulatory therapy.MethodsHuman arginase-1-specific T cells were isolated and expanded for functional characterization of reactivity against arginase-1 expressing target cells as well as subsequent phenotyping of the targeted arginase-1 positive cells. Syngeneic C57BL/6 mouse tumor models were used to assess the therapeutic efficacy of IO112.ResultsWe show that arginase-1-specific memory T cells specifically recognize arginase-1 expressing cells, such as mRNA transfected autologous dendritic cells (DCs) and B cells as well as M2 polarized macrophages in vitro. In addition, activated arginase-1-specific T cells produce pro-inflammatory cytokines IFNγ and TNFα. Secretion of TH1 cytokines by these T cells suggests that they may act as potent immune modulators in the tumor microenvironment, since many arginase-1 expressing myeloid cells are not terminally differentiated and they can be re-polarized to an immunostimulatory, M1-like phenotype. We also observed that targeting of M2-polarized arginase-1 expressing monocytic leukemia cell line THP-1 with arginase-1-specific CD4+ T cells induces upregulation of PD-L1 on the THP-1 cells. Furthermore, we demonstrate anti-tumor activity of IO112 in syngeneic mouse tumor models (B16 and MC38), both as monotherapy and in combination with anti-PD-1 treatment. The therapeutic effect was associated with increased immune infiltration in the IO112-treated mice compared to the control.ConclusionsWe demonstrate that arginase-1 specific T cells can influence the polarization of arginase-1-expressing immune cells. Our study provides evidence that IO112 immune therapy against arginase-1 is an attractive way of modulating the immune suppressive tumor microenvironment for therapeutic benefit. With this rationale, we are currently undertaking Investigational New Drug (IND) application enabling studies to explore this approach in a clinical setting.ReferencesMartinenaite E, Mortensen REJ, Hansen M, Holmström MO, Ahmad SM, Jørgensen NGD, Met Ö, Donia M, Svane IM, Andersen MH. Frequent adaptive immune responses against arginase-1. Oncoimmunology 2018;7(3):e1404215.Martinenaite E, Ahmad SM, Svane IM, Andersen MH. Peripheral memory T cells specific for Arginase-1. Cell Mol Immunol 2019;16(8):718–719.Martinenaite E, Ahmad SM, Bendtsen SK, Jørgensen MA, Weis-Banke SE, Svane IM, Andersen MH. Arginase-1-based vaccination against the tumor microenvironment: the identification of an optimal T-cell epitope. Cancer Immunol Immunother 2019;68(11):1901–1907.Ethics ApprovalThis study was approved by the Scientific Ethics Committee for The Capital Region of Denmark and Danish Ethics Committee on experimental animal welfare.

L-arginine depletion by regulatory cells and cancer cells expressing arginase-1 (Arg-1) is a vital contributor to the immunosuppressive tumor microenvironment in patients with cancer. We have recently described the existence of pro-inflammatory effector T cells that recognize Arg-1. Hence, Arg-1-specific self-reactive T cells are a naturally occurring part of the memory T-cell repertoire of the human immune system. Here, we further characterize a highly immunogenic epitope from Arg-1. We describe frequent T-cell-based immune responses against this epitope in patients with cancer, as well as in healthy donors. Furthermore, we show that Arg-1-specific T cells expand in response to the TH2 cytokine interleukin (IL)-4 without any specific stimulation. Arg-1-specific memory TH1 cells that respond to increased IL-4 concentration may, therefore, drive the immune response back into the TH1 pathway. Arg-1-specific T cells thus appear to have an important function in immune regulation. Because Arg-1 plays an important role in the immunosuppressive microenvironment in most cancers, an immune modulatory vaccination approach can readily be employed to tilt the balance away from immune suppression in these settings.

Mutations in exon 9 of the calreticulin gene (CALR) frequently occur in patients with chronic myeloproliferative neoplasms (MPN). Patients exhibit spontaneous cellular immune responses to epitopes derived from the mutant CALR C-terminus, and CALR-mutant-specific T cells recognize autologous CALR-mutant malignant cells. This study investigated whether CALR-mutant-specific T cells occur naturally in CALRwt MPN-patients and in healthy individuals. Specific immune responses against epitopes in the mutant CALR peptide sequence were detected in both CALRwt MPN-patients and in healthy individuals. Healthy donors displayed more frequent and stronger CALR-mutant specific T-cell responses compared to the responses identified in CALR-mutant MPN-patients. Several T-cell responses were identified in healthy donors directly ex vivo. Importantly, by running functional analyses on live-sorted immune cells from healthy donors, we showed that circulating CALR-mutant-specific immune cells are T-memory cells. These findings suggest, that healthy individuals acquire a CALR exon 9 mutation, but the immune system reacts and clears the mutant cells, and during this reaction generates CALR-mutant specific T-memory cells. We believe that these findings provide the evidence for tumor immune surveillance in MPN.

The JAK2 V617F mutation is a target for specific T cells in the JAK2 V617F-positive myeloproliferative neoplasms

Recently, there has been an increased focus on the immune checkpoint protein PD-1 and its ligand PD-L1 due to the discovery that blocking the PD-1/PD-L1 pathway with monoclonal antibodies elicits striking clinical results in many different malignancies. We have described naturally occurring PD-L1-specific T cells that recognize both PD-L1-expressing immune cells and malignant cells. Thus, PD-L1-specific T cells have the ability to modulate adaptive immune reactions by reacting to regulatory cells. Thus, utilization of PD-L1-derived T cell epitopes may represent an attractive vaccination strategy for targeting the tumor microenvironment and for boosting the clinical effects of additional anticancer immunotherapy. This review summarizes present information about PD-L1 as a T cell antigen, depicts the initial findings about the function of PD-L1-specific T cells in the adjustment of immune responses, and discusses future opportunities.

The enzyme arginase-1 reduces the availability of arginine to tumor-infiltrating immune cells, thus reducing T-cell functionality in the tumor milieu. Arginase-1 is expressed by some cancer cells and by immune inhibitory cells, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), and its expression is associated with poor prognosis. In the present study, we divided the arginase-1 protein sequence into overlapping 20-amino-acid-long peptides, generating a library of 31 peptides covering the whole arginase-1 sequence. Reactivity towards this peptide library was examined in PBMCs from cancer patients and healthy individuals. IFNγ ELISPOT revealed frequent immune responses against multiple arginase-1-derived peptides. We further identified a hot-spot region within the arginase-1 protein sequence containing multiple epitopes recognized by T cells. Next, we examined in vitro-expanded tumor-infiltrating lymphocytes (TILs) isolated from melanoma patients, and detected arginase-1-specific T cells that reacted against epitopes from the hot-spot region. Arginase-1-specific CD4+T cells could be isolated and expanded from peripheral T cell pool of a patient with melanoma, and further demonstrated the specificity and reactivity of these T cells. Overall, we showed that arginase-1-specific T cells were capable of recognizing arginase-1-expressing cells. The activation of arginase-1-specific T cells by vaccination is an attractive approach to target arginase-1-expressing malignant cells and inhibitory immune cells. In the clinical setting, the induction of arginase-1-specific immune responses could induce or increase Th1 inflammation at the sites of tumors that are otherwise excluded due to infiltration with MDSCs and TAMs.

The calreticulin (CALR) exon 9 mutations are found in ∼30% of patients with essential thrombocythemia and primary myelofibrosis. Recently, we reported spontaneous immune responses against the CALR mutations. Here, we describe that CALR-mutant (CALRmut)-specific T cells are able to specifically recognize CALRmut cells. First, we established a T-cell culture specific for a CALRmut epitope. These specific T cells were able to recognize several epitopes in the CALRmut C terminus. Next, we established a CALRmut-specific CD4+ T-cell clone by limiting dilution. These CD4+ T cells recognized autologous CALRmut monocytes and hematopoietic stem cells, and T-cell recognition of target cells was dependent on the presence of CALR. Furthermore, we showed that the CALRmut response was human leukocyte antigen (HLA)-DR restricted. Finally, we demonstrated that the CALRmut-specific CD4+ T cells, despite their phenotype, were cytotoxic to autologous CALRmut cells, and that the cytotoxicity was mediated by degranulation of the T cells. In conclusion, the CALR exon 9 mutations are targets for specific T cells and thus are promising targets for cancer immune therapy such as peptide vaccination in patients harboring CALR exon 9 mutations.

The B-cell lymphoma-2 (Bcl-2) family of proteins play a crucial role in multiple myeloma (MM), contributing to lacking apoptosis which is a hallmark of the disease. This makes the Bcl-2 proteins interesting targets for therapeutic peptide vaccination. We report a phase I trial of therapeutic vaccination with peptides from the proteins Bcl-2, Bcl-XL and Mcl-1 in patients with relapsed MM. Vaccines were given concomitant with bortezomib. Out of 7 enrolled patients, 4 received the full course of 8 vaccinations. The remaining 3 patients received fewer vaccinations due to progression, clinical decision of lacking effect and development of hypercalcemia, respectively. There were no signs of toxicity other than what was to be expected from bortezomib. Immune responses to the peptides were seen in all 6 patients receiving more than 2 vaccinations. Three patients had increased immune responses after vaccination. Vaccination against Bcl-2 was well tolerated and was able to induce immune responses in patients with relapsed MM.

Tumor cells and tumor-infiltrating macrophages produce the chemokine CCL22, which attracts regulatory T cells (Tregs) into the tumor microenvironment, decreasing anticancer immunity. Here, we investigated the possibility of targeting CCL22-expressing cells by activating specific T cells. We analyzed the CCL22 protein signal sequence, identifying a human leukocyte antigen A2- (HLA-A2-) restricted peptide epitope, which we then used to stimulate peripheral blood mononuclear cells (PMBCs) to expand populations of CCL22-specific T cells in vitro. T cells recognizing an epitope derived from the signal-peptide of CCL22 will recognize CCL22-expressing cells even though CCL22 is secreted out of the cell. CCL22-specific T cells recognized and killed CCL22-expressing cancer cells. Furthermore, CCL22-specific T cells lysed acute monocytic leukemia cells in a CCL22 expression-dependent manner. Using the Enzyme-Linked ImmunoSPOT assay, we examined peripheral blood mononuclear cells from HLA-A2+ cancer patients and healthy volunteers for reactivity against the CCL22-derived T-cell epitope. This revealed spontaneous T-cell responses against the CCL22-derived epitope in cancer patients and in healthy donors. Finally, we performed tetramer enrichment/depletion experiments to examine the impact of HLA-A2-restricted CCL22-specific T cells on CCL22 levels among PMBCs. The addition or activation of CCL22-specific T cells decreased the CCL22 level in the microenvironment. Activating CCL22-specific T cells (e.g., by vaccination) may directly target cancer cells and tumor-associated macrophages, thereby modulating Treg recruitment into the tumor environment and augmenting anticancer immunity.

Programmed death 1 (PD-1) is an inhibitory molecule expressed on the surface of T cells. The PD-1 ligand (PD-L1 (B7-H1)) is expressed on nonhematopoietic cells as well as antigen-presenting cells and placental cells located in an inflammatory microenvironment, as PD-L1 is in general upregulated by interferons. The PD-1/PD-L1 pathway is thought to assure peripheral T-cell tolerance and is involved in controlling the proliferation and cytokine production of T cells.1 PD-L1 has been described to be expressed on cancer cells in many different hematological malignancies, where it contributes to protection of the malignant cells from immune destruction.2, 3 As an example, in aggressive B-cell lymphomas both malignant cells and infiltrating immune cells have been depicted to express PD-L1.4 Likewise, myeloma cells upregulate PD-L1 to escape antitumor immunity.5 Expression of PD-L1 has even been correlated to violent characteristics of myeloma cells.6 PD-L1 has furthermore been described to be involved in antileukemia immune escape in myeloid leukemias.7, 8, 9 Thus, chronic myeloid leukemia (CML) cells express elevated levels of PD-L1, whereas CML-specific T cells express PD-1.8 PD-1 signaling on such T cells results in T-cell exhaustion and disease progression. PD-1 expression on T cells among peripheral blood mononuclear cells (PBMCs) from patients is in general elevated in comparison with healthy donors. Likewise, in bone marrow biopsies from acute myeloid leukemia (AML) as well as myelodysplastic syndrome patients, blasts have been found to be positive for PD-L1, whereas stroma/non-blast cellular compartment was positive for PD-1. The potential of targeting of the PD-L1/PD-1 pathway was recently demonstrated in a phase I clinical trial with patients suffering from different hematopoietic malignancies (AML, chronic lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma or multiple myeloma), who were treated with anti-PD-1-blocking antibodies.9 No severe toxicity was reported and the treatment seemed to induce clinical effects. At present, a PD-1-blocking antibody is being investigated in AML patients in combination with a cancer vaccine ({"type":"clinical-trial","attrs":{"text":"NCT01096602","term_id":"NCT01096602"}}NCT01096602). We have described that natural existing PD-L1-specific cytotoxic T lymphocytes (CTLs) are able to recognize and kill both malignant lymphoma cells as well as normal PD-L1-expressing immune cells.10, 11 Furthermore, we recently described that the addition of PD-L1-specific CTLs 1 week after stimulation of PBMCs with viral epitopes from Epstein–Barr virus (EBV) or cytomegalovirus (CMV) resulted in an immense increase in the number of virus-specific CD8+ T cells in vitro.12 Hence, PD-L1-specific CTLs may effectively enhance the effector phase of the immune response. To further examine the potential of using PD-L1-specific T cells in the treatment of hematological malignancies, we here stimulated PBMCs from 14 human leukocyte antigen (HLA)-A2+ donors with a well-known HLA-A2-restricted CMV epitope either in co-culture with the HLA-A2-restricted epitope PD-L115–23 or an irrelevant HLA-A2-restricted epitope from HIV-1 in the presence of IL-2, as depicted in Figure 1a. After four in vitro stimulations the T-cell reactivity toward the CMV epitope was examined for each donor by the use of HLA-A2/CMV tetramers (Figure 1b). Notably, we observed a significant increase in the numbers of virus-specific T cells in the cultures that had been co-stimulated with the PD-L115–23 peptide epitope. Examples of three donors, where co-activation of PD-L1-specific T cells significantly boosted T-cell immunity toward CMV are illustrated in Figure 1c. Thus, the stimulation of PD-L1-specific CTLs by vaccination may additionally boost other effector T cells by removing PD-L1-positive, immune-suppressive cells that inhibit the activation and proliferation of PD-1-positive T cells. Next, to examine how PD-L1-specific CTLs can influence antileukemia immunotherapy in general, we examined the ability of PD-L115–23-specific CTLs11 to kill well-characterized PD-L1+ AML cells—UKE-1,13 SET-2 (ref. 13) and THP-1 (ref. 14)—in standard 51Cr release assays. PD-L115–23-specific CTLs efficiently killed UKE-1 and THP-1 cells (Figures 2a and b). In contrast, the SET-2 cells were not killed by the PD-L115–23-specific CTLs (Figure 2a). Likewise, a control EBV-positive B-lymphoblastoid cell line RPMI6666 (ref. 15) was not killed by the PD-L1-specific CTLs (Figure 2b). As a further control, the PD-L115–23-specific CTLs efficiently lysed TAP-deficient T2 cells pulsed with PD-L115–23 efficiently, whereas no cytotoxicity was observed against T2 cells pulsed with an irrelevant peptide from HIV (Figure 2c). Our observations on one hand show that PD-L1-specific CTLs are able to react directly toward AML cells and kill the malignant cells. However, not all AML cells were killed, as the PD-L1-specific CTLs were not able to kill SET-2 cells. To examine whether the activation of PD-L1-specific CTLs could have an indirect effect on the immunity against SET-2 cells, we stimulated PBMCs from the three donors in which we had observed an increased CMV response after co-stimulation with the PD-L115–23 peptide (as depicted in Figure 2d) with SET-2 cells. Thus, after four in vitro stimulations of PBMCs with SET-2 cells either in co-culture with the PD-L115–23 epitope or an irrelevant HLA-A2-restricted epitope from HIV-1 in the presence of IL-2, we examined the ability of the resulting T-cell cultures to recognize and kill SET-2 cells in standard 51Cr release assays. As illustrated in Figure 2e the T-cell cultures from all three donors co-stimulated with PD-L115–23 epitope more efficiently lysed SET-2 cells compared with the cultures co-stimulated with an irrelevant HIV epitope. Hence, although PD-L115–23-specific CTLs do not recognize SET-2 cells, the activation of these by stimulation boosted additional T-cell immunity toward SET-2 cells. This could point to a scenario were PD-L1-based vaccination might be beneficial even in leukemia patients where PD-L1-specific CTLs do not react toward the leukemia cells themselves. Thus, the enhancement of PD-L1-specific CTLs in patients might be valuable both by the direct killing of leukemia cells as well as indirectly by the reinforcement of antileukemic T cells. The addition of PD-L1 vaccination should be easily implementable and highly synergistic with other immune-based therapies. The induction of specific T cells represents a new and attractive immune therapeutic approach, in which the specific depletion of target cells is not limited to targeting proteins that are expressed on the cell surface. This is important, as the PD-L1 epitope used in this study is located near the N-terminal of the PD-L1 sequence as part of the signal peptide, and is therefore not part of the extracellular domain. An additional principal difference between therapeutically induced T cells and surface blockade by antibodies is that the former reduces not only the target protein-mediated immune suppression but also other immune-suppressive effects mediated by the target cells. Taken together, vaccination against PD-L1 and antibody-mediated PD-1/PD-L1 blockade should therefore be considered complementary rather than combative. In fact, an exciting therapeutic strategy would be to combine anti-PD-L1 vaccination with, for example, anti-CTLA4- or anti-LAG3-blocking antibodies. Taken together, we believe that the findings justify and warrant clinical testing to evaluate the efficiency and safety of PD-L1-based vaccinations in hematological malignancies. Hence, we are in the process of initiating a phase I vaccination study at the Center for Caner Immune Therapy, Copenhagen University Hospital, Herlev. Figure 1 Co-stimulation with a PD-L1-restricted epitope enhances the frequency of virus-specific T cells. (a) PBMCs (5 × 10e6) from HLA-A2+ donors were stimulated in vitro with the HLA-A2-restricted epitope CMV pp65495–504 (NLVPMVATV) peptide either ... Figure 2 PD-L1-specific CTLs directly kill AML cells and enhance additional antileukemic immunity. (a) Functional capacity of I PD-L115–23-specific CTLs assayed by 51Cr release assay. Specific lysis of the PD-L1+, HLA-A2+ AML cells UKE-1 ...

Tryptophan-2,3-dioxygenase (TDO) physiologically regulates systemic tryptophan levels in the liver. However, numerous studies have linked cancer with activation of local and systemic tryptophan metabolism. Indeed, similar to other heme dioxygenases TDO is constitutively expressed in many cancers. In the present study, we detected the presence of both CD8+ and CD4+ T-cell reactivity toward TDO in peripheral blood of patients with malignant melanoma (MM) or breast cancer (BC) as well as healthy subjects. However, TDO-reactive CD4+ T cells constituted distinct functional phenotypes in health and disease. In healthy subjects these cells predominately comprised interferon (IFN)γ and tumor necrosis factor (TNF)-α producing Th1 cells, while in cancer patients TDO-reactive CD4+ T-cells were more differentiated with release of not only IFNγ and TNFα, but also interleukin (IL)-17 and IL-10 in response to TDO-derived MHC-class II restricted peptides. Hence, in healthy donors (HD) a Th1 helper response was predominant, whereas in cancer patients CD4+ T-cell responses were skewed toward a regulatory T cell (Treg) response. Furthermore, MM patients hosting a TDO-specific IL-17 response showed a trend toward an improved overall survival (OS) compared to MM patients with IL-10 producing, TDO-reactive CD4+ T cells. For further characterization, we isolated and expanded both CD8+ and CD4+ TDO-reactive T cells in vitro. TDO-reactive CD8+ T cells were able to kill HLA-matched tumor cells of different origin. Interestingly, the processed and presented TDO-derived epitopes varied between different cancer cells. With respect to CD4+ TDO-reactive T cells, in vitro expanded T-cell cultures comprised a Th1 and/or a Treg phenotype. In summary, our data demonstrate that the immune modulating enzyme TDO is a target for CD8+ and CD4+ T cell responses both in healthy subjects as well as patients with cancer; notably, however, the functional phenotype of these T-cell responses differ depending on the respective conditions of the host.

In the present study, we describe forkhead box O3 (FOXO3)-specific, cytotoxic CD8+ T cells existent among peripheral-blood mononuclear cells (PBMCs) of cancer patients. FOXO3 immunogenicity appears specific, as we did not detect reactivity toward FOXO3 among T cells in healthy individuals. FOXO3 may naturally serve as a target antigen for tumor-reactive T cells as it is frequently over-expressed in cancer cells. In addition, expression of FOXO3 plays a critical role in immunosuppression mediated by tumor-associated dendritic cells (TADCs). Indeed, FOXO3-specific cytotoxic T lymphocytes (CTLs) were able to specifically recognize and kill both FOXO3-expressing cancer cells as well as dendritic cells. Thus, FOXO3 was processed and presented by HLA-A2 on the cell surface of both immune cells and cancer cells. As FOXO3 programs TADCs to become tolerogenic, FOXO3 signaling thereby comprises a significant immunosuppressive mechanism, such that FOXO3 targeting by means of specific T cells is an attractive clinical therapy to boost anticancer immunity. In addition, the natural occurrence of FOXO3-specific CTLs in the periphery suggests that these T cells hold a function in the complex network of immune regulation in cancer patients.