Therapeutic cancer vaccination against mutant calreticulin in myeloproliferative neoplasms induces expansion of specific T cells in the periphery but specific T cells fail to enrich in the bone marrow.

Background: Therapeutic cancer vaccination against mutant calreticulin (CALR) in patients with CALR-mutant (CALRmut) myeloproliferative neoplasms (MPN) induces strong T-cell responses against mutant CALR yet fails to demonstrate clinical activity. Infiltration of tumor specific T cells into the tumor microenvironment is needed to attain a clinical response to therapeutic cancer vaccination. Aim: Determine if CALRmut specific T cells isolated from vaccinated patients enrich in the bone marrow upon completion of vaccination and explore possible explanations for the lack of enrichment. Methods: CALRmut specific T cells from four of ten vaccinated patients were expanded, enriched, and analyzed by T-cell receptor sequencing (TCRSeq). The TCRs identified were used as fingerprints of CALRmut specific T cells. Bone marrow aspirations from the four patients were acquired at baseline and at the end of trial. T cells were enriched from the bone marrow aspirations and analyzed by TCRSeq to identify the presence and fraction of CALRmut specific T cells at the two different time points. In silico calculations were performed to calculate the ratio between transformed cells and effector cells in patients with CALRmut MPN. Results: The fraction of CALRmut specific T cells in the bone marrow did not increase upon completion of the vaccination trial. In general, the T cell repertoire in the bone marrow remains relatively constant through the vaccination trial. The enriched and expanded CALRmut specific T cells recognize peripheral blood autologous CALRmut cells. In silico analyses demonstrate a high imbalance in the fraction of CALRmut cells and CALRmut specific effector T-cells in peripheral blood. Conclusion: CALRmut specific T cells do not enrich in the bone marrow after therapeutic cancer peptide vaccination against mutant CALR. The specific T cells recognize autologous peripheral blood derived CALRmut cells. In silico analyses demonstrate a high imbalance between the number of transformed cells and CALRmut specific effector T-cells in the periphery. We suggest that the high burden of transformed cells in the periphery compared to the number of effector cells could impact the ability of specific T cells to enrich in the bone marrow.

Anti-PD-L1/PD-L2 therapeutic vaccination in untreated chronic lymphocytic leukemia patients with unmutated IgHV.

Chronic lymphocytic leukemia (CLL) patients with unmutated immunoglobulin heavy chain (IgHV) are at risk of early disease progression compared to patients with mutated IgHV. As a preventive strategy, we treated 19 previously untreated CLL patients with unmutated IgHV in a phase 1/2 trial (clinicaltrials.gov, NCT03939234) exploring the efficacy and toxicity of a therapeutic cancer vaccine containing peptides derived from programmed death ligand 1 (PD-L1) and ligand 2 (PD-L2), hoping to restore immunological control of the disease. According to the International Workshop on Chronic lymphocytic Leukemia (iwCLL) response criteria, no patients obtained a response; however, during follow-up, one patient had complete normalization of the peripheral lymphocyte count and remained in biochemical remission after a follow-up time of 15 months. At the end of treatment, one patient had progressed, and 17 patients had stable disease. During follow-up with a median time of 23.5 months since inclusion, seven patients had progressed, and eight patients had stable disease. The median time to first treatment (TTFT) from diagnosis was 90.3 months with a median follow-up time of 50.1 months. This apparent favorable outcome in TTFT needs to be investigated in a randomized setting, as our population may have been biased. More than 80% of patients obtained vaccine-specific immune responses, confirming the immunogenicity of the vaccine. The vaccine was generally well tolerated with only grade I-II adverse events. Although there were some signs of clinical effects, the vaccine seems to be insufficient as monotherapy in CLL, possibly due to a high tumor burden. The efficacy of the vaccine should preferably be tested in combination with novel targeted therapies or as a consolidating treatment.

Peptide Vaccination Against PD-L1 With IO103 a Novel Immune Modulatory Vaccine in Multiple Myeloma: A Phase I First-in-Human Trial.

Background: Immune checkpoint blockade with monoclonal antibodies targeting programmed death 1 (PD-1) and its ligand PD-L1 has played a major role in the rise of cancer immune therapy. We have identified naturally occurring self-reactive T cells specific to PD-L1 in both healthy donors and cancer patients. Stimulation with a PD-L1 peptide (IO103), activates these cells to exhibit inflammatory and anti-regulatory functions that include cytotoxicity against PD-L1-expressing target cells. This prompted the initiation of the present first-in-human study of vaccination with IO103, registered at clinicaltrials.org (NCT03042793). Methods: Ten patients with multiple myeloma who were up to 6 months after high dose chemotherapy with autologous stem cell support, were enrolled. Subcutaneous vaccinations with IO103 with the adjuvant Montanide ISA 51 was given up to fifteen times during 1 year. Safety was assessed by the common toxicity criteria for adverse events (CTCAE). Immunogenicity of the vaccine was evaluated using IFNγ enzyme linked immunospot and intracellular cytokine staining on blood and skin infiltrating lymphocytes from sites of delayed-type hypersensitivity. The clinical course was described. Results: All adverse reactions to the PD-L1 vaccine were below CTCAE grade 3, and most were grade 1-2 injection site reactions. The total rate of adverse events was as expected for the population. All patients exhibited peptide specific immune responses in peripheral blood mononuclear cells and in skin-infiltrating lymphocytes after a delayed-type hypersensitivity test. The clinical course was as expected for the population. Three of 10 patients had improvements of responses which coincided with the vaccinations. Conclusion: Vaccination against PD-L1 was associated with low toxicity and high immunogenicity. This study has prompted the initiation of later phase trials to assess the vaccines efficacy. Clinical Trial Registration: clinicaltrials.org, identifier NCT03042793.

Arginase-1-based vaccination against the tumor microenvironment: the identification of an optimal T-cell epitope.

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.

The inhibitory checkpoint, PD-L2, is a target for effector T cells: Novel possibilities for immune therapy.

Cell surface molecules of the B7/CD28 family play an important role in T-cell activation and tolerance. The relevance of the PD-1/PD-L1 pathway in cancer has been extensively studied whereas PD-L2 has received less attention. However, recently the expression of PD-L2 was described to be independently associated with clinical response in anti-PD1-treated cancer patients. Here, we investigated whether PD-L2 might represent a natural target that induces specific T cells. We identified spontaneous specific T-cell reactivity against two epitopes located in the signal peptide of PD-L2 from samples from patients with cancer as well as healthy individuals ex vivo. We characterized both CD8+ and CD4+ PD-L2-specific T cells. Interestingly, the epitope in PD-L2 that elicited the strongest response was equivalent to a potent HLA-A2-restricted epitope in PD-L1. Importantly, PD-L1-specific and PD-L2-specific T cells did not cross-react; therefore, they represent different T-cell antigens. Moreover, PD-L2-specific T cells reacted to autologous target cells depending on PD-L2 expression. These results suggested that activating PD-L2 specific T cells (e.g., by vaccination) might be an attractive strategy for anti-cancer immunotherapy. Accordingly, PD-L2 specific T cells can directly support anti-cancer immunity by killing of target cells, as well as, indirectly, by releasing pro-inflammatory cytokines at the microenvironment in response to PD-L2-expressing immune supressive cells.

PD-L1-specific T cells.

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.

Peptide vaccination against multiple myeloma using peptides derived from anti-apoptotic proteins: a phase I trial.

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.

Tryptophan 2,3-dioxygenase (TDO)-reactive T cells differ in their functional characteristics in health and cancer.

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.

Spontaneous presence of FOXO3-specific T cells in cancer patients.

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.