Patterns of immune-cell infiltration in murine models of melanoma: roles of antigen and tissue site in creating inflamed tumorsAbstractImmune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/β-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/β-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes. |
An HER2 DNA vaccine with evolution-selected amino acid substitutions reveals a fundamental principle for cancer vaccine formulation in HER2 transgenic miceAbstractEnhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general. |
Pembrolizumab as first-line treatment for metastatic uveal melanomaAbstractBackgroundNo standard treatment has been defined for metastatic uveal melanoma (mUM). Although clinical trials testing Nivolumab/Pembrolizumab for cutaneous melanoma did not include mUM, anti PD-1 agents are commonly used for this disease. Patients and methodsIn this prospective observational cohort single arm study, we investigated efficacy and safety of Pembrolizumab as first-line therapy for mUM. The efficacy was evaluated in terms of progression-free survival (PFS), response rate and overall survival (OS). Toxicity was also assessed. ResultsSeventeen patients were enrolled. A median of 8 cycles were administered (range 2–28). Two patients achieved partial response (11.7%), 6 a disease stabilization (35.3%), whereas 9 (53%) had a progression. No complete response was observed. PFS of the overall population was 3.8 months. PFS was 9.7 months for patients with an interval higher than 5 years from diagnosis of primary tumor to metastatic disease and 2.6 months for patients with an interval lower than 5 years [p = 0.039, HR 0.2865 (95% CI 0.0869–0.9443)]. Median OS was not reached. The two responding patients were still on treatment with Pembrolizumab at the time of data analysis. Survival was 12.8 months for patients with clinical benefit, while OS for progressive patients was 3.1 months. PD-L1 expression and genomic abnormalities predictive of relapse after diagnosis of primary tumor were not associated with PFS. Toxicity was mild, without grade 3–4 side effects. ConclusionsThe efficacy of Pembrolizumab does not seem particularly different when compared to other agents for mUM, but responding patients had a remarkable disease control. |
Impact of combination immunochemotherapies on progression of 4NQO-induced murine oral squamous cell carcinomaAbstractAdvanced oral squamous cell carcinomas (OSCC) have limited therapeutic options. Although immune therapies are emerging as a potentially effective alternative or adjunct to chemotherapies, the therapeutic efficacy of combination immune chemotherapies has yet to be determined. Using a 4-nitroquinolone-N-oxide (4NQO) orthotopic model of OSCC in immunocompetent mice, we evaluated the therapeutic efficacy of single- and combined-agent treatment with a poly-epitope tumor peptide vaccine, cisplatin and/or an A2AR inhibitor, ZM241385. The monotherapies or their combinations resulted in a partial inhibition of tumor growth and, in some cases, a significant but transient upregulation of systemic anti-tumor CD8+ T cell responses. These responses eroded in the face of expanding immunoregulatory cell populations at later stages of tumor progression. Our findings support the need for the further development of combinatorial therapeutic approaches that could more effectively silence dominant immune inhibitory pathways operating in OSCC and provide novel, more beneficial treatment options for this tumor. |
First-in-human phase 1 dose-escalating trial of G305 in patients with advanced solid tumors expressing NY-ESO-1AbstractHuman tumor cells express antigens that serve as targets for the host cellular immune system. This phase 1 dose-escalating study was conducted to assess safety and tolerability of G305, a recombinant NY-ESO-1 protein vaccine mixed with glucopyranosyl lipid A (GLA), a synthetic TLR4 agonist adjuvant, in a stable emulsion (SE). Twelve patients with solid tumors expressing NY-ESO-1 were treated using a 3 + 3 design. The NY-ESO-1 dose was fixed at 250 µg, while GLA-SE was increased from 2 to 10 µg. Safety, immunogenicity, and clinical responses were assessed prior to, during, and at the end of therapy. G305 was safe and immunogenic at all doses. All related AEs were Grade 1 or 2, with injection site soreness as the most commonly reported event (100%). Overall, 75% of patients developed antibody response to NY-ESO-1, including six patients with increased antibody titer ( ≥ 4-fold rise) and three patients with seroconversion from negative (titer < 100) to positive (titer ≥ 100). CD4 T-cell responses were observed in 44.4% of patients; 33.3% were new responses and 1 was boosted ( ≥ 2-fold rise). Following treatment, 8 of 12 patients had stable disease for 3 months or more; at the end of 1 year, three patients had stable disease and nine patients were alive. G305 is a potent immunotherapeutic agent that can stimulate NY-ESO-1-specific antibody and T-cell responses. The vaccine was safe at all doses of GLA-SE (2–10 µg) and showed potential clinical benefit in this population of patients. |
PLAC1: biology and potential application in cancer immunotherapyAbstractThe emergence of immunotherapy has revolutionized medical oncology with unprecedented advances in cancer treatment over the past two decades. However, a major obstacle in cancer immunotherapy is identifying appropriate tumor-specific antigens to make targeted therapy achievable with fewer normal cells being impaired. The similarity between placentation and tumor development and growth has inspired many investigators to discover antigens for effective immunotherapy of cancers. Placenta-specific 1 (PLAC1) is one of the recently discovered placental antigens with limited normal tissue expression and fundamental roles in placental function and development. There is a growing body of evidence showing that PLAC1 is frequently activated in a wide variety of cancer types and promotes cancer progression. Based on the restricted expression of PLAC1 in testis, placenta and a wide variety of cancers, we have designated this molecule with new terminology, cancer–testis–placenta (CTP) antigen, a feature that PLAC1 shares with many other cancer testis antigens. Recent reports from our lab provide compelling evidence on the preferential expression of PLAC1 in prostate cancer and its potential utility in prostate cancer immunotherapy. PLAC1 may be regarded as a potential CTP antigen for targeted cancer immunotherapy based on the available data on its promoting function in cancer development and also its expression in cancers of different histological origin. In this review, we will summarize current data on PLAC1 with emphasis on its association with cancer development and immunotherapy. |
Checkpoint blockade immunotherapy enhances the frequency and effector function of murine tumor-infiltrating T cells but does not alter TCRβ diversityAbstractCheckpoint blockade immunotherapy is now a first-line treatment option for patients with melanoma. Despite achieving objective responses in about half of patients, the exact immune mechanisms elicited and those required for therapeutic success have not been clearly identified. Insight into these mechanisms is key for improving outcomes in a broader range of cancer patients. We used a murine melanoma model to track responses by different subsets of tumor-infiltrating lymphocytes (TIL) during checkpoint blockade immunotherapy. Tumors from treated mice had increased frequencies of both CD4+ and CD8+ T cells, which also showed evidence of functional reinvigoration and elevated effector cytokine production after immunotherapy. We predicted that increased T cell numbers and function within tumors reflected either infiltration by new T cells or clonal expansion by a few high-affinity tumor-reactive T cells. To address this, we compared TIL diversity before and after immunotherapy by sequencing the complementarity determining region 3 (CDR3) of all T cell receptor beta (TCRβ) genes. While checkpoint blockade effectively slowed tumor progression and increased T cell frequencies, the diversity of intratumoral T cells remained stable. This was true when analyzing total T cells and when focusing on smaller subsets of effector CD4+ and CD8+ TIL as well as regulatory T cells. Our study suggests that checkpoint blockade immunotherapy does not broaden the T cell repertoire within murine melanoma tumors, but rather expands existing T cell populations and enhances effector capabilities. |
Combination of denosumab and immune checkpoint inhibition: experience in 29 patients with metastatic melanoma and bone metastasesAbstractBackgroundPD-1 inhibition (PD-1i) is the standard of care in melanoma and other malignancies. In patients with bone metastases of solid tumors, the monoclonal antibody denosumab directed against RANKL is approved for the prevention of skeletal-related events. However, RANKL is not only relevant in osteoclastogenesis, but also has immunological effects. Hence, we aimed at investigating, whether the combination of PD-1i and denosumab produces synergistic effects in metastatic melanoma treatment. MethodsWe retrospectively collected and analyzed clinical data of metastatic melanoma patients with bone metastases, who received PD-1i and denosumab therapy. Results29 patients were identified with a median age of 60.7 years: 20 were male and 9 were female. 20 patients (69%) were in stage IV M1c and 9 (31%) in stage IV M1d; 52% had an increased serum LDH. 24 patients (83%) received PD-1i as first-line therapy and five patients (17%) as second- or third-line therapy. 13 patients received the triple combination nivolumab, ipilimumab and denosumab (N + I+D), 16 patients received PD-1i and denosumab (PD-1i + D). Within a median follow-up time of 19.8 months, 17 patients progressed with a median time to progression of 6 months. The objective response rate was 54% in the N + I + D group and 50% in the PD-1i + D group. Recalcification of bone metastases was radiologically observed in 18 (62%) patients. No unexpected treatment-related adverse events emerged. ConclusionsThe combination therapy of metastatic melanoma with PD-1i and denosumab was feasible without unexpected safety issues and showed a promising efficacy signal. Further investigation in prospective studies is needed. |
Clinicopathological implications of TIM3 + tumor-infiltrating lymphocytes and the miR-455-5p/Galectin-9 axis in skull base chordoma patientsAbstractChordoma is difficult to eradicate due to high local recurrence rates. The immune microenvironment is closely associated with tumor prognosis; however, its role in skull base chordoma is unknown. The expression of Galectin-9 (Gal9) and tumor-infiltrating lymphocyte (TIL) markers was assessed by immunohistochemistry. Kaplan–Meier and multivariate Cox analyses were used to assessing local recurrence-free survival (LRFS) and overall survival (OS) of patients. MiR-455-5p was identified as a regulator of Gal9 expression. Immunopositivity for Gal9 was associated with tumor invasion (p = 0.019), Karnofsky performance status (KPS) score (p = 0.017), and total TIL count (p < 0.001); downregulation of miR-455-5p was correlated with tumor invasion (p = 0.017) and poor prognosis; and the T-cell immunoglobulin and mucin-domain 3 (TIM3)+ TIL count was associated with chordoma invasion (p = 0.010) and KPS score (p = 0.037). Furthermore, multivariate analysis indicated that only TIM3+ TIL density was an independent prognostic factor for LRFS (p = 0.010) and OS (p = 0.016). These results can be used to predict clinical outcome and provide a basis for immune therapy in skull base chordoma patients. |
Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancerAbstractWe evaluated the analytical and clinical performance of a novel circulating tumor cell (CTC)-based blood test for determination of programmed death ligand 1 (PD-L1) protein expression status in real time in treatment-naïve non-small cell lung cancer (NSCLC) patients. CTCs were detected in 86% of patients with NSCLC (I–IV) at the time of diagnosis, with a 67% PD-L1 positivity rate (≥ 1 PDL + CTC). Among 33 NSCLC patients with PD-L1 results available via both tissue immunohistochemistry (IHC) and CTC assays, 78.9% were positive according to both methods. The CTC test identified an additional ten cases that were positive for PD-L1 expression but that tested negative via IHC analysis. Detection of higher PD-L1 expression on CTCs compared to that in the corresponding tissue was concordant with data obtained using other platforms in previously treated patients. The concordance in PD-L1 expression between tissue and CTCs was approximately 57%, which is higher than that reported by others. In summary, evaluation of PD-L1 protein expression status on CTCs isolated from NSCLC patients is feasible. PD-L1 expression status on CTCs can be determined serially during the disease course, thus overcoming the myriad challenges associated with tissue analysis. |
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