Targeted BRAF inhibitors: Immunological effects and combination with immunotherapy

<p>Metastatic melanoma is the most aggressive form of skin cancer, associated with a poor prognosis, and the incidence worldwide is increasing. Recently, selective mutant BRAF inhibitors and checkpoint blockade immunotherapy have advanced clinical treatment of metastatic melanoma. However, efficacy of these therapies individually is limited. Combining treatments may allow BRAF inhibition to augment immunotherapy by increasing tumour antigen availability and improving immune system targeting of tumours. The success of this approach depends upon fully elucidating immunological interactions of BRAF inhibitors, and optimizing combination strategies. To study the immunological effects of BRAF inhibitors and their combination with immunotherapy, novel murine BrafV600E Pten-/- Cdkn2a cell lines were characterized. These were found to be moderately sensitive to BRAF inhibition compared with the widely used human BRAFV600E cell lines A375 and SK-mel-5. In vitro targeted BRAF inhibition was shown to induce cell death through apoptosis, and partially reverse melanoma-mediated immunosuppression by human melanoma cell lines. Utilising subcutaneously injected syngeneic, murine BRAFV600E cell lines, the BRAF inhibitor PLX4720 was shown to decrease tumour growth in vivo. Host immune involvement in BRAF inhibitor efficacy was determined by comparing PLX4720 treatment in NOD/Scid and C57BL/6 mice. PLX4720 control of tumour growth was significantly less effective in immunocompromised mice, resulting in reduced survival advantage. These findings demonstrate that the anti-tumour effects of mutant BRAF inhibitors are partially immune dependent, although the nature of this immune involvement remains to be defined. It was further shown that BRAFV600E inhibition directly affected immune responses. In vitro, both human and murine T cell activation were boosted by low concentrations of mutant BRAF inhibitors. This was confirmed in vivo, with antigen-specific T cell proliferation significantly increased by PLX4720 treatment. The final chapters of this thesis explored the combination of active immunotherapy with targeted BRAF inhibition. A vaccine was devised that consisted of irradiated, autologous tumour cells loaded with the adjuvant α-galactosylceramide. This vaccine was shown to be effective in both prophylactic and therapeutic settings in a BRAFV600E melanoma model. Mechanistically, vaccine increased effector T cell responses and decreased frequencies of Tregs. Vaccine efficacy was CD4⁺ T cell-dependent, and did not require CD8⁺ T cells. Combination of vaccine with targeted BRAF inhibition was investigated in different settings. A combination therapy strategy was developed that achieved additive, but not synergistic benefit. Additionally, targeting specific aspects of the tumour microenvironment that may confer tumour resistance to BRAF inhibitor-mediated cell death was investigated. Both depletion of Tregs and inhibition of TNFα were explored, but did not result in a significant improvement in therapy. In summary, the studies undertaken in this thesis demonstrate that BRAF inhibitors can augment vaccine-induced T cell responses. Moreover, this research revealed the anti-tumour efficacy of BRAFV600E inhibition is partially immune dependent and can be improved by combination with active immunotherapy. These discoveries generated a combination therapy strategy with improved efficacy over single agent treatment. Further studies are needed to realise the full potential of this combination therapy approach, and achieve a synergistic benefit.</p>

Targeted BRAF inhibitors: Immunological effects and combination with immunotherapy

<p>Metastatic melanoma is the most aggressive form of skin cancer, associated with a poor prognosis, and the incidence worldwide is increasing. Recently, selective mutant BRAF inhibitors and checkpoint blockade immunotherapy have advanced clinical treatment of metastatic melanoma. However, efficacy of these therapies individually is limited. Combining treatments may allow BRAF inhibition to augment immunotherapy by increasing tumour antigen availability and improving immune system targeting of tumours. The success of this approach depends upon fully elucidating immunological interactions of BRAF inhibitors, and optimizing combination strategies. To study the immunological effects of BRAF inhibitors and their combination with immunotherapy, novel murine BrafV600E Pten-/- Cdkn2a cell lines were characterized. These were found to be moderately sensitive to BRAF inhibition compared with the widely used human BRAFV600E cell lines A375 and SK-mel-5. In vitro targeted BRAF inhibition was shown to induce cell death through apoptosis, and partially reverse melanoma-mediated immunosuppression by human melanoma cell lines. Utilising subcutaneously injected syngeneic, murine BRAFV600E cell lines, the BRAF inhibitor PLX4720 was shown to decrease tumour growth in vivo. Host immune involvement in BRAF inhibitor efficacy was determined by comparing PLX4720 treatment in NOD/Scid and C57BL/6 mice. PLX4720 control of tumour growth was significantly less effective in immunocompromised mice, resulting in reduced survival advantage. These findings demonstrate that the anti-tumour effects of mutant BRAF inhibitors are partially immune dependent, although the nature of this immune involvement remains to be defined. It was further shown that BRAFV600E inhibition directly affected immune responses. In vitro, both human and murine T cell activation were boosted by low concentrations of mutant BRAF inhibitors. This was confirmed in vivo, with antigen-specific T cell proliferation significantly increased by PLX4720 treatment. The final chapters of this thesis explored the combination of active immunotherapy with targeted BRAF inhibition. A vaccine was devised that consisted of irradiated, autologous tumour cells loaded with the adjuvant α-galactosylceramide. This vaccine was shown to be effective in both prophylactic and therapeutic settings in a BRAFV600E melanoma model. Mechanistically, vaccine increased effector T cell responses and decreased frequencies of Tregs. Vaccine efficacy was CD4⁺ T cell-dependent, and did not require CD8⁺ T cells. Combination of vaccine with targeted BRAF inhibition was investigated in different settings. A combination therapy strategy was developed that achieved additive, but not synergistic benefit. Additionally, targeting specific aspects of the tumour microenvironment that may confer tumour resistance to BRAF inhibitor-mediated cell death was investigated. Both depletion of Tregs and inhibition of TNFα were explored, but did not result in a significant improvement in therapy. In summary, the studies undertaken in this thesis demonstrate that BRAF inhibitors can augment vaccine-induced T cell responses. Moreover, this research revealed the anti-tumour efficacy of BRAFV600E inhibition is partially immune dependent and can be improved by combination with active immunotherapy. These discoveries generated a combination therapy strategy with improved efficacy over single agent treatment. Further studies are needed to realise the full potential of this combination therapy approach, and achieve a synergistic benefit.</p>

Dendritic Cell Vaccination of Glioblastoma: Road to Success or Dead End

Glioblastomas (GBM) are the most frequent and aggressive malignant primary brain tumor and remains a therapeutic challenge: even after multimodal therapy, median survival of patients is only 15 months. Dendritic cell vaccination (DCV) is an active immunotherapy that aims at inducing an antitumoral immune response. Numerous DCV trials have been performed, vaccinating hundreds of GBM patients and confirming feasibility and safety. Many of these studies reported induction of an antitumoral immune response and indicated improved survival after DCV. However, two controlled randomized trials failed to detect a survival benefit. This raises the question of whether the promising concept of DCV may not hold true or whether we are not yet realizing the full potential of this therapeutic approach. Here, we discuss the results of recent vaccination trials, relevant parameters of the vaccines themselves and of their application, and possible synergies between DCV and other therapeutic approaches targeting the immunosuppressive microenvironment of GBM.

TMOD-14. CONJOINED CLASS I AND II EPITOPES ENHANCE NEOANTIGEN-TARGETED ACTIVE IMMUNOTHERAPY

INTRODUCTION Cancer vaccines involve the administration of tumor-associated antigens into the host to generate anti-tumor T cell responses. Glioblastoma (GBM) is a disease with poor prognosis. GBM has a limited number of immunotherapeutic targets due to low mutational load, and is also highly heterogeneous; targeting a single antigen leads to antigen escape and tumor growth. METHODS VMDK mice were subcutaneously implanted with 750,000 SMA560 cells and on days 1 and 8 post implantation, mice were treated with 15 nmol of the universal helper epitope, P30, conjoined to the MHCI-restricted neoepitopes Odc1MHCI-P30 or Topbp1MHCI-P30. Human CD27 (hCD27) transgenic mice were intracranially implanted with CT2A-Odc1, followed by anti-CD27 and 15 nmol of Odc1MHCI-P30. B16.OVA or B16.F10 tumor cells were intracranially implanted in hCD27 mice and received SIINFEKL-P30 or Trp2-P30 conjoined peptides. Tumor growth, survival, or IFNγ secretion of splenic or tumor-infiltrating cells was assessed. RESULTS Unlike Odc1MHCI mixed with P30, conjoined Odc1MHCI-P30 had equivalent immunogenicity and anti-tumor efficacy to that observed with native long Odc1 peptide. Native long peptide of Topbp1 did not elicit an antitumor response, yet Topbp1MHCI-P30 caused an increase in numbers of IFNγ-secreting splenocytes and a decrease in tumor growth and similar to that seen with Odc1MHCI-P30 . Anti-CD27 treatment significantly increased numbers of IFNγ secreting splenocytes in Odc1MHCI-P30 vaccinated hCD27 mice, and the use of anti-CD27 with Odc1MHCI-P30 achieved long-term survival in 90% of tumor bearing hCD27 mice. Anti-CD27 synergized with SIINFEKL-P30 and Trp2-P30 to significantly improve survival after administration of these peptides. CONCLUSIONS Our work shows that poorly immunogenic neoantigens can be conjoined to P30 and used to generate an anti-tumor response in mouse models of GBM, and anti-tumor responses of conjoined peptides can be enhanced with anti-CD27 treatment. Together, these data demonstrate the efficacy of neoantigen vaccines for the treatment of heterogeneous GBM.

948 Final results from AIPAC: A phase IIb comparing eftilagimod alpha (a soluble LAG-3 protein) vs. placebo in combination with weekly paclitaxel in HR+ HER2- MBC

BackgroundEftilagimod alpha (efti; IMP321) is a soluble LAG-3 protein (LAG-3Ig) that binds to a subset of MHC class II molecules and mediates activation of antigen-presenting cells followed by CD8 T-cells. Weekly paclitaxel is a standard of care chemo-regimen after failure of endocrine-based therapy for metastatic breast carcinoma (MBC). AIPAC (Active Immunotherapy PAClitaxel) investigated the addition of efti to weekly paclitaxel in these patients (pts).MethodsThis placebo-controlled, double-blinded, 1:1 randomized phase IIb trial enrolled pts with measurable disease, HR+ HER2- MBC after endocrine-based therapy. Pts received paclitaxel (80 mg/m² IV on D1, D8, D15) + efti (30 mg) or placebo on D2, D16 (every 2 weeks) for up to 24 weeks following efti/placebo for up to 52 weeks. The primary endpoint (EP) was progression-free survival (RECIST1.1) by BICR. Secondary EPs included overall survival (OS), PFS (local read), overall response rate (ORR), biomarker, quality of life. Exploratory EPs included univariate/multivariate analyses.Results227 pts were randomized (Jan2017-Jul2019). All except 1 received ≥1 treatment and were included in the full analysis set [efti (n=114); placebo (n=112)]. Data cut-off was 14May2021 (min. follow-up= 22 months). Median age was 60 yrs with ECOG 0 in 61.5%. 91.6% had visceral disease. Pts were mostly endocrine resistant (84%) and partially pre-treated with CDK4/6 inhibitors (44.2%). Post-study treatment was similar. Median OS was 20.4 (95% CI: 14.3-25.1) months in the efti group vs. 17.5 (95% CI: 12.9-21.9) in the placebo group. HR was 0.88 (95%CI: 0.64-1.19; p=0.197). In predefined univariate analyses, younger pts, low baseline monocytes and luminal B showed significant/clinically meaningful improvement in OS (table 1).Efti increased PBMC/T cell (CD4/CD8) count vs. placebo, correlating with improved OS (Spearman Rho=0.6, p=0.02 for CD8 T cells). In a whole population multivariate cox regression model, increasing BMI and prior treatment with CDK4/6 were independent significant poor prognostic markers for PFS and OS.TEAEs leading to discontinuation were similar at 5.3%(efti) & 6.3%(placebo). PFS (Primary EP) and safety were reported at SABCS 2020 (Abstract#132).Abstract 948 Table 1Overall survival by subgroups at final analysisConclusionsEfti added to paclitaxel led to a non-significant 2.9 months median OS increase in HR+ HER2- MBC pts after endocrine-based therapy. Effects were significant in pts <65yrs, with low monocytes and more aggressive disease (luminal B). Efti increased circulating CD4/CD8 T cells, which significantly correlated to improved OS. Weekly paclitaxel + efti should be further investigated in MBC.Trial RegistrationThe trial identifiers are IMP321-P011 (code for sponsor), 2015-002541-63 (EudraCT) and NCT02614833 (ClinicalTrials.gov).Ethics ApprovalThe study was approved by relevant ethic committees and institutional review boards.

Effect of Prepregnancy Lymphocyte Active Immunotherapy on Unexplained Recurrent Miscarriage, Pregnancy Success Rate, and Maternal-Infant Outcome

Objective. To evaluate the effect of prepregnancy lymphocyte active immunotherapy on unexplained recurrent miscarriage, pregnancy success rate, and maternal-infant outcome. Methods. A total of 124 patients with recurrent miscarriage admitted to our hospital from January 2018 to December 2020 were selected as the research objects and divided into the experimental group and the control group according to the random number table method, with 62 patients in each group. The experimental group was treated with lymphocyte active immunotherapy, and the control group was given conventional treatment. The pregnancy success rate, estrogen indexes, hemorheology indexes, and psychological state of the two groups were compared. Results. The experimental group garnered a notably higher pregnancy success rate and a prominently lower miscarriage rate than the control group ( P < 0.05 ). Better results of self-rating anxiety scale (SAS) and self-rating depression scale (SDS) were observed in the experimental group, as compared to the control group ( P < 0.05 ). The experimental group yielded more desirable results in terms of treatment satisfaction, estrogen indexes, and hemorheology indexes in comparison with the control group ( P < 0.05 ). Conclusion. The use of lymphocyte active immunotherapy for patients with unexplained recurrent miscarriage can significantly increase the pregnancy success rate, optimize the maternal-infant outcome, drive down the miscarriage rate, and ameliorate the patient’s estrogen levels and hemorheology indicators, which is worthy of promotion and application in clinical practice.

Cancer-Associated Glycosphingolipids as Tumor Markers and Targets for Cancer Immunotherapy

Aberrant expression of glycosphingolipids is a hallmark of cancer cells and is associated with their malignant properties. Disialylated gangliosides GD2 and GD3 are considered as markers of neuroectoderm origin in tumors, whereas fucosyl-GM1 is expressed in very few normal tissues but overexpressed in a variety of cancers, especially in small cell lung carcinoma. These gangliosides are absent in most normal adult tissues, making them targets of interest in immuno-oncology. Passive and active immunotherapy strategies have been developed, and have shown promising results in clinical trials. In this review, we summarized the current knowledge on GD2, GD3, and fucosyl-GM1 expression in health and cancer, their biosynthesis pathways in the Golgi apparatus, and their biological roles. We described how their overexpression can affect intracellular signaling pathways, increasing the malignant phenotypes of cancer cells, including their metastatic potential and invasiveness. Finally, the different strategies used to target these tumor-associated gangliosides for immunotherapy were discussed, including the use and development of monoclonal antibodies, vaccines, immune system modulators, and immune effector-cell therapy, with a special focus on adoptive cellular therapy with T cells engineered to express chimeric antigen receptors.

Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients

Given the emergence of SARS-CoV-2 virus as a life-threatening pandemic, identification of immunodominant epitopes of the viral structural proteins, particularly the nucleocapsid (NP) protein and receptor binding domain (RBD) of spike protein, is important to determine targets for immunotherapy and diagnosis. In this study, epitope screening was performed using a panel of overlapping peptides spanning the entire sequences of the RBD and NP proteins of SARS-CoV-2 in the sera from 66 COVID-19 patients and 23 healthy subjects by enzyme-linked immunosorbent assay (ELISA). Also, three non-overlapping peptides belonging to the S2 domain of spike protein were assessed. Our results showed that while reactivity of patients’ sera with reduced recombinant RBD protein was significantly lower than the native form of RBD (p<0.001), no significant differences were observed for reactivity of patients’ sera with reduced and non-reduced NP protein. Pepscan analysis revealed weak to moderate reactivity towards different RBD peptide pools, which was more focused on peptides encompassing aa 181-223 of RBD. NP peptides, however, displayed strong reactivity with a single peptide covering aa 151-170. These findings were confirmed by peptide depletion experiments using both ELISA and Western blotting. Altogether, our data suggest the involvement of mostly conformational disulfide bond-dependent immunodominant epitopes in RBD-specific antibody response, while the IgG response to NP is dominated by linear epitopes. Identification of antigenic epitope in NP and RBD of SARS-CoV-2 could provide important advances for the development of passive and active immunotherapy as well as diagnostic tools for the control of COVID-19 infection.