Innovative therapy, monoclonal antibodies, and beyond: Highlights from the eighth annual meeting.

The eighth annual conference of "Innovative therapy, monoclonal antibodies, and beyond" was held in Milan on Jan. 26, 2018, and hosted by Fondazione IRCCS-Istituto Nazionale dei Tumori (Fondazione IRCCS INT). The conference was divided into two main scientific sessions, of i) pre-clinical assays and novel biotargets, and ii) clinical translation, as well as a third session of presentations from young investigators, which focused on recent achievements within Fondazione IRCCS INT on immunotherapy and targeted therapies. Presentations in the first session addressed the issue of cancer immunotherapy activity with respect to tumor heterogeneity, with key topics addressing: 1) tumor heterogeneity and targeted therapy, with the definition of the evolutionary Index as an indicator of tumor heterogeneity in both space and time; 2) the analysis of cancer evolution, with the introduction of the TRACERx Consortium-a multi-million pound UK research project focused on non-small cell lung cancer (NSCLC); 3) the use of anti-estrogen agents to boost immune recognition of breast cancer cells; and 4) the high degree of functional plasticity within the NK cell repertoire, including the expansion of adaptive NK cells following viral challenges. The second session addressed: 1) the effectiveness of radiotherapy to enhance the proportion of patients responsive to immune-checkpoint blockers (ICBs); 2) the use of MDSC scores in selecting melanoma patients with high probability to be responsive to ICBs; and 3) the relevance of the gut microbiome as a predictive factor, and the potential of its perturbation in increasing the immune response rate to ICBs. Overall, a picture emerged of tumor heterogeneity as the main limitation that impairs the effectiveness of anti-cancer therapies. Thus, the choice of a specific therapy based on reproducible and selective predictive biomarkers is an urgent unmet clinical need that should be addressed in order to increase the proportion of long-term responding patients and to improve the sustainability of novel drugs.

Safety and immunogenicity of the PRAME cancer immunotherapeutic in metastatic melanoma: results of a phase I dose escalation study.

PURPOSE: The PRAME tumour antigen is expressed in several tumour types but in few normal adult tissues. A dose-escalation phase I/II study (NCT01149343) assessed the safety, immunogenicity and clinical activity of the PRAME immunotherapeutic (recombinant PRAME protein (recPRAME) with the AS15 immunostimulant) in patients with advanced melanoma. Here, we report the phase I dose-escalation study segment. PATIENTS AND METHODS: Patients with stage IV PRAME-positive melanoma were enrolled to 3 consecutive cohorts to receive up to 24 intramuscular injections of the PRAME immunotherapeutic. The RecPRAME dose was 20, 100 or 500 µg in cohorts 1, 2 and 3, respectively, with a fixed dose of AS15. Adverse events (AEs), including predefined dose-limiting toxicity (DLT) and the anti-PRAME humoral response (ELISA), were coprimary end points. Cellular immune responses were evaluated using in vitro assays. RESULTS: 66 patients were treated (20, 24 and 22 in the respective cohorts). AEs considered by the investigator to be causally related were mostly grade 1 or 2 injection site symptoms, fatigue, chills, fever and headache. Two DLTs (grade 3 brain oedema and proteinuria) were recorded in two patients in two cohorts (cohorts 2 and 3). All patients had detectable anti-PRAME antibodies after four immunisations. Percentages of patients with predefined PRAME-specific-CD4+T-cell responses after four immunisations were similar in each cohort. No CD8+ T-cell responses were detected. CONCLUSIONS: The PRAME immunotherapeutic had an acceptable safety profile and induced similar anti-PRAME-specific humoral and cellular immune responses in all cohorts. As per protocol, the phase II study segment was initiated to further evaluate the 500 µg PRAME immunotherapeutic dose. TRIAL REGISTRATION NUMBER: NCT01149343, Results.

Melanoma cells homing to the brain: an in vitro model.

We developed an in vitro contact through-feet blood brain barrier (BBB) model built using type IV collagen, rat astrocytes, and human umbilical vein endothelial cells (HUVECs) cocultured through Transwell porous polycarbonate membrane. The contact between astrocytes and HUVECs was demonstrated by electron microscopy: astrocytes endfeet pass through the 8.0 µm pores inducing HUVECs to assume a cerebral phenotype. Using this model we evaluated transmigration of melanoma cells from two different patients (M1 and M2) selected among seven melanoma primary cultures. M2 cells showed a statistically significant higher capability to pass across the in vitro BBB model, compared to M1. Expression of adhesion molecules was evaluated by flow cytometry: a statistically significant increased expression of MCAM, αvß3, and CD49b was detected in M1. PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1. Specifically, data suggest that MMP2 and MMP9 could be directly involved in BBB permeability and that brain invasion by melanoma cells could be related to the overexpression of many MMPs. Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

Dose-finding/phase II trial: bevacizumab, immunotherapy, and chemotherapy (BIC) in metastatic renal cell cancer (mRCC). Antitumor effects and variations of circulating T regulatory cells (Treg).

The aim of this study was to explore the efficacy and toxicities of a combined regimen of bevacizumab plus immunotherapy and chemotherapy (BIC) and the circulating T regulatory cells (Treg) in metastatic renal cell cancer (mRCC). Nephrectomized mRCC patients were enrolled into a multicenter single-arm dose-finding study with five escalated dose levels of chemotherapy with intravenous gemcitabine and 5-fluorouracil associated with fixed intravenous doses of bevacizumab, subcutaneous low doses of interleukin-2, and interferon-α-2a. An expanded cohort (phase II study) was treated at the recommended dose for additional safety and efficacy information according to minimax Simon two-stage design. Blood samples for Treg were collected and evaluated by fluorescence-activated cell sorting (FACS) analysis on cycle 1. Fifty-one patients were entered to receive one of five dose levels. Median age was 58 years (male 67 %, pretreated 49 %): 15 patients were low risk according to Memorial Sloan-Kettering Cancer Center (MSKCC) criteria, while 27 and nine were respectively intermediate- and high-risk patients. More frequent grade 3 and 4 toxicities included nonfebrile neutropenia, thrombocytopenia, and fever. Among patients evaluable for response (49), 29.5 % had partial response and 37 % stable disease. Overall median time to progression and median overall survival were 8.8 and 22.67 months, respectively. We observed a rapid increase in the percentage of Treg after immunotherapy and a reduction after bevacizumab only in patient who obtained a partial response or stable disease. The BIC was feasible, well tolerated, and shown interesting activity. Further studies are needed to explore if Treg could have a role in clinical response in mRCC treated with bevacizumab.

Adaptive immune contexture at the tumour site and downmodulation of circulating myeloid-derived suppressor cells in the response of solitary fibrous tumour patients to anti-angiogenic therapy.

BACKGROUND: Host immunity is emerging as a key player in the prognosis and response to treatment of cancer patients. However, the impact of the immune system and its modulation by therapies are unknown in rare soft tissue sarcomas such as solitary fibrous tumours (SFTs), whose management in the advanced forms includes anti-angiogenic therapy. Here, we studied the in situ and systemic immune status of advanced SFT patients and the effects of sunitinib malate (SM) in association with the clinical efficacy. METHODS: Immune contexture of SFTs was assessed by immunohistochemistry in lesions from untreated or SM-treated patients. Frequency of circulating myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and T-cell functions was assessed ex vivo in SFT patients prior and during anti-angiogenic therapy. Patients with long-term tumour control were included to correlate immune profiles and clinical responses. RESULTS: Anti-angiogenic naïve SFT lesions were heavily infiltrated by CD163(+)CD14(+)CD68(-) and CD163(+)CD14(-)CD68(-) myeloid cells but devoid of T cells. Conversely, post-SM tumours acquired a new subset of CD68(+)CD14(+) myeloid cells and displayed traits of an on-going adaptive immunity, strongly enriched in activated CD8(+) and CD4(+) T cells. These changes at the tumour site paralleled the alleviation of systemic immunosuppression and the drop in the frequency of circulating monocytic MDSCs (mMDSCs) and granulocytic MDSCs (gMDSCs). Rebound in the number of mMDSCs, but not of gMDSCs occurred at disease progression, and a reduced percentages of mMDSCs, comparable to those found in healthy donors (HDs), endured only in the SM-responsive patients. CONCLUSIONS: The immune contexture of SFT patients is heavily involved in anti-angiogenic therapy and it could be exploited to achieve more durable disease control through immune-based combination strategies.

Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells.

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl-L-cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition.

Tumour-released exosomes and their implications in cancer immunity.

Tumour cells release vesicular structures, defined as microvesicles or exosomes, carrying a large array of proteins from their originating cell. The expression of antigenic molecules recognized by T cells has originally suggested a role for these organelles as a cell-free antigen source for anticancer vaccines. However, recent evidence shows that tumour exosomes may also exert a broad array of detrimental effects on the immune system, ranging from apoptosis in activated antitumour T cells to impairment of monocyte differentiation into dendritic cells and induction of myeloid suppressive cells. Immunosuppressive exosomes of tumour origin can be found in neoplastic lesions and sera from cancer patients, implying a potential role of this pathway in in vivo tumour progression. Through the expression of molecules involved in angiogenesis promotion, stromal remodelling, delivery of signalling pathways through growth factor/receptor transfer, chemoresistance and genetic intercellular exchange, tumour exosomes could represent a versatile tool for moulding host environment. Hence, their secretion by neoplastic cells may in the future become a novel pathway to target for therapeutic intervention in cancer patients.

Opposite immune functions of GM-CSF administered as vaccine adjuvant in cancer patients.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been and is still widely used as an adjuvant in clinical trials of vaccination with autologous tumor cells, peptides and/or dendritic cells in a variety of human neoplasms. This cytokine was administered either as product of gene-transduced tumor cells or as recombinant protein together with the vaccine given subcutaneously or intradermally. Results of these trials were heterogeneous in terms of induction of vaccine-specific immune response and of clinical response. Though in some of these studies GM-CSF appeared to help in generating an immune response, in others no effect or even a suppressive effect was reported. Here, we review the literature dealing with the immune adjuvant activity of GM-CSF both in animal models and clinical trials. As a consequence of such analysis, we conclude that GM-CSF may increase the vaccine-induced immune response when administered repeatedly at relatively low doses (range 40-80 microg for 1-5 days) whereas an opposite effect was often reported at dosages of 100-500 microg. The potential mechanisms of the GM-CSF-mediated immune suppression are discussed at the light of studies describing the activation and expansion of myeloid suppressor cells by endogenous tumor-derived or exogenous GM-CSF.

Vaccination of patients with solid tumours.

The molecular characterisation of human tumour antigens recognised by T cells has provided new impetus for immunisation of patients bearing tumours expressing well-defined antigens. After evaluating the immunogenicity of the new, molecularly characterised antigens in vitro, several clinical studies were conducted to assess the in vivo immunogenicity and the clinical efficacy of vaccines including these antigens. The findings generated by trials based on the administration of peptides or DNA-encoding antigens are discussed to highlight the limits of this therapeutic approach; however, this approach has resulted in some complete and durable regressions, although still in a unsatisfactory small number of cases (5-25%). The recent use of dendritic cells loaded ex vivo with tumour antigens suggests that a high frequency of tumour-specific immune responses can be achieved. Possible means of overcoming the clinical limits and improving the outcome of previous studies are also discussed.

Cytotoxic T-lymphocyte responses in melanoma through in vitro stimulation with the Melan-A peptide analogue A27L: a qualitative analysis.

Modifications in tumour antigen-derived epitopes that stabilize the major histocompatibility complex (MHC)-peptide complex result in enhanced stimulatory capacity and improved immunogenicity of the altered peptide. These epitope analogues are attractive candidates for the development of peptide-based vaccine trials. Any modification, however, in tumour antigens may induce T-cell responses that could either fail to react against the naturally occurring peptides or represent only a subset of the total antigen-specific repertoire. In the present study, we performed a critical analysis of the ability of cytotoxic T-lymphocyte (CTL) clones, derived from two melanoma patients through stimulation with the A27L peptide analogue, to cross-react with the naturally processed Melan-A/MART-1 (Melan-A) peptides in terms of T-cell receptor (TCR) affinity, functional avidity and fine antigen specificity. We found that all the A27L-specific clones analysed possessed a very low avidity for the natural Melan-A peptides, and that their binding affinity for human leukocyte antigen (HLA) tetramers complexed with both the modified and the natural Melan-A peptides did not strictly correlate with their functional avidity. We also observed that these clones were able to cross-recognize both natural Melan-A peptides in one patient, but only one peptide in the second patient. We discuss the capability of the A27L peptide analogue to stimulate all the available Melan-A-specific repertoire.

Tumor necrosis factor-alpha induces coordinated changes in major histocompatibility class I presentation pathway, resulting in increased stability of class I complexes at the cell surface.

It is demonstrated that similar to interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) induces coordinated changes at different steps of the major histocompatibility complex (MHC) class I processing and presentation pathway in nonprofessional antigen-presenting cells (APCs). TNF-alpha up-regulates the expression of 3 catalytic immunoproteasome subunits--LMP2, LMP7, and MECL-1--the immunomodulatory proteasome activator PA28 alpha, the TAP1/TAP2 heterodimer, and the total pool of MHC class I heavy chain. It was also found that in TNF-alpha--treated cells, MHC class I molecules reconstitute more rapidly and have an increased average half-life at the cell surface. Biochemical changes induced by TNF-alpha in the MHC class I pathway were translated into increased sensitivity of TNF-alpha--treated targets to lysis by CD8(+) cytotoxic T cells, demonstrating improved presentation of at least certain endogenously processed MHC class I--restricted peptide epitopes. Significantly, it was demonstrated that the effects of TNF-alpha observed in this experimental system were not mediated through the induction of IFN-gamma. It appears to be likely that TNF-alpha--mediated effects on MHC class I processing and presentation do not involve any intermediate messengers. Collectively, these data demonstrate the existence of yet another biologic activity exerted by TNF-alpha, namely its capacity to act as a coordinated multi-step modulator of the MHC class I pathway of antigen processing and presentation. These results suggest that TNF-alpha may be useful when a concerted up-regulation of the MHC class I presentation machinery is required but cannot be achieved by IFN-gamma. (Blood. 2001;98:1108-1115)

Simultaneous transduction of B7-1 and IL-2 genes into human melanoma cells to be used as vaccine: enhancement of stimulatory activity for autologous and allogeneic lymphocytes.

In order to construct an immunogenic cellular vaccine, we transduced three HLA-A*0201 human melanoma lines, selected for expression of classes I and II HLA, adhesion molecules and the T cell-defined melanoma antigens Melan/MART-1, gp100 and tyrosinase, with both interleukin-2 (IL-2) and B7-1 genes by the use of a polycistronic retroviral vector. The lines were selected to share only the HLA-A*0201 allele to avoid generation of strong alloreactivity in case of their multiple in vivo use in HLA-A*0201 + patients. Phenotypic and functional analysis of B7-1-IL2 transduced melanoma lines in comparison with B7-1 transduced and/or parental untransduced counterparts were then carried out. Tumor cells expressing either B7-1 or both genes did not change their original antigenic profile. From a functional point of view, expression of both genes in melanoma lines: (1) improved the response of anti-melanoma cytotoxic T lymphocytes (CTL) over singly transduced or untransduced melanoma cells when subthreshold levels of MHC-peptide complexes were expressed by melanoma cells; (2) conferred a distinct advantage in the ability to stimulate cytotoxicity and interferon-gamma release by autologous and/or HLA-A*0201-compatible allogeneic lymphocytes; (3) allowed the generation of a high number of specific CTL by in vitro stimulation of lymphocytes of HLA-A*0201-melanoma patients. Thus, B7-IL2 gene-transduced melanoma lines appear to display a high immunogenicity and could be used as vaccine in melanoma patients.

Human heat shock protein 70 peptide complexes specifically activate antimelanoma T cells.

Members of the heat shock protein 70 (HSP70) family display a broad cellular localization and thus bind a repertoire of chaperoned peptides potentially derived from proteins of different cellular compartments. In this report, we show that HSP70 purified from human melanoma can activate T cells recognizing melanoma differentiation antigens in an antigen- and HLA class I-dependent fashion. HLA class I-restricted anti-melanoma T cells were susceptible to MHC-restricted, HSP70-dependent stimulation, indicating that HSP70 complexed peptides were able to gain access to the class I HLA presentation pathway. In addition, MHC matching between the melanoma cells used as a source of HSP and the responding T cells were not required, indicating that HSP70 activation may occur across MHC barriers. Besides the MHC-restricted and peptide-dependent activation pathway, HSP70 with no endogenous complexed peptides or HSP70 purified from antigen-negative cells was also able to induce IFN-gamma release by antimelanoma T cells by a MHC-independent mechanism. In this case, however, higher doses of HSP70 were required. The capacity to activate class I-restricted, antitumor T cells as well as antigen-presenting cells, together with the finding that the HSP70 chaperoned peptide repertoire includes melanoma-shared epitopes, holds promise for a HSP70-based cancer vaccine.

Quantitation of antigen-reactive T cells in peripheral blood by IFNgamma-ELISPOT assay and chromium-release assay: a four-centre comparative trial.

The ELISPOT assay is increasingly being used for the monitoring of the induction of antigen-reactive T cells in cancer vaccination trials. In order to evaluate the reliability of T cell frequency analysis with the ELISPOT assay, a comparative study was performed in four European laboratories. Six samples from healthy subjects were analyzed for the frequency of influenza-reactive CD8+ T cells in peripheral blood mononuclear cells (PBMC) by IFNgamma-ELISPOT assay. In addition, one laboratory determined cytotoxic T cell precursor (CTL) frequencies in these samples by limiting dilution chromium-release assay (LDA), and three laboratories performed a variant of the LDA, the multiple microculture assay (MMA). Consistent frequencies of influenza peptide-reactive T cells were obtained with the ELISPOT assay in all four laboratories. The numbers detected by ELISPOT assay correlated closely with those determined by LDA. In contrast, the frequencies obtained with the MMA differed considerably and showed little correlation with the other two assays. This study shows that it is possible to use the ELISPOT assay to determine with reliability antigen-reactive T cells in a multicenter setting. We suggest that this assay may be suitable for monitoring cancer vaccine trials.

Natural T-cell response against MHC class I epitopes of epithelial cell adhesion molecule, her-2/neu, and carcinoembryonic antigen in patients with colorectal cancer.

The antigens epithelial cell adhesion molecule (Ep-CAM), her-2/neu, and carcinoembryonic antigen (CEA) are potential T-cell targets in antigen-specific vaccination-based cancer therapy. We performed this study to evaluate whether a natural specific T-cell response against these tumor-associated antigens (TAAs) already exists in patients with colorectal carcinoma (CRC). We used the IFN-gamma ELISPOT assay to detect circulating TAA-reactive T cells directly ex vivo in unstimulated peripheral blood mononuclear cells. We analyzed the T-cell response in peripheral blood mononuclear cells of 22 HLA-A2-positive patients with CRC and 8 HLA-A2-positive healthy subjects against 3 HLA A2-restricted peptide epitopes of the TAAs Ep-CAM (GLKAGVIAV), her-2/neu (IISAVVGIL), and CEA (YLSGANLNL). Seven of 22 patients but none of the 8 healthy subjects had T cells specifically secreting IFN-gamma in response to one to three of these antigens (n = 4, Ep-CAM; n = 5, her-2/neu; n = 6, CEA). In three of the seven responding patients, TAA-reactive T cells were further characterized by flow cytometry. In all three patients, the majority of these T cells have a CD3+CD8+IFN-gamma+CD69+CD45RA+ phenotype, resembling activated effector-type T cells. T-cell responses occurred only in patients with metastatic disease (Dukes' stages C and D). The results of this study indicate that natural T-cell responses against TAAs occur in approximately one-half of CRC patients with involvement of lymph nodes or distant metastases, but not in CRC patients with disease confined to the intestinum.

Cytokines in cancer therapy.

Cytokines are crucial factors in the activation and development of immune response, including responses against tumor cells. Interleukin (IL)-2, a T-cell growth factor, has been largely used to activate T and NK cells in vivo and to maintain such an activation for therapeutic purposes. When given to patients, IL-2 was shown to cause clinical responses, especially in metastatic melanoma and renal cancer patients, though its mechanism of action could not be completely elucidated. Cytokines (IL-2, IL-12, GM-CSF) are also used as natural adjuvants of vaccines of various formulation to help in activating and maintaining an antitumor immune response. This review summarizes findings deriving from the use of cytokines in cancer therapy and provides insights into future approaches when a more appropriate use of cytokines, together with new vaccines, is likely to improve clinical outcome.

Amino acid substitutions in the melanoma antigen recognized by T cell 1 peptide modulate cytokine responses in melanoma-specific T cells.

Single amino acid substitutions in melanoma-associated peptides dramatically enhance T-cell cytotoxicity against target cells presenting the modified peptides (often referred to as heteroclitic peptides). The authors tried to determine whether peptide modifications influence other aspects of T-cell immunity toward malignant melanoma. A heteroclitic peptide, E26F, with an E to F substitution in melanoma antigen recognized by T cell 1 (MART-1)26-35, triggers an enhanced tyrosine phosphorylation response when compared with the native- and other-modified MART-1 peptides. Similarly, the E26F peptide enhances the production of mRNA for interleukin (IL)-5, IL-10, IL-13, IL-15, and interferon-gamma and significantly enhances release of IL-13 and IL-10 from anti-MART-1 cytotoxic T cells. Another heteroclitic peptide, 1L, with an A to L substitution in MART-1(27-35), also enhances the tyrosine phosphorylation response in anti-MART-1 cytotoxic CD8+ T cells. Yet, 1L does not enhance the production of T helper cell type 2-like cytokines (IL-10 and IL-13). Together these data show that minor amino acid modifications of immunodominant melanoma peptides profoundly influence the cytokine response in melanoma-specific T cells.

Murine granulocytes control human tumor growth in SCID mice.

Severe combined immunodeficient (SCID) mice generally do not reject allogeneic or xenogeneic organ grafts and represent a unique model for investigating in vivo the behaviour of both normal and neoplastic human cells. However, cells from human primary tumors often do not grow in SCID mice. We have previously shown that the major reaction of SCID mice to the engraftment of human peripheral blood leukocytes is a massive granulocyte recruitment into the site of transplantation. In this study, we have investigated the role of murine granulocytes in the control of human tumor cell growth in SCID mice. We report here that murine granulocytes infiltrate and delimit the human tumor mass and that treatment of SCID mice with anti-murine granulocyte antibody markedly improves the growth of human tumor cell lines of different origin through suppression of the host granulocyte reaction. This finding provides a new tool for improving the human tumor take in SCID mice, thus opening new perspectives for a practical in vivo preclinical test of anti-tumor strategies. Moreover, this study, even with the limits of the known natural reaction against xenotransplants, further supports the importance of granulocytes in the control of tumor take and growth.