Stereotactic ablative body radiotherapy (SABR) combined with immunotherapy (L19-IL2) versus standard of care in stage IV NSCLC patients, ImmunoSABR: a multicentre, randomised controlled open-label phase II trial.

BACKGROUND: About 50% of non-small cell lung cancer (NSCLC) patients have metastatic disease at initial diagnosis, which limits their treatment options and, consequently, the 5-year survival rate (15%). Immune checkpoint inhibitors (ICI), either alone or in combination with chemotherapy, have become standard of care (SOC) for most good performance status patients. However, most patients will not obtain long-term benefit and new treatment strategies are therefore needed. We previously demonstrated clinical safety of the tumour-selective immunocytokine L19-IL2, consisting of the anti-ED-B scFv L19 antibody coupled to IL2, combined with stereotactic ablative radiotherapy (SABR). METHODS: This investigator-initiated, multicentric, randomised controlled open-label phase II clinical trial will test the hypothesis that the combination of SABR and L19-IL2 increases progression free survival (PFS) in patients with limited metastatic NSCLC. One hundred twenty-six patients will be stratified according to their metastatic load (oligo-metastatic: ≤5 or poly-metastatic: 6 to 10) and randomised to the experimental-arm (E-arm) or the control-arm (C-arm). The C-arm will receive SOC, according to the local protocol. E-arm oligo-metastatic patients will receive SABR to all lesions followed by L19-IL2 therapy; radiotherapy for poly-metastatic patients consists of irradiation of one (symptomatic) to a maximum of 5 lesions (including ICI in both arms if this is the SOC). The accrual period will be 2.5-years, starting after the first centre is initiated and active. Primary endpoint is PFS at 1.5-years based on blinded radiological review, and secondary endpoints are overall survival, toxicity, quality of life and abscopal response. Associative biomarker studies, immune monitoring, CT-based radiomics, stool collection, iRECIST and tumour growth rate will be performed. DISCUSSION: The combination of SABR with or without ICI and the immunocytokine L19-IL2 will be tested as 1st, 2nd or 3rd line treatment in stage IV NSCLC patients in 14 centres located in 6 countries. This bimodal and trimodal treatment approach is based on the direct cytotoxic effect of radiotherapy, the tumour selective immunocytokine L19-IL2, the abscopal effect observed distant from the irradiated metastatic site(s) and the memory effect. The first results are expected end 2023. TRIAL REGISTRATION: ImmunoSABR Protocol Code: NL67629.068.18; EudraCT: 2018-002583-11; Clinicaltrials.gov: NCT03705403; ISRCTN ID: ISRCTN49817477; Date of registration: 03-April-2019.

A phase II study of the L19IL2 immunocytokine in combination with dacarbazine in advanced metastatic melanoma patients.

Engineered cytokine products represent promising agents for the treatment of immunogenic tumors, such as malignant melanoma, in addition to immune checkpoint inhibitors. Here we describe the results of a controlled, randomized phase II clinical trial, aimed at assessing the therapeutic potential of L19IL2, a fully human fusion protein consisting of the L19 antibody specific to the alternatively spliced extra-domain B of fibronectin, fused to human interleukin-2 in advanced metastatic melanoma. In one arm, patients received dacarbazine (DTIC; 1000 mg/m2 of body surface on day 1 of 21-day cycles) as single agent, while in two other arms L19IL2 (22.5 million international units of IL2 equivalents) was added, based on two different schedules of administration. In total, 69 patients with stage IV melanoma were enrolled (24 in the dacarbazine arm, 23 and 22 in the other combination arms, respectively) and 67 received treatment. Analyses of efficacy results show a statistically significant benefit in terms of overall response rate and median progression-free survival for patients receiving L19IL2 in combination with DTIC, compared to DTIC as single agent. In light of these results, further clinical investigations with L19IL2 (alone or in combination with other agents) are warranted.

Intralesional treatment of metastatic melanoma: a review of therapeutic options.

Intralesional therapy of melanoma patients with locally advanced metastatic disease is attracting increasing interest, not least due to its ability to lead to both direct tumor cell killing and the stimulation of both a local and a systemic immune response. An obvious pre-requisite for this type of approach is the presence of accessible metastases that are amenable to direct injection with the therapeutic agent of interest. Patients who present with these characteristics belong to stages IIIB/C or IV of the disease. Surgical resection with intention to cure is the standard of care for patients with limited tumor burden and confined spread of disease (resectable patients). However, this category of patients is at a high risk of further recurrences until the disease becomes inoperable (unresectable) or progresses to a more advanced stage with visceral organ involvement, after which the prognosis is particularly grim. Most of the intralesional treatments tested so far, including the recently approved oncolytic virus talimogene laherparepvec, target the subpopulation of patients with unresectable disease, but the possibility to use the intralesional treatment in a neoadjuvant setting for fully resectable patients is attracting considerable interest. The present article reviews approved products and advanced stage pharmaceutical agents in development for the intralesional treatment of melanoma patients.

Armed antibodies for cancer treatment: a promising tool in a changing era.

Advances in the understanding of tumor immunology and molecular biology of melanoma cells have favored a larger application of immunotherapy and targeted therapies in the clinic. Several selective mutant gene inhibitors and immunomodulating antibodies have been reported to improve overall survival or progression-free survival in metastatic melanoma patients. However, despite impressive initial responses, patients treated with selective inhibitors relapse quickly, and toxicities associated to the use of immunomodulating antibodies are not easily manageable. In this sense, the concept of using antibodies as delivery vehicles for the preferential in vivo localization of the drug at the site of disease with reduction of side effects has raised particular interest. Antibody-cytokine fusion proteins (termed immunocytokines) represent a new simple and effective way to deliver the immunomodulatory payload at the tumor site, with the aim of inducing both local and systemic antitumoral immune responses and limiting systemic toxicities. Several clinical trials have been conducted and are actually ongoing with different immunocytokines, in several tumor histotypes. In metastatic melanoma patients, different drug delivery modalities such as systemic, loco-regional and intratumoral are under investigation. In this review, the rationale for the use of L19-IL2 and L19-TNF, two clinical stage immunocytokines produced by the Philogen group, as well as opportunities for their future development will be discussed.

Intralesional administration of L19-IL2/L19-TNF in stage III or stage IVM1a melanoma patients: results of a phase II study.

The intratumoral injection of cytokines, in particular IL2, has shown promise for cutaneous melanoma patients with unresectable disease or continuous recurrence despite surgery. We recently reported that the intralesional injection of L19-IL2, an immunocytokine combining IL2 and the human monoclonal antibody fragment L19, resulted in efficient regional control of disease progression, increased time to distant metastasis and evidence of effect on circulating immune cell populations. We have also shown in preclinical models of cancer a remarkable synergistic effect of the combination of L19-IL2 with L19-TNF, a second clinical-stage immunocytokine, based on the same L19 antibody fused to TNF. Here, we describe the results of a phase II clinical trial based on the intralesional administration of L19-IL2 and L19-TNF in patients with stage IIIC and IVM1a metastatic melanoma, who were not candidate to surgery. In 20 efficacy-evaluable patients, 32 melanoma lesions exhibited complete responses upon intralesional administration of the two products, with mild side effects mainly limited to injection site reactions. Importantly, we observed complete responses in 7/13 (53.8 %) non-injected lesions (4 cutaneous, 3 lymph nodes), indicating a systemic activity of the intralesional immunostimulatory treatment. The intralesional administration of L19-IL2 and L19-TNF represents a simple and effective method for the local control of inoperable melanoma lesions, with a potential to eradicate them or make them suitable for a facile surgical removal of the residual mass.

Blood biocompatibility of surface-bound multi-walled carbon nanotubes.

Blood clots when it contacts foreign surfaces following platelet activation. This can be catastrophic in clinical settings involving extracorporeal circulation such as during heart-lung bypass where blood is circulated in polyvinyl chloride tubing. Studies have shown, however, that surface-bound carbon nanotubes may prevent platelet activation, the initiator of thrombosis. We studied the blood biocompatibility of polyvinyl chloride, surface-modified with multi-walled carbon nanotubes in vitro and in vivo. Our results show that surface-bound multi-walled carbon nanotubes cause platelet activation in vitro and devastating thrombosis in an in vivo animal model of extracorporeal circulation. The mechanism of the pro-thrombotic effect likely involves direct multi-walled carbon nanotube-platelet interaction with Ca(2+)-dependant platelet activation. These experiments provide evidence, for the first time, that modification of surfaces with nanomaterials modulates blood biocompatibility in extracorporeal circulation.

Preclinical evaluation of IL2-based immunocytokines supports their use in combination with dacarbazine, paclitaxel and TNF-based immunotherapy.

Antibody-cytokine fusion proteins ("immunocytokines") represent a promising class of armed antibody products, which allow the selective delivery of potent pro-inflammatory payloads at the tumor site. The antibody-based selective delivery of interleukin-2 (IL2) is particularly attractive for the treatment of metastatic melanoma, an indication for which this cytokine received marketing approval from the US Food and drug administration. We used the K1735M2 immunocompetent syngeneic model of murine melanoma to study the therapeutic activity of F8-IL2, an immunocytokine based on the F8 antibody in diabody format, fused to human IL2. F8-IL2 was shown to selectively localize at the tumor site in vivo, following intravenous administration, and to mediate tumor growth retardation, which was potentiated by the combination with paclitaxel or dacarbazine. Combination treatment led to a substantially more effective tumor growth inhibition, compared to the cytotoxic drugs used as single agents, without additional toxicity. Analysis of the immune infiltrate revealed a significant accumulation of CD4(+) T cells 24 h after the administration of the combination. The fusion proteins F8-IL2 and L19-IL2, specific to the alternatively spliced extra domain A and extra domain B of fibronectin respectively, were also studied in combination with tumor necrosis factor (TNF)-based immunocytokines. The combination treatment was superior to the action of the individual immunocytokines and was able to eradicate neoplastic lesions after a single intratumoral injection, a procedure that is being clinically used for the treatment of Stage IIIC melanoma. Collectively, these data reinforce the rationale for the use of IL2-based immunocytokines in combination with cytotoxic agents or TNF-based immunotherapy for the treatment of melanoma patients.

Regulator of G-protein signaling 18 integrates activating and inhibitory signaling in platelets.

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-α-q and G-α-i subunits of heterotrimeric G-proteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3γ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3-deficient RGS18 is more active compared with 14-3-3-bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA- and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq.

Predicting response to vascular endothelial growth factor inhibitor and chemotherapy in metastatic colorectal cancer.

BACKGROUND: Bevacizumab improves progression free survival (PFS) and overall survival (OS) in metastatic colorectal cancer patients however currently there are no biomarkers that predict response to this treatment. The aim of this study was to assess if differential protein expression can differentiate patients who respond to chemotherapy and bevacizumab, and to assess if select proteins correlate with patient survival. METHODS: Pre-treatment serum from patients with metastatic colorectal cancer (mCRC) treated with chemotherapy and bevacizumab were divided into responders and nonresponders based on their progression free survival (PFS). Serum samples underwent immunoaffinity depletion and protein expression was analysed using two-dimensional difference gel electrophoresis (2D-DIGE), followed by LC-MS/MS for protein identification. Validation on selected proteins was performed on serum and tissue samples from a larger cohort of patients using ELISA and immunohistochemistry, respectively (n = 68 and n = 95, respectively). RESULTS: 68 proteins were identified following LC-MS/MS analysis to be differentially expressed between the groups. Three proteins (apolipoprotein E (APOE), angiotensinogen (AGT) and vitamin D binding protein (DBP)) were selected for validation studies. Increasing APOE expression in the stroma was associated with shorter progression free survival (PFS) (p = 0.0001) and overall survival (OS) (p = 0.01), DBP expression (stroma) was associated with shorter OS (p = 0.037). Increasing APOE expression in the epithelium was associated with a longer PFS and OS, and AGT epithelial expression was associated with a longer PFS (all p < .05). Increasing serum AGT concentration was associated with shorter OS (p = 0.009). CONCLUSIONS: APOE, DBP and AGT identified were associated with survival outcomes in mCRC patients treated with chemotherapy and bevacizumab.

2D DIGE proteomic analysis of early post mortem muscle exudate highlights the importance of the stress response for improved water-holding capacity of fresh pork meat.

Variation in water-holding capacity (WHC), which presents a major economic burden to the swine industry, is considered to be underpinned by variation at a molecular and biochemical level. High-resolution 2D DIGE followed by MS analysis and Western blot were used to unravel the proteome of muscle exudate, collected following centrifugation, in the pH 4-7 range. A first 2DE-based protein map of this substrate was produced where 89 spots were successfully characterised. Two phenotypes divergent for WHC plus one intermediate were compared with a view to deciphering the biochemical processes impacting on variation in WHC. Twenty spots were observed to be altered across the phenotypes. Of these, 14 represented sixteen proteins including metabolic enzymes, stress response proteins and structural proteins. Triosephosphate isomerase and transferrin showed a major difference between the two extreme phenotypes, and may have potential as biological markers for WHC prediction. Several members of the HSPs family were highlighted. This proteomic study makes an important contribution towards a more detailed molecular view of the processes behind WHC and will provide a valuable resource for future investigations.

Composition of the bovine uterine proteome is associated with stage of cycle and concentration of systemic progesterone.

Early embryonic loss accounts for over 70% of total embryonic and foetal loss in dairy cattle. Early embryonic development and survival is associated with the concentration of systemic progesterone. To determine if the uterine proteome is influenced by stage of cycle or systemic progesterone concentrations, uterine flushings were collected from the ipsi- and contralateral uterine horns of beef heifers on Days 7 (n = 10) and 15 (n = 10) of the oestrous cycle. Animals were separated into low or high progesterone groups based on plasma progesterone concentrations on Day 5 of the cycle. Samples were albumin depleted before iTRAQ R labeling and subsequent strong cation exchange-LC-MS/MS analyses. A total of 20 proteins were up to 5.9-fold higher (p<0.05) and 20 were up to 2.3-fold lower on Day 15 compared toDay 7. In addition, the expression of a number of proteins on Day 7 and/or 15 of the cycle was correlated with progesterone concentrations during Days 3­7 or the rate of change in progesterone between Days 3 and 7. This study highlights the dynamic changes occurring in the microenvironment surrounding the embryo during this period. The findings here also support the hypothesis that progesterone supports embryonic development by altering the maternal uterine environment.

The presence of outer arm fucose residues on the N-glycans of tissue inhibitor of metalloproteinases-1 reduces its activity.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of N-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the N-glycan structures showed that SF9 TIMP-1 has the simplest N-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their N-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the N-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to N-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its N-glycans.

Monitoring post mortem changes in porcine muscle through 2-D DIGE proteome analysis of Longissimus muscle exudate.

BACKGROUND: Meat quality is a complex trait influenced by a range of factors with post mortem biochemical processes highly influential in defining ultimate quality. High resolution two-dimensional DIfference Gel Electrophoresis (2-D DIGE) and Western blot were applied to study the influence of post mortem meat ageing on the proteome of pork muscle. Exudate collected from the muscle following centrifugation was analysed at three timepoints representing a seven day meat ageing period. RESULTS: The intensity of 136 spots varied significantly (p < 0.05) across this post mortem period and 40 spots were identified using mass spectrometry. The main functional categories represented were metabolic proteins, stress-related proteins, transport and structural proteins. Metabolic and structural proteins were generally observed to increase in abundance post mortem and many likely represent the accumulation of the degradation products of proteolytic enzyme activity. In contrast, stress-related proteins broadly decreased in abundance across the ageing period. Stress response proteins have protective roles in maintaining cellular integrity and a decline in their abundance over time may correlate with a reduction in cellular integrity and the onset of meat ageing. Since cellular conditions alter with muscle ageing, changes in solubility may also contribute to observed abundance profiles. CONCLUSIONS: Muscle exudate provided valuable information about the pathways and processes underlying the post mortem ageing period, highlighting the importance of post mortem modification of proteins and their interaction for the development of meat quality traits.

Exploring the glycosylation of serum CA125.

Ovarian cancer is the most lethal gynaecologic cancer affecting women. The most widely used biomarker for ovarian cancer, CA125, lacks sensitivity and specificity. Here, we explored differences in glycosylation of CA125 between serum from patients with ovarian cancer and healthy controls. We found differences between CA125 N-glycans from patient sera compared to controls. These include increases in core-fucosylated bi-antennary monosialylated glycans, as well as decreases in mostly bisecting bi-antennary and non-fucosylated glycans in patients compared to controls. Measurement of the glycosylated state of CA125 may therefore provide a more specific biomarker for patients with ovarian cancer.

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation, and accurate within-gel matching. 2D-DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS (sodium dodecyl sulfate)-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometric techniques and further analyzed by other biochemical methods. In this chapter, we describe application of this methodology to separation and quantitation of protein mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in meat industry.

Crystallin distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei lenses.

The eye lens remains transparent because of soluble lens proteins known as crystallins. For years γ-crystallins have been known as the main lens proteins in lower vertebrates such as fish and amphibians. The unique growth features of the lens render it an ideal structure to study ageing; few studies have examined such changes in anuran lenses. This study aimed to investigate protein distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei species. Lenses were fractionated into concentric layers by controlled dissolution. Water-soluble proteins were separated into high (HMW), middle (MMW) and low molecular weight (LMW) fractions by size-exclusion HPLC and constituents of each protein class revealed by 1DE and 2DE. Spots were selected from 2DE gels on the basis of known ranges of subunit molecular weights and pH ranges and were identified by MALDI-TOF/TOF MS following trypsin digestion. Comparable lens distribution patterns were found for each species studied. Common crystallins were detected in both species; the most prominent of these was γ-crystallin. Towards the lens centre, there was a decrease in α- and ß-crystallin proportions and an increase in γ-crystallins. Subunits representing taxon-specific crystallins demonstrating strong sequence homology with ζ-crystallin/quinone oxidoreductase were found in both L. infrafrenata and P. sauvagei lenses. Further work is needed to determine which amphibians have taxon-specific crystallins, their evolutionary origins, and their function.

A comparison of the bovine uterine and plasma proteome using iTRAQ proteomics.

Early embryo loss is a key factor affecting fertility in dairy and beef herds. Prior to implantation, the bovine embryo spends around 16 days free-floating in the uterine environment and is dependent on the composition of uterine fluid for normal growth and development. However, there is a lack of information regarding the protein composition of the bovine uterus and how it relates to plasma. In this study, uterine flushings (UF) (n = 6) and blood plasma (n = 4) were collected from beef heifers on day 7 of the oestrous cycle, albumin depleted and compared using iTRAQ proteomics. A total of 35 proteins were higher and 18 were lower in UF including metabolic enzymes, proteins with anti-oxidant activity and those involved in modulation of the immune response. This study confirms the dynamic nature of the bovine uterine proteome and that it differs from plasma. Factors affecting the uterine proteome and how it impacts on embryo survival warrant further study.

Proteomic characterization of histotroph during the preimplantation phase of the estrous cycle in cattle.

Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC-MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy.

Immunodepletion of albumin and immunoglobulin G from bovine plasma.

Current MS-based proteomics has facilitated the identification of large numbers of proteins from complex mixtures. The bovine plasma proteome has the potential to provide a wealth of information concerning the biological state of an animal. However, during MS-based experiments, higher abundance proteins such as albumin and immunoglobulin G (IgG) can hinder the identification of potentially important proteins that are present in much lower abundance. While a variety of readily available technologies exist for the depletion of multiple high-abundance proteins from human, mouse and rat samples, there are few available for bovine. In this study, we report the depletion of >97% of albumin and >92% of IgG from bovine plasma.