"Innovative strategies for the generation of novel human therapeutic anti-tumor and immunomodulatory antibodies"

Immunotherapy, based on the use of novel human mAbs endowed with antitumor or immunomodulatory activity, is an increasingly important strategy for cancer therapy. Monoclonal antibodies can be directed against Tumor Associated Antigens (TAAs), to inhibit their oncogenic function, or against Immune Checkpoints (IC), to modulate specific T cell responses against cancer. Furthermore, ongoing clinical trials in oncology are currently testing combinatorial treatments of anti-TAA and immunomodulatory antibodies. In our laboratory novel human antitumor immunoagents have been successfully produced against the TAA ErbB2, which is a Tyrosine Kinase Receptor, overexpressed in breast cancer and several other carcinomas. In particular, a fully human single chain antibody fragment (scFv), named Erbicin, able to bind to an epitope of ErbB2 different from those recognized by the clinically validated mAbs Trastuzumab and Pertuzumab, was isolated by phage display selection on live cells. A human anti-ErbB2 “compact antibody” (100kDa) was also generated by the fusion of Erbicin with a human IgG1 Fc, which was found to efficiently inhibit ErbB2-positive tumor growth both in vitro and in vivo. As a further progress, a trispecific antibody derivative, named Tribody, targeting three noncompeting epitopes on the extracellular domain of ErbB2 was obtained by fusing 3 binding moieties derived from Erb-hcAb, Trastuzumab and Pertuzumab, respectively. The triparatopic Tribody significantly downregulates ErbB2 and inhibits the cell growth of tumor cells, including those resistant to Trastuzumab. We show here that the multiparatope tribody combines and potentiates the therapeutic effects of the 3 different antibodies in 1 single antibody construct, thus allowing for the reduction of costs of antibody production and overcoming the limits related to monotherapy associated drug resistance. On the other hand, we performed a massive parallel screening of phage antibody library to obtain a large repertoire of fully human immunomodulatory antibodies against several-immune regulatory checkpoints to be used in monotherapy or in combinatorial treatments for cancer therapy. We used for the first time an innovative selection strategy on human activated lymphocytes to generate a large collection of scFvs against 10 different IC, called “Immunome Library”, from which scFvs specifically recognizing a given receptor could be pulled out by subsequent affinity selection cycles on recombinant purified proteins used as baits. By Next Generation Sequencing and bioinformatic analysis we ranked individual scFvs in each collection and identified those with the highest level of enrichment. Human IgGs from three of these collections (i.e. PD-1, PD-L1 and LAG-3) were generated and tested for their binding and biological activity. In particular, they were found to specifically bind to their targets with high affinity, to efficiently activate T cell proliferation, induce cytokine secretion and inhibit in vivo tumor growth. Interestingly, the novel isolated mAbs have comparable or even better binding affinity and biological activity than the clinically validated anti-PD-1 mAb Nivolumab. Finally, in order to explore the possibility to enhance the antitumor effects of the anti-TAA and immunomodulatory antibodies we tested some combinatorial treatments. We found that the anti-ErbB2 antibody Erb-hcAb and the novel anti-PD-L1 mAb, generated in our laboratory, showed additive antitumor effects on a panel of ErbB2-positive tumor cells, thus suggesting novel therapeutic approaches for the therapy of breast cancer

Congenital Diarrheal Disorders: An Updated Diagnostic Approach

Congenital diarrheal disorders (CDDs) are a group of inherited enteropathies with a typical onset early in the life. Infants with these disorders have frequently chronic diarrhea of sufficient severity to require parenteral nutrition. For most CDDs the disease-gene is known and molecular analysis may contribute to an unequivocal diagnosis. We review CDDs on the basis of the genetic defect, focusing on the significant contribution of molecular analysis in the complex, multistep diagnostic work-up

Oncolytic Adenoviral Vector-Mediated Expression of an Anti-PD-L1-scFv Improves Anti-Tumoral Efficacy in a Melanoma Mouse Model

Oncolytic virotherapy is an emerging therapeutic approach based on replication-competent viruses able to selectively infect and destroy cancer cells, inducing the release of tumor-associated antigens and thereby recruiting immune cells with a subsequent increase in antitumoral immune response. To increase the anticancer activity, we engineered a specific oncolytic adenovirus expressing a single-chain variable fragment of an antibody against PD-L1 to combine blockage of PD-1/PD-L1 interaction with the antitumoral activity of Onc.Ad5. To assess its efficacy, we infected B16.OVA cells, a murine model of melanoma, with Ad5 Delta 24 -anti-PD-L1-scFv and then co-cultured them with C57BL/6J naive splenocytes. We observed that the combinatorial treatments were significantly more effective in inducing cancer cell death. Furthermore, we assessed the efficacy of intratumoral administrations of Ad5 Delta 24-anti-PD-L1-scFv in C57BL/6J mice engrafted with B16.OVA and compared this treatment to that of the parental Ad5 Delta 24 or placebo. Treatment with the scFv-expressing Onc.Ad induced a marked reduction of tumor growth concerning the parental Onc.Ad. Additionally, the evaluation of the lymphocytic population infiltrating the treated tumor reveals a favorable immune profile with an enhancement of the CD8(+) population. These data suggest that Onc.Ad-mediated expression of immune checkpoint inhibitors increases oncolytic virotherapy efficacy and could be an effective and promising tool for cancer treatments, opening a new way into cancer therapy.Peer reviewe

Niveles altos del mar durante el Último Máximo Interglacial deducidos mediante muescas de abrasión y márgenes internos de terrazas marinas en Puerto Deseado, Provincia de Santa Cruz, Argentina

A detailed geomorphological survey was undertaken in the area of Puerto Deseado (Santa Cruz Province, Argentina) to reconstruct the Relative Sea-level (RSL) position during the Last Interglacial highstand. The presence of active and well-preserved abrasive notches and inner margins of terraces related to the MIS5e and to the Holocene, measured with DGPS, allowed to accurately estimate the RSL change from the present to the MIS5e highstand at ca. 21 m. The geomorphological and geochronological analyses support the notion of the presence of a significant regional tectonic uplift in the Atlantic Patagonia, which can be locally estimated at ca. 0.12 mm/yr.Se realizó un estudio geomorfológico detallado en la zona de Puerto Deseado (provincia de Santa Cruz, Argentina) para reconstruir la posición relativa del nivel (RNM) del mar durante el Último Máximo Interglacial. La presencia de muescas de abrasión activas y bien conservadas, y la posición de los márgenes internos de las terrazas relacionadas con el MIS5e y el Holoceno, medido con GPS diferencial, permitió estimar con precisión el cambio RNM desde el presente hasta la transgresión MIS5e en aproximadamente 21 m. Los análisis geomorfológicos y geocronológicos sustentan la noción de la presencia de un significativo levantamiento tectónico regional en la Patagonia Atlántica, que puede estimarse localmente en alrededor de 0,12 mm/año.Facultad de Ciencias Naturales y Muse

Niveles altos del mar durante el Último Máximo Interglacial deducidos mediante muescas de abrasión y márgenes internos de terrazas marinas en Puerto Deseado, Provincia de Santa Cruz, Argentina

A detailed geomorphological survey was undertaken in the area of Puerto Deseado (Santa Cruz Province, Argentina) to reconstruct the Relative Sea-level (RSL) position during the Last Interglacial highstand. The presence of active and well-preserved abrasive notches and inner margins of terraces related to the MIS5e and to the Holocene, measured with DGPS, allowed to accurately estimate the RSL change from the present to the MIS5e highstand at ca. 21 m. The geomorphological and geochronological analyses support the notion of the presence of a significant regional tectonic uplift in the Atlantic Patagonia, which can be locally estimated at ca. 0.12 mm/yr.Se realizó un estudio geomorfológico detallado en la zona de Puerto Deseado (provincia de Santa Cruz, Argentina) para reconstruir la posición relativa del nivel (RNM) del mar durante el Último Máximo Interglacial. La presencia de muescas de abrasión activas y bien conservadas, y la posición de los márgenes internos de las terrazas relacionadas con el MIS5e y el Holoceno, medido con GPS diferencial, permitió estimar con precisión el cambio RNM desde el presente hasta la transgresión MIS5e en aproximadamente 21 m. Los análisis geomorfológicos y geocronológicos sustentan la noción de la presencia de un significativo levantamiento tectónico regional en la Patagonia Atlántica, que puede estimarse localmente en alrededor de 0,12 mm/año.Facultad de Ciencias Naturales y Muse

Bifidobacterium affects antitumor efficacy of oncolytic adenovirus in a mouse model of melanoma

Gut microbiota plays a key role in modulating responses to cancer immunotherapy in melanoma patients. Oncolytic viruses (OVs) represent emerging tools in cancer therapy, inducing a potent immunogenic cancer cell death (ICD) and recruiting immune cells in tumors, poorly infiltrated by T cells. We investigated whether the antitumoral activity of oncolytic adenovirus Ad5D24-CpG (Ad-CpG) was gut microbiota-mediated in a syngeneic mouse model of melanoma and observed that ICD was weakened by vancomycin-mediated perturbation of gut microbiota. Ad-CpG efficacy was increased by oral supplementation with Bifidobacterium, reducing melanoma progression and tumor-infiltrating regulatory T cells. Fecal microbiota was enriched in bacterial species belonging to the Firmicutes phylum in mice treated with both Bifidobacterium and Ad-CpG; furthermore, our data suggest that molecular mimicry between melanoma and Bifidobacterium-derived epitopes may favor activation of cross-reactive T cells and constitutes one of the mechanisms by which gut microbiota modulates OVs response

Beta-Blocker Use in Older Hospitalized Patients Affected by Heart Failure and Chronic Obstructive Pulmonary Disease: An Italian Survey From the REPOSI Register

Beta (β)-blockers (BB) are useful in reducing morbidity and mortality in patients with heart failure (HF) and concomitant chronic obstructive pulmonary disease (COPD). Nevertheless, the use of BBs could induce bronchoconstriction due to β2-blockade. For this reason, both the ESC and GOLD guidelines strongly suggest the use of selective β1-BB in patients with HF and COPD. However, low adherence to guidelines was observed in multiple clinical settings. The aim of the study was to investigate the BBs use in older patients affected by HF and COPD, recorded in the REPOSI register. Of 942 patients affected by HF, 47.1% were treated with BBs. The use of BBs was significantly lower in patients with HF and COPD than in patients affected by HF alone, both at admission and at discharge (admission, 36.9% vs. 51.3%; discharge, 38.0% vs. 51.7%). In addition, no further BB users were found at discharge. The probability to being treated with a BB was significantly lower in patients with HF also affected by COPD (adj. OR, 95% CI: 0.50, 0.37-0.67), while the diagnosis of COPD was not associated with the choice of selective β1-BB (adj. OR, 95% CI: 1.33, 0.76-2.34). Despite clear recommendations by clinical guidelines, a significant underuse of BBs was also observed after hospital discharge. In COPD affected patients, physicians unreasonably reject BBs use, rather than choosing a β1-BB. The expected improvement of the BB prescriptions after hospitalization was not observed. A multidisciplinary approach among hospital physicians, general practitioners, and pharmacologists should be carried out for better drug management and adherence to guideline recommendations

New Insights on the Role of Anti-PD-L1 and Anti-CTLA-4 mAbs on Different Lymphocytes Subpopulations in TNBC

Antibody-based cancer immunotherapy includes monoclonals against immune checkpoints (ICs), to modulate specific T cell responses against cancer. NK cells are a newly emerging target for immune checkpoint receptor inhibition in cancer immunotherapy, as ICs are also expressed on NK cells in various cancers. The latter cells are becoming attractive targets for cancer immunotherapy, as they are effector cells similar to CTLs, exerting natural cytotoxicity against primary tumor cells and metastasis, and they are able to distinguish tumor cells from healthy ones, leading to more specific anti-tumor cytotoxicity and reduced off-target effects. Thus, we decided to test the effects on isolated NK cells and T cell subpopulations of novel immunomodulatory mAbs, recently generated in our lab, in comparison with those in clinical use, such as ipilimumab and atezolizumab. Interestingly, we found that the novel anti-CTLA-4 (ID-1) and anti-PD-L1 (PD-L1_1) antibodies are able to induce NK cell activation and exert anti-tumor effects on TNBC cells co-cultured with NK cells more efficiently than the clinically validated ones, either when used as single agents or in combinatorial treatments. On the other hand, ipilimumab was found to be more effective in activating T cells with respect to ID-1. These findings indicate that antibodies targeting different epitopes can have differential effects on different lymphocytes subpopulations and that novel combinations of mAbs could be suitable for therapeutic approaches aimed at activating not only T cells but also NK cells, especially for tumors lacking MHC

A Comparison of the Antitumor Efficacy of Novel Multi-Specific Tribodies with Combinations of Approved Immunomodulatory Antibodies

Many advances in antitumor therapies have been achieved with antagonistic antibodies targeting the programmed cell death protein 1 (PD-1) or its ligand (PD-L1); however, many cancer patients still develop resistance to anti–PD-1/PD-L1 treatments often associated with the upregulation of other immune checkpoints such as Lymphocyte Activation Gene-3 (LAG-3). In order to verify whether it is possible to overcome these limits, we analyzed and compared the effects of combinations of the clinically validated anti-LAG-3 mAb (Relatlimab) with anti-PD-1 (Pembrolizumab) or anti-PD-L1 (Atezolizumab) monoclonal antibodies (mAbs) with those of novel bispecific tribodies (TRs), called TR0304 and TR0506, previously generated in our lab by combining the binding moieties of novel human antibodies targeting the same ICs of the mentioned mAbs. In particular, TR0304, made up of a Fab derived from an anti-PD-L1 mAb and two single-chain variable fragments (scFvs) derived from an anti-LAG-3 mAb, was tested in comparison with Relatlimab plus Atezolizumab, and TR0506, made up of an antigen-binding fragment (Fab) derived from the same anti-LAG-3 mAb and two scFvs derived from an anti-PD-1 mAb, was tested in comparison with Relatlimab and Pembrolizumab. We found that the two novel TRs showed similar binding affinity to the targets with respect to validated mAbs, even though they recognized distinct or only partially overlapping epitopes. When tested for their functional properties, they showed an increased ability to induce lymphocyte activation and stronger in vitro cytotoxicity against tumor cells compared to combinatorial treatments of clinically validated mAbs. Considering that tribodies also have other advantages with respect to combinatorial treatments, such as reduced production costs and lower dose requirements, we think that these novel immunomodulatory TRs could be used for therapeutic applications, particularly in monotherapy-resistant cancer patients