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Article ; Online: Novel Human Anti-PD-L1 mAbs Inhibit Immune-Independent Tumor Cell Growth and PD-L1 Associated Intracellular Signalling

Margherita Passariello / Anna Morena D’Alise / Annachiara Esposito / Cinzia Vetrei / Guendalina Froechlich / Elisa Scarselli / Alfredo Nicosia / Claudia De Lorenzo

Scientific Reports, Vol 9, Iss 1, Pp 1-

2019 Volume 13

Abstract: Abstract The novel antibody-based immunotherapy in oncology exploits the activation of immune system mediated by immunomodulatory antibodies specific for immune checkpoints. Among them, the programmed death ligand-1 (PD-L1) is of particular interest as ... ...

Abstract	Abstract The novel antibody-based immunotherapy in oncology exploits the activation of immune system mediated by immunomodulatory antibodies specific for immune checkpoints. Among them, the programmed death ligand-1 (PD-L1) is of particular interest as it is expressed not only on T-cells, but also on other immune cells and on a large variety of cancer cells, such as breast cancer cells, considering its high expression in both ErbB2-positive and Triple Negative Breast Cancers. We demonstrate here that PD-L1_1, a novel anti-PD-L1 T -cell stimulating antibody, inhibits PD-L1-tumor cell growth also by affecting the intracellular MAPK pathway and by activating caspase 3. Similar in vitro results were obtained for the first time here also with the clinically validated anti-PD-L1 mAb Atezolizumab and in vivo with another validated anti-mouse anti-PD-L1 mAb. Moreover, we found that two high affinity variants of PD-L1_1 inhibited tumor cell viability more efficiently than the parental PD-L1_1 by affecting the same MAPK pathways with a more potent effect. Altogether, these results shed light on the role of PD-L1 in cancer cells and suggest that PD-L1_1 and its high affinity variants could become powerful antitumor weapons to be used alone or in combination with other drugs such as the anti-ErbB2 cAb already successfully tested in in vitro combinatorial treatments.
Keywords	Medicine ; R ; Science ; Q
Subject code	570
Language	English
Publishing date	2019-09-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Generation of a Retargeted Oncolytic Herpes Virus Encoding Adenosine Deaminase for Tumor Adenosine Clearance

Chiara Gentile / Arianna Finizio / Guendalina Froechlich / Anna Morena D’Alise / Gabriella Cotugno / Sara Amiranda / Alfredo Nicosia / Elisa Scarselli / Nicola Zambrano / Emanuele Sasso

International Journal of Molecular Sciences, Vol 22, Iss 13521, p

2021 Volume 13521

Abstract: Background: Oncolytic viruses are immunotherapeutic agents that can be engineered to encode payloads of interest within the tumor microenvironment to enhance therapeutic efficacy. Their therapeutic potential could be limited by many avenues for immune ... ...

Abstract	Background: Oncolytic viruses are immunotherapeutic agents that can be engineered to encode payloads of interest within the tumor microenvironment to enhance therapeutic efficacy. Their therapeutic potential could be limited by many avenues for immune evasion exerted by the tumor. One such is mediated by adenosine, which induces pleiotropic immunosuppression by inhibiting antitumor immune populations as well as activating tolerogenic stimuli. Adenosine is produced starting from the highly immunostimulatory ATP, which is progressively hydrolyzed to ADP and adenosine by CD39 and CD73. Cancer cells express high levels of CD39 and CD73 ectoenzymes, thus converting immunostimulatory purinergic signal of ATP into an immunosuppressive signal. For this reason, CD39, CD73 and adenosine receptors are currently investigated in clinical trials as targets for metabolic cancer immunotherapy. This is of particular relevance in the context of oncovirotherapy, as immunogenic cell death induced by oncolytic viruses causes the secretion of a high amount of ATP which is available to be quickly converted into adenosine. Methods: Here, we took advantage of adenosine deaminase enzyme that naturally converts adenosine into the corresponding inosine derivative, devoid of immunoregulatory function. We encoded ADA into an oncolytic targeted herpes virus redirected to human HER2. An engineered ADA with an ectopic signal peptide was also generated to improve enzyme secretion (ADA-SP). Results: Insertion of the expression cassette was not detrimental for viral yield and cancer cell cytotoxicity. The THV_ADA and THV_ADA-SP successfully mediated the secretion of functional ADA enzyme. In in vitro model of human monocytes THP1, this ability of THV_ADA and THV_ADA-SP resulted in the retrieval of eADO-exposed monocytes replication rate, suggesting the proficiency of the viruses in rescuing the immune function. Conclusions: Encoding ADA into oncolytic viruses revealed promising properties for preclinical exploitation.
Keywords	oncolytic virus ; adenosine ; adenosine deaminase ; targeted therapy ; immunometabolism ; immunotherapy ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2021-12-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Generation of a Novel Mesothelin-Targeted Oncolytic Herpes Virus and Implemented Strategies for Manufacturing

Guendalina Froechlich / Chiara Gentile / Luigia Infante / Carmen Caiazza / Pasqualina Pagano / Sarah Scatigna / Gabriella Cotugno / Anna Morena D’Alise / Armin Lahm / Elisa Scarselli / Alfredo Nicosia / Massimo Mallardo / Emanuele Sasso / Nicola Zambrano

International Journal of Molecular Sciences, Vol 22, Iss 477, p

2021 Volume 477

Abstract: Background: HER2-based retargeted viruses are in advanced phases of preclinical development of breast cancer models. Mesothelin (MSLN) is a cell-surface tumor antigen expressed in different subtypes of breast and non-breast cancer. Its recent ... ...

Abstract	Background: HER2-based retargeted viruses are in advanced phases of preclinical development of breast cancer models. Mesothelin (MSLN) is a cell-surface tumor antigen expressed in different subtypes of breast and non-breast cancer. Its recent identification as a marker of some triple-negative breast tumors renders it an attractive target, presently investigated in clinical trials employing antibody drug conjugates and CAR-T cells. The availability of MSLN-retargeted oncolytic viruses may complement the current immunotherapeutic panel of biological drugs against HER2-negative breast and non-breast tumors. Methods: A fully virulent, tumor-targeted oncolytic Herpes simplex virus-1 (MSLN-THV) with a selectivity for mesothelin-expressing cancer cells was generated. Recombineering technology was used to replace an essential moiety of the viral glycoprotein D with antibody fragments derived from clinically validated MSLN monoclonal antibodies, and to allow IL12 cargo expression in infected cells. Panels of breast and female reproductive system cell lines were used to verify the oncolytic potential of the viral constructs. A platform for production of the retargeted viruses was developed in HEK 293 cells, providing stable expression of a suitable chimeric receptor. Results: We demonstrated the selectivity of viral infection and cytotoxicity by MSLN-retargeted viruses in a panel of mesothelin-positive cancer cells, originating from breast and female reproductive system tumors. We also developed a second-generation oncolytic MSLN-THV, encoding IL12, to enhance the immunotherapeutic potential of the viral backbone. A non-tumor cell line expressing a chimeric MSLN/Nectin-1 receptor, de-sensitized from antiviral responses by genetic inactivation of the Stimulator of Interferon Genes ( STING )-dependent pathway was engineered, to optimize viral yields. Conclusions: Our proof-of-concept study proposes MSLN-retargeted herpesviruses as potential cancer immunotherapeutics for assessments in preclinical models of MSLN-positive ...
Keywords	oncolytic virus ; triple negative breast cancer ; malignant mesothelioma ; targeted therapy ; MSLN ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	616
Language	English
Publishing date	2021-01-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Replicative conditioning of Herpes simplex type 1 virus by Survivin promoter, combined to ERBB2 retargeting, improves tumour cell-restricted oncolysis

Emanuele Sasso / Guendalina Froechlich / Gabriella Cotugno / Anna Morena D’Alise / Chiara Gentile / Veronica Bignone / Maria De Lucia / Biljana Petrovic / Gabriella Campadelli-Fiume / Elisa Scarselli / Alfredo Nicosia / Nicola Zambrano

Scientific Reports, Vol 10, Iss 1, Pp 1-

2020 Volume 12

Abstract: Abstract Oncolytic virotherapy is emerging as a promising therapeutic option for solid tumours. Several oncolytic vectors in clinical testing are based on attenuated viruses; thus, efforts are being taken to develop a new repertoire of oncolytic viruses, ...

Abstract	Abstract Oncolytic virotherapy is emerging as a promising therapeutic option for solid tumours. Several oncolytic vectors in clinical testing are based on attenuated viruses; thus, efforts are being taken to develop a new repertoire of oncolytic viruses, based on virulent viral genomes. This possibility, however, raises concerns dealing with the safety features of the virulent phenotypes. We generated a double regulated Herpes simplex type-1 virus (HSV-1), in which tumour cell restricted replicative potential was combined to selective entry via ERBB2 receptor retargeting. The transcriptional control of the viral alpha4 gene encoding for the infected cell protein-4 (ICP4) by the cellular Survivin/BIRC5 promoter conferred a tumour cell-restricted replicative potential to a virulent HSV-1 genome. The combination of the additional ERBB2 retargeting further improved the selectivity for tumour cells, conferring to the double regulated virus a very limited ability to infect and propagate in non-cancerous cells. Accordingly, a suitable replicative and cytotoxic potential was maintained in tumour cell lines, allowing the double regulated virus to synergize in vivo with immune checkpoint (anti-PD-1) blockade in immunocompetent mice. Thus, restricting the replicative spectrum and tropism of virulent HSV-1 genomes by combination of conditional replication and retargeting provides an improved safety, does not alter the oncolytic strength, and is exploitable for its therapeutic potential with immune checkpoint blockade in cancer.
Keywords	Medicine ; R ; Science ; Q
Subject code	616 ; 570
Language	English
Publishing date	2020-03-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: VENUS, a Novel Selection Approach to Improve the Accuracy of Neoantigens’ Prediction

Guido Leoni / Anna Morena D’Alise / Fabio Giovanni Tucci / Elisa Micarelli / Irene Garzia / Maria De Lucia / Francesca Langone / Linda Nocchi / Gabriella Cotugno / Rosa Bartolomeo / Giuseppina Romano / Simona Allocca / Fulvia Troise / Alfredo Nicosia / Armin Lahm / Elisa Scarselli

Vaccines, Vol 9, Iss 880, p

2021 Volume 880

Abstract: Neoantigens are tumor-specific antigens able to induce T-cell responses, generated by mutations in protein-coding regions of expressed genes. Previous studies demonstrated that only a limited subset of mutations generates neoantigens in microsatellite ... ...

Abstract	Neoantigens are tumor-specific antigens able to induce T-cell responses, generated by mutations in protein-coding regions of expressed genes. Previous studies demonstrated that only a limited subset of mutations generates neoantigens in microsatellite stable tumors. We developed a method, called VENUS (Vaccine-Encoded Neoantigens Unrestricted Selection), to prioritize mutated peptides with high potential to be neoantigens. Our method assigns to each mutation a weighted score that combines the mutation allelic frequency, the abundance of the transcript coding for the mutation, and the likelihood to bind the patient’s class-I major histocompatibility complex alleles. By ranking mutated peptides encoded by mutations detected in nine cancer patients, VENUS was able to select in the top 60 ranked peptides, the 95% of neoantigens experimentally validated including both CD8 and CD4 T cell specificities. VENUS was evaluated in a murine model in the context of vaccination with an adeno vector encoding the top ranked mutations prioritized in the MC38 cell line. Efficacy studies demonstrated anti tumoral activity of the vaccine when used in combination with checkpoint inhibitors. The results obtained highlight the importance of a combined scoring system taking into account multiple features of each tumor mutation to improve the accuracy of neoantigen prediction.
Keywords	neoantigen ; cancer vaccine ; VENUS ; prediction ; MC38 ; Medicine ; R
Subject code	616
Language	English
Publishing date	2021-08-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Correction

PLoS Neglected Tropical Diseases, Vol 15, Iss 4, p e

A next generation vaccine against human rabies based on a single dose of a chimpanzee adenovirus vector serotype C.

2021 Volume 0009348

Abstract: This corrects the article DOI:10.1371/journal.pntd.0008459.]. ...

Abstract	[This corrects the article DOI:10.1371/journal.pntd.0008459.].
Keywords	Arctic medicine. Tropical medicine ; RC955-962 ; Public aspects of medicine ; RA1-1270
Language	English
Publishing date	2021-04-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

Emanuele Sasso / Rolando Paciello / Francesco D’Auria / Gennaro Riccio / Guendalina Froechlich / Riccardo Cortese / Alfredo Nicosia / Claudia De Lorenzo / Nicola Zambrano

BioMed Research International, Vol

2015 Volume 2015

Abstract: Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver ... ...

Abstract	Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications.
Keywords	Medicine ; R
Language	English
Publishing date	2015-01-01T00:00:00Z
Publisher	Hindawi Limited
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Absence of systemic toxicity changes following intramuscular administration of novel pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines to BALB/c mice

Ondondo, Beatrice / Alfredo Nicosia / Caroline Brennan / Steven J. Crome / Tomáš Hanke

Vaccine. 2013 Nov. 12, v. 31, no. 47

2013

Abstract: The systemic toxicity of three candidate HIV-1 vaccines plasmid pSG2.HIVconsv DNA (D), ChAdV63.HIVconsv (C) and MVA.HIVconsv (M) expressing chimeric immunogen derived from the most conserved regions of the HIV-1 proteome was evaluated in two repeat-dose ... ...

Abstract	The systemic toxicity of three candidate HIV-1 vaccines plasmid pSG2.HIVconsv DNA (D), ChAdV63.HIVconsv (C) and MVA.HIVconsv (M) expressing chimeric immunogen derived from the most conserved regions of the HIV-1 proteome was evaluated in two repeat-dose studies in the male and female BALB/c mice.In study UNO011, mice received three doses of 2×107 plaque-forming units of MVA.HIVconsv vaccine (MMM). In study UNO012, mice received 3 doses of 50μg of pSG2.HIVconsv DNA followed by a single dose of 5.95×109 virus particles of ChAdV63.HIVconsv vaccine (DDDC). Similarly constituted control groups received the vehicle alone (phosphate buffered saline) at the same volume-dose. All vaccines were administered by intramuscular needle injection into the right hind limb at 14-day intervals and animals were sacrificed 7 days after the last dose. Assessment of local and systemic toxicity was made. Induction of HIV-1-specific responses was confirmed. Parameters assessed included clinical condition, body weight, food consumption, ophthalmoscopy, haematology, blood chemistry, organ weight and macroscopic and microscopic pathology.In both studies, treatment with the candidate vaccines elicited strong HIV-1-specific T-cell responses. The vaccine treatment was well-tolerated without any adverse systemic toxicological changes. The local toxicity findings observed in these studies were consistent with the predicted response to a vaccine/substance administration by intramuscular injection.The three novel anti-HIV-1 vaccines were well tolerated when administered by intramuscular injection to BALB/c mice. These results supported an application for authorisation by the Medicines and Healthcare Products Regulatory Agency of the UK to test these vaccines for the first time in phase I clinical trials in healthy both uninfected subjects and HIV-1-infected patients stable on antiretroviral treatment.
Keywords	antigens ; antiretroviral agents ; blood chemistry ; chimerism ; clinical trials ; food consumption ; health services ; hematology ; HIV infections ; Human immunodeficiency virus 1 ; intramuscular injection ; mice ; patients ; plasmids ; proteome ; tissue weight ; T-lymphocytes ; toxicity ; vaccines ; viruses ; United Kingdom
Language	English
Dates of publication	2013-1112
Size	p. 5594-5601.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	605674-x
ISSN	1873-2518 ; 0264-410X
ISSN (online)	1873-2518
ISSN	0264-410X
DOI	10.1016/j.vaccine.2013.06.068
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries

Biancamaria Cembrola / Valentino Ruzza / Fulvia Troise / Maria Luisa Esposito / Emanuele Sasso / Valeria Cafaro / Margherita Passariello / Feliciano Visconte / Maddalena Raia / Luigi Del Vecchio / Anna Morena D’Alise / Riccardo Cortese / Elisa Scarselli / Nicola Zambrano / Claudia De Lorenzo / Alfredo Nicosia

BioMed Research International, Vol

2019 Volume 2019

Abstract: The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we ... ...

Abstract	The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.
Keywords	Medicine ; R
Subject code	570
Language	English
Publishing date	2019-01-01T00:00:00Z
Publisher	Hindawi Limited
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A next generation vaccine against human rabies based on a single dose of a chimpanzee adenovirus vector serotype C.

PLoS Neglected Tropical Diseases, Vol 14, Iss 7, p e

2020 Volume 0008459

Abstract: Rabies, caused by RNA viruses in the Genus Lyssavirus, is the most fatal of all infectious diseases. This neglected zoonosis remains a major public health problem in developing countries, causing the death of an estimated 25,000-159,000 people each year, ...

Abstract	Rabies, caused by RNA viruses in the Genus Lyssavirus, is the most fatal of all infectious diseases. This neglected zoonosis remains a major public health problem in developing countries, causing the death of an estimated 25,000-159,000 people each year, with more than half of them in children. The high incidence of human rabies in spite of effective vaccines is mainly linked to the lack of compliance with the complicated administration schedule, inadequacies of the community public health system for local administration by the parenteral route and the overall costs of the vaccine. The goal of our work was the development of a simple, affordable and effective vaccine strategy to prevent human rabies virus infection. This next generation vaccine is based on a replication-defective chimpanzee adenovirus vector belonging to group C, ChAd155-RG, which encodes the rabies glycoprotein (G). We demonstrate here that a single dose of this vaccine induces protective efficacy in a murine model of rabies challenge and elicits strong and durable neutralizing antibody responses in vaccinated non-human primates. Importantly, we demonstrate that one dose of a commercial rabies vaccine effectively boosts the neutralizing antibody responses induced by ChAd155-RG in vaccinated monkeys, showing the compatibility of the novel vectored vaccine with the current post-exposure prophylaxis in the event of rabies virus exposure. Finally, we demonstrate that antibodies induced by ChAd155-RG can also neutralize European bat lyssaviruses 1 and 2 (EBLV-1 and EBLV-2) found in bat reservoirs.
Keywords	Arctic medicine. Tropical medicine ; RC955-962 ; Public aspects of medicine ; RA1-1270
Language	English
Publishing date	2020-07-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
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Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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