Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies

Sasso, Emanuele; D’Avino, Chiara; Passariello, Margherita; D’Alise, Anna Morena; Siciliano, Daniela; Esposito, Maria Teresa; Froechlich, Guendalina; Cortese, Riccardo; Scarselli, Elisa; Zambrano, Nicola; Nicosia, Alfredo; Lorenzo, Claudia De

doi:10.1080/19420862.2018.1496772

Cited by 33 publications

(92 citation statements)

References 31 publications

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“…An untreated and a α-mPD-1 treated group were used as control. The growth of tumours was measured by caliper every 3 or 4 days using the formula (LxW 2 )/2 31 . Animals were sacrificed as soon as signs of distress or a tumour volume above 1500 mm 3 occurred.…”

Section: Viral Rescue Production and Titration And Realtime Pcr Analmentioning

confidence: 99%

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

Sasso

Froechlich

Cotugno

et al. 2020

Sci Rep

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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.Oncolytic viruses (OVs) represent a class of natural or engineered viral species, possessing the ability to infect cancer cells, while sparing healthy tissues 1 . This selectivity is usually conferred by attenuating mutations, resulting in preferential replication of viral genomes in tumour cells 2 . Besides reducing the tumour bulk, OVs also act as immunotherapeutic agents. This feature is principally due to the immunogenic cell death (ICD) mechanisms induced by OVs, including immunogenic apoptosis, necrosis, necroptosis, pyroptosis, and autophagic cell death [3][4][5] . Viral infection also induces the release of stimulating cytokines, such as IL-1, IL-6, IL-12, IL-18, IFN-γ. Along with these molecules, lysed cancer cells release tumour-associated antigens and cancer-related proteins. These mechanisms allow the immunosuppressed tumour microenvironment (TME) to turn into an immunocompetent habitat. This effect is potentiated in OVs, in which immunostimulatory cytokines or chemokines are encoded by engineered viral genomes, and can synergize with immune checkpoint blockade 6-8 . This may translate into a therapeutic opportunity to potentiate the effects of immune checkpoint blockade in patients refractory to immunotherapy 9,10 . Talimogene laherparepvec (T-VEC), a Herpes simplex-based OV (oHSV), was approved by FDA in 2015 for treatment of recurrent melanoma after initial surgery 11,12 . It holds a ICP34.5-deleted genome, resulting in attenuated neurovirulence, and diminished infection of normal cells. Transgen...

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Section: Viral Rescue Production and Titration And Realtime Pcr Analmentioning

confidence: 99%

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

Sasso

Froechlich

Cotugno

et al. 2020

Sci Rep

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“…Mutants. An scFv specific for PD-L1 protein, named "PD-L1_1," was previously identified from a phagemid library [62]. To improve the affinity of this scFv (hereafter called wild type) for PD-L1, we generated repertoires of mutants by CDR-targeted random mutagenesis, just restricted to the CDR3 (33 bp in length) of its VH.…”

Section: Multistep Random Mutagenesis Of An Anti-pd-l1 Scfv and High-mentioning

confidence: 99%

“…e Fc fragment only worked as tag for the detection of the protein by flow cytometry (see Materials and Methods). Although PD-L1 is a monomer in its physiological context, that is, when expressed on the plasma membrane of human cells, and although monomeric PD-L1 is commercially available, we preferred to use the same PD-L1 format as that used for the de novo isolation of the wild type PD-L1 specific scFv in a phage display system [62].…”

Section: Generation Of Scfv-displaying Yeast Libraries and Cell Sortimentioning

confidence: 99%

“…Our research group identified by phage display a novel, fully human anti-PD-L1 antibody with low nanomolar apparent affinity for PD-L1; this mAb was able to induce T cell proliferation in vitro and showed in vivo antitumor activity [62]. In this work, we generated novel anti-PD-L1 single chain variable fragments (scFvs) by affinity maturation of heavy chain complementarity determining region 3 (HCDR3) of the mAb previously characterized, using yeast surface display.…”

Section: Introductionmentioning

confidence: 99%

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

Cembrola

Ruzza

Troise

et al. 2019

BioMed Research International

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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.

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“…As both PDGFRβ and PD-L1 are crucial targets for TNBC treatments [31,43,44], we tested whether their co-inhibition would enhance TNBC cells killing. To this aim, we used an aptamer and a mAb that we previously validated as high a nity binders and inhibitors of PDGFRβ [31] and PD-L1 [40,45], respectively, in different tumor types including TNBC. Speci cally, Gint4.T is a 2'F-Py RNA aptamer that binds to the extracellular domain of PDGFRβ and inhibits receptor activation and downstream phosphatidyl-3-kinase (PI3K)/Akt and ERK1/2 pathways [22,31,35], thus affecting growth, migration and invasion of mesenchymal TNBC cell lines [31].…”

Section: Resultsmentioning

confidence: 99%

Aptamer targeted therapy potentiates immune checkpoint blockade in triple-negative breast cancer

Camorani

Passariello

Agnello

et al. 2020

Preprint

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Background: Triple-negative breast cancer (TNBC) is a uniquely aggressive cancer with high rates of relapse due to resistance to chemotherapy. TNBC expresses higher levels of programmed cell death-ligand 1 (PD-L1) compared to other breast cancers, providing the rationale for the recently approved immunotherapy with anti-PD-L1 monoclonal antibodies (mAbs). A huge effort is dedicated to identify actionable biomarkers allowing for combination therapies with immune-checkpoint blockade. Platelet-derived growth factor receptor β (PDGFRβ) is highly expressed in invasive TNBC, both on tumor cells and tumor microenvironment. We recently proved that tumor growth and lung metastases are impaired in mouse models of human TNBC by a high efficacious PDGFRβ aptamer. Hence, we aimed at investigating the effectiveness of a novel combination treatment with the PDGFRβ aptamer and anti-PD-L1 mAbs in TNBC.Methods: The targeting ability of the anti-human PDGFRβ aptamer toward the murine receptor was verified by streptavidin-biotin assays and confocal microscopy, and its inhibitory function by transwell migration assays. The anti-proliferative effects of the PDGFRβ aptamer/anti-PD-L1 mAbs combination was assessed in human MDA-MB-231 and murine 4T1 TNBC cells, both grown as monolayer or co-cultured with lymphocytes. Tumor cell lysis and cytokines secretion by lymphocytes were analyzed by LDH quantification and ELISA, respectively. Orthotopic 4T1 xenografts in syngeneic mice were used for dissecting the effect of aptamer/mAb combination on tumor growth, metastasis and lymphocytes infiltration. Ex vivo analyses through immunohistochemistry, RT-qPCR and immunoblotting were performed. Results: We show that the PDGFRβ aptamer potentiates the anti-proliferative activity of anti-PD-L1 mAbs on both human and murine TNBC cells, according to its human/mouse cross-reactivity. Further, by binding to activated human and mouse lymphocytes, the aptamer enhances the anti-PD-L1 mAb-induced cytotoxicity of lymphocytes against tumor cells. Importantly, the aptamer heightens the antibody efficacy in inhibiting tumor growth and lung metastases in mice. It acts on both tumor cells, inhibiting Akt and ERK1/2 signaling pathways, and immune populations, increasing intratumoral CD8+T cells and reducing FOXP3+Treg cells. Conclusion: Co-treatment of PDGFRβ aptamer with anti-PD-L1 mAbs is a viable strategy, thus providing for the first an evidence of the efficacy of PDGFRβ/PD-L1 co-targeting combination therapy in TNBC.

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Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies

Cited by 33 publications

References 31 publications

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

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

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

Aptamer targeted therapy potentiates immune checkpoint blockade in triple-negative breast cancer

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