FoxP3, CTLA-4, and IDO in Canine Melanocytic Tumors

Porcellato, Ilaria; Brachelente, Chiara; Cappelli, Katia; Menchetti, Laura; Silvestri, Serenella; Sforna, Monica; Mecocci, Samanta; Iussich, Selina; Leonardi, Leonardo; Mechelli, Luca

doi:10.1177/0300985820960131

Cited by 27 publications

(34 citation statements)

References 84 publications

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“…The presence of few CTLA-4 positive cells within the tumor microenvironment in the context of a negativity of tumor cells seems to indicate that this pathway is not particularly involved in the immunoescape of epSCCs. Still, further studies on CTLA-4 expression in veterinary neoplastic diseases are necessary as currently available data are still few [34,39,40]. To the best of the authors' knowledge, this was the first time CTLA-4 was investigated in horses.…”

Section: Discussionmentioning

confidence: 96%

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PD-L1/PD-1 and CTLA-4 Expression in Equine Penile Squamous Cell Carcinomas

Porcellato

Mecocci

Brachelente

et al. 2021

Animals

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In horses, penile squamous cell carcinomas (epSCCs) are among the most common cutaneous neoplastic lesions. These tumors usually arise in benign lesions such as viral plaques and papillomas frequently induced by Equus caballus papillomavirus type 2 (EcPV2) infection. In the last decade, the introduction of immune checkpoint inhibitors (ICI) for the treatment of human cancers has demonstrated promising results. Among the most commonly targeted pathways, there is PD-1/PD-L1 and CTLA-4. The aim of this study is to investigate the expression of the PD-1/PD-L1 pathway and CTLA-4 in the tumor microenvironment of epSCCs to assess the feasibility of an immunotherapeutic approach. Twenty equine epithelial tumors were retrospectively selected and submitted to RT-qPCR for PD-1 and PD-L1 genes. After testing antibodies cross-reactivity by western blotting, immunohistochemistry for PD-L1 and CTLA-4 was performed. Results from RT-qPCR demonstrated that 3/20 cases expressed the PD-L1 gene, whereas the PD-1 gene was not detected. Immunohistochemical positivity for PD-L1 was found only in one case. CTLA-4-positive cells were observe in all cases but were few (Mdn = 4.8; IQR = 2.3–7.1 cells/HPF). In this study group, PD-1/PD-L1 and CTLA-4 do not appear to be highly expressed and therefore the use of ICI in epSCCs may not have promising rates of response.

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Section: Discussionmentioning

confidence: 96%

“…Then, slides were washed with PBS and incubated in 3% H 2 O 2 for 10 min. After a protein blocking step, the incubation with rabbit polyclonal anti-PD-L1 and mouse monoclonal anti-CTLA-4 [34] antibodies was performed. After secondary biotinylated antibody and streptavidin incubation, immunolabeling was revealed with AEC.…”

Section: Immunohistochemistrymentioning

confidence: 99%

PD-L1/PD-1 and CTLA-4 Expression in Equine Penile Squamous Cell Carcinomas

Porcellato

Mecocci

Brachelente

et al. 2021

Animals

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“…The primer set is reported in Table 2. As previously described [31,32] a Real-Time PCR amplification using SsoFast EvaGreen Supermix (BioRad, Hercules, CA, USA) was performed in a CFX96 Real-Time System with 5 µL of 1:5 diluted cDNA. Each sample was tested in triplicate and fluorescence data were collected at the end of the second step of each cycle.…”

Section: Rt-qpcr For Host Gene Studymentioning

confidence: 99%

Equine Genital Squamous Cell Carcinoma Associated with EcPV2 Infection: RANKL Pathway Correlated to Inflammation and Wnt Signaling Activation

Mecocci

Porcellato

Armando

et al. 2021

Biology

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Equine genital squamous cell carcinomas (egSCCs) are among the most common equine tumors after sarcoids, severely impairing animal health and welfare. Equus caballus papillomavirus type 2 (EcPV2) infection is often related to these tumors. The aim of this study was to clarify the molecular mechanisms behind egSCCs associated with EcPV2 infection, investigating receptor activator of nuclear factor-kappa B ligand (RANKL) signaling in NF-kB pathway, together with the Wnt and IL17 signaling pathways. We analyzed the innate immune response through gene expression evaluation of key cytokines and transcription factors. Moreover, Ki67 index was assessed with immunohistochemistry. EcPV2-E6 DNA was checked, and viral presence was confirmed in 21 positive out to 23 cases (91%). Oncogene expression was confirmed in 14 cases (60.8%) for E6 and in 8 (34.7%) for E2. RANKL, nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB)-p50, NFKBp65, interleukin (IL)-6, IL17, IL23p19, IL8, IL12p35, IL12p40, β-catenin (BCATN1), FOS like 1 (FOSL1), and lymphoid enhancer binding factor 1 (LEF1) showed a significant upregulation in tumor samples compared to healthy tissues. Our results describe an inflammatory environment characterized by the activation of RANKL/RANK and IL17 with the relative downstream pathways, and a positive modulation of inflammatory cytokines genes such as IL6 and IL8. Moreover, the increase of BCATN1, FOSL1, and LEF1 gene expression suggests an activation of both canonical and non-canonical Wnt signaling pathway that could be critical for carcinogenesis and tumor progression.

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“…Therefore, naturally occurring cancer in pet dogs is an ideal animal model for studying complex immune interactions during cancer treatment (27)(28)(29). Various studies have shown that the CTLA-4 plays a similar role in canine cancer (30). However, ICIs for CTLA4 blockade are not commercially available for canine cancer patients due to the lack of species-speci c mAbs.…”

Section: Introductionmentioning

confidence: 99%

“…Nanobodies (Nbs), representing the variable fragments (VHH) of camelid heavy-chain-only antibodies (HcAbs), bind to antigens in the absence of a light chain (14). Nbs are small in size (~ 15kDa) and bind to antigens with high speci city and a nity like conventional mAbs (30,31,33). Nbs or Nb-based molecules are in different stages of development for cancer diagnosis and therapy (31)(32)(33)(34).…”

Section: Introductionmentioning

confidence: 99%

Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients

Marable

Ruiz

Jaiswal

et al. 2021

Preprint

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Background Cancer is the leading cause of death in the geriatric dog population. Currently, the use of immune checkpoint inhibitors (ICIs) such as anti-CTLA4 antibodies has markedly improved the prognosis of several cancers in their advanced stages. However, ICIs targeting CTLA4 blockade to treat canine cancer patients are yet to define. In this study, we sought to develop, characterize and assess whether chimeric heavy chain only antibodies (cHcAbs) against CTLA4 are viable therapeutic candidates for the treatment of canine cancers.Methods Anti-CTLA4 nanobodies (Nbs) were identified from a yeast nanobody (Nb) library using magnetic-assisted cell sorting (MACS) and flow cytometry. cHcAbs were engineered by genetically fusing the DNA sequences coding for anti-CTLA4 Nbs with the Fc domain of the subclass B of canine IgG. Recombinant cHcAbs were purified from ExpiCHO-S cells. Stable cell lines expressing canine CTLA4 and FcγRI were used to elucidate the binding ability and specificity of cHcAbs. PBMCs isolated from healthy dogs were used to evaluate the ability of cHcAbs to activate canine PBMCs.Results Novel Nbs were identified using the extracellular domain of canine CTLA4 protein to screen a fully synthetic yeast nanobody library. Purified Nbs bind specifically to natïve canine CTLA4. We report that chimeric HcAbs, which were engineered by fusing the anti-CTLA4 Nbs and Fc region of subclass B of canine IgG, were half the size of a conventional mAb and formed dimers. The chimeric HcAbs specifically binds both with canine CTLA4 and FcƳ receptors. As the binding of Nbs overlapped with the MYPPPY motif of canine CTLA4, these Nbs were expected to sterically disrupt the interaction of canine CTLA4 to B-7s. Like their human counterpart, canine CTLA4 was expressed on helper T cells and a small subset of cytotoxic T cells. Canine Tregs also constitutively expressed CTLA4, and stimulation with PMA/Ionomycin dramatically increased expression of CTLA4 on the cell surface. Stimulation of canine PBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control.Conclusions This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients. Furthermore, this approach provides a critical proof-of-concept for developing nanobody-based humanized anti-CTLA4 therapy for advanced stages of cancers.

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FoxP3, CTLA-4, and IDO in Canine Melanocytic Tumors

Cited by 27 publications

References 84 publications

PD-L1/PD-1 and CTLA-4 Expression in Equine Penile Squamous Cell Carcinomas

PD-L1/PD-1 and CTLA-4 Expression in Equine Penile Squamous Cell Carcinomas

Equine Genital Squamous Cell Carcinoma Associated with EcPV2 Infection: RANKL Pathway Correlated to Inflammation and Wnt Signaling Activation

Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients

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