Drug conjugation to hyaluronan widens therapeutic indications for ovarian cancer

Management of ovarian cancer still requires improvements in therapeutic options. A drug delivery strategy was tested that allows specific targeting of tumor cells in combination with a controlled release of a cytotoxic molecule. To this aim, the efficacy of a loco-regional intraperitoneal treatment with a bioconjugate (ONCOFID-S) derived by chemical linking of SN-38, the active metabolite of irinotecan (CPT-11), to hyaluronan was assessed in a mouse model of ovarian carcinomatosis. In vitro, the bioconjugate selectively interacted with ovarian cancer cells through the CD44 receptor, disclosed a dose-dependent tumor growth inhibition efficacy comparable to that of free SN-38 drug, and inhibited Topoisomerase I function leading to apoptosis by a mechanism involving caspase-3 and -7 activation and PARP cleavage. In vivo, the intraperitoneal administration of ONCOFID-S in tumor-bearing mice did not induce inflammation, and evidenced an improved therapeutic efficacy compared with CPT-11. In conclusion, SN-38 conjugation to hyaluronan significantly improved the profile of in vivo tolerability and widened the field of application of irinotecan. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of ovarian cancer

P06.09 Anti-hPSMA CAR engineered NK-92 cells: An off-the-shelf cellular therapeutic for targeted elimination of prostate cancer cells

Background Adoptive cell therapy of malignant diseases takes advantages of the cellular immune system to recognize and destroy cancer cells. Despite the remarkable success in B cell malignancies after adoptive transfer of CD19 CAR T cells, CAR T cell therapy in solid tumors has shown less encouraging clinical results, above all caused by tumor escape mechanisms.In order to overcome such limitations, NK-92, a permanent and IL-2-dependent cell line with a high cytotoxicity in vitro, has been engineered in preclinical models with CAR. In this project, we exploited a CAR directed against the human antigen hPSMA that is overexpressed in prostate tumors. This project aimed at transducing NK-92 cell line to obtain a hPSMA-specific CAR NK-92 cell population, to be thereafter characterized in vitro and in vivo for antigen-specific functional activity. Materials and Methods NK-92 cell line was transduced with a lentiviral vector (LV) carrying a CAR anti-hPSMA. The cell population obtained was then sorted and analyzed for degranulation capacity, IFNγ production and lytic activity against hPSMA+ (PC3-hPSMA, LNCaP) or hPSMA-tumor cell lines. In vivo therapeutic efficacy of CAR-transduced NK-92 was evaluated initially using Winn-Assay and than in subcutaneous and orthotopic tumor models. Results CAR-expressing LV efficiently transduced NK-92 cells, which in turn produced cytokines, degranulated and exerted a relevant cytotoxic upon challenge with PSMA+ prostate tumor cells, irrespective of 10 Gy γ-irradiation. In all the in vivo, tumor models CAR-transduced NK-92 shown a statistically significant inhibition of tumor growth. Conclusions Chimeric antigen receptor-engineered NK-92 could offer a valid and cost-effective alternative to primary CAR NK or T cells, in particular in cases, where a suitable donor is not available or the sophisticated infrastructure needed for cell isolation, expansion and genetic modification is missing. This work demonstrates that CAR-engineered NK-92 cells display a high and specific recognition of hPSMA+ PC both in vitro as is in vivo, and could represent an efficient strategy as a new therapeutic intervention against prostate carcinoma, thus paving the way to an Off-The-Shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective antitumor immunity. Disclosure Information G. Zuccolotto: None. A. Penna: None. I.M. Montagner: None. D. Carpanese: None. A. Rosato: None

Anti-PSMA CAR-engineered NK-92 Cells: An Off-the-shelf Cell Therapy for Prostate Cancer

Prostate cancer (PCa) has become the most common cancer among males in Europe and the USA. Adoptive immunotherapy appears a promising strategy to control the advanced stages of the disease by specifically targeting the tumor, in particular through chimeric antigen receptor T (CAR-T) cell therapy. Despite the advancements of CAR-T technology in the treatment of hematological malignancies, solid tumors still represent a challenge. To overcome current limits, other cellular effectors than T lymphocytes are under study as possible candidates for CAR-engineered cancer immunotherapy. A novel approach involves the NK-92 cell line, which mediates strong cytotoxic responses against a variety of tumor cells but has no effect on non-malignant healthy counterparts. Here, we report a novel therapeutic approach against PCa based on engineering of NK-92 cells with a CAR recognizing the human prostate-specific membrane antigen (PSMA), which is overexpressed in prostatic neoplastic cells. More importantly, the potential utility of NK-92/CAR cells to treat PCa has not yet been explored. Upon CAR transduction, NK-92/CAR cells acquired high and specific lytic activity against PSMA-expressing prostate cancer cells in vitro, and also underwent degranulation and produced high levels of IFN-\u3b3 in response to antigen recognition. Lethal irradiation of the effectors, a safety measure requested for the clinical application of retargeted NK-92 cells, fully abrogated replication but did not impact on phenotype and short-term functionality. PSMA-specific recognition and antitumor activity were retained in vivo, as adoptive transfer of irradiated NK-92/CAR cells in prostate cancer-bearing mice restrained tumor growth and improved survival. Anti-PSMA CAR-modified NK-92 cells represent a universal, off-the-shelf, renewable, and cost-effective product endowed with relevant potentialities as a therapeutic approach for PCa immunotherapy

P09.03 Hyaluronic acid as a new immunologic adjuvant in cancer: design of effective preventive and therapeutic vaccination strategies for HER2/neu-positive breast tumors

Background The use of proteins as immunogens is attractive for the development of vaccines, but requires efficient adjuvants to overcome their weak immunogenicity. Recently, we investigated the potential of the TLR2/4 agonist hyaluronan (HA) as an immunological adjuvant for protein-based vaccines.1 2 Conjugation of HA to antigens strongly increased their immunogenicity and promoted their rapid translocation to draining lymph nodes, resulting in robust and long-lasting humoral responses.1 On these bases, we investigated the potentiality of HA-based technology in the design of cancer vaccines. To this aim, HA was conjugated to the extracellular domain of rat HER2/neu (rHER2/neu) and validated in the preventive and therapeutic vaccination settings. Materials and Methods Female BALB/c or BALB-neuT mice were immunized with rHER2/neu-HA. In vivo depletion of CD4+, CD8+ T and B cells was performed, and sera and spleens were collected to characterized antigen-specific humoral and cellular responses. Vaccinated BALB/c mice were challenged and re-challenged with rHER2/neu-overexpressing TUBO cells to assess the protective or therapeutic activity of rHER2/neu-HA vaccination strategy, as well as immunological memory. Results HA performed efficiently as robust and long-lasting humoral (IgG1, IgG2a, and IgG2b) and cellular responses were detected using very low antigen doses and number of boosters. Outstandingly, at 1-year post-vaccination, anti-rHER2/neu specific antibodies showed even improved effector functions (maturation of affinity for the receptor and increased complement-derived cytotoxicity functions). HA vaccination turned out effective in both the prophylactic (100% mice survived) and therapeutic (tumor regression in 2/12 mice) settings, and broke tolerance against rHER2/neu, delaying spontaneous tumor growth in BALB-neuT mice. Both humoral and cellular responses contributed to the success of HA-based vaccination, but CD8+ T cells played only a marginal role. Conclusions Cancer vaccines have not yet achieved significant clinical efficacy due to their poor immunogenicity, and the validation of more effective adjuvants occurred sometimes at the expense of safety. HA combines the unique immunomodulatory features of a TLR agonist with the tolerability of a fully natural polymer, proving to be a promising adjuvant for the creation of effective and safe cancer vaccines with the potential for rapid clinical translation. References Dalla Pieta A, Carpanese D, et al. Hyaluronan is a natural and effective immunological adjuvant for protein-based vaccines. Cell Mol Immunol 2021;18(5):1197–1210. Rosato A, Montagner IM, Carpanese D, Dalla Pieta A. Hyaluronic acid as a natural adjuvant for protein and peptide-based vaccines. 30.04.2020. WO/2020/084558, PCT/IB2019/059122. Disclosure Information D. Carpanese: None. I. Montagner: None. A. Dalla Pieta: None. V. Rossi: None. A. Penna: None. G. Zuccolotto: None. G. Pasut: None. A. Grigoletto: None. A. Rosato: None

Chimeric Antigen Receptor Engineered T cells for adoptive immunotherapy of Prostate Cancer

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. In this regard, however, results from clinical trials have shown that a single-approach immunotherapy (antibodymediated or cell-mediated) might be insufficient to eradicate tumor cells and allows them to survive and adapt to the body’s defence mechanisms or to passively administered cures. The aim of my PhD project was the generation of CARengineered T cells specific for the human Prostate Specific Membrane Antigen (hPSMA), and their funtional evaluation in vitro and in vivo. We exploited the concept that combination of two powerful tools, by endowing T cells with recognition capacity and high specificity of antibodies, can lead to results superior to those obtained by single-approach treatments. To this aim, we developed a CAR containing both the CD3zeta and CD28 signalling moieties fused to a scFv targeting the hPSMA, to engineer human PBMC for the immunotherapy of prostate cancer. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter, capable of robust and coordinated expression of two transgenes, thus allowing to co-express the CAR in conjunction with a reporter gene (luciferase), to track the transgenic T cell population to the tumor site by in vivo optical imaging after adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC that, in turn, were readily stimulated to produce cytokines and exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Then, we exploited our in vivo imaging know-how and set up some experiments to precisely define the effect of the Tbodies in tumour-grafted mice and to visualize their effect in a mouse model of prostate cancer. To this end, tumour cells were transduced with a lentiviral vector coding for the reporter gene luciferase. Stably transduced cells were then used for the in vivo experiments as described above. For the analysis, mice were anaesthetized and injected with luciferin, the substrate of the enzime luciferase: the catabolyc reaction produced bioluminescence that can be visualized using Ivis Lumina II instrument (Xenogen). Last, for the setting up of a prostate cancer mouse model, we injected s.c. and i.v. luciferase-transduced PC3-hPSMA cells or PC3-WT as control. Than we evaluated the therapeutic efficacy of the Tbodies after local and systemical injection. Mice were analyzed weekly, using the Ivis Lumina II platform. Upon in vivo transfer in tumor-bearing mice, CAR-engineered T cells survived shortly but were nonetheless capable of inducing striking therapeutic effects, thus supporting the transfer of this approach to clinical settings.Nell'ambito dell'immunoterapia cellulare adottiva, l’ingegnerizzazione di linfociti T con recettori diretti contro antigeni tumorali rappresenta un'efficace strategia per generare in tempi rapidi un elevato numero di linfociti tumore-specifici. In alternativa al TCR fisiologico, la cellula T può essere ingegnerizzata con recettori chimerici per l'antigene (CAR) costituiti da un dominio di riconoscimento antigenico (derivato da anticorpi monoclonali) fuso a domini di trasduzione del segnale derivati dal complesso TCR: questo tipo di struttura combina la specificità del riconoscimento anticorpale (MHC-indipendente) con le potenzialità anti-tumorali dei linfociti T. L’attività di ricerca svolta nel mio corso di Dottorato si è focalizzata sullo sviluppo di un protocollo per la generazione di linfociti T ingegnerizzati con un CAR di seconda generazione (scFv-CD28-CD3ζ) diretto contro l'antigene hPSMA (Prostate Specific Membrane Antigen) per il trattamento del carcinoma prostatico. L'utilizzo di vettori lentivirali (LV) come alternativa ai più diffusi vettori oncoretrovirali, consente di trasdurre in modo efficace cellule T scarsamente differenziate, con positive implicazioni sulla loro funzionalità in vivo; in questo studio, inoltre, la presenza nel vettore LV di un promotore bidirezionale recentemente descritto ha permesso l'espressione coordinata del CAR e del gene reporter Firefly Luciferase con l'obiettivo di monitorare il destino biologico dei linfociti trasferiti adottivamente in vivo, mediante imaging di bioluminescenza (BLI). La popolazione di PBMC ingegnerizzati con recettore chimerico (T-body) è caratterizzata da un'elevata percentuale di espressione del CAR anti-hPSMA (superiore al 50%), da un fenotipo di memoria e dalla capacità di riconoscere e lisare in modo specifico cellule esprimenti l'antigene. In un modello murino di tumore prostatico sottocutaneo i T-body si sono dimostrati efficaci in diversi protocolli terapeutici: co-inoculati con cellule tumorali, inoculati a livello locale, o inoculati per via sistemica in topi portatori di tumori disseminati. Contrariamente il trasferimento adottivo per via sistemica non ha determinato alcun risultato terapeutico nei tumori sottocutanei. Il monitoraggio della distribuzione in vivo dei linfociti mediante BLI ha infatti evidenziato una scarsa capacità di sopravvivenza e di homing al sito tumorale. I risultati positivi ottenuti in questo lavoro, in particolare lo sviluppo di un recettore chimerico anti-hPSMA di seconda generazione, l'utilizzo di vettori LV e la generazione di T-body funzionali in vitro e in vivo, costituiscono il razionale per ulteriori studi e per future applicazioni cliniche di questo tipo di approccio in pazienti con carcinoma prostatico; rimane tuttavia fondamentale chiarire le dinamiche di ricircolazione e distribuzione delle cellule T con l'obiettivo di implementarne la capacità di sopravvivere, ricircolare e raggiungere il sito tumorale, fattori indispensabili per mediare l'effettiva regressione della neoplasia

New 99mTc-radioimmunoconjugates for pancreatic carcinoma detection

Pancreas carcinoma is responsible for more than 30% of tumor-related death because it is notoriously difficult to diagnose; thus, new diagnostic approaches are imperatively needed. Recently, prostate stem cell antigen (PSCA) and mesothelin demonstrated high expression and wide distribution in pancreatic cancer, but not in normal pancreas. This research aims to develop new radioimmunoconjugates (RICs) for pancreatic cancer detection based on monoclonal antibodies (mAb) to PSCA and mesothelin, which are heavily overexpressed in this tumor histotype. Either mAbs labeled with fluorophore exhibited high recognition capacity for Ag + tumor-cells both in vitro and in vivo, as assessed by cytometry analysis and optical imaging. 99mTc-radioimmunoconjugates were obtained with high labelling efficiency (98%), by reduction of both mAbs with 2-mercaptoethanol and incubation with 99mTcO4 12 in presence of a weak competition ligand. Stability tests performed in saline solution and human serum demonstrated that RICs are stable for at least 48 h. Both mAbs were radiolabeled by direct method with a high radiochemical efficiency and stability, and could be used for pancreatic cancer detection

PSMA-Specific Car-Engineered T Cells for Prostate Cancer: CD28 outperforms combined CD28-41BB “Super- stimulation”

Prostate cancer (PCa) is the second leading cause of malignancy-related mortality in males in the Western world. Although treatment like prostatectomy and radiotherapy for localized cancer have good results, similar positive outcomes are not achieved in metastatic PCa. Consequently, these aggressive and metastatic forms of PCa urgently need new methods of treatment. We already described an efficient and specific secondgeneration (2G) Chimeric Antigen Receptor (CAR) against Prostate Specific Membrane Antigen (PSMA), a glycoprotein overexpressed in prostate cancer and also present on neovasculature of several tumor entities. In an attempt to improve efficacy and in vivo survival of anti-PSMA 2G CAR-T cells, we developed a third generation (3G) CAR containing two costimulatory elements, namely CD28 and 4-1BB co-signaling domains, in addition to CD3z. Differently from what described for other 3G receptors, our third generation CAR disclosed an antitumor activity in vitro similar to the related 2G CAR that comprises the CD28 co-signaling domain only. Moreover, the additional costimulatory domain produced detrimental effects, which could be attributed to an increased activation-induced cell death (AICD). Indeed, such “superstimulation” resulted in an exhausted phenotype of CAR-T cells, after prolonged in vitro restimulation, a higher frequency of cell death, and an impairment in yielding sufficient numbers of transgenic T lymphocytes. Thus, the optimal combination of costimulatory domains for CAR development should be assessed cautiously and evaluated case-by-case

Drug conjugation to hyaluronan widens therapeutic indications for ovarian cancer

ABSTRACT Management of ovarian cancer still requires improvements in therapeutic In vitro, the bioconjugate selectively interacted with ovarian cancer cells through the CD44 receptor, disclosed a dose-dependent tumor growth inhibition efficacy comparable to that of free SN-38 drug, and inhibited Topoisomerase I function leading to apoptosis by a mechanism involving caspase-3 and -7 activation and PARP cleavage. In vivo, the intraperitoneal administration of ONCOFID-S in tumor-bearing mice did not induce inflammation, and evidenced an improved therapeutic efficacy compared with CPT-11. In conclusion, SN-38 conjugation to hyaluronan significantly improved the profile of in vivo tolerability and widened the field of application of irinotecan. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of ovarian cancer

Hematopoietic and non-hematopoietic p66Shc differentially regulates stem cell traffic and vascular response to ischemia in diabetes

Peripheral artery disease (PAD) is a severe complication of diabetes, characterized by defective traffic of hematopoietic stem/progenitor cells (HSPCs). We examined the hematopoietic versus non-hematopoietic role of p66Shc in regulating HSPC traffic and blood flow recovery after ischemia in diabetic mice

Collagen VI regulates peripheral nerve regeneration by modulating macrophage recruitment and polarization

Macrophages contribute to peripheral nerve regeneration and produce collagen VI, an extracellular matrix protein involved in nerve function. Here, we show that collagen VI is critical for macrophage migration and polarization during peripheral nerve regeneration. Nerve injury induces a robust upregulation of collagen VI, whereas lack of collagen VI in Col6a1(-/-) mice delays peripheral nerve regeneration. In vitro studies demonstrated that collagen VI promotes macrophage migration and polarization via AKT and PKA pathways. Col6a1(-/-) macrophages exhibit impaired migration abilities and reduced antiinflammatory (M2) phenotype polarization, but are prone to skewing toward the proinflammatory (M1) phenotype. In vivo, macrophage recruitment and M2 polarization are impaired in Col6a1(-/-) mice after nerve injury. The delayed nerve regeneration of Col6a1(-/-) mice is induced by macrophage deficits and rejuvenated by transplantation of wild-type bone marrow cells. These results identify collagen VI as a novel regulator for peripheral nerve regeneration by modulating macrophage function