Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate

<p>Abstract</p> <p>Background</p> <p>Dendritic cells (DCs) are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines.</p> <p>Methods</p> <p>Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1) culture media; 2) culture surface; 3) duration of activating DCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma; 4) method of DC harvest; and 5) cryomedia and final DC product formulation.</p> <p>Results</p> <p>DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning^®tissue-culture treated surface and Corning^®ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC) Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs), and high IL-12p70 production <it>in vitro </it>that continued for 24 hours after the cryopreserved DCs were thawed and replated in fresh media.</p> <p>Conclusions</p> <p>This study examined criteria including DC phenotype, viability, IL-12p70 production and the ability to stimulate MLR as metrics of whole oxidized tumor lysate-pulsed DC immunogenicity and functionality. Development and optimization of this unique method is now being tested in a clinical trial of autologous oxidized tumor lysate-pulsed DC in clinical-scale in recurrent ovarian, primary peritoneal or fallopian tube cancer (NCT01132014).</p

Targeting the tumor vasculature to enhance T cell activity.

T cells play a critical role in tumor immune surveillance as evidenced by extensive mouse-tumor model studies as well as encouraging patient responses to adoptive T cell therapies and dendritic cell vaccines. It is well established that the interplay of tumor cells with their local cellular environment can trigger events that are immunoinhibitory to T cells. More recently it is emerging that the tumor vasculature itself constitutes an important barrier to T cells. Endothelial cells lining the vessels can suppress T cell activity, target them for destruction, and block them from gaining entry into the tumor in the first place through the deregulation of adhesion molecules. Here we review approaches to break this tumor endothelial barrier and enhance T cell activity

Development of immunotherapeutic cellular models for the treatment of ovarian cancer

T cell-based adoptive immunotherapy is a promising approach for the therapy of patients with advanced epithelial ovarian cancer (EOC). However, adoptive T cell therapy currently requires the isolation and characterization of rare antigen-specific T cells from each patient thus limiting its broad application. However, large amounts of tumor-reactive T cells with redirected specificity toward tumor associated antigens (TAA) can be generated following their genetic engineering with: A. Antigen-specific T cell receptors (TCRs) that bind to endogenously processed antigen-derived peptides that are presented by the major histocompatibility complex (MHC) molecules. B. Chimeric antigen receptors (CARs) that couple the high affinity binding of antibodies with the signaling domains of the T cell receptor and can bind to extracellular antigens in a non-MHC dependent manner.Ovarian cancers express a variety of TAA. Amongst those antigens ErbB2 (HER2/neu) represents an attractive candidate for the immunotherapy of cancer patients. However, the breadth of ErbB2 expression by primary human ovarian cancers remains controversial, which questions the suitability of this antigen for T cell based therapies. To address this issue, we performed extensive ErbB2 expression analysis on a wide panel of primary tumors as well as established and short-term human ovarian cancer cell lines. Sensitive detection methods including flow cytometry, western blot analysis and q-PCR revealed ErbB2 expression in all fresh tumor cells derived from primary ascites or solid tumors as well as all established cancer cell lines.In order to develop T cell immunotherapeutic strategies against ErbB2-expressing tumors we isolated a novel ErbB2-specific TCR from highly avid ErbB2-specific CD8+ T cells. T cells genetically modified to express the ErbB2 TCR recognized some ovarian tumor cell lines that efficiently processed and presented the ErbB2 peptide 369-377 through their MHC class I molecules (HLA-A2). In order to improve the targeting of ErbB2-expressing ovarian cancer and we sought toemploy CAR T cell therapy. Genetically-engineered human T cells expressing an ErbB2-specific chimeric antigen receptor (CAR) recognized and reacted against all established or primary ovarian cancer cells.For the development of immunotherapeutic approaches against other tumor antigens we constructed and evaluated a fully-human CAR with specificity for mesothelin which is widely expressed in EOC. Primary human T cells expressing the P4 CAR specifically produced exerted potent cytolytic functions when co-cultured with mesothelin-expressing tumors in vitro. Of note P4 CAR reactivity was not abrogated by soluble tumor-secreted or recombinant mesothelin protein even at supraphysiological levels. Importantly, adoptive transfer of P4 CAR-expressing T cells mediated the regression of large, established tumor in a xenogenic model of human ovarian cancer.A caveat of the P4 CAR approach is the possible on-target toxicity in normal mesothelial tissues that express low levels of mesothelin. To specify T cells for robust effector function that is selective for tumor but not normal tissue, we developed a trans-signaling CAR strategy that employs two CARs of differing antigen specificity; one recognizing mesothelin and the other the folate receptor alpha (FRα). T cells were simultaneously transduced with a CAR that provides suboptimal activation upon binding of mesothelin and a chimeric costimulatory receptor that recognizes a FRα. Trans-signaling CAR T cells demonstrated strong reactivity only against tumor cells co-expressing both antigens. Only weak reactivity was observed against mesothelin-expressing target cells both in vitro and in vivo. Thus, a dual specificity, trans-signaling CAR approach can potentiate the therapeutic efficacy of CAR T cells against cancer while minimizing "on target " toxicity against normal tissues bearing a single antigen.In summary, we have successfully constructed, developed and characterized physiological (TCR) and chimeric antigen receptors (CAR) of human origin. Finally to retain the powerful capacity of a conventional CAR T cell approach and increase the safety of the CAR T cells we developed a novel trans signaling CAR T cell platform. The above mentioned T cell engineering strategies will improve the anti-cancer effectiveness of T cell adoptive transfer therapies and inaugurate avenues for the successful treatment of ovarian cancer.Η μεταφορά αυτόλογων Τ λεμφοκυττάρων που έχουν υψηλή ικανότητα αναγνώρισης και δραστικότητας κατά συγκεκριμένων αντιγόνων του καρκίνου των ωοθηκών αποτελεί μια ισχυρή θεραπευτική προσέγγιση για τη θεραπεία του καρκίνου των ωοθηκών. Ένας περιοριστικός παράγοντας στις τρέχουσες ανοσοθεραπευτικές προσεγγίσεις είναι ότι τα καρκινικά κύτταρα αναπτύσσουν μηχανισμούς καταστολής της ανοσολογικής επιτήρησης τους. Έτσι τα Τ κύτταρα που έχουν υψηλή ειδικότητα και δραστικότητα κατά διαφόρων καρκινικών αντιγόνων απουσιάζουν από τους όγκους των ωοθηκών. Με στόχο την δημιουργία Τ κυττάρων με ισχυρή αντικαρκινική δραστικότητα προχωρήσαμε στη γενετική τροποποίηση Τ κυττάρων με: Α. Φυσιολογικούς Τ κυτταρικούς υποδοχείς (TCRs) με ειδικότητα για καρκινικά πεπτίδια προσδεδεμένα σε MHC μόρια τάξεως Ι. Β. Χιμαιρικούς Τ ανοσοϋποδοχείς (CARs) οι οποίοι αναγνωρίζουν εξωκυττάριες πρωτεΐνες των καρκινικών κυττάρων ανεξάρτητα από την έκφραση των MHC μορίων.Τα ωοθηκικά καρκινικά κύτταρα εκφράζουν ένα πλήθος αντιγόνων τα οποία είναι στενά συνδεδεμένα με την εξέλιξη και την επιθετικότητα του όγκου. Ένα από τα ελκυστικότερα αντιγόνα-στόχους είναι το ErbB2 (HER2/neu). Το εύρος όμως της έκφρασης αυτού του υποδοχέα στην επιφάνεια των καρκινικών κυττάρων παραμένει αμφισβητούμενο. Γι' αυτό το λόγο πραγματοποιήσαμε εκτενή ανάλυση της έκφρασης του ErbB2 σε ένα μεγάλο αριθμό πρωτογενών όγκων και καρκινικών σειρών. Με εφαρμογή πιο ευαίσθητων μεθόδων ανίχνευσης και ανάλυσης, όπως είναι η κυτταρομετρία ροής, η ανοσοαποτύπωση και η ποσοτική PCR αποδείξαμε την ευρεία έκφραση του ErbB2 σε όλα τα καρκινικά κύτταρα που προήλθαν από πρωτοπαθείς στερεούς όγκους και ασκίτες καθώς επίσης και από ωοθηκικές καρκινικές σειρές.Για τη στόχευση του υποδοχέα του ErbB2 κλωνοποιήσαμε ένα καινοτόμο Τ κυτταρικό υποδοχέα με ειδικότητα για τον ErbB2 επίτοπο 369-377 από ErbB2-ειδικά Τ κύτταρα. Γενετικά τροποποιημένα Τ κύτταρα που εξέφραζαν τον ανασυνδυασμένο ErbB2-ειδικό Τ κυτταρικό υποδοχέα αντέδρασαν κατά ορισμένων καρκινικών σειρών Οι συγκεκριμένες σειρές παρουσιάζαν σε επαρκή ποσότητα τον ErbB2 αντιγονικό επιτόπο 369-377 με μόρια MHC τάξεως I (HLA-A2) πάνω στην κυτταρική επιφάνεια. Αντιθέτως, γενετικά τροποποιημένα Τ κύτταρα που εξέφραζαν ένα ErbB2-χιμαιρικό ανοσοϋποδοχέα αντέδρασαν κατά όλων των πρωτοπαθών καρκινικών κυττάρων και καρκινικών σειρών με επακόλουθο την έκκριση κυτταροκινών και τον θάνατο των καρκινικών κυττάρων.Για την ανάπτυξη ανοσοθεραπευτικών προσέγγισεων έναντι άλλων αντιγόνων κατασκευάσαμε έναν χιμαιρικό ανοσοϋποδοχέα (P4) με ειδικότητα για τη μεσοθηλίνη η οποία εκφράζεται ευρέως στον καρκίνο των ωοθηκών. T κύτταρα που είχαν τροποποιηθεί γενετικά ώστε να εκφράζουν το P4 χιμαιρικό ανοσουποδοχέα παρουσίασαν ισχυρή δραστικότητα κατά καρκινών κυττάρων που εκφράζουν μεσοθηλίνη και ικανότητα εξολόθρευσης συμπαγών όγκων in vivo. Επίσης βρήκαμε ότι η ενεργότητα των P4 CAR Τ κυττάρων δεν επηρεάστηκε από τα υψηλά επίπεδα μεσοθηλίνης που εκκρίνονται από τα καρκινικά κύτταρα.Λόγω του χαμηλού αλλά ανιχνεύσιμου επιπέδου έκφρασης της μεσοθηλίνης στα φυσιολογικά μεσοθηλιακά κύτταρα, η ασφάλεια της χρήσης γενετικά τροποποιημένων P4 Τ κυττάρων εγκυμονεί τον κίνδυνο καταστροφής φυσιολογικών ιστών. Με στόχο την αύξηση της επιλεκτικότητας των T κυττάρων έναντι των καρκινικών κυττάρων αναπτύξαμε μια καινοτόμο πλατφόρμα γενετικής τροποποίησης αυτών με δύο χιμαιρικούς ανοσοϋποδοχείς ειδικότητας για τα καρκινικά αντιγόνα μεσοθηλίνη και υποδοχέα του φυλλικού οξέος (FRα). Για τηπλήρη λειτουργία αυτών των Τ κυττάρων πρέπει να υπάρχει δυνατότητα ταυτόχρονης πρόσδεσης και των δύο υποδοχέων στους αντιγονικούς τους στόχους. Τα διπλής ειδικότητας T κύτταρα παρουσιάσαν ισχυρή δραστικότητα μόνο κατά καρκινικών κύτταρων τα οποία εκφράζουν και τις δύο πρωτεΐνες. Αντιθέτως, τα Τ αυτά κύτταρα επέδειξαν δραματικά μειωμένο βαθμό λειτουργιών έναντι κυττάρων που εξέφραζαν μόνο μεσοθηλίνη. Συνεπώς η στρατηγική αυτή καθιστά δυνατή την αποτελεσματική εξολόθρευση καρκινικών κυττάρων και την ελαχιστοποίηση της ανεπιθύμητης στόχευσης φυσιολογικών ιστών.Ανακεφαλαιώνοντας, στη παρούσα διατριβή περιγράψαμε την ανάπτυξη και δοκιμή διαφόρων προσεγγίσεων με τις οποίες μπορούν να δημιουργηθούν Τ κύτταρα με ισχυρή αντικαρκινική δραστικότητα για τη θεραπεία του καρκίνου των ωοθηκών. Τέλος για την ασφαλή εφαρμογή αυτών των προσεγγίσεων σε μελλοντικές κλινικές δοκιμές με ασθενείς καρκίνου των ωοθηκών αναπτύξαμε μια μέθοδο για την ελαχιστοποίηση της Τ κυτταρικής δραστικότητας κατά των φυσιολογικών ιστών

Overexpression of GPC6 and TMEM132D in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p=0.0067) and 0.861 (p<0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p=0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling

Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels

<div><p>The breadth of HER2 expression by primary human ovarian cancers remains controversial, which questions its suitability as a universal antigen in this malignancy. To address these issues, we performed extensive HER2 expression analysis on a wide panel of primary tumors as well as established and short-term human ovarian cancer cell lines. Conventional immunohistochemical (IHC) analysis of multiple tumor sites in 50 cases of high-grade ovarian serous carcinomas revealed HER2 overexpression in 29% of evaluated sites. However, more sensitive detection methods including flow cytometry, western blot analysis and q-PCR revealed HER2 expression in all fresh tumor cells derived from primary ascites or solid tumors as well as all established and short-term cultured cancer cell lines. Cancer cells generally expressed HER2 at higher levels than that found in normal ovarian surface epithelial (OSE) cells. Accordingly, genetically-engineered human T cells expressing an HER2-specific chimeric antigen receptor (CAR) recognized and reacted against all established or primary ovarian cancer cells tested with minimal or no reactivity against normal OSE cells. In conclusion, all human ovarian cancers express immunologically-detectable levels of HER2, indicating that IHC measurement underestimates the true frequency of HER2-expressing ovarian cancers and may limit patient access to otherwise clinically meaningful HER2-targeted therapies.</p> </div

HER2 surface protein expression in established and primary ovarian cell lines.

<p>HER2 is expressed in the cell surface of all ascites and solid tumor-derived tumor cells. HER2 expression was assessed using flow cytometry in a large panel of clinical ascites and solid tumor specimens. A breast cancer cell line (MDA 468) and a leukemia cell line (CEM) which do not express any HER2 were used as negative controls. HER2 was found to be expressed in all tested ascites and solid tumor specimens albeit at different levels. HER2 cell surface levels are expressed as specific MFI. <i>P</i> = 0.03 when comparing percentage of HER2 protein-expressing cells in established vs. short-term lines. <i>P</i> = 0.43 when comparing MFI of HER2 protein detection in established vs. to short-term cell lines. <i>P</i> values were calculated using student’s unpaired t-test analysis.</p

Normal established and primary ovarian surface epithelial cells (OSE) express HER2 protein and <i>ERBB2</i> mRNA.

<p><b><i>A.</i></b><i> ERBB2</i> mRNA quantitation in normal OSE cells (398, IOSE-4, IOSE-6 and 1744) by q-PCR. SKOV-3 and CEM were used as positive and negative HER2 expressing cell lines respectively. Bars show the mean ± SD value of triplicate wells. <b><i>B.</i></b> Surface HER2 protein expression (solid histograms) by normal ovarian epithelial cells by flow cytometry; isotype antibody control (open histograms). <b><i>C–D.</i></b> Comparison of the <i>ERBB2</i> mRNA and protein levels between ovarian cancer ascites, solid tumor and normal OSE cells. (<b><i>C</i></b>) Vertical scatter plots of the <i>ERBB2</i> mRNA in ascites, solid tumor and normal OSE determined by q-PCR. The mean of each group is indicated by the horizontal line. <i>P</i> = 0.0498 when comparing <i>ERBB2</i> mRNA in ascites vs normal OSE cells; <i>P</i> = 0.0210 when comparing <i>ERBB2</i> mRNA in solid tumors vs normal OSE cells. (<b><i>D</i></b>) Vertical scatter plots of the protein levels in ascites, solid tumor and normal OSE determined by flow cytometry. The mean of each group is indicated by the horizontal line <i>P</i> = 0.0616 when comparing HER2 protein in ascites vs normal OSE cells; <i>P</i> = 0.1749 when comparing HER2 protein in solid tumors vs normal OSE cells. <i>P</i> values were calculated using unpaired student’s t-test analysis.</p

Ubiquitous HER2 expression in ovarian cancer cell lines.

<p><b><i>A.</i></b><i>ERBB2</i> mRNA levels in ovarian cancer cell lines by q-PCR. <i>ERBB2</i> mRNA levels of ovarian cancer cell lines are relative to that of ErbB2-negative CEM cells. All ovarian cancer cell lines express <i>ERBB2</i> mRNA. B-actin was used as an endogenous gene control. Results depict the mean ± SD of triplicate wells. Mean relative <i>ERBB2</i> mRNA expression amongst established and short-term OvCa cell lines was not statistically different (<i>P</i> = 0.45). <i>P</i> value was calculated using unpaired student’s t-test analysis. <b><i>B.</i></b> Detection of surface HER2 protein expression (filled histograms) by human ovarian cancer cell lines by flow cytometry; isotype antibody control (open histograms). <b><i>C.</i></b> Western blot analysis of HER2 protein expression in representative cell lines expressing differential amounts of HER2. HER2 protein is expressed at variable levels in all the ovarian cell lines tested. B-actin was used as control.</p