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Background: Effective chimeric antigen receptor (CAR) T cell therapy against solid tumors must overcome a hostile, tumor microenvironment that includes tumor-associated macrophages (TAM). Pancreatic adenocarcinomas (PDAC) are commonly infiltrated with TAMs polarized to a tumor-promoting M2 phenotype rather than a T cell-stimulatory and tumor-inhibitory M1 phenotype. The GoCAR platform combines an inducible MyD88/CD40 (iMC) costimulation protein with a 1st generation CAR. Our previously published results demonstrated that iMC costimulation, activated by the small molecule dimerizer, rimiducid (Rim), enhanced CAR-T proliferation and anti-tumor efficacy. Here, we examined the extrinsic effects of iMC signaling on CAR-T cell immune-activating ligands, cytokine production and their ability to polarize M2 macrophage to an anti-tumor phenotype. Methods: Macrophages were prepared from peripheral blood monocytes of four or more random blood donors from the Gulf Coast Regional Blood Center (Houston, TX) and differentiated in vitro to an M2 phenotype with TGF-β and IL-10, or an M1 phenotype (as a positive control). GoCAR-T cells targeting prostate stem cell antigen (PSCA) were prepared by retroviral transduction from the autologous donors. To test the effects of iMC activation on macrophage polarization, contact-dependent or independent (i.e., separation of cell populations with transwell inserts) coculture assays were performed with and without activation of iMC with 1 nM Rim and/or surface-bound PSCA. Anti-tumor cytotoxicity was measured by coculture with PSCA+ Panc1-GFP cells. Results: CAR activation by PSCA antigen recognition or iMC activation with Rim decreased CD163, an M2 macrophage marker, and increased CD80, expressed on M1 macrophage. Full activation of the GoCAR-T cells with both Rim and the target antigen fully repolarized M2 macrophage to an M1 marked phenotype (CD163lowCD80high). This repolarization could be directed partially in the absence of cell-cell contact by diffusion of soluble factors through Transwell membranes. M2 macrophages repolarized by conditioning media from activated GoCAR-T cells also exhibited the functionality of M1 macrophages and acquired cytotoxicity against tumor cells. Furthermore, cultures of conditioned macrophages and limiting dilutions of GoCAR-T cells demonstrated a cooperative enhancement of the cytotoxicity of PSCA GoCAR-T cells toward Panc-1 targets. This cooperation was more effective for GoCAR-T cells than CD28- or 4-1BB-enhanced 2nd generation PSCA CAR-T cells. Conclusions: These results predict that GoCAR-T activation with Rim will convert TAMs within a solid tumor microenvironment from T cell inhibitors to tumor-caustic agents. Citation Format: Xiaohong Wang, Konrad Gabrusiewicz, David M. Spencer, Aaron E. Foster, J. Henri Bayle. Small molecule inducible MyD88/CD40 (iMC) in CAR-T cells can repolarize M2 macrophage to an anti-tumor M1 phenotype [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2193.

The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlie the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/β-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found to be coactivated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible β-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-β (TGF-β) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-β signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1–WNT–TGF-β signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets. Cancer Res; 74(2); 609–20. ©2013 AACR.

Despite the potency of dendritic cells (DC) as antigen-presenting cells for priming adaptive immunity, DC-based cancer vaccines have been largely insufficient to effectively reduce tumor burden or prevent tumor progression in most patients. To enhance DC-based vaccines, we used the combination of a synthetic ligand-inducible CD40 receptor (iCD40) along with Toll-like receptor-4 (TLR-4) ligation in human monocyte-derived DCs. The iCD40 receptor permits targeted, reversible activation of CD40 in vivo, potentially bypassing the essential role of CD4+ T cells for activation of DCs. As a rigorous preclinical study of this approach, we evaluated key parameters of DC activation and function. Whereas neither iCD40 nor TLR-4 signaling alone led to high levels of interleukin (IL)-12p70 and IL-6, using iCD40 in combination with lipopolysaccharide (LPS) or monophosphoryl lipid A led to strongly synergistic production of both. Furthermore, this approach led to high expression of DC maturation markers, epitope-specific CTL and T helper 1 responses, as well as DC migration in vitro and in vivo. Moreover, use of iCD40-modified and LPS-stimulated DCs led to targeted expansion of autologous T cells against tumor-associated antigens, including prostate-specific membrane antigen, and elimination of preestablished tumors, supporting this technology as a potent strategy for DC-based cancer immunotherapy. [Cancer Res 2007;67(21):10528–10]

To establish optimized conditions for immunity against prostate cancer, we compared the efficacy of multiple approaches in autochthonous and s.c. transgenic adenocarcinoma of the mouse prostate (TRAMP)-based models. Mice immunized with interleukin (IL)-12–containing apoptotic, but not necrotic TRAMP-C2 cell–based, vaccines were resistant to TRAMP-C2 tumor challenge and re-challenge, independently of the route of vaccination (s.c. or i.p.). Administration of γ-irradiated TRAMP-C2 cells preinfected with adenovirus containing both B7-1 and IL-12 genes, unlike adenovirus containing B7-1 alone, considerably protected C57BL/6 mice from TRAMP-C2 tumor growth and extended the life span of TRAMP mice. Vaccines that included dendritic cells, instead of IL-12, were equally efficient. Whereas injections of ligand-inducible caspase-1– and IL-12–containing adenoviruses cured small s.c. TRAMP-C2 tumors, nanopump-regulated delivery of viruses led to elimination of much larger tumors. The antitumor immune responses involved CD4+-, CD8+-, and natural killer cells and were strengthened by increasing the number of vaccinations. Intraprostatic administration of inducible caspase-1– and IL-12–containing adenoviruses resulted in local cell death and improved survival of adenocarcinoma-bearing TRAMP mice. Thus, tumor cell apoptosis induced by caspase in situ and accompanied by IL-12 is efficient against prostate cancer in a preclinical model.

Mice with a single copy of the retinoblastoma gene (Rb+/−) develop a syndrome of multiple neuroendocrine neoplasia. They usually succumb to fast-growing, Rb-deficient melanotroph tumors of the pituitary intermediate lobe, which are extremely rare in humans. Thus, full assessment of Rb role in other, more relevant to human pathology, neoplasms is complicated. To prevent melanotroph neoplasia while preserving spontaneous carcinogenesis in other types of cells, we have prepared transgenic mice in which 770-bp fragment of pro-opiomelanocortin promoter directs expression of the human RB gene to melanotrophs (TgPOMC-RB). In three independent lines, transgenic mice crossed to Rb+/− background are devoid of melanotroph tumors but develop the usual spectrum of other neoplasms. Interestingly, abrogation of melanotroph carcinogenesis results in accelerated progression of pituitary anterior lobe tumors and medullary thyroid carcinomas. A combination of immunologic tests, cell culture studies, and tumorigenicity assays indicates that α-melanocyte–stimulating hormone, which is overproduced by melanotroph tumors, attenuates neoplastic progression by decreasing cell proliferation and inducing apoptosis. Taken together, we show that cell lineage–specific complementation of Rb function can be successfully used for refining available models of stochastic carcinogenesis and identify α-melanocyte–stimulating hormone as a potential attenuating factor during progression of neuroendocrine neoplasms.

Background: While chimeric antigen receptor (CAR)-T immunotherapies have shown remarkable efficacy against leukemias and lymphomas, improved CAR-T efficacy and persistence are needed to overcome solid tumors, without compromising safety. Here, we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to their cognate ligands. Cell activation is controlled by the homodimerizer rimiducid that triggers signaling cascades downstream of MyD88 and CD40 via an engineered chimeric protein termed iMC. A rapamycin-controlled pro-apoptotic switch, iRC9, is co-expressed, which induces dimerization of the caspase-9 domain to mitigate possible toxicity from excessive CAR-T function. When combined with a first generation CAR, these molecular switches allow for specific and efficient regulation of engineered T cells. Methods & Results: T cells were activated and co-transduced with the HER2 GoCAR (SFG-iMC.2A-CAR.ζ) and RapaCIDe (SFG-iRC9.2A-ΔCD19) vectors to generate “Dual-switch GoCAR-T” cells. Combined transduction of RapaCIDe and HER2 GoCAR vectors into T cells did not adversely affect the antitumor efficacy of the GoCAR-T cells, which eliminated OE19 esophageal tumor cells in a 7-day coculture assay at a 1:20 effector to target ratio (3.9±4.3% OE19-eGFPFluc cells remained in GoCAR-modified cultures vs. 1.1±0.1% for the dual-switch GoCAR), and promoted T cell expansion (53.4±9.4% CAR+ for GoCAR vs. 44.6±13.2% for the dual-switch). When challenged in an OE19 tumor-bearing mouse model, rimiducid stimulation of the dual-switch GoCAR-T cells enhanced tumor killing and T cell expansion. Deployment of the off-switch induced fast (½ Vmax ~ 8 hours) and efficient elimination of T cells (Dual-switch GoCAR-T = 94.1% AnnV+/PI+ vs. GoCAR-T = 5.1%) in a caspase-3 activation assay with real-time (IncuCyte) monitoring as well as AnnV/PI detection by flow cytometry. In vivo assessment of the RapaCIDe switch was performed via eGFPluciferase (eGFPfluc)-labeled RapaCIDe-T cells in NSG mice. Rapamycin, but not rimiducid, treatment efficiently eliminated RapaCIDe-T cells within 24 hours, which is similar to the rate observed by the clinically validated rimiducid-regulated CaspaCIDe switch. Summary: Dual-switch GoCAR-T, a novel platform comprising a first-generation CAR combined with regulated costimulation and apoptotic signaling elements, effectively controlled tumor growth and T cell expansion and elimination in vitro and in vivo. This dual-switch technology provides a user-controlled system for managing persistence and safety of tumor antigen-specific CAR-T cells. Citation Format: MyLinh T. Duong, Matthew R. Collinson-Pautz, Eva Morschl, Mary E. Brandt, Ming Zhang, Kevin W. Slawin, Aaron E. Foster, J. Henri Bayle, David M. Spencer. Dual-switchGoCAR-T cells: small molecule-regulated “GO” and “STOP” switches to target solid cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-184. doi:10.1158/1538-7445.AM2017-LB-184

Introduction: Use of tumor antigen-specific T cell receptors (TCRs) to refocus T cell killing has shown tantalizing clinical efficacy; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Also, MHC class I downregulation in tumors further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a small molecule dimerizer (rimiducid)-dependent “activation switch”, called inducible MyD88/CD40 (iMC), along with tumor-targeted TCRs to regulate T cell expansion and activation, while affecting upregulation of MHC class I on tumors. Methods: Human T cells were CD3/CD28-activated and transduced with αβTCR-encoding γ-retroviruses recognizing either the CT antigen, PRAME (HLA-A*02:01/SLLQHLIGL), or the B-cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*07:02/APAPTAVVL). Parallel “Go-TCR” vectors co-expressed iMC, comprising MyD88 and CD40 signaling domains along with rimiducid-binding FKBP12-V36. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed T2 cells (PRAME only) or against PRAME+/Bob1+, HLA-A2+ -B7+ U266 myeloma cells +/- 10 nM rimiducid. MHC class I induction was measured using transwell assays and flow cytometry. In vitro tumor killing was analyzed by T cell and tumor coculture assays at various effector to target ratios over a 7-day period. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 1×107 transduced T cells. iMC was activated in vivo by weekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo BLI imaging and flow cytometry. Results: All vectors efficiently (∼85%) transduced activated T cells and showed antigen-specific IFN-γ production and cytotoxicity against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 cells. However, both iMC signaling and TCR ligation of PRAME peptide-pulsed T2 Cells were required for IL-2 production. Coculture assays with U266 cells showed that tumor elimination, IL-2 secretion and robust (∼ 50-fold) T cell proliferation (vs TCR signaling alone) was optimized with concurrent rimiducid-driven iMC activation in both “Go-PRAME” and “Go-Bob1” constructs. Further, iMC activation produced TCR-independent IFN-γ that increased (∼100-fold) MHC class I expression on tumor cells. In NSG mice engrafted with U266 tumors, iMC-PRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ∼1000-fold on day 81 post-injection vs T cells expressing only the PRAME TCR (p < 0.001). Summary: The novel rimiducid-regulated “Go” switch, iMC, greatly augments activation and expansion of TCR-engineered T cells while sensitizing tumors to T cells via cytokine-induced MHC class I upregulation. iMC-enhanced TCRs are prototypes of novel “Go-TCR” engineered T cell therapies that increase efficacy, safety and durability of adoptive T cell therapies. Citation Format: David M. Spencer, Tsvetelina P. Hoang, Aaron Foster, Tania Rodriguez, David Torres, An Lu, Jeannette Crisostomo, Lorenz Jahn, Mirjam H.M. Heemskerk. Go-TCR™: Inducible MyD88/CD40 (iMC) enhances proliferation and survival of tumor-specific TCR-modified T cells, increasing anti-tumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-084.

TMPRSS2/ERG gene fusions are found in the majority of prostate cancers; however, there is significant heterogeneity in the 5′ region of the alternatively spliced fusion gene transcripts. We have found that there is also significant heterogeneity within the coding exons as well. There is variable inclusion of a 72-bp exon and other novel alternatively spliced isoforms. To assess the biological significance of these alternatively spliced transcripts, we expressed various transcripts in primary prostatic epithelial cells (PrEC) and in an immortalized PrEC line, PNT1a. The fusion gene transcripts promoted proliferation, invasion, and motility with variable activities that depended on the structure of the 5′ region encoding the TMPRSS2/ERG fusion and the presence of the 72-bp exon. Cotransfection of different isoforms further enhanced biological activity, mimicking the situation in vivo, in which multiple isoforms are expressed. Finally, knockdown of the fusion gene in VCaP cells resulted in inhibition of proliferation in vitro and tumor progression in an in vivo orthotopic mice model. Our results indicate that TMPRSS2/ERG fusion isoforms have variable biological activities promoting tumor initiation and progression and are consistent with our previous clinical observations indicating that certain TMPRSS2/ERG fusion isoforms are significantly correlated with more aggressive disease. [Cancer Res 2008;68(20):8516–24]

In vivo imaging advances have greatly expanded the use of animal cancer models. Herein, we describe two new models that permit prostate imaging ex vivo, in vivo, and in utero. Further, we show the use of these models for detecting small metastasis and testing reagents that modulate the androgen receptor (AR) axis. A luciferase reporter gene was directed to the prostate epithelium using three composite promoters called human kallikrein 2 (hK2)-E3/P, PSA-E2/P, and ARR2PB, derived from hK2, PSA, and rat probasin regulatory elements, to generate the EZC1, EZC2, and EZC3-prostate mice, respectively. EZC2 and EZC3-prostate display robust expression in the prostate with only minimal detectable expression in other organs, including testes and epididymis. Luciferase expression was detected as early as embryonic day 13 (E13) in the urogenital track. To image prostate cancer progression, lines of EZC mice were bred with prostate cancer models TRAMP and JOCK1, and imaged longitudinally. When crossed with prostate cancer models, EZC3 facilitated detection of metastatic lesions although total prostate luciferase expression was static or reduced due to weakening of AR-regulated promoters. Castration reduced luciferase expression by 90% and 97% in EZC2 and EZC3 mice, respectively, and use of GnRH antagonist also led to extensive inhibition of reporter activity. The EZC-prostate model permits prostate imaging in vivo and should be useful for imaging prostate development, growth, metastasis, and response to treatment noninvasively and longitudinally. These models also provide powerful new reagents for developing improved drugs that inhibit the AR axis. (Cancer Res 2006; 66(12): 6199-209)

Mice with a single copy of the retinoblastoma gene (Rb(+/-)) develop a syndrome of multiple neuroendocrine neoplasia. They usually succumb to fast-growing, Rb-deficient melanotroph tumors of the pituitary intermediate lobe, which are extremely rare in humans. Thus, full assessment of Rb role in other, more relevant to human pathology, neoplasms is complicated. To prevent melanotroph neoplasia while preserving spontaneous carcinogenesis in other types of cells, we have prepared transgenic mice in which 770-bp fragment of pro-opiomelanocortin promoter directs expression of the human RB gene to melanotrophs (TgPOMC-RB). In three independent lines, transgenic mice crossed to Rb(+/-) background are devoid of melanotroph tumors but develop the usual spectrum of other neoplasms. Interestingly, abrogation of melanotroph carcinogenesis results in accelerated progression of pituitary anterior lobe tumors and medullary thyroid carcinomas. A combination of immunologic tests, cell culture studies, and tumorigenicity assays indicates that alpha-melanocyte-stimulating hormone, which is overproduced by melanotroph tumors, attenuates neoplastic progression by decreasing cell proliferation and inducing apoptosis. Taken together, we show that cell lineage-specific complementation of Rb function can be successfully used for refining available models of stochastic carcinogenesis and identify alpha-melanocyte-stimulating hormone as a potential attenuating factor during progression of neuroendocrine neoplasms.

The constitutive activation of signal transducer and activator of transcription (STAT) proteins has been demonstrated in many diverse human cancer cell lines and clinical tumors including prostate cancer. The STAT family has at least seven members, and the two forms of the STAT5 protein, STAT5A and STAT5B, exhibit a high degree of sequence similarity. We have reported previously that expression of STAT5B, but not STAT5A, could enhance the transforming potential of v-src and induces cell cycle progression and motility in fibroblasts. In the current study we demonstrate specific activation of STAT5B in epithelial cells representing invasive and metastatic prostate cancer. We also demonstrate that the naturally occurring dominant-negative isoform STAT5DeltaB can block cell cycle progression through G(1) and inhibit the growth, invasive potential, and clonogenic ability of these prostate cancer cell lines. Furthermore, we report that the dominant-negative isoform STAT5DeltaB can inhibit the growth of prostate cancer cells in grafting studies. These results support our hypothesis that specific activation of STAT5, in particular STAT5B, facilitates the progression of prostate cancer.

Accurate determination of the contributions of oncogenes toward tumor progression requires their regulation. Herein, we created transgenic mice with prostate-specific expression of ligand-inducible FGFR1 or FGFR2, based on lipid-permeable dimerizing molecules, called chemical inducers of dimerization. Despite extensive homology and equivalent expression by both chimeric receptors in the ventral prostate gland, only FGFR1 triggers detectable nuclear translocation of Erk and progression to prostatic intraepithelial neoplasia (PIN). Induction of PIN grade I-II, indicated by multiple layers of atypical cells, is seen consistently by 12 weeks of chemical inducers of dimerization treatment. By 6 months, more extensive nuclear atypia, thickened "reactive" stroma, and basement membrane herniation occurs, corresponding to PIN IV. By timed removal of FGFR1 signaling, we show that induced hyperplasia is reversible until extensive intraductal vascularization occurs, but continued progression requires prolonged FGFR1 signaling. Additionally, by highlighting differences between the two receptors and creating the foundation for controlling FGFR1 signaling during prostate cancer progression, a model of early stage prostate cancer is established for developing targeted intervention directed toward the FGFR signaling axis.

Changes in the fibroblast growth factor receptor (FGFR) axis are often associated with prostate cancer (CaP) progression. We have used chemically induced dimerization (CID) to elucidate the individual contributions of FGFR1 and FGFR2 to tumor etiology. Novel CaP cell lines stably expressing CID/AP20187-inducible FGFR1 (iFGFR1) and iFGFR2 were made using the tumorigenic transgenic adenocarcinoma of the murine prostate (TRAMP)-derived clone, TRAMP-C2N (C2N), to generate C2N.iFGFR1 or C2N.iFGFR2 cells. To test the effects of iFGFR activation on tumor growth, mice bearing s.c. C2N.iFGFR1- or C2N.iFGFR2-derived tumors were treated biweekly with CID. Activation of iFGFR1 led to rapid tumor growth as a result of increased proliferation. In contrast, expression of iFGFR2 inhibited tumor growth. Furthermore, we have ascertained that FGFR1 activation appears to be most important during the early stages of tumor development, but once established, tumors become rapidly CID independent. In these C2N-based lines, quantitative signaling differences were seen between the two receptors, with iFGFR1 leading to more robust extracellular signal-regulated kinase activation. Additionally, activation of iFGFR1, but not iFGFR2, led to strong up-regulation of osteopontin, a secreted glycoprotein involved in integrin activation and associated with CaP progression and metastasis. These studies support the hypothesis that observed changes in the FGFR axis in mammals during CaP progression are causally important.

Recent advances in dendritic cell (DC)-based cancer vaccines have highlighted the potential utility of arming the immune system to target neoplastic self. A strong, yet controlled, TH1 response has typically been the goal of DC-based protocols, as it is cell-based adaptive immunity that possesses the ability to most-efficiently destroy the nascent tumor or occult micrometastases. Nonetheless, many DC-intrinsic factors have impeded the development of a vaccine strong enough to impart meaningful clinical results, and there remains a need for further study of the parameters required for maximal, targeted DC activity. Previously, we have shown that contemporaneous loading of DC with overlapping MHC class I and MHC class II peptide antigens leads to an enhanced TH1 response when compared to canonical DC stimulation as assayed by release of IL-12, sCTLA-4, proliferation of CD8+ CD25+, INF-γ release, and specific killing of target both in vitro and in vivo. These and other data have lead to the hypothesis that DC possess a sensor mechanism capable of discerning amino acid sequence similarities between peptides bound by MHC class I and class II and that tRNA synthetases may serve as critical components of the putative recognition machinery due to their inherent ability to recognize and distinguish between individual amino acid side chains. Here we report that the aminoacyl-tRNA synthetase complex (MACS), a large molecular complex comprised of at least nine aminoacyl-tRNA synthetases, possess interesting characteristics that might link it to this phenomenon. Components of the MACS complex, including the p43 structural subunit, were found in DC exosomes, the contents of which mimic those of the late endosome, a known cross-presentation compartment. MARS (methionyl-tRNA synthetase), a component of the MACS, as well as non-MACS components VARS (valyl-tRNA synthetase) and GARS (glycyl-tRNA synthetase) were found within exosomes as well. UVC cross-linking of exosomal extracts resulted in the incorporation of individual tRNA-synthetase isoforms into a very large complex of indeterminate size. p43 was differentially released from the MACS in response to the loading of DC only with matched class I (GFP mRNA) and class II (recombinant GFP protein) determinants. siRNA knockdown of MACS structural component p38 was able to ablate the ability of doubly-loaded DC to enhance the generation of CD8+CD25+ cells as well as the ability of these cells to secrete IFN-γ. Further, siRNA knockdown of p38 abrogated the release of p43 from DC loaded with matched class I and II determinants. With an enhanced understanding of these regulatory phenomena, the development of small molecule TH1 agonists and/or immunotherapeutic protocols with enhanced clinical efficacy become realistic and achievable goals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-132. doi:1538-7445.AM2012-LB-132

Cancer growth and metastasis requires proliferation, cell migration, and stromal remodeling, functions also found during organogenesis and wound repair. The fibroblast growth factor (FGF) and canonical Wnt/β-catenin signaling pathways are two pathways central to these functions. FGFs and their receptors (FGFRs) are expressed in most tissues and play pivotal roles in development, wound healing and neovascularization, and are also upregulated in many solid cancers, including prostate, mammary, renal, kidney, bladder, and testicular tumors. We previously demonstrated in the JOCK1 (juxtaposition of CID and kinase) prostate cancer (PCa) mouse model that chemically induced dimerization (CID) of FGFR1 signaling in the prostate is sufficient for tumor initiation and early tumor maintenance and prolonged (42 weeks) FGFR1 activation results in PCa with distant metastasis. The Wnt pathway is also vital for proper embryogenesis and homeostasis of adult tissues by regulating stem cell self-renewal, pluripotency, and differentiation in several tissues, including colon, hair shaft, and chondrocyte stem/progenitor cells. The uncontrolled activation of this important stem pathway is also associated with various cancers, including colon, breast and prostate. Recently, we have discovered that induced crosslinking of the Wnt co-receptor LRP5 is sufficient to induce canonical Wnt signaling and nuclear localization of β-catenin. We subsequently developed two novel mouse models where Wnt can be specifically activated in the prostate epithelium, Pro-Cat (prostate-targeted inducible β-catenin), or in virtually every tissue sub-layer, Ubi-Cat (ubiquitously expressed inducible β-catenin). Consistent with previous literature, induced Pro-Cat mice never progressed beyond prostatic hyperplasia, however, after a year of pathway induction, 2 out of 6 Ubi-Cat mice developed adenocarcinoma, indicating a yet unexplored role for stromal Wnt signaling in tumorigenesis. In order to delineate possible crosstalk and synergism between FGF and Wnt pathways, we bred JOCK1 mice unto Pro-Cat and Ubi-Cat transgenic lines. Both Pro-Cat/JOCK1 and Ubi-Cat/JOCK1 mice developed widespread hyperplasia, high-grade PIN and adenocarcinoma with an extensive reactive stroma by 24 weeks of CID injections. Fascinatingly, Pro-Cat/JOCK1 mice do not continue to progress beyond the timeline established by JOCK1 induction alone whereas Ubi-Cat/JOCK1 mice continue to progress to advanced transitional sarcomatoid lesions. These results suggest that intracellular crosstalk between Wnt and FGFR1 in the epithelia is sufficient to accelerate tumor initiation, however accelerated progression requires intercellular crosstalk between the stroma and the epithelia. Additional experiments necessary to definitively implicate the stroma in accelerated tumorigenesis are underway and will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3287. doi:1538-7445.AM2012-3287

Dendritic cell (DC) vaccines are a rapidly progressing area of translational research intended for developing new cancer treatment modalities. Although ex vivo DC-based vaccines have been extensively studied and clinically tested for the treatment of cancer, they are inherently limited by short DC lifespan after activation, labor-intensive production (requiring ex vivo manipulation of autologous cells), high cost and consequent limited accessibility to the patient. Therefore, we have attempted to develop portable, regulatable adjuvants that could be targeted to DCs and activated in situ. Development by our lab of an inducible variant of the costimulatory molecule, CD40 (iCD40), and a composite, inducible MyD88/CD40 (iMC) adjuvant, that also incorporates the universal Toll like receptor adapter, MyD88, have been shown to increase the potency and lifespan of DC vaccines. These inducible receptors act as in vivo DC “switches” that lead to the priming and robust expansion of antigen (Ag)-specific T-cells capable of eliminating pre-established tumors in mice. Despite the success of these DC “switch” systems, practicality and scalability of patient-tailored ex vivo DC vaccines remains a major hindrance to their widespread applicability. Therefore, the future of DC vaccines lies in the development of “off-the-shelf” methodologies, such as viral or non-viral vectors that can deliver adjuvants along with tumor antigens. DNA vaccines are attractive for this purpose owing to their elegant simplicity, ease of production, and lack of anti-vector immune responses. In order to successfully vaccinate patients with DNA, one must be able to deliver plasmid encoding therapeutic genes to target cells efficiently. In vivo electroporation is a relatively new technology just starting to be tested in clinical trials, and provides a safe, simple, and effective means by which to administer DNA vaccines. Data from our preliminary studies of the electroporative delivery of plasmids encoding the model antigen β-galactosidase (LacZ) in mice suggests that LacZ-specific T-cell responses are induced, and that upon tumor challenge, mice receiving prophylaxis via electroporation had slower tumor growth kinetics when compared to controls. Together, this preliminary data supports the notion that DNA vaccination with tumor Ag by electroporation may be a simple and effective “off-the-shelf” cancer vaccine strategy. Additionally, this vaccination strategy may be enhanced by the addition of the iMC adjuvant. Therefore, further investigation of intradermal electroporation is warranted by these findings, to better characterize the induced immune responses and better optimize vaccination Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1568. doi:1538-7445.AM2012-1568

Background: We report interim clinical and immune monitoring results for the low dose cohort of patients enrolled in an ongoing Phase I/IIa clinical trial of BPX-101, a novel, autologous dendritic cell (DC) vaccine for mCRPC based on AP1903-inducible, iCD40-expressing DCs. Methods: Men with progressive mCRPC were enrolled in a 3+3 dose escalation Phase I/IIa trial evaluating BPX-101 and AP1903. BPX-101 is produced from a single leukapheresis procedure by elutriation, differentiation of monocytes into DCs, transduction with Ad5f35-inducible human (ih)-CD40, antigen loading with a form of Prostate Specific Membrane Antigen (PSMA), and pre-activation with lipopolysaccharide and activating agent, AP1903. BPX-101 was administered intradermally every 2 weeks for 6 doses. AP1903 (0.4 mg/kg) was infused 24 hours after each BPX-101 dose. Blood samples were collected weekly, cryopreserved and batched for immune monitoring assays after completion of the first 12-weeks of therapy. IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-12, GMCSF, TNFα, IFNγ, IP-10, MCP-1, MIP-1α, MIP-1ß, and RANTES levels were evaluated, using a LINCOplex Immunoassay Panel. Results: Of six subjects enrolled to date, three patients enrolled in the low dose cohort (4 × 106 cells) have completed at least 12 weeks of therapy (median 23, range 22-27), and all remain on study with objective stable disease. No unexpected drug-related adverse events have been observed in any patient to date. Changes in levels of MCP-1, MIP-1α, MIP- 1ß, and RANTES tracked closely for all three low dose-cohort subjects. In one subject who experienced a 46% PSA decline after 8 weeks and a 20% measurable metastatic disease reduction per RECIST 1.1 after 12 weeks of therapy, MCP-1 levels spiked 17.5-, 17.2-, 4.2- and 6.8-fold over baseline levels one week after vaccinations #1, 2, 3 and 5 respectively, and returned to within 8-30% of baseline levels the following week. In this same patient, IFNγ and GM-CSF levels followed a similar pattern; GM-CSF spiked from undetectable baseline levels to 22.0, 16.2, 9.9, and 11.7 pg/ml one week after vaccinations #1, 2, 3 and 5 respectively, and returned to undetectable levels the following week. In a second patient, who experienced a 6% PSA decline after 12 weeks of therapy, GM-CSF levels spiked from undetectable levels to 63.2 pg/mL at week 5, returning to undetectable levels over the next 7 weeks. Serum IL-6 levels for the low dose cohort fell from a mean of 9.3 pg/mL at baseline (med 5.2, range 4-18.8), to a mean of 1.6 pg/mL (med 1.2, range 1.0-2.6) at 6 weeks and a mean of 0.6 pg/mL (med 0.09, range 0-1.8) at 12 weeks. Conclusions: In two of three subjects completing 12 weeks of therapy at the lowest dose, dramatic spikes in serum inflammatory cytokine levels correlated with PSA declines in both and measurable disease decline in one. Serum IL-6 levels, which may have prognostic value in patients with mCRPC, fell significantly (65-99%) in all three subjects. By the end of March, 2010, three patients in the mid-dose cohort (12.5 × 106 cells) will have completed 12 weeks of therapy, and enrollment of up to six patients in the high dose cohort (25 × 106 cells) will have begun. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-80.

Introduction: Given the pre-eminent role of dendritic cells (DCs) as antigen-presenting cells, their exploitation as natural adjuvants in vaccination protocols for the treatment of various malignancies and infectious diseases is not surprising. Nevertheless, inadequate or unsustained activation has likely limited response rates in clinical trials. We previously reported a potent synthetic dimerizer drug-inducible CD40 (iCD40) receptor that permits temporally controlled DC-specific activation within the context of an immunological synapse (Hanks et al, Nat Med, 2005). We showed that when combined with Toll-like Receptor (TLR) adjuvants, iCD40-modified DCs (iCD40-DCs) have enhanced survival, co-stimulatory marker expression, migration, IL-12 production, antigen (Ag)-specific T cell stimulatory capacity and anti-tumor responses (Lapteva et al, Cancer Res, 2007). A phase I/II clinical trial based on iCD40-DCs to treat castration-resistant, metastatic prostate cancer began enrolling patients in August 2009. Procedures: To simplify this approach, we have developed a unified, inducible DC switch, called “iCD40. MyD88”, which combines signaling elements from costimulatory molecule, CD40, and TLR adapter, MyD88, fused to tandem FK506-binding binding proteins (FKBP12v36). Activation is initiated in vitro or in vivo by lipid-permeable dimerizing ligands (i.e. AP1903 or AP20187). Results: Both murine and human iCD40. MyD88-DCs secrete high (ng) levels of IL-12 and maturation markers in a ligand-dependent fashion without the need for exogenous adjuvants. iCD40. MyD88 activation is associated with synergistic activation of IKKα/β and p38. Moreover, tumor-bearing mice treated with antigen-pulsed, iCD40. MyD88-DCs show a potent anti-tumor immune response, consistent with expansion of antigen-specific CTLs in vivo. Bicistronic vectors combining iCD40. MyD88 along with tumor-associated antigens are underway. Conclusion: This “combo-switch” permits potent, targeted activation of antigen-pulsed DCs in vivo, leading to improved anti-tumor immunity with a high likelihood for reduced side effects. Production of a single vector that combines a potent DC switch with tumor antigen is the first requisite step leading to economically viable, “off-the-shelf”, vaccine therapies for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4761.