Colon Carcinoma Xenograft Model Research Articles

Abstract 1356: The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation

Abstract VISTA, a predominantly myeloid immune checkpoint, promotes an immunosuppressive tumor microenvironment and has been associated with resistance to approved checkpoint therapies. While blockade of VISTA with antibodies has shown anti-tumor efficacy in preclinical models, the biological mechanisms that underlie this effect remain poorly understood. To investigate the mechanistic basis of anti-tumor responses, we performed single-cell RNA sequencing of tumor infiltrating leukocytes from the cell-derived murine colon carcinoma xenograft model, CT26, which is known to be sensitive to VISTA blockade. We used HMBD-002, a uniquely blocking but non-depleting clinical-stage IgG4 anti-VISTA antibody, to identify the key immune cells and pathways impacted by VISTA blockade in the absence of Fc-mediated cell depletion. The translatability of these findings was validated in a human PBMC mixed leukocyte reaction assay. Subsequently, targeted depletion or inhibition of the identified immune populations and pathways was performed to confirm their contribution to the anti-tumor efficacy of HMBD-002.The blockade of VISTA by HMBD-002 significantly increased CD8+ T cells in the CT26 tumors, promoting an activated cytotoxic Tc1 program without inducing exhaustion. Concurrently, a substantial increase in proinflammatory M1 tumor-associated macrophages (TAMs) was noted within treated tumors, while other immune cell populations remained unaffected. Among the signaling pathways, the type-I interferon (IFN) response exhibited a significant upregulation, particularly in TAMs, indicating a type-I IFN-driven M1 reprogramming by HMBD-002. Validation experiments further demonstrated that the depletion of TAMs or CD8+ T cells, but not CD4+ T cells, abrogated the efficacy of HMBD-002. Moreover, blocking the type-I IFN signaling effectively impeded HMBD-002-induced TAM reprogramming and subsequent anti-tumor efficacy, thus confirming the pivotal roles of CD8+ T cells, TAMs, and type-I IFN signaling in driving the anti-tumor response to VISTA blockade. In conclusion, VISTA blockade with HMBD-002 promotes an anti-tumor response by type-I IFN mediated reprogramming of TAMs and activation of cytotoxic CD8+ T cells. Citation Format: Bhushan Dharmadhikari, Olga Zharkova, Dipti Thakkar, Roberto Tirado-Magallanes, Konrad Paszkiewicz, Piers J. Ingram, Jerome D. Boyd-Kirkup. The VISTA blocking antibody HMBD-002 promotes type-I interferon signaling and drives anti-tumor responses through macrophage reprograming and cytotoxic CD8+ T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1356.

Molecularly Imprinted Nanomedicine for Anti-angiogenic Cancer Therapy via Blocking Vascular Endothelial Growth Factor Signaling.

The VEGF-VEGFR2 (VEGF = vascular endothelial growth factor) signaling has been a promising target in cancer therapy. However, because conventional anti-angiogenic therapeutics suffer from drawbacks, particularly severe side effects, novel anti-angiogenic strategies are much needed. Herein, we report the rational engineering of VEGF-targeted molecularly imprinted polymer nanoparticles (nanoMIP) for anti-angiogenic cancer therapy. The anti-VEGF nanomedicine was prepared via a state-of-the-art molecular imprinting approach using the N-terminal epitope of VEGF as the template. The nanoMIP could target the two major pro-angiogenic isoforms (VEGF165 and VEGF121) with high affinity and thereby effectively block the VEGF-VEGFR2 signaling, yielding a potent anti-angiogenic effect of "killing two birds with one stone". In vivo experiments demonstrated that the anti-VEGF nanoMIP effectively suppressed tumor growth via anti-angiogenesis in a xenograft model of human colon carcinoma without apparent side effects. Thus, this study not only proposes an unprecedented anti-angiogenic strategy for cancer therapy but also provides a new paradigm for the rational development of MIPs-based "drug-free" nanomedicines.

Co-delivery of PDL1-blocking scFv and chemotherapeutics using engineered exosomes for cancer therapy

Poor clinical outcomes after immune checkpoint blockers alone may be offset by the combination of chemotherapeutic agents. However, safe, effective and simultaneous delivery of immune checkpoint blockers and chemotherapeutics to tumor cells poses a great challenge. In this study, we took advantage of the low biotoxicity, cargo shielding and immune privilege of exosomes to construct an engineered exosome that can co-deliver chemotherapeutic paclitaxel (PTX) and PDL1-blocking single-chain variable fragments (scFv) specifically to the cancer cells. We demonstrated that the PDL1-blocking scFv expressed on the surface of the exosomes well bound to PD-L1 and significantly reduced the inhibitory effect of PD-L1 on T cells. Moreover, engineered exosomes loaded PTX were more effectively absorbed by the recipient cells as compared to free PTX or control exosomes loaded PTX. In addition, the intravenous injection of APLG-EXO-PTX also inhibited the tumor growth in colon carcinoma xenograft model mice, and showed a significantly increased expression of effector cytokine, IFN-γ indicating that the engineered exosome loaded with PTX could significantly upregulate T-cell activation. The strategy of co-delivering functional scFv and anti-cancer drugs via exosomes heralds a potential approach to improve the effectiveness of cancer treatment in the near future.

Novel biphenyl-based scaffold as potent and selective histone deacetylase 6 (HDAC6) inhibitors: Identification, development and pharmacological evaluation

A series of novel biphenyl-based scaffold derivatives were identified as selective histone deacetylase 6 (HDAC6) inhibitors through an in-house compound library screening approach. The biological evaluation indicated that most of target compounds exhibited moderate to good inhibitory activity and selectivity against HDAC6. Especially, compound C10 was identified as a potent and highly selective HDACs inhibitor, with HDAC1 IC50 value of 3600 nM, HDAC6 IC50 value of 23 nM, and the HDAC1/6 selectivity index of 157. Moreover, C10 displayed robust anti-proliferative activity, induced cancer cells apoptosis, increased the level of acetylated α-tubulin and inhibited cancer cells migration in vitro. C10 showed significant antitumor efficacy (TGI: 75%) in CT26 colon carcinoma xenograft model in mice with no considerable toxicity in vivo. More importantly, C10 could also activate antitumor immunity so as to synergistically exert antitumor effects in vivo. Overall, our findings have provided a new avenue for design, development and investigation into the mechanism underlying the antitumor efficacy of selective HDAC6 inhibitors.

Abstract 1890: A solution to T-cell engager toxicity: An anti-CD3 Prodrug DARPin (CD3-PDD) shows no toxicity, but potent anti-tumor activity in a humanized mouse model

Abstract T-cell engagers (TCEs) direct cytotoxic T-cell response towards tumor cells by binding simultaneously to a tumor-associated antigen (TAA) on target cells and to CD3 on T-cells, thereby forming an artificial immune synapse. They have been shown to be very potent anti-tumor drugs, as exemplified by blinatumomab, an α-CD19 x α-CD3 bispecific. However, the development of TCEs for hematological and solid tumors has been hampered by several factors, amongst them severe toxicity, elicited by on-target/off-tumor recruitment of T-cells and cytokine release syndrome (CRS). In order to overcome this challenge, an anti-CD3 Prodrug DARPin® (CD3-PDD) has been developed, consisting of a mouse cross-reactive EGFR-binder and a CD3-binder, linked via a protease-cleavable linker to an anti-idiotypic anti-CD3 binder (termed blocker hereafter). This α-EGFR x α-CD3 x blocker Prodrug is unable to bind and recruit T-cells in its non-cleaved state, but is designed to become activated in the tumor microenvironment upon cleavage of the linker by tumor-associated proteases. A control Prodrug DARPin® with a non-cleavable linker showed neither tumor cell killing, nor T-cell activation at concentrations >1'000-fold over the EC50 of the active, non-blocked TCE DARPin® in in vitro tumor cell killing and T-cell activation assays. In contrast, a CD3-PDD containing a cleavable linker was partially activated by proteases secreted from the tumor cells (HCT 116). Pre-treatment of the CD3-PDD with recombinant protease prior to the in vitro assay fully activated the molecule, with EC50 values comparable to the active, non-blocked TCE. Next, an in vivo proof-of-principle study was performed in a human colon carcinoma xenograft model (HCT 116) using immunodeficient mice humanized with hematopoietic stem cells (CD34+) and optimized for the presence of human myeloid cells. Due to the mouse cross-reactivity of the EGFR-binder, this animal model allowed to assess both anti-tumor efficacy and safety (therapeutic window). The cleavable CD3-PDD demonstrated a robust anti-tumor activity, similar to the one observed with active, non-blocked TCE. Most importantly, while the active, non-blocked TCE elicited strong toxicity, leading to loss of animals and requiring treatment stop, the cleavable CD3-PDD could be dosed without significant safety findings. In summary, a conditionally activated CD3-PDD shows similar efficacy but none of the toxicity of the active, non-blocked TCE. Our approach therefore holds great promise for the development of future CD3-PDD as therapeutics, enabling the utilization of less tumor-specific targets for highly potent TCEs. Ultimately, the ability of the versatile DARPin® technology to generate tailor-made anti-idiotypic DARPin® molecules can unlock novel therapeutic design spaces, which we are exploring beyond the conditionally activated CD3-PDD format. Citation Format: Andreas Bosshart, Julia Katharina Ahlskog, Aline Eggenschwiler, Dieter Schiegg, Yvonne Grübler, Sandra Wandel, Simon Fontaine, Maria Paladino, Susanne Mangold, Tanja Hospodarsch, Alexandra Neculcea, Chloé Iss, Christel Herzog, Bernd Schlereth. A solution to T-cell engager toxicity: An anti-CD3 Prodrug DARPin (CD3-PDD) shows no toxicity, but potent anti-tumor activity in a humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1890.

Abstract 2273: Selection of first-in-human clinical dose range for the tumor-targeted 4-1BB agonist MP0310 (AMG 506) using a pharmacokinetic/pharmacodynamics modeling approach

Abstract Following the clinical success of checkpoint inhibitors, cancer immunotherapy is rapidly expanding into combination treatments to enhance response rates and duration. Engagement of co-stimulatory molecules from the tumor necrosis factor receptor (TNFR) superfamily, including 4-1BB, may be a promising approach to enhance the benefits of cancer immunotherapy. Agonistic antibodies against the costimulatory receptor 4-1BB (CD137) have been shown to effectively enhance the anti-tumor activity of checkpoint inhibitors and other agents in preclinical animal models. However, the clinical development of 4-1BB agonistic antibodies has met with limited success thus far. Anti-4-1BB monoclonal antibodies have either been reported to cause significant dose-limiting hepatotoxicity or demonstrated limited efficacy as single agent therapeutics. We generated a DARPin® therapeutic candidate, MP0310 (AMG 506), which comprises domains binding to 4-1BB and fibroblast activation protein (FAP). MP0310 triggers 4-1BB activation only if bound and clustered via FAP which is abundantly expressed by cancer associated fibroblasts present in many solid tumors. In vitro functional assays indicate that MP0310 is a potent T cell co-stimulator in the presence of FAP-expressing cells. In vivo activity of tumor-targeted 4-1BB agonism was assessed in the HT-29 colon carcinoma xenograft model in PBMC humanized mice in combination with a T cell engager. Consistent with tumor targeting, MP0310 enhanced intra-tumoral CD8 T cell expansion while showing only limited systemic activity. We used a translational pharmacokinetic-pharmacodynamic (PK-PD) modeling approach to integrate in vitro and in vivo data to support the estimation of a minimal anticipated biological effect level (MABEL) and the relevant dose range for first in human (FIH) studies. In addition to a PK model describing the MP0310 concentrations over time in mouse and monkey, direct and indirect response models were used to describe receptor occupancy (RO), intra-tumoral CD8 T cell infiltration and peripheral CD8 count kinetics. Predictions from the combined PK and PD models using average intra-tumoral and peripheral drug concentrations (Cav) provide a MABEL dose with minimal expected systemic PD effects at 20% RO as well as the anticipated therapeutic dose range in humans. In conclusion, our PK/PD-modeling approach provides support for the selection of a safe starting dose for MP0310 (AMG506) in patients. It provides a rationale for selecting the maximum tested dose, and may help reduce the number of cancer patients receiving sub-therapeutic doses. MP0310 (AMG 506) is currently being evaluated in a Ph1 clinical study. Citation Format: Alexander Link, Laurent Juglair, Heïdi Poulet, Guy Lemaillet, Christian Reichen, Patricia Schildknecht, Ivana Tosevski, Joanna Robinson, Niina Veitonmäki, Jörg Herbst, Keith Dawson, Christof Zitt, Camila de Almeida, Rik de Greef, Victor Levitsky, Michael T. Stumpp, Hong Ji, Elmar Vom Baur. Selection of first-in-human clinical dose range for the tumor-targeted 4-1BB agonist MP0310 (AMG 506) using a pharmacokinetic/pharmacodynamics modeling approach [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 2273.

Injectable pegylated niclosamide (polyethylene glycol-modified niclosamide) for cancer therapy.

Niclosamide is an antihelminthic drug. Recent studies show that niclosamide exerts antitumor activity through inhibiting multiple signals including Wnt/β-catenin, mTORC1, signal transducer and activator of transcription 3, NF-κB, notch signals; however, the insolubility and poor bioavailability limits its potential clinic use, the aim of the present work is to synthesize an injectable pegylated niclosamide (polyethylene glycol-modified niclosamide) and investigate its antitumor activity in vitro and in vivo. The pegylated niclosamide (mPEG5000-Nic) was synthesized and the chemical structure was identified by Fourier transform infrared spectra and 1 H nuclear magnetic resonance spectra. The antitumor activity was evaluated in CT26 and HCT116 colon cancer cells in vitro and nude mouse xenograft model of CT26 cells in vivo. The water solubility of niclosamide in mPEG5000-Nic was significantly increased. Niclosamide could be released from mPEG5000-Nic nanoparticles in PBS solution. mPEG5000-Nic inhibited the cell viability of CT26 and HCT116 cells in vitro. No animal death was observed in mice with intraperitoneal injection of mPEG5000-Nic (equivalent to 1000 mg/kg niclosamide) within 24 hr, indicating that mPEG5000-Nic was less toxic. In nude mouse, xenograft model of CT26 colon carcinoma, intraperitoneal injection of mPEG5000-Nic (equivalent to niclosamide 50 mg/kg) inhibited tumor growth but had no effect on animal body weight and heart, liver, kidney, and lung weight in vivo. Meanwhile, in the same model, intraperitoneal injection of the positive clinic drug 5-fluorouracil not only inhibited the tumor growth, but also reduced the animal body weight. Our study demonstrates that pegylated niclosamide is novel niclosamide delivery system with clinical perspective for cancer therapy.

Abstract 1530: Polymorphonuclear myeloid derived suppressor cells secrete IL6 to promote colon tumorigenesis in an inflammatory vicious cycle

Abstract It is widely accepted now that chronic inflammation can create a favorable environment for neoplastic transformation and tumor progression. Tumor Microenvironment consists of various innate and adaptive immune cells, which secrete numerous inflammatory mediating and immunoregulatory cytokines. Among all immune cells infiltrated to the tumor site, myeloid cells have been considered as major players in the tumor associated inflammation. Recently, a subpopulation named myeloid-derived suppressor cells (MDSCs) have gain a great attention as a potential link between inflammation and tumor progression. MDSCs are heterogeneous population of immature myeloid cells that is subdivided into monocytic (M-MDSC) and polymorphonuclear (PMN-MDSC) subsets depending on different markers expressed differentially on their surface. Both PMN-MDSC and M-MDSC have been shown to be immune-suppressive and to exert an immunoregulatory role in the tumor microenvironment. However, a clear distinction in their functional and molecular properties has remained a matter of debate. Here we report that PMN-MDSC but not M-MDSC is the major secreting immune subset of the proinflammatory and tumor-promoting cytokine Interleukin 6 (IL6) in colon cancer. IL6 has been shown in literature to function as a tumor promoter through enhancing tumor growth, invasion and chemoresistance. We observed that IL6 upregulates DNMT1 and DNMT3b in colon carcinoma cells to boost their growth and survival in a STAT3 dependent mechanism. We also observed that tumor-expressed IL6 acts as a key modulator of the immune profile in colon tumor microenvironment. We generated an IL6-overexpressing colon carcinoma cell lines. We observed that IL6-overexpressing tumor triggers a significant increment in PMN-MDSCs but not M-MDSCs infiltration and accumulation in mouse colon carcinoma xenograft model. This remarkable PMN-MDSC tumor enrichment is associated with less effector T lymphocytes infiltration and more progressive and proliferative tumor. Moreover, DSS-induced colitis murine model is characterized with higher infiltration of PMN-MDSC but not M-MDSC, which secrete significant amount of IL6. Therefore, inflammation-derived IL6 induces neoplastic transformation of colonic epithelial cells through upregulation of DNMT1 and DNMT3b. Collectively; our data indicate that PMN-MDSC is the primary IL6 producer among all tumor-infiltrated leukocytes. PMN-MDSC-derived IL6 has a dual tumor favoring function through nurturing colon tumor cells in DNA-methylation dependent mechanism, and raising an immunosuppressive context enriched with more PMN-MDSCs, which secrete more IL6 in an endless vicious cycle. Thus, targeting this circuit, may have a promising implication in colon cancer immunotherapy. Citation Format: Mohammed L. Ibrahim, John D. Klement, Priscilla S. Redd, Chunwan Lu, Kebin Liu. Polymorphonuclear myeloid derived suppressor cells secrete IL6 to promote colon tumorigenesis in an inflammatory vicious cycle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1530.

Abstract 2380: Preclinical safety and efficacy of a tumor-directed T cell activating 4-1BB x 5T4 ADAPTIR™ bispecific antibody

Abstract The ability to induce potent anti-tumor activity by stimulating 4-1BB (CD137), a key co-stimulatory receptor, makes 4-1BB an attractive immunotherapeutic target. However, a clinically tested, 4-1BB targeting monospecific antibody has been hampered by dose-limiting hepatic toxicities. To improve safety of 4-1BB targeting therapies we have developed a 4-1BB x 5T4 bispecific antibody designed to direct tumor-specific T cell responses to the tumor by stimulating 4-1BB only when co-engaged with 5T4, a tumor-associated antigen. The preclinical dataset presented here provides an overview of the mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic. ALG.APV-527 was built using the ADAPTIR™ platform with binding domains from the ALLIGATOR-GOLD® human scFv library. Its 5T4-dependent agonistic function was assessed using primary CD8+ T cells or NK cells in the presence of 5T4-expressing cells. Secretion of IFN-γ or granzyme B was measured at 72 hrs using ELISA. To measure proliferation, PBMCs were labelled with Cell Trace™ and gated CD8+ T cells were analyzed using flow cytometry. For tumor inhibition studies, the human 5T4-expressing colon carcinoma HCT116 xenograft model was used. 5T4 expression was evaluated in normal human tissues and different human tumors by immunohistochemistry. The preclinical safety profile of ALG.APV-527 was evaluated in a single and repeated dose, dose-range finding toxicology study in non-human primates (NHP). The study design included all the standard repeated dose toxicity parameters and in addition, pharmacokinetics, immunogenicity, and pharmacodynamic end-points. ALG.APV-527 induces a 5T4-dependent increase in IFN-γ and granzyme B production and enhances proliferation of T cells and NK cells. Furthermore, ALG.APV-527 inhibits tumor growth in a human 5T4-expressing colon carcinoma xenograft model. 5T4 is overexpressed in multiple solid tumors, potentially directing the activity of 4-1BB induced by ALG.APV-527 to 5T4-expressing tumors, improving the risk/benefit profile. Four doses (administered once weekly) did not cause any adverse events in the NHP toxicity study. In conclusion, ALG.APV-527 induces potent CD8+ T cell and NK co-stimulation but only in the presence of 5T4. Based on its efficacy and preclinical safety profile, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of multiple 5T4-expressing solid tumors. Citation Format: Anna Dahlman, Michelle Nelson, Jeannette Bannink, Starrla Johnson, Doreen Werchau, Anneli Nilsson, Lill Ljung, Gabriele Blahnik-Fagan, Robert Bader, Adnan Deronic, Peter Ellmark, Maria Askmyr, Gabriela Hernandez-Hoyos, Cathy McMahan, Sara Fritzell. Preclinical safety and efficacy of a tumor-directed T cell activating 4-1BB x 5T4 ADAPTIR™ bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2380.

Chimeric Antigen Receptor-modified T Cells Repressed Solid Tumors and Their Relapse in an Established Patient-derived Colon Carcinoma Xenograft Model.

Adoptive transfer of T cells engineered with a chimeric antigen receptor (CAR) is deemed as the silver bullet to overcome the barriers of solid tumor treatment; however, the therapeutic application against solid tumors faces major challenges largely owing to the complex heterogeneity and immunosuppressive microenvironment of solid tumors. Preclinical development of CAR-T-cell products necessitates an appropriate animal model for the evaluation and improvement of their therapeutic capacities. Patient-derived xenograft (PDX) resembles real patients in several ways, and may serve as an attractive alternative to generate and evaluate the efficacy of CAR-T-cell products. In this study, we established and characterized a PDX mouse model implanted with colorectal cancer (CRC) xenograft. Human epidermal growth factor receptor 2 (HER2) expression in CRC specimens was detected by immunohistochemistry. The fragments of patient tumors were subcutaneously implanted into immunodeficient NOD-NPG mice after surgery. Furthermore, HER2-specific CAR-T cells were engineered and tested in our model to show their effectiveness in tumor clearance. Adoptive transfer of HER2-specific CAR-T cells resulted in the regression or even elimination of CRC xenograft and protection of relapse from rechallenged colon cancer tissue in PDX model. Significant survival advantage was achieved in these mice as compared with those transplanted with green fluorescent protein-T cells. Thus, this study showed that CAR-T-cell treatment may be a promising approach for solid tumor clearance and that the PDX model may be useful to evaluate the effects of CAR-T cells.

Theranostic Role of 32P-ATP as Radiopharmaceutical for the Induction of Massive Cell Death within Avascular Tumor Core.

Drug inaccessibility to vast areas of the tumor parenchyma is amongst the major hurdles for conventional therapies. Treatment efficacy rapidly decreases with distance from vessels and most of the tumor cells survive therapy. Also, between subsequent cycles of treatment, spared cancer cells replace those killed near the vessels, improving their access to nutrients, boosting their proliferation rate, and thus enabling tumor repopulation. Because of their property of "acting at a distance," radioisotopes are believed to overcome the physical barrier of vascular inaccessibility.Methods A novel molecular imaging tool called Cerenkov Luminescence Imaging (CLI) was employed for the detection of Cerenkov radiation emitted by beta particles, allowing in vivo tracking of beta-emitters. More precisely we investigated using a xenograft model of colon carcinoma the potential use of 32P-ATP as a novel theranostic radiopharmaceutical for tracing tumor lesions while simultaneously hampering their growth.Results Our analyses demonstrated that 32P-ATP injected into tumor-bearing mice reaches tumor lesions and persists for days and weeks within the tumor parenchyma. Also, the high-penetrating beta particles of 32P-ATP exert a "cross-fire" effect that induces massive cell death throughout the entire tumor parenchyma including core regions.Conclusion Our findings suggest 32P-ATP treatment as a potential approach to complement conventional therapies that fail to reach the tumor core and to prevent tumor repopulation.

Peginterferon Beta-1a Shows Antitumor Activity as a Single Agent and Enhances Efficacy of Standard of Care Cancer Therapeutics in Human Melanoma, Breast, Renal, and Colon Xenograft Models.

Because of its tumor-suppressive effect, interferon-based therapy has been used for the treatment of melanoma. However, limited data are available regarding the antitumor effects of pegylated interferons, either alone or in combination with approved anticancer drugs. We report that treatment of human WM-266-4 melanoma cells with peginterferon beta-1a induced apoptotic markers. Additionally, peginterferon beta-1a significantly inhibited the growth of human SK-MEL-1, A-375, and WM-266-4 melanoma xenografts established in immunocompromised mice. Peginterferon beta-1a regressed large, established WM-266-4 xenografts in nude mice. Treatment of SK-MEL-1 tumor-bearing mice with a combination of peginterferon beta-1a and the MEK inhibitor PD325901 ((R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide) significantly improved tumor growth inhibition compared with either agent alone. Examination of the antitumor activity of peginterferon beta-1a in combination with approved anticancer drugs in breast and renal carcinomas revealed improved antitumor activity in these preclinical xenograft models, as did the combination of peginterferon beta-1a and bevacizumab in a colon carcinoma xenograft model.

Abstract 2215: Pembrolizumab and ipilimumab reduce human RKO colon carcinoma tumor growth in HLA matched CD34-NSG humanized mice

Abstract Over the past decade there has been an increasing demand for preclinical models useful for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. The recently developed humanized mouse models composed of various types of human immune cells has the potential to recapitulate the human anti-tumor response modulated by checkpoint inhibitors. Tumor cells may develop multiple mechanisms to evade immune surveillance, including PD-L1 upregulation which suppresses T-cell effector responses towards the tumor. In the present study, we evaluated the efficacy of the checkpoint inhibitors pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4) in the human RKO colon carcinoma xenograft model in CD34-NSG humanized mice. These mice were engrafted with human hematopoietic stem cells (HSC) that matched the HLA haplotype of the RKO cells (HLA-A*01). Flow cytometry analysis confirmed high level of expression of PD-L1 in RKO colon carcinoma cells. The results from this study revealed that pembrolizumab and ipilimumab monotherapies significantly inhibited tumor growth. Surprisingly, combination therapy did not provide additive or synergistic effects and resulted in the same level of efficacy as the individual regimens. In order to understand the impact of these therapies on distinct immune cell populations, we analyzed the distribution of CD4 and CD8 T lymphocytes, Treg cells, NK cells and B cells in peripheral blood, spleen and tumor samples from tumor bearing humanized mice, via flow cytometry. We found that these checkpoint inhibitors can enhance effector functions of CD4+ and CD8+ T cells associated with increased expression of IFN-Γ. A parallel study with these checkpoint inhibitors carried out with humanized mice engrafted with HSC of the same haplotype as the RKO cells but from a different donor resulted in identical anti-tumor efficacy. In summary, these studies validate the use of humanized mouse models to test the tumor response to immune-checkpoint based therapies as it shows significant anti-tumor growth as a result of pembrolizumab and ipilimumab monotherapies associated with activation of T lymphocytes. Citation Format: Marcio Lasaro, Jason M. Davis, Mark Ellison, Martin R. Graf, Vivek Mahajan, Aidan Synnott, Robert J. Mullin, Paula L. Miliani De Marval. Pembrolizumab and ipilimumab reduce human RKO colon carcinoma tumor growth in HLA matched CD34-NSG humanized mice. [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 2215.

Targeting of cancer‑associated fibroblasts enhances the efficacy of cancer chemotherapy by regulating the tumor microenvironment.

Cancer-associated fibroblasts (CAFs), key components of the tumor stroma, can regulate tumorigenesis by altering the tumor microenvironment in variety of ways to promote angiogenesis, recruit inflammatory immune cells and remodel the extracellular matrix. Using a murine xenograft model of colon carcinoma, the present study observed that oxaliplatin increased the accumulation of CAFs and stimulated the production of cytokines associated with CAFs. When oxaliplatin was combined with the small-molecule dipeptidyl peptidase inhibitor PT-100, which inhibits CAFs by targeting fibroblast activation protein (FAP), the accumulation of CAFs was markedly reduced, xenograft tumor growth was significantly suppressed and the survival of the mice increased, compared to those of mice treated with oxaliplatin or PT-100 alone. Furthermore, the xenograft tumor tissues of mice treated with oxaliplatin and PT-100 contained lower numbers of tumor-associated macrophages and dendritic cells, expressed lower levels of cytokines associated with CAFs and had a lower density of CD31+ endothelial cells. The present study demonstrated that pharmacological inhibition of CAFs improved the response to chemotherapy, reduced the recruitment of immune tumor-promoting cells and inhibited angiogenesis. Combining chemotherapy with agents which target CAFs may represent a novel strategy for improving the efficacy of chemotherapy and reducing chemoresistance.

Abstract 295: Delivery of checkpoint inhibitor antibodies and other therapeutics directly to tumors by encoding them within the oncolytic adenovirus enadenotucirev

Abstract We are developing “armed” versions of the oncolytic adenovirus, enadenotucirev (EnAd), that will selectively infect and deliver immunotherapeutics to tumours following systemic dosing. EnAd is a potent, chimeric Ad11p/Ad3 adenovirus active against a range of epithelial cancer cells. In normal cells, EnAd is attenuated and shows little or no activity by either cytotoxicity assay or qPCR. In vivo, EnAd shows efficacy in a range of xenograft human tumor models following intra-tumoural, intravenous and intra-peritoneal injection and is currently being evaluated clinically for treatment of several different epithelial cancers. Data from ongoing clinical studies have shown that i.v. dosed EnAd infects and selectively replicates in tumor cells, producing significant amounts of viral protein (hexon). This is associated with CD8 cell accumulation and also indicates that transgene encoded proteins will be made in significant amounts by tumors following i.v. delivery of an armed EnAd virus. To develop armed EnAd variants we have developed a novel, efficient cloning system for rapid generation of viruses that can produce antibodies and other payloads under the control of the virus replication cycle or exogenous promoters. As an initial exemplification of the platform we have successfully produced EnAd variants encoding full-length (NG-135) and ScFv (NG-76) forms of anti-human VEGF antibodies. These have similar virus activity profiles to EnAd in cancer cell lines in vitro (virus replication, gene expression and oncolytic action), but also express and release the respective anti-VEGF antibody forms into the culture supernatant of tumor cells but not non-transformed cells. Using HCT-116 or DLD human colon carcinoma xenograft models we have shown that the virus infection profile following intra-tumoral injection is also similar to the parental EnAd virus (virus replication and hexon gene expression). Anti-VEGF antibody expression could be detected in the tumor tissue as both mRNA and functional antibody. Antibodies were detectable early (within 3 days of infection) and sustained over several weeks. Using an orthotopic A549 lung tumor model, NG-135 virus dosed i.v. following the development of tumors was able to decrease tumor burden by >90%. Viruses similarly expressing IgG1 or ScFv versions of anti-PDL1 and anti-CTLA4 checkpoint inhibitor antibodies have also been made and shown to produce functional antibodies that inhibit the respective receptor-ligand interactions in a range of in vitro assays, including upregulation of T-cell responses. In conclusion, our data show that EnAd can be modified to produce different antibody “payloads” following infection of human tumor cells in vitro and in vivo. Evaluation of the in vivo impact of these armed oncolytic viruses on the growth and microenvironment of tumors is now in progress. Citation Format: Brian R. Champion, Prithvi Kodialbail, Sam Illingworth, Nalini Rasiah, Daniel Cochrane, John Beadle, Kerry Fisher, Alice Brown. Delivery of checkpoint inhibitor antibodies and other therapeutics directly to tumors by encoding them within the oncolytic adenovirus enadenotucirev. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2015-295

Preclinical activity of nanoliposomal irinotecan is governed by tumor deposition and intratumor prodrug conversion.

A major challenge in the clinical use of cytotoxic chemotherapeutics is maximizing efficacy in tumors while sparing normal tissue. Irinotecan is used for colorectal cancer treatment but the extent of its use is limited by toxic side effects. Liposomal delivery systems offer tools to modify pharmacokinetic and safety profiles of cytotoxic drugs. In this study, we defined parameters that maximize the antitumor activity of a nanoliposomal formulation of irinotecan (nal-IRI). In a mouse xenograft model of human colon carcinoma, nal-IRI dosing could achieve higher intratumoral levels of the prodrug irinotecan and its active metabolite SN-38 compared with free irinotecan. For example, nal-IRI administered at doses 5-fold lower than free irinotecan achieved similar intratumoral exposure of SN-38 but with superior antitumor activity. Tumor response and pharmacokinetic modeling identified the duration for which concentrations of SN-38 persisted above a critical intratumoral threshold of 120 nmol/L as determinant for antitumor activity. We identified tumor permeability and carboxylesterase activity needed for prodrug activation as critical factors in achieving longer duration of SN-38 in tumors. Simulations varying tumor permeability and carboxylesterase activity predicted a concave increase in tumor SN-38 duration, which was confirmed experimentally in 13 tumor xenograft models. Tumors in which higher SN-38 duration was achieved displayed more robust growth inhibition compared with tumors with lower SN-38 duration, confirming the importance of this factor in drug response. Overall, our work shows how liposomal encapsulation of irinotecan can safely improve its antitumor activity in preclinical models by enhancing accumulation of its active metabolite within the tumor microenvironment.

"Arming" the chimeric oncolytic adenovirus enadenotucirev to deliver checkpoint inhibitors and other therapeutics directly to tumours

Enadenotucirev (EnAd; formerly called ColoAd1) is a potent, chimeric Ad11p/Ad3 adenovirus active against a range of epithelial cancer cells, with a shorter time-to-lysis than either wild type Ad11p, Ad3 or Ad5. In normal cells, EnAd is attenuated and shows little or no activity by either cytotoxicity or by qPCR. In vivo, EnAd shows efficacy in a range of xenograft human tumour models following intra-tumoural, intravenous and intra-peritoneal injection [1] and is currently being evaluated clinically for treatment of several different epithelial cancers. Data from an ongoing clinical mechanism of action study have shown that i.v. dosed EnAd infects and replicates in tumour cells, producing significant amounts of viral protein (hexon), indicating that transgene encoded proteins will also be made in significant amounts by tumours following i.v. delivery of an armed EnAd virus. To develop 'armed' variants for delivery of therapeutic agents that enhance EnAd's anti-tumour activity, we have developed a novel, efficient cloning system for rapid generation of modified viruses that can be dosed systemically to deliver immunomodulatory antibodies into tumours. We chose to first encode anti-VEGF antibodies since, unlike immunomodulators, they could be readily evaluated in vivo in immunodeficient mouse human tumour xenograft models. We have successfully produced EnAd variants encoding full-length (NG-135) and ScFv (NG-76) forms of anti-human VEGF antibodies which have similar virus activity profiles to EnAd in cancer cell lines in vitro (virus replication, gene expression and oncolytic action), but also express and release the respective anti-VEGF antibody forms into the culture supernatant. Using either HCT-116 or DLD human colon carcinoma xenograft models we have shown that the virus infection profile following intra-tumoural injection is similar to the parental EnAd virus (virus replication and hexon gene expression). Anti-VEGF antibody expression by these tumours could be detected in the tissue as both mRNA and functional antibody. Antibodies were detectable early (within 3 days of infection) and expression was sustained over several weeks. Furthermore, low levels of anti-VEGF antibody were detectable in the blood. Production and evaluation of viruses similarly expressing checkpoint inhibitor antibodies is now in progress, together with evaluation of anti-VEGF armed oncolytic viruses for their impact on the growth and microenvironment of tumour xenografts.

Dianthin-EGF is an effective tumor targeted toxin in combination with saponins in a xenograft model for colon carcinoma.

The intention of this work was to lift saponin supported tumor targeted therapies onto the next level by using targeted toxins in nude mice xenotransplant models. Combined application of dianthin coupled to EGF and saponin SO-1861 was tested in a xenograft model of colon carcinoma. In vitro cytotoxicity was tested in real-time in NIH3T3 cells (no human EGF receptor expression), HER14 and human colon carcinoma HCT116 (both EGF receptor overexpressing) cells. A xenograft model was established using HCT116 cells and tumor-bearing animals were treated with SO-1861 (30 µg/treatment) and dianthin coupled to EGF (0.35 µg/treatment). Tumor progression was monitored, using (18)F-2-fluor-2-desoxy-d-glucose, by small animal PET and by x-ray computed tomography. In vitro results demonstrated a high-receptor specificity and the in vivo experiment showed a progressive reduction of the tumor volume and glycolytic activity in the treated group (>95% reduction; p < 0.05). This therapy has great advantage because of high specificity, low side effects and great effectiveness for future development in the treatment of colon cancer.

Molecular imaging of cell death in tumors. Increasing annexin A5 size reduces contribution of phosphatidylserine-targeting function to tumor uptake.

ObjectiveAnnexin A5 is a phosphatidylserine binding protein that binds dying cells in vivo. Annexin A5 is a potential molecular imaging agent to determine efficacy of anti-cancer therapy in patients. Its rapid clearance from circulation limits tumor uptake and, hence, its sensitivity. The aim of this study is to determine if non-invasive imaging of cell death in tumors will benefit from increasing circulation time of annexin A5 by increasing its size.ProceduresAnnexin A5 size was increased by complexation of biotinylated annexin A5 with Alexa-Fluor680-labeled streptavidin. The non-binding variant of annexin A5, M1234, was used as negative control. The HT29 colon carcinoma xenograft model in NMRI nude mice was used to measure tumor uptake in vivo. Tumor uptake of fluorescent annexin A5-variants was measured using non-invasive optical imaging.ResultsThe annexin A5-streptavidin complex (4∶1, moles:moles, Mw ∼200 kDa) binds phosphatidylserine-expressing membranes with a Hill-coefficient of 5.7±0.5 for Ca2+-binding and an EC50 of 0.9±0.1 mM Ca2+ (EC50 is the Ca2+ concentration required for half maximal binding)(annexin A5: Hill-coefficient 3.9±0.2, EC50 1.5±0.2 mM Ca2+). Circulation half-life of annexin A5-streptavidin is ±21 minutes (circulation half-life of annexin A5 is ±4 min.). Tumor uptake of annexin A5-streptavidin was higher and persisted longer than annexin A5-uptake but depended less on phosphatidylserine binding.ConclusionIncreasing annexin A5 size prolongs circulation times and increases tumor uptake, but decreases contribution of PS-targeting to tumor uptake and abolishes power to report efficacy of therapy.

Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.