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Supplementary Figure from DNA-PK Inhibitor Peposertib Amplifies Radiation-Induced Inflammatory Micronucleation and Enhances TGFβ/PD-L1 Targeted Cancer Immunotherapy

Radiotherapy is the most widely used cancer treatment and improvements in its efficacy and safety are highly sought-after. Peposertib (also known as M3814), a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, effectively suppresses the repair of radiation-induced DNA double-strand breaks (DSB) and regresses human xenograft tumors in preclinical models. Irradiated cancer cells devoid of p53 activity are especially sensitive to the DNA-PK inhibitor, as they lose a key cell-cycle checkpoint circuit and enter mitosis with unrepaired DSBs, leading to catastrophic consequences. Here, we show that inhibiting the repair of DSBs induced by ionizing radiation with peposertib offers a powerful new way for improving radiotherapy by simultaneously enhancing cancer cell killing and response to a bifunctional TGFβ “trap”/anti-PD-L1 cancer immunotherapy. By promoting chromosome misalignment and missegregation in p53-deficient cancer cells with unrepaired DSBs, DNA-PK inhibitor accelerated micronuclei formation, a key generator of cytosolic DNA and activator of cGAS/STING-dependent inflammatory signaling as it elevated PD-L1 expression in irradiated cancer cells. Triple combination of radiation, peposertib, and bintrafusp alfa, a fusion protein simultaneously inhibiting the profibrotic TGFβ and immunosuppressive PD-L1 pathways was superior to dual combinations and suggested a novel approach to more efficacious radioimmunotherapy of cancer.Implications:Selective inhibition of DNA-PK in irradiated cancer cells enhances inflammatory signaling and activity of dual TGFβ/PD-L1 targeted therapy and may offer a more efficacious combination option for the treatment of locally advanced solid tumors.

NHS-muIL12 and avelumab combination treatment enhanced the necrotic fraction in EMT-6 tumors.

Purpose: To determine whether combination therapy with NHS-muIL12 and the anti-programmed death ligand 1 (PD-L1) antibody avelumab can enhance antitumor efficacy in preclinical models relative to monotherapies.Experimental Design: BALB/c mice bearing orthotopic EMT-6 mammary tumors and μMt− mice bearing subcutaneous MC38 tumors were treated with NHS-muIL12, avelumab, or combination therapy; tumor growth and survival were assessed. Tumor recurrence following remission and rechallenge was evaluated in EMT-6 tumor-bearing mice. Immune cell populations within spleen and tumors were evaluated by FACS and IHC. Immune gene expression in tumor tissue was profiled by NanoString® assay and plasma cytokine levels were determined by multiplex cytokine assay. The frequency of tumor antigen–reactive IFNγ-producing CD8+ T cells was evaluated by ELISpot assay.Results: NHS-muIL12 and avelumab combination therapy enhanced antitumor efficacy relative to either monotherapy in both tumor models. Most EMT-6 tumor–bearing mice treated with combination therapy had complete tumor regression. Combination therapy also induced the generation of tumor-specific immune memory, as demonstrated by protection against tumor rechallenge and induction of effector and memory T cells. Combination therapy enhanced cytotoxic NK and CD8+ T-cell proliferation and T-bet expression, whereas NHS-muIL12 monotherapy induced CD8+ T-cell infiltration into the tumor. Combination therapy also enhanced plasma cytokine levels and stimulated expression of a greater number of innate and adaptive immune genes compared with either monotherapy.Conclusions: These data indicate that combination therapy with NHS-muIL12 and avelumab increased antitumor efficacy in preclinical models, and suggest that combining NHS-IL12 and avelumab may be a promising approach to treating patients with solid tumors. Clin Cancer Res; 23(19); 5869–80. ©2017 AACR.

Supplementary Materials and Methods

Radiotherapy is the most widely used cancer treatment and improvements in its efficacy and safety are highly sought-after. Peposertib (also known as M3814), a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, effectively suppresses the repair of radiation-induced DNA double-strand breaks (DSB) and regresses human xenograft tumors in preclinical models. Irradiated cancer cells devoid of p53 activity are especially sensitive to the DNA-PK inhibitor, as they lose a key cell-cycle checkpoint circuit and enter mitosis with unrepaired DSBs, leading to catastrophic consequences. Here, we show that inhibiting the repair of DSBs induced by ionizing radiation with peposertib offers a powerful new way for improving radiotherapy by simultaneously enhancing cancer cell killing and response to a bifunctional TGFβ “trap”/anti-PD-L1 cancer immunotherapy. By promoting chromosome misalignment and missegregation in p53-deficient cancer cells with unrepaired DSBs, DNA-PK inhibitor accelerated micronuclei formation, a key generator of cytosolic DNA and activator of cGAS/STING-dependent inflammatory signaling as it elevated PD-L1 expression in irradiated cancer cells. Triple combination of radiation, peposertib, and bintrafusp alfa, a fusion protein simultaneously inhibiting the profibrotic TGFβ and immunosuppressive PD-L1 pathways was superior to dual combinations and suggested a novel approach to more efficacious radioimmunotherapy of cancer. Implications: Selective inhibition of DNA-PK in irradiated cancer cells enhances inflammatory signaling and activity of dual TGFβ/PD-L1 targeted therapy and may offer a more efficacious combination option for the treatment of locally advanced solid tumors.

Highlights • Combination therapy induced both adaptive and innate antitumor immunity. • Combination therapy significantly enhanced antitumor activity. • Combination therapy generated tumor antigen specific immune memory. • Bintrafusp alfa mediated the reduction of lung metastases., Combinatorial immunotherapy approaches are emerging as viable cancer therapeutic strategies for improving patient responses and outcomes. This study investigated whether two such immunotherapies, with complementary mechanisms of action, could enhance antitumor activity in murine tumor models. The immunocytokine NHS-IL12, and surrogate NHS-muIL12, are designed to deliver IL-12 and muIL-12, respectively, to the tumor microenvironment (TME) to activate NK cells and CD8+ T cells and increase their cytotoxic functions. Bintrafusp alfa (BA) is a bifunctional fusion protein composed of the extracellular domains of the TGF-β receptor II to function as a TGF-β “trap” fused to a human IgG1 antibody blocking PD-L1. With this dual-targeting strategy, BA enhances efficacy over that of monotherapies in preclinical studies. In this study, NHS-muIL12 and BA combination therapy enhanced antitumor activity, prolonged survival, and induced tumor-specific antitumor immunity. This combination therapy increased tumor-specific CD8+ T cells and induced immune profiles, consistent with the activation of both adaptive and innate immune systems. In addition, BA reduced lung metastasis in the 4T1 model. Collectively, these findings could support clinical trials designed to investigate NHS-IL12 and BA combination therapy for patients with advanced solid tumors

BackgroundBintrafusp alfa (BA) is a bifunctional fusion protein designed for colocalized, simultaneous inhibition of two immunosuppressive pathways, transforming growth factor-β (TGF-β) and programmed death-ligand 1 (PD-L1), within the tumor microenvironment (TME). We hypothesized that targeting PD-L1 to the tumor by BA colocalizes the TGF-β trap (TGF-βRII) to the TME, enabling it to sequester TGF-β in the tumor more effectively than systemic TGF-β blockade, thereby enhancing antitumor activity.MethodsMultiple technologies were used to characterize the TGF-β trap binding avidity. BA versus combinations of anti-PD-L1 and TGF-β trap or the pan-TGF-β antibody fresolimumab were compared in proliferation and two-way mixed lymphocyte reaction assays. Immunophenotyping of tumor-infiltrating lymphocytes (TILs) and RNA sequencing (RNAseq) analysis assessing stromal and immune landscape following BA or the combination therapy were performed in MC38 tumors. TGF-β and PD-L1 co-expression and their associated gene signatures in MC38 tumors and human lung carcinoma tissue were studied with single-cell RNAseq (scRNAseq) and immunostaining. BA-induced internalization, degradation, and depletion of TGF-β were investigated in vitro.ResultsBA and fresolimumab had comparable intrinsic binding to TGF-β1, but there was an ~80× avidity-based increase in binding affinity with BA. BA inhibited cell proliferation in TGF-β-dependent and PD-L1-expressing cells more potently than TGF-β trap or fresolimumab. Compared with the combination of anti-PD-L1 and TGF-β trap or fresolimumab, BA enhanced T cell activation in vitro and increased TILs in MC38 tumors, which correlated with efficacy. BA induced distinct gene expression in the TME compared with the combination therapy, including upregulation of immune-related gene signatures and reduced activities in TGF-β-regulated pathways, such as epithelial-mesenchymal transition, extracellular matrix deposition, and fibrosis. Regulatory T cells, macrophages, immune cells of myeloid lineage, and fibroblasts were key PD-L1/TGF-β1 co-expressing cells in the TME. scRNAseq analysis suggested BA modulation of the macrophage phenotype, which was confirmed by histological assessment. PD-L1/TGF-β1 co-expression was also seen in human tumors. Finally, BA induced TGF-β1 internalization and degradation in the lysosomes.ConclusionBA more effectively blocks TGF-β by targeting TGF-β trap to the tumor via PD-L1 binding. Such colocalized targeting elicits distinct and superior antitumor responses relative to single agent combination therapy.

Summary Localized radiotherapy (RT) induces an immunogenic antitumor response that is in part counterbalanced by activation of immune evasive and tissue remodeling processes, e.g., via upregulation of programmed cell death-ligand 1 (PD-L1) and transforming growth factor β (TGF-β). We report that a bifunctional fusion protein that simultaneously inhibits TGF-β and PD-L1, bintrafusp alfa (BA), effectively synergizes with radiotherapy, leading to superior survival in multiple therapy-resistant murine tumor models with poor immune infiltration. The BA + RT (BART) combination increases tumor-infiltrating leukocytes, reprograms the tumor microenvironment, and attenuates RT-induced fibrosis, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Consistently, the beneficial effects of BART are in part reversed by depletion of cytotoxic CD8+ T cells. Intriguingly, targeting of the TGF-β trap to PD-L1+ endothelium and the M2/lipofibroblast-like cell compartment by BA attenuated late-stage RT-induced lung fibrosis. Together, the results suggest that the BART combination has the potential to eradicate therapy-resistant tumors while sparing normal tissue, further supporting its clinical translation.

The targeting of pathways that regulate tumor angiogenesis is an effective strategy for the treatment of solid tumors. Transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) are master regulators of tumor angiogenesis that limit the efficacy of cancer therapies and suppress the anti-tumor immune response. Combination therapies consisting of immune checkpoint inhibitors and tumor angiogenesis inhibitors are emerging as effective treatments for solid cancers. Bintrafusp alfa (M7824) is a bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 antibody blocking PD-L1. Here, we tested the antitumor efficacy of a combination therapy consisting of bintrafusp alfa plus a VEGF receptor kinase inhibitor (pazopanib, lenvatinib, regorafenib, axitinib, or nintedanib), or a VEGF neutralizing antibody (B20) in multiple preclinical tumor models. The combination of bintrafusp alfa with lenvatinib, regorafenib, axitinib, or nintedanib showed significantly greater antitumor efficacy than the respective monotherapies in the CT26 mouse syngeneic colon carcinoma model. In the 4T1 mouse breast carcinoma model, the combination of bintrafusp alfa with anti-VEGF, lenvatinib, or regorafenib was significantly more efficacious than the respective monotherapies. Additionally, the combination of bintrafusp alfa with pazopanib also showed superior antitumor efficacy in an orthotopic Renca mouse tumor model. These data indicate that bintrafusp alfa may be a viable combination partner with multiple angiogenesis inhibitors and provide a rational for the continued development of these combination therapies to treat solid cancers. Citation Format: Tsz-Lun Yeung, Adam Lazorchak, Huakui Yu, Jin Qi, Guozhong Qin, Bo Marelli, Kai Fu, Yan Lan. Bintrafusp alfa enhances the antitumor efficacy of anti-VEGF and VEGF receptor kinase inhibitors in preclinical solid tumor models [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 443.

Ionizing radiation therapy (RT) is a standard of care for many cancer indications. RT promotes the activation of transforming growth factor (TGF)-β, a pleotropic cytokine that mediates RT-induced fibrosis and suppresses anti-tumor immunity. Additionally, RT induces the expression of the immunosuppressive checkpoint PD-L1 on tumor cells. Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor, functioning as a TGF-β “trap”, fused to a human IgG1 antibody blocking PD-L1. Bintrafusp alfa enhances anti-tumor immunity elicited by RT and reverses RT-induced fibrosis in preclinical models. Inhibition of ataxia telangiectasia mutated (ATM) increases the cancer cell's sensitivity to radiotherapy by blocking cellular pathways which facilitate the response to, and repair of, RT induced DNA double strand breaks. We hypothesize that a combination therapy consisting of bintrafusp alfa, RT and the ATM kinase inhibitor M4076 will show greater anti-tumor efficacy than the respective mono- or dual-combination therapies in immune competent preclinical solid tumor models. The anti-tumor efficacy of bintrafusp alfa, RT, and M4076 triple combination therapy was explored in the immune therapy resistant 4T1 murine syngeneic breast carcinoma model. This triple combination therapy significantly increased anti-tumor efficacy and prolonged overall survival relative to the bintrafusp alfa + RT or M4076 + RT dual combination therapies. The triple combination was further evaluated in the more immunogenic MC38 colon carcinoma model. MC38 tumor bearing mice treated with the triple combination therapy showed a significantly greater tumor growth inhibition and long term tumor free survival when compared with the bintrafusp alfa + RT or M4076 + RT treatment groups. Finally, the bintrafusp alfa + RT + M4076 combination therapy also showed significantly greater tumor growth inhibition and long term tumor free survival when compared to the combination of anti-PD-L1 + RT + M4076. The bintrafusp alfa triple combination resulted in 8 of 10 mice showing long term tumor free survival, whereas the anti-PD-L1 triple combination failed to elicit tumor free survival (0 of 10 mice tumor free). These studies demonstrate the superior anti-tumor efficacy of the Bintrafusp alfa + RT + M4076 triple combination therapy over that of the dual combination therapies of RT + bintrafusp alfa or RT + M4076. These data in the MC38 model also suggest that bintrafusp alfa may promote a superior anti-tumor immune response relative to that of anti-PD-L1 when in combination with RT and M4076. Together, these data support the rational to develop a bintrafusp alfa + RT + M4076 combination therapy for the treatment of solid tumors. Citation Format: Adam Lazorchak, Huakui Yu, Guozhong Qin, Jin Qi, Bo Marelli, Yan Lan. Bintrafusp alfa enhances anti-tumor efficacy in combination with radiation therapy plus the ataxia telangiectasia mutated (ATM) inhibitor, M4076 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-088.

Background: Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor, to function as a TGF-β “trap”, fused to a human IgG1 antibody blocking PD-L1, which has shown early evidence of clinical activity in phase 1 studies in patients with advanced solid tumors. We recently reported high expression of HMGA2 in patients with triple negative breast cancer (TNBC) who experienced disease control (response or stable disease) with bintrafusp alfa treatment compared with those who had progressive disease, suggesting that HMGA2 may predict response to bintrafusp alfa in this indication. HMGA2 is a transcriptional regulator whose expression is upregulated by TGF-β signaling and is known to be an important factor in mediating TGF-β–induced epithelial–mesenchymal transition (EMT). The purpose of this study was to evaluate the association between HMGA2 expression and TGF-β signaling in TNBC and the effect of bintrafusp alfa on HMGA2/TGF-β signaling. Methods: The syngeneic murine 4T1 tumor model was utilized for this study because it closely mimics human stage IV TNBC, has high baseline levels of HMGA2 expression, and bintrafusp alfa directly modulates canonical TGF-β signaling and induces antitumor activity in this model. Results: Tumors from mice treated with isotype control, anti–PD-L1, anti–PD-L1(mut)/TGF-β trap (trap control), or bintrafusp alfa were analyzed by RNAseq and both bintrafusp alfa or trap control treatment decreased HMGA2 expression relative to anti–PD-L1 or isotype control treatment. In isotype control-treated 4T1 tumors Spearman's rho correlation coefficient showed that 29% (26/89) of the TGF-β signaling-related genes significantly correlated with HMGA2 expression, indicating that there is a strong association between HMGA2 and TGF-β signaling. In bintrafusp alfa-treated tumors, the association of HMGA2 and TGF-β signaling was even stronger, with 55% (49/89) of TGF-β signaling-related genes significantly associated with HMGA2, suggesting that there is a relationship between HMGA2, TGF-β signaling, and bintrafusp alfa pharmacodynamic effects in this TNBC model. Analysis of individual TGF-β signaling-related genes from this signature showed significant correlation between HMGA2 expression and the expression of TGF-β receptors, ligands, collagen, and EMT-related genes. Furthermore, both bintrafusp alfa and trap control treatment significantly reduced expression of these HMGA2-correlated TGF-β signaling-related genes relative to anti–PD-L1 or isotype control treatment in the 4T1 model, suggesting that HMGA2 is associated with TGF-β-related signaling more strongly in the presence of TGF-β sequestration. Conclusions: Taken together, these observations warrant further analysis of the potential link between HMGA2 and bintrafusp alfa antitumor efficacy. Citation Format: Tsz-Lun Yeung, George Locke, Adam Lazorchak, Guozhong Qin, Huakui Yu, Jin Qi, Bo Marelli, Molly Jenkins, Alex Rolfe, Laureen S. Ojalvo, Isabelle Dussault, Yan Lan. Association between TGF-β signaling and HMGA2, a potential biomarker for bintrafusp alfa in triple negative breast cancer [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 1573.

Antibodies targeting immune checkpoints are emerging as potent and viable cancer therapies, but not all patients respond to these as single agents. Concurrently targeting additional immunosuppressive pathways is a promising approach to enhance immune checkpoint blockade, and bifunctional molecules designed to target two pathways simultaneously may provide a strategic advantage over the combination of two single agents. M7824 (MSB0011359C) is a bifunctional fusion protein composed of a monoclonal antibody against programmed death ligand 1 (PD-L1) fused to the extracellular domain of human transforming growth factor-β (TGF-β) receptor II, which functions as a "trap" for all three TGF-β isoforms. We demonstrate that M7824 efficiently, specifically, and simultaneously binds PD-L1 and TGF-β. In syngeneic mouse models, M7824 suppressed tumor growth and metastasis more effectively than treatment with either an anti-PD-L1 antibody or TGF-β trap alone; furthermore, M7824 extended survival and conferred long-term protective antitumor immunity. Mechanistically, the dual anti-immunosuppressive function of M7824 resulted in activation of both the innate and adaptive immune systems, which contributed to M7824's antitumor activity. Finally, M7824 was an effective combination partner for radiotherapy or chemotherapy in mouse models. Collectively, our preclinical data demonstrate that simultaneous blockade of the PD-L1 and TGF-β pathways by M7824 elicits potent and superior antitumor activity relative to monotherapies.

Purpose: To determine whether combination therapy with NHS-muIL12 and the anti-programmed death ligand 1 (PD-L1) antibody avelumab can enhance antitumor efficacy in preclinical models relative to monotherapies. Experimental Design: BALB/c mice bearing orthotopic EMT-6 mammary tumors and μMt− mice bearing subcutaneous MC38 tumors were treated with NHS-muIL12, avelumab, or combination therapy; tumor growth and survival were assessed. Tumor recurrence following remission and rechallenge was evaluated in EMT-6 tumor-bearing mice. Immune cell populations within spleen and tumors were evaluated by FACS and IHC. Immune gene expression in tumor tissue was profiled by NanoString® assay and plasma cytokine levels were determined by multiplex cytokine assay. The frequency of tumor antigen–reactive IFNγ-producing CD8+ T cells was evaluated by ELISpot assay. Results: NHS-muIL12 and avelumab combination therapy enhanced antitumor efficacy relative to either monotherapy in both tumor models. Most EMT-6 tumor–bearing mice treated with combination therapy had complete tumor regression. Combination therapy also induced the generation of tumor-specific immune memory, as demonstrated by protection against tumor rechallenge and induction of effector and memory T cells. Combination therapy enhanced cytotoxic NK and CD8+ T-cell proliferation and T-bet expression, whereas NHS-muIL12 monotherapy induced CD8+ T-cell infiltration into the tumor. Combination therapy also enhanced plasma cytokine levels and stimulated expression of a greater number of innate and adaptive immune genes compared with either monotherapy. Conclusions: These data indicate that combination therapy with NHS-muIL12 and avelumab increased antitumor efficacy in preclinical models, and suggest that combining NHS-IL12 and avelumab may be a promising approach to treating patients with solid tumors. Clin Cancer Res; 23(19); 5869–80. ©2017 AACR.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

We have developed a presenilin-1 ( PS1 ) conditional knockout mouse (cKO), in which PS1 inactivation is restricted to the postnatal forebrain. The PS1 cKO mouse is viable and exhibits no gross abnormalities. The carboxy-terminal fragments of the amyloid precursor protein differentially accumulate in the cerebral cortex of cKO mice, while generation of β-amyloid peptides is reduced. Expression of Notch downstream effector genes, Hes1 , Hes5 , and Dll1 , is unaffected in the cKO cortex. Although basal synaptic transmission, long-term potentiation, and long-term depression at hippocampal area CA1 synapses are normal, the PS1 cKO mice exhibit subtle but significant deficits in long-term spatial memory. These results demonstrate that inactivation of PS1 function in the adult cerebral cortex leads to reduced Aβ generation and subtle cognitive deficits without affecting expression of Notch downstream genes.

Generation of a floxed Presenilin-1 (PS1) allele involved two recombination events in the embryonic stem (ES) cells. First, a targeting vector containing a loxP site in intron 1 and a floxed CMV-HYG/TK double selection cassette in intron 3 was integrated into the PS1 locus by homologous recombination. The use of a negative selection cassette, PGK-DTA, dramatically increased the recombination efficiency within the targeted locus (75-fold). Second, an expression vector encoding Cre recombinase was introduced to excise the floxed CMV-HYG/TK cassette via site-specific recombination. However, all five ES cell clones testing positive for the proper removal of the CMV-HYG/TK cassette also contained a proportion of ES cells in which recombination had occurred between the distal loxP sites in introns 1 and 3, resulting in excision of the entire floxed region. It is therefore critical to screen for possible recombination events involving all 3 loxP sites, in order to identify ES cells clones bearing high proportions of the desired ES cells. genesis 26:5-8, 2000.

The promoter region of human ryudocan was isolated from a human λ dash genomic library and cloned into pBluescript. The 5′ flanking region contained a classical TATA box and GC rich regions that are commonly found in constituitively expressed genes. Two fusion gene constructs, one of 898 bp and the other of 480 bp of the 5′ flanking DNA coupled to the luciferase gene,were transiently expressed in a mouse endothelioma cell line and in human umbilical endothelial cell cultures. Both constructs were capable of driving luciferase expression; the 898 bp construct produced greater levels of luciferase activity than the 480 bp construct in both cell types examined. Analysis of the sequence revealed the presence of several potential sites for nuclear transcription factor binding; the relevance of these sites is presently unknown.

Background. L-BLP25 is an investigational antigen-specific cancer immunotherapeutic agent targeting human mucin 1 (MUC1). A single low dose of cyclophosphamide (CPA) is known to be immunostimulatory and may enhance the anti-tumor effects of L-BLP25 when given 3 days before treatment initiation. Here we report the immune and anti-tumor efficacy of L-BLP25 with CPA pretreatment in subcutaneous (s.c.) and orthotopic models of murine pancreatic cancer. These models allow for reproduction of the clinical scenario, where 8 weekly doses of L-BLP25 are administered. Methods. Murine pancreatic tumor cells Panc02 transfected with human MUC1 were injected for comparison s.c. or orthotopically into human MUC1 transgenic mice. Mice received either vehicle control (PBS), CPA (100 mg/kg), L-BLP25 (100 μg), or L-BLP25 (100 μg) + CPA (100 mg/kg). CPA was administered intravenously (i.v.) 3 days prior to initiation of 8 weekly intradermal (i.d.) or s.c. L-BLP25 injections. Tumor volume was monitored and splenic cell populations were quantified by FACS. In the s.c. tumor model, the contribution of CD4+ and CD8+ T cell subsets to the inhibition of tumor growth was studied. In the orthotopic model, antigen-specific IFN-γ secretion was assessed via ex vivo T cell re-stimulation with antigen alone. Results. In the s.c. model of pancreatic cancer, tumor volume was reduced compared to vehicle at day 49 after i.d. administration of L-BLP25 (41.4%, p Citation Format: Kenneth Hance, Bo Marelli, Jin Qi, Guozhong Qin, Huakui Yu, Hong Wang, Xiaomei Xu, Robert Tighe, Beatrice Brunkhorst, Yan Lan, Robert Hofmeister, Helen Sabzevari, Michael Wolf. Mechanism of action of L-BLP25 in preclinical pancreatic tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1237. doi:10.1158/1538-7445.AM2013-1237

Background Tecemotide is a MUC1 antigen-specific therapeutic cancer vaccine. In phase III clinical studies, delivery of tecemotide is preceded by a single low dose of cyclophosphamide (CPA) to inhibit regulatory T cells (Tregs) and enhance the response to the tumor-associated antigen. Here, we investigated effects of CPA on the immune environment and tumor growth in preclinical murine models. Methods The effect of CPA was investigated in tumor-free human MUC1 transgenic mice and anti-tumor responses were evaluated in the mice engrafted with syngeneic colorectal and ovarian cancer cells expressing human MUC1. The mice received either vehicle control, CPA (100 mg/ kg), tecemotide (100 μg) or CPA + tecemotide. The immune cell phenotype and function in the spleen were assessed on Days 1, 3 and 7 post-administration.