Large Established Tumors Research Articles

Abstract B3: Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer immunotherapy

Abstract It remains unclear why the clinically used anti-CTLA-4 antibodies, popularly called checkpoint inhibitors, have severe immunotherapy-related adverse effects (irAEs) and yet suboptimal cancer immunotherapeutic effects (CITE). Here we report that while irAE-prone Ipilimumab and TremeIgG1 rapidly direct cell surface CTLA-4 for lysosomal degradation, the non-irAE-prone antibodies we generated, HL12 or HL32, dissociate from CTLA-4 after endocytosis and allow CTLA-4 recycling to cell surface by the LRBA-dependent mechanism. Disrupting CTLA-4 recycling results in robust CTLA-4 downregulation by all anti-CTLA-4 antibodies and confers toxicity to a non-irAE-prone anti-CTLA-4 mAb. Conversely, increasing the pH sensitivity of TremeIgG1 by introducing designed tyrosine-to-histidine mutations prevents antibody-triggered lysosomal CTLA-4 downregulation and dramatically attenuates irAE. Surprisingly, by avoiding CTLA-4 downregulation and due to their increased bioavailability, pH-sensitive anti-CTLA-4 antibodies are more effective in intratumor regulatory T-cell depletion and rejection of large established tumors. Our data establish a new paradigm for cancer research that allows for abrogating irAE while increasing CITE of anti-CTLA-4 antibodies. Citation Format: Yan Zhang, Xuexiang Du, Mingyue Liu, Fei Tang, Peng Zhang, Chunxia Ai, James K. Fields, Eric J. Sundberg, Olga S. Latinovic, Martin Devenport, Pan Zheng, Yang Liu. Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B3.

Abstract P5-04-03: Conversion of non-T cell-inflamed triple-negative breast cancer by in situ immunomodulation of induced intratumoral cross-presenting dendritic cells

Abstract Checkpoint inhibitor (CPI)-based immunotherapy such as PD-1/PD-L1 blockade has shown an unprecedented success for the treatment of a variety of malignancies; however, meaningful clinical activity is seen only in a subset of breast cancer patients. This is due, at least in part, to poor T-cell infiltration into the tumor prior to CPI therapy, which is negatively correlated with the treatment response (non-T cell-inflamed tumor). Effective priming and trafficking of tumor-specific T cells to the tumor microenvironment (TME) can theoretically overcome current limitations of immunotherapy for non-T cell-inflamed tumors such as breast cancer. Accumulating evidence indicates that tumor-residing Batf3-dependent CD103+ dendritic cells (DCs) play critical roles in not only priming of tumor-specific CD8+ T cells, but also trafficking of CD8+ T cells to the TME, thus enhancing responses to PD-1/PD-L1 blockade. In the present study, we hypothesized that mobilization and activation of cross-presenting CD103+ DCs in the TME would increase CD8+ T cell infiltrate to the tumor, and facilitate effective regression of breast cancer. To this end, we utilized two triple-negative syngeneic mammary cancer models, 4T1 on a H-2d background and AT3 derived from the PyMT-MMTV model on a H-2b background, and evaluated antitumor efficacy of a combinatorial therapeutic regimen comprised of intratumoral administration of Fms-like tyrosine kinase 3 receptor ligand (Flt3L) to mobilize CD103+ DCs to the TME, local radiotherapy (RT) to induce immunogenic cell death and maturation of DCs, and peritumoral administration of a toll-like receptor 3 (TLR3) agonist to activate DCs. We found intratumoral administration of Flt3L increased the number of CD103+ DCs in the tumor, blood and lymph nodes, and local RT induced upregulation of TLR3, CD40 and CD86 on the tumor-residing CD103+ DCs. Following intratumoral administration of Flt3L, local RT, and in situ delivery of a TLR3 agonist significantly increased CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), mediated effective regression of large established tumors, and significantly improved survival in multiple mouse tumor models. Phenotypic analysis of circulating T cells revealed that the combinatorial regimen increased the frequency of effector memory and central memory T cell subsets with upregulation of PD-1 and C-X3-C motif chemokine receptor 1 (CX3CR1), suggesting effective activation and differentiation of T cells. Interestingly, we found the frequency of macrophages significantly decreased in the tumor after combinatorial treatment, indicating remodeling of both the myeloid and lymphoid compartments in the TME. Furthermore, increased PD-1 expression on CD4+ and CD8+ TILs, and PD-L1 expression in the TME suggest that antitumor efficacy of the combinatorial regimen can be additionally enhanced by PD-1/PD-L1 blockade. Taken together, combinatorial in situ immunomodulation with intratumoral Flt3L administration, RT and, peritumoral administration of a TLR3 agonist can convert non-T cell-inflamed tumors to T-cell inflamed tumors, remodel an immunosuppressive TME, and potentially overcome primary resistance to PD-1/PD-L1 blockade for triple-negative breast cancer. Acknowledgement: This work was supported by Uehara Memorial Foundation (T.O.), NIH/NCI K08CA197966 (F.I.) and P30CA016056 involving the use of Roswell Park Comprehensive Cancer Center’s Flow Cytometry Core. Citation Format: Takaaki Oba, Tibor Keler, Henry C Marsh, Scott Abrams, Fumito Ito. Conversion of non-T cell-inflamed triple-negative breast cancer by in situ immunomodulation of induced intratumoral cross-presenting dendritic cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-03.

Safe eradication of large established tumors using neovasculature‐targeted tumor necrosis factor‐based therapies

Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity‐on‐Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF‐based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo. Upregulation of adhesion markers supports enhanced T‐cell infiltration leading to control or elimination of solid tumors by, respectively, CAR T cells or a combination therapy with CD8‐targeted type I interferon AcTakine. Co‐treatment with a CD13‐targeted type II interferon AcTakine leads to very rapid destruction of the tumor neovasculature and complete regression of large, established tumors. As no tumor markers are needed, safe and efficacious elimination of a broad range of tumor types becomes feasible.

Sensitization to immune checkpoint blockade through activation of a STAT1/NK axis in the tumor microenvironment.

Cancer immunotherapy using antibodies that target immune checkpoints has delivered outstanding results. However, responses only occur in a subset of patients, and it is not fully understood what biological processes determine an effective outcome. This lack of understanding hinders the development of rational combination treatments. We set out to define the pretreatment microenvironment associated with an effective outcome by using the fact that inbred mouse strains bearing monoclonal cancer cell line-derived tumors respond in a dichotomous manner to immune checkpoint blockade (ICB). We compared the cellular composition and gene expression profiles of responsive and nonresponsive tumors from mice before ICB and validated the findings in cohorts of patients with cancer treated with ICB antibodies. We found that responsive tumors were characterized by an inflammatory gene expression signature consistent with up-regulation of signal transducer and activator of transcription 1 (STAT1) and Toll-like receptor 3 (TLR3) signaling and down-regulation of interleukin-10 (IL-10) signaling. In addition, responsive tumors had more infiltrating-activated natural killer (NK) cells, which were necessary for response. Pretreatment of mice with large established tumors using the STAT1-activating cytokine interferon-γ (IFNγ), the TLR3 ligand poly(I:C), and an anti-IL-10 antibody sensitized tumors to ICB by attracting IFNγ-producing NK cells into the tumor, resulting in increased cure rates. Our results identify a pretreatment tumor microenvironment that predicts response to ICB, which can be therapeutically attained. These data suggest a biomarker-driven approach to patient management to establish whether a patient would benefit from treatment with sensitizing therapeutics before ICB.

Hijacking antibody-induced CTLA-4 lysosomal degradation for safer and more effective cancer immunotherapy

It remains unclear why the clinically used anti-CTLA-4 antibodies, popularly called checkpoint inhibitors, have severe immunotherapy-related adverse effects (irAEs) and yet suboptimal cancer immunotherapeutic effects (CITE). Here we report that while irAE-prone Ipilimumab and TremeIgG1 rapidly direct cell surface CTLA-4 for lysosomal degradation, the non-irAE-prone antibodies we generated, HL12 or HL32, dissociate from CTLA-4 after endocytosis and allow CTLA-4 recycling to cell surface by the LRBA-dependent mechanism. Disrupting CTLA-4 recycling results in robust CTLA-4 downregulation by all anti-CTLA-4 antibodies and confers toxicity to a non-irAE-prone anti-CTLA-4 mAb. Conversely, increasing the pH sensitivity of TremeIgG1 by introducing designed tyrosine-to-histidine mutations prevents antibody-triggered lysosomal CTLA-4 downregulation and dramatically attenuates irAE. Surprisingly, by avoiding CTLA-4 downregulation and due to their increased bioavailability, pH-sensitive anti-CTLA-4 antibodies are more effective in intratumor regulatory T-cell depletion and rejection of large established tumors. Our data establish a new paradigm for cancer research that allows for abrogating irAE while increasing CITE of anti-CTLA-4 antibodies.

Abstract 3050: Multimeric IgM antibodies targeting DR5 are potent and rapid inducers of tumor cell apoptosis and cell death in vitro and in vivo

Abstract The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors, death receptor 4 (DR4) and death receptor 5 (DR5) are attractive anti-tumor targets because of their ability to directly induce apoptosis in cancer cells. Though several agonistic IgG antibodies targeting DR5 have demonstrated evidence of preclinical efficacy, little evidence of clinical efficacy was observed likely due to insufficient receptor crosslinking in the tumor microenvironment. We have developed a novel multivalent IgM antibody targeting DR5 that effectively clusters the receptor and rapidly induces apoptosis and tumor cell cytotoxicity. In Colo205 colorectal tumor cells, anti-DR5 IgM induced caspase activation was detected within 30 minutes and by 2 hours nearly all cells had undergone apoptosis, demonstrating a more rapid and greater magnitude of apoptotic induction than anti-DR5 IgG. In an in vitro cytotoxicity screen of solid and hematologic tumor cell lines, an IgM antibody targeting DR5 ranged from 100-fold to greater than 10,000-fold more potent compared to IgG targeting DR5, and IgM was potent on cell lines resistant to IgG crosslinking mediated cell death. In vivo, anti-DR5 IgM induced tumor eradication in the Colo205 model, tumor regression in the IgG-resistant colorectal HCT15 model, and significantly extended overall survival in the B-cell leukemia model Nalm-6 even though dosing was only within the first week of study. In all in vivo studies the IgM was significantly more efficacious than IgG targeting DR5. The DR5 targeting IgM antibody was also efficacious in large established Colo205 tumors up to 600 mm3 in volume and in colorectal patient-derived xenograft (PDX) models as well. When administered in conjunction with standard of care chemotherapy Irinotecan, anti-DR5 IgM induced durable tumor regression in the HCT15 colorectal carcinoma model with 14 out of 30 animals from 2 separate studies tumor free at the end of study (day 71); no animals dosed with IgG were tumor free. In a less sensitive pancreatic model BxPC3, combination of standard of care Gemcitabine with anti-DR5 IgM resulted in an additive effect, while the IgG plus Gemcitabine efficacy was more modest and not statistically significant compared to Gemcitabine alone. Taken together, these results demonstrate that efficient IgM clustering of DR5 is significantly more potent in vitro and in vivo than IgGs and support the development of a human IgM antibody therapeutic targeting DR5 with the potential to treat both solid and hematologic tumors. Citation Format: Beatrice Wang, Tasnim Kothambawala, Ling Wang, Avneesh Saini, Ramesh Baliga, Angus Sinclair, Bruce Keyt. Multimeric IgM antibodies targeting DR5 are potent and rapid inducers of tumor cell apoptosis and cell death in vitro and in vivo [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 3050.

Abstract 5003: Vaccibody DNA vaccine platform VB10.NEO induces strong neo-antigen specific CD8+ T cell responses critical to cure established tumors in pre-clinical models

Abstract BACKGROUND: Recent advances in the field of cancer immunotherapy have identified CD8+ T cell responses against tumor-specific neoantigens as a key driver of tumor regression and prolonged survival. VB10.NEO is a highly potent DNA plasmid vaccine with intrinsic adjuvant effect designed for efficient delivery of personalized tumor-specific neoantigens. VB10.NEO plasmid is translated in vivo and the secreted protein will covalently bind to endocytic receptors on APC by a targeting unit expressing CCL3 (MIP-1α) allowing efficient uptake and presentation of the neoantigens. In addition, CCL3 will attract immune cells by chemotaxis and induce maturation of APC locally. The objective of this study is to demonstrate the potential of VB10.NEO to induce tumor-specific T cell responses and control tumor growth. METHODS: VB10.NEO was delivered i.m to study CD8+ and CD4+ neoantigen-specific T cell responses in four different pre-clinical mouse models. Immunogenicity was compared with delivery of neoantigen as traditional peptide-adjuvant immunization. The tumor protective effect of VB10.NEO in the presence or absence of anti-PD-1 therapy was investigated in the CT26 colon carcinoma model. RESULTS: Vaccibody DNA Vaccine Platform VB10.NEO is flexible and can hold up to at least 40 neoepitopes. VB10.NEO vaccination induced strong neoantigen-specific T cell responses. Homologous boost vaccinations further augmented the response. T cell responses against predicted CD8+ T cell epitopes previously reported as non-immunogenic or activating only weak T cell responses using a conventional peptide-adjuvant or RNA immunization showed strong CD8+ T cell responses when delivered in the Vaccibody format, demonstrating a unique and strong priming of CD8+ T cells using VB10.NEO. The response was also accompanied by CD4+ T cell responses. In a therapeutic tumor setting VB10.NEO vaccinated mice (monotherapy) induced tumor protective responses. The effect was augmented when combining VB10.NEO with anti-PD-1 where complete regression of large established tumors was observed. All tumour-free mice rechallenged with a lethal tumor dose were protected indicating induction of long-lasting memory responses. Furthermore, the critical role of CD8+ T cells for the observed tumour protection was confirmed when depleting CD8+ T cells. CONCLUSION: VB10.NEO immunotherapy induce strong CD8+ T cell responses critical for anti-tumor effect which demonstrate the unique characteristic of the Vaccibody platform to potentiate activation of CD8+ T cells. VB10.NEO in combination with anti-PD-1 further synergize the potent immune responses resulting in durable complete tumor regression in pre-clinical models supporting the scientific rational for the current ongoing clinical trial investigating VB10.NEO in combination with CPI in patients with advanced solid tumors. Citation Format: Elisabeth Stubsrud, Stine Granum, Helene Zell-Flagstad, Audun Bersaas, Lise Madelene Skullerud, Monika Sekelja, Karoline Schjetne, Agnete Fredriksen. Vaccibody DNA vaccine platform VB10.NEO induces strong neo-antigen specific CD8+ T cell responses critical to cure established tumors in pre-clinical models [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 5003.

Bacterium‐Mimicking Vector with Enhanced Adjuvanticity for Cancer Immunotherapy and Minimized Toxicity

AbstractBacteria are utilized as adjuvants for anticancer immunotherapy. However, immunotherapy with bacteria or their extracts is limited due to their toxicity. On the basis of the innate molecular interactions of foreign molecules from the bacterial components with the host pattern recognition receptors (PRRs), a synthetic adjuvant vector morphologically mimicking the bacterium is engineered, which comprises an optimal combination of components derived from bacterial cell wall, flagellum, and nucleoid. The bacterium‐mimicking vectors (BMVs) cooperatively trigger multiple signaling pathways of PRRs and display superior antitumor therapeutic and prophylactic effects to either that of the reported synthetic or bacterium‐derived adjuvant. Significantly, BMVs improve the efficacy of photothermal ablation therapy to eradicate 50% of large established tumor in mice that completely reject tumor rechallenge. The synthetic BMVs with the detoxified and controllable composition exhibit minimized toxicity. Such a bacterium‐mimicking engineering strategy provides a rational approach to select pathogen‐associated molecular patterns, which drives the desired antitumor immune response. The engineered BMVs offer a promising alternative to the bacterial adjuvant for cancer immunotherapy.

C-105 Clinical trial of HER2 cancer vaccine shows benefit in 45% of evaluable patients in phase I study

We have developed and tested therapeutic cancer vaccine targeting HER2-expressing cancers based on autologous dendritic cells (DCs) transduced with an adenovirus expressing the non-signaling extracellular and transmembrane domains of HER2, a driver oncogene in many cancers including breast, ovarian, lung, colorectal, gastroesophageal, and bladder, among others. In mice, the homologous vaccine cured virtually all mice with large established tumors up to 2 cm and with established macroscopic lung metastases. In mice, the protection was dependent on antibodies to HER2 that inhibited HER2 phosphorylation, but was FcR (ADCC) independent, and thus different from the main recognized mechanism of the clinically approved anti-HER2 monoclonal antibody, trastuzumab. A phase I trial is ongoing in patients with advanced metastatic cancers who have failed at least one line of standard therapy, whose tumors scored HER2 1+, 2+ or 3+ by immunohistochemistry or have a HER2 FISH score ≥1.8. Part 1 of the trial was carried out in patients naïve to trastuzumab or other HER2-directed therapies, as it is important to demonstrate safety in the absence of complicating effects of prior HER2-targeted therapies. In Part 1, although no responses were seen in 6 patients at the lowest dose of 5 million DCs, at the second and third dose escalations (10 and 20 million DCs), of 11 evaluable patients, 5 (45%) had clinical benefit, including one CR lasting 89 weeks, one PR lasting 24 weeks, and 3 cases of stable disease, seen in patients with metastatic ovarian, gastroesophageal or colorectal cancers. Adverse reactions were mainly injection site reactions that did not require treatment in most patients. Serial echocardiograms did not show any evidence of cardiotoxicity. The number of circulating tumor cells also decreased in 40% (8/20), 83% (5/6) and 100% (2/2) at 12, 28 and 48 weeks on study, respectively. Based on these results and safety data, the FDA and IRB have approved increasing the maximum dose to 40 million DCs and opening Part 2 of the trial in patients who have failed prior HER2-directed therapies like trastuzumab, mostly metastatic breast and gastric cancer patients. Early results in Part II starting at 20 million DCs already showed 2/6 patients with stable disease. Thus, we have translated a cancer vaccine from mice to human clinical trials with very promising early results, and intend to combine this vaccine with checkpoint inhibitors, as vaccines can induce T cell responses that turn “cold” tumors into “hot” ones amenable to checkpoint blockade immunotherapy.

Synergistic STING activation by PC7A nanovaccine and ionizing radiation improves cancer immunotherapy

Solid cancers are able to escape immune surveillance and are resistant to current treatment in immunotherapy. Recent evidence indicates the critical role of the stimulator of interferon genes (STING) pathway in antitumor immunity. STING-targeted activation is extensively investigated as a new strategy for cancer therapy. Previously, we reported a safe and efficacious STING-activating nanovaccine to boost systemic tumor-specific T cell responses in multiple tumor models. Local radiotherapy has been reported to not only reduce tumor burden but also enhance local antitumor immunity in a STING-dependent manner. In this study, we demonstrate that combination of these two modalities leads to a synergistic response with long-term regression of large established tumors in two mouse tumor models. The percentage of CD8+ T cells increased significantly in primary tumors after combination therapy. Mechanistically, the augmented T cell responses of radiotherapy and nanovaccine is STING pathway dependent. Furthermore, nanovaccine synergizes with radiotherapy to achieve a better therapeutic effect in distal tumors. These findings suggest that combination of local radiotherapy with systemic PC7A nanovaccine offers a useful strategy to improve the therapeutic outcome of late stage solid cancers.

A Sampling of Highlights from the Literature

Human lung tumors upregulate FGL1 (from Armed Forces Institute of Pathology via Wikimedia Commons)LAG-3 is a T-cell inhibitory receptor that also contributes to suppression mediated by regulatory T cells. A high-affinity ligand for LAG-3 is reported by Wang et al. to be fibrinogen-like protein 1 (FGL1), which is normally produced in the liver. T cells' effector functions are restored both in vitro and in vivo by blocking FGL1. When it is absent completely, mice acquire autoimmune syndromes. Upregulated FGL1 gene expression in solid tumors is also associated with poorer outcomes. Inhibiting both FGL1 and PD-1 pathways has synergistic effects, suggesting that combination blockade has antitumor therapeutic potential.Wang J, …, Chen L. Cell 2019 Jan;176:334–47.e12.Change can be good (by Lln.gyenes lfv via Wikimedia Commons)Patients with various solid tumors are currently being treated with immune checkpoint inhibitors, but not all responses are durable. The tumor mutational burden of multiple types of solid tumors was determined by sequencing 468 cancer-related genes from tumors and normal tissues. Patients with tumors of any type, except gliomas, have better overall survival after checkpoint blockade therapy if their mutational burden is in the upper 20th percentile for that tumor, which illustrates the prognostic value of this approach.Samstein RM, …, Morris LGT. Nat Genet 2019 Jan 14. DOI: 10.1038/s41588-018-0312-8.Regulating translation of PD-L1 (by LadyofHats via Wikimedia Commons)Immune evasion by tumors relies on changes in protein expression that were found to emanate from alterations in translation in a murine model of aggressive liver cancer. Tumors that both overexpress MYC and harbor the KRASG12D mutation produce PD-L1, translation of which is repressed in tumors harboring the KRASG12D mutation alone, because an upstream translational repressor is bypassed. Metastasis and immune evasion is dependent upon relief of this translational repression.Xu Y, …, Ruggero D. Nat Med 2019 Jan 14. DOI: 10.1038/s41591-018-0321-2.18th-century tumor educating a B cell (from J. Ashton's Chap-books of the Eighteenth Century, 1834)B cells accumulate in the draining lymph nodes (dLNs) of breast tumors. These B cells secrete IgG that targets the glycosylated form of heatshock protein HSPA4 on breast cancer cells. Ligation of HSPA4 induces the NF-κB pathway, which in turn activates the CXCR4-SDF1α chemotactic system, attracting cancer cells into the dLN and promoting metastasis. These events form a premetastatic niche in the dLNs and ultimately correlate with worse progression-free and overall survival.Gu Y, …, Cao X. Nat Med 2019 Jan 14. DOI: 10.1038/s41591-018-0309-y.STING agonist (by J. Couperus via flickr)Soluble (s)IL15/IL15R complexes are produced in the tumor microenvironment and enhance the numbers of infiltrating CD8+ T cells. Large established tumors are low in sIL15/IL15R, but by delivering STING agonists intratumorally as an inflammatory signal, soluble complexes are upregulated and released, which then drives tumor regression.Santana Carrero RM, …, Schluns KS. Proc Natl Acad Sci USA 2019 Jan;116:599–608.Tregs and conventional T cells use distinct subsets of TCRs (by Seanbatty via Pixabay)Regulatory T cells (Tregs) use a repertoire of T-cell receptors (TCRs) distinct from conventional T cells. Tumor-infiltrating Tregs from different tumor types are oligoclonal by deep sequencing of TCRB. The most expanded clones are tumor antigen–specific, shown by paired TCRA/B chain–transfection into autologous T cells. These Treg clones are also in circulation, but their TCRs are not found on other conventional T cells in the tumor or normal tissue.Ahmadzadeh M, …, Rosenberg SA. Sci Immunol 2019 Jan;4:eaao4310.

Abstract A004: HER2 cancer vaccine phase I clinical trial shows clinical benefit in 54% of evaluable patients

Abstract We developed and tested in mice and human patients a therapeutic cancer vaccine targeting HER2-expressing cancers comprising autologous dendritic cells (DCs) transduced with an adenovirus expressing the non-signaling extracellular and transmembrane domains of HER2, a driver oncogene in many cancers including breast, ovarian, lung, colorectal, gastroesophageal, and bladder, and others. In mice, the homologous vaccine cured virtually all mice with large established tumors up to 2 cm and with established macroscopic lung metastases, and the protection was dependent on antibodies to HER2 that inhibited HER2 phosphorylation, but was FcR (ADCC) independent, and thus distinct from the main recognized mechanism of the clinically-approved anti-HER2 monoclonal antibody, trastuzumab. We are carrying out a phase I clinical trial in patients with advanced metastatic cancers who had progressed on treatment with at least one line of standard therapy, with tumors that are HER2 1+, 2+ or 3+ by immunohistochemistry or have a HER2 FISH score ≥ 1.8. Part 1 of the trial was carried out in patients naïve to trastuzumab or other HER2-directed therapies, to be able to demonstrate safety in the absence of complicating effects of prior HER2-targeted therapies. In Part 1, although no responses were seen in 6 patients at the lowest dose of 5 million DCs, at the second and third dose escalations (10 and 20 million DCs), of 11 evaluable patients, 6 (54%) had clinical benefit, including one complete response (CR) lasting 89 weeks, one partial response (PR) lasting 24 weeks, and 4 cases of stable disease (SD), seen in patients with metastatic ovarian, gastroesophageal, prostate or colorectal cancers. Adverse reactions were mainly injection site reactions that generally did not require treatment. Serial echocardiograms did not reveal any evidence of cardiotoxicity. The number of circulating tumor cells also decreased in 40% (8/20), 83% (5/6) and 100% (2/2) at 12, 28 and 48 weeks on study, respectively. Based on these results and safety data, the FDA and IRB approved increasing the maximum dose to 40 million DCs and opening Part 2 of the trial in patients who have progressed after prior HER2-directed therapies such as trastuzumab, mostly metastatic breast and gastric cancer patients. Early results in Part II already showed 2 patients with SD. Thus, we have translated a cancer vaccine from mice to human clinical trials with very promising early results, and intend to combine this vaccine with checkpoint inhibitors, as vaccines can induce T-cell responses that turn “cold” tumors into “hot” ones amenable to checkpoint blockade immunotherapy. Citation Format: Jay A. Berzofsky, Lauren V. Wood, Hoyoung Maeng, Jane Trepel, David Stroncek, John C. Morris. HER2 cancer vaccine phase I clinical trial shows clinical benefit in 54% of evaluable patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A004.

Abstract B133: Implantable synthetic immune hydrogel for spatiotemporal modulation of tumor-derived immunosuppression and systemic antitumor immunity

Abstract The development of biomaterial-based immune niches that can shape tumor-induced immunosuppressive factors will play an important role to improve current cancer immunotherapy. Herein, we developed an engineered 3-dimensional porous scaffold for creating synergistic action between myeloid-derived suppressor cell (MDSC)-depleting drugs, which can revert an immunosuppressive tumor microenvironment to one supporting antitumor immunity, and cancer vaccines (whole tumor lysate-based antigens and nano-adjuvants carrying Toll-like receptor 3 agonists), which can induce tumor antigen-specific T-cell responses. The local implantation of the scaffold (called immuneCare-DISC, iCD) as a post-surgical treatment in large established 4T1 breast tumors induced systemic antitumor immunity and prevented relapse and metastasis, resulting in survival of 100%. These therapeutic results were obtained from the depletion of MDSCs by control releasing of gemcitabine and the recruitment/activation of dendritic cells, increased infiltrating of T-cells and enhanced level of IFN-γ production by sustained releasing of cancer vaccine from the iCD. This synthetic immune niche iCD as a spatiotemporal modulation of tumor-induced immunosuppression and cancer vaccine-induced immune stimulation is proposed to generate favorable environment to prevent recurrence and metastasis following the surgical resection. Citation Format: Chanyoung Song, Il Woo Shin, Long Ren, Yong Taik Lim. Implantable synthetic immune hydrogel for spatiotemporal modulation of tumor-derived immunosuppression and systemic antitumor immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B133.

Abstract PR09: Intratumoral delivery of engineered modified vaccinia virus Ankara expressing Flt3L and OX40L for "in situ" therapeutic cancer vaccination

Abstract Modified vaccinia virus Ankara (MVA) is a highly attenuated, effective, and safe vaccinia strain that is an important vaccine vector for infectious diseases and cancers. We have previously shown that intratumoral (IT) injection of inactivated MVA elicits stronger antitumor immunity compared with live MVA in murine B16-F10 melanoma and MC38 colon cancer models, indicating that viral immune inhibitory factors might be involved in attenuating antitumor effects. Here we provide evidence that vaccinia virulence factor C7 interacts with transcription factors IRF3 and STAT2, which attenuates both type I IFN production and IFN receptor signaling. Infection of conventional dendritic cells (cDCs) with MVAΔC7L in which C7L gene is deleted results in higher levels of IFNB gene induction and IRF3 phosphorylation compared with MVA. IT MVAΔC7L also induced stronger antitumor responses compared with MVA, which correlates with higher levels of tumor infiltrating activated CD4+ and CD8+ T-cells in both injected and noninjected tumors in a bilateral B16-F10 tumor implantation model. We engineered a recombinant MVA in which the C7L gene was replaced with human Flt3L (hFlt3L) and the J2R gene (a.k.a. TK gene) was replaced with murine OX40L (mOX40L) or human OX40L (hOX40L). Using B16-F10 and MC38 tumor models, we show that IT MVAΔC7L-hFlt3L-ΔJ2R-mOX40L is more effective than MVAΔC7L-hFlt3L, or MVAΔC7L, or heat-inactivated MVAΔC7L in eradicating injected tumors, delaying the growth of the noninjected tumors, and prolonging mice survival. ELISPOT analysis demonstrate that IT MVAΔC7L-hFlt3L-ΔJ2R-mOX40L generates the most tumor-specific CD8+ and CD4+ T-cells compared with the other viruses mentioned above. The combination of IT MVAΔC7L-hFlt3L-ΔJ2R-mOX40L with systemic delivery of anti-CTLA-4 or anti-PD-L1 antibodies results in more efficient eradication of injected tumors, higher survival rate compared with IT virus alone in both bilateral tumor implantation and unilateral large established tumor models. MVAΔC7L-hFlt3L-ΔJ2R-hOX40L has been generated and will be investigated in clinical trials for patients with metastatic cancers in the near future. Taken together, we provide proof-of-concept results for the development of immune activating recombinant MVA to alter tumor immunosuppressive microenvironment through the induction of type I IFN via the cytosolic DNA-sensing pathway, activation of CD103+ dendritic cells, as well as direct activation of both CD4 and CD8 T-cells. This approach enhances tumor antigen presentation and T-cell activation through "in situ" therapeutic vaccination effects, either used as monotherapy or in combination with anti-CTLA-4 or anti-PD-L1 antibody therapy. Citation Format: Liang Deng, Ning Yang, Yi Wang, Wei Yan, Jiahu Wang, John Choi, Stewart Shuman, Taha D. Merghoub, Jedd D. Wolchok. Intratumoral delivery of engineered modified vaccinia virus Ankara expressing Flt3L and OX40L for "in situ" therapeutic cancer vaccination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR09.

Abstract B119: DC-recruiting biomaterial vaccine to enhance antitumor immunity

Abstract Biomaterials have shown substantial potential to integrate synergistically with current cancer vaccine strategies and enhance their effectiveness. We recently developed an injectable biomaterial vaccine via spontaneous assembly of mesoporous silica (MPS) microparticles into a 3D scaffold in vivo. When formulated with GM-CSF and the TLR-9 agonist CpG, the MPS vaccine modulates host dendritic cell (DC) activation and trafficking. Here we demonstrate that a single injection of the MPS vaccine induced persistent germinal center B cell activity for over 30 days. Consequently, when immunized with a small linear Her2/neu peptide within the Trastuzumab binding domain, the MPS vaccine elicited over 2 orders of magnitude higher IgG1 and IgG2a antibody titer compared to a traditional bolus vaccine, and the antibody exhibited immunoreactivity on the native Her2 structure on breast cancer cells. To further enhance CTL responses against tumor antigens, we co-presented the antigen with polyethylenimine (PEI) in the MPS vaccine. PEI increased antigen cross-presentation in murine DCs, and TNF-a and IL-6 production in both murine and human DCs in vitro. Compared to the MPS vaccine, the MPS-PEI vaccine enhanced activated and antigen+ DCs in the vaccine and the vaccine draining lymph node by ~2 fold. Systemically, using both OVA and a HPV-E7 peptide as antigens, the MPS-PEI vaccine induced ~2.5 fold higher IFN-y producing antigen specific circulating CD8+ T-cells compared to the MPS vaccine. Impressively, using a HPV-E7 expressing tumor model, we demonstrated that a single injection of the MPS-PEI vaccine completely eradicated large established tumors in over 80% of mice. Finally, when immunized with a pool of recently sequenced B16 melanoma neoantigen peptides, the MPS-PEI vaccine induced potent therapeutic tumor growth control and synergy with anti-CTLA4 checkpoint blockade therapy. These findings suggest that the MPS vaccine may serve as a facile multifunctional and multi-epitope platform to modulate host immune cell function and augment personalized antitumor immunity. Citation Format: Aileen W. Li, Maxence O. Dellacherie, Miguel Sobral, Omr O. Ali, Jaeyun Kim, David J. Mooney. DC-recruiting biomaterial vaccine to enhance antitumor immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B119.

Amphiphilic nanoparticle delivery enhances the anticancer efficacy of a TLR7 ligand via local immune activation

Although immunotherapy shows great promise for the long-term control of cancer, many tumors still fail to respond to treatment. To improve the outcome, the delivery of immunostimulants to the lymph nodes draining the tumor, where the antitumor immune response is initiated, is key. Efforts to use nanoparticles as carriers for cancer immunotherapy have generally required targeting agents and chemical modification of the drug, and have unfortunately resulted in low delivery and therapeutic efficiency. Here, we report on the efficacy of gold nanoparticles with approximately 5 nm hydrodynamic diameter coated with a mixture of 1-octanethiol and 11-mercaptoundecanesulfonic acid for the delivery of an immunostimulatory TLR7 ligand to tumor-draining lymph nodes. The drug was loaded without modification through nonspecific adsorption into the ligand shell of the nanoparticles, taking advantage of their amphiphilic nature. After loading, nanoparticles retained their stability in solution without significant premature release of the drug, and the drug cargo was immunologically active. Upon subcutaneous injection into tumor-bearing mice, the drug-loaded particles were rapidly transported to the tumor-draining lymph nodes. There, they induced a local immune activation and fostered a cytotoxic T-cell response that was specific for the tumor. Importantly, the particle-delivered TLR7 ligand blocked the growth of large established tumors and significantly prolonged survival compared to the free form of the drug. Thus, we demonstrate for the first time that nanoparticle delivery of a TLR7 immunostimulant to the tumor-draining lymph nodes enhances antitumor immunity and improves the outcome of cancer immunotherapy.

Optimized Gemcitabine Therapy in Combination with E7 Peptide Immunization Elicits Tumor Cure by Preventing Ag-Specific CTL Inhibition in Animals with Large Established Tumors.

The role of chemotherapeutic agents in tumor immunotherapy is still controversial. In this study, we test using a TC-1 tumor model whether gemcitabine plus E7 peptide vaccine regimens (E7 peptides+CpG-ODN+anti-4-1BB Abs) may result in tumor cure in mice with large established tumors, with a focus on their effects on Ag-specific cytotoxic T lymphocyte (CTL) and myeloid-derived suppressor cell levels. Gemcitabine inhibited tumor growth by its direct cytotoxicity to tumor cells in vivo. E7 peptide vaccine regimens enhanced Ag-specific CTL lytic and antitumor therapeutic activity. Initial combination therapy using gemcitabine and E7 peptide vaccine regimens resulted in tumor regression with tumor relapse in animals with large established tumors, which appeared to result from the suppression of Ag-specific CTL activity by gemcitabine treatment. However, optimization of gemcitabine therapy by reducing its dose and frequency led to complete tumor regression without any recurring tumors in all tested mice even after discontinuation of therapy, possibly due to Ag-specific CTL responses. Thus, this study shows that the optimal dose and therapy frequency of gemcitabine are critical for achieving tumor cure in tumor-bearing animals undergoing E7 peptide vaccine regimen therapy, mainly by preventing CTL suppression. These findings may have implications for designing peptide-based therapeutic vaccines in cancer patients undergoing chemotherapy.

Abstract IA06: Enhancing combination immunotherapy and adoptive cell therapy of cancer via lymph node-targeted vaccines

Abstract Lymph node targeting is achieved clinically is sentinel lymph node mapping in cancer patients, where small-molecule dyes are efficiently delivered to lymph nodes by binding to serum albumin. To mimic this process in vaccine delivery, we synthesized amphiphile-vaccines (lipid-polymer conjugates of peptides, proteins, or molecular adjuvants) designed to non-covalently bind vaccine antigens and adjuvants to endogenous albumin. These amphiphile conjugates both protect peptide antigens from premature degradation and greatly increase their delivery to lymph nodes. Such “albumin-hitchhiking” amphiphiles elicited as much as 30-fold amplified cellular immune responses and antitumor immunity against multiple antigens, and further enable mucosal delivery of therapeutic vaccines through albumin-mediated transport across epithelial barriers via the neonatal Fc receptor. When combined with complementary immunotherapy agents, these lymph node-targeted vaccines are capable of eradicating large established tumors in several mouse models, providing a blueprint for curative immunotherapies. We have further demonstrated that amph-vaccines can be used to deliver ligands for chimeric antigen receptor (CAR) T cells to lymph nodes, creating efficient booster vaccines for CAR T cells. Citation Format: Darrell J. Irvine. Enhancing combination immunotherapy and adoptive cell therapy of cancer via lymph node-targeted vaccines [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA06.

Combination of radiation and interleukin 12 eradicates large orthotopic hepatocellular carcinoma through immunomodulation of tumor microenvironment

ABSTRACTImmunotherapies have shown promising results in certain cancer patients. For hepatocellular carcinoma (HCC), the multiplicity of an immunotolerant microenvironment within both the tumor, and the liver per se may limit the efficacy of cancer immunotherapies. Since radiation induces immunogenic cell death and inflammatory reactions within the tumor microenvironment, we hypothesized that a combination therapy of radiation and lasting local immunostimulating agents, achieved by intratumoral injection of an adenoviral vector encoding interleukin 12, may reverse the immunotolerant microenvironment within a well-established orthotopic HCC toward a state favorable for inducing antitumor immunities. Our data showed that radiation and IL-12 combination therapy (RT/IL-12) led to dramatic tumor regression in animals bearing large subcutaneous or orthotopic HCC, induced systemic effect against distant tumor, and significantly prolonged survival. Radiation monotherapy induced tumor regression at early times but afterwards most tumors regained exponential growth, while IL-12 monotherapy only delayed tumor growth. Mechanistic studies revealed that RT/IL-12 increased expression of MHC class II and co-stimulatory molecules CD40 and CD86 on tumor-infiltrating dendritic cells, suggesting an improvement of their antigen presentation activity. RT/IL-12 also significantly reduced accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and impaired their suppressive functions by reducing production of reactive oxygen species. Accordingly, tumor-infiltrating CD8+ T cells and NK cells were significantly activated toward the antitumor phenotype, as revealed by increased expression of CD107a and TNF-α. Together, our data showed that RT/IL-12 treatment could reset the intratumoral immunotolerant state and stimulate activation of antitumor cellular immunity that is capable of eliminating large established HCC tumors.

Abstract 5009: Defined factors overcome T-cell exhaustion via abscopal effect

Abstract Irreversible exhaustion of tumor-associated T cells is an important factor limiting the efficacy of PD-1 blockade in a number of cancers. Recent data suggest that PD-1 blockade functions largely by augmenting T cell costimulation. Given that activated APCs provide necessary T-cell costimulation, we hypothesized that enforced activation of intratumoral APCs would prime tumor-specific T cells and synergize with PD-1 blockade to overcome T-cell exhaustion. After screening various agents in syngeneic murine models, we found that dual CD40/TLR4 activation within a single tumor triggers a systemic tumor-specific CD8 T-cell response that is dependent on BATF3+ dendritic cells. Remarkably, this approach abolishes exhausted PD-1+ intratumoral T cells in treated as well as distant tumors while sparing more proximal T cells outside the tumors. In addition to treating large established tumors, this approach also confers persistent immunity allowing animals to reject reimplanted tumors 90 days after treatment. Dual CD40/TLR4 activation within a single tumor is thus a promising method for overcoming tumor-associated T-cell exhaustion in a manner that may provide durable systemic control of metastatic human cancers while sparing healthy tissues. Citation Format: Danny Khalil, Luis Felipe Campesato, Sadna Budhu, Yanyun Li, Caitlin Jones, Nathan Suek, Cailian Liu, Billel Gasmi, Rachel Giese, Stephane Pourpe, Taha Merghoub, Jedd D. Wolchok. Defined factors overcome T-cell exhaustion via abscopal effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5009.