Intratumoral Immunotherapy Research Articles

Intratumoral immunotherapy with anti-PD-1 and TLR9 agonist induces systemic antitumor immunity without accelerating rejection of cardiac allografts.

Immune checkpoint inhibitors, such as programmed cell death 1 (PD-1) blockades, have revolutionized the field of cancer immunotherapy. However, there is a growing concern whether PD-1 inhibitors can be administered safely to transplant recipients with advanced cancer, as the T cells activated by checkpoint inhibitors may become reactive not only toward tumor antigens but also toward donor alloantigen, thereby resulting in allograft rejection. Here, immunotherapy with anti-PD-1/toll like receptor 9 agonist was administered to C57BL/6 mice bearing a cardiac allograft that were receiving maintenance immunosuppression or a PI4KIIIβ inhibitor-based tolerogenic regimen. Intratumoral (i.t.), but not systemic, immunotherapy promoted potent anti-tumor responses, but did not accelerate allograft rejection. This effect was associated with a pro-immunogenic effect induced by i.t. immunotherapy resulting in systemic cellular and humoral immune anti-tumor responses. Furthermore, when the tumor and cardiac allograft shared major histocompatibility complex (MHC) antigens, i.t. immunotherapy promoted immune responses directed against tumor and the cardiac allograft resulting in allograft rejection. The anti-tumor effect was compromised by maintenance immunosuppression with cyclosporin A, indicating that an optimal balance between enhanced anti-tumor immunity and decreased transplant immunoreactivity is critical. A clinically relevant approach could be to temporarily withdraw maintenance immunosuppression and/or replace it with a PI4KIIIβ inhibitor-based tolerance-inducing regimen to allow for effective immunotherapy to take place.

Intratumoral Immunotherapy for Early-stage Solid Tumors.

The unprecedented benefits of immunotherapy in advanced malignancies have resulted in increased interests in exploiting immune stimulatory agents in earlier-stage solid tumors in the neoadjuvant setting. However, systemic delivery of immunotherapies may cause severe immune-related side-effects and hamper the development of combination treatments. Intratumoral delivery of neoadjuvant immunotherapy provides a promising strategy in harnessing the power of immunotherapy while minimizing off-target toxicities. The direct injection of immune stimulating agents into the tumor primes the local tumor-specific immunity to generate a systemic, durable clinical response. Intratumoral immunotherapy is a highly active area of investigation resulting in a plethora of agents, for example, immune receptor agonists, non-oncolytic and oncolytic viral therapies, being tested in preclinical and clinical settings. Currently, more than 20 neoadjuvant clinical trials exploring distinct intratumoral immune stimulatory agents and their combinations are ongoing. Practical considerations, including appropriate timing and optimal local delivery of immune stimulatory agents play an important role in safety and efficacy of this approach. Here, we discuss promising approaches in drug delivery technologies and opportunity for combining intratumoral immunotherapy with other cancer treatments and summarize the recent preclinical and clinical evidences that highlighted its promise as a part of routine oncologic care.

Response Criteria for Intratumoral Immunotherapy in Solid Tumors: itRECIST.

3141Background: The approval of intratumoral (IT) immunotherapy for metastatic melanoma and the active development of numerous novel IT drugs have created a need for standardized evaluation of resp...

Response criteria for intratumoral immunotherapy in solid tumors: ItRECIST.

3141 Background: The approval of intratumoral (IT) immunotherapy for metastatic melanoma and the active development of numerous novel IT drugs have created a need for standardized evaluation of response to this unique treatment strategy. The Response Evaluation Criteria in Solid Tumors (RECIST) is not suitable for assessing responses separately for injected and noninjected tumors. Building on RECIST concepts, we propose an IT immunotherapy RECIST (itRECIST) to capture data and assess local and systemic responses in a standardized fashion for clinical trials involving IT immunotherapies. Methods: itRECIST will address the unique needs of IT immunotherapy trials but, where possible, aligns with RECIST 1.1 and iRECIST. It does not dictate which lesions to inject but provides guidelines for collecting data and assessing response as treatment evolves. Results: itRECIST enables overall response assessment, separate response assessments in injected and noninjected lesions, and continued assessment following modifications of therapy at initial progression. At baseline, lesions are classified into 4 categories: target injected, target noninjected, nontarget injected, and nontarget noninjected. After baseline, lesions can be reclassified from noninjected to injected if the investigator decides to change the lesions to inject, but target and nontarget designations never change. Overall response at each assessment is based on target lesion response (injected and noninjected), nontarget lesion response, and absence/appearance of new lesions. Noninjected lesion response is determined by comparing tumor burden with baseline and nadir values. Injected lesion assessment is based on visit-to-visit changes in the lesions injected during treatment and on a combined assessment once the patient is off treatment. A new response category is defined to capture progression that would be “confirmed” per iRECIST even though injected lesions are responding and therapy continues. Multiple examples have been created to aid in training and adoption. Conclusions: itRECIST is an important step toward a standardized method of response assessment for this promising and evolving therapeutic modality. The proposed guidelines can be adopted into trial protocols and routine clinical practice without the need for complex additional assessments by treating physicians. Until there is evidence to support wider use, itRECIST is intended only to support standardized collection of data and to facilitate exploratory analysis. Authors G.V.G. and A.D.K. contributed equally to this work.

Intratumor immunotherapy utilizing real time radiological image guidance: Early experience from a tertiary cancer center.

e15223 Background: Intratumoral (IT) delivery of immunotherapeutic agents is a compelling approach to overcoming the systemic barriers of toxicity and lack of efficacy associated with many novel agents. In this context, the application of IT delivery is growing rapidly, however the feasibility and safety profile of these interventions for lesions that require real time radiological image-guidance remains unknown. Methods: Patients who underwent IT injections of immunotherapeutic agents across several clinical trials over a 5 year period at a single tertiary cancer center were included in this analysis. Technical success & procedure related adverse events of the IT injection and the frequently accompanying concomitant or sequential image-guided biopsy of injected (adscopal) and non-injected (abscopal) sites were analyzed. Results: A total of 256 patients with metastatic &/or unresectable solid malignancies underwent a total of 1096 (median 5) image-guided IT investigational agent injections during the study period. There were no adverse events attributable to the technical component of the procedure, specifically during the needle insertions or 3862 concomitant biopsies. Soft tissue and nodal lesions in the extraparietal locations & visceral lesions in deeper, intraparietal locations and solid organs were also injected (adrenal, liver and lung). The median injected lesion tumor length was 3.3cm. Serious adverse events (NCI CTC AE ≥ 3) including dyspnea and severe flu-like symptoms developing within 24 hours of the injection & required hospitalization following 1.5% of injections. Conclusions: Intratumoral immunotherapeutic injections & tissue sampling with real time radiological image guidance are feasible across a wide range of regions and organs irrespective of agent and histology. Post-delivery anticipated & adverse events were rare & manageable.

Abstract No. 430 Self-assembling hydrogel platform improves intratumoral drug delivery and retention

Direct intratumoral (IT) delivery of immunotherapeutics is an increasingly important intersection between interventional radiology and oncology. However, the challenge of IT drug deposition and retention has become apparent as drugs delivered in low viscosity fluids localize poorly in solid tumors. We hypothesized that drugs embedded in a multidomain peptide (MDP) hydrogel would exhibit significantly improved delivery and retention within tumors following percutaneous delivery. The cationic lysine-based MDP K2 (SL)6K2 (or K2-MDP) was used to load and test the biodistribution of the contrast agent iohexol. The radio-opacity of various hydrogel formulations containing 24, 48, and 120 mgI/mL iohexol and 1% wtK2-MDP stock peptide prepared in a buffered solution were tested via CT. Next, we generated a tumor model by subcutaneously implanting 5x106 McA-7777 cells in the 2 flanks of female Buffalo rats (n = 10). Once the tumors reached 1 cm in diameter, the MDP-iohexol hydrogel was injected using a 29G needle at a rate of 50 uL/sec under fluoroscopy, followed by immediate post injection CT scan. Injections of free iohexol in the contralateral tumors were used as controls. All formulations tested here were fully homogeneous and successfully formed hydrogels. The radio-opacity intensity curve showed that the optimal concentration for in vivo experiments was 60 mgI/mL within 1% wt K2-MDP. Fluoroscopic imaging during in vivo injection of free iohexol demonstrated leakage along the tumor capsule as well as venous intravasation. In marked contrast, there was substantially improved localization of the MDP-iohexol hydrogel within the tumor without intravasation. This was confirmed by subsequent CT imaging which demonstrated an approximately 5-fold improvement in the percentage of tumor volume containing the agent (38% vs. 8%, P < 0.001). Our K2-MDP hydrogel significantly improve intratumoral delivery of iohexol, suggesting that embedding immunotherapeutics in this hydrogel has the potential to improve the delivery and outcome in patients receiving intratumoral immunotherapy.

Potency of CD8+ T-cell mediated antitumor immunity from intratumoral immunotherapy with STING agonist, ADU-S100, in an esophageal adenocarcinoma model.

408 Background: Esophageal adenocarcinoma (EAC) is a deadly disease with poor prognosis due to limited treatment options. STING is a transmembrane protein that activates the transcription of type I IFN genes, resulting in the stimulation of APCs and enhanced CD8+ T-cell infiltration. Recently, STING agonists have demonstrated durable anticancer activity in solid tumors when used alone and in combination with either chemotherapy, radiation or immunotherapy. In this study we evaluated the efficacy and immunomodulatory effects of STING agonist +/- radiation in an established EAC model. Methods: Esophagojejunostomy was performed on rats to induce reflux leading to the development EAC. At 32 weeks post operatively, rats received either STING (ADU-S100) or placebo (PBS), +/- 16Gy radiation. Drug efficacy was evaluated by pre- and post- treatment MRI, serial biopsies, histology and RT-PCR. Additionally, immunofluorescence was performed using CD8 and PD-L1 antibodies. Results: A comparison of MRIs in the study groups between 32 and 40 weeks demonstrated a mean increase in percentage tumor volume of 76.7 % and 152.4% in the P and P+R arms and a decrease of 30.1 % and 50.8% in the S and S+R arms, respectively (ANOVA test p &lt; 0.0001) Overall, the S+R group demonstrated the best results with maximum mean volume reduction with all cases responding. Downstream gene expression, pre, on, and post- treatment demonstrated significant upregulation of IFNβ, TNFα, IL-6 and CCL-2 in the treatment groups compared with placebo. On and post treatment, radiation alone, ADU-S100 alone and ADU-S100 + radiation groups demonstrated enhanced PD-LI expression, induced by higher densities of IFNγ producing CD8+ T-cells (p &lt; 0.01). Conclusions: ADU-S100 +/- radiation exhibits potent anti-tumor efficacy and a promising immunomodulatory profile in a de novo EAC model providing the rationale for clinical testing, likely concurrently in combination with immune checkpoint inhibitors.

Intratumoral Immunotherapy-Update 2019.

Intratumoral immunotherapies aim to trigger local and systemic immunologic responses via direct injection of immunostimulatory agents with the goal of tumor cell lysis, followed by release of tumor‐derived antigens and subsequent activation of tumor‐specific effector T cells. In 2019, a multitude of intratumoral immunotherapies with varied mechanisms of action, including nononcolytic viral therapies such as PV‐10 and toll‐like receptor 9 agonists and oncolytic viral therapies such as CAVATAK, Pexa‐Vec, and HF10, have been extensively evaluated in clinical trials and demonstrated promising antitumor activity with tolerable toxicities in melanoma and other solid tumor types. Talimogene laherparepvec (T‐VEC), a genetically modified herpes simplex virus type 1–based oncolytic immunotherapy, is the first oncolytic virus approved by the U.S. Food and Drug Administration for the treatment of unresectable melanoma recurrent after initial surgery. In patients with unresectable metastatic melanoma, T‐VEC demonstrated a superior durable response rate (continuous complete response or partial response lasting ≥6 months) over subcutaneous GM‐CSF (16.3% vs. 2.1%; p < .001). Responses were seen in both injected and uninjected lesions including visceral lesions, suggesting a systemic antitumor response. When combined with immune checkpoint inhibitors, T‐VEC significantly improved response rates compared with single agent; similar results were seen with combinations of checkpoint inhibitors and other intratumoral therapies such as CAVATAK, HF10, and TLR9 agonists. In this review, we highlight recent results from clinical trials of key intratumoral immunotherapies that are being evaluated in the clinic, with a focus on T‐VEC in the treatment of advanced melanoma as a model for future solid tumor indications.Implications for PracticeThis review provides oncologists with the latest information on the development of key intratumoral immunotherapies, particularly oncolytic viruses. Currently, T‐VEC is the only U.S. Food and Drug Administration (FDA)‐approved oncolytic immunotherapy. This article highlights the efficacy and safety data from clinical trials of T‐VEC both as monotherapy and in combination with immune checkpoint inhibitors. This review summarizes current knowledge on intratumoral therapies, a novel modality with increased utility in cancer treatment, and T‐VEC, the only U.S. FDA‐approved oncolytic viral therapy, for medical oncologists. This review evaluates approaches to incorporate T‐VEC into daily practice to offer the possibility of response in selected melanoma patients with manageable adverse events as compared with other available immunotherapies.

Repurposing the yellow fever vaccine for intratumoral immunotherapy

Live 17D is widely used as a prophylactic vaccine strain for yellow fever virus that induces potent neutralizing humoral and cellular immunity against the wild‐type pathogen. 17D replicates and kills mouse and human tumor cell lines but not non‐transformed human cells. Intratumoral injections with viable 17D markedly delay transplanted tumor progression in a CD8 T‐cell‐dependent manner. In mice bearing bilateral tumors in which only one is intratumorally injected, contralateral therapeutic effects are observed consistent with more prominent CD8 T‐cell infiltrates and a treatment‐related reduction of Tregs. Additive efficacy effects were observed upon co‐treatment with intratumoral 17D and systemic anti‐CD137 and anti‐PD‐1 immunostimulatory monoclonal antibodies. Importantly, when mice were preimmunized with 17D, intratumoral 17D treatment achieved better local and distant antitumor immunity. Such beneficial effects of prevaccination are in part explained by the potentiation of CD4 and CD8 T‐cell infiltration in the treated tumor. The repurposed use of a GMP‐grade vaccine to be given via the intratumoral route in prevaccinated patients constitutes a clinically feasible and safe immunotherapy approach.

Repurposing rotavirus vaccines for intratumoral immunotherapy can overcome resistance to immune checkpoint blockade.

Although immune checkpoint-targeted therapies are currently revolutionizing cancer care, only a minority of patients develop durable objective responses to anti-PD-1, PD-L1, and CTLA-4 therapy. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors and overcome the resistance to these immunotherapies. Oncolytic properties of common viruses can be exploited for the priming of antitumor immunity, and such oncolytic viruses are currently in active clinical development in combination with immune checkpoint-targeted therapies. However, the routine implementation of these therapies is limited by their manufacturing constraints, the risk of exposure of clinical staff, and the ongoing regulations on genetically modified organisms. We sought to determine whether anti-infectious disease vaccines could be used as a commercially available source of immunostimulatory agents for cancer immunotherapy. We found that rotavirus vaccines have both immunostimulatory and oncolytic properties. In vitro, they can directly kill cancer cells with features of immunogenic cell death. In vivo, intratumoral rotavirus therapy has antitumor effects that are dependent on the immune system. In several immunocompetent murine tumor models, intratumoral rotavirus overcomes resistance to and synergizes with immune checkpoint-targeted therapy. Heat- and UV-inactivated rotavirus lost their oncolytic activity but kept their synergy with immune checkpoint-targeted antibodies through the up-regulation of the double-stranded RNA receptor retinoic acid-induced gene 1 (RIG-I). Rotavirus vaccines are clinical-grade products used in pediatric and adult populations. Therefore, in situ immunization strategies with intratumoral-attenuated rotavirus could be implemented quickly in the clinic.

Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies

BackgroundImmune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically “cold” tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated.MethodsThe response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models.ResultsTargeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models.ConclusionLocalized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.

Abstract 1474: Repurposing the yellow fever vaccine for intratumoral immunotherapy

Abstract Introduction: Yellow Fever attenuated virus vaccine (17D) induces very potent cellular and humoral immunity. Its delivery in situ in the tumor bed may induce an immune response leading to the killing of infected cells. Experimental procedures: 17D virus was propagated in Vero cells and infectious supernatants were harvested. To evaluate in vitro sensitivity of tumors to 17D infection, a panel of human and mouse tumor cell lines were infected with increasing MOIs and viability was quantified. To evaluate the antitumor efficacy of intratumor (i.t.) injection of 17D, 4x106pfu/50μl were injected (4 doses) in MC38- and in B16-OVA-tumor bearing C57BL/6 mice. Systemic antitumor effects of i.t 17D were examined in bilateral MC38 models in which one of the tumors was left uninjected. Depletion experiments were performed to assess the contribution of CD4, CD8, Gr1 and NK cells. To determine the role of conventional type-I dendritic cells (cDC1), identical experiments were performed in BATF3-/- mice. Tumor and lymph node immune infiltrates were examined by multiparametric flow cytometry. Combination with intraperitoneal anti-PD1 or anti-CD137 was performed to improve antitumor efficacy. To evaluate the effects of 17D-preexisting immunity, mice were preimmunized with 17D prior to MC38 bilateral inoculation and were subsequently treated. To assess the individual cell type contribution to the antitumor effects induced upon 17D pre-immunization, CD8, CD4, CD8+CD4 or sera from preimmunized mice were passively transferred 24h prior to first 17D i.t injection to MC38-tumor bearing mice. Results: 17D live attenuated strain is able to induce cell death of mouse and human tumor cell lines. In MC38 or B16-OVA mice models, repeated i.t. injections of 17D clearly delayed tumor progression. Regarding the systemic effect of i.t. 17D, certain contralateral therapeutic efficacy was induced upon i.t injection in bilateral MC38 models. These local and distant effects were dependent on CD8+ and cDC1 cells. 17D i.t injection increased in CD8+ T cell infiltrates and decreased in Tregs in injected tumors, while in contralateral tumors induced an increase of CD8+ and NK cells expressing key immunomodulatory receptors. I.t. 17D with anti-CD137 monoclonal antibody (mAb) induced a strong synergistic effect, while combination with anti-PD-1 mAb showed mild local additive efficacy compared to 17D monotherapy. Both combinatorial treatments delayed contralateral tumor growth. Notably, 17D preimmunization induced an important improvement of local and distant antitumor immunity. Such beneficial effects of preimmunization were transferred along with CD8+ T cells. Conclusions: 17D can be safely injected i.t. in preclinical models, giving rise to immune-mediate antitumor effects that can be combined with other immunotherapy agents. The repurposed use of this GMP-grade vaccine in preimmunized patients represents a promising and feasible immunotherapy approach. Citation Format: M. Angela Aznar, Carmen Molina, Alvaro Teijeira, Arantza Azpilikueta, Saray Garasa, Alfonso R. Sanchez-Paulete, Iñaki Etxeberria, Maite Alvarez, Pedro Berraondo, Ignacio Melero. Repurposing the yellow fever vaccine for intratumoral immunotherapy [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 1474.

Talimogene Laherparepvec: Review of its Mechanism of Action and Clinical Efficacy and Safety

Intratumoral immunotherapy inhibits tumor growth by killing injected tumor cells, thus initiating local and systemic immune responses. Oncolytic viruses are a novel class of intratumoral immunotherapies that show promise for treating solid tumors. Talimogene laherparepvec is a first-in-class, genetically modified, herpes simplex virus type 1-based oncolytic immunotherapy approved for the local treatment of unresectable cutaneous, subcutaneous and nodal lesions in patients with melanoma recurrent after initial surgery. This review highlights the dual mechanism of action for talimogene laherparepvec (direct tumor cell lysis and stimulation of local response in tumor microenvironment and systemic immune response in distant metastases), summarizes key preclinical and clinical trials evaluating efficacy and safety of talimogene laherparepvec in melanoma, and describes studies ongoing in other solid tumors.

Localizing radioimmunotherapy via nanochannel device for sustained intratumoral drug delivery for solid tumor treatment.

37 Background: Immune related adverse events still occur in up to 91% of patients receiving immunotherapy. Intratumoral administration is a potential solution to limit these events by reducing systemic exposure while increasing local concentration. However, intratumoral injection is hindered by rapid clearance of drug from the tumor. Our group has developed an intratumoral nanochannel implant for sustained delivery of immunotherapy (NDES) as a solution. Recent clinical and preclinical studies have found success in the combination of radiotherapy and immunotherapy in improving both local and distant tumor response. We propose a combination of radiotherapy followed by sustained intratumoral delivery of immunotherapy as a treatment regimen to improve tumor response and reduce side effects. Methods: Mice were implanted with the 4T1 murine mammary carcinoma and separated into treatment and control groups. CD40 agonist antibody was delivered intratumorally via NDES. Radiotherapy was administered at 8 Gy for three consecutive days and implantation of the NDES occurred the day after the final fraction of radiation. The tumor growth curve was plotted and tumor, spleen, lymph nodes, lungs, and blood were collected at the study endpoint for further flow cytometry and histological analysis. Results: The combination of radiotherapy and sustained intratumoral CD40 antibody showed significantly decreased tumor growth rates over radiotherapy or sustained intratumoral immunotherapy alone. Both intratumoral and systemic T-cell activation was observed. Mice treated with sustained intratumoral CD40 antibody showed decreased toxicity when compared to those receiving comparable systemic dosage. Conclusions: Radiotherapy combined with local sustained intratumoral immunotherapy is a potential strategy for improving treatment of solid tumors which currently show suboptimal response to current immunotherapy regimens and for inducing a systemic response to distant metastases while minimizing adverse side effects. The sustained delivery profile of the NDES presents an opportunity for practical intratumoral administration and avoidance of repeated invasive procedures in patients.

Intratumoral Immunotherapy with XCL1 and sFlt3L Encoded in Recombinant Semliki Forest Virus-Derived Vectors Fosters Dendritic Cell-Mediated T-cell Cross-Priming.

: Multiple lines of evidence indicate a critical role of antigen cross-presentation by conventional BATF3-dependent type 1 classical dendritic cells (cDC1) in CD8-mediated antitumor immunity. Flt3L and XCL1, respectively, constitute a key growth/differentiation factor and a potent and specific chemoattractant for cDC1. To exploit their antitumor functions in local immunotherapy, we prepared Semliki Forest Virus (SFV)-based vectors encoding XCL1 and soluble Flt3L (sFlt3L). These vectors readily conferred transgene expression to the tumor cells in culture and when engrafted as subcutaneous mouse tumor models. In syngeneic mice, intratumoral injection of SFV-XCL1-sFlt3L (SFV-XF) delayed progression of MC38- and B16-derived tumors. Therapeutic activity was observed and exerted additive effects in combination with anti-PD-1, anti-CD137, or CTLA-4 immunostimulatory mAbs. Therapeutic effects were abolished by CD8β T-cell depletion and were enhanced by CD4 T-cell depletion, but not by T regulatory cell predepletion with anti-CD25 mAb. Antitumor effects were also abolished in BATF3- and IFNAR-deficient mice. In B16-OVA tumors, SFV-XF increased the number of infiltrating CD8 T cells, including those recognizing OVA. Consistently, following the intratumoral SFV-XF treatment courses, we observed increased BATF3-dependent cDC1 among B16-OVA tumor-infiltrating leukocytes. Such an intratumoral increase was not seen in MC38-derived tumors, but both resident and migratory cDC1 were boosted in SFV-XF-treated MC38 tumor-draining lymph nodes. In conclusion, viral gene transfer of sFlt3L and XCL1 is feasible, safe, and biologically active in mice, exerting antitumor effects that can be potentiated by CD4 T-cell depletion. SIGNIFICANCE: These findings demonstrate that transgenic expression of sFLT3L and XCL1 in tumor cells mediates cross-priming of, and elicits potent antitumor activity from, CD8 T lymphocytes, particularly in combination with CD4 T-cell depletion.

Starting the fight in the tumor: expert recommendations for the development of human intratumoral immunotherapy (HIT-IT)

A European Society for Medical Oncology (ESMO)-sponsored expert meeting was held in Paris on 8 March 2018 which comprised 11 experts from academia, 11 experts from the pharmaceutical industry and 2 clinicians who were representatives of ESMO. The focus of the meeting was exclusively on the intratumoral injection/delivery of immunostimulatory agents with the aim of harmonizing the standard terms and methodologies used in the reporting of human intratumoral immunotherapy (HIT-IT) clinical trials to ensure quality assurance and avoid a blurring of the data reported from different studies. The goal was to provide a reference document, endorsed by the panel members that could provide guidance to clinical investigators, pharmaceutical companies, ethics committees, independent review boards, patient advocates and the regulatory authorities and promote an increase in the number and quality of HIT-IT clinical trials in the future. Particular emphasis was placed not only on the development of precise definitions to facilitate a better understanding between investigators but also on the importance of systematic serial biopsies as a driver for translational research and the need for the recording and reporting of data, to facilitate a better understanding of the key processes involved.

Addressing the Adult Soft Tissue Sarcoma Microenvironment with Intratumoral Immunotherapy

Sarcoma is comprised of a heterogeneous group of tumors originating from the mesenchyme. Sarcoma is also the first tumor that responded to immunotherapeutic agents often termed as “Coley's toxins.” However, immunotherapy is yet to establish its presence in sarcomas. Complex interactions between tumor and immune cells in the tumor microenvironment play a crucial role in response to immunotherapy. There is a dynamic equilibrium created by the immune cells infiltrating the tumor, and this forms the basis of tumor evasion. Manipulating the intratumoral microenvironment will help overcome tumor evasion.

Nanofluidic drug-eluting seed for sustained intratumoral immunotherapy in triple negative breast cancer

Conventional systemic immunotherapy administration often results in insufficient anti-tumor immune response and adverse side effects. Delivering immunotherapeutics intratumorally could maximize tumor exposure, elicit efficient anti-tumor immune response, and minimize toxicity. To fulfill the unmet clinical need for sustained local drug delivery and to avoid repeated intratumoral injections, we developed a nanofluidic-based device for intratumoral drug delivery called the nanofluidic drug-eluting seed (NDES). The NDES is inserted intratumorally using a minimally invasive trocar method similar to brachytherapy seed insertion and offers a clinical advantage of drug elution. Drug diffusion from the NDES is regulated by physical and electrostatic nanoconfinement, thereby resulting in constant and sustained immunotherapeutic delivery without the need for injections or clinician intervention. In this study, the NDES was used to deliver immunotherapeutics intratumorally in the 4 T1 orthotopic murine mammary carcinoma model, which recapitulates triple negative breast cancer. We demonstrated that NDES-mediated intratumoral release of agonist monoclonal antibodies, OX40 and CD40, resulted in potentiation of local and systemic anti-tumor immune response and inhibition of tumor growth compared to control mice. Further, mice treated with NDES-CD40 demonstrated minimal liver damage compared to systemically treated mice. Collectively, our study highlights the NDES as an effective platform for sustained intratumoral immunotherapeutic delivery. The potential clinical impact is tremendous given that the NDES is applicable to a broad spectrum of drugs and solid tumors.

Abstract 711: Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies

Abstract Despite the recent breakthrough of checkpoint blockade antibodies targeting T cells to combat cancer, it has become apparent that the majority of patients are not fully responsive. Engaging innate immune cells is a promising way to fully exploit the anti-cancer potential of the immune system. Our strategy is to stimulate TOLL-like receptors (TLRs) with MEDI9197, a potent TLR7 and TLR8 agonist, which stimulates the release of interferon-alpha (IFN-α) from plasmacytoid dendritic cells (pDCs) and interleukin-12 (IL-12) and other cytokines from myeloid DCs. While several compounds including MEDI9197, CpG and STING agonists can induce the production of IFN-α, we show that only MEDI9197 is able to induce high levels of interferon-gamma (IFN-γ) and IL-12p70 in human PBMCs, a hallmark of TLR 7/8 agonists. We also demonstrate in vitro that MEDI9197 upregulates activation markers on both innate and adaptive human immune cells, repolarizes macrophages and skews immunity from a Th2 towards a Th1 phenotype in a PHA assay, thereby increasing the functional activity of NK and cytotoxic CD8 cells. MEDI9197 is an imidazoquinoline with a unique lipid tail and formulation designed for intratumoral (IT) dosing and retention at the site of injection to minimize unwanted systemic exposure. Mass spectrometry imaging of MEDI9197 in mouse tumors and whole-body autoradiography in rats confirm local retention of MEDI9197, with &amp;lt; 5% of the initial IT dose detectable in the rat serum. We show in the B16-OVA mouse syngeneic tumor model that a single IT dose increases the percentage of tumor infiltrating lymphocytes (TILs), enhances activation of T cells, and leads to a qualitative shift from single to dual cytokine production (IFN-γ and Tumor necrosis factor alpha, TNF-α). Gene expression analysis further reveals increased expression of activatory innate and adaptive immune gene signatures consistent with increased inflammation. As our mouse flow cytometry studies show elevated levels of PD-1 and PD-L1 on TILs, the combination of MEDI9197 with anti-PD-L1 mAb was also evaluated and resulted in enhanced efficacy compared to MEDI9197 alone. We further demonstrate in vitro that combining MEDI9197 with durvalumab (anti-PD-L1) augments cytokine production (e.g. IL-2, IFN-γ) in a co-culture of human DCs and allogenic T cells. These data showcase MEDI9197 as a potent modulator of the innate and adaptive immune system in the fight against cancer and support the rationale for evaluating MEDI9197 in combination with checkpoint blockade and costimulatory agonists in the clinic. MEDI9197 is currently being evaluated in human clinical trials for safety and efficacy (NCT02556463). Citation Format: Stefanie R. Mullins, John Vasilakos, Katharina Deschler, Iwen Grigsby, Song Ren, Matthew J. Elder, Simon J. Dovedi, Andrew J. Leishman, Patricia Ryan, Zachary Cooper, James Elvecrog, Ronald Herbst, Rakesh Kumar, Mark Tomai, Robert W. Wilkinson. Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies [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 711.

Abstract 1708: Nanofluidic drug-eluting seed for sustained intratumoral immunotherapy in triple negative breast cancer

Abstract Conventional systemic immunotherapy administration often results in insufficient anti-tumor immune response and adverse side effects. Delivering immunotherapeutics intratumorally could maximize tumor exposure, elicit robust anti-tumor immune response, and minimize toxicity. To fulfill the unmet clinical need for sustained local drug delivery and to avoid repeated intratumoral injections, we developed a nanofluidic-based device for intratumoral drug delivery called the nanofluidic drug-eluting seed (NDES). The NDES is inserted intratumorally using a minimally invasive trocar method similar to brachytherapy seed insertion and offers a clinical advantage of intratumoral drug elution. Drug diffusion from the NDES is regulated by physical and electrostatic nanoconfinement, thereby resulting in constant and sustained immunotherapeutic delivery without the need for repeated injections or clinician intervention. In this study, the NDES was utilized to deliver immunotherapeutics intratumorally in the 4T1 orthotopic murine mammary carcinoma model, which recapitulates triple negative breast cancer. We demonstrated that NDES-mediated intratumoral release of agonist immunotherapeutic monoclonal antibodies, OX40 and CD40, resulted in potentiation of local and systemic anti-tumor immune response and significant inhibition of tumor growth compared to control mice. Further, mice treated with NDES-CD40 demonstrated minimal liver damage compared to systemically treated mice. Collectively, our study highlights the NDES as an effective platform for sustained intratumoral immunotherapeutic delivery. The potential clinical impact is tremendous given that the NDES is applicable to a broad spectrum of drugs and solid tumors. Citation Format: Corrine Ying Xuan Chua, Priya Jain, Marco Folci, Andrea Ballerini, Jessica Rhudy, April Gilbert, Shail Shaibala, Giacomo Bruno, Carly S. Filgueira, Cassian Yee, E. Brian Butler, Alessandro Grattoni. Nanofluidic drug-eluting seed for sustained intratumoral immunotherapy in triple negative breast cancer [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 1708.