Linda Hammerich

In situ vaccination: Cancer immunotherapy both personalized and off‐the‐shelf

As cancer immunotherapy continues to benefit from novel approaches which cut immune ‘brake pedals’ (e.g. anti‐PD1 and anti‐CTLA4 antibodies) and push immune cell gas pedals (e.g. IL2, and IFNα) there will be increasing need to develop immune ‘steering wheels’ such as vaccines to guide the immune system specifically toward tumor associated antigens. Two primary hurdles in cancer vaccines have been: identification of universal antigens to be used in ‘off‐the‐shelf’ vaccines for common cancers, and 2) logistical hurdles of ex vivo production of individualized whole tumor cell vaccines. Here we summarize approaches using ‘in situ vaccination’ in which intratumoral administration of off‐the‐shelf immunomodulators have been developed to specifically induce (or amplify) T cell responses to each patients individual tumor. Clinical studies have confirmed the induction of systemic immune and clinical responses to such approaches and preclinical models have suggested ways to further potentiate the translation of in situ vaccine trials for our patients.

Lymphoma: Immune Evasion Strategies

While the cellular origin of lymphoma is often characterized by chromosomal translocations and other genetic aberrations, its growth and development into a malignant neoplasm is highly dependent upon its ability to escape natural host defenses. Neoplastic cells interact with a variety of non-malignant cells in the tumor milieu to create an immunosuppressive microenvironment. The resulting functional impairment and dysregulation of tumor-associated immune cells not only allows for passive growth of the malignancy but may even provide active growth signals upon which the tumor subsequently becomes dependent. In the past decade, the success of immune checkpoint blockade and adoptive cell transfer for relapsed or refractory lymphomas has validated immunotherapy as a possible treatment cornerstone. Here, we review the mechanisms by which lymphomas have been found to evade and even reprogram the immune system, including alterations in surface molecules, recruitment of immunosuppressive subpopulations, and secretion of anti-inflammatory factors. A fundamental understanding of the immune evasion strategies utilized by lymphomas may lead to better prognostic markers and guide the development of targeted interventions that are both safer and more effective than current standards of care.

In situ vaccination for the treatment of cancer

Vaccination has had a tremendous impact on human health by harnessing the immune system to prevent and eradicate infectious diseases and this same approach might be used in cancer therapy. Cancer vaccine development has been slowed hindered by the paucity of universal tumor-associated antigens and the difficulty in isolating and preparing individualized vaccines ex vivo. Another approach has been to initiate or stimulate an immune response in situ (at the tumor site) and thus exploit the potentially numerous tumor-associated antigens there. Here, we review the many approaches that have attempted to accomplish effective in situ vaccination, using intratumoral administration of immunomodulators to increase the numbers or activation state of either antigen present cells or T cells within the tumor.

Local Immunotherapies of Cancer

The past decade has seen clinical cancer research turn away from refining cytotoxic therapies to identifying immunomodulatory therapies that can harness the immune system to do the work of fighting cancer for us. The first revolution in cancer therapy brought us potent but toxic chemotherapies that were often initially good at controlling tumors; however, in the metastatic setting, recurrence was typically inevitable and more difficult to control. Immunotherapy has now emerged as among the most promising class of therapeutics for the treatment of cancer. These therapies harness the potent tumoricidal potential of cytotoxic effector cells of the patient’s own immune system. Although checkpoint-blocking antibodies approved in the past decade offer great promise, they non-specifically release the brakes on immune cells, and the majority of cancer patients are, so far, non-responders, while some patients experience toxic autoimmune adverse effects. One approach to improve on these limitations is with localized, in situ therapies, inducing a vaccinal response within the tumor microenvironment. These approaches alter the tumor microenvironment by inducing local immunogenic cell death to release tumor antigen, recruiting inflammatory leukocytes, priming of the adaptive immune response, and inhibiting tolerogenic mechanisms through which tumors dampen the immune response. Through a combined approach using some or all of these features, we are working to develop an in situ vaccine strategy that will result in systemic elimination of tumor cells as well as long-term remissions.

Immunomodulation Within a Single Tumor Site to Induce Systemic Antitumor Immunity: In Situ Vaccination for Cancer

Prophylactic vaccinations have been one of the greatest advances in modern medicine, both eradicating disease and reducing mortality. The translation of this advance into cancer therapy has been challenging and dates back to the turn of the twentieth century (Currie, Br J Cancer 26: 141–153, 1972). Cancer cells, derived from an aberrant clone, bear predominantly self-antigens and thus avoid alerting the immune system. In addition, the tumor microenvironment can be severely immunosuppressive; adding an extra layer of protection against the host immune response. Tumor cells can actively suppress immune responses through the downregulation of antigen presentation and the production of membrane-bound and secreted immuneregulatory molecules (Upadhyay et al, Cancers (Basel), 7: 736-62, 2015). To overcome such obstacles, a successful cancer vaccine must be able to induce a powerful immune response against tumor-associated antigens (TAAs) while avoiding normal host cells. This strategy has proven difficult because TAAs are highly variable in their immunogenicity and undergo immune editing to escape recognition. In addition, they can differ between tumor types and more importantly between individuals (Escors, New J Sci, 2014: 25, 2014). The presence of antigen-presenting cells (APCs) is generally low in the tumor microenvironment. Some efficacy in the treatment of cancer has been demonstrated by the use of autologous dendritic cells (DCs) pulsed with tumor cell lysates containing a whole array of antigens as well as single TAAs (Reichardt et al, Blood Rev: 18, 235-43, 2004). DC can be differentiated and expanded from peripheral blood ex vivo, and a resected tumor mass can be used to subsequently load the DC with TAAs. These strategies, while successful in developing a patient-specific vaccine, are labor and time intensive limiting the ability to experiment with numerous iterations to optimize the approach.

Abstract A012: Combining in situ vaccination with immune checkpoint blockade induces long-term regression of lymphoma tumors

Background: Low-grade non-Hodgkin9s B-cell lymphomas are generally incurable, with standard therapies inducing only temporary remissions. Preliminary results with anti-PD-1 therapy have yielded low response rates, though tumor-targeted vaccines represent promising, novel treatment strategies. In a pre-clinical mouse model, we attempt to develop and optimize an in situ vaccine combining recruitment of dendritic cells (DC) and low-dose local radiotherapy (XRT) with intratumoral (i.t) administration of a toll-like receptor (TLR) agonist Methods: A20 lymphoma-bearing mice were injected i.t. with FMS-like tyrosine kinase-3 ligand (Flt3L) daily for 9 days (30ug/mouse), followed by local XRT (9Gy) and i.t. injections of poly-ICLC (50ug/mouse) for 5 days. Leukocyte accumulation in tumors, lymph nodes, and spleens was analyzed by flow cytometry and animals were monitored for tumor growth and survival. To assess uptake of tumor antigens by DC, mCherry-expressing A20 cells were used. For assessment of systemic anti-tumor response tumors were inoculated on both flanks, but only one site was treated as described before. In some groups, anti-PD-1 blocking antibody was injected systemically during vaccination. Results: Injection of Flt3L induced potent accumulation of DC at the tumor site, tumor-draining lymph node (TDLN) and the spleen, with intratumoral injection being superior to systemic injection in increasing intratumoral and TDLN DCs. Interestingly, Flt3L-treatment led to an 8-fold increase in TLR3+ DC in the tumor. Local XRT increased the amount of mCherry+ DC in the tumor, indicating enhanced uptake of dying tumor cells. XRT of A20 cells also induced activation of Flt3L-treated splenic DC in vitro. While combination of FLt3L and local XRT was not able to cure established tumors, the combination of Flt3L and XRT with poly-ICLC induced long-lasting tumor regression in 40% of mice as well as regression of untreated tumors. This was accompanied by induction of tumor-reactive, Interferon γ (IFN γ)-producing T cells. Of note, the combination of Flt3L and XRT increased expression of PD-1 and PD-L1 on tumor infiltrating T cells and tumor cells, respectively. Consistently, systemic treatment with a PD-1 blocking antibody significantly enhanced the efficacy of the Flt3L-primed in situ vaccine leading to complete tumor regression at the treated site and a significant survival benefit compared to the in situ vaccine without PD-1 blockade. PD-1 blockade also increased the number of tumor-reactive T cells. Conclusions: In situ vaccination combining intratumoral Flt3L injection with local XRT, poly-ICLC and anti-PD-1 induces a potent anti-tumor immune response able to induce long-term regression of established lymphoma tumors. Citation Format: Linda Hammerich, Thomas Davis, Tibor Keler, Andres M. Salazar, Joshua D. Brody. Combining in situ vaccination with immune checkpoint blockade induces long-term regression of lymphoma tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A012.

Natural toll-like receptor agonists in prophylactic vaccines for dendritic cell activation

There is an unmet need for novel and effective treatments for lymphoma, the fifth most common malignancy in the US. In situ vaccination—an immunotherapeutic maneuver involving local irradiation, intratumoral (i.t.) injections of Flt3L and Toll-like receptor (TLR) agonist—has been shown in recent clinical trials (NCT00185965, NCT00880581, NCT00226993) to induce partial and complete remissions in patients with low-grade lymphoma[1]. The strength of anti-tumor response correlates with the potency of immunogenic dendritic cells (DCs) to efficiently uptake and present tumor antigens to T cells[2]. While the latest clinical trial NCT01976585 employs Poly-ICLC—a synthetic TLR3 agonist—to activate DCs, we hypothesize that “natural” TLR agonists (nTLRa) contained within prophylactic vaccines could simultaneously target multiple TLRs and be repurposed as clinical-grade DC activators for the in situ vaccination maneuver[3].