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Summary: Dose-limiting toxicities are thought to temper the efficacy of single-agent 4-1BB agonists. To overcome this hurdle, in this issue of Cancer Discovery, Muik and colleagues report preclinical and clinical studies describing a first-in-class bispecific fusion protein targeting 4-1BB and PD-L1. See related article by Muik et al., p. 1248 (9).

BackgroundAdoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies, yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients, but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo, circumventing TLR-related toxicity.MethodsIn this study we investigated how tumor-specific murine CD8+T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG.ResultsHerein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist, CpG, commonly used in the clinic, to bolster T cell—B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination, which are classically required for effective ACT therapy. CpG-conditioned CD8+T cells acquired a unique proteomic signature hallmarked by an IL-2RαhighICOShighCD39lowphenotype and an altered metabolic profile, all reliant on B cells transiently present in the culture. Likewise, human TILs benefitted from expansion with CpG ex vivo, as they also possessed the IL-2RαhighICOShighCD39lowphenotype. CpG fostered the expansion of potent CD8+T cells with the signature phenotype and antitumor ability via empowering a direct B–T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture.ConclusionsOur results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors.

Following its emergence in late November of 2020, the SARS-CoV-2 Omicron (B.1.1.529) variant has caused major global public health concerns. We recently demonstrated that in healthy adults the Omicron variant exhibits strong resistance to immunity induced by two doses of the mRNA vaccines, but a booster mRNA vaccine dose can provide strong protection against Omicron. However, it is currently unknown how well these mRNA vaccine boosters protect immunocompromised groups, including cancer patients, from the Omicron variant. Here we show that (1) neutralizing antibody (nAb) titers against the Delta and Omicron variants in cancer patients after two-dose mRNA vaccines are 4.2-fold and 21.3-fold lower, respectively, compared to the ancestral D614G, and (2) nAb titers against the Delta and Omicron variants in boosted cancer patients are 3.6-fold and 5.1-fold lower, respectively, compared to D614G. Our findings highlight the effectiveness and need for booster vaccination strategies in immunocompromised groups including cancer patients to protect from the Omicron variant.

There is currently a critical need to determine the efficacy of SARS-CoV-2 vaccination for immunocompromised patients. In this study, we determined the neutralizing antibody response in 160 cancer patients diagnosed with chronic lymphocytic leukemia (CLL), lung cancer, breast cancer, and various non-Hodgkin’s lymphomas (NHL), after they received two doses of mRNA vaccines. Serum from 46 mRNA vaccinated health care workers (HCWs) served as healthy controls. We discovered that (1) cancer patients exhibited reduced neutralizing antibody titer (NT50) compared to HCWs; (2) CLL and NHL patients exhibited the lowest NT50 levels, with 50-60% of them below the detection limit; (3) mean NT50 levels in patients with CLL and NHL was ~2.6 fold lower than those with solid tumors; and (4) cancer patients who received anti-B cell therapy exhibited significantly reduced NT50 levels. Our results demonstrate an urgent need for novel immunization strategies for cancer patients against SARS-CoV-2, particularly those with hematological cancers and those on anti-B cell therapies.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) elicits the regression of metastatic malignancies, yet a low proportion of patients achieve complete durable responses. The high incidence of relapse in these patients highlights the need to better understand mechanisms of tumor escape from T cell control. While melanoma has provided the foundation for developing TIL therapy, much less is known about TIL efficacy and relapse in other malignancies. We sought to investigate TIL characteristics in mouse tumors which have not been studied in this setting. Here, we expanded murine TIL ex vivo in IL-2 from fragments of multiple tumor models, including oral cavity cancer models of varying immunogenicity. Additionally, TIL was expanded from pmel-1 mice bearing B16F10 melanoma, yielding an enriched population of tumor-infiltrating TCR transgenic T cells. Murine TIL are similar to human TIL in that they express high levels of inhibitory receptors (PD-1, Tim-3, etc.) and can be expanded ex vivo in IL-2 extensively. Of clinical relevance, we draw parallels between murine and human oral cavity cancer TIL, evaluating relationships between inhibitory receptor expression and function. This platform can be used by labs even in the absence of clinical specimens or clean cell facilities and will be important to more broadly understand TIL phenotypes across many different malignancies.

Background Disparities in colon cancer (CC) outcomes may be due to a more aggressive phenotype in African American patients in the setting of a decreased tumor immunity, though the precise mechanism for this result has not been well elucidated. To explore the molecular factors underlying CC disparities, we compared the immunogenomic signatures of CC from African American and European American patients. Methods We identified all CC patients from the publicly available Cancer Genome Atlas for whom race and survival data are available. Immunophenotype signatures were established for African American and European American patients. Comparisons were made regarding survival and a multivariable linear regression model was created to determine the association of immune cellular components with race. Differential gene expression was also assessed. Results Of the 254 patients identified, 58 (23%) were African American and 196 (77%) were European American. African American patients had a decreased progression free survival (p = 0.04). Tumors from African American patients displayed a reduced fraction of macrophages and CD8+ T cells and an increased fraction of B cells compared with tumors from European Americans. Differences persisted when controlling for sex, age, and disease stage. Immunostimulatory and immunoinhibitory gene profiles including major histocompatibility complex expression differed by race. Conclusions Differences in the tumor immune microenvironment of African American as compared to European American CC specimens may play a role in the survival differences between the groups. These differences may provide targeted therapeutic opportunities., We uncovered key molecular factors underlying racial disparities in African American versus European American patients. Immunogenomic signatures in these patient cohorts were identified using a publicly available tumor repository and robust bioinformatic tools. Significant differences in gene expression were found in the tumor microenvironment which may underlie key clinical differences in these patients and reveal new therapeutic targets to effective treat all patients in the future.

Background: Disparities in colorectal cancer outcomes may be due to a more aggressive phenotype in African American patients in the setting of a decreased tumor immunity, though the precise mechanism for this result has not been well elucidated. To explore the molecular factors underlying colorectal cancer disparities, we compared the immunogenomic signatures of colorectal cancer from African American and European American patients.Methods: We identified all colorectal cancer patients from the publicly available Cancer Genome Atlas for whom race and survival data are available. Immunophenotype signatures were established for African American and European American patients. Comparisons were made regarding survival and a multivariable linear regression model was created to determine the association of immune cellular components with race. Differential gene expression was also assessed. Results: Of the 254 patients identified, 58 (23%) were African American and 196 (77%) were European American. African American patients had a decreased progression free survival (p=0.04). Tumors from African American patients displayed a reduced fraction of macrophages and CD8+ T cells and an increased fraction of B cells compared with tumors from European Americans. Differences persisted when controlling for sex, age and disease stage. Immunostimulatory and immunoinhibitory gene profiles including MHC expression differed by race. Conclusions: Differences in the tumor immune microenvironment of African American as compared to European American colorectal cancer specimens may play a role in the survival differences between the groups. These differences may provide targeted therapeutic opportunities.

The immune system is the hard-wired host defense mechanism against pathogens as well as cancer. Five years ago, we pondered the question if the era of cancer immunotherapy was upon us (Li et al., Exp Hem Oncol 2013). Exciting progresses have been made at all fronts since then, including (1) sweeping approval of six agents by the US Food and Drug Administration (FDA) to block the PD-1/PD-L1 pathway for treatment of 13 cancer types; (2) a paradigm shifting indication of PD-1 and CTLA4 blockers for the management of a broad class of cancers with DNA mismatch repair defect, the first-ever tissue agnostic approval of cancer drugs; (3) real world practice of adoptive T cell therapy with two CD19-directed chimeric antigen receptor T cell products (CAR-T) for relapsed and/or refractory B cell malignancies including acute lymphoid leukemia and diffuse large B cell lymphoma, signaling the birth of a field now known as synthetic immunology; (4) the award of 2018 Nobel Prize in Physiology and Medicine from the Nobel Committee to Tasuku Honjo and James Allison “for their discovery of cancer medicine by inhibition of negative immune regulation” ( www.nobelprize.org/prizes/medicine/2018 ); and (5) the emerging new concept of normalizing rather than amplifying anti-tumor immunity for guiding the next wave of revolution in the field of immuno-oncology (IO) (Sanmamed and Chen, Cell 2018). This article will highlight the significant developments of immune-oncology as of October 2018. The US FDA approved indications of all seven immune checkpoint blockers, and two CD19-directed CAR-T products are tabulated for easy references. We organized our discussion into the following sections: introduction, cell therapy, emerging immunotherapeutic strategies, expediting oncology drug development in an era of breakthrough therapies, new concepts in cancer immunology and immunotherapy, and concluding remarks. Many of these topics were covered by the 2018 China Cancer Immunotherapy Workshop in Beijing, the fourth annual conference co-organized by the Chinese American Hematologist and Oncologist Network (CAHON), China FDA (CFDA; now known as China National Medical Product Administration (NMPA)), and the Tsinghua University. We significantly expanded our discussion of important IO developments beyond what were covered in the conference, and proposed a new Three Rs conceptual framework for cancer immunotherapy, which is to reverse tolerance, rejuvenate the immune system, and restore immune homeostasis. We conclude that the future of immuno-oncology as a distinct discipline of cancer medicine has arrived.

Summary Background Immunotherapy with PD-1 or PD-L1 blockade fails to induce a response in about 80% of patients with unselected non-small cell lung cancer (NSCLC), and many of those who do initially respond then develop resistance to treatment. Agonists that target the shared interleukin-2 (IL-2) and IL-15Rβγ pathway have induced complete and durable responses in some cancers, but no studies have been done to assess the safety or efficacy of these agonists in combination with anti-PD-1 immunotherapy. We aimed to define the safety, tolerability, and activity of this drug combination in patients with NSCLC. Methods In this non-randomised, open-label, phase 1b trial, we enrolled patients (aged ≥18 years) with previously treated histologically or cytologically confirmed stage IIIB or IV NSCLC from three academic hospitals in the USA. Key eligibility criteria included measurable disease, eligibility to receive anti-PD-1 immunotherapy, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received the anti-PD-1 monoclonal antibody nivolumab intravenously at 3 mg/kg (then 240 mg when US Food and Drug Administration [FDA]-approved dosing changed) every 14 days (either as new treatment or continued treatment at the time of disease progression) and the IL-15 superagonist ALT-803 subcutaneously once per week on weeks 1–5 of four 6-week cycles for 6 months. ALT-803 was administered at one of four escalating dose concentrations: 6, 10, 15, or 20 μg/kg. The primary endpoint was to define safety and tolerability and to establish a recommended phase 2 dose of ALT-803 in combination with nivolumab. Analyses were per-protocol and included any patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02523469; phase 2 enrolment of patients is ongoing. Findings Between Jan 18, 2016, and June 28, 2017, 23 patients were enrolled and 21 were treated at four dose levels of ALT-803 in combination with nivolumab. Two patients did not receive treatment because of the development of inter-current illness during enrolment, one patient due to leucopenia and one patient due to pulmonary dysfunction. No dose-limiting toxicities were recorded and the maximum tolerated dose was not reached. The most common adverse events were injection-site reactions (in 19 [90%] of 21 patients) and flu-like symptoms (15 [71%]). The most common grade 3 adverse events, occurring in two patients each, were lymphocytopenia and fatigue. A grade 3 myocardial infarction occurred in one patient. No grade 4 or 5 adverse events were recorded. The recommended phase 2 dose of ALT-803 is 20 μg/kg given once per week subcutaneously in combination with 240 mg intravenous nivolumab every 2 weeks. Interpretation ALT-803 in combination with nivolumab can be safely administered in an outpatient setting. The promising clinical activity observed with the addition of ALT-803 to the regimen of patients with PD-1 monoclonal antibody relapsed and refractory disease shows evidence of anti-tumour activity for a new class of agents in NSCLC. Funding Altor BioScience (a NantWorks company), National Institutes of Health, and Medical University of South Carolina Hollings Cancer Center.

Colorectal cancer (CRC) remains the third most common malignancy and the second-leading cause of cancer-related deaths in the United States. Large multi-omic databases, such as The Cancer Genome Atlas and the International Colorectal Cancer Subtyping Consortium, have identified distinct molecular subtypes related to anatomy. The identification of genomic alterations in CRC is now critical because of the recent success and US Food and Drug Administration approval of pembrolizumab and nivolumab for microsatellite-instable tumors. In parallel, landmark studies have established the prognostic significance of the CRC tumor-infiltrating lymphocyte and the clinical impact of the tumor immune microenvironment. As a result, there is a growing appreciation for immunogenomics, the interconnected relation between tumor genomics and the immune microenvironment. The clinical implications of CRC immunogenomics continue to expand, and it will likely serve as a guide for next-generation immunotherapy strategies for improving outcomes for this disease. Cancer 2018;124:1650-9. © 2018 American Cancer Society.

BackgroundThe use of immunotherapies such as immune checkpoint blockade or adoptive cell therapy has substantially improved outcomes for many patients with advanced malignancies. However, a majority of these patients still do not respond or relapse. Thus, extensive efforts are being made to improve these therapies. We and others have demonstrated that adoptive cell therapy can be improved by the co-administration of TLR agonists intratumorally. TLR agonists have also been administered to patients alongside a number of other immunotherapies, but often induce toxic side effects that may be exacerbated by cell therapy. We hypothesized that TLR agonists could alternatively be used in ex vivo cell culture to propagate a more potent cell therapy product.MethodsTo address our question, we used a transgenic mouse model of tumor-infiltrating lymphocyte (TIL) therapy, pmel-1, in which CD8+ T cells express a TCR specific for a melanoma/melanocyte antigen. We activated CD8+ pmel-1 T cells with APCs and the Toll-like receptor 9 agonist, CpG. Cell therapy efficacy was determined against mice bearing established B16F10 melanoma. Mechanisms underlying TLR-improved T cell products were determined using ex vivo cell depletion strategies and blocking antibodies.ResultsCpG-expanded pmel-1 T cell products were much more effective against B16F10 melanoma in vivo than traditionally expanded T cells; they conferred more durable antitumor responses and improved survival of mice. CD8+ T cells generated in the presence of CpG also had heightened engraftment and persistence in the mice. We found that CpG did not act directly on T cells in culture, but on B cells, as depletion of B cells alone (not DCs, macrophages, NK cells, or CD4 cells) ablated the effects of CpG. B cells in CpG-treated cultures expressed a unique cytokine profile and upregulated several costimulatory markers on their cell surface, so we next questioned whether CpG improved the B cell/T cell axis via a direct or indirect (soluble) factor. Together, cell supernatant transfer experiments and costimulatory blockade experiments revealed that the direct interaction between B and T cells was required for the CpG-mediated improvement of the T cell product.ConclusionsOur findings reveal a novel role for B cells in the generation of potent CD8+ T cell therapies against an aggressive solid tumor. These findings highlight a unique way B cells can become powerful APCs for generating effective antitumor CD8+ T cells and can be directly translated to improve cell therapy products for patients with advanced malignancies.Ethics ApprovalAll animal procedures performed at the Medical University of South Carolina or Emory University were approved by each university’s Institutional Animal Care & Use Committee, protocol number 0488 or 201900225, respectively.

Summary Oral cavity squamous cell carcinoma (OCSCC) is a prevalent surgically treated subset of head and neck cancer with frequent recurrence and poor survival. Immunotherapy has demonstrated efficacy in recurrent/metastatic head and neck cancer. However, whether antitumor responses could be fostered by neoadjuvant presurgical immunotherapy remains unclear. Using a Simon’s two-stage design, we present results of a single-arm phase-II trial where 12 patients with stage II-IVA OCSCC received 3 to 4 biweekly doses of 3 mg/kg nivolumab followed by definitive surgical resection with curative intent. Presurgical nivolumab therapy in this cohort shows an overall response rate of 33% (n = 4 patients; 95% CI: 12%–53%). With a median follow up of 2.23 years, 10 out of 12 treated patients remain alive. Neoadjuvant nivolumab is safe, well-tolerated, and is not associated with delays in definitive surgical treatment in this study. This work demonstrates feasibility and safety for incorporation of nivolumab in the neoadjuvant setting for OCSCC (ClinicalTrials.gov: NCT03021993)., Graphical abstract, Highlights • Presurgical nivolumab in patients with resectable oral cancer has an ORR of 33% • 10 out of 12 treated patients remain alive with a median follow up of 2.23 years • Neoadjuvant nivolumab is safe, well-tolerated, and not associated with treatment delays • This shows feasibility of nivolumab in the neoadjuvant setting for OCSCC (NCT03021993), Oral cavity squamous cell carcinoma is a prevalent subset of head and neck cancer with high recurrence rates and poor survival outcomes despite complex multimodality therapy. The current study by Knochelmann et al. demonstrates that presurgical nivolumab therapy has an overall response rate of 33% in this patient population.

The accessibility of adoptive T-cell transfer therapies (ACT) is hindered by the cost and time required for product development. Here we describe a streamlined ACT protocol using Th17 cells expanded only 4 days ex vivo. While shortening expansion compromised cell yield, this method licensed Th17 cells to eradicate large tumors to a greater extent than cells expanded longer term. Day 4 Th17 cells engrafted, induced release of multiple cytokines including IL6, IL17, MCP-1, and GM-CSF in the tumor-bearing host, and persisted as memory cells. IL6 was a critical component for efficacy of these therapies via its promotion of long-term immunity and resistance to tumor relapse. Mechanistically, IL6 diminished engraftment of FoxP3+ donor T cells, corresponding with robust tumor infiltration by donor effector over regulatory cells for the Day 4 Th17 cell product relative to cell products expanded longer durations ex vivo. Collectively, this work describes a method to rapidly generate therapeutic T-cell products for ACT and implicates IL6 in promoting durable immunity of Th17 cells against large, established solid tumors. Significance: An abbreviated, 4-day ex vivo expansion method licenses Th17 cells to confer long-lived immunity against solid malignancies via induction of systemic IL6 in the host. See related commentary by Fiering and Ho, p. 3795

Pancreatic adenocarcinoma (PDAC) remains a formidable disease that needs improved therapeutic strategies. Even though immunotherapy has revolutionized treatment for various solid tumor types, it remains largely ineffective in treating individuals with PDAC. This review describes how the application of genome-wide analysis is revitalizing the field of PDAC immunotherapy. Major themes include new insights into the body’s immune response to the cancer, and key immunosuppressive elements that blunt that antitumor immunity. In particular, new evidence indicates that T cell-based antitumor immunity against PDAC is more common, and more easily generated, than previously thought. However, equally common are an array of cellular and molecular defenses employed by the tumor against those T cells. These discoveries have changed how current immunotherapies are deployed and have directed development of novel strategies to better treat this disease. Thus, the impact of genomic analysis has been two-fold: both in demonstrating the heterogeneity of immune targets and defenses in this disease, as well as providing a powerful tool for designing and identifying personalized therapies that exploit each tumor’s unique phenotype. Such personalized treatment combinations may be the key to developing successful immunotherapies for pancreatic adenocarcinoma.

2596 Background: There is currently a paucity of treatment options for checkpoint relapsed patients who have had an initial response but subsequently progress. N803, a novel IgG1 Fc-engineered IL-15-complexed protein may rescue checkpoint activity in a checkpoint independent manner via its selective enhancement of natural killer cell (NK) and CD8+ T cell number and function, without stimulation of T regs and MDSCs. Methods: QUILT 3.055, (NCT03228667) a phase 2b study of N803 plus investigator choice CPI in 11 tumor types: NSCLC, SCLC, Urothelial carcinoma, HNSCC, Merkel cell carcinoma, Melanoma (single PD-1/PD-L1 CPI or w/ ipilimumab), Renal cell carcinoma (RCC), Gastric cancer, Cervical cancer, Hepatocellular carcinoma, Microsatellite instability-high (MSI-H)/ mismatch repair deficient (dMMR) solid tumors, with a heterogeneous mix of prior therapies. We present interim data for 135 patients treated with CPI alone or in combination with chemotherapy as their most recent prior therapy. Trial inclusion required investigator assessed progression on last line of therapy, patients had either CR with relapse or partial response or stable disease for 6 months with progression as their most recent result of checkpoint therapy. Patients with hyperprogression or best initial response of progression were excluded. Subjects received N803 15mck/kg SC q 3 weeks in combination with the same checkpoint inhibitor on which they had their most recent progression. Results: Preliminary data from 135 patients (60% NSCLC) with treatment with checkpoint and N-803 following progression on the same checkpoint show CR 0%, PR 8%, Stable Disease 51%, Progression 29%, response unevaluable 12% to date. A PR or SD was seen in all subgroups. Median PFS 3.9 months (95% CI: 2.6,5.0). Median OS 13.8 months (95% CI: 11.8, 16.3) N-803 is well tolerated with grade 1-2 common N-803 treatment related adverse events (TRAE) were injection site reaction (68%), chills (32%) fatigue (26%), pyrexia (26%), flu-like illness (14%), nausea (12%) and no other individual AE > 10%. Grade 3 N-803 TRAE were 12% but no individual grade 3 AEs were greater than 5%. Conclusions: N803 demonstrates low toxicity in patients previously treated with CPI and promising efficacy of cessation of progression and induction of response and durable stable disease in patients who had previously progressed on a CPI containing regimen in multiple tumor types and different CPIs. Clinical trial information: NCT03228667.

Repetitive stimulation of T-cell receptor (TCR) with cognate antigen results in robust proliferation and expansion of the T cells, and also imprints them with replicative senescence signatures. Our previous studies have shown that life-span and antitumor function of T cells can be enhanced by inhibiting reactive oxygen species (ROS) or intervening with ROS-dependent JNK activation that leads to its activation-induced cell death. Because tumor suppressor protein p53 is also a redox active transcription factor that regulates cellular ROS generation that triggers downstream factor–mediating apoptosis, we determined if p53 levels could influence persistence and function of tumor-reactive T cells. Using h3T TCR transgenic mice, with human tyrosinase epitope–reactive T cells developed on p53 knockout (KO) background, we determined its role in regulating antitumor T-cell function. Our data show that as compared with h3T cells, h3T-p53 KO T cells exhibited enhanced glycolytic commitment that correlated with increased proliferation, IFNγ secretion, cytolytic capacity, expression of stemness gene signature, and decreased TGF-β signaling. This increased effector function correlated to the improved control of subcutaneously established murine melanoma after adoptive transfer of p53-KO T cells. Pharmacological inhibition of human TCR-transduced T cells using a combination of p53 inhibitors also potentiated the T-cell effector function and improved persistence. Thus, our data highlight the key role of p53 in regulating the tumor-reactive T-cell response and that targeting this pathway could have potential translational significance in adoptive T-cell therapy. Cancer Res; 76(18); 5229–40. ©2016 AACR.

e15008 Background: The oncotherapeutic promise of IL-15, a potent immune stimulant, is limited by short serum half-life. The fusion protein N-803 is an IL-15 superagonist complex that has > 20-fold longer half-life in vivo vs IL-15. This study characterized the pharmacokinetic (PK) profile, biological activity, and safety of N-803 after subcutaneous administration to healthy human volunteers. Methods: Volunteers were randomized 1:1 to receive 1 mg/mL or 2 mg/mL N-803. Each subject received 2 doses of N-803: 10 µg/kg followed 24 days later by 20 µg/kg. After each dose, PK and safety measures were assayed for 9 successive days. Primary endpoint was the PK profile of N-803; secondary was safety; and exploratory endpoints were cytokine levels, immune cell characterization, and immunogenicity. Results: N-803 resulted in no grade ≥3 or serious adverse events (AEs). Mild injection site reactions, chills, and pyrexia were the most common AEs. Serum N-803 concentrations peaked 10.3-15.4 hours after administration and declined with a half-life of 20.0-20.7 hours. Peak N-803 serum concentrations were dose-dependent, with a 1.5-fold increase in Cmax after administration of 20 µg/kg vs 10 µg/kg. In the peripheral blood, N-803 induced a transient decline, followed by a significant increase (3-fold) in NK cell number that persisted for ≥24 days. N-803 also caused a significant proliferation of NK (22-fold increase in Ki-67+ cells), CD8+ (27-fold) and CD4+ T (11-fold) cells; however, increased cell number occurred only in NK cells. N-803 administration also increased serum levels of interferon gamma, IL-10, and IL-6. One of 14 evaluable subjects had measureable anti-N-803 antibodies at the end-of-study visit. Conclusions: N-803 results in prolonged elevation of drug serum concentrations, contrasting with rapid clearance of recombinant human IL-15 (ie, half-life of ~20 vs < 1 hour). N-803 administration was well-tolerated in healthy volunteers, without evidence of adverse systemic inflammatory responses, and resulted in proliferation of NK cells and CD8+ T cells, as well as sustained increases in NK cell number. Findings in this study are consistent with published results from N-803 administration in treating liquid tumors and lung cancer. Our results demonstrate N-803 administration potentiates the proliferation and activity of lymphocytes with antitumor and antivirus properties, and suggest this investigational product holds promise in treatment of cancer as well as infectious disease such as HIV. Clinical trial information: NCT03381586 .

The emergence of immuno-oncology (IO) agents has led to unprecedented successes in the treatment of deadly cancers and sometimes even cures. These agents act by “releasing the brakes” and thereby unleashing the full potential of cells that fight tumors. Sadly, not every patient benefits from these new treatments. We need to know who will benefit and who may not. Here we approached the problem by designing a customized workflow by using high-dimensional single-cell mass cytometry (CyTOF) combined with machine-learning bioinformatics for the in-depth characterization of single immune cells in predicting and monitoring immune responses. The analysis is data driven, can be adapted to high-throughput approaches, and can model arbitrary trial designs such as batch effects and paired designs. We tested our workflow on two studies: a) Predict response to anti-PD-1 immunotherapy in melanoma. In our discovery cohort peripheral blood mononuclear cells (PBMCs) from 20 patients with stage IV melanoma before and after 12 weeks of anti-PD-1 therapy were analyzed. We observed a clear T-cell response on therapy. The most evident difference in responders before therapy was an enhanced frequency of CD14+ CD16+HLA-DRhi classical monocytes. We validated our results using conventional flow cytometry in an independent exploratory cohort of 31 patients before therapy. Finally, we correlated enhanced monocyte frequencies before therapy initiation with clinical response and could show association with lower hazard, extended progression-free and overall survival. b) Monitoring the immune response in anti-PD-1 immunotherapy-refractory non-small cell lung cancer to a novel combination immunotherapy of anti-PD-1 and an IL-15 superagonist. Twenty-one patients with non-small cell lung cancer who mostly were refractory to previous anti-PD-1 treatment received a novel combination therapy of IL-15 superagonist plus anti-PD-1. A response in the CD8+ T cell compartment was observed. Unexpectedly, our high-dimensional approach was able to detect and characterize a strong expansion of innate tumor-reactive effector NK cells starting around day 4 of therapy. Taken together, our unbiased artificial intelligence-driven immune workflow might support patient selection prior to therapy, select the right drug combination, and identify new druggable cell populations. Citation Format: Luis Cardenas, Silvia Guglietta, John Wrangle, Mark Rubinstein, Mark Robinson, Carsten Krieg. Using high-dimensional machine-assisted analysis for biomarkers detection and guidance of immunotherapy to cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA09.

Mouse CD8(+) T cells conditioned with interleukin (IL)-12 ex vivo mediate the potent regression of established melanoma when transferred into lymphodepleted mice. However, the quantitative and qualitative changes induced by IL-12 in the responding mouse CD8(+) T cells have not been well defined. Moreover, the mechanisms by which IL-12-conditioning impacts human CD8(+) T cells, and how such cells might be expanded prior to infusion into patients is not known. We found that ex vivo IL-12-conditioning of mouse CD8(+) T cells led to a tenfold-100-fold increase in persistence and anti-tumor efficacy upon adoptive transfer into lymphodepleted mice. The enhancing effect of IL-12 was associated with maintenance of functional avidity. Importantly, in the context of ongoing ACT clinical trials, human CD8(+) T cells genetically modified with a tyrosinase-specific T cell receptor (TCR) exhibited significantly enhanced functional activity when conditioned with IL-12 as indicated by heightened granzyme B expression and elevated peptide-specific CD107a degranulation. This effect was sustainable despite the 20 days of in vitro cellular expansion required to expand cells over 1,000-fold allowing adequate cell numbers for administration to cancer patients. Overall, these findings support the efficacy and feasibility of ex vivo IL-12-conditioning of TCR-modified human CD8(+) T cells for adoptive transfer and cancer therapy.

Effector CD8+ T cells conditioned with IL12 during activation mediate enhanced antitumor efficacy after adoptive transfer into lymphodepleted hosts; this is due in part to improved IL7 responsiveness. Therefore, we hypothesized that increasing the intensity or type of lymphodepletion would deplete more IL7-consuming host cells and improve the persistence and antitumor activity of IL12-conditioned CD8+ T cells. Using cyclophosphamide, fludarabine, and total body irradiation (TBI, 6 Gy) either individually or in combination, we found that combined lymphodepletion best enhanced T-cell engraftment in mice. This improvement was strongly related to the extent of leukopenia, as posttransfer levels of donor T cells inversely correlated to host cell counts after lymphodepletion. Despite the improvement in engraftment seen with combination lymphodepletion, dual-agent lymphodepletion did not augment the antitumor efficacy of donor T cells compared with TBI alone. Similarly, IL7 supplementation after TBI and transfer of tumor-reactive T cells failed to improve persistence or antitumor immunity. However, IL15 or IL2 supplementation greatly augmented the persistence and antitumor efficacy of donor tumor-reactive T cells. Our results indicate that the amount of host IL7 induced after single agent lymphodepletion is sufficient to potentiate the expansion and antitumor activity of donor T cells, and that the efficacy of future regimens may be improved by providing posttransfer support with IL2 or IL15. Significance: The relationship between lymphodepletion and cytokine support plays a critical role in determining donor T-cell engraftment and antitumor efficacy. Cancer Res; 78(11); 3067–74. ©2018 AACR.

Background Dendritic cells (DCs) enhance the quality of anti-tumor immune response in patients with cancer. Thus, we posit that DC-based immunotherapy, in conjunction with toll-like receptor (TLR)-3 agonist poly-ICLC, is a promising approach for harnessing immunity against metastatic or locally advanced unresectable pancreatic cancer (PC). Methods We generated autologous DCs from the peripheral blood of HLA-A2+ patients with PC. DCs were pulsed with three distinct A2-restricted peptides: 1) human telomerase reverse transcriptase (hTERT, TERT572Y), 2) carcinoembryonic antigen (CEA; Cap1-6D), and 3) survivin (SRV.A2). Patients received four intradermal injections of 1 × 107 peptide-pulsed DC vaccines every 2 weeks (Day 0, 14, 28, and 42). Concurrently, patients received intramuscular administration of Poly-ICLC at 30 μg/Kg on vaccination days (i.e., day 0, 14, 28, and 42), as well as on days 3, 17, 21, 31, 37, and 45. Our key objective was to assess safety and feasibility. The effect of DC vaccination on immune response was measured at each DC injection time point by enumerating the phenotype and function of patient T cells. Results Twelve patients underwent apheresis: nine patients with metastatic disease, and three patients with locally advanced unresectable disease. Vaccines were successfully manufactured from all individuals. We found that this treatment was well-tolerated, with the most common symptoms being fatigue and/or self-limiting flu-like symptoms. Among the eight patients who underwent imaging on day 56, four patients experienced stable disease while four patients had disease progression. The median overall survival was 7.7 months. One patient survived for 28 months post leukapheresis. MHC class I –tetramer analysis before and after vaccination revealed effective generation of antigen-specific T cells in three patients with stable disease. Conclusion Vaccination with peptide-pulsed DCs in combination with poly-ICLC is safe and induces a measurable tumor specific T cell population in patients with advanced PC. Trial registration NCT01410968; Name of registry: clinicaltrials.gov; Date of registration: 08/04/2011). Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0459-2) contains supplementary material, which is available to authorized users.

Background: The presence of tumor metastases in standard of care lymph node biopsies during surgical resection is used to help determine clinical prognosis. Given the importance of T cells in mediating anti-tumor responses, we sought to assess the clinical significance of T-Cell Receptor (TCR) usage in matched hilar and mediastinal lymph nodes from non-small cell lung cancer (NSCLC) patients. Methods: TCR-Beta chains were quantified by the immunoSEQ® Assay (Adaptive Biotechnologies) in primary early-stage NSCLC tissue, matched hilar lymph nodes (proximal), and matched mediastinal lymph nodes (distal) for 27 patients, who were treated with surgical resection. Patients were selected such that half experienced recurrence and half remained recurrence-free over the 30+ months of patient follow-up. Results: T-cell clonality in resected tumors is significantly correlated (Spearman’s Rho = 0.52, p = 0.0005) but poorly concordant (Pearson’s R2 = 0.14, p = 0.05) with hilar and mediastinal lymph node clonality. The clonality of hilar and mediastinal lymph nodes is both significantly correlated and moderately concordant (Spearman’s Rho = 0.7, p << 0.01; Pearson’s R2 = 0.44, p = 0.0001). 70-80% of top T-cell clones in tumors were detected in at least one lymph node, and the 100 most abundant tumor clones comprised a median 8.4% and 7.5% of the hilar and mediastinal lymph node repertoires of progression-free subjects, and 7.6% and 6.4% of subjects with tumor progression. Aside from the traditional lymphvascular invasion assessment by histopathology and PET SUVs, the abundance of the top 100 tumor clones and the fraction of top 100 tumor clones detected in the mediastinal lymph node were both independently predictive of early-stage NSCLC progression-free survival (Cox Likelihood p = 0.031 and 0.017, respectively). A 10% increase in the abundance of top 100 tumor clones was associated with a 2.3x increase in risk for progression, suggesting that greater tumor repertoire diversity is favorable in the surgical resection setting. In contrast, for every 10 tumor clones detected in the mediastinal lymph node, the risk of progression decreased by 0.32x, suggesting that increased T-cell trafficking between the tumor and more distant lymph nodes is a favorable prognostic biomarker. Conclusion: The representation of top tumor T-cell clones is fairly similar in hilar and mediastinal lymph nodes. However, the detection of more top tumor clones in the mediastinal lymph nodes is strongly associated with less risk of progression after surgical tumor resection. This biomarker is likely a surrogate for more robust T-cell trafficking between the tumor and lymph circulation and may be a novel hallmark of better patient outcomes. Disclaimers: For Research Use Only. Not for use in diagnostic procedures. Citation Format: Julie A. Rytlewski, Mark P. Rubinstein, Chadrick E. Delinger, Barry Gibney, Erik C. Yusko, Catherine Sanders, John M. Wrangle, Kathryn Lindsey. Detection of tumor T-cell clones in mediastinal lymph nodes is associated with lower risk of tumor progression [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 3175.

Checkpoint inhibitors have significantly accelerated cancer treatment but still a majority of patients do not respond. Biomarker driven patient stratification early to the right immunotherapeutic might enhance response and patient survival. Here we used high-dimensional mass cytometry (CyTOF) combined with machine-learning bioinformatics for the in-depth characterization of immune responses before and during anti-PD-1 immunotherapy. CyTOF allows us to monitor protein expression of 34 markers on a single cell while running 20 samples simultaneously. The analysis is data driven, can be adapted to high throughput approaches and can model arbitrary trial designs such as batch effects and paired designs and is quantitative over millions of events. Using CyTOF as a precision medicine tool we could predict response to anti-PD-1 using liquid blood biopsies. Biobanked peripheral blood mononuclear cells (PBMCs) from 51 patients with stage IV melanoma before and after 12 weeks of anti-PD-1 therapy was analyzed. We observed a clear T cell response on therapy. The most evident difference in responders before therapy was an enhanced frequency of CD14+ CD16+HLA-DRhi classical monocytes. We validated our results using conventional flow and found a clear correlation of enhanced monocyte frequencies before therapy initiation with clinical response such as lower hazard and extended progression-free and overall survival. In a second study we used CyTOF to monitor immune response in 21 non small cell lung cancer (NSCLC) patients that initially responded and then progressed under anti-PD-1 to a novel combination immunotherapy of anti-PD-1 plus an IL-15 super-agonist (ALT-803). In this phase Ib clinical study a response in the CD8+ T cell compartment was observed. Unexpected our high dimensional unbiased analysis was able to detect and characterize a strong expansion of innate tumor-reactive effector NK cells starting around day 4 of therapy. Taken together, our unbiased artificial intelligence driven immune workflow might support patient selection prior to therapy, and serve as a novel tool for precision medicine to select the right drug combination and identify new drug-able cell populations. Citation Format: Carsten Krieg, Luis Cardenas, Silvia Guglietta, John Wrangle, Mark Rubinstein, Mark Robinson. Is biomarker-driven precision medicine possible by using high dimensional augmented intelligence assisted analysis of cancer immune responses [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 4225.

6055 Background: PD-1 inhibition therapy has revolutionized clinical medicine as it can mediate durable responses in a small cohort of patients. Yet, it remains incompletely understood why these patients respond. To address this question, we studied patients with oral cavity squamous cell carcinoma (OCSCC) to elucidate immune phenotypes associated with response to nivolumab. Methods: We defined the immune profile from the blood and tumor of patients on neoadjuvant nivolumab. We tested if tumor-infiltrating lymphocytes (TIL) could be preferentially expanded ex vivo from nivolumab-responsive patients versus those who were either non-responsive or had never received nivolumab. During the course of therapy, we comprehensively profiled a number of surface markers on patients’ T cells to define their activation status, cytotoxic capacity and memory phenotype. Moreover, the immune profile of the peripheral blood was assessed pre- and post-nivolumab using high dimensional mass cytometry. Results: Regardless of PD-1 therapy, TIL were successfully expanded from 11 of the 12 patients. TIL were comprised of both CD4+ and CD8+ T cells. Additional investigation revealed that the frequency of CD4+ T cells and effector memory T cells in TIL correlated with disease progression (CD4: p = 0.04, r = 0.74, effector memory: p = 0.046, r = 0.74). TILs from responders expressed higher CD26 (p = 0.007, r = -0.88) and Tim3 (p = 0.045, r = -0.74) while PD-1, Lag3, and Ox40 were not differentially expressed based on response. Spearman correlation and Mann Whitney U test were used to assess phenotypic differences. Conclusions: We demonstrate, for the first time, that TIL can be reliably expanded from OCSCC patients on neoadjuvant nivolumab. Moreover, individuals who were responsive to PD-1 blockade had TIL expressing high levels of CD26 and Tim3. Future studies will explore if these markers are predictive of responses and if they contribute to treatment outcome.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR05) of the Conference Proceedings. Citation Format: John Wrangle, Vamshidar Velchetti, Manish Patel, Samantha Suriano, Marzena Swiderska-syn, Kate Anderton, James Ravenel, Elisabeth Hill, Amy Rock, Hing Wong, Mark Rubinstein. Safety and activity of the IL-15/sIL-15Rα complex ALT-803 in combination with the anti-PD1 mAb nivolumab in metastatic non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B34.

Background: Despite the unprecedented efficacy of immunotherapy with PD-1/PD-L1 blockade in non-small cell lung cancer, treatment failure is common and an area of unmet clinical need. IL-2/IL-15Rbg agonists, which potently augment the activity of CD8+ T and NK cells, have shown the ability to induce complete and durable responses in select cancers. However, this class of drugs has been neither combined with PD-1 targeted immunotherapies nor examined extensively in NSCLC. We hypothesized that the addition of an IL-15 superagonist (ALT-803) would be safe and improve the efficacy of anti-PD-1 mAb (nivolumab) in metastatic NSCLC patients in second-line therapy. Methods: In this phase Ib trial, patients with metastatic NSCLC who were eligible to initiate or to continue receiving nivolumab (anti-PD-1 mAb) after first-line therapy were enrolled to trial, including patients with disease relapsed on or refractory to PD-1 blockade. Nivolumab was administered in combination with ALT-803 at four dose levels. The primary objective of the study was to define safety and tolerability and a recommended phase II dose of ALT-803 in combination with nivolumab, with secondary objectives including immune correlates and clinical parameters including response rate. Results: In a first-in-human combination, 21 NSCLC patients were treated at 4 dose levels of ALT-803 in combination with the approved dose of nivolumab. No dose-limiting toxicities were observed, and the most common adverse events were fever, flu-like symptoms, and injection site reactions. The recommended phase II dose (RP2D) of therapy is weekly subcutaneous ALT-803 at 20 mcg/kg in combination with intravenous nivolumab 240 mg every 14 days. Multiple objective responses were observed, including when ALT-803 was added to a failing PD-1 mAb regimen. Discussion: The recommended phase II dose of ALT-803 plus nivolumab was determined and the regimen can safely be administered in the outpatient setting. Responses among patients with PD-1 mAb relapsed disease with the addition of ALT-803 to their regimen demonstrate for the first time evidence of antitumor activity for a new class of agents for NSCLC and in patients with disease progression on PD-1/PD-L1 pathway blockade. This abstract is also being presented as Poster B34. Citation Format: John Wrangle, Vamshidar Velchetti, Manish Patel, Samantha Suriano, Marzena Swiderska-syn, Kate Anderton, James Ravenel, Elisabeth Hill, Amy Rock, Hing Wong, Mark Rubinstein. Safety and activity of the IL-15/sIL-15Rα complex ALT-803 in combination with the anti-PD1 mAb nivolumab in metastatic non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR05.

e24141Background: Current treatments for SCLC produce transient responses – 2-year survival is 5%. One reason is that the treatments apply stress upstream of p53 to upregulate it (cytotoxicity), bu...

e24134Background: Most advanced non-small cell lung cancer (NSCLC) cases do not respond to anti-PD1 therapy, because most NSCLC avoids immune-recognition in the 1st place, e.g, by epigenetic suppre...

IL-7 and IL-15 are critical for supporting T cells transferred into a lymphopenic environment. As activated CD8(+) T cells downregulate IL-7Rα, it is thought IL-15 is more important. However, we find that CD8(+) T cells activated with IL-12 have elevated IL-7Rα and rely on IL-7 for persistence and antitumor immunity.

Meeting abstracts IL-12 is a heterodimeric, pro-inflammatory cytokine that enhances the cytotoxic activity of natural killer (NK) cells and cytotoxic CD8+ T-lymphocytes, and induces an IFN-α-dominated Th1 CD4+ T-lymphocyte response. IL-12 as an immunotherapeutic agent administered subcutaneously

Cancer immunotherapy is one the most effective approaches for treating patients with tumors, as it bolsters the generation and persistence of memory T cells. In preclinical work, it has been reported that adoptively transferred CD4+ and CD8+ lymphocytes that secrete IL-17A (i.e., Th17 and Tc17 cells) regress tumors to a greater extent than IFN-γ(+)Th1 or Tc1 cells in vivo. Herein, we review the mechanisms underlying how infused Th17 and Tc17 cells regress established malignancies in clinically relevant mouse models of cancer. We also discuss how unique signaling cues--such as co-stimulatory molecules (ICOS and 41BB), cytokines (IL-12 and IL-23) or pharmaceutical reagents (Akt inhibitors, etc.)--can be exploited to bolster the therapeutic potential of IL-17(+) lymphocytes with an emphasis on using this knowledge to improve next-generation clinical trials for patients with cancer.

Successful development of immunotherapeutic strategies for hepatocellular cancer (HCC) has been impeded by limited understanding of tumor-induced profound tolerance and lack of a clinically faithful HCC model. Recently, we developed a novel model that recapitulates typical features of human HCC. Using this clinically relevant model, we demonstrate that tumor growth impairs host immunity and causes a profound exhaustion of tumor antigen-specific (TAS) CD8+ T cells. Increase in frequency and suppressive function of regulatory T cells (Tregs) is critically involved in this tumor-induced immune dysfunction. We further demonstrate that sunitinib suppresses Tregs and prevents tumor-induced immune tolerance, allowing TAS immunization to activate endogenous CD8+ T cells. As a result, this combinational strategy delays tumor growth. Importantly, the additional integration of exogenous naïve TAS CD8+ T cells by adoptive cell transfer (ACT) leads to the elimination of the established tumors without recurrence and promotes long-term survival of the treated mice. Mechanistically, sunitinib treatment primes the antitumor immune response by significantly decreasing Treg frequency, reducing TGF-β and IL-10 production by Tregs, and also protecting TAS CD8+ T cells from tumor-induced deletion in the setting of HCC. Taken together, sunitinib quantitatively and qualitatively modifies Tregs to overcome tumor-induced immune deficiency, suggesting the potential of sunitinib as a therapeutic immune activator for HCC control.

Purpose: The adoptive cell transfer (ACT) of CD8+ T cells is a promising treatment for advanced malignancies. Lymphodepletion before ACT enhances IFNγ+CD8+ T cell (Tc0)–mediated tumor regression. Yet, how lymphodepletion regulates the function and antitumor activity of IL17A+CD8+ T cells (Tc17) is unknown. Experimental Design: To address this question, pmel-1 CD8+ T cells were polarized to secrete either IL17A or IFNγ. These subsets were then infused into mice with B16F10 melanoma that were lymphoreplete [no total body irradiation (TBI)], or lymphodepleted with nonmyeloablative (5 Gy) or myeloablative (9 Gy with hematopoietic stem cell transplantation) TBI. The activation of innate immune cells and function of donor T-cell subsets were monitored in recipient mice. Results: Tc17 cells regress melanoma in myeloablated mice to a greater extent than in lymphoreplete or nonmyeloablated mice. TBI induced functional plasticity in Tc17 cells, causing conversion from IL17A to IFNγ producers. Additional investigation revealed that Tc17 plasticity and antitumor activity were mediated by IL12 secreted by irradiated host dendritic cells (DC). Neutralization of endogenous IL12 reduced the antitumor activity of Tc17 cells in myeloablated mice, whereas ex vivo priming with IL12 enhanced their capacity to regress melanoma in nonmyeloablated animals. This, coupled with exogenous administration of low-dose IL12, obviated the need for host preconditioning, creating curative responses in nonirradiated mice. Conclusions: Our findings indicate that TBI-induced IL12 augments Tc17 cell–mediated tumor immunity and underline the substantial implications of in vitro preparation of antitumor Tc17 cells with IL12 in the design of T-cell immunotherapies. Clin Cancer Res; 21(11); 2546–57. ©2015 AACR.

Ex vivo IL-12 priming of tumor-reactive CD8+ T-cells enhances their persistence and subsequent anti-tumor efficacy upon adoptive cell transfer (ACT) into lymphodepleted mice. Since IL-7 and IL-15 are considered critical for the persistence of adoptively transferred T-cells, we investigated the responsiveness of IL-12 primed CD8+ T-cells to these homeostatic cytokines to help elucidate the mechanisms behind their superior anti-tumor abilities. Using pmel-1 T-cell receptor transgenic mice, we found that pmel CD8+ T cells activated in the presence of IL-12 (pmelIL-12) showed much greater expansion in an irradiated (6 Gy) host compared to cells activated without IL-12 (pmelsham). This expansion was dependent on host IL-7, but not IL-15. Compared to pmelsham, pmelIL-12 demonstrated greatly enhanced in vitro IL-7 responsiveness, as measured by proliferation and phosphorylation of signaling molecules STAT5, S6 and AKT. These striking differences were not seen with IL-15. Despite not playing a major role in pmelIL-12 effector phase expansion, IL-15 was critical for maximum anti-tumor efficacy of pmelIL12, as irradiated mice devoid of IL-15, like mice receiving IL-7 neutralizing antibody (clone M25), showed reduced survival compared to irradiated mice receiving pmelIL-12 only. This decreased tumor control likely occurred because IL-15 was needed for long-term persistence of pmelIL-12. Together, these findings suggest that IL-12 priming of CD8 + T-cells augments the efficacy of ACT protocols in part by conferring an enhanced ability to respond to IL-7, thereby enabling transferred cells to persist in the IL-7 rich post-lymphodepletion environment.

ALT-803, a complex of an interleukin (IL)-15 superagonist mutant and a dimeric IL-15 receptor αSu/Fc fusion protein, was found to exhibit significantly stronger in vivo biologic activity on NK and T cells than IL-15. In this study, we show that a single dose of ALT-803, but not IL-15 alone, eliminated well-established 5T33P and MOPC-315P myeloma cells in the bone marrow of tumor-bearing mice. ALT-803 treatment also significantly prolonged survival of myeloma-bearing mice and provided resistance to rechallenge with the same tumor cells through a CD8+ T-cell–dependent mechanism. ALT-803 treatment stimulated CD8+ T cells to secrete large amounts of IFN-γ and promoted rapid expansion of CD8+CD44high memory T cells in vivo. These memory CD8+ T cells exhibited ALT-803–mediated upregulation of NKG2D (KLRK1) but not PD-1 (PDCD1) or CD25 (IL2RA) on their cell surfaces. ALT-803–activated CD8+ memory T cells also exhibited nonspecific cytotoxicity against myeloma and other tumor cells in vitro, whereas IFN-γ had no direct effect on myeloma cell growth. ALT-803 lost its antimyeloma activity in tumor-bearing IFN-γ knockout mice but retained the ability to promote CD8+CD44high memory T-cell proliferation, indicating that ALT-803–mediated stimulation of CD8+CD44high memory T cells is IFN-γ–independent. Thus, besides well-known IL-15 biologic functions in host immunity, this study shows that IL-15–based ALT-803 could activate CD8+CD44high memory T cells to acquire a unique innate-like phenotype and secrete IFN-γ for nonspecific tumor cell killing. This unique immunomodulatory property of ALT-803 strongly supports its clinical development as a novel immunotherapeutic agent against cancer and viral infections. Cancer Res; 73(10); 3075–86. ©2013 AACR.

e14579Background: Dendritic cells (DCs) shape the quantity and quality of anti-tumor immunity in patients with cancer. We hypothesized that DC-based immunotherapy, in conjunction with a novel long-...

T cells can inhibit tumor growth, but their function in the tumor microenvironment is often suppressed. Many solid tumors exhibit abundant macrophage infiltration and low oxygen tension, yet how hypoxic conditions may affect innate immune cells and their role in tumor progression is poorly understood. Targeted deletion of the hypoxia-responsive transcription factor hypoxia-inducible factor-1α (HIF-1α) in macrophages in a progressive murine model of breast cancer resulted in reduced tumor growth, although vascular endothelial growth factor-A levels and vascularization were unchanged. Tumor-associated macrophages can suppress tumor-infiltrating T cells by several mechanisms, and we found that hypoxia powerfully augmented macrophage-mediated T-cell suppression in vitro in a manner dependent on macrophage expression of HIF-1α. Our findings link the innate immune hypoxic response to tumor progression through induction of T-cell suppression in the tumor microenvironment. Cancer Res; 70(19); 7465–75. ©2010 AACR.

Cancer researchEpardaud, MathieuElpek, Kutlu GRubinstein, Mark PYonekura, Ai-risBellemare-Pelletier, AngeliqueBronson, RoderickHamerman, Jessica AGoldrath, Ananda WTurley, Shannon JengR01 AI067545/AI/NIAID NIH HHS/Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't2008/04/17 09:00Cancer Res. 2008 Apr 15;68(8):2972-83. doi: 10.1158/0008-5472.CAN-08-0045.; Tumors often escape immune-mediated destruction by suppressing lymphocyte infiltration or effector function. New approaches are needed that overcome this suppression and thereby augment the tumoricidal capacity of tumor-reactive lymphocytes. The cytokine interleukin-15 (IL-15) promotes proliferation and effector capacity of CD8(+) T cells, natural killer (NK) cells, and NKT cells; however, it has a short half-life and high doses are needed to achieve functional responses in vivo. The biological activity of IL-15 can be dramatically increased by complexing this cytokine to its soluble receptor, IL-15R alpha. Here, we report that in vivo delivery of IL-15/IL-15R alpha complexes triggers rapid and significant regression of established solid tumors in two murine models. Despite a marked expansion of IL-2/IL-15R beta(+) cells in lymphoid organs and peripheral blood following treatment with IL-15/IL-15R alpha complexes, the destruction of solid tumors was orchestrated by tumor-resident rather than newly infiltrating CD8(+) T cells. Our data provide novel insights into the use of IL-15/IL-15R alpha complexes to relieve tumor-resident T cells from functional suppression by the tumor microenvironment and have significant implications for cancer immunotherapy and treatment of chronic infections.

Expression of the MHC I-chain related molecules A and B (MICA/B) on epithelial cell surface in response to transformation or DNA damage can signal the immune system of the abnormality and thus initiate active immune surveillance by Natural Killer (NK) cells and cytotoxicity T cells. We and others have shown that malignant tumor cells can shed MICA/B to down regulate NKG2D expression and negatively impact NK and CD8 T cell function in cancer patients. Increased tumor-specific shedding of cell surface NKG2D ligand, MHC I chain related molecule (MIC), is associated with advanced stage and metastasis in many types of epithelial cancer. High serum levels of soluble MIC (sMIC) insults the immune system not only by down-regulating NKG2D expression on natural killer (NK) cells and effector T cells but also perturbing NK cell peripheral maintenance. Whether sMIC is an effective cancer therapeutic target has not been addressed due to the lack of specific sMIC-blocking reagent and clinically relevant pre-clinical animal models. Using a “humanized” clinically relevant spontaneous prostate carcinoma TRAMP/MIC bi-transgenic mouse model and multiple engineered syngeneic transplantable tumor models, we unprecedentedly show that therapy with a sMIC-specific monoclonal antibody induced rapid regression of primary tumors and metastasis without systemic toxicity. The therapy revamped a myriad of anti-tumor immune responses including NK cell homeostatic renewal and function, CD4 T cells to Th1 and Th17 responses. Using the well-defined melanoma model, we further show that anti-sMIC therapy augments Pmel-1 antigen-specific CD8 T cell responses. Notably, depletion of NK cells mitigated the therapeutic effect of anti-sMIC antibody and the effector function of both CD4 and CD8 T cells. We conclude that sMIC is an effective therapeutic target to potentiate innate and adoptive immune responses against MIC+ malignancies. Citation Format: Jennifer D. Wu, Fahmin Basher, Mark Rubinstein. Antibody targeting soluble NKG2D ligand sMIC induces regression of primary tumors and eliminates metastasis in multiple pre-clinical cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2468. doi:10.1158/1538-7445.AM2015-2468

Cytokine-based and antibody-targeted immunotherapies have both been important approaches in the treatment of malignant cancers. However, combinational therapies of cytokines and tumor-targeting antibodies remain to be further explored, especially in advanced solid tumors. In this study, C57BL/6 mice bearing established subcutaneous B16F10 melanoma were treated with mouse melanoma targeting anti-gp75 monoclonal antibody (mAb), TA99, combined with interleukin (IL)-15 based superagonist ALT-803. This soluble protein complex consists of an IL-15 superagonist mutant (IL-15N72D) associated with an IL-15 receptor α Sushi domain - human IgG1 Fc fusion protein. Compared to native IL-15, ALT-803 possesses superior in vivo biologic activity for stimulating NK and CD8+ memory T cells. The combined ALT-803+TA99 therapy significantly exceeded either monotherapy in inhibiting melanoma tumor growth (p < 0.001) and prolonging survival (p < 0.01) of B16F10 tumor-bearing mice. Through immune cell depletion studies and immunophenotyping of peripheral cells as well as tumor-infiltrating leukocyte subsets, we found that ALT-803 enhances TA99-mediated antitumor immunity through activation of NK cells and expansion of the CD8+CD44high memory T cell arm. In contrast, CD4+ T cells were shown to play more of a suppressive role in the therapeutic effect of ALT-803+TA99, possibly through involvement of regulatory T cells or ALT-803-mediated induction of PD-L1 on CD4+ T cells in the periphery and tumor microenvironment. Addition of anti-PD-L1 mAb to ALT-803+TA99 therapy resulted in a further increase in antitumor activity against subcutaneous B16F10 tumors. Furthermore, tumor-bearing mice that survived due to ALT-803+TA99 combination therapy exhibited long term antitumor memory against B16F10 tumor cell rechallenge. Immune-depletion studies revealed that this antitumor memory was associated with CD4+ T cells, CD8+ T cells and NK cells. Our findings suggest a therapeutic opportunity for ALT-803 in combination with tumor-targeting antibodies to simultaneously augment targeted antitumor activities of therapeutic antibodies and induce a long-term vaccinal effect which will provide durable responses in the treated host.

The adoptive transfer of tumor-reactive T cells has shown great promise in treating patients with metastatic cancer. However, effective T cell responses are limited by the availability of T cell growth factors such as IL-2 and tumor-induced suppressive pathways. As tumor-induced suppression may hamper cytokine responsiveness, we hypothesized that combinatorial therapy providing exogenous cytokine with blockade of inhibitory pathways would lead to synergistic anti-tumor responses. We evaluated this hypothesis by treating mice with palpable B16 melanoma tumors with lymphodepletion and transfer of activated, tumor-reactive CD8 + T cells (pmel-1 TCR transgenic). The persistence of the adoptively transferred tumor-reactive CD8 + T cells was dramatically augmented in the recipient mice with injections of an IL-15 superagonist (ALT-803) which, compared with IL-2, has greater biological activity and does not expand T regulatory cells. The ALT-803-treated mice also survived significantly longer than the untreated mice. B16 melanoma tumor cells were found to express PD-L1 and activated CD8 + T cells have PD-1 on their surface. Thus, we also gave mice anti-PD-L1 mAb treatment to block this PD-1/PD-L-1 inhibitory pathway. Our preliminary data suggest that combinatorial therapy with anti-PD-L1 mAb led to synergistic improvement in anti-tumor efficacy. We are now determining the optimal timing and dosing of ALT-803 and anti-PD-L1 mAb therapy to confirm these results. Currently, ALT-803 is in clinical trials for treating patients with various solid and hematologic tumors. Our findings suggest combinatorial therapy relieving T cell dysfunction using checkpoint inhibitors and providing ALT-803 cytokine therapy may lead to substantially improved outcomes over currently available therapies for patients with metastatic cancer.

The immune system is the built-in host defense mechanism against infectious agents as well as cancer. Protective immunity against cancer was convincingly demonstrated in the 1940s with syngeneic animal models (JNCI 18:769-778, 1976; Cancer Immun 1:6, 2001). Since then, the last century’s dream has been to effectively prevent and cure cancers by immunological means. This dream has slowly but surely become a reality (Nature 480:480-489, 2011). The successful examples of immunoprophylaxis and therapy against cancers include: (i) targeted therapy using monoclonal antibodies (Nat Rev Cancer 12:278-287, 2012); (ii) allogeneic hematopoietic stem cell transplantion to elicit graft-versus-cancer effect against a variety of hematopoietic malignancies (Blood 112:4371-4383, 2008); (iii) vaccination for preventing cancers with clear viral etiology such as hepatocellular carcinoma and cervical cancer (Cancer J Clin 57:7-28, 2007; NEJM 336:1855-1859, 1997); (iv) T cell checkpoint blockade against inhibitory pathways including targeting CTLA-4 and PD-1 inhibitory molecules for the treatment of melanoma and other solid tumors (NEJM 363:711-723, 2010; NEJM 366:2443-2454, 2012; NEJM 369:122-133, 2013; NEJM 366:2455-2465, 2012); (v) antigen-pulsed autologous dendritic cell vaccination against prostate cancer (NEJM 363:411-422, 2010); and (vi) the transfer of T cells including those genetically engineered with chimeric antigen receptors allowing targeting of B cell neoplasms (NEJM 365:725-733, 2011; NEJM 368:1509-1518, 2013; Blood 118:4817-4828, 2013; Sci Transl Med 5:177ra138, 2013). This article provides an overview on the exciting and expanding immunological arsenals against cancer, and discusses critical remaining unanswered questions of cancer immunology. The inherent specificity and memory of the adaptive immune response towards cancer will undoubtedly propel cancer immunotherapy to the forefront of cancer treatment in the immediate near future. Study of the fundamental mechanisms of the immune evasion of cancer shall also advance the field of immunology towards the development of effective immunotherapeutics against a wide spectrum of human diseases.

Adoptive T-cell transfer therapy (ACT) is a promising approach for the treatment of metastatic melanoma. Using the pmel-1 transgenic mouse as a source of melanoma-reactive CD8 T-cells, our lab has shown that priming these tumor-specific cells ex vivo with IL-12 (pmelIL-12) prior to ACT results in significant anti-tumor synergy and enhanced persistence in combination with cyclophosphamide (CTX). Here we demonstrate that other currently accepted lymphodepletion approaches, total body irradiation (TBI) alone or in combination with CTX, are also capable of synergizing with ACT of pmelIL-12. Interestingly, all 3 regimens (CTX, TBI and CTX + TBI) showed a similar ability to synergize with pmelIL-12, suggesting a common underlying mechanism. To elucidate the mechanism(s) behind this observed synergy, we evaluated whether the persistence of pmelIL-12 cells transferred into a lymphopenic host required the cytokines IL-7 and IL-15, which are known to be increased in the host post lymphodepletion. We found that the early expansion of pmelIL-12 in an irradiated host was partially dependent on IL-7, but not IL-15, as both the percentage and total number of donor cells recovered from spleens of irradiated hosts 1-week after ACT were greatly diminished in mice treated with an antibody against the IL-7 receptor alpha, CD127, or with an IL-7 neutralizing antibody (clone M25). In vitro these pmelIL-12 cells were able to proliferate in response to IL-7 to a much greater extent than pmel CD8 T-cells activated without IL-12. Together, these findings demonstrate that activated CD8 T cells primed with IL-12 display enhanced responsiveness to IL-7, and that IL-7 is necessary for maximal persistence of these cells in an irradiated host. Therefore, we conclude that the observed synergy between IL-12 priming of CD8 T cells and lymphodepletion is due in part to increased availability of host IL-7 after lymphodepletion. Citation Format: Christopher Bryce Johnson, Bennett R. May, Colleen A. Cloud, Mark P. Rubinstein, David J. Cole. The synergy between IL-12 priming of CD8 T cells and lymphodepletion is mediated by host IL-7. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A6.

IL-15/IL-15Rα complexes induce expansion and activation of cytotoxic lymphocytes including NK and CD8 T cells with higher activity compared to IL-15 alone in vivo, and therefore, have therapeutic potential against cancer and chronic infections. Recent studies demonstrated that continuous treatment with IL-15/IL-15Rα complexes improves the survival of tumor bearing mice, however fail to control tumor outgrowth. Given the limited efficacy of long-term cytokine therapy, we sought to investigate the impact of continuous stimulations with IL-15/IL-15Rα complexes on NK and CD8 T cells in tumor-free mice in vivo. Long-term treatment with IL-15/IL-15Rα complexes induced massive expansion of both NK and CD8 T cells. While the functional capacity of CD8 T cells was enhanced after long-term treatments, NK cells exhibited marked defects in activation, IFNγ production, degranulation, and cytotoxicity. In addition, long-term treatment generated a suppressive environment and resulted in marked increase in Treg and CD11c+Gr1+ cell numbers. However, impaired NK cell function was not due to regulatory T cells or various myeloid cell mediated-suppressive factors. Our results have important clinical implications for the design of immunotherapies and vaccines using multiple doses of immunostimulatory agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3829.