Chordoma cancer stem cell subpopulation characterization may guide targeted immunotherapy approaches to reduce disease recurrence.

Introduction: Cancer stem cells (CSCs), a group of tumor-initiating and tumor-maintaining cells, may be major players in the treatment resistance and recurrence distinctive of chordoma. Characterizing CSCs is crucial to better targeting this subpopulation. Methods: Using flow cytometry, six chordoma cell lines were evaluated for CSC composition. In vitro, cell lines were stained for B7H6, HER2, MICA-B, ULBP1, EGFR, and PD-L1 surface markers. Eighteen resected chordomas were stained using a multispectral immunofluorescence (mIF) antibody panel to identify CSCs in vivo. HALO software was used for quantitative CSC density and spatial analysis. Results: In vitro, chordoma CSCs express more B7H6, MICA-B, and ULBP1, assessed by percent positivity and mean fluorescence intensity (MFI), as compared to non-CSCs in all cell lines. PD- L1 percent positivity is increased by >20% in CSCs compared to non-CSCs in all cell lines except CH22. In vivo, CSCs comprise 1.39% of chordoma cells and most are PD-L1+ (75.18%). A spatial analysis suggests that chordoma CSCs cluster at an average distance of 71.51 mm (SD 73.40 mm) from stroma. Discussion: To our knowledge, this study is the first to identify individual chordoma CSCs and describe their surface phenotypes using in vitro and in vivo methods. PD-L1 is overexpressed on CSCs in chordoma human cell lines and operative tumor samples. Similarly, potential immunotherapeutic targets on CSCs, including B7H6, MICA-B, ULBP1, EGFR, and HER2 are overexpressed across cell lines. Targeting these markers may have a preferential role in combating CSCs, an aggressive subpopulation likely consequential to chordoma's high recurrence rate.

Augmentation of tumor expression of HLA-DR, CXCL9, and CXCL10 may improve olfactory neuroblastoma immunotherapeutic responses.

BACKGROUND: Olfactory neuroblastoma is a rare malignancy of the anterior skull base typically treated with surgery and adjuvant radiation. Although outcomes are fair for low-grade disease, patients with high-grade, recurrent, or metastatic disease oftentimes respond poorly to standard treatment methods. We hypothesized that an in-depth evaluation of the olfactory neuroblastoma tumor immune microenvironment would identify mechanisms of immune evasion in high-grade olfactory neuroblastoma as well as rational targetable mechanisms for future translational immunotherapeutic approaches. METHODS: Multispectral immunofluorescence and RNAScope evaluation of the tumor immune microenvironment was performed on forty-seven clinically annotated olfactory neuroblastoma samples. A retrospective chart review was performed and clinical correlations assessed. RESULTS: A significant T cell infiltration was noted in olfactory neuroblastoma samples with a stromal predilection, presence of myeloid-derived suppressor cells, and sparse natural killer cells. A striking decrease was observed in MHC-I expression in high-grade olfactory neuroblastoma compared to low-grade disease, representing a mechanism of immune evasion in high-grade disease. Mechanistically, the immune effector stromal predilection appears driven by low tumor cell MHC class II (HLA-DR), CXCL9, and CXCL10 expression as those tumors with increased tumor cell expression of each of these mediators correlated with significant increases in T cell infiltration. CONCLUSION: These data suggest that immunotherapeutic strategies that augment tumor cell expression of MHC class II, CXCL9, and CXCL10 may improve parenchymal trafficking of immune effector cells in olfactory neuroblastoma and augment immunotherapeutic responses.

Targeting sinonasal undifferentiated carcinoma with a combinatory immunotherapy approach.

PURPOSE: Sinonasal undifferentiated carcinoma (SNUC) is a rare, aggressive malignancy of the sinonasal cavity with poor prognosis and limited treatment options. To investigate the potential for SNUC sensitivity to combinatory immunotherapy, we performed in vitro studies with SNUC cell lines and used multi-spectral immunofluorescence to characterize the in vivo patient SNUC tumor immune microenvironment (TIME). EXPERIMENTAL DESIGN: Human-derived SNUC cell lines were used for in vitro studies of tumor cell susceptibility to natural killer (NK) cell-based immunotherapeutic strategies. Tumor samples from 14 treatment naïve SNUC patients were examined via multi-spectral immunofluorescence and clinical correlations assessed. RESULTS: Anti-PD-L1 blockade enhanced NK cell lysis of SNUC cell lines ∼5.4 fold (P ≤ 0.0001). This effect was blocked by a CD16 neutralizing antibody demonstrating activity through an antibody-dependent cellular cytotoxicity (ADCC) mediated pathway. ADCC-dependent lysis of SNUC cells was further enhanced by upregulation of PD-L1 on tumor cells by exogenous interferon-gamma (IFN-γ) administration or interleukin-15 (IL-15) stimulated IFN-γ release from NK cells. Combination treatment with anti-PD-L1 blockade and IL-15 superagonism enhanced NK-cell killing of SNUC cells 9.6-fold (P ≤ 0.0001). Untreated SNUC patient tumor samples were found to have an NK cell infiltrate and PD-L1+ tumor cells at a median of 5.4 cells per mm2. A striking 55.7-fold increase in CKlow tumor cell/NK cell interactions was observed in patients without disease recurrence after treatment (P = 0.022). Patients with higher CD3+CD8+ in the stroma had a significantly improved 5-year overall survival (P = 0.0029) and a significant increase in CKlow tumor cell/CD8+ cytotoxic T cell interactions was noted in long-term survivors (P = 0.0225). CONCLUSION: These data provide the pre-clinical rationale for ongoing investigation into combinatory immunotherapy approaches for SNUC.

A landscape of checkpoint blockade resistance in cancer: underlying mechanisms and current strategies to overcome resistance.

The discovery of immune checkpoints and the development of immune checkpoint inhibitors (ICI) have achieved a durable response in advanced-stage cancer patients. However, there is still a high proportion of patients who do not benefit from ICI therapy due to a lack of response when first treated (primary resistance) or detection of disease progression months after objective response is observed (acquired resistance). Here, we review the current FDA-approved ICI for the treatment of certain solid malignancies, evaluate the contrasting responses to checkpoint blockade in different cancer types, explore the known mechanisms associated with checkpoint blockade resistance (CBR), and assess current strategies in the field that seek to overcome these mechanisms. In order to improve current therapies and develop new ones, the immunotherapy field still has an unmet need in identifying other molecules that act as immune checkpoints, and uncovering other mechanisms that promote CBR.

Targeting Immunosuppressive Adenosine Signaling: A Review of Potential Immunotherapy Combination Strategies.

The tumor microenvironment regulates many aspects of cancer progression and anti-tumor immunity. Cancer cells employ a variety of immunosuppressive mechanisms to dampen immune cell function in the tumor microenvironment. While immunotherapies that target these mechanisms, such as immune checkpoint blockade, have had notable clinical success, resistance is common, and there is an urgent need to identify additional targets. Extracellular adenosine, a metabolite of ATP, is found at high levels in the tumor microenvironment and has potent immunosuppressive properties. Targeting members of the adenosine signaling pathway represents a promising immunotherapeutic modality that can potentially synergize with conventional anti-cancer treatment strategies. In this review, we discuss the role of adenosine in cancer, present preclinical and clinical data on the efficacy adenosine pathway inhibition, and discuss possible combinatorial approaches.

Exploiting docetaxel-induced tumor cell necrosis with tumor targeted delivery of IL-12.

There is strong evidence that chemotherapy can induce tumor necrosis which can be exploited for the targeted delivery of immuno-oncology agents into the tumor microenvironment (TME). We hypothesized that docetaxel, a chemotherapeutic agent that induces necrosis, in combination with the bifunctional molecule NHS-IL-12 (M9241), which delivers recombinant IL-12 through specific targeting of necrotic regions in the tumor, would provide a significant antitumor benefit in the poorly inflamed murine tumor model, EMT6 (breast), and in the moderately immune-infiltrated tumor model, MC38 (colorectal). Docetaxel, as monotherapy or in combination with NHS-IL-12, promoted tumor necrosis, leading to the improved accumulation and retention of NHS-IL-12 in the TME. Significant antitumor activity and prolonged survival were observed in cohorts receiving docetaxel and NHS-IL-12 combination therapy in both the MC38 and EMT6 murine models. The therapeutic effects were associated with increased tumor infiltrating lymphocytes and were dependent on CD8+ T cells. Transcriptomics of the TME of mice receiving the combination therapy revealed the upregulation of genes involving crosstalk between innate and adaptive immunity factors, as well as the downregulation of signatures of myeloid cells. In addition, docetaxel and NHS-IL-12 combination therapy effectively controlled tumor growth of PD-L1 wild-type and PD-L1 knockout MC38 in vivo, implying this combination could be applied in immune checkpoint refractory tumors, and/or tumors regardless of PD-L1 status. The data presented herein provide the rationale for the design of clinical studies employing this combination or similar combinations of agents.

Dying of Stress: Chemotherapy, Radiotherapy, and Small-Molecule Inhibitors in Immunogenic Cell Death and Immunogenic Modulation.

Innovative strategies to re-establish the immune-mediated destruction of malignant cells is paramount to the success of anti-cancer therapy. Accumulating evidence suggests that radiotherapy and select chemotherapeutic drugs and small molecule inhibitors induce immunogenic cell stress on tumors that results in improved immune recognition and targeting of the malignant cells. Through immunogenic cell death, which entails the release of antigens and danger signals, and immunogenic modulation, wherein the phenotype of stressed cells is altered to become more susceptible to immune attack, radiotherapies, chemotherapies, and small-molecule inhibitors exert immune-mediated anti-tumor responses. In this review, we discuss the mechanisms of immunogenic cell death and immunogenic modulation and their relevance in the anti-tumor activity of radiotherapies, chemotherapies, and small-molecule inhibitors. Our aim is to feature the immunological aspects of conventional and targeted cancer therapies and highlight how these therapies may be compatible with emerging immunotherapy approaches.

Exploiting the immunogenic potential of standard of care radiation or cisplatin therapy in preclinical models of HPV-associated malignancies.

BACKGROUND: While radiation and chemotherapy are primarily purposed for their cytotoxic effects, a growing body of preclinical and clinical evidence demonstrates an immunogenic potential for these standard therapies. Accordingly, we sought to characterize the immunogenic potential of radiation and cisplatin in human tumor models of HPV-associated malignancies. These studies may inform rational combination immuno-oncology (IO) strategies to be employed in the clinic on the backbone of standard of care, and in so doing exploit the immunogenic potential of standard of care to improve durable responses in HPV-associated malignancies. METHODS: Retroviral transduction with HPV16 E7 established a novel HPV-associated sinonasal squamous cell carcinoma (SNSCC) cell line. Three established HPV16-positive cell lines were also studied (cervical carcinoma and head and neck squamous cell carcinoma). Following determination of sensitivities to standard therapies using MTT assays, flow cytometry was used to characterize induction of immunogenic cell stress following sublethal exposure to radiation or cisplatin, and the functional consequence of this induction was determined using impedance-based real time cell analysis cytotoxicity assays employing HPV16 E7-specific cytotoxic lymphocytes (CTLs) with or without N803 (IL-15/IL-15-Rα superagonist) or exogenous death receptor ligands. In vitro observations were translated using an in vivo xenograft NSG mouse model of human cervical carcinoma evaluating cisplatin in combination with CTL adoptive cell transfer. RESULTS: We showed that subpopulations surviving clinically relevant doses of radiation or cisplatin therapy were more susceptible to CTL-mediated lysis in four of four tumor models of HPV-associated malignancies, serving as a model for HPV therapeutic vaccine or T-cell receptor adoptive cell transfer. This increased killing was further amplified by IL-15 agonism employing N803. We further characterized that radiation or cisplatin induced immunogenic cell stress in three of three cell lines, and consequently demonstrated that upregulated surface expression of Fas and TRAIL-R2 death receptors at least in part mediated enhanced CTL-mediated lysis. In vivo, cisplatin-induced immunogenic cell stress synergistically potentiated CTL-mediated tumor control in a human model of HPV-associated malignancy. CONCLUSION: Standard of care radiation or cisplatin therapy induced immunogenic cell stress in preclinical models of HPV-associated malignancies, presenting an opportunity poised for exploitation by employing IO strategies in combination with standard of care.

Multi-spectral immunofluorescence evaluation of the myeloid, T cell, and natural killer cell tumor immune microenvironment in chordoma may guide immunotherapeutic strategies.

Background: Chordoma is a rare, invasive, and devastating bone malignancy of residual notochord tissue that arises at the skull base, sacrum, or spine. In order to maximize immunotherapeutic approaches as a potential treatment strategy in chordoma it is important to fully characterize the tumor immune microenvironment (TIME). Multispectral immunofluorescence (MIF) allows for comprehensive evaluation of tumor compartments, molecular co-expression, and immune cell spatial relationships. Here we implement MIF to define the myeloid, T cell, and natural killer (NK) cell compartments in an effort to guide rational design of immunotherapeutic strategies for chordoma. Methods: Chordoma tumor tissue from 57 patients was evaluated using MIF. Three panels were validated to assess myeloid cell, T cell, and NK cell populations. Slides were stained using an automated system and HALO software objective analysis was utilized for quantitative immune cell density and spatial comparisons between tumor and stroma compartments. Results: Chordoma TIME analysis revealed macrophage infiltration of the tumor parenchyma at a significantly higher density than stroma. In contrast, helper T cells, cytotoxic T cells, and T regulatory cells were significantly more abundant in stroma versus tumor. T cell compartment infiltration more commonly demonstrated a tumor parenchymal exclusion pattern, most markedly among cytotoxic T cells. NK cells were sparsely found within the chordoma TIME and few were in an activated state. No immune composition differences were seen in chordomas originating from diverse anatomic sites or between those resected at primary versus advanced disease stage. Conclusion: This is the first comprehensive evaluation of the chordoma TIME including myeloid, T cell, and NK cell appraisal using MIF. Our findings demonstrate that myeloid cells significantly infiltrate chordoma tumor parenchyma while T cells tend to be tumor parenchymal excluded with high stromal infiltration. On average, myeloid cells are found nearer to target tumor cells than T cells, potentially resulting in restriction of T effector cell function. This study suggests that future immunotherapy combinations for chordoma should be aimed at decreasing myeloid cell suppressive function while enhancing cytotoxic T cell and NK cell killing.

Immune targeting of three independent suppressive pathways (TIGIT, PD-L1, TGFß) provides significant antitumor efficacy in immune checkpoint resistant models.

Immune checkpoint blockade (ICB) therapy, while groundbreaking, must be improved to promote enhanced durable responses and to prevent the development of treatment-refractory disease. Cancer therapies that engage, enable, and expand the antitumor immune response will likely require rationally designed combination strategies. Targeting multiple immunosuppressive pathways simultaneously may provide additional therapeutic benefit over singular targeting. We therefore hypothesized that the use of two molecules which inhibit three independent, but overlapping, pathways (TIGIT:CD155, PD-1/PD-L1, and TGFß) would provide significant antitumor efficacy in the syngeneic ICB resistant colorectal tumor model MC38 expressing human carcinoembryonic antigen (CEA) in CEA transgenic mice. This novel combination treatment strategy has significant antitumor activity and survival benefit in two models of murine carcinomas, MC38-CEA (CRC) and TC1 (HPV+ lung carcinoma). MC38-CEA mice that responded to αTIGIT and bintrafusp alfa combination therapy generated memory responses and were protected from rechallenge. These effects were dependent on CD4+ and CD8+ T cells, as well as increased immune infiltration into the TME. This combination induced production of tumor-specific CD8+ T cells, and an increase in activation and cytotoxicity resulting in an overall activated immune landscape in the tumor. Data presented herein demonstrate the αTIGIT and bintrafusp alfa combination has efficacy across multiple tumor models, including the checkpoint-resistant model of murine colon carcinoma, MC38-CEA and the HPV+ model TC-1.

Tipping the scales: Immunotherapeutic strategies that disrupt immunosuppression and promote immune activation.

Immunotherapy has emerged as an effective therapeutic approach for several cancer types. However, only a subset of patients exhibits a durable response due in part to immunosuppressive mechanisms that allow tumor cells to evade destruction by immune cells. One of the hallmarks of immune suppression is the paucity of tumor-infiltrating lymphocytes (TILs), characterized by low numbers of effector CD4+ and CD8+ T cells in the tumor microenvironment (TME). Additionally, the proper activation and function of lymphocytes that successfully infiltrate the tumor are hampered by the lack of co-stimulatory molecules and the increase in inhibitory factors. These contribute to the imbalance of effector functions by natural killer (NK) and T cells and the immunosuppressive functions by myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the TME, resulting in a dysfunctional anti-tumor immune response. Therefore, therapeutic regimens that elicit immune responses and reverse immune dysfunction are required to counter immune suppression in the TME and allow for the re-establishment of proper immune surveillance. Immuno-oncology (IO) agents, such as immune checkpoint blockade and TGF-ß trapping molecules, have been developed to decrease or block suppressive factors to enable the activity of effector cells in the TME. Therapeutic agents that target immunosuppressive cells, either by direct lysis or altering their functions, have also been demonstrated to decrease the barrier to effective immune response. Other therapies, such as tumor antigen-specific vaccines and immunocytokines, have been shown to activate and improve the recruitment of CD4+ and CD8+ T cells to the tumor, resulting in improved T effector to Treg ratio. The preclinical data on these diverse IO agents have led to the development of ongoing phase I and II clinical trials. This review aims to provide an overview of select therapeutic strategies that tip the balance from immunosuppression to immune activity in the TME.

The emerging role of off-the-shelf engineered natural killer cells in targeted cancer immunotherapy.

Natural killer (NK) cells are innate lymphocytes that recognize and clear infected and transformed cells. The importance of NK cells in tumor surveillance underlies the development of NK cell therapy as cancer treatment. The NK-92 cell line has been successfully modified to express high-affinity CD16 receptor for antibody-dependent cellular cytotoxicity and/or chimeric antigen receptors (CARs) that can recognize antigens expressed on tumor cells and mediate NK cell activation. Since there is no need for human leukocyte antigen matching or prior exposure to the tumor antigens, NK-92 provides an opportunity for the development of next-generation off-the-shelf cell therapy platforms. CAR-engineered NK-92 cells have demonstrated robust antitumor activity in in vitro and in vivo preclinical studies, propelling the clinical development of CAR NK-92 cells. Preliminary phase 1 data indicate that CAR NK-92 can be safely administered in the clinic. In this review, we provide an overview of recent advances in the research and clinical application of this novel cell immunotherapy.

From Immunogenic Cell Death to Immunogenic Modulation: Select Chemotherapy Regimens Induce a Spectrum of Immune-Enhancing Activities in the Tumor Microenvironment.

Cancer treatment has rapidly entered the age of immunotherapy, and it is becoming clear that the effective therapy of established tumors necessitates rational multi-combination immunotherapy strategies. But even in the advent of immunotherapy, the clinical role of standard-of-care chemotherapy regimens still remains significant and may be complementary to emerging immunotherapeutic approaches. Depending on dose, schedule, and agent, chemotherapy can induce immunogenic cell death, resulting in the release of tumor antigens to stimulate an immune response, or immunogenic modulation, sensitizing surviving tumor cells to immune cell killing. While these have been previously defined as distinct processes, in this review we examine the published mechanisms supporting both immunogenic cell death and immunogenic modulation and propose they be reclassified as similar effects termed "immunogenic cell stress." Treatment-induced immunogenic cell stress is an important result of cytotoxic chemotherapy and future research should consider immunogenic cell stress as a whole rather than just immunogenic cell death or immunogenic modulation. Cancer treatment strategies should be designed specifically to take advantage of these effects in combination immunotherapy, and novel chemotherapy regimens should be designed and investigated to potentially induce all aspects of immunogenic cell stress.

Exploiting off-target effects of estrogen deprivation to sensitize estrogen receptor negative breast cancer to immune killing.

BACKGROUND: There are highly effective treatment strategies for estrogen receptor (ER)+, progesterone receptor (PR)+, and HER2+ breast cancers; however, there are limited targeted therapeutic strategies for the 10%-15% of women who are diagnosed with triple-negative breast cancer. Here, we hypothesize that ER targeting drugs induce phenotypic changes to sensitize breast tumor cells to immune-mediated killing regardless of their ER status. METHODS: Real-time cell analysis, flow cytometry, qRT-PCR, western blotting, and multiplexed RNA profiling were performed to characterize ER+ and ER- breast cancer cells and to interrogate the phenotypic effects of ER targeting drugs. Sensitization of breast cancer cells to immune cell killing by the tamoxifen metabolite 4-hydroxytamoxifen (4-OHT) and fulvestrant was determined through in vitro health-donor natural killer cell 111IN-release killing assays. A syngeneic tumor study was performed to validate these findings in vivo. RESULTS: Pretreatment with tamoxifen metabolite 4-OHT or fulvestrant resulted in increased natural killer (NK)-mediated cell lysis of both ER+ and ER- breast cancer cells. Through multiplexed RNA profiling analysis of 4-OHT-treated ER+ and ER- cells, we identified increased activation of apoptotic and death receptor signaling pathways and identified G protein-coupled receptor for estrogen (GPR30) engagement as a putative mechanism for immunogenic modulation. Using the specific GPR30 agonist G-1, we demonstrate that targeted activation of GPR30 signaling resulted in increased NK cell killing. Furthermore, we show that knockdown of GPR30 inhibited 4-OHT and fulvestrant mediated increases to NK cell killing, demonstrating this is dependent on GPR30 expression. Moreover, we demonstrate that this mechanism remains active in a 4-OHT-resistant MCF7 cell line, showing that even in patient populations with ER+ tumors that are resistant to the cytotoxic effects of tamoxifen, 4-OHT treatment sensitizes them to immune-mediated killing. Moreover, we find that fulvestrant pretreatment of tumor cells synergizes with the IL-15 superagonist N-803 treatment of NK cells and sensitizes tumor cells to killing by programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK) cells. Finally, we demonstrate that the combination of fulvestrant and N-803 is effective in triple-negative breast cancer in vivo. CONCLUSION: Together, these findings demonstrate a novel effect of ER targeting drugs on the interaction of ER+ and, surprisingly, ER- tumors cells with the immune system. This study is the first to demonstrate the potential use of ER targeting drugs as immunomodulatory agents in an ER agnostic manner and may inform novel immunotherapy strategies in breast cancer.

Stay on Target: Reengaging Cancer Vaccines in Combination Immunotherapy.

Effective treatment of established tumors requires rational multicombination immunotherapy strategies designed to target all functions of the patient immune system and tumor immune microenvironment. While these combinations build on the foundation of successful immune checkpoint blockade antibodies, it is increasingly apparent that successful immunotherapy will also require a cancer vaccine backbone to engage the immune system, thereby ensuring that additional immuno-oncology agents will engage a tumor-specific immune response. This review summarizes ongoing clinical trials built upon the backbone of cancer vaccines and focusing on those clinical trials that utilize multicombination (3+) immuno-oncology agents. We examine combining cancer vaccines with multiple checkpoint blockade antibodies, novel multifunctional molecules, adoptive cell therapy and immune system agonists. These combinations and those yet to enter the clinic represent the future of cancer immunotherapy. With a cancer vaccine backbone, we are confident that current and coming generations of rationally designed multicombination immunotherapy can result in effective therapy of established tumors.

Vaccine Increases the Diversity and Activation of Intratumoral T Cells in the Context of Combination Immunotherapy.

Resistance to immune checkpoint blockade therapy has spurred the development of novel combinations of drugs tailored to specific cancer types, including non-inflamed tumors with low T-cell infiltration. Cancer vaccines can potentially be utilized as part of these combination immunotherapies to enhance antitumor efficacy through the expansion of tumor-reactive T cells. Utilizing murine models of colon and mammary carcinoma, here we investigated the effect of adding a recombinant adenovirus-based vaccine targeting tumor-associated antigens with an IL-15 super agonist adjuvant to a multimodal regimen consisting of a bifunctional anti-PD-L1/TGF-ßRII agent along with a CXCR1/2 inhibitor. We demonstrate that the addition of vaccine induced a greater tumor infiltration with T cells highly positive for markers of proliferation and cytotoxicity. In addition to this enhancement of cytotoxic T cells, combination therapy showed a restructured tumor microenvironment with reduced Tregs and CD11b+Ly6G+ myeloid cells. Tumor-infiltrating immune cells exhibited an upregulation of gene signatures characteristic of a Th1 response and presented with a more diverse T-cell receptor (TCR) repertoire. These results provide the rationale for the addition of vaccine-to-immune checkpoint blockade-based therapies being tested in the clinic.

Differential combination immunotherapy requirements for inflamed (warm) tumors versus T cell excluded (cool) tumors: engage, expand, enable, and evolve.

BACKGROUND: Different types of tumors have varying susceptibility to immunotherapy and hence require different treatment strategies; these cover a spectrum ranging from 'hot' tumors or those with high mutational burden and immune infiltrates that are more amenable to targeting to 'cold' tumors that are more difficult to treat due to the fewer targetable mutations and checkpoint markers. We hypothesized that an effective anti-tumor response requires multiple agents that would (1) engage the immune response and generate tumor-specific effector cells; (2) expand the number and breadth of the immune effector cells; (3) enable the anti-tumor activity of these immune cells in the tumor microenvironment; and (4) evolve the tumor response to widen immune effector repertoire. METHODS: A hexatherapy combination was designed and administered to MC38-CEA (warm) and 4T1 (cool) murine tumor models. The hexatherapy regimen was composed of adenovirus-based vaccine and IL-15 (interleukin-15) superagonist (N-803) to engage the immune response; anti-OX40 and anti-4-1BB to expand effector cells; anti-PD-L1 (anti-programmed death-ligand 1) to enable anti-tumor activity; and docetaxel to promote antigen spread. Primary and metastatic tumor growth inhibition were measured. The generation of anti-tumor immune effector cells was analyzed using flow cytometry, ELISpot (enzyme-linked immunospot), and RNA analysis. RESULTS: The MC38-CEA and 4T1 tumor models have differential sensitivities to the combination treatments. In the 'warm' MC38-CEA, combinations with two to five agents resulted in moderate therapeutic benefit while the hexatherapy regimen outperformed all these combinations. On the other hand, the hexatherapy regimen was required in order to decrease the primary and metastatic tumor burden in the 'cool' 4T1 model. In both models, the hexatherapy regimen promoted CD4+ and CD8+ T cell proliferation and activity. Furthermore, the hexatherapy regimen induced vaccine-specific T cells and stimulated antigen cascade. The hexatherapy regimen also limited the immunosuppressive T cell and myeloid derived suppressor cell populations, and also decreased the expression of exhaustion markers in T cells in the 4T1 model. CONCLUSION: The hexatherapy regimen is a strategic combination of immuno-oncology agents that can engage, expand, enable, and evolve the immune response and can provide therapeutic benefits in both MC38-CEA (warm) and 4T1 (cool) tumor models.

Combinatorial Natural Killer Cell-based Immunotherapy Approaches Selectively Target Chordoma Cancer Stem Cells.

Chordoma is a rare tumor derived from notochord remnants that has a propensity to recur and metastasize despite conventional multimodal treatment. Cancer stem cells (CSC) are implicated in chordoma's resistant and recurrent behavior; thus strategies that target CSCs are of particular interest. Using in vitro cytotoxicity models, we demonstrated that anti-programmed death-ligand 1 (N-601) and anti-epidermal growth factor receptor (cetuximab) antibodies enhanced lysis of chordoma cells by healthy donor and chordoma patient NK cells through antibody-dependent cellular cytotoxicity (ADCC). Treatment of NK cells with an IL-15 superagonist complex (N-803) increased their cytotoxicity against chordoma cells, which was further enhanced by treatment with N-601 and/or cetuximab. PD-L1-targeted chimeric antigen receptor NK cells (PD-L1 t-haNKs) were also effective against chordoma cells. CSCs were preferentially vulnerable to NK cell killing in the presence of N-601 and N-803. Flow cytometric analysis of a chordoma CSC population showed that CSCs expressed significantly more NK activating ligand B7-H6 and PD-L1 than non-CSCs, thus explaining a potential mechanism of selective targeting. These data suggest that chordoma may be effectively targeted by combinatorial NK cell-mediated immunotherapeutic approaches and that the efficacy of these approaches in chordoma and other CSC-driven tumor types should be investigated further in clinical studies.

Therapy of Established Tumors with Rationally Designed Multiple Agents Targeting Diverse Immune-Tumor Interactions: Engage, Expand, Enable.

Immunotherapy of immunologically cold solid tumors may require multiple agents to engage immune effector cells, expand effector populations and activities, and enable immune responses in the tumor microenvironment (TME). To target these distinct phenomena, we strategically chose five clinical-stage immuno-oncology agents, namely, (i) a tumor antigen-targeting adenovirus-based vaccine (Ad-CEA) and an IL15 superagonist (N-803) to activate tumor-specific T cells, (ii) OX40 and GITR agonists to expand and enhance the activated effector populations, and (iii) an IDO inhibitor (IDOi) to enable effector-cell activity in the TME. Flow cytometry, T-cell receptor (TCR) sequencing, and RNA-sequencing (RNA-seq) analyses showed that in the CEA-transgenic murine colon carcinoma (MC38-CEA) tumor model, Ad-CEA + N-803 combination therapy resulted in immune-mediated antitumor effects and promoted the expression of costimulatory molecules on immune subsets, OX40 and GITR, and the inhibitory molecule IDO. Treatment with Ad-CEA + N-803 + OX40 + GITR + IDOi, termed the pentatherapy regimen, resulted in the greatest inhibition of tumor growth and protection from tumor rechallenge without toxicity. Monotherapy with any of the agents had little to no antitumor activity, whereas combining two, three, or four agents had minimal antitumor effects. Immune analyses demonstrated that the pentatherapy combination induced CD4+ and CD8+ T-cell activity in the periphery and tumor, and antitumor activity associated with decreased regulatory T-cell (Treg) immunosuppression in the TME. The pentatherapy combination also inhibited tumor growth and metastatic formation in 4T1 and LL2-CEA murine tumor models. This study provides the rationale for the combination of multimodal immunotherapy agents to engage, enhance, and enable adaptive antitumor immunity.

Natural Born Killers: NK Cells in Cancer Therapy.

Cellular therapy has emerged as an attractive option for the treatment of cancer, and adoptive transfer of chimeric antigen receptor (CAR) expressing T cells has gained FDA approval in hematologic malignancy. However, limited efficacy was observed using CAR-T therapy in solid tumors. Natural killer (NK) cells are crucial for tumor surveillance and exhibit potent killing capacity of aberrant cells in an antigen-independent manner. Adoptive transfer of unmodified allogeneic or autologous NK cells has shown limited clinical benefit due to factors including low cell number, low cytotoxicity and failure to migrate to tumor sites. To address these problems, immortalized and autologous NK cells have been genetically engineered to express high affinity receptors (CD16), CARs directed against surface proteins (PD-L1, CD19, Her2, etc.) and endogenous cytokines (IL-2 and IL-15) that are crucial for NK cell survival and cytotoxicity, with positive outcomes reported by several groups both preclinically and clinically. With a multitude of NK cell-based therapies currently in clinic trials, it is likely they will play a crucial role in next-generation cell therapy-based treatment. In this review, we will highlight the recent advances and limitations of allogeneic, autologous and genetically enhanced NK cells used in adoptive cell therapy.