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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.
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Antígeno B7-H1 , Neoplasias , Camundongos , Animais , Docetaxel , Linfócitos T CD8-Positivos , Interleucina-12/farmacologia , Necrose , Microambiente Tumoral , Linhagem Celular Tumoral , ImunoterapiaRESUMO
Circulating tumor-derived exosomes (TEX) interact with a variety of cells in cancer-bearing hosts, leading to cellular reprogramming which promotes disease progression. To study TEX effects on the development of solid tumors, immunosuppressive exosomes carrying PD-L1 and FasL were isolated from supernatants of murine or human HNSCC cell lines. TEX were delivered (IV) to immunocompetent C57BL/6 mice bearing premalignant oral/esophageal lesions induced by the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Progression of the premalignant oropharyngeal lesions to malignant tumors was monitored. A single TEX injection increased the number of developing tumors (6.2 versus 3.2 in control mice injected with phosphate-buffered saline; P < 0.0002) and overall tumor burden per mouse (P < 0.037). The numbers of CD4+ and CD8+ T lymphocytes infiltrating the developing tumors were coordinately reduced (P < 0.01) in mice injected with SCCVII-derived TEX relative to controls. Notably, TEX isolated from mouse or human tumors had similar effects on tumor development and immune cells. A single IV injection of TEX was sufficient to condition mice harboring premalignant OSCC lesions for accelerated tumor progression in concert with reduced immune cell migration to the tumor.
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Carcinogênese/patologia , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Exossomos/patologia , Neoplasias Bucais/patologia , 4-Nitroquinolina-1-Óxido/toxicidade , Animais , Apoptose , Antígeno B7-H1/metabolismo , Carcinogênese/induzido quimicamente , Carcinogênese/metabolismo , Carcinógenos/toxicidade , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/metabolismo , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Bucais/induzido quimicamente , Neoplasias Bucais/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Advanced oral squamous cell carcinomas (OSCC) have limited therapeutic options. Although immune therapies are emerging as a potentially effective alternative or adjunct to chemotherapies, the therapeutic efficacy of combination immune chemotherapies has yet to be determined. Using a 4-nitroquinolone-N-oxide (4NQO) orthotopic model of OSCC in immunocompetent mice, we evaluated the therapeutic efficacy of single- and combined-agent treatment with a poly-epitope tumor peptide vaccine, cisplatin and/or an A2AR inhibitor, ZM241385. The monotherapies or their combinations resulted in a partial inhibition of tumor growth and, in some cases, a significant but transient upregulation of systemic anti-tumor CD8+ T cell responses. These responses eroded in the face of expanding immunoregulatory cell populations at later stages of tumor progression. Our findings support the need for the further development of combinatorial therapeutic approaches that could more effectively silence dominant immune inhibitory pathways operating in OSCC and provide novel, more beneficial treatment options for this tumor.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Imunoterapia/métodos , Neoplasias Bucais/terapia , Neoplasias Experimentais/terapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , 4-Nitroquinolina-1-Óxido/toxicidade , Animais , Cisplatino/uso terapêutico , Terapia Combinada/métodos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Bucais/induzido quimicamente , Neoplasias Bucais/imunologia , Neoplasias Bucais/patologia , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/induzido quimicamente , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Resultado do Tratamento , Triazinas/uso terapêutico , Triazóis/uso terapêutico , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
Tumor blood vessels are frequently inefficient in their design and function, leading to high interstitial fluid pressure, hypoxia, and acidosis in the tumor microenvironment (TME), rendering tumors refractory to the delivery of chemotherapeutic agents and immune effector cells. Here we identified the NOTCH antagonist delta-like 1 homologue (DLK1) as a vascular pericyte-associated antigen expressed in renal cell carcinomas (RCC), but not in normal kidney tissues in mice and humans. Vaccination of mice bearing established RCC against DLK1 led to immune-mediated elimination of DLK1(+) pericytes and to blood vessel normalization (i.e., decreased vascular permeability and intratumoral hypoxia) in the TME, in association with tumor growth suppression. After therapeutic vaccination, tumors displayed increased prevalence of activated VCAM1(+)CD31(+) vascular endothelial cells (VECs) and CXCL10, a type-1 T cell recruiting chemokine, in concert with increased levels of type-1 CD8(+) tumor-infiltrating lymphocytes (TIL). Vaccination against DLK1 also yielded (i) dramatic reductions in Jarid1B(+), CD133(+), and CD44(+) (hypoxia-responsive) stromal cell populations, (ii) enhanced tumor cell apoptosis, and (iii) increased NOTCH signaling in the TME. Coadministration of a γ-secretase inhibitor (N-[N-(3,5-Difluorophenacetyl-l-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT)) that interferes with canonical NOTCH signaling resulted in the partial loss of therapeutic benefits associated with lentivirus encoding full-length murine (lvDLK1)-based vaccination.
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Vacinas Anticâncer/uso terapêutico , Carcinoma de Células Renais/irrigação sanguínea , Carcinoma de Células Renais/terapia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Animais , Apoptose , Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação ao Cálcio , Vacinas Anticâncer/imunologia , Permeabilidade Capilar , Carcinoma de Células Renais/genética , Hipóxia Celular , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lentivirus/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Pericitos/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Microambiente Tumoral , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
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.
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Neoplasias , Humanos , ImunoterapiaRESUMO
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.
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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.
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Vacinas Anticâncer , Neoplasias , Antígenos de Neoplasias , Humanos , Inibidores de Checkpoint Imunológico , Terapia de Imunossupressão , Imunoterapia/métodos , Fator de Crescimento Transformador beta , Microambiente TumoralRESUMO
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.
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Morte Celular Imunogênica , Neoplasias , Humanos , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , ImunidadeRESUMO
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.
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Carcinoma , Neoplasias do Colo , Infecções por Papillomavirus , Animais , Antígeno B7-H1 , Linfócitos T CD8-Positivos , Antígeno Carcinoembrionário/farmacologia , Antígeno Carcinoembrionário/uso terapêutico , Carcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Camundongos , Infecções por Papillomavirus/tratamento farmacológico , Receptor de Morte Celular Programada 1/uso terapêutico , Receptores Imunológicos/metabolismo , Receptores Imunológicos/uso terapêutico , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/uso terapêuticoRESUMO
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.
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Antineoplásicos , Carcinoma , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Animais , Camundongos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Interleucina-15/farmacologia , Linfócitos T Citotóxicos , Infecções por Papillomavirus/complicações , Padrão de Cuidado , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
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.
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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.
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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.
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Antineoplásicos Imunológicos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Docetaxel/administração & dosagem , Inibidores de Checkpoint Imunológico/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Vacinas de DNA/administração & dosagem , Animais , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Terapia Combinada , Docetaxel/farmacologia , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Interleucina-15/agonistas , Camundongos , Receptores OX40/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Vacinas de DNA/genética , Vacinas de DNA/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
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Antígeno B7-H1 , Cordoma , Humanos , Cetuximab , Cordoma/terapia , Linhagem Celular Tumoral , Recidiva Local de Neoplasia/patologia , Células Matadoras Naturais , Imunoterapia , Anticorpos , Células-Tronco NeoplásicasRESUMO
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.
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Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Neoplasias do Colo/terapia , Imunoterapia/métodos , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Although immune checkpoint inhibitors have revolutionized cancer treatment, clinical benefit with this class of agents has been limited to a subset of patients. Hence, more effective means to target tumor cells that express immune checkpoint molecules should be developed. For the first time, we report a novel natural killer (NK) cell line, programmed death-ligand 1 (PD-L1) targeting high-affinity natural killer (t-haNK), which was derived from NK-92 and was engineered to express high-affinity CD16, endoplasmic reticulum-retained interleukin (IL)-2, and a PD-L1-specific chimeric antigen receptor (CAR). We show that PD-L1 t-haNK cells also retained the expression of native NK receptors and carried a high content of granzyme and perforin granules. METHODS: NanoString, flow cytometry, and immunofluorescence analyses were performed to characterize the phenotype of irradiated PD-L1 t-haNK cells. In vitro PD-L1 t-haNK cell activity against cancer cell lines and human peripheral blood mononuclear cells (PBMCs) was determined via flow-based and 111In-release killing assays. The antitumor effect of PD-L1 t-haNK cells in vivo was investigated using MDA-MB-231, H460, and HTB1 xenograft models in NOD-scid IL2Rgammanull (NSG) mice. Additionally, the antitumor effect of PD-L1 t-haNK cells, in combination with anti-PD-1 and N-803, an IL-15 superagonist, was evaluated using mouse oral cancer 1 syngeneic model in C57BL/6 mice. RESULTS: We show that PD-L1 t-haNK cells expressed PD-L1-targeting CAR and CD16, retained the expression of native NK receptors, and carried a high content of granzyme and perforin granules. In vitro, we demonstrate the ability of irradiated PD-L1 t-haNK cells to lyse 20 of the 20 human cancer cell lines tested, including triple negative breast cancer (TNBC) and lung, urogenital, and gastric cancer cells. The cytotoxicity of PD-L1 t-haNK cells was correlated to the PD-L1 expression of the tumor targets and can be improved by pretreating the targets with interferon (IFN)-γ. In vivo, irradiated PD-L1 t-haNK cells inhibited the growth of engrafted TNBC and lung and bladder tumors in NSG mice. The combination of PD-L1 t-haNK cells with N-803 and anti-PD-1 antibody resulted in superior tumor growth control of engrafted oral cavity squamous carcinoma tumors in C57BL/6 mice. In addition, when cocultured with human PBMCs, PD-L1 t-haNK cells preferentially lysed the myeloid-derived suppressor cell population but not other immune cell types. CONCLUSION: These studies demonstrate the antitumor efficacy of PD-L1 t-haNK cells and provide a rationale for the potential use of these cells in clinical studies.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/transplante , Células Supressoras Mieloides/imunologia , Neoplasias/terapia , Animais , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Terapia Combinada/métodos , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Células Supressoras Mieloides/efeitos dos fármacos , Neoplasias/imunologia , Neoplasias/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Engenharia de Proteínas , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de IgG/genética , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Natural killer (NK) cells are immune cells capable of killing virally infected cells and tumor cells without the need for antigen stimulation. Tumors, however, can create a suppressive microenvironment that decreases NK function. A feature of many tumors is hypoxia (low oxygen perfusion), which has been previously shown to decrease NK function. A high affinity NK (haNK) cell has been engineered to express a high affinity CD16 receptor as well as internal interleukin (IL)-2 for increased antibody-dependent cellular cytotoxicity (ADCC) and activation, respectively. We sought to investigate the tolerance of NK cells versus haNK cells to hypoxia. METHODS: We exposed healthy donor (HD) NK and X-irradiated haNK cells to normoxia (20% oxygen) as well as hypoxia (0% oxygen) and investigated their ability to kill prostate, breast and lung tumor cell lines after 5 hours. We also used monoclonal antibodies cetuximab (anti-EGFR) or avelumab (antiprogrammed death-ligand 1) to investigate the effects of hypoxia on NK ADCC. Genomic and proteomic analyzes were done to determine the effect of hypoxia on the expression of factors important to NK cell function. RESULTS: While HD NK cell cytolytic abilities were markedly and significantly impaired under hypoxic conditions, haNK cells maintained killing capacity under hypoxic conditions. NK killing, serial killing and ADCC were maintained under hypoxia in haNK cells. IL-2 has been previously implicated in serial killing and perforin regeneration and thus the endogenous IL-2 produced by haNK cells is likely a driver of the maintained killing capacity of haNK cells under hypoxic conditions. Activation of signal transducer and activator of transcription 3 (STAT3) is not seen in haNKs under hypoxia but is significant in HD NK cells. Pharmaceutical activation of STAT3 in haNKs led to reduced killing, implicating active STAT3 in reduced NK cell function. CONCLUSIONS: In contrast to HD NK cells, haNK cells are resistant to acute hypoxia. The potent cytolytic function of haNK cells was maintained in an environment comparable to what would be encountered in a tumor. The data presented here provide an additional mechanism of action for haNK cells that are currently being evaluated in clinical trials for several tumor types.
Assuntos
Hipóxia Celular/imunologia , Células Matadoras Naturais/metabolismo , Proteômica/métodos , Linhagem Celular Tumoral , HumanosRESUMO
Failed T cell-based immunotherapies in the presence of genomic alterations in antigen presentations pathways may be overcome by NK cell-based immunotherapy. This approach may still be limited by the presence of immunosuppressive myeloid populations. Here, we demonstrate that NK cells (haNKs) engineered to express a PD-L1 chimeric antigen receptor (CAR) haNKs killed a panel of human and murine head and neck cancer cells at low effector-to-target ratios in a PD-L1-dependent fashion. Treatment of syngeneic tumors resulted in CD8 and PD-L1-dependent tumor rejection or growth inhibition and a reduction in myeloid cells endogenously expressing high levels of PD-L1. Treatment of xenograft tumors resulted in PD-L1-dependent tumor growth inhibition. PD-L1 CAR haNKs reduced levels of macrophages and other myeloid cells endogenously expressing high PD-L1 in peripheral blood from patients with head and neck cancer. The clinical study of PD-L1 CAR haNKs is warranted.
Assuntos
Antígeno B7-H1/metabolismo , Células Matadoras Naturais/fisiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Receptores de Antígenos Quiméricos/metabolismoRESUMO
Very late antigen-4 (VLA-4; also known as integrin α4ß1) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radionuclide therapy (TRT). 177Lu-DOTA-PEG4-LLP2A (177Lu-LLP2A) is a TRT that shows high affinity for VLA-4 and high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of 177Lu-LLP2A, alone and combined with immune checkpoint inhibitors (ICIs) (anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies), in B16F10 tumor-bearing mice. Methods: Tumor cells (1 × 106) were implanted subcutaneously in C57BL/6 mice. After 8-10 d, the mice were randomized into 8 groups. 177Lu-LLP2A was injected intravenously on day 8 or 9 (single dose), and ICI antibodies were administered intraperitoneally in 3 doses. Tumor growth was monitored over time via calipers. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3-scrambled LLP2A was injected in tumor-bearing mice, and tumors were collected 4 h after injection and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results: TRT alone showed efficacy comparable to the dual-ICI anti-PD-1 + anti-CTLA-4 or anti-PD-L1 + anti-CTLA-4, whereas TRT + ICIs significantly enhanced survival. TUNEL staining showed that the highest levels of apoptosis were in the TRT + ICI groups. In addition to targeting tumor cells, TRT also bound immune cells in the tumor microenvironment. Flow cytometry data showed that the tumors consisted of about 77% tumor cells and fibroblasts (CD45-negative/CD49d-positive) and about 23% immune cells (CD45-positive/CD49d-positive) and that immune cells expressed higher levels of VLA-4. Cy3-LLP2A and CD49d colocalized with macrophages (CD68), T cells (CD8, CD4), and B cells (CD19). Immunohistochemical analysis identified a significant colocalization of Cy3-LLP2A and CD68. Conclusion: Combination treatment with TRT + ICIs targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.
Assuntos
Integrina alfa4beta1/antagonistas & inibidores , Lutécio/uso terapêutico , Melanoma Experimental/imunologia , Melanoma Experimental/radioterapia , Radioimunoterapia/métodos , Radioisótopos/uso terapêutico , Compostos Radiofarmacêuticos/uso terapêutico , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular Tumoral , Dipeptídeos/química , Feminino , Radioisótopos de Gálio , Compostos Heterocíclicos com 1 Anel/química , Humanos , Lutécio/farmacocinética , Masculino , Melanoma Experimental/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Compostos de Fenilureia/química , Polietilenoglicóis/química , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Dosagem Radioterapêutica , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Dendritic cells (DC) play a pivotal role in the induction and regulation of immune responses. In cancer, DC-based vaccines have proven to be safe and to elicit protective and therapeutic immunological responses. Recently, we showed that specific mTORC2 (mechanistic target of rapamycin complex 2) deficiency in DC enhances their ability to promote Th1 and Th17 responses after LPS stimulation. In the present study, bone marrow-derived mTORC2-deficient (Rictor(-/-)) DC were evaluated as a therapeutic modality in the murine B16 melanoma model. Consistent with their pro-inflammatory profile (enhanced IL-12p70 production and low PD-L1 expression versus control DC), intratumoral (i.t.) injection of LPS-activated Rictor(-/-) DC slowed B16 melanoma growth markedly in WT C57BL/6 recipient mice. This antitumor effect was abrogated when Rictor(-/-) DC were injected i.t. into B16-bearing Rag(-/-) mice, and also after selective CD8(+) T cell depletion in wild-type hosts in vivo, indicating that CD8(+) T cells were the principal regulators of tumor growth after Rictor(-/-) DC injection. I.t. administration of Rictor(-/-) DC also reduced the frequency of myeloid-derived suppressor cells within tumors, and enhanced numbers of IFNγ(+) and granzyme-B(+) cytotoxic CD8(+) T cells both in the spleens and tumors of treated animals. These data suggest that selective inhibition of mTORC2 activity in activated DC augments their pro-inflammatory and T cell stimulatory profile, in association with their enhanced capacity to promote protective CD8(+) T cell responses in vivo, leading to slowed B16 melanoma progression. These novel findings may contribute to the design of more effective DC-based vaccines for cancer immunotherapy.