Very late antigen-4 (VLA-4; also known as integrin α
β
) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radionuclide ...therapy (TRT).
Lu-DOTA-PEG
-LLP2A (
Lu-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
Lu-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.
Tumor cells (1 × 10
) were implanted subcutaneously in C57BL/6 mice. After 8-10 d, the mice were randomized into 8 groups.
Lu-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.
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.
Combination treatment with TRT + ICIs targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.
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.
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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NK-92 cells engineered to express high-affinity CD16 and/or chimeric antigen receptors are potential allogeneic off-the-shelf cell therapy products that show a favorable safety profile based on preliminary results of phase 1 studies.
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 pre-malignant 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 vs 3.2 in control mice injected with PBS; 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 pre-malignant OSCC lesions for accelerated tumor progression in concert with reduced immune cell migration to the tumor.
BackgroundAlthough 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.MethodsNanoString, 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.ResultsWe 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.ConclusionThese 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.
BackgroundDifferent 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.MethodsA 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.ResultsThe 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.ConclusionThe 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.
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.
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-β 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.
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.
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 TGFβ) 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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