A chimeric antigen receptor uniquely recognizing MICA/B stress proteins provides an effective approach to target solid tumors.

BACKGROUND: The advent of chimeric antigen receptor (CAR) T cell therapies has transformed the treatment of hematological malignancies; however, broader therapeutic success of CAR T cells has been limited in solid tumors because of their frequently heterogeneous composition. Stress proteins in the MICA and MICB (MICA/B) family are broadly expressed by tumor cells following DNA damage but are rapidly shed to evade immune detection. METHODS: We have developed a novel CAR targeting the conserved α3 domain of MICA/B (3MICA/B CAR) and incorporated it into a multiplexed-engineered induced pluripotent stem cell (iPSC)-derived natural killer (NK) cell (3MICA/B CAR iNK) that expressed a shedding-resistant form of the CD16 Fc receptor to enable tumor recognition through two major targeting receptors. FINDINGS: We demonstrated that 3MICA/B CAR mitigates MICA/B shedding and inhibition via soluble MICA/B while simultaneously exhibiting antigen-specific anti-tumor reactivity across an expansive library of human cancer cell lines. Pre-clinical assessment of 3MICA/B CAR iNK cells demonstrated potent antigen-specific in vivo cytolytic activity against both solid and hematological xenograft models, which was further enhanced in combination with tumor-targeted therapeutic antibodies that activate the CD16 Fc receptor. CONCLUSIONS: Our work demonstrated 3MICA/B CAR iNK cells to be a promising multi-antigen-targeting cancer immunotherapy approach intended for solid tumors. FUNDING: Funded by Fate Therapeutics and NIH (R01CA238039).

MICA/B antibody induces macrophage-mediated immunity against acute myeloid leukemia.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem and progenitor cell malignancy characterized by poor clinical outcomes. Major histocompatibility complex class I polypeptide-related sequence A and B (MICA/B) are stress proteins expressed by cancer cells, and antibody-mediated inhibition of MICA/B shedding represents a novel approach to stimulate immunity against cancers. We found that the MICA/B antibody 7C6 potently inhibits the outgrowth of AML in 2 models in immunocompetent mice. Macrophages were essential for therapeutic efficacy, and 7C6 triggered antibody-dependent phagocytosis of AML cells. Furthermore, we found that romidepsin, a selective histone deacetylase inhibitor, increased MICB messenger RNA in AML cells and enabled subsequent stabilization of the translated protein by 7C6. This drug combination substantially increased surface MICA/B expression in a human AML line, pluripotent stem cell-derived AML blasts and leukemia stem cells, as well as primary cells from 3 untreated patients with AML. Human macrophages phagocytosed AML cells following treatment with 7C6 and romidepsin, and the combination therapy lowered leukemia burden in a humanized model of AML. Therefore, inhibition of MICA/B shedding promotes macrophage-driven immunity against AML via Fc receptor signaling and synergizes with an epigenetic regulator. These results provide the rationale for the clinical testing of this innovative immunotherapeutic approach for the treatment of AML.

NKG2D and MICA/B shedding: a 'tag game' between NK cells and malignant cells.

Natural killer (NK) cells are innate lymphocytes with cytotoxic functions and recognise target cells with the NK group 2D (NKG2D) receptor. Tumor cells are marked for NK-cell-mediated destruction upon expression of MICA and MICB (MICA/B), which are NKG2D ligands upregulated by many human cancers in response to cellular stress pathways associated with malignant transformation such as DNA damage and accumulation of misfolded proteins. However, MICA/B proteins are downregulated by tumor cells via intriguing molecular mechanisms, such as post-translational modifications in which the external domains of MICA/B are proteolytically cleaved by surface proteases and shed into the extracellular space. MICA/B shedding by cancer cells causes effective escape from NKG2D recognition and allows the development of cancers. Patients frequently have increased concentrations of soluble MICA/B molecules shed in the blood plasmas and sera, thus indicating that MICA/B shedding is a therapeutic target in immune-oncology. Here, we review the clinical significance of MICA/B shedding in cancer as well as novel immunotherapeutic approaches that aim to restore NKG2D-mediated surveillance. We also briefly discuss potential roles of MICA/B shedding beyond oncology, such as in viral infections and immune tolerance. This review will help to inform the future developments of NKG2D-based immunotherapies.

Inhibition of MICA and MICB Shedding Elicits NK-Cell-Mediated Immunity against Tumors Resistant to Cytotoxic T Cells.

Resistance to cytotoxic T cells is frequently mediated by loss of MHC class I expression or IFNγ signaling in tumor cells, such as mutations of B2M or JAK1 genes. Natural killer (NK) cells could potentially target such resistant tumors, but suitable NK-cell-based strategies remain to be developed. We hypothesized that such tumors could be targeted by NK cells if sufficient activating signals were provided. Human tumors frequently express the MICA and MICB ligands of the activating NKG2D receptor, but proteolytic shedding of MICA/B represents an important immune evasion mechanism in many human cancers. We showed that B2M- and JAK1-deficient metastases were targeted by NK cells following treatment with a mAb that blocks MICA/B shedding. We also demonstrated that the FDA-approved HDAC inhibitor panobinostat and a MICA/B antibody acted synergistically to enhance MICA/B surface expression on tumor cells. The HDAC inhibitor enhanced MICA/B gene expression, whereas the MICA/B antibody stabilized the synthesized protein on the cell surface. The combination of panobinostat and the MICA/B antibody reduced the number of pulmonary metastases formed by a human melanoma cell line in NOD/SCID gamma mice reconstituted with human NK cells. NK-cell-mediated immunity induced by a mAb specific for MICA/B, therefore, provides an opportunity to target tumors with mutations that render them resistant to cytotoxic T cells.

NK Cell Interaction With Platelets and Myeloid Cells in the Tumor Milieu.

Natural killer (NK) cells recognize and kill tumor cells via germ-line encoded receptors and polarized degranulation of cytotoxic molecules, respectively. As such, NK cells help to inhibit the development of cancers. The activating receptor NKG2D induces NK cell-mediated killing of metastasizing tumor cells by recognition of the stress-induced ligands MICA, MICB, and ULBP1-6. However, platelets enable escape from this immune surveillance mechanism by obstructing the interactions between NK cells and tumor cells or by cleaving the stress-induced ligands. It is also being increasingly appreciated that NK cells play additional roles in cancer immunity, including chemokine-mediated recruitment of antigen presenting cells in the tumor microenvironment that is followed by generation of adaptive immunity. However, the NK cell interplays with dendritic cells, and macrophages are extremely complex and involve molecular interactions via NKG2D and cytokine receptors. Specifically, NKG2D-mediated chronic interaction between NK cells and tumor-infiltrating macrophages causes immune suppression by differentiating NK cells toward a dysfunctional state. Here we discuss the underlying mechanisms of NK cell control by platelets and myeloid cells with focus on NKG2D and its ligands, and provide a timely perspective on how to harness these pathways with novel immunotherapeutic approaches.

Radiotherapy induces responses of lung cancer to CTLA-4 blockade.

Focal radiation therapy enhances systemic responses to anti-CTLA-4 antibodies in preclinical studies and in some patients with melanoma1-3, but its efficacy in inducing systemic responses (abscopal responses) against tumors unresponsive to CTLA-4 blockade remained uncertain. Radiation therapy promotes the activation of anti-tumor T cells, an effect dependent on type I interferon induction in the irradiated tumor4-6. The latter is essential for achieving abscopal responses in murine cancers6. The mechanisms underlying abscopal responses in patients treated with radiation therapy and CTLA-4 blockade remain unclear. Here we report that radiation therapy and CTLA-4 blockade induced systemic anti-tumor T cells in chemo-refractory metastatic non-small-cell lung cancer (NSCLC), where anti-CTLA-4 antibodies had failed to demonstrate significant efficacy alone or in combination with chemotherapy7,8. Objective responses were observed in 18% of enrolled patients, and 31% had disease control. Increased serum interferon-ß after radiation and early dynamic changes of blood T cell clones were the strongest response predictors, confirming preclinical mechanistic data. Functional analysis in one responding patient showed the rapid in vivo expansion of CD8 T cells recognizing a neoantigen encoded in a gene upregulated by radiation, supporting the hypothesis that one explanation for the abscopal response is radiation-induced exposure of immunogenic mutations to the immune system.

Rapid CLIP dissociation from MHC II promotes an unusual antigen presentation pathway in autoimmunity.

A number of autoimmunity-associated MHC class II proteins interact only weakly with the invariant chain-derived class II-associated invariant chain peptide (CLIP). CLIP dissociates rapidly from I-Ag7 even in the absence of DM, and this property is related to the type 1 diabetes-associated ß57 polymorphism. We generated knock-in non-obese diabetic (NOD) mice with a single amino acid change in the CLIP segment of the invariant chain in order to moderately slow CLIP dissociation from I-Ag7 These knock-in mice had a significantly reduced incidence of spontaneous type 1 diabetes and diminished islet infiltration by CD4 T cells, in particular T cells specific for fusion peptides generated by covalent linkage of proteolytic fragments within ß cell secretory granules. Rapid CLIP dissociation enhanced the presentation of such extracellular peptides, thus bypassing the conventional MHC class II antigen-processing pathway. Autoimmunity-associated MHC class II polymorphisms therefore not only modify binding of self-peptides, but also alter the biochemistry of peptide acquisition.

Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity.

MICA and MICB are expressed by many human cancers as a result of cellular stress, and can tag cells for elimination by cytotoxic lymphocytes through natural killer group 2D (NKG2D) receptor activation. However, tumors evade this immune recognition pathway through proteolytic shedding of MICA and MICB proteins. We rationally designed antibodies targeting the MICA α3 domain, the site of proteolytic shedding, and found that these antibodies prevented loss of cell surface MICA and MICB by human cancer cells. These antibodies inhibited tumor growth in multiple fully immunocompetent mouse models and reduced human melanoma metastases in a humanized mouse model. Antitumor immunity was mediated mainly by natural killer (NK) cells through activation of NKG2D and CD16 Fc receptors. This approach prevents the loss of important immunostimulatory ligands by human cancers and reactivates antitumor immunity.

A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing.

Many human cancers are resistant to immunotherapy, for reasons that are poorly understood. We used a genome-scale CRISPR-Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of antitumor immunity. Inactivation of >100 genes-including Pbrm1, Arid2, and Brd7, which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex-sensitized mouse B16F10 melanoma cells to killing by T cells. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T cell cytotoxicity genes, and Pbrm1-deficient murine melanomas were more strongly infiltrated by cytotoxic T cells.

Inhalation therapy with M1 inhibits experimental melanoma development and metastases in mice.

BACKGROUND: M1 is a homeopathic medicine with immunostimulatory properties used mainly by cancer patients to complement current therapies. Metastatic melanoma is a skin-originated form of cancer without a single therapy able to produce high rate and sustained responses, which attracts the use of complementary therapies such as M1. However, M1's anti-melanoma effects remain to be pre-clinically demonstrated. Therefore in the present work, we utilized a pulmonary metastatic melanoma model and a subcutaneous melanoma growth model to investigate the potential benefits of treatment with M1. METHODS: C57BL/6 mice were injected intravenously or subcutaneously with B16F10 mouse melanoma cells. After 24 h, mice were treated with either M1 or vehicle (water) for 14 days, euthanized and harvested for multi-parameter pulmonary and tumor analyses. RESULTS: Mice treated with M1 had significantly lower tumor burden in the lungs and subcutaneous tissue than control mice. Furthermore, tumors were impaired in proliferation and tumor related angiogenesis by the inhibition of myeloid derived suppressor cells (MDSC) positive for angiotensin II type 1 receptor (AT1R). CONCLUSION: Altogether these data suggest M1 is an efficient candidate for melanoma therapy to be considered for future clinic studies as this study is the first supporting the idea that melanoma patients may benefit with the treatment. The treatment with M1 provides advantages considering the highly-diluted properties and a cost effective alternative to costly chemotherapeutic approaches with, if any, lower toxicity.

Immunosurveillance and therapy of multiple myeloma are CD226 dependent.

Multiple myeloma (MM) is an age-dependent hematological malignancy. Evaluation of immune interactions that drive MM relies on in vitro experiments that do not reflect the complex cellular stroma involved in MM pathogenesis. Here we used Vk*MYC transgenic mice, which spontaneously develop MM, and demonstrated that the immune system plays a critical role in the control of MM progression and the response to treatment. We monitored Vk*MYC mice that had been crossed with Cd226 mutant mice over a period of 3 years and found that CD226 limits spontaneous MM development. The CD226-dependent anti-myeloma immune response against transplanted Vk*MYC MM cells was mediated both by NK and CD8+ T cells through perforin and IFN-γ pathways. Moreover, CD226 expression was required for optimal antimyeloma efficacy of cyclophosphamide (CTX) and bortezomib (Btz), which are both standardly used to manage MM in patients. Activation of costimulatory receptor CD137 with mAb (4-1BB) exerted strong antimyeloma activity, while inhibition of coinhibitory receptors PD-1 and CTLA-4 had no effect. Taken together, the results of this study provide in vivo evidence that CD226 is important for MM immunosurveillance and indicate that specific immune components should be targeted for optimal MM treatment efficacy. As progressive immunosuppression associates with MM development, strategies aimed to increase immune functions may have important therapeutic implications in MM.

DNAM-1 expression marks an alternative program of NK cell maturation.

Natural killer (NK) cells comprise a heterogeneous population of cells important for pathogen defense and cancer surveillance. However, the functional significance of this diversity is not fully understood. Here, we demonstrate through transcriptional profiling and functional studies that the activating receptor DNAM-1 (CD226) identifies two distinct NK cell functional subsets: DNAM-1(+) and DNAM-1(-) NK cells. DNAM-1(+) NK cells produce high levels of inflammatory cytokines, have enhanced interleukin 15 signaling, and proliferate vigorously. By contrast, DNAM-1(-) NK cells that differentiate from DNAM-1(+) NK cells have greater expression of NK-cell-receptor-related genes and are higher producers of MIP1 chemokines. Collectively, our data reveal the existence of a functional program of NK cell maturation marked by DNAM-1 expression.

Natural killer cells are essential for the ability of BRAF inhibitors to control BRAFV600E-mutant metastatic melanoma.

BRAF(V600E) is a major oncogenic mutation found in approximately 50% of human melanoma that confers constitutive activation of the MAPK pathway and increased melanoma growth. Inhibition of BRAF(V600E) by oncogene targeting therapy increases overall survival of patients with melanoma, but is unable to produce many durable responses. Adaptive drug resistance remains the main limitation to BRAF(V600E) inhibitor clinical efficacy and immune-based strategies could be useful to overcome disease relapse. Tumor microenvironment greatly differs between visceral metastasis and primary cutaneous melanoma, and the mechanisms involved in the antimetastatic efficacy of BRAF(V600E) inhibitors remain to be determined. To address this question, we developed a metastatic BRAF(V600E)-mutant melanoma cell line and demonstrated that the antimetastatic properties of BRAF inhibitor PLX4720 (a research analogue of vemurafenib) require host natural killer (NK) cells and perforin. Indeed, PLX4720 not only directly limited BRAF(V600E)-induced tumor cell proliferation, but also affected NK cell functions. We showed that PLX4720 increases the phosphorylation of ERK1/2, CD69 expression, and proliferation of mouse NK cells in vitro. NK cell frequencies were significantly enhanced by PLX4720 specifically in the lungs of mice with BRAF(V600E) lung metastases. Furthermore, PLX4720 also increased human NK cell pERK1/2, CD69 expression, and IFNγ release in the context of anti-NKp30 and IL2 stimulation. Overall, this study supports the idea that additional NK cell-based immunotherapy (by checkpoint blockade or agonists or cytokines) may combine well with BRAF(V600E) inhibitor therapy to promote more durable responses in melanoma.