Lipid-Encapsulated mRNAs Encoding Complex Fusion Proteins Potentiate Antitumor Immune Responses.

Lipid nanoparticle (LNP)-encapsulated mRNA has been used for in vivo production of several secreted protein classes, such as IgG, and has enabled the development of personalized vaccines in oncology. Establishing the feasibility of delivering complex multispecific modalities that require higher-order structures important for their function could help expand the use of mRNA/LNP biologic formulations. Here, we evaluated whether in vivo administration of mRNA/LNP formulations of SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT could achieve oligomerization and extend exposure, on-target activity, and antitumor responses comparable with that of the corresponding recombinant fusion proteins. Intravenous infusion of the formulated LNP-encapsulated mRNAs led to rapid and sustained production of functional hexameric proteins in vivo, which increased the overall exposure relative to the recombinant protein controls by ∼28 to 140 fold over 96 hours. High concentrations of the mRNA-encoded proteins were also observed in secondary lymphoid organs and within implanted tumors, with protein concentrations in tumors up to 134-fold greater than with the recombinant protein controls 24 hours after treatment. In addition, SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT mRNAs induced a greater increase in antigen-specific CD8+ T cells in the tumors. These mRNA/LNP formulations were well tolerated and led to a rapid increase in serum and intratumoral IL2, delayed tumor growth, extended survival, and outperformed the activities of benchmark mAb controls. Furthermore, the mRNA/LNPs demonstrated improved efficacy in combination with anti-PD-L1 relative to the recombinant fusion proteins. These data support the delivery of complex oligomeric biologics as mRNA/LNP formulations, where high therapeutic expression and exposure could translate into improved patient outcomes. SIGNIFICANCE: Lipid nanoparticle-encapsulated mRNA can efficiently encode complex fusion proteins encompassing immune checkpoint blockers and costimulators that functionally oligomerize in vivo with extended pharmacokinetics and durable exposure to induce potent antitumor immunity.

Shining a LIGHT on myeloid cell targeted immunotherapy.

Despite over a decade of clinical trials combining inhibition of emerging checkpoints with a PD-1/L1 inhibitor backbone, meaningful survival benefits have not been shown in PD-1/L1 inhibitor resistant or refractory solid tumours, particularly tumours dominated by a myelosuppressive microenvironment. Achieving durable anti-tumour immunity will therefore likely require combination of adaptive and innate immune stimulation, myeloid repolarisation, enhanced APC activation and antigen processing/presentation, lifting of the CD47/SIRPα (Cluster of Differentiation 47/signal regulatory protein alpha) 'do not eat me' signal, provision of an apoptotic 'pro-eat me' or 'find me' signal, and blockade of immune checkpoints. The importance of effectively targeting mLILRB2 and SIRPAyeloid cells to achieve improved response rates has recently been emphasised, given myeloid cells are abundant in the tumour microenvironment of most solid tumours. TNFSF14, or LIGHT, is a tumour necrosis superfamily ligand with a broad range of adaptive and innate immune activities, including (1) myeloid cell activation through Lymphotoxin Beta Receptor (LTßR), (2) T/NK (T cell and natural killer cell) induced anti-tumour immune activity through Herpes virus entry mediator (HVEM), (3) potentiation of proinflammatory cytokine/chemokine secretion through LTßR on tumour stromal cells, (4) direct induction of tumour cell apoptosis in vitro, and (5) the reorganisation of lymphatic tissue architecture, including within the tumour microenvironment (TME), by promoting high endothelial venule (HEV) formation and induction of tertiary lymphoid structures. LTBR (Lymphotoxin beta receptor) and HVEM rank highly amongst a range of costimulatory receptors in solid tumours, which raises interest in considering how LIGHT-mediated costimulation may be distinct from a growing list of immunotherapy targets which have failed to provide survival benefit as monotherapy or in combination with PD-1 inhibitors, particularly in the checkpoint acquired resistant setting.

LIGHT (TNFSF14) Costimulation Enhances Myeloid Cell Activation and Antitumor Immunity in the Setting of PD-1/PD-L1 and TIGIT Checkpoint Blockade.

Coinhibition of TIGIT (T cell immunoreceptor with Ig and ITIM domains) and PD-1/PD-L1 (PD-1/L1) may improve response rates compared with monotherapy PD-1/L1 blockade in checkpoint naive non-small cell lung cancer with PD-L1 expression >50%. TIGIT mAbs with an effector-competent Fc can induce myeloid cell activation, and some have demonstrated effector T cell depletion, which carries a clinical liability of unknown significance. TIGIT Ab blockade translates to antitumor activity by enabling PVR signaling through CD226 (DNAM-1), which can be directly inhibited by PD-1. Furthermore, DNAM-1 is downregulated on tumor-infiltrating lymphocytes (TILs) in advanced and checkpoint inhibition-resistant cancers. Therefore, broadening clinical responses from TIGIT blockade into PD-L1low or checkpoint inhibition-resistant tumors, may be induced by immune costimulation that operates independently from PD-1/L1 inhibition. TNFSF14 (LIGHT) was identified through genomic screens, in vitro functional analysis, and immune profiling of TILs as a TNF ligand that could provide broad immune activation. Accordingly, murine and human bifunctional fusion proteins were engineered linking the extracellular domain of TIGIT to the extracellular domain of LIGHT, yielding TIGIT-Fc-LIGHT. TIGIT competitively inhibited binding to all PVR ligands. LIGHT directly activated myeloid cells through interactions with LTßR (lymphotoxin ß receptor), without the requirement for a competent Fc domain to engage Fcγ receptors. LIGHT costimulated CD8+ T and NK cells through HVEM (herpes virus entry mediator A). Importantly, HVEM was more widely expressed than DNAM-1 on T memory stem cells and TILs across a range of tumor types. Taken together, the mechanisms of TIGIT-Fc-LIGHT promoted strong antitumor activity in preclinical tumor models of primary and acquired resistance to PD-1 blockade, suggesting that immune costimulation mediated by LIGHT may broaden the clinical utility of TIGIT blockade.

IL26, a Noncanonical Mediator of DNA Inflammatory Stimulation, Promotes TNBC Engraftment and Progression in Association with Neutrophils.

IL26 is a unique amphipathic member of the IL10 family of cytokines that participates in inflammatory signaling through a canonical receptor pathway. It also directly binds DNA to facilitate cellular transduction and intracellular inflammatory signaling. Although IL26 has almost no described role in cancer, our in vivo screen of inflammatory and cytokine pathway genes revealed IL26 to be one of the most significant inflammatory mediators of mammary engraftment and lung metastatic growth in triple-negative breast cancer (TNBC). Examination of human breast cancers demonstrated elevated IL26 transcripts in TNBC specimens, specifically in tumor cells as well as in Th17 CD4+ T cells within clinical TNBC specimens. IL26 did not have an autocrine effect on human TNBC cells, but rather its effect on engraftment and growth in vivo required neutrophils. IL26 enhanced mouse-derived DNA induction of inflammatory cytokines, which were collectively important for mammary and metastatic lung engraftment. To neutralize this effect, we developed a novel IL26 vaccine to stimulate antibody production and suppress IL26-enhanced engraftment in vivo, suggesting that targeting this inflammatory amplifier could be a unique means to control cancer-promoting inflammation in TNBC and other autoimmune diseases. Thus, we identified IL26 as a novel key modulator of TNBC metastasis and a potential therapeutic target in TNBC as well as other diseases reliant upon IL26-mediated inflammatory stimulation. SIGNIFICANCE: These findings identify IL26 as a unique, clinically relevant, inflammatory amplifier that enhances TNBC engraftment and dissemination in association with neutrophils, which has potential as a therapeutic target. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/15/3088/F1.large.jpg.

CD40 Enhances Type I Interferon Responses Downstream of CD47 Blockade, Bridging Innate and Adaptive Immunity.

Disrupting the binding of CD47 to SIRPα has emerged as a promising immunotherapeutic strategy for advanced cancers by potentiating antibody-dependent cellular phagocytosis (ADCP) of targeted antibodies. Preclinically, CD47/SIRPα blockade induces antitumor activity by increasing the phagocytosis of tumor cells by macrophages and enhancing the cross-presentation of tumor antigens to CD8+ T cells by dendritic cells; both of these processes are potentiated by CD40 signaling. Here we generated a novel, two-sided fusion protein incorporating the extracellular domains of SIRPα and CD40L, adjoined by a central Fc domain, termed SIRPα-Fc-CD40L. SIRPα-Fc-CD40L bound CD47 and CD40 with high affinity and activated CD40 signaling in the absence of Fc receptor cross-linking. No evidence of hemolysis, hemagglutination, or thrombocytopenia was observed in vitro or in cynomolgus macaques. Murine SIRPα-Fc-CD40L outperformed CD47 blocking and CD40 agonist antibodies in murine CT26 tumor models and synergized with immune checkpoint blockade of PD-1 and CTLA4. SIRPα-Fc-CD40L activated a type I interferon response in macrophages and potentiated the activity of ADCP-competent targeted antibodies both in vitro and in vivo These data illustrated that whereas CD47/SIRPα inhibition could potentiate tumor cell phagocytosis, CD40-mediated activation of a type I interferon response provided a bridge between macrophage- and T-cell-mediated immunity that significantly enhanced durable tumor control and rejection.

CD47 blockade augmentation of trastuzumab antitumor efficacy dependent on antibody-dependent cellular phagocytosis.

The HER2-specific monoclonal antibody (mAb), trastuzumab, has been the mainstay of therapy for HER2+ breast cancer (BC) for approximately 20 years. However, its therapeutic mechanism of action (MOA) remains unclear, with antitumor responses to trastuzumab remaining heterogeneous and metastatic HER2+ BC remaining incurable. Consequently, understanding its MOA could enable rational strategies to enhance its efficacy. Using both murine and human versions of trastuzumab, we found its antitumor activity dependent on Fcγ receptor stimulation of tumor-associated macrophages (TAMs) and antibody-dependent cellular phagocytosis (ADCP), but not cellular cytotoxicity (ADCC). Trastuzumab also stimulated TAM activation and expansion, but did not require adaptive immunity, natural killer cells, and/or neutrophils. Moreover, inhibition of the innate immune ADCP checkpoint, CD47, significantly enhanced trastuzumab-mediated ADCP and TAM expansion and activation, resulting in the emergence of a unique hyperphagocytic macrophage population, improved antitumor responses, and prolonged survival. In addition, we found that tumor-associated CD47 expression was inversely associated with survival in HER2+ BC patients and that human HER2+ BC xenografts treated with trastuzumab plus CD47 inhibition underwent complete tumor regression. Collectively, our study identifies trastuzumab-mediated ADCP as an important antitumor MOA that may be clinically enabled by CD47 blockade to augment therapeutic efficacy.

An unbiased in vivo functional genomics screening approach in mice identifies novel tumor cell-based regulators of immune rejection.

The clinical successes of immune checkpoint therapies for cancer make it important to identify mechanisms of resistance to anti-tumor immune responses. Numerous resistance mechanisms have been identified employing studies of single genes or pathways, thereby parsing the tumor microenvironment complexity into tractable pieces. However, this limits the potential for novel gene discovery to in vivo immune attack. To address this challenge, we developed an unbiased in vivo genome-wide RNAi screening platform that leverages host immune selection in strains of immune-competent and immunodeficient mice to select for tumor cell-based genes that regulate in vivo sensitivity to immune attack. Utilizing this approach in a syngeneic triple-negative breast cancer (TNBC) model, we identified 709 genes that selectively regulated adaptive anti-tumor immunity and focused on five genes (CD47, TGFß1, Sgpl1, Tex9 and Pex14) with the greatest impact. We validated the mechanisms that underlie the immune-related effects of expression of these genes in different TNBC lines, as well as tandem synergistic interactions. Furthermore, we demonstrate the impact of different genes with previously unknown immune functions (Tex9 and Pex14) on anti-tumor immunity. Thus, this innovative approach has utility in identifying unknown tumor-specific regulators of immune recognition in multiple settings to reveal novel targets for future immunotherapies.

Antibody-based immunotherapy of cancer.

By targeting surface antigens expressed on tumor cells, monoclonal antibodies have demonstrated efficacy as cancer therapeutics. Recent successful antibody-based strategies have focused on enhancing antitumor immune responses by targeting immune cells, irrespective of tumor antigens. We discuss these innovative strategies and propose how they will impact the future of antibody-based cancer therapy.

Monoclonal antibodies for the treatment of cancer.

Over the past decade, the clinical utility of monoclonal antibodies has been realized and antibodies are now a mainstay for the treatment of cancer. Antibodies have the unique capacity to target and kill tumor cells while simultaneously activating immune effectors to kill tumor cells through the complement cascade or antibody-dependent cellular cytotoxicity (ADCC). This multifaceted mechanism of action combined with target specificity underlies the capacity of antibodies to elicit anti-tumor responses while minimizing the frequency and magnitude of adverse events. This review will focus on mechanisms of action, clinical applications and putative mechanisms of resistance to monoclonal antibody therapy in the context of cancer.

Capillary arterialization requires the bone-marrow-derived cell (BMC)-specific expression of chemokine (C-C motif) receptor-2, but BMCs do not transdifferentiate into microvascular smooth muscle.

Chemokine (C-C motif) receptor-2 (CCR2) regulates arteriogenesis and angiogenesis, facilitating the MCP-1-dependent recruitment of growth factor-secreting bone marrow-derived cells (BMCs). Here, we tested the hypothesis that the BMC-specific expression of CCR2 is also required for new arteriole formation via capillary arterialization. Following non-ischemic saphenous artery occlusion, we measured the following in gracilis muscles: monocyte chemotactic protein-1 (MCP-1) in wild-type (WT) C57Bl/6J mice by ELISA, and capillary arterialization in WT-WT and CCR2(-/-)-WT (donor-host) bone marrow chimeric mice, as well as BMC transdifferentiation in EGFP(+)-WT mice, by smooth muscle (SM) alpha-actin immunochemistry. MCP-1 levels were significantly elevated 1 day after occlusion in WT mice. In WT-WT mice at day 7, compared to sham controls, arterial occlusion induced a 34% increase in arteriole length density, a 46% increase in SM alpha-actin(+) vessels, and a 45% increase in the fraction of vessels coated with SM alpha-actin, indicating significant capillary arterialization. However, in CCR2(-/-)-WT mice, no differences were observed between arterial occlusion and sham surgery. In EGFP(+)-WT mice, EGFP and SM alpha-actin never colocalized. We conclude that BMC-specific CCR2 expression is required for skeletal muscle capillary arterialization following arterial occlusion; however, BMCs do not transdifferentiate into smooth muscle.

Bone marrow-derived cell-specific chemokine (C-C motif) receptor-2 expression is required for arteriolar remodeling.

OBJECTIVE: Bone marrow-derived cells (BMCs) and inflammatory chemokine receptors regulate arteriogenesis and angiogenesis. Here, we tested whether arteriolar remodeling in response to an inflammatory stimulus is dependent on BMC-specific chemokine (C-C motif) receptor 2 (CCR2) expression and whether this response involves BMC transdifferentiation into smooth muscle. METHODS AND RESULTS: Dorsal skinfold window chambers were implanted into C57Bl/6 wild-type (WT) mice, as well as the following bone marrow chimeras (donor-host): WT-WT, CCR2(-/-)-WT, WT-CCR2(-/-), and EGFP(+)-WT. One day after implantation, tissue MCP-1 levels rose from "undetectable" to 463 pg/mg, and the number of EGFP(+) cells increased more than 4-fold, indicating marked inflammation. A 66% (28 microm) increase in maximum arteriolar diameter was observed over 7 days in WT-WT mice. This arteriolar remodeling response was completely abolished in CCR2(-/-)-WT mice but largely rescued in WT-CCR2(-/-) mice. EGFP(+) BMCs were numerous throughout the tissue, but we found no evidence that EGFP(+) BMCs transdifferentiate into smooth muscle, based on examination of >800 arterioles and venules. CONCLUSIONS: BMC-specific CCR2 expression is required for injury/inflammation-associated arteriolar remodeling, but this response is not characterized by the differentiation of BMCs into smooth muscle.

Influence of poly(D,L-lactic-co-glycolic acid) microsphere degradation on arteriolar remodeling in the mouse dorsal skinfold window chamber.

Poly(D,L-lactic-co-glycolic acid) (PLGA) is a biodegradable polymer that is widely used for drug delivery. However, the degradation of PLGA alters the local microenvironment and may influence tissue structure and/or function. Here, we studied whether PLGA degradation affects the structure of the arteriolar microcirculation through arteriogenic expansion of maximum lumenal diameters and/or the formation of new smooth muscle-coated vessels. Single microspheres comprised of 50:50 PLGA (521 +/- 52.7 microm diameter), 50:50 PLGA with bovine serum albumin (BSA) (547 +/- 62.2 microm), 85:15 PLGA (474 +/- 52.6 microm), or 85:15 PLGA with BSA (469 +/- 57.2 microm) were implanted into mouse dorsal skinfold window chambers, and longitudinal arteriolar diameter measurements were made in the presence of a vasodilator (10(-4)M adenosine) over 7 days. At the end of the 7-day period, the length density of all smooth muscle-coated microvessels was also determined. Implantation of the window chamber alone elicited a 22% increase in maximum arteriolar diameter. However, the addition of 85:15 and 50:50 PLGA microspheres, bearing either BSA or no protein, elicited a significant enhancement of this arteriogenic response, with final maximum arteriolar diameters ranging from 36 to 46% more than their original size. Interestingly, the influence of PLGA degradation on microvascular structure was limited to lumenal arteriolar expansion, as we observed no significant differences in length density of smooth muscle-coated microvessels. We conclude that the degradation of PLGA microspheres may elicit an arteriogenic response in subcutaneous tissue in the dorsal skinfold window chamber; however, it has no apparent effect on the total length of smooth muscle-coated microvasculature.