Positive allosteric modulation of CD11b as a novel therapeutic strategy against lung cancer

Lung cancer is one of the leading causes of cancer-related deaths in the United States. A major hurdle for improved therapies is immune suppression mediated by the tumor and its microenvironment. The lung tumor microenvironment (TME) contains large numbers of tumor-associated macrophages (TAMs), which suppress the adaptive immune response, increase neo-vascularization of the tumor, and provide pro-tumor factors to promote tumor growth. CD11b is highly expressed on myeloid cells, including TAMs, where it forms a heterodimeric integrin receptor with CD18 (known as CD11b/CD18, Mac-1, CR3, and αMβ2), and plays an important role in recruitment and biological functions of these cells, and is a validated therapeutic target. Here, we describe our pre-clinical studies targeting CD11b in the context of lung cancer, using pharmacologic and genetic approaches that work via positive allosteric modulation of CD11b function. GB1275 is a novel small molecule modulator of CD11b that is currently in Phase 1/2 clinical development. We assess GB1275 treatment effects on tumor growth and immune infiltrates in the murine Lewis Lung Carcinoma (LLC) syngeneic tumor model. Additionally, as an orthogonal approach to determine mechanisms of action, we utilize our recently developed novel CD11b knock-in (KI) mouse that constitutively expresses CD11b containing an activating isoleucine to glycine substitution at residue 332 in the ligand binding CD11b A-domain (I332G) that acts as a positive allosteric modulator of CD11b activity. We report that pharmacologic modulation of CD11b with GB1275 significantly reduces LLC tumor growth. CD11b KI mice similarly show significant reduction in both the size and rate of LLC tumor growth, as compared to WT mice, mimicking our observed treatment effects with GB1275. Tumor profiling revealed a significant reduction in TAM infiltration in GB1275-treated and in CD11b KI mice, increase in the ratio of M1/M2-like TAMs, and concomitant increase in cytotoxic T cells. The profiling also showed a significant decrease in CCL2 levels and a concomitant reduction in Ly6

Integrin CD11b activation drives anti-tumor innate immunity

Myeloid cells are recruited to damaged tissues where they can resolve infections and tumor growth or stimulate wound healing and tumor progression. Recruitment of these cells is regulated by integrins, a family of adhesion receptors that includes integrin CD11b. Here we report that, unexpectedly, integrin CD11b does not regulate myeloid cell recruitment to tumors but instead controls myeloid cell polarization and tumor growth. CD11b activation promotes pro-inflammatory macrophage polarization by stimulating expression of microRNA Let7a. In contrast, inhibition of CD11b prevents Let7a expression and induces cMyc expression, leading to immune suppressive macrophage polarization, vascular maturation, and accelerated tumor growth. Pharmacological activation of CD11b with a small molecule agonist, Leukadherin 1 (LA1), promotes pro-inflammatory macrophage polarization and suppresses tumor growth in animal models of murine and human cancer. These studies identify CD11b as negative regulator of immune suppression and a target for cancer immune therapy

Promoting CD8+ Memory for Cancer Immunotherapy using Aptamer-Targeted siRNA Therapeutics

Cancer is among the leading causes of death worldwide. According to the National Cancer Institute, in 2012, there were 8.2 million cancer-related deaths worldwide, 14 million new cases and the number of new cancer cases is expected to rise to 22 million within the next two decades. Currently, Standard therapies for most cancers include surgical removal, radiotherapy using ionizing radiation or chemotherapy using drugs such as doxorubicin etc. Although these therapies help control the disease severity, they lack specificity and elicit significant side effects through their targeting of normal tissues. These off-target effects range from blood disorders, hair loss, diarrhea and sometimes long-term damage to organs including lung, liver etc., thus affecting the patients’ lifestyle severely. In addition, these therapies are not curative, where patients eventually have recurrence of cancer. Immunotherapy for cancer is an attractive alternative as it has the potential to circumvent these pitfalls. The immune system is capable of recognizing cancer cells specifically, thus minimizing the off-target effects on healthy tissue. Immune system can also prevent recurrence of the disease due to its ability to generate long-term memory responses, thus conferring life-long protection. This feature of the immune system has helped in the eradication of deadly diseases such as small pox and can be harnessed against cancer to control the disease. However, immune response elicited against cancer even in response to vaccine or radiation is often weak and not curative. One approach to potentiate antitumor immunity is to enhance the long-term persistence of vaccine or radiation-induced anti-tumor CD8+ T cells. A characteristic CD8+ T cell response involves parallel generation of short-lived effector cells (SLEC) and memory precursor effector cells (MPEC) from naïve responding cells. Upon activation, about 95-98% of the T cells differentiate into SLECs, giving rise to immediate effectors, while only 2-5% differentiate into MPECs, giving rise to persistent memory subsets. The ability of these CD8+ memory subsets to confer host protection has been attributed to their enhanced proliferative capacity upon antigen re-encounter and their ability to mediate systemic immunity by residing in lymph nodes, helping to control the metastatic load in cancers. To this end, generation of these CD8+ memory subsets during a vaccine induced response, suggests an approach to mount an optimal anti-tumor immune response. Over the last decade, profiling of the transcriptional factors expressed by different T cell subsets upon activation has provided important insights into memory T cell differentiation. High levels of transcription factors Tcf-7, Eomes, Bcl-6 have been shown to favor MPECs while high levels of T-bet, Blimp-1 and mTOR have been shown to favor SLECs. Increased IL-2 signaling in activated CD8+ T cells, via the binding of IL-2 cytokine to CD25 (IL-2Ra), promotes the development of SLEC via the coordinated expression of intracellular mediators such as Blimp-1, whereas reduced IL-2 signaling was shown to favor the development of memory CD8+ T cells. Although blocking anti-CD25 antibodies can be used to reduce IL-2 signaling, systemic administration of CD25 antibodies could exert off-target effects such as interfering with the function of CD25 expressing CD4+Foxp3+ regulatory cells and therefore increase the risk of autoimmune pathology. it’s also desirable to achieve a modest reduction in the strength of IL-2 signaling in activated CD8+ T cells rather than a complete shut-down. To achieve this, we took advantage of RNA interference. We used siRNAs designed against CD25 mRNA to downregulate the levels of CD25 to achieve a decrease in IL-2 signaling. These CD25 siRNA were conjugated to a 4-1BB aptamer which targets activated CD8+ T cells. Aptamers are single-stranded DNA, RNA or peptide molecules capable of binding their target protein with equal or greater affinities than antibodies. The 4-1BB RNA aptamer binds 4-1BB, a Tumor Necrosis Factor Receptor that gets upregulated on CD8+ T cells upon TCR activation. 4-1BB targeted delivery of CD25 siRNAs downregulated levels of CD25 at mRNA and protein level. Using 4-1BB aptamer as a targeting agent was crucial in delivering these siRNAs to activated CD8+ T cells as no effect was observed when these siRNAs were conjugated to a scrambled aptamer. Downregulation of IL-2 signaling in activated CD8+ T cells enabled their differentiation into MPECs. This was evident from the change in transcriptional profile where activated CD8+ T cells treated with 4-1BB-CD25 had reduced levels of Blimp-1 and increased levels of Bcl-6 and Tcf-7. A higher proportion of 4-1BB-CD25 treated cells re-expressed CD62L and also exhibited improved response to IL-7 cytokine. Systemic administration of the 4-1BB aptamer-CD25 siRNA conjugate via tail vein injection downregulated CD25 mRNA only in 4-1BB activated CD8+ T cells. Treatment of mice bearing 4T-1 mammary carcinoma with the 4-1BB aptamer-CD25 siRNA conjugates enhanced the antitumor response of a cellular vaccine as well as that of local radiation therapy. To validate the approach of using 4-1BB targeted delivery of siRNAs to activated CD8+ T cells to enhance their persistence, we generated siRNAs against Axin-1. Axin-1 is the rate-limiting component of the b-catenin destruction complex. Reduction in Axin-1 levels decreases the activity of the destruction complex, thus turning on the WNT pathway, leading to activation of b-catenin/Tcf-7 complex and transcription of genes responsible for CD8+ memory generation. We tested the ability of 4-1BB aptamer targeted delivery of an Axin-1 siRNA to CD8+ T cells to enhance CD8+ T cell memory development and antitumor activity. Indicative of the generality of the approach, 4-1BB aptamer targeted reduction of Axin-1 in CD8+ T cells, enhanced persistence of activated CD8+ T cells, potentiating vaccine mediated anti-tumor immunity. This study shows that aptamer-targeted siRNA therapeutics can be used to specifically modulate the function of circulating CD8+ T cells, skewing their development into long-lasting memory CD8+ T cells, and thereby potentiate antitumor immunity

A clinically useful approach to enhance immunological memory and antitumor immunity

Persistence of vaccine-induced immune responses, not the initial magnitude, best correlates with protective antitumor immunity. In mice, oligonucleotide aptamer-targeted siRNA inhibition of mammalian target of rapamycin (mTOR) activity in activated CD8+ T cells promotes their differentiation into functionally competent memory cells leading to enhanced antitumor immunity, a protective effect superior to that of non-targeted administration of the mTOR inhibitor rapamycin

Abstract A23: Potentiating immunological memory in mice using aptamer targeted siRNA delivery to inhibit mediators of effector differentiation in CD8+ cytotoxic T lymphocytes

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR11) of the Conference Proceedings. Citation Format: Alexey Berezhnoy, Rajagopalan Anugraha, Thomas Malek, Eli Gilboa. Potentiating immunological memory in mice using aptamer targeted siRNA delivery to inhibit mediators of effector differentiation in CD8+ cytotoxic T lymphocytes. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A23.</jats:p

Abstract PR11: Potentiating immunological memory in mice using aptamer targeted siRNA delivery to inhibit mediators of effector differentiation in CD8+ cytotoxic T lymphocytes

Abstract Recent studies in mice, nonhuman primates, and clinical trials in human patients have emphasized the importance of the persistence of the vaccine-induced immune response, immunological memory, in mediating protective immunity against infectious diseases and cancer. Inhibition of mediators of effector differentiation like mTOR, Blimp-1, CD25, glycolysis, or GSK3β, using genetic means or whenever available pharmacological agents, not only prevented the accumulation of the short-lived effectors but also redirected the activated T cells to differentiate along the memory pathway. For example, pharmacological inhibition of mTOR, or GSK3β with rapamycin, or TSW119, respectively, led to the differentiation of antigen activated CD8+ T cells into long lasting memory cells that exhibited enhanced antiviral and antitumor immunity. Nevertheless, pharmacological agents often exhibit undesirable effects reflecting the broad distribution of their targets. For example, rapamycin inhibition of mTOR promotes the development of Treg, and GSK3β inhibition polarizes dendritic cells to a tolerogenic state, arguably counterproductive in the setting of vaccination. In addition, development of pharmacological agents to modulate the function of intracellular targets that are not accessible to antibodies (undruggable targets) is highly challenging, and their availability especially for clinical use is limited. We are developing a versatile, broadly applicable, and clinically feasible approach to promote the generation of memory T cell responses that addresses the main limitations of pharmacological agents. RNAi is used to downregulate intracellular mediators of effector differentiation, that are targeted to CD8+ T cells by conjugation to an oligonucleotide aptamer ligand. We have shown that a CD8+ T cell targeted 4-1BB aptamer-raptor siRNA conjugate administered to mice by tail vein injection downregulated mTORC1, while preserving mTORC2, activity in at least 60% of adoptively transferred OVA-specific transgenic OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced memory response in mice. Whereas the aptamer-raptor siRNA generated CTL exhibited normal cytotoxic effector functions and enhanced vaccine-induce protective antitumor immunity in both prophylactic and therapeutic tumor models, the rapamycin generated CTL were defective in their cytotoxic effector functions and failed to elicit protective immunity against a tumor challenge. Underscoring the lack of cell specificity of rapamycin action, and providing a potential mechanisms underlying the effector defect of rapamycin generated memory CD8+ T cells, dendritic cells from mice treated with rapamycin, but not with aptamer-raptor siRNA conjugate, exhibited reduced alloMLR activity, consistent with the known suppressive effects of rapamycin induced mTOR inhibition on DC. Given that inhibition of mTORC1 may not be the optimal way of enhancing memory, we have used the aptamer targeting platform to inhibit other mediators of effector differentiation in activated CD8+ T cells. In preliminary studies 4-1BB aptamer-targeted inhibition of Axin-1, to promote wnt signaling, or reducing IL-2 signaling by downregulating CD25 expression, enhanced vaccine-induced protective antitumor immunity in tumor-bearing mice. Using the OT-I adoptive transfer model, in preliminary experiments we have shown that 4-1BB aptamer targeted co-inhibition of both raptor and PD-1 increased the formation of memory OT-I cells; while not superior to that of inhibiting raptor alone, the OT-I cells expressed significantly less PD-1 on cell surface, and were, therefore, resistant to PD-1-mediated immune suppression. Overall, these studies demonstrate the feasibility and efficiency of aptamer targeted delivery of siRNAs to immune cells, and underscore the potential advantages of aptamer-targeted siRNA delivery over nontargeted administration of pharmacological agents. This abstract is also presented as Poster A23. Citation Format: Alexey Berezhnoy, Rajagopalan Anugraha, Thomas Malek, Eli Gilboa. Potentiating immunological memory in mice using aptamer targeted siRNA delivery to inhibit mediators of effector differentiation in CD8+ cytotoxic T lymphocytes. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr PR11.</jats:p

Abstract 3147: Generation of exhaustion-resistant memory CD8 T-cells by simultaneous downmodulation of mTORC1 and PD-1 in activated CD8 T-cells

Abstract CD8 memory T cells play a crucial role in controlling chronic infections and certain malignancies. It is possible to expand the population redirecting early-activated T-cells toward memory differentiation by altering intracellular signaling of mTORC1 pathway. Indeed, treatment with mTORC1 inhibitor rapamycin increased formation of memory cells as published previously; however, animals treated with the drug exhibited signs of immune suppression and rapamycin-generated CD8 memory T-cells failed to protect animals against melanoma challenge. We introduced highly efficient RNAi-based method to downmodulate mTORC1 signaling specifically in target cells. siRNA against raptor, a critical component of the signaling complex, was delivered in vivo into activated T-cells by conjugating to the 4-1BB-binding RNA aptamer resulting in expansion of CD8+ memory T-cells formation. Memory T-cells are generally resistant to immune suppression, yet in vivo, the population suffers from the gradual attrition mediated by PD-1 signalling. We developed the conjugate with two highly effective siRNAs against raptor and PD-1 linked to the same 4-1BB aptamer molecule. In vitro, both siRNAs efficiently dissociated from the conjugate inside target cells and simultaneously downregulated its respective mRNAs. In vivo, using OT-I adoptive transfer model, we demonstrated that treatment with 4-1BB-raptor-PD1 conjugates increased formation of memory CD8+ T-cells (as measured by absolute number of OT-1 cells 30 days after the treatment) in a rate similar to the 4-1BB-raptor conjugate. There was, however, qualitative difference between two populations, as CD8+ memory T-cells expanded with 4-1BB-raptor-PD1 conjugates expressed significantly less PD-1 on cell surface, and were, therefore, resistant to PD-1-mediated immune suppression. Thus, we introduced a new method to generate memory cells with enhanced functional potential and currently exploring its therapeutic usefulness in various models. Citation Format: Alexey Berezhnoy, Agata Levay, Anugraha Rajagopalan, Yvonne Puplampu-Dove, Eli Gilboa. Generation of exhaustion-resistant memory CD8 T-cells by simultaneous downmodulation of mTORC1 and PD-1 in activated CD8 T-cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2015-3147</jats:p

Aptamer-Targeted Attenuation of IL-2 Signaling in CD8 + T Cells Enhances Antitumor Immunity

Immune responses elicited against cancer using existing therapies such as vaccines or immune stimulatory antibodies are often not curative. One way to potentiate antitumor immunity is to enhance the long-term persistence of anti-tumor CD8 T cells. Studies have shown that the persistence of activated CD8 T cells is negatively impacted by the strength of interleukin 2 (IL-2) signaling. Here, we used small interfering RNAs (siRNAs) against CD25 (IL-2Rα) to attenuate IL-2 signaling in CD8 T cells. The siRNAs were targeted to 4-1BB-expressing CD8 T cells by conjugation to a 4-1BB-binding oligonucleotide aptamer. Systemic administration of the 4-1BB aptamer-CD25 siRNA conjugate downregulated CD25 mRNA only in 4-1BB-expressing CD8 T cells promoting their differentiation into memory cells. Treatment with the 4-1BB aptamer-CD25 siRNA conjugates enhanced the antitumor response of a cellular vaccine or local radiation therapy. Indicative of the generality of this approach, 4-1BB aptamer-targeted delivery of an Axin-1 siRNA, a rate-limiting component of the β-catenin destruction complex, enhanced CD8 T cell memory development and antitumor activity. These findings show that aptamer-targeted siRNA therapeutics can be used to modulate the function of circulating CD8 T cells, skewing their development into long-lasting memory CD8 T cells, and thereby potentiating antitumor immunity

Potentiating tumor immunity using aptamer-targeted RNAi to render CD8 +

TGFβ secreted by tumor cells and/or tumor infiltrating stromal cells is a key mediator of tumor growth and immune suppression at the tumor site. Nonetheless, clinical trials in cancer patients targeting the TGFβ pathway exhibited at best a modest therapeutic benefit. A likely reason, a common limitation of many cancer drugs, is that the physiologic roles of TGFβ in tissue homeostasis, angiogenesis, and immune regulation precluded the dose escalation necessary to achieve a profound clinical response. Murine studies have suggested that countering immune suppressive effects of TGFβ may be sufficient to inhibit tumor growth. Here we describe an approach to render vaccine-activated CD8 + T cells transiently resistant to TGFβ inhibition using an siRNA against Smad4 to inhibit a key step in the canonical TGFβ signaling pathway. The siRNA was targeted to vaccine activated CD8 + T cells in the mouse by conjugation to a 4-1BB binding oligonucleotide (ODN) aptamer ligand (4-1BB-Smad4 conjugate). In vitro the 4-1BB-Smad4 conjugate rendered T cells partially resistant to TGFβ inhibition, and treatment of tumor bearing mice with systemically administered 4-1BB-Smad4 conjugate enhanced vaccine- and irradiation-induced antitumor immunity. Limiting the inhibitory effects of TGFβ to tumor-specific T cells will not interfere with its multiple physiologic roles and hence reduce the risk of toxicity