CD3 aptamers promote expansion and persistence of tumor-reactive T cells for adoptive T cell therapy in cancer.

The CD3/T cell receptor (TCR) complex is responsible for antigen-specific pathogen recognition by T cells, and initiates the signaling cascade necessary for activation of effector functions. CD3 agonistic antibodies are commonly used to expand T lymphocytes in a wide range of clinical applications, including in adoptive T cell therapy for cancer patients. A major drawback of expanding T cell populations ex vivo using CD3 agonistic antibodies is that they expand and activate T cells independent of their TCR antigen specificity. Therapeutic agents that facilitate expansion of T cells in an antigen-specific manner and reduce their threshold of T cell activation are therefore of great interest for adoptive T cell therapy protocols. To identify CD3-specific T cell agonists, several RNA aptamers were selected against CD3 using Systematic Evolution of Ligands by EXponential enrichment combined with high-throughput sequencing. The extent and specificity of aptamer binding to target CD3 were assessed through surface plasma resonance, P32 double-filter assays, and flow cytometry. Aptamer-mediated modulation of the threshold of T cell activation was observed in vitro and in preclinical transgenic TCR mouse models. The aptamers improved efficacy and persistence of adoptive T cell therapy by low-affinity TCR-reactive T lymphocytes in melanoma-bearing mice. Thus, CD3-specific aptamers can be applied as therapeutic agents which facilitate the expansion of tumor-reactive T lymphocytes while conserving their tumor specificity. Furthermore, selected CD3 aptamers also exhibit cross-reactivity to human CD3, expanding their potential for clinical translation and application in the future.

Modulating T Cell Responses by Targeting CD3.

Harnessing the immune system to fight cancer has become a reality with the clinical success of immune-checkpoint blockade (ICB) antibodies against PD(L)-1 and CTLA-4. However, not all cancer patients respond to ICB. Thus, there is a need to modulate the immune system through alternative strategies for improving clinical responses to ICB. The CD3-T cell receptor (TCR) is the canonical receptor complex on T cells. It provides the "first signal" that initiates T cell activation and determines the specificity of the immune response. The TCR confers the binding specificity whilst the CD3 subunits facilitate signal transduction necessary for T cell activation. While the mechanisms through which antigen sensing and signal transduction occur in the CD3-TCR complex are still under debate, recent revelations regarding the intricate 3D structure of the CD3-TCR complex might open the possibility of modulating its activity by designing targeted drugs and tools, including aptamers. In this review, we summarize the basis of CD3-TCR complex assembly and survey the clinical and preclinical therapeutic tools available to modulate CD3-TCR function for potentiating cancer immunotherapy.

IL-6/STAT3 signaling in tumor cells restricts the expression of frameshift-derived neoantigens by SMG1 induction.

BACKGROUND: The quality and quantity of tumor neoantigens derived from tumor mutations determines the fate of the immune response in cancer. Frameshift mutations elicit better tumor neoantigens, especially when they are not targeted by nonsense-mediated mRNA decay (NMD). For tumor progression, malignant cells need to counteract the immune response including the silencing of immunodominant neoantigens (antigen immunoediting) and promoting an immunosuppressive tumor microenvironment. Although NMD inhibition has been reported to induce tumor immunity and increase the expression of cryptic neoantigens, the possibility that NMD activity could be modulated by immune forces operating in the tumor microenvironment as a new immunoediting mechanism has not been addressed. METHODS: We study the effect of SMG1 expression (main kinase that initiates NMD) in the survival and the nature of the tumor immune infiltration using TCGA RNAseq and scRNAseq datasets of breast, lung and pancreatic cancer. Different murine tumor models were used to corroborate the antitumor immune dependencies of NMD. We evaluate whether changes of SMG1 expression in malignant cells impact the immune response elicited by cancer immunotherapy. To determine how NMD fluctuates in malignant cells we generated a luciferase reporter system to track NMD activity in vivo under different immune conditions. Cytokine screening, in silico studies and functional assays were conducted to determine the regulation of SMG1 via IL-6/STAT3 signaling. RESULTS: IL-6/STAT3 signaling induces SMG1, which limits the expression of potent frameshift neoantigens that are under NMD control compromising the outcome of the immune response. CONCLUSION: We revealed a new neoantigen immunoediting mechanism regulated by immune forces (IL-6/STAT3 signaling) responsible for silencing otherwise potent frameshift mutation-derived neoantigens.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression.

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Therapeutic Strategies to Enhance Tumor Antigenicity: Making the Tumor Detectable by the Immune System.

Cancer immunotherapy has revolutionized the oncology field, but many patients still do not respond to current immunotherapy approaches. One of the main challenges in broadening the range of responses to this type of treatment is the limited source of tumor neoantigens. T cells constitute a main line of defense against cancer, and the decisive step to trigger their activation is mediated by antigen recognition. Antigens allow the immune system to differentiate between self and foreign, which constitutes a critical step in recognition of cancer cells and the consequent development or control of the malignancy. One of the keystones to achieving a successful antitumor response is the presence of potent tumor antigens, known as neoantigens. However, tumors develop strategies to evade the immune system and resist current immunotherapies, and many tumors present a low tumor mutation burden limiting the presence of tumor antigenicity. Therefore, new approaches must be taken into consideration to overcome these shortcomings. The possibility of making tumors more antigenic represents a promising front to further improve the success of immunotherapy in cancer. Throughout this review, we explored different state-of-the-art tools to induce the presentation of new tumor antigens by intervening at protein, mRNA or genomic levels in malignant cells.

ICOS Costimulation at the Tumor Site in Combination with CTLA-4 Blockade Therapy Elicits Strong Tumor Immunity.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) blockade therapy is able to induce long-lasting antitumor responses in a fraction of cancer patients. Nonetheless, there is still room for improvement in the quest for new therapeutic combinations. ICOS costimulation has been underscored as a possible target to include with CTLA-4 blocking treatment. Herein, we describe an ICOS agonistic aptamer that potentiates T cell activation and induces stronger antitumor responses when locally injected at the tumor site in combination with anti-CTLA-4 antibody in different tumor models. Furthermore, ICOS agonistic aptamer was engineered as a bi-specific tumor-targeting aptamer to reach any disseminated tumor lesions after systemic injection. Treatment with the bi-specific aptamer in combination with CTLA-4 blockade showed strong antitumor immunity, even in a melanoma tumor model where CTLA-4 treatment alone did not display any significant therapeutic benefit. Thus, this work provides strong support for the development of combinatorial therapies involving anti-CTLA-4 blockade and ICOS agonist tumor-targeting agents.

Intratumoral injection of activated B lymphoblast in combination with PD-1 blockade induces systemic antitumor immunity with reduction of local and distal tumors.

In spite of the success of PD-1 blocking antibodies in the clinic their benefits are still restricted to a small fraction of patients. Immune-desert tumors and/or the highly immunosuppressive tumor milieu might hamper the success of PD-1/PD-L1 blocking therapies into a broader range of cancer patients. Although still under debate, there is a cumulative body of evidence that indicates B tumor-infiltrating lymphocytes are a good prognostic marker in most types of cancer, especially in those that form ectopic lymphoid tissue structures. Taking this into account, we reason that the adoptive transfer of activated B lymphoblasts (ABL) in the tumor could be a feasible therapeutic approach to shift the immunosuppressive tumor microenvironment into an immune-permissive one. In this work we show the antitumor effect of ABL therapy in two different tumor models: colon carcinoma (CT26) and melanoma (B16/F10). The ABL transfer in the most relevant non-immunogenic B16/F10 melanoma model depicts synergism with anti-PD-1 antibody therapy. Furthermore, systemic antitumor immunity was detected in mice treated with PD-1 antibody/ABL combination which was able to reach distal metastatic lesions.

Diacylglycerol kinase α inactivation is an integral component of the costimulatory pathway that amplifies TCR signals.

The arsenal of cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. T cells rely on diacylglycerol (DAG) to carry out their functions. DAG availability and signaling are regulated by the enzymes diacylglycerol kinase (DGK) α and ζ, whose excess function drives T cells into hyporesponsive states. Targeting DGKα is a promising strategy for coping with cancer; its blockade could reinstate T-cell attack on tumors while limiting tumor growth, due to positive DGKα functions in several oncogenic pathways. Here, we made a side-by-side comparison of the effects of commercial pharmacological DGK inhibitors on T-cell responses with those promoted by DGKα and DGKζ genetic deletion or silencing. We show the specificity for DGKα of DGK inhibitors I and II and the structurally similar compound ritanserin. Inhibitor treatment promoted Ras/ERK (extracellular signal-regulated kinase) signaling and AP-1 (Activator protein-1) transcription, facilitated DGKα membrane localization, reduced the requirement for costimulation, and cooperated with enhanced activation following DGKζ silencing/deletion. DGKiII and ritanserin had similar effects on TCR proximal signaling, but ritanserin counteracted long-term T-cell activation, an effect that was potentiated in DGKα-/- cells. In contrast with enhanced activation triggered by pharmacological inhibition, DGKα silencing/genetic deletion led to impaired Lck (lymphocyte-specific protein tyrosine kinase) activation and limited costimulation responses. Our results demonstrate that pharmacological inhibition of DGKα downstream of the TCR provides a gain-of-function effect that amplifies the DAG-dependent signaling cascade, an ability that could be exploited therapeutically to reinvigorate T cells to attack tumors.