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.

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.

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.

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.

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.

Idiotype vaccines produced with a non-cytopathic alphavirus self-amplifying RNA vector induce antitumor responses in a murine model of B-cell lymphoma.

A promising therapy for patients with B-cell lymphoma is based on vaccination with idiotype monoclonal antibodies (mAbs). Since idiotypes are different in each tumor, a personalized vaccine has to be produced for each patient. Expression of immunoglobulins with appropriate post-translational modifications for human use often requires the use of stable mammalian cells that can be scaled-up to reach the desired level of production. We have used a noncytopathic self-amplifying RNA vector derived from Semliki Forest virus (ncSFV) to generate BHK cell lines expressing murine follicular lymphoma-derived idiotype A20 mAb. ncSFV/BHK cell lines expressed approximately 2 mg/L/24 h of A20 mAb with proper quaternary structure and a glycosylation pattern similar to that of A20 mAb produced by hybridoma cells. A20 mAb purified from the supernatant of a ncSFV cell line, or from the hybridoma, was conjugated to keyhole limpet hemocyanin and used to immunize Balb/c mice by administration of four weekly doses of 25 µg of mAb. Both idiotype mAbs were able to induce a similar antitumor protection and longer survival compared to non-immunized mice. These results indicate that the ncSFV RNA vector could represent a quick and efficient system to produce patient-specific idiotypes with potential application as lymphoma vaccines.

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.

Identification of LAG3 high affinity aptamers by HT-SELEX and Conserved Motif Accumulation (CMA).

LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.

Targeting inhibition of Foxp3 by a CD28 2'-Fluro oligonucleotide aptamer conjugated to P60-peptide enhances active cancer immunotherapy.

The specific inhibition of Treg function has long been a major technical challenge in cancer immunotherapy. So far no single cell-surface marker has been identified that could be used to distinguish Treg cells from other lymphocytes. The only available specific marker mostly expressed in Treg is Foxp3, which is an intracellular transcription factor. A targeting molecule able to penetrate the membrane and inhibit Foxp3 within the cell is needed. P60-peptide is able to do that, but due to lack of target specificity, the doses are extremely high. In this study we have shown as a proof of concept that P60 Foxp3 inhibitor peptide can be conjugated with a CD28 targeting aptamer to deliver the peptide to CD28-expressing cells. The AptCD28-P60 construct is a clinically feasible reagent that improves the efficacy of the unconjugated P60 peptide very significantly. This approach was used to inhibit Treg function in a vaccination context, and it has shown a significant improvement in the induced immune response, entailing a lower tumor load in an antigen-specific cancer vaccine protocol.

In Silico Aptamer Docking Studies: From a Retrospective Validation to a Prospective Case Study-TIM3 Aptamers Binding.

Complementing Systematic Evolution of Ligands by EXponential Enrichment (SELEX) technologies with in silico prediction of aptamer binders has attracted a lot of interest in the recent years. We propose a workflow involving 2D structure prediction, 3D RNA modeling using Rosetta and docking to the target protein with 3dRPC for: (i) prediction of the binding mode of our two previously reported potent (Kd < 50 nmol/l) murine TIM3 aptamers, and (ii) the prioritization of TIM3 aptamers that were enriched in the SELEX workflow. The procedure was first validated in five different study cases. As a novelty, cluster analysis of the docked poses was carried out and shown to be useful in reproducing the binding mode or at least in identifying the binding site and the experimental aptamer-protein interactions. For TIM3, our therapeutic target of interest, a plausible binding site and binding mode was identified that might explain the lack of cross-reactivity in murine over human TIM-3. Concerning the prioritization of the aptamers, the inclusion of the cluster analysis as an additional criterion following a rank-by-rank approach is discussed and compared with the performance of the docking scoring function alone for two validation cases and for the prospective assessment of the novel aptamers as TIM3 binders.

Identification of TIM3 2'-fluoro oligonucleotide aptamer by HT-SELEX for cancer immunotherapy.

TIM3 belongs to a family of receptors that are involved in T-cell exhaustion and Treg functions. The development of new therapeutic agents to block this type of receptors is opening a new avenue in cancer immunotherapy. There are currently several clinical trials ongoing to combine different immune-checkpoint blockades to improve the outcome of cancer patients. Among these combinations we should underline PD1:PDL1 axis and TIM3 blockade, which have shown very promising results in preclinical settings. Most of these types of therapeutic agents are protein cell-derived products, which, although broadly used in clinical settings, are still subject to important limitations. In this work we identify by HT-SELEX TIM3 non-antigenic oligonucleotide aptamers (TIM3Apt) that bind with high affinity and specificity to the extracellular motives of TIM3 on the cell surface. The TIM3Apt1 in its monomeric form displays a potent antagonist capacity on TIM3-expressing lymphocytes, determining the increase of IFN-γ secretion. In colon carcinoma tumor-bearing mice, the combinatorial treatment of TIM3Apt1 and PDL1-antibody blockade is synergistic with a remarkable antitumor effect. Immunotherapeutic aptamers could represent an attractive alternative to monoclonal antibodies, as they exhibit important advantages; namely, lower antigenicity, being chemically synthesized agents with a lower price of manufacture, providing higher malleability, and antidote availability.

MRP1-CD28 bi-specific oligonucleotide aptamers: target costimulation to drug-resistant melanoma cancer stem cells.

In this work we show a clinically feasible strategy to convert in situ the own tumor into an endogenous vaccine by coating the melanoma cancerous cells with CD28 costimulatory ligands. This therapeutic approach is aimed at targeting T-cell costimulation to chemotherapy-resistant tumors which are refractory and been considered as untreatable cancers. These tumors are usually defined by an enrichment of cancer stem cells and characterized by the higher expression of chemotherapy-resistant proteins. In this work we develop the first aptamer that targets chemotherapy-resistant tumors expressing MRP1 through a novel combinatorial peptide-cell SELEX. With the use of the MRP1 aptamer we engineer a MRP1-CD28 bivalent aptamer that is able to bind MRP1-expressing tumors and deliver the CD28 costimulatory signal to tumor-infiltrating lymphocytes. The bi-specific aptamer is able to enhance costimulation in chemotherapy-resistant tumors. Melanoma-bearing mice systemically treated with MRP1-CD28 bivalent aptamer show reduced growth, thus proving an improved mice survival.Besides, we have designed a technically feasible and translational whole-cell vaccine (Aptvax). Disaggregated cells from tumors can be directly decorated with costimulatory ligand aptamers to generate the vaccine Aptvax. CD28Aptvax made of irradiated tumor cells coated with the CD28-agonistic aptamer attached to MRP1 elicits a strong tumor- cell immune response against melanoma tumors reducing tumor growth.

2-fluoro-RNA oligonucleotide CD40 targeted aptamers for the control of B lymphoma and bone-marrow aplasia.

Recent studies have underscored the importance of immunomodulatory antibodies in the treatment of solid and hematological tumors. ODN-Aptamers are rising as a novel class of drugs that can rival therapeutic antibodies. The success of some of the current cancer immunotherapy approaches in oncological patients depends on the intrinsic antigenicity of each tumor as has recently been disclosed, and it is hampered in those patients that are treated with myeloablative chemotherapy or radiotherapy, which induce profound immunosuppression. CD40 agonist and antagonist molecules offer a new therapeutic alternative which has the potential to generate anticancer effects by different mechanisms. HS-SELEX was performed to identify high-affinity aptamers against CD40, and three therapeutic CD40 constructs were engineered as: CD40 agonist aptamer, CD40 antagonist aptamer and CD40 agonistic aptamer-shRNA chimera. It is shown that CD40 agonist aptamers can be used to promote bone-marrow aplasia recovery. CD40 antagonist aptamers are revealed to have a direct antitumor effect on CD40-expressing B-cell lymphoma in vitro and in vivo. Further, in order to identify a therapeutic reagent that will generate the optimal conditions for cancer immunotherapy (antigen-presenting cell activation, tumor antigenicity enhancement and bone-marrow aplasia recovery), CD40 agonist aptamer-shRNA chimera was generated to target the inhibition of the Nonsense mRNA Mediated Decay (NMD) to tumor cells.

Variations in "rescuability" of immunoglobulin molecules from different forms of human lymphoma: implications for anti-idiotype vaccine development.

Idiotypic (Id) vaccination has shown promising results in patients with follicular lymphoma (FL). However, it still remains unclear whether the same approach might be suitable for the treatment of other B-cell malignancies. For this reason, we recently performed an interim analysis of patients proposed to receive this treatment at our center. The feasibility of employing idiotype vaccines was evaluated for five different B-cell malignancies in their first relapse, both in terms of induction and fusion, as well as overall treatment. Our data suggest that, unlike follicular lymphoma (87%), this approach is not feasible to treat other B-cell malignancies (0-20%) such as mantle cell, small lymphocytic, diffuse large cell and Burkitt's lymphoma (P < 0.01). The main difficulties encountered were technical problems related to the survival of idiotype-producing hybridomas (83%) and the early loss of idiotype production by growing hybridomas (17%). However, it remains possible that an idiotype vaccine might still be produced through molecular means for most, if not all cases of relapsing B-cell malignancies.

Idiotype vaccine production using hybridoma technology.

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy both in Europe and in the United States. Follicular lymphoma (FL), a tumor comprised of mature B cells, represents one fourth of all NHL and, despite good response rates to standard treatments, tends to frequently relapse to such an extent that it is still considered incurable. Among several alternative therapeutic options actively being pursued, immunotherapy by idiotypic vaccination is in the forefront of clinical experimental medicine. The idiotype vaccine consists of the tumor-specific immunoglobulin conjugated with keyhole limpet hemocyanin (KLH) and administered together with an adjuvant. Over the last 20 years, researchers have proven that this vaccine can induce specific immune responses. Too, those patients with such responses experience a disease-free survival longer than normally achievable, although these latter results require further confirmation in large clinical trials. Traditionally, idiotype vaccines have been produced through hybridoma technology. In this chapter this technology is described.

CD28 aptamers as powerful immune response modulators.

CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. We have isolated two aptamers that bind to the CD28 receptor. As a monomer, one of them interfered with the binding of CD28 to its ligand (B7), precluding the costimulatory signal, whereas the other one was inactive. However, dimerization of any of the anti-CD28 aptamers was sufficient to provide an artificial costimulatory signal. No antibody has featured a dual function (i.e., the ability to work as agonist and antagonist) to date. Two different agonistic structures were engineered for each anti-CD28 aptamer. One showed remarkably improved costimulatory properties, surpassing the agonistic effect of an anti-CD28 antibody. Moreover, we showed in vivo that the CD28 agonistic aptamer is capable of enhancing the cellular immune response against a lymphoma idiotype and of prolonging survival of mice which receive the aptamer together with an idiotype vaccine. The CD28 aptamers described in this work could be used to modulate the immune response either blocking the interaction with B7 or enhancing vaccine-induced immune responses in cancer immunotherapy.Molecular Therapy - Nucleic Acids (2013) 2, e98; doi:10.1038/mtna.2013.26; published online 11 June 2013.

Dendritic cell vaccination in glioblastoma after fluorescence-guided resection.

AIM: To assess whether the addition of a customized, active immunotherapy to standard of care including fluorescence-guided surgery, may provide hints of an improved survival for patients with poor-prognosis, incurable glioblastoma multiform. METHODS: Preliminary to our ongoing, phase-II clinical trial, we conducted a small pilot study enrolling five consecutive patients with resectable glioblastoma. In terms of Recursive Partitioning Analysis, four patients were class V and one was class IV. In all five cases, fluorescence-guided surgery was employed, followed by rapid steroid discontinuation. Patients were then treated with a combination of standard radio-chemotherapy with temozolomide and tumor lysate-pulsed, mature dendritic cell-based vaccinations. RESULTS: Though all five patients ultimately progressed, with any further treatment left to the sole decision of the treating oncologist, active immunotherapy was very well tolerated and induced specific immune responses in all three patients for whom enough material was available for such an assessment. Median progression-free survival was 16.1 mo. Even more important, median and mean overall survival were 27 mo and 26 mo, respectively. Three patients have died with an overall survival of 9 mo, 27 mo and 27.4 mo, while the other two are still alive at 32 mo and 36 mo, the former receiving treatment with bevacizumab, while the latter has now been off therapy for 12 mo. Four of five patients were alive at two years. CONCLUSION: Active immunotherapy with tumor lysate-pulsed, autologous dendritic cells is feasible, safe, well tolerated and biologically efficacious. A phase-II study is ongoing to possibly improve further on our very encouraging clinical results.

Idiotype vaccines for lymphoma therapy.

Despite having been the first cancer vaccine to provide clear-cut evidence of biological and clinical efficacy as well as of clinical benefit in humans, idiotype vaccines have failed to become the first therapeutic cancer vaccine to be granted regulatory approval. Indeed, idiotypic vaccination is still an experimental therapeutic option for some types of B-cell malignancy over 20 years after its first use in patients with lymphoma. The ultimate reason for this situation lies in the recent failure of three large-scale, independent, randomized trials to achieve their respective main clinical end points. Interestingly, each trial had been designed with intrinsic pitfalls that are likely to have influenced, and perhaps even entirely compromized, all chances each study had to succeed. Therefore, it is difficult to conclude whether any of the different idiotype vaccines employed so far may still represent an ideal candidate for further trials. Meanwhile, other idiotype vaccine formulations are under active investigation.

Idiotype vaccines for lymphoma: Potential factors predicting the induction of immune responses.

Over the last two decades, lymphoma idiotype vaccines have been the first human cancer vaccines to show striking evidence of biological and clinical efficacy on the one hand, as well as clinical benefit on the other. More recently, however, three large-scale, independent, randomized clinical trials on idiotypic vaccination have failed to achieve their main clinical endpoints for reasons likely to depend more on flaws in each clinical trial's study design than on each vaccination strategy per se. Independently of these considerations, a major hurdle for the development of this substantially innocuous and yet potentially very effective type of treatment has been the fact that, even to date, no factors ascertainable before vaccination have been prospectively singled out as predictors of subsequently vaccine-induced, idiotype-specific immune as well as clinical responses. The aim of this review article is precisely to analyze what has been and what could be done in this respect in order to give a greater chance of success to future trials aimed at regulatory approval of idiotype vaccines.