Immune Checkpoint Blocking Antibodies Research Articles

A versatile platform for the tumor-targeted delivery of immune checkpoint-blocking immunoglobin G

Immunotherapies based on immune checkpoint-blocking antibodies have been considered the most attractive cancer treatments in recent years. However, the systemic administration of immune checkpoint-blocking antibodies is limited by low response rates and high risk of inducing immune-related adverse events (irAEs), which might be overcome by the tumor-targeted delivery of these antibodies. To achieve tumor-targeted delivery, immune checkpoint-blocking antibodies are usually modified with tumor-homing ligands through difficult genetic fusion or chemical conjugation. As most immune checkpoint-blocking antibodies are immunoglobin G (IgG) antibodies, we hypothesize that these IgG antibodies might be noncovalently modified with a tumor-homing ligand fused to an IgG-binding domain (IgBD). To test this hypothesis, the tumor-homing ZPDGFRβ affibody, which targets platelet-derived growth factor receptor β (PDGFRβ), was fused to the Fab-selective IgBD in a trimeric format. After mixing ZPDGFRβ fused to the IgBD with immune checkpoint-blocking IgG against programmed death-ligand 1 (αPD-L1), a novel homogenous complex was formed, indicating that αPD-L1 had been successfully modified with ZPDGFRβ fused to the IgBD. ZPDGFRβ-modified αPD-L1 bound to both PDGFRβ and PD-L1, thus leading to greater tumor uptake and antitumor effects in mice bearing PDGFRβ+PD-L1+ tumor grafts. In addition, due to the broad spectrum of IgBD for IgG, immune checkpoint-blocking IgG antibodies against cytotoxic T-lymphocyte-associated protein 4 (αCTLA-4) and signal regulatory protein alpha (αSIRPα) were also modified with ZPDGFRβ fused to the IgBD. These results demonstrated that a tumor-homing ligand fused to the IgBD might be developed as a versatile platform for the modification of immune checkpoint-blocking IgG antibodies to achieve tumor-targeted delivery.

Abstract 1642: A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity

Abstract Therapeutic antibodies have improved survival of both hematologic and solid cancer patients, inducing long-lasting responses and even cures. Clinically successful antibodies exert antitumor activity by targeting tumor cells directly e.g. anti-CD20 mAb in B cell cancer, or by targeting and activating immune cells that seek and destroy cancer cells in the tumor microenvironment (immune checkpoint blocking antibodies;ICB). Still, many patients fail to respond or acquire resistance to these therapies. Understanding mechanisms and overcoming resistance to distinct classes of antibody drugs, is therefore clearly warranted and has the potential to further improve cancer outcomes. The inhibitory Fc gamma receptor (FcγR) IIB may precipitate resistance to cancer immunotherapy by several mechanisms, acting both on tumor and immune effector cells. We previously developed an FcγRIIB blocking antibody (BI-1206). Based on its cytolytic activity against B cell cancer cells and ability to block rituximab internalization from tumor cells, as well as enhance rituximab therapeutic activity in mice humanized for CD20 and FcγRIIB or bearing relapsed/refractory CLL in vivo, BI-1206 is being explored in clinical trials (NCT03571568; NCT04219254). Emerging data demonstrate that FcγRs can modulate the therapeutic activity of ICB antibodies (anti-PD-1/PD-L1 and anti-CTLA-4). While both anti-CTLA-4 and anti-PD-1/L1 mAb are effective and approved for cancer therapy, and combined checkpoint blockade improves therapeutic responses, anti-CTLA-4 therapies are associated with tolerability issues. Means of enhancing the anti-CTLA-4 therapeutic window are therefore desirable in order to deliver this therapy to more patients. Recent preclinical and retrospective clinical data indicate that anti-CTLA-4 antibodies may promote antitumor immunity by dual mechanisms; “releasing the brakes” on T effector cells and depleting immune suppressive intratumoral T regulatory (Treg) cells. Anti-CTLA-4 induced Treg depletion was shown to be FcγR-dependent. Here, we report the generation of a novel, fully human FcγRIIB-blocking antibody (BI-1607) engineered to eliminate Fc-mediated FcγR binding and function (Fc-null anti-FcγRIIB). Using a mechanism-of-action-matched surrogate antibody, we demonstrate that Fc-null anti-FcγRIIB enhances therapeutic efficacy of submaximally efficacious doses of anti-CTLA-4 in responsive (MC38) or resistant (CT26) syngeneic immune competent mouse models. Of relevance to the clinical setting where low dose anti-CTLA-4/anti-PD-1 combination therapies have been approved for different indications, we further demonstrate that triplet therapy significantly enhanced survival in the checkpoint blockade-poorly responsive B16 tumor model. Collectively our results provide proof-of-concept for Fc-null anti-FcγRIIB to enhance anti-CTLA-4 ICB in the treatment of cancer. Citation Format: Linda Mårtensson, Robert Oldham, Marie Borggren, Mathilda Kovacek, Therese Blidberg, Ali Roghanian, Ulla-Carin Thornberg, Ingrid Karlsson, Stephen A. Beers, Mark S. Cragg, Ingrid Teige, Bjorn L. Frendéus. A novel FcγRIIB-blocking antibody to enhance FcγR-dependent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1642.

Injectable Hydrogels as Local Depots at Tumor Sites for Antitumor Immunotherapy and Immune-Based Combination Therapy.

Despite the encouraging clinical responses of several human cancers to immunotherapy, the efficacy of this treatment remains limited by variable objective response rates and severe systemic immune-related adverse events. To overcome these issues, injectable hydrogels have been developed as local depots that permit the sustained release of single or multiple immunotherapy agents, including traditional immunomodulatory factors, immune checkpoint blocking antibodies, and exogenous immune cells. The antitumor efficacy of immunotherapy can also be enhanced by its combination with other therapeutic approaches, including chemotherapy, radiotherapy, and phototherapy. Despite local treatment strategies, potent systemic antitumor immune responses with low systemic toxicity can be obtained, leading to significant local and abscopal tumor-killing, reduced tumor metastasis, and the prevention of tumor recurrence. This review highlights recent progress in injectable hydrogel-based local depots for tumor immunotherapy and immune-based combination therapy. Moreover, the proposed mechanisms responsible for these antitumor effects are discussed.

MiRNAs and lncRNAs as Novel Therapeutic Targets to Improve Cancer Immunotherapy.

Simple SummaryCancer onset and progression are promoted by high deregulation of the immune system. Recently, major advances in molecular and clinical cancer immunology have been achieved, offering new agents for the treatment of common tumors, often with astonishing benefits in terms of prolonged survival and even cure. Unfortunately, most tumors are still resistant to current immune therapy approaches, and basic knowledge of the resistance mechanisms is eagerly awaited. We focused our attention on noncoding RNAs, a class of RNA that regulates many biological processes by targeting selectively crucial molecular pathways and that, recently, had their role in cancer cell immune escape and modulation of the tumor microenvironment identified, suggesting their function as promising immunotherapeutic targets. In this scenario, we point out that noncoding RNAs are progressively emerging as immunoregulators, and we depict the current information on the complex network involving the immune system and noncoding RNAs and the promising therapeutic options under investigation. Novel opportunities are emerging from noncoding-RNAs for the treatment of immune-refractory tumors.Immunotherapy is presently one of the most promising areas of investigation and development for the treatment of cancer. While immune checkpoint-blocking monoclonal antibodies and chimeric antigen receptor (CAR) T-cell-based therapy have recently provided in some cases valuable therapeutic options, the goal of cure has not yet been achieved for most malignancies and more efforts are urgently needed. Noncoding RNAs (ncRNA), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), regulate several biological processes via selective targeting of crucial molecular signaling pathways. Recently, the key roles of miRNA and lncRNAs as regulators of the immune-response in cancer have progressively emerged, since they may act (i) by shaping the intrinsic tumor cell and microenvironment (TME) properties; (ii) by regulating angiogenesis, immune-escape, epithelial-to-mesenchymal transition, invasion, and drug resistance; and (iii) by acting as potential biomarkers for prognostic assessment and prediction of response to immunotherapy. In this review, we provide an overview on the role of ncRNAs in modulating the immune response and the TME. We discuss the potential use of ncRNAs as potential biomarkers or as targets for development or clinical translation of new therapeutics. Finally, we discuss the potential combinatory approaches based on ncRNA targeting agents and tumor immune-checkpoint inhibitor antibodies or CAR-T for the experimental treatment of human cancer.

A Three-in-one ZIFs-Derived CuCo(O)/GOx@PCNs Hybrid Cascade Nanozyme for Immunotherapy/Enhanced Starvation/Photothermal Therapy.

Glucose oxidase (GOx) is regarded as an ideal endogenous natural enzyme for tumor starvation therapy and photothermal therapy (PTT) is a promising strategy for the ablation of primary tumor. In this work, Cu-doped cobalt oxide and porous carbon nanocomposites (CuCo(O)@PCNs) were synthesized from double-layered ZIF-8@ZIF-67 and GOx was loaded in the porous carbon to form a CuCo(O)/GOx@PCNs hybrid nanozyme. CuCo(O) was characterized as the Cu0.3Co2.7O4 phase through X-ray diffraction analysis and it can react with H2O2 to generate O2 and alleviate tumor hypoxia, resulting in the recovered enzymatic activity of GOx and the enhanced starvation therapy. The porous nanocarbon can ablate the primary tumor because of its high photothermal conversion efficiency of 40.04%. The three-in-one functions of oxygen supply, glucose consumption, and photothermal conversion were realized in the ZIFs-derived CuCo(O)/GOx@PCNs nanozyme and the starvation therapy effect was improved by PTT and oxygen supplement. Furthermore, the inhibition effect of CuCo(O)/GOx@PCNs on metastatic tumor is similar to combined therapy of the nanozyme and the immune checkpoint-blocking antibody, α-PD-1. The related antitumor immune mechanism was studied through the analysis of immune-related proinflammatory cytokines and the activated T cells. This work may provide new ideas for the development and application of the ZIFs-derived hybrid nanozyme in tumor therapy and the CuCo(O)/GOx@PCNs nanozyme may be a promising alternative to immune checkpoint inhibitors.

Vaccination against PD-L1 with IO103 a Novel Immune Modulatory Vaccine in Basal Cell Carcinoma: A Phase IIa Study.

Simple SummaryBasal cell carcinoma is the most common skin cancer and new treatments for patients with widespread and numerous tumors are lacking. In a previous study treating patients with multiple myeloma with a peptide vaccine called IO103 against an immune checkpoint molecule called programmed death ligand 1, two cases of basal cell carcinoma regressed. The aim of the present study was to assess the effect of vaccination with IO103 in ten patients with basal cell carcinoma. Patients were vaccinated with Montanide as adjuvant up to nine times during six months. Regression in tumor size of at least 30% was seen in five of 18 tumors, two of which showed complete regression. Vaccinations resulted in immune responses against the vaccine in blood samples from nine of ten patients and in skin samples from five of nine patients. The findings suggest that the vaccine may be effective against some basal cell carcinomas.Antitumor activity of immune checkpoint blocking antibodies against programmed death 1 (PD-1) in basal cell carcinoma (BCC) has been described. IO103 is a peptide vaccine against the major PD-1 ligand PD-L1. A phase IIa study of vaccination with IO103 and Montanide adjuvant was conducted in patients with resectable BCC (NCT03714529). Vaccinations were given six times every 2 weeks (q2w), followed by three vaccines q4w in responders. Primary endpoints were clinical responses of target tumors, change in target tumor size and immune responses to the vaccine. Secondary endpoint was safety. One tumor per patient was designated target tumor and biopsied twice during the course of vaccination. Synchronous non-target BCCs were not biopsied during vaccinations. Ten patients were vaccinated (six patients received six vaccinations and four patients received nine vaccinations). A partial response (PR) was seen in two target tumors. Two complete responses (CR) and one PR were observed in eight non-target tumors in four patients. No tumors progressed. Related adverse events were grade 1 and reversible. Immune responses against IO103 were induced in blood samples from nine of ten and skin-infiltrating lymphocytes from five of the nine patients. The regressions seen in non-target tumors suggest that IO103 may be effective against a subtype of BCC.

Next Generation Therapeutic Strateg‐Es: Evolving cancer immunotherapy through agents that Engage, Expand and Enable the anti‐tumor immune response

AbstractThe development and FDA approval of immune checkpoint blocking antibodies have brought new light to cancer immunotherapy. While immune checkpoint blockade (ICB) has demonstrated clinical benefit in certain tumors as monotherapy, effective therapy of established tumors necessitates a combination of multiple immuno‐oncology agents targeting diverse functions of the immune system. These combination strategies should be tactically designed to Engage the immune system by inducing a tumor‐antigen specific T‐cell population, Expand the number of antigen‐specific cytotoxic T cells and increase their migration to the tumor microenvironment, and once there, Enable prolonged and persistent effector function. Although viral therapeutic cancer vaccines have demonstrated little efficacy as monotherapies, they have substantial potential to Engage the immune system as one branch of a multipronged treatment strategy. This review will summarize prior and ongoing Phase II and III clinical trials built upon the foundation of viral therapeutic cancer vaccines. We examine their efficacy as a monotherapy, and more importantly, when combined with additional agents that Expand and Enable the immune system. It is clear that the future of cancer immunotherapy will include evolving treatment strategies made up of multiple agents, and we are optimistic that in this context viral therapeutic cancer vaccines will emerge as an important part of next generation effective therapeutic strategies.

Targeting cancer metastasis with antibody therapeutics.

Cancer metastasis, the spread of disease from a primary to a distal site through the circulatory or lymphatic systems, accounts for over 90% of all cancer related deaths. Despite significant progress in the field of cancer therapy in recent years, mortality rates remain dramatically higher for patients with metastatic disease versus those with local or regional disease. Although there is clearly an urgent need to develop drugs that inhibit cancer spread, the overwhelming majority of anticancer therapies that have been developed to date are designed to inhibit tumor growth but fail to address the key stages of the metastatic process: invasion, intravasation, circulation, extravasation, and colonization. There is growing interest in engineering targeted therapeutics, such as antibody drugs, that inhibit various steps in the metastatic cascade. We present an overview of antibody therapeutic approaches, both in the pipeline and in the clinic, that disrupt the essential mechanisms that underlie cancer metastasis. These therapies include classes of antibodies that indirectly target metastasis, including anti-integrin, anticadherin, and immune checkpoint blocking antibodies, as well as monoclonal and bispecific antibodies that are specifically designed to interrupt disease dissemination. Although few antimetastatic antibodies have achieved clinical success to date, there are many promising candidates in various stages of development, and novel targets and approaches are constantly emerging. Collectively, these efforts will enrich our understanding of the molecular drivers of metastasis, and the new strategies that arise promise to have a profound impact on the future of cancer therapeutic development. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Unique challenges for glioblastoma immunotherapy-discussions across neuro-oncology and non-neuro-oncology experts in cancer immunology. Meeting Report from the 2019 SNO Immuno-Oncology Think Tank.

Cancer immunotherapy has made remarkable advances with over 50 separate Food and Drug Administration (FDA) approvals as first- or second-line indications since 2015. These include immune checkpoint blocking antibodies, chimeric antigen receptor-transduced T cells, and bispecific T-cell-engaging antibodies. While multiple cancer types now benefit from these immunotherapies, notable exceptions thus far include brain tumors, such as glioblastoma. As such, it seems critical to gain a better understanding of unique mechanistic challenges underlying the resistance of malignant gliomas to immunotherapy, as well as to acquire insights into the development of future strategies. An Immuno-Oncology Think Tank Meeting was held during the 2019 Annual Society for Neuro-Oncology Scientific Conference. Discussants in the fields of neuro-oncology, neurosurgery, neuro-imaging, medical oncology, and cancer immunology participated in the meeting. Sessions focused on topics such as the tumor microenvironment, myeloid cells, T-cell dysfunction, cellular engineering, and translational aspects that are critical and unique challenges inherent with primary brain tumors. In this review, we summarize the discussions and the key messages from the meeting, which may potentially serve as a basis for advancing the field of immune neuro-oncology in a collaborative manner.

Corn-like Au/Ag nanorod-mediated NIR-II photothermal/photodynamic therapy potentiates immune checkpoint antibody efficacy by reprogramming the cold tumor microenvironment

Immune checkpoint blocking (ICB) antibodies have shown great success in the clinic, but their low response rate in patients with immunosuppressive cold tumors remains a huge challenge. Inspired by the capability of immunogenic cell death (ICD) to convert tumors from cold to hot, we developed a corn-like Au/Ag nanorod (NR) that can induce the ICD of tumor cells under 1064-nm light irradiation. The corn-like Au/Ag NRs plus NIR-II light irradiation strikingly increased the tumor infiltration of T cells and provoked a systemic immune response to reprogram the immunosuppressive cold tumor microenvironment; these NRs synergized with ICB antibodies to efficiently inhibit distant tumor growth. Encouragingly, the combination of aCTLA4 and Au/Ag NRs plus 1064-nm light irradiation elicited a strong immunological memory effect that protected against tumor recurrence.

The role of dendritic cells for therapy of B-cell lymphoma with immune checkpoint inhibitors

Immune checkpoint blocking (ICB) is a promising new tool of cancer treatment. Yet, the underlying therapeutic mechanisms are not fully understood. Here we investigated the role of dendritic cells (DCs) for the therapeutic effect of ICB in a λ-MYC-transgenic mouse model of endogenously arising B-cell lymphoma. The growth of these tumors can be effectively delayed by antibodies against CTLA-4 and PD-1. Tumor-infiltrating DCs from mice having received therapy showed an upregulation of costimulatory molecules as well as an augmented IL-12/IL-10 ratio as compared to untreated controls. Both alterations seemed to be induced by interferon-γ (IFN-γ), which is upregulated in T cells and natural killer cells upon ICB. Furthermore, the enhanced IL-12/IL-10 ratio, which favors Th1-prone antitumor T-cell responses, was a consequence of direct interaction of ICB antibodies with DCs. Importantly, the capability of tumor-infiltrating DCs of stimulating peptide-specific or allogeneic T-cell responses in vitro was improved when DCs were derived from ICB-treated mice. The data indicate that ICB therapy is not only effective by directly activating T cells, but also by triggering a complex network, in which DCs play a pivotal role at the interface between innate and adaptive antitumor responses.

Bifidobacterium alters the gut microbiota and modulates the functional metabolism of T regulatory cells in the context of immune checkpoint blockade.

Immune checkpoint-blocking antibodies that attenuate immune tolerance have been used to effectively treat cancer, but they can also trigger severe immune-related adverse events. Previously, we found that Bifidobacterium could mitigate intestinal immunopathology in the context of CTLA-4 blockade in mice. Here we examined the mechanism underlying this process. We found that Bifidobacterium altered the composition of the gut microbiota systematically in a regulatory T cell (Treg)-dependent manner. Moreover, this altered commensal community enhanced both the mitochondrial fitness and the IL-10-mediated suppressive functions of intestinal Tregs, contributing to the amelioration of colitis during immune checkpoint blockade.

Efficacy and safety of IO103 a novel anti PD-L1 vaccine in basal cell carcinoma.

e22070 Background: Basal cell carcinoma (BCC) is the most frequent malignancy. Case reports have described effect of immune checkpoint blocking antibodies (ICB) against PD-1 in BCC (Cannon et al. 2018, Chang et al. 2017, Ikeda et al. 2016, Winkler et al. 2017). Autoimmune side effects of ICB are well known and limits a wider use in BCC. IO103 is a peptide vaccine against major programmed death (PD) ligand 1 (PD-L1). In a first-in-man study of IO103 in patients with multiple myeloma (NCT03042793) regression of co-existing BCC was observed in two patients which prompted the current study. Methods: A single arm phase IIa study of subcutaneous IO103 (q2w six times followed optionally by q4w an additional 3 times) with the adjuvant Montanide for patients with BCC (NCT03714529). Eligible patients had at least one biopsy verified BCC suitable for sequential biopsies, and no severe autoimmune comorbidity. The primary endpoints were categorical clinical responses of target tumors, percent change in largest diameter of target tumor and immune responses to the vaccine. Secondary endpoint was safety. One tumor per patient was designated target tumor, if more than one tumor the remaining were designated non-target tumors. Only target tumors were sequentially biopsied during vaccinations. Results: Ten patients were included at the department of Dermatology, Herlev and Gentofte Hospital, Denmark between November 19th, 2018 and October 21st, 2019. Based on target lesion assessment, one patient achieved partial response. Two additional patients achieved CR in non-target lesions, including one lesion that had recurred after previous surgery. Categorical clinical responses are given in table. Adverse events were max CTCAE Grade 2 and related events were only CTCAE Grade 1 reversible reactions. Vaccine induced immune responses against PD-L1 were detected in blood samples from eight of ten patients and vaccine specific skin-infiltrating lymphocytes from five of eight tested patients. Conclusions: In this exploratory study, one target tumor achieved partial response. Nonetheless, the two clearances and one partial response of non-target tumors suggest that IO103 may be effective against BCCs. The vaccine was well tolerated. Understanding the underlying molecular mechanisms would be needed to select patients for further studies with IO103. Clinical trial information: NCT03714529. [Table: see text]

SAT-491 Transient Thyrotoxicosis with Immune Checkpoint Inhibitors Therapy: The Importance of Endocrine Evaluation

IntroductionThe immune checkpoint-blocking antibody nivolumab is recognized as having a crucial role in different malignancies by blocking programmed death-1 (PD-1) receptor immune cells. Besides its benefits, nivolumab may cause endocrine immuno-related adverse events (irAEs), including thyroid dysfunction (TD).Clinical caseA 71-year-old man, with an uneventful past medical history, was diagnosed with stage IIIB (T4N2M0), epithelial-growth-factor-receptor (EGFR) wild type, lung adenocarcinoma.The patient underwent 4 cycles of first line chemotherapy with cisplatin/vinorelbine and then radiotherapy, obtaining a partial response.After 4 months, tumor progression was identified, as assessed by whole-body 18-Fluorodeoxyglucose positron emission tomography (FDG-PET) scan, showing pleural and nodal metastasis. Nivolumab, 3 mg/kg every 2 weeks, was started at this point.While pre-nivolumab thyroid function was normal, 3 months after starting the therapy, a low serum TSH level of 0.04 mUI/mL (0.38-5.33) was found, associated with a normal level of FT4, of 10.8 pmol/L (7.9-14.4). Thyroid antibody (Ab) tests, including TSH-receptor Ab, were negative. At ultrasound examination, thyroid gland parenchyma was normo-echoic, demonstrating an isoechoic thyroid nodule in the right lobe, with regular margins, measuring 14mm diameter. Previous medical history was negative for thyroid disease.One week after the referred thyroid function tests, nivolumab was discontinued due to progressive disease as assessed by abdominal magnetic resonance, demonstrating right adrenal metastasis and patient started cisplatin/permetrexed chemotherapy. One month after nivolumab suspension, patient had already normalized thyroid tests, with TSH 2.27 mUI/mL and FT4 9.1 pmol/L. More recently (6 months after nivolumab discontinuation), thyroid function tests continued stable, with TSH 1.05 mUI/ml and T4L 9.4 mmol/l. At this point, patient was receiving permetrexed maintenance therapy.ConclusionsImmune checkpoint molecules as nivolumab, play a crucial role in anti-tumor immunity evasion. Besides its benefits, it may cause irAEs, including TD.We believe it’s essential to perform thyroid function tests at baseline and before the administration of each nivolumab dose, if possible. Additionally, large prospective studies are required in order to assess, the impact of autoimmunity on the development of TD induced by nivolumab, and its potential effect on overall survival and specific cancer survival data.

Neurologic Complications of Immunotherapy

Immunotherapy enhances a patient's own immune system to fight against malignancy and has become increasingly popular during the last decade. Tumor cells can escape destruction by the patient's immune system by overexpression of immunosuppressive molecules such as cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), programmed cell death-1 (PD-1) receptor, and its ligand PD-L1. Immunotherapy agents target these inhibitors and basically reactivate the cytotoxic lymphocytes to destroy the tumor cells. Immunotherapy agents currently are FDA-approved for advanced malignancies including melanoma, Hodgkin lymphoma, head and neck squamous cell carcinoma, non-small cell lung cancer, and renal cell carcinoma. In addition, they have shown beneficial promise in the treatment of other malignancies such as high-grade gliomas.1 As of 2018, the common immune checkpoint blocking antibodies approved by the FDA for immunotherapy include ipilimumab, nivolumab, and pembrolizumab; however, exponential growth of these agents is expected in the next few years when many new agents will receive approval and cover a wider range of malignancies. Considering that immunotherapy seeks to harness the patient's own immune system to fight cancer, increased inflammatory changes are expected. The inflammatory changes may involve the skin, gastrointestinal tract, liver, endocrine, and neurologic systems and together are known as immune-related adverse events (irAEs).

Abstract A43: Identifying a role for Ntrk1 in regulating immune functionality and checkpoint blockade resistance in Kras/p53 mutant lung cancer

Abstract Lung cancer is the leading cause of cancer-related deaths in the United States, with the 5-year survival rate of all lung cancer patients combined at only 18%. The implementation of cancer immunotherapeutics for solid tumors such as lung cancers has shown great promise for improving clinical outcome. Immune checkpoint blocking antibodies targeting the PD-1/PD-L1 axis have been FDA approved for the treatment of late-stage metastatic disease, and clinical trial results indicate durable response in a small percentage of patients (~15-20%) compared to standard-of-care chemotherapy. However, the majority of patients show little to no response, or they acquire resistance during treatment with checkpoint inhibitors delivered as a monotherapy. Therefore, identifying resistance mechanisms and potential combination therapy strategies is imperative to improve response rates to immune checkpoint inhibitors and patient prognosis. To address this, we developed a clinically relevant in vivo shRNA screen that focuses on genes encoding for FDA-approved drug targets (FDAome). We implanted Kras/p53 (KP) mutant murine lung cancer cells expressing the FDAome shRNA library into syngeneic mice treated with isotype control or anti-PD-1 antibody. Sequencing for the unique barcoded shRNAs revealed that neurotrophic receptor tyrosine kinase 1 (Ntrk1) was significantly depleted from tumors challenged with PD-1 blockade, suggesting that it provides a survival advantage to tumor cells when under immune system pressure. To confirm Ntrk1 as a hit from the screen, we analyzed tumors treated with anti-PD-1 antibody and found that transcript levels of Ntrk1 are significantly upregulated with treatment, and cell lines derived from tumors with acquired resistance to anti-PD-1 treatment also have higher levels of Ntrk1 expression. Additionally, in vitro co-culture assays revealed that Ntrk1 overexpressing cells upregulate PD-L1 and other immunosuppressive markers when cultured with splenocytes, and this upregulation was inhibited through pharmacologic targeting of either Trk or Jak signaling. Stable knockdown of Ntrk1 in murine KP mutant lung cancer cells reduced Jak signaling and downregulation of PD-L1. In vivo, Ntrk1 knockdown tumors demonstrated significantly reduced tumor growth, and analysis of tumor-infiltrating T cell populations via flow cytometry revealed significantly fewer CD25+/FoxP3+ T regulatory cells in Ntrk1 knockdown tumors, as well as reduced exhaustion of CD8+ cytotoxic T cells. In contrast, Ntrk1 overexpressing tumors had a significant increase in tumor growth, as well as a hyperexhaustive phenotype in the intratumoral CD8+ T cells. Together, these data suggest that Ntrk1 activity can modulate Jak/Stat signaling to regulate PD-L1 expression as well as the expression of other immunosuppressive molecules, thereby promoting an exhausted and suppressed tumor microenvironment. Citation Format: Jessica Konen, Bertha L. Rodriguez, Laura Gibson, Rosalba Minelli, Jeffrey Kovacs, Alessandro Carugo, Christopher Bristow, Timothy Heffernan, Don L. Gibbons. Identifying a role for Ntrk1 in regulating immune functionality and checkpoint blockade resistance in Kras/p53 mutant lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A43.

Human ovarian cancer intrinsic mechanisms regulate lymphocyte activation in response to immune checkpoint blockade

Immune checkpoint blocking antibodies are currently being tested in ovarian cancer (OC) patients and have shown some responses in early clinical trials. However, it remains unclear how human OC cancer cells regulate lymphocyte activation in response to therapy. In this study, we have established and optimised an in vitro tumour-immune co-culture system (TICS), which is specifically designed to quantify the activation of multiple primary human lymphocyte subsets and human cancer cell killing in response to PD-1/L1 blockade. Human OC cell lines and treatment naïve patient ascites show differential effects on lymphocyte activation and respond differently to PD-1 blocking antibody nivolumab in TICS. Using paired OC cell lines established prior to and after chemotherapy relapse, our data reveal that the resistant cells express low levels of HLA and respond poorly to nivolumab, relative to the treatment naïve cells. In accordance, knockdown of IFNγ receptor expression compromises response to nivolumab in the treatment naïve OC cell line, while enhanced HLA expression induced by a DNA methyltransferase inhibitor promotes lymphocyte activation in TICS. Altogether, our results suggest a ‘cross resistance’ model, where the acquired chemotherapy resistance in cancer cells may confer resistance to immune checkpoint blockade therapy through down-regulation of antigen presentation machinery. As such, agents that can restore HLA expression may be a suitable combination partner for immunotherapy in chemotherapy-relapsed human ovarian cancer patients.

Development and characterization of a novel anti-OX40 antibody for potent immune activation.

With the great success of anti-CTLA-4 and anti-PD-1 therapeutics in cancer immunotherapy, tumor necrosis factor receptor superfamily members have been recognized as ideal targets to provide co-stimulatory signals in combination with immune checkpoint blocking antibodies. Among these is OX40 (CD134), a co-stimulatory molecule expressed by activated immune cells. Recently, several anti-OX40 agonistic monoclonal antibodies, pogalizumab as the most advanced, have entered early phase clinical trials. Using ayeast platform and multiple screening methods, we identified a fully human anti-OX40 antibody (IBI101) with distinct modes of action. Unlike pogalizumab, IBI101 partially blocks the binding of OX40 to its ligand OX40L and exhibits both FcγR-dependent and independent agonistic activities in NF-κB luciferase reporter assays. IBI101 also promotes T cell activation and proliferation in vitro. These unique properties partially explain the more potent anti-tumor activity of IBI101 than that of pogalizumab in humanized NOG mice bearing LoVo tumors. In addition, IBI101 shows efficacious anti-tumor activity in mice when administrated alone or in combination with anti-PD-1 antibodies. In human OX40 knock-in mice bearing MC38 colon carcinoma, IBI101 treatment induces tumor antigen-specific CD8+ T-cell responses, decreases immunosuppressive regulatory T cells in tumor, and enhances the immune response to PD-1 inhibition. Preclinical studies of IBI101 in non-human primates demonstrate typical pharmacokinetic characteristics of an IgG antibody and no drug-related toxicity. Collectively, IBI101 has desirable preclinical attributes which support its clinical development for cancer treatment.

Initial development of biosimilar immune checkpoint blockers using HEK293 cells

Antibodies that block interaction of immune checkpoint receptors with its ligands have revolutionized the treatment of several cancers. Despite the success of this approach, the high cost has been restricted the use of this class of drugs. In this context, the development of biosimilar can be an important strategy for reducing prices and expanding access after patent has been dropped. Here, we evaluated the use of HEK293 cells for transient expression of an immune checkpoint-blocking antibody as a first step for biosimilar development. Antibody light and heavy chain genes were cloned into pCI-neo vector and transiently expressed in HEK293 cells. The culture supernatant was then subjected to protein A affinity chromatography, which allowed to obtain the antibody with high homogeneity. For physicochemical comparability, biosimilar antibody and reference drug were analyzed by SDS-PAGE, isoelectric focusing, circular dichroism and fluorescence spectroscopy. The results indicated that the both antibodies have a high degree of structural similarity. Lastly, the biosimilar antibody binding capacity to target receptor was shown to be similar to reference product in ELISA and flow cytometry assays. These data demonstrate that the HEK293 system can be used as an important tool for candidate selection and early development of biosimilar antibodies.

TGF-β inhibition combined with cytotoxic nanomedicine normalizes triple negative breast cancer microenvironment towards anti-tumor immunity.

Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored.Methods: Here, we re-purposed the TGF-β inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors.Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4.Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.