CTLA-4 Blocking Antibodies Research Articles

Abstract 2721: Sustained delivery of low-dose anti-CTLA-4 by genetically engineered encapsulated cells to the tumor microenvironment drives tumor response and prolongs survival in a colorectal cancer model

Abstract Introduction: Systemic therapy with CTLA-4 blocking antibody (aCTLA-4) restores endogenous antitumor immunity and induces remarkable long-term clinical benefits in patients with melanoma. Yet immune-related side effects remain a major hurdle to extending its label to many more types of cancer. Intra- and peritumoral administration of aCTLA-4 has recently emerged as a means to optimize its dose/efficacy ratio while preventing its off-target, systemic toxicities. Sustained peritumoral delivery of low-dose aCTLA-4 by genetically engineered encapsulated cells could offer a promising option for cancer treatment addressing the shortcomings of systemic therapy. Material and Methods: The efficacy of sustained, low-dose aCTLA-4 delivered by genetically engineered encapsulated cells was assessed in human CTLA4 knock-in mice subcutaneously engrafted with MC38 colon adenocarcinoma cells. In a therapeutic model, mice were treated with either encapsulated human myoblast cells secreting aCTLA-4 (MVX-3) in the vicinity of the tumor, or i.p. injections of Ipilimumab (10mg/kg BIW x 4). Encapsulated wild-type cells (WT) and PBS were used as respective controls. A subgroup of mice was terminated at day 7 post-treatment for early pharmacological characterization. Tumor size and survival were measured to evaluate the efficacy. Secretion of aCTLA-4 by MVX-3 and aCTLA-4 titer in plasma was quantified by ELISA. Tumors, spleens, and draining lymph nodes were collected for flow cytometry analyses. Results: Peritumoral administration of MVX-3 induced durable complete tumor rejection (2/7) and tumor growth control (4/7) when administered at doses 1’000 times lower than i.p ipilimumab, whereas rapid tumor growth without any tumor rejection was observed in mice treated with control vehicles. I.p. ipilimumab induced durable complete tumor rejection (9/12), while treatment-related toxicities upon dosing led to premature mice termination (3/12). MVX-3 was found as equally effective as i.p. ipilimumab in decreasing the proportion of CTLA4+ helper and regulatory T cells in the tumor at Day 7 post-treatment. Survival was also improved by MVX-3 compared to control vehicles. Conclusions: These findings suggest that sustained delivery of low-dose aCTLA-4 by genetically engineered encapsulated cells to the tumor microenvironment could achieve similar therapeutic benefits as systemic therapy, without the commonly associated toxicities. The safety and biological efficacy profile of MVX-3 encourage further preclinical and clinical explorations. Citation Format: Julien Grogg, Emily Charrier, Remi Vernet, Muriel Urwyler, Olivier Von Rohr, Valentin Saingier, Fabien Courtout, Aurelien Lathuiliere, Adrien Engel, Nicolas Mach. Sustained delivery of low-dose anti-CTLA-4 by genetically engineered encapsulated cells to the tumor microenvironment drives tumor response and prolongs survival in a colorectal cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2721.

Abstract CT013: S1616: Ipilimumab plus nivolumab versus ipilimumab alone in patients with metastatic or unresectable melanoma that did not respond to anti-PD-1 therapy

Abstract Background: Patients with advanced melanoma primarily refractory to single agent PD-1 blockade therapy have an option of receiving the CTLA-4 blocking antibody ipilimumab, but if ipilimumab should be given as a single agent or in combination with the anti-PD-1 nivolumab has not been established prospectively. Methods: Patients aged >18 with metastatic or unresectable melanoma without objective response to anti-PD-1 therapy given without CTLA-4 therapy were randomized 3:1 to receive either ipilimumab 3mg/kg + nivolumab 1mg/kg q3 wks x4 cycles followed by nivolumab 480mg q4wks (ipi/nivo) up to 2 years, or ipilimumab 3mg/kg q3weeks x4 cycles (ipi). Additional key eligibility criteria included ECOG Performance Statue (PS) 0-2, no active central nervous system metastases, autoimmune disease, or need for steroids at doses of >10 mg of prednisone or the equivalent. The primary endpoint was progression free survival (PFS). Disease assessments were performed every 12 weeks until progression. Secondary endpoints included overall survival (OS), objective response rate (ORR), and toxicity. All patients were to submit a fresh tumor biopsy and whole blood for correlative endpoints prior to cycle 1 and again at week 5. Results: 92 eligible patients were enrolled, 69 to ipi/nivo, 23 to ipi. Median age was 64 and 69 in the ipi/nivo and ipi arm respectively. 67% and 65% were male. 65% of patients in both arms had ECOG PS of 0. With a median follow up of 25.3 months, the hazard ratio (HR) for PFS was 0.63 (90% CI 0.41, 0.97) with a statistically significant 1-sided p-value of 0.04 favoring ipi/nivo. The 6-month PFS estimates were 34% (90% CI: 25%-44%) and 13% (4%-27%) for ipi/nivo and ipi respectively. ORR was 28% for ipi/nivo (95% CI 17%-40%) and 9% for ipi (95% CI: 3%-34%). With a median follow up of 24.4 months, 39/69 patients in the ipi/nivo arm and 12/23 patients in the ipi arm had died. 12-month OS was 63% (90% CI 52%-72%) in the ipi/nivo arm and 57% (38%-71%) months in the ipi arm. HR for OS was 0.94 (90% CI 0.54, 1.62) in favor of ipi/nivo with a p-value of 0.42. Adverse event rates were similar in both arms. One treatment related death was reported in the ipi/nivo arm due to disseminated intravascular coagulation and one treatment related death was reported in the ipi arm due to colonic perforation. Conclusions: This is the first prospective randomized study comparing ipi/nivo to ipi alone in patients with melanoma without response to anti-PD1 therapy. Ipi/nivo was associated with improved progression free survival as compared to ipi alone. The response rate of 28% to ipi/nivo as compared to 9% to ipi alone implies that patients who do not respond to PD-1 alone can be rescued with ipi/nivo. The toxicity of combination therapy was manageable. Ipi/nivo is an appropriate standard in patients with metastatic melanoma who do not respond to single-agent PD-1 therapy. ClinicalTrials.gov Identifier: NCT03033576 Funding: NIH/NCI grants: U10CA180888, U10CA180819, U10CA180821, U10CA180868; Other grants: SU2C-AACR-CT06-17 Citation Format: Ari M. Vanderwalde, James Moon, Kari Kendra, Nikhil I. Khushalani, Frances Collichio, Jeffrey A. Sosman, Alexandra Ikeguchi, Adrienne I. Victor, Thach-Giao Truong, Bartosz Chmielowski, David C. Portnoy, Michael C. Wu, Kenneth F. Grossmann, Antoni Ribas. S1616: Ipilimumab plus nivolumab versus ipilimumab alone in patients with metastatic or unresectable melanoma that did not respond to anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT013.

NCT04552223: A phase II study of nivolumab plus BMS-986016 (relatlimab) in patients with metastatic uveal melanoma (UM) (CA224-094).

TPS9590 Background: Uveal melanoma (UM) is a rare disease but 50% of patients will eventually develop metastatic disease, for which not effective therapy is available. Liver-directed therapies, immunotherapy, targeted therapy and chemotherapy have limited activity [1]. Lymphocyte activation gene 3(LAG-3) is an immune checkpoint receptor associated with decreased T-cell effector function and tumor escape. Preclinical models have shown that dual inhibition of LAG-3 and PD-1 blockade generates synergistic anti-tumor activity [2]. Recent preclinical data indicates that uveal melanoma CD8+ T cells express the checkpoint receptor LAG3 to a greater extent than PD1 or CTLA4 [3,4]. Therefore, LAG3 is a potential candidate for checkpoint inhibitor immunotherapy in UM. Relatlimab is a human LAG-3-specific antibody isolated from immunized transgenic mice which binds to a defined epitope on LAG-3 with high affinity and specificity and potently blocks the interaction of LAG-3 with its ligand, MHC Class II. Methods: This is an open-label, single arm, single site investigator-initiated phase II study, NCT04552223. Based on Simon two-stage minimax design, 13 patients will be enrolled in Stage 1. If at least one response is noted, the study will proceed to Stage 2 and enroll an additional 14 patients. The null hypothesis will be rejected if 4 or more responses are observed among 27 patients. This design achieves 5% type I error and 80% power when the true ORR is 20%. The trial opened to accrual in December 2020. As of February 15, 2021 four patients had been enrolled the first stage of accrual. Main eligibility criteria include patients with biopsy proven metastatic uveal melanoma, previously untreated with PD-1, CTLA-4 and/or LAG-3 blocking antibodies and in good performance status. Enrolled patients are treated in the outpatient setting. Nivolumab 480 mg is mixed in the same bag with relatlimab 160 mg and administered intravenously over 60 minutes every 4 weeks until disease progression or intolerable toxicity for up to 24 months. The primary endpoint is best objective response rate (ORR). Secondary endpoints include disease control rate (DCR), progression-free survival (PFS), overall survival (OS), median duration of response (mDOR), and adverse events (AEs). Correlative studies will evaluate pre- and post-treatment characteristics of T cells in the tumor microenvironment and blood. Clinical trial information: NCT04552223.

Abstract 5734: Gut microbiota predicts response and toxicity with neoadjuvant immunotherapy

Abstract Introduction: Checkpoint blockade immunotherapy has revolutionized melanoma treatment. However, immune related adverse events (irAEs) are frequent, particularly when PD-1 and CTLA-4 blocking antibodies are combined. There is an urgent need for biomarkers to predict response as well as toxicity. The intestinal microbiome influences immune processes throughout the body and is therefore hypothesised to influence both immunotherapy response and well as development of irAEs. Methods: This study utilized sequencing to profile the fecal microbiome from Stage III melanoma patients (n= 38) treated with combination anti-PD1 and anti-CTLA4 antibodies in the neoadjuvant setting (OpacinNeo Trial; NCT02977052) at Melanoma Institute Australia. Absolute bacterial loads were determined with qPCR. Global microbial gene abundance was determined through metagenomic sequencing, allowing analysis of microbial metabolic capcity, and actual short chain fatty acid output (butyrate, acetate, propionate) was assessed by NMR. Matched PBMC were profiled in detail using mass cytometry and immune populations were correlated with microbial metrics. We tested the ability of machine learning to predict therapeutic response and irAEs from microbial data. Results: Patients who failed to respond (n=6), and those who developed severe (G3-G5) irAEs (n=11), had low microbial diversity before treatment (p=0.0234 and p=0.0226 respectively). Four of the six non-responders in this cohort failed to complete treatment and received immunosuppression before pathologic assessment of response, because of the development of severe irAEs. Thus, non-response may, in fact, have been a consequence of irAE development in those cases. High pre-treatment loads of Ruminococcaceae and methanogenic archaea, and greater relative abundance of butyrate production pathways, were significantly associated with response and protection from irAEs. Patients who had low bacterial diversity had increased proportions of effector and memory CD4 and CD8 T cells in the blood and increased numbers of circulating MAIT, NK and gdT cells prior to immunotherapy. Importantly, microbial taxa were predictive of patients who subsequently failed to respond to therapy while also developing severe irAEs with overall accuracy of 92.1%. Conclusions: A gut ecosystem enriched with beneficial microbial functions including production of butyrate is associated with protection from irAEs with neoadjuvant combination immunotherapy. The findings allow prediction of outcomes with combination immunotherapy and will inform development of therapeutic microbial interventions. Citation Format: Marcel Batten, Erin Shanahan, Rebecca Simpson, Mark Read, Ines P. Silva, Alexandra Angelatos, Jian Tan, Chandra Adhikari, Alexander M. Menzies, Robyn P. Saw, Laurence Macia, Maria Gonzalez, Kerwin Shannon, Rebecca Velickovic, Irene L. Reijers, Christian U. Blank, James S. Wilmott, Andrew J. Holmes, Richard A. Scolyer, Georgina V. Long. Gut microbiota predicts response and toxicity with neoadjuvant immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5734.

Combined Anti-Cancer Strategies Based on Anti-Checkpoint Inhibitor Antibodies.

Therapeutic monoclonal antibodies for the treatment of cancer came of age in 1997, with the approval of anti-CD20 Rituximab. Since then, a wide variety of antibodies have been developed with many different formats and mechanisms of action. Among these, antibodies blocking immune checkpoint inhibitors (ICI) have revolutionized the field, based on the novelty of their concept and their demonstrated efficacy in several types of cancer otherwise lacking effective immunotherapy approaches. ICI are expressed by tumor, stromal or immune cells infiltrating the tumor microenvironment, and negatively regulate anti-tumor immunity. Antibodies against the first discovered ICI, CTLA-4, PD-1 and PD-L1, have shown significant activity in phase III studies against melanoma and other solid cancers, alone or in combination with chemotherapy or radiotherapy. However, not all cancers and not all patients respond to these drugs. Therefore, novel antibodies targeting additional ICI are currently being developed. In addition, CTLA-4, PD-1 and PD-L1 blocking antibodies are being combined with each other or with other antibodies targeting novel ICI, immunostimulatory molecules, tumor antigens, angiogenic factors, complement receptors, or with T cell engaging bispecific antibodies (BsAb), with the aim of obtaining synergistic effects with minimal toxicity. In this review, we summarize the biological aspects behind such combinations and review some of the most important clinical data on ICI-specific antibodies.

Genomic landscape of young ATLL patients identifies frequent targetable CD28 fusions

AbstractAdult T-cell leukemia/lymphoma (ATLL) in Japan presents at a median age of 70 years and only 5% of patients are <50 years of age. We conducted RNA and targeted DNA sequencing of 8 ATLLs from Japanese patients <50 years of age and identified 3 (37.5%) with both CTLA4-CD28 and inducible costimulator (ICOS)-CD28 fusions. Mutations of PLCG1, PRKCB, and STAT3, which were frequent in other ATLL-sequencing studies, were not identified. Differential expression analysis identified the negative checkpoint molecule LAG3 as the most downregulated gene among cases with the fusions. Immunohistochemistry demonstrated expression of CD80 and CD86, the ligands for CTLA4 and CD28, on ATLL cells and tumor-associated macrophages, respectively. Expression of CTLA4-CD28 in Ba/F3 cells conferred cytokine-independent growth when cocultured with Raji cells that express CD80 and CD86. Growth was associated with recruitment of the p85 subunit of phosphatidylinositol 3-kinase to CTLA4-CD28 and phosphorylation of AKT and extracellular signal-regulated kinase. A CTLA4-blocking antibody reduced cytokine-independent growth in a dose-dependent manner. Together, these results suggest that young Japanese ATLL cases have a unique biology dependent on cell-nonautonomous interactions that drive CD28 signaling. Assessment for CD28 fusions and treatment with CTLA4 blockade should be considered in younger patients with relapsed/refractory ATLL.

CTLA-4 blockade boosts the expansion of tumor-reactive CD8+ tumor-infiltrating lymphocytes in ovarian cancer

Adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) can induce durable complete tumor regression in patients with advanced melanoma. Efforts are currently underway to expand this treatment modality to other cancer types. In the microenvironment of ovarian cancer, the engagement of co-inhibitory immune checkpoint molecules such as CTLA-4 can lead to the inactivation of TILs. Thus, approaches that directly manipulate co-inhibitory pathways within the tumor microenvironment might improve the expansion of tumor-reactive TILs. The initial expansion of TILs for ACT from tumor fragments provides a window of opportunity to manipulate an intact tumor microenvironment and improve CD8+ T-cell output and TIL tumor reactivity. To exploit this, we used a CTLA-4-blocking antibody, added during the initial TIL culture, and found that the blockade of CTLA-4 favored the propagation of CD8+ TILs from ovarian tumor fragments. Interestingly, adding the CTLA-4 blocking antibody in the initial phase of the TIL culture resulted in more potent anti-tumor TILs in comparison to standard TIL cultures. This phenotype was preserved during the rapid expansion phase. Thus, targeting CTLA-4 within the intact tumor microenvironment of tumor fragments enriches tumor-reactive TILs and may improve clinical outcome of TIL-based ACT in ovarian cancer.

Immune checkpoint modulation enhances HIV-1 antibody induction

Eliciting protective titers of HIV-1 broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development, but current vaccine strategies have yet to induce bnAbs in humans. Many bnAbs isolated from HIV-1-infected individuals are encoded by immunoglobulin gene rearrangments with infrequent naive B cell precursors and with unusual genetic features that may be subject to host regulatory control. Here, we administer antibodies targeting immune cell regulatory receptors CTLA-4, PD-1 or OX40 along with HIV envelope (Env) vaccines to rhesus macaques and bnAb immunoglobulin knock-in (KI) mice expressing diverse precursors of CD4 binding site HIV-1 bnAbs. CTLA-4 blockade augments HIV-1 Env antibody responses in macaques, and in a bnAb-precursor mouse model, CTLA-4 blocking or OX40 agonist antibodies increase germinal center B and T follicular helper cells and plasma neutralizing antibodies. Thus, modulation of CTLA-4 or OX40 immune checkpoints during vaccination can promote germinal center activity and enhance HIV-1 Env antibody responses.

Genomic Landscape of Young ATLL Patients Identifies Frequent Targetable CD28 Fusions

Introduction Adult T-cell leukemia/lymphoma (ATLL) is a T-cell neoplasm induced by human T-cell leukemia virus type 1 (HTLV-1). In endemic areas, HTLV-1 infection typically occurs during breastfeeding but the median age of ATLL presentation in Japan is &gt;70. Approximately 5% of ATLL patients in Japan present at age &lt;50. Here, we hypothesized that ATLL in younger patients will have distinct genetic alterations. Methods DNA and RNA samples were extracted from frozen lymph nodes. Targeted capture sequencing for 98 genes and analysis of RNA sequencing (RNAseq) were performed in 8 samples of young patients with ATL (defined as ≤ 50 years in the current study) Results and discussion The most frequently mutated genes were CCR4 and CARD11, each in 3 of the analyzed cases (37.5%). Mutations of PLCG1, PRKCB and STAT3 were frequent in a previous genomic analysis (Kataoka et al. Nat Genet. 2015) that were not age-selected, but were not identified in our 8 cases. Three (37.5%) cases harbored concurrent CTLA4-CD28 and ICOS-CD28 fusions. This contrasts with previous reports, in which cases with both fusions were found in &lt;1% of PTCL and ATLL patients. We confirmed the presence of both fusions in all 3 cases by RT-PCR. The structure of the CTLA4-CD28 fusion suggests that the extracellular portion of CTLA4 is expressed on the cell surface where it can interact with CD80 and CD86 to activate signaling through the intracellular CD28 portion. In contrast to CTLA4-CD28, the ICOS-CD28 fusion links the N-terminal signal peptide of ICOS with the extracellular and intracellular portions of CD28. This fusion should simply result in overexpression of CD28 and haploinsufficiency of ICOS. To evaluate the function of CTLA4-CD28, we used Ba/F3 cells, which are strictly dependent on exogenous IL-3 for proliferation. We transduced Ba/F3 cells with CTLA4-CD28 or a CTLA4-CD28 mutant (mut) with three amino acid substitutions that abrogate CD28 signaling. We then cultured these cells in the presence or absence of Raji cells, which express CD80 and CD86. In the absence of Raji cells, neither CTLA4-CD28 nor CTLA4-CD28mut expression conferred IL-3-independent growth. However, Ba/F3 cells expressing CTLA4-CD28 cultured in the presence of Raji cells achieved IL-3 independence. This was not the case for Ba/F3 cells expressing CTLA4-CD28mut, indicating a requirement on functional CTLA4-CD28 signaling. Western-blot analysis identified activation of CD28 signal in Ba/F3 cells with CTLA4-CD28 co-cultured with Raji cells. These data indicate that ligand-bound CTLA4-CD28 can promote downstream signaling in the absence of an endogenous TCR complex. Gene set enrichment analysis (GSEA) comparing the 3 ATLL cases with CD28 fusions to the 5 cases that lacked fusions demonstrated enrichment of previously defined gene signatures associated with AKT and RAF signaling, both of which are downstream of CD28 activation. GSEA also identified enrichments of T-cell function-related signatures, including pathways involved in interferon responses, in cases with CD28 fusions Cases with CTLA4-CD28 and ICOS-CD28 fusions become haploinsufficient for CTLA4 and ICOS. As expected, these cases had lower expression of both genes by RNAseq. Interestingly, these cases also had higher expression of CD80. By immunohistochemistry, ATLL cells with CTLA4-CD28 and ICOS-CD28 fusions expressed CD80 and macrophages in the tumor microenvironment expressed CD86. Thus, both intra- and intercellular interactions could drive CTLA4-CD28 and ICOS-CD28 signaling in these cases. Treatment of Ba/F3 cells expressing CTLA4-CD28 and cultured in the presence of Raji cells with a CTLA4 blocking antibody suppressed proliferation in a dose-dependent fashion. A previous case report (Mol Genet Genomic Med, 2015) described a patient with Sezary Syndrome and CTLA4-CD28 fusion who had a deep but transient response to the anti-CTLA4 antibody ipilimumab. This study did not clarify the extent to which response resulted from blocking of cell-autonomous CTLA4-CD28 signaling versus activation of a cell non-autonomous immune response. Nonetheless, it strongly supports the testing of CTLA4 blockade in additional cases of T-cell malignancies, including ATLL, that harbor CTLA4-CD28 fusions. Disclosures Stevenson: Celgene: Research Funding. Ohshima:Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding; SRL, Inc.: Consultancy. Weinstock:Verastem Oncology: Research Funding; Celgene: Research Funding.

Insuffisance surrénalienne secondaire : actualités diagnostiques et thérapeutiques: News in diagnosis and therapeutics of secondary adrenal insufficiency

Immunotherapy and opioids treatment are new causes of secondary adrenal insufficiency (SAI). Prevalence of SAI with immunotherapy is more frequent with combined therapy (8% vs 4 to 10% with CTLA4 blocking antibody and 1% with PD1 blocking antibody). Although hypophysitis are more frequently observed with CTLA4 blocking antibody, some cases of Isolated SAI have been reported in patients treated by PD1 blocking antibody. SAI could be transient, requiring long-term monitoring. The use of opioid analgesics is increasing in many countries, thus becoming a public health problem. Prevalence of opioid-related SAI is unclear but recent prospective studies reveal a prevalence between 5 and 20%. The main risk factor to develop this pathology is morphine-equivalent daily dose. Diagnosis relies on 8.00 am plasma cortisol measurement and cortisol increase after Synacthen® administration. Recent cortisol immuno-assays, in agreement with mass spectrometry, give lower reference values, encouraging reevaluation of the current cut-off of 500 nmol/L. New modified-release hydrocortisone preparations have been recently developed to better mimic the physiological cortisol rhythm and to improve compliance in adrenocortical deficient patients. Nowadays, continuous subcutaneous hydrocortisone infusion seems to be a unique replacement therapy allowing adequate circadian biorhythm but should be restricted to specific patients due to the complexity of this substituting strategy.© 2019 Published by Elsevier Masson SAS. All rights reserved.Cet article fait partie du numéro supplément Les Must de l’Endocrinologie 2019 réalisé avec le soutien institutionnel de Ipsen-Pharma.

Oral tolerance to peanut allergy is mediated by CTLA-4-positive regulatory T cells

Abstract Peanut allergies are life-long diseases that cause severe anaphylactic responses to affected individuals. Oral ingestion of peanut products prevents infants from developing peanut allergy. However, little information is currently available regarding the immunologic mechanisms for this protection. Therefore, the goal of this project was to establish an oral tolerance model to peanut allergy in mice and to determine the immunologic mechanisms. Naïve BALB/c mice that were exposed intranasally (i.n.) to peanut flour (PNf) developed peanut allergy as demonstrated by increased serum levels of IgE antibody as well as acute anaphylactic response when challenged with peanut extract. When mice were fed peanut butter (PNB) prior to i.n PNf exposure, they were protected from developing peanut allergy; these mice showed decreased serum levels of IgE antibody and lacked anaphylactic response. T follicular helper (Tfh) cells and germinal center (GC) B cells were decreased in lung draining lymph nodes of mice fed PNB. Furthermore, scRNSseq revealed 2 distinct CD4+ T cell subpopulations that are increased in those protected mice. CTLA-4, a checkpoint molecule, was highly expressed in one of these subpopulations. Systemic administration of a CTLA-4 blocking antibody to mice fed PNB increased the numbers of Tfh cells and GC B cells and increased serum levels of IgE antibody. Finally, anti-CTLA-4 reversed the clinical efficacy of oral PNB administration, resulting in manifestation of acute anaphylaxis when challenged with peanut extract. Thus, oral administration of peanut product(s) prevents development of peanut allergy in mice. CTLA4+CD4+ T cells likely play a pivotal role in this process by regulating the development of Tfh cells.

The CTLA-4 x OX40 bispecific antibody ATOR-1015 induces anti-tumor effects through tumor-directed immune activation

BackgroundThe CTLA-4 blocking antibody ipilimumab has demonstrated substantial and durable effects in patients with melanoma. While CTLA-4 therapy, both as monotherapy and in combination with PD-1 targeting therapies, has great potential in many indications, the toxicities of the current treatment regimens may limit their use. Thus, there is a medical need for new CTLA-4 targeting therapies with improved benefit-risk profile.MethodsATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody generated by linking an optimized version of the Ig-like V-type domain of human CD86, a natural CTLA-4 ligand, to an agonistic OX40 antibody. In vitro evaluation of T-cell activation and T regulatory cell (Treg) depletion was performed using purified cells from healthy human donors or cell lines. In vivo anti-tumor responses were studied using human OX40 transgenic (knock-in) mice with established syngeneic tumors. Tumors and spleens from treated mice were analyzed for CD8+ T cell and Treg frequencies, T-cell activation markers and tumor localization using flow cytometry.ResultsATOR-1015 induces T-cell activation and Treg depletion in vitro. Treatment with ATOR-1015 reduces tumor growth and improves survival in several syngeneic tumor models, including bladder, colon and pancreas cancer models. It is further demonstrated that ATOR-1015 induces tumor-specific and long-term immunological memory and enhances the response to PD-1 inhibition. Moreover, ATOR-1015 localizes to the tumor area where it reduces the frequency of Tregs and increases the number and activation of CD8+ T cells.ConclusionsBy targeting CTLA-4 and OX40 simultaneously, ATOR-1015 is directed to the tumor area where it induces enhanced immune activation, and thus has the potential to be a next generation CTLA-4 targeting therapy with improved clinical efficacy and reduced toxicity. ATOR-1015 is also expected to act synergistically with anti-PD-1/PD-L1 therapy. The pre-clinical data support clinical development of ATOR-1015, and a first-in-human trial has started (NCT03782467).

A pilot study of interferon-alpha-2b dose reduction in the adjuvant therapy of high-risk melanoma

Melanoma is a highly immunogenic tumor and consequently, efforts have been centered on the development of immune-based treatments for this malignancy [1]. Interferons were initially described in the mid-1950s as proteins that interfere with viral replication [2, 3]. Interferons are cytokines that activate Janus kinases (Jak), which lead to phosphorylation and activation of transcription factors belonging to the signal transducer and activator of transcription (STAT) family [4, 5]. Interferon-alpha (IFN-α) became available for use in clinical trials in the mid-1980s [6]. Results suggested that IFN-α inhibited the proliferation of malignant cells and stimulated immune effectors; therefore, IFN-α was initially used in patients with advanced disease [7, 8]. Since then, several meta-analyses have demonstrated that high-dose adjuvant IFN-α (daily 20 MU/m2 intravenous induction therapy for 1 month followed by maintenance subcutaneous 10 MU/m2 three times per week for at least 1 year) can prolong the disease-free interval in high-risk melanoma patients [9]. The introduction of checkpoint inhibitor therapy has revolutionized the adjuvant therapy of melanoma; however, there remains a role for IFN-α in this setting based on the potential for cancer immune escape or autoimmune events with CTLA-4 and PD-1 blocking antibodies [10–14]. There has also been significant advances in mitogen-activated protein kinase (MAPK) targeted therapies, particularly for BRAF (an intracellular signaling kinase) and MEK (signaling molecule downstream of BRAF). A recent clinical trial demonstrated significant improvement in both relapse-free survival and overall survival with adjuvant dabrafenib (BRAF inhibitor) plus trametinib (MEK inhibitor) in patients with stage III melanoma. These therapies are now approved for adjuvant therapy in BRAF mutated tumors [15]. However, since only approximately 40–50% of melanoma cells harbor an activating BRAF mutation, there still remains a role for IFN-α in this setting as the remaining 50–60% of melanomas would not be susceptible to BRAF-targeted therapies. IFN-α activates the Jak-STAT signaling pathway and induces synthesis of hundreds of different proteins [4, 5]. Our group has shown that STAT1-mediated gene regulation within immune effectors is necessary for mediating the anti-tumor effects of IFN-α and also that the amount IFN-α administered to melanoma patients is likely in excess of the optimal biological dose [4]. Indeed, high doses of IFN-α appear to be no more effective in the induction of phosphorylated STAT1 (p-STAT1) and in the transcription of interferon-stimulated genes (ISGs) than intermediate doses [16, 17]. Our group’s studies in genetically manipulated mice have shown that suppressors of cytokine signaling-1 (SOCS1) and SOCS3 negatively regulate IFN-induced Jak-STAT signal transduction, gene regulation and anti-melanoma activity, and that high doses of IFN-α can induce SOCS proteins [18, 19]. We hypothesized that lower doses of IFN-α would be superior for induction of IFN signal transduction in patient immune cells. A prospective clinical trial was performed wherein patients eligible for adjuvant IFN-α-2b received 1 month of standard intravenous high-dose IFN-α-2b (20 MU/m2) followed by subcutaneous IFN-α-2b at a dose of 10 MU/m2 with dose reductions at set intervals down to a level of 4 MU/m2. Jak-STAT signal transduction and transcription of ISGs in patient peripheral blood mononuclear cells (PBMCs) were monitored during the course of adjuvant IFN-α therapy. The objective of this pilot study was to determine if lower doses of IFN-α were as effective in the induction of IFN signal transduction and gene expression as the standard high dose regimen.

Abstract 3778: Combination immunotherapy intercepts murine pancreatic cancer progression

Abstract Immunotherapy agents that activate T cells are changing clinical practice for some metastatic cancer patients. Vaccines targeting viral oncogenes prevent the development of hepatocellular and cervical cancers. We therefore tested the hypothesis that a vaccine targeting the oncogene that initiates pancreatic cancer (PDA) given in combination with immunotherapy agents that reprogram the earliest procarcinogenic inflammatory changes will slow (intercept) PDA development. The KRAS proto-oncogene is a driver mutation expressed in &amp;gt;90% of pancreatic intraepithelial neoplasms (PanINs) and an ideal candidate target for early vaccination against PDA. However, even early PanINs have immunosuppressive microenvironments highly expressing inhibitory immune checkpoints like CTLA-4 and infiltrating immunosuppressive cells such as regulatory T cells (Tregs). The overall goal of this research is to reprogram the immunosuppressive environment while specifically activating immunity against early premalignancies to prevent PDA development. We previously reported that early dosing of KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, which endogenously recapitulate human PDA tumorigenesis, with the Treg depleting and inactivating agents cyclophosphamide (cy) and PC61 antibody in combination with an attenuated Listeria monocytogenes vaccine expressing mutant KrasG12D (LM-Kras) can slow tumorigenesis in 40% of mice. In this study we attempted to improve the efficacy of a vaccine approach by specifically inhibiting the immunosuppressive effects of the CTLA-4 immune checkpoint on Tregs with a CTLA-4 blocking antibody. KPC mice at 4-5 weeks with early PanINs received 2 doses of LM-Kras and cy/PC61 and 8 doses of anti-CTLA-4. Control mice received isotype antibody with LM-Kras and cy/PC61 or no treatment. At 10-11 weeks, pancreata were pathologically evaluated and assigned grades normal, low (1) and high-grade (2 and 3) PanINs, and PDA. Mice receiving LM-Kras + anti-CTLA-4 had a significantly lower incidence of low- and high-grade PanINs and PDA compared to controls. Furthermore, mice receiving LM-Kras, cy/PC61, and anti-CTLA-4 did not develop lesions beyond PanIN 1, indicating that the combination therapy may halt PDA development at early stages. To investigate the immune profiles of mice receiving treatment, flow cytometry was performed on tumor-infiltrating lymphocytes (TILs) isolated from pancreata to assess abundance of Tregs, infiltration of effector and memory T cell populations, and expression of inhibitory immune checkpoint molecules. Mice receiving combination therapy had decreased numbers of Tregs, increased numbers of effector and memory T cells, and increased CD8+ T cells to Tregs ratio in the microenvironment compared to controls. Further studies using immunohistochemistry and RNA expression profiles are under way to further characterize the immune changes in the microenvironment following treatment. Citation Format: Aleksandra Popovic, Nina J. Chu, Todd Armstrong, Elizabeth M. Jaffee. Combination immunotherapy intercepts murine pancreatic cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3778.

CTLA-4 blockade induces indoleamine 2,3-dioxygenase where treatment with 1- methyl-d-tryptophan displays greater treatment effect in tumors with high ido expression

Background: As hepatocellular carcinoma (HCC) traditionally occurs in chronically inflamed livers, these tumors are often immunogenic and therefore potential targets for immunotherapy. CTLA-4 blockade has been utilized in two trails in HCC with promising results. However, not all patients have the same response to therapy by a largely unknown mechanism. Indoleamine 2,3- dioxygenase (IDO) with its two forms, IDO1 and IDO2, is part of kynurenine pathway for the metabolism of the essential amino acid tryptophan. It has been demonstrated that IDO acts as an immunosuppressive enzyme altering the tumor microenvironment and is upregulated in inflammatory states with the most potent inducer being IFN-γ. High levels of IDO in tumors including HCC have been associated with a poor prognosis. This study aimed at identifying IDO as a potential mechanism of immune escape from CTLA-4 directed therapy. Methods: The drug 1-methyl-D-tryptophan (1-D-MT), which has been shown to inhibit IDO2 activity, was utilized alone or in combination with a CTLA-4 blocking antibody in treatment of mice with subcutaneous and orthotropic HCC. Induction of IDO RNA in tumor cells was evaluated by quantitative polymerase chain reaction (qPCR). Results: Combination therapy with 1-D-MT and an anti-CTLA-4 blocking antibody produces a greater anti-tumor effect in both a subcutaneous and orthotropic model than either drug alone. Additionally, tumors with high IDO induction showed a better response to combination treatment. Mice injected with anti-CTLA-4 antibody showed higher IDO2 expression in tumors. Conclusion: These results provide evidence that IDO expression induces an additional effect of immunosuppression in the tumor microenvironment, especially after anti-CTLA-4 therapy, and support a combination therapy with IDO inhibitors for a successful immunotherapy treatment.

Targeting CD38 to improve anti-PD-1/CTLA-4 combination therapy in lung cancer.

144 Background: The combination of anti-PD-1 and anti-CTLA-4 is a promising strategy that is being clinically explored to treat a variety of cancer types. Some patients display primary resistance to this combination treatment, while others relapse after treatment. Although some cancer patients have long-term durable responses to the combination therapy of anti-PD-1 and anti-CTLA-4, physicians have been looking for biomarkers that predict response. Methods: Using multiple immunocompetent syngeneic and K-rasLA1/+p53R172H?g/+ spontaneous animal models of lung cancer, we have explored the mechanisms of resistance to anti-PD-1/CTLA-4 combination therapy by evaluating the molecular and cellular immune profiles of the tumor microenvironment. Antibody-mediated cell depletion assays were also conducted to validate the mechanisms. Lastly, to determine the applicability to patients, we analyzed 791 lung cancer patients’ specimens with immunohistochemistry staining and mined many immune markers in multiple large independent patient databases (~1900 tumors). Results: We observed that tumor bearing mice treated with combined PD-1 and CTLA-4 blocking antibodies developed resistance through the up-regulation of CD38, and that targeting CD38 abolished the resistance in a manner dependent on B7-costimulation. Additionally, combined PD-1 and CTLA-4 blockade eradicates CD38-deficient tumors in mice. Further study revealed that control of tumors by triple blockade of CD38, PD-1, and CTLA-4 depended on improved functionality of CD4+/CD8+ TILs, which were reactivated by enriched CD103+ dendritic cells in the tumor microenvironment. Pathologic analysis revealed positive immunohistochemical staining for CD38 on tumor cells in 15-23% of cases and bioinformatic analyses revealed a strong correlation between CD38 expression and an immune inflammatory signature. Conclusions: Targeting CD38 improves the efficacy of anti-PD-1/CTLA-4 combination therapy in lung cancer. CD38 on tumor cells could potentially serve as a novel biomarker of resistance for immune checkpoint inhibition.

Development of anti-drug antibodies is associated with shortened survival in patients with metastatic melanoma treated with ipilimumab

ABSTRACTIntroduction: Checkpoint inhibitors, including the CTLA-4 blocking antibody ipilimumab, have become the new standard therapy for many metastatic cancers. Development of anti-drug antibodies (ADAs) after treatment with other biopharmaceuticals has been thoroughly described, but the induction of ADAs after treatment with checkpoint inhibitors has been inadequately investigated. In this retrospective study, we relate ipilimumab serum levels and anti-ipilimumab antibody levels to clinical outcomes in patients with metastatic melanoma (MM).Method: Serum samples from 31 patients with MM were analyzed for serum levels of ipilimumab and ADAs to ipilimumab at baseline, and before the 2nd and 4th infusion using an in-house bead-based assay. The results were correlated with progression-free survival (PFS) and overall survival (OS).Results: Low serum levels of ipilimumab before the 2nd infusion correlated significantly with a shorter OS (p = 0.01) and PFS (p = 0.02). Eight patients (26%) were ADA-positive at either timepoint. ADA positivity correlated significantly with a shorter OS (p = 0.03) with a hazard ratio (HR) of 3.0 (95% CI: 1.2-7.8). Four of 8 ADA-positive patients (50%) discontinued therapy before the 4th infusion due to disease progression, compared to three of 23 (13%) ADA-negative patients.Conclusion: We confirm that low serum levels of ipilimumab are associated with a shortened OS, and we show for the first time that ADAs to ipilimumab are associated with shorter OS in patients with MM.

Abstract IA05: Selective pressure by activated T cells identifies de novo and adaptive resistance mechanisms in melanoma

Abstract Melanoma has been the tumor type in which immune checkpoint antibodies have been directed for the longest time in clinical trials and clinical practice. Compared to many other advanced solid tumors, a significant minority of patients have manifestations of metastatic disease that are amenable to minimally invasive biopsies to fuel translational research. With CTLA-4 blocking antibodies, the response rate was only 10% and insights into predictive biomarkers only began to emerge once large numbers of patients had been treated and with long-term follow-up. However, with PD-1/PD-L1 blocking antibodies response rates as high as 40% have been observed in previously untreated melanoma patients, making it far more tractable to glean insights into differences between responders and nonresponders in smaller number of patients. Emerging data from analysis of pretreatment tumor biopsies strongly support the greater, but still not high, likelihood of patients responding to PD-1/PD-L1 antibody-based therapy if there are robust numbers of CD8+ T cells infiltrating into a tumor prior to the start of therapy. Response prediction appears to be improved by assessment of T-cell infiltration into tumors early in the course of therapy. Modest improvement in response prediction appears to be achieved by accounting for the presence of regulatory T cells, with those tumors having the highest effector CD8+/Treg ratio being the most likely to respond. Additional predictive accuracy is contributed by a marker of T-cell activation, such as granzyme B, which would only be scored as positive if there are T cells present in the first place. While many additional modulators of T-cell entry and function have been implicated as mechanistically related to immune checkpoint antibody efficacy/resistance, none have been shown to have add predictive accuracy for response. A parallel body of evidence has developed indicating that the presence of predicted mutated neoantigens can predict response to immune checkpoint antibodies in melanoma. It is notable that melanoma has the highest number of mutations per megabase of any common solid tumor, which is thought to associate with the high response rate (40%) compared to other cancers in which PD-1/PD-L1 antibodies have demonstrated efficacy leading to regulatory approvals (15-20% response rates). The current state-of-the-art approach for predicting the presence of mutated antigens is algorithm-based, matching altered peptide sequences with a patient's HLA type to rank potential high-affinity binders. A very small number of such antigens have been validated by the presence of specific T-cell clones that recognize those antigens in patients. Nonetheless, response prediction appears robust and roughly compared to that achieved with phenotypic markers of tumor recognition described above, particularly when predicted mutated neoantigens are clonal. The most recent area of active investigation pertains to the phenotype of tumors with de novo resistance to immune checkpoint antibodies and mechanisms of acquire resistance. Early work suggests that the same phenotype of resistance associated with MAP kinase pathway targeted therapy (tumor cells that lose expression of melanocyte markers in favor of neural crest markers) predicts nonresponse to PD-1 antibodies. Very few patients with progression following response have been interrogated to date, but genetic loss of components of the MHC complex and inactivating mutations in the interferon signaling pathway in tumor cells appear to account for a significant proportion of cases. Citation Format: Keith T. Flaherty. Selective pressure by activated T cells identifies de novo and adaptive resistance mechanisms in melanoma [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr IA05.

Photodynamic-Immune Checkpoint Therapy Eradicates Local and Distant Tumors by CD8+ T Cells

Photodynamic therapy (PDT) is a clinically applied tumor ablation method that reduces tumor burden and may induce T-cell responses, providing a therapeutic option for mutated tumors. In this study, we applied PDT in two mouse tumor models and assessed its effect on outgrowth of PDT-treated and distant untreated tumors. PDT of established tumors resulted in complete tumor eradication in most mice, which were then protected against tumor rechallenge. Correspondingly, the therapeutic effect was abrogated upon systemic depletion of CD8+ T cells, indicating PDT-induced tumor antigen cross-presentation and T-cell activation. In a double-tumor model, PDT of primary tumors induced enhanced infiltration of untreated distant tumors by CD8+ T cells, which significantly delayed their outgrowth. Combination therapy of PDT and CTLA-4-blocking antibodies significantly improved therapeutic efficacy and survival of double-tumor-bearing mice. These results show that local tumor ablation by PDT induces CD8+ T-cell responses crucial for systemic tumor eradication, which can be further enhanced by combination with immune checkpoint blockade. This combination of two clinically applied therapies may be a treatment strategy for advanced cancer without previous knowledge of tumor-specific antigens. Cancer Immunol Res; 5(10); 832-8. ©2017 AACR.

Ipilimumab-induced thrombotic thrombocytopenic purpura (TTP)

BackgroundCTLA-4 (Cytotoxic T-lymphocyte-associated protein 4) was the first immune checkpoint receptor clinically targeted for use in cancer treatment. It is expressed exclusively on T-cells where its primary role is to regulate the amplitude of the early stages of T-cell activation.1 Ipilimumab, a CTLA-4 blocking antibody, has been widely used for the treatment of patients with high risk and metastatic melanoma. Given its mechanism of action and consequent immune activation, the side effect profile of this drug greatly differs from that of standard cytotoxic chemotherapy. Adverse events are from the most part immune-mediated, ranging from the more common, such as rash and fatigue, to the less common, such as immune endocrinopathy and colitis.Case presentationWe describe a case of immune-mediated thrombotic thrombocytopenic purpura (TTP) in a 68 year-old woman with high risk, stage III melanoma occurring after 3 cycles of adjuvant treatment with ipilimumab as part of a clinical trial.ConclusionThe range of immune-mediated adverse events during treatment with ipilimumab is wide and varied and clinicians should have a high degree of suspicion when managing these patients.