Howard Streicher

Antigen Processing for Presentation to T Lymphocytes: Function, Mechanisms, and Implications for the T-Cell Repertoire

Antigen processing encompasses the metabolic events that a protein antigen must undergo in or on the antigen-presenting cell before it can be recognized by the T lymphocyte. It appears that a primary goal of these events is to unfold the protein to expose residues that are buried in the native conformation, which is designed to be soluble in water. The APC usually accomplishes this task by proteolytic cleavage of the protein, but we have found that artificial unfolding without proteolysis is sufficient. The purpose of unfolding may be to allow different faces of the antigenic site to bind simultaneously to the T-cell receptor and the MHC molecule on the APC, or to interact with other structures on the membrane of the APC. This requirement for unfolding appears to apply to everything from small peptides to large multimeric proteins. We have found that the way the antigen is processed and the structure of the fragments produced can greatly affect the availability of antigenic sites. For instance, some antigenic sites are not recognized when the native protein is used as immunogen, despite the fact that immunization with a small peptide corresponding to that site reveals both the ability of the site to bind to MHC molecules of the animal in question and the presence of a T-cell repertoire specific for that site. The antigenic site is not destroyed by processing, since it can be presented by the same F1 APC to T cells of another MHC type. Similarly, cross-reactivity between homologous epitopes of related proteins may occur at the peptide level even though the native proteins do not crossreact for the same T-cell clone. Since these events occur with monoclonal T cells, they cannot be due to suppressor cells specific for other sites on the native molecule. The best explanation is that the products of natural processing of the protein are larger than the peptides corresponding to the minimal antigenic sites, and contain hindering structures that interfere with binding to some MHC molecules and not others, or to some T-cell receptors and not others. Thus, antigen processing is a third factor that can lead to apparent Ir gene defects - in addition to MHC specificity and holes in the T-cell repertoire - and can significantly influence which antigenic sites are immunodominant.(ABSTRACT TRUNCATED AT 400 WORDS)

Melanoma: a model for testing new agents in combination therapies.

Treatment for both early and advanced melanoma has changed little since the introduction of interferon and IL-2 in the early 1990s. Recent data from trials testing targeted agents or immune modulators suggest the promise of new strategies to treat patients with advanced melanoma. These include a new generation of B-RAF inhibitors with greater selectivity for the mutant protein, c-Kit inhibitors, anti-angiogenesis agents, the immune modulators anti-CTLA4, anti-PD-1, and anti-CD40, and adoptive cellular therapies. The high success rate of mutant B-RAF and c-Kit inhibitors relies on the selection of patients with corresponding mutations. However, although response rates with small molecule inhibitors are high, most are not durable. Moreover, for a large subset of patients, reliable predictive biomarkers especially for immunologic modulators have not yet been identified. Progress may also depend on identifying additional molecular targets, which in turn depends upon a better understanding of the mechanisms leading to response or resistance. More challenging but equally important will be understanding how to optimize the treatment of individual patients using these active agents sequentially or in combination with each other, with other experimental treatment, or with traditional anticancer modalities such as chemotherapy, radiation, or surgery. Compared to the standard approach of developing new single agents for licensing in advanced disease, the identification and validation of patient specific and multi-modality treatments will require increased involvement by several stakeholders in designing trials aimed at identifying, even in early stages of drug development, the most effective way to use molecularly guided approaches to treat tumors as they evolve over time.

A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers"

The International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) a programmatic look at innovative avenues for the identification of relevant parameters to assist clinical and basic scientists who study the natural course of host/tumor interactions or their response to immune manipulation. The task force has two primary goals: 1) identify best practices of standardized and validated immune monitoring procedures and assays to promote inter-trial comparisons and 2) develop strategies for the identification of novel biomarkers that may enhance our understating of principles governing human cancer immune biology and, consequently, implement their clinical application. Two working groups were created that will report the developed best practices at an NCI/FDA/iSBTc sponsored workshop tied to the annual meeting of the iSBTc to be held in Washington DC in the Fall of 2009. This foreword provides an overview of the task force and invites feedback from readers that might be incorporated in the discussions and in the final document.

A phase I study of indoximod in patients with advanced malignancies

Purpose Indoximod is an oral inhibitor of the indoleamine 2,3-dioxygenase pathway, which causes tumor-mediated immunosuppression. Primary endpoints were maximum tolerated dose (MTD) and toxicity for indoximod in patients with advanced solid tumors. Secondary endpoints included response rates, pharmacokinetics, and immune correlates. Experimental Design Our 3+3 phase I trial comprised 10 dose levels (200, 300, 400, 600, and 800 mg once/day; 600, 800, 1200, 1600, and 2000 mg twice/day). Inclusion criteria were measurable metastatic solid malignancy, age ≥18 years, and adequate organ/marrow function. Exclusion criteria were chemotherapy ≤ 3 weeks prior, untreated brain metastases, autoimmune disease, or malabsorption. Results In 48 patients, MTD was not reached at 2000 mg twice/day. At 200 mg once/day, 3 patients previously treated with checkpoint inhibitors developed hypophysitis. Five patients showed stable disease >6 months. Indoximod plasma AUC and Cmax plateaued above 1200mg. Cmax (∼12 μM at 2000 mg twice/day) occurred at 2.9 hours, and half-life was 10.5 hours. C reactive protein (CRP) levels increased across multiple dose levels. Conclusions Indoximod was safe at doses up to 2000 mg orally twice/day. Best response was stable disease >6 months in 5 patients. Induction of hypophysitis, increased tumor antigen autoantibodies and CRP levels were observed.

Phase I Clinical Trial of Ipilimumab in Pediatric Patients with Advanced Solid Tumors

Purpose: Ipilimumab is a first-in-class immune checkpoint inhibitor approved for treatment of metastatic melanoma but not studied in children until this phase I protocol. Experimental Design: This study examined safety, pharmacokinetics, and immunogenicity, and immune correlates of ipilimumab administered to subjects ≤21 years old with recurrent or progressive solid tumors. Dose escalation cohorts received 1, 3, 5, or 10 mg/m2 intravenously every 3 weeks in a 3 + 3 design. Response was assessed after 6 weeks and 12 weeks, and then every 3 months. Treatment was continued until disease progression or unacceptable toxicity. Results: Thirty-three patients received 72 doses of ipilimumab. Patients enrolled had melanoma (n = 12), sarcoma (n = 17), or other refractory solid tumors (n = 4). Immune-related adverse events included pancreatitis, pneumonitis, colitis, endocrinopathies, and transaminitis with dose-limiting toxicities observed at 5 and 10 mg/kg dose levels. Pharmacokinetics revealed a half-life of 8 to 15 days. At day 21, subjects had increased levels of cycling T cells, but no change in regulatory T-cell populations. Six subjects had confirmed stable disease for 4 to 10 cycles (melanoma, osteosarcoma, clear cell sarcoma, and synovial sarcoma). Conclusions: Ipilimumab was safely administered to pediatric patients using management algorithms for immune-related toxicities. The spectrum of immune-related adverse events is similar to those described in adults; however, many of the pediatric toxicities were evident after a single dose. Although no objective tumor regressions were observed with ipilimumab as a single agent, subjects with immune-related toxicities had an increased overall survival compared with those who showed no evidence of breaking tolerance. Clin Cancer Res; 22(6); 1364–70. ©2015 AACR.

Immune checkpoint inhibitors in clinical trials

Immunology-based therapy is rapidly developing into an effective treatment option for a surprising range of cancers. We have learned over the last decade that powerful immunologic effector cells may be blocked by inhibitory regulatory pathways controlled by specific molecules often called “immune checkpoints.” These checkpoints serve to control or turn off the immune response when it is no longer needed to prevent tissue injury and autoimmunity. Cancer cells have learned or evolved to use these mechanisms to evade immune control and elimination. The development of a new therapeutic class of drugs that inhibit these inhibitory pathways has recently emerged as a potent strategy in oncology. Three sets of agents have emerged in clinical trials exploiting this strategy. These agents are antibody-based therapies targeting cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed cell death 1 (PD-1), and programmed cell death ligand 1 (PD-L1). These inhibitors of immune inhibition have demonstrated extensive activity as single agents and in combinations. Clinical responses have been seen in melanoma, renal cell carcinoma, non-small cell lung cancer, and several other tumor types. Despite the autoimmune or inflammatory immune-mediated adverse effects which have been seen, the responses and overall survival benefits exhibited thus far warrant further clinical development.

Validation of biomarkers to predict response to immunotherapy in cancer: Volume II — clinical validation and regulatory considerations

There is growing recognition that immunotherapy is likely to significantly improve health outcomes for cancer patients in the coming years. Currently, while a subset of patients experience substantial clinical benefit in response to different immunotherapeutic approaches, the majority of patients do not but are still exposed to the significant drug toxicities. Therefore, a growing need for the development and clinical use of predictive biomarkers exists in the field of cancer immunotherapy. Predictive cancer biomarkers can be used to identify the patients who are or who are not likely to derive benefit from specific therapeutic approaches. In order to be applicable in a clinical setting, predictive biomarkers must be carefully shepherded through a step-wise, highly regulated developmental process. Volume I of this two-volume document focused on the pre-analytical and analytical phases of the biomarker development process, by providing background, examples and “good practice” recommendations. In the current Volume II, the focus is on the clinical validation, validation of clinical utility and regulatory considerations for biomarker development. Together, this two volume series is meant to provide guidance on the entire biomarker development process, with a particular focus on the unique aspects of developing immune-based biomarkers. Specifically, knowledge about the challenges to clinical validation of predictive biomarkers, which has been gained from numerous successes and failures in other contexts, will be reviewed together with statistical methodological issues related to bias and overfitting. The different trial designs used for the clinical validation of biomarkers will also be discussed, as the selection of clinical metrics and endpoints becomes critical to establish the clinical utility of the biomarker during the clinical validation phase of the biomarker development. Finally, the regulatory aspects of submission of biomarker assays to the U.S. Food and Drug Administration as well as regulatory considerations in the European Union will be covered.

Nivolumab with or without ipilimumab treatment for metastatic sarcoma (Alliance A091401): two open-label, non-comparative, randomised, phase 2 trials

BACKGROUND Patients with metastatic sarcoma have limited treatment options. Nivolumab and ipilimumab are monoclonal antibodies targeting PD-1 and CTLA-4, respectively. We investigated the activity and safety of nivolumab alone or in combination with ipilimumab in patients with locally advanced, unresectable, or metastatic sarcoma. METHODS We did a multicentre, open-label, non-comparative, randomised, phase 2 study that enrolled patients aged 18 years or older and had central pathology confirmation of sarcoma with at least one measurable lesion by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, evidence of metastatic, locally advanced or unresectable disease, an ECOG performance status of 0-1, and received at least one previous line of systemic therapy. Patients were assigned to treatment in an unblinded manner, as this trial was conducted as two independent, non-comparative phase 2 trials. Enrolled patients were assigned (1:1) via a dynamic allocation algorithm to intravenous nivolumab 3 mg/kg every 2 weeks, or nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses. Thereafter, all patients received nivolumab monotherapy (3 mg/kg) every 2 weeks for up to 2 years. The primary endpoint was the proportion of patients with locally advanced, unresectable or metastatic soft tissue sarcoma achieving a confirmed objective response. Analysis was per protocol. This study is ongoing although enrolment is closed. It is registered with ClinicalTrials.gov, number NCT02500797. FINDINGS Between Aug 13, 2015, and March 17, 2016, 96 patients from 15 sites in the USA underwent central pathology review for eligibility and 85 eligible patients, including planned over-enrolment, were allocated to receive either nivolumab monotherapy (43 patients) or nivolumab plus ipilimumab (42 patients). The primary endpoint analysis was done according to protocol specifications in the first 76 eligible patients (38 patients per group). The number of confirmed responses was two (5% [92% CI 1-16] of 38 patients) in the nivolumab group and six (16% [7-30] of 38 patients) in the nivolumab plus ipilimumab group. The most common grade 3 or worse adverse events were anaemia (four [10%] patients), decreased lymphocyte count (three [7%]), and dehydration, increased lipase, pain, pleural effusion, respiratory failure, secondary benign neoplasm, and urinary tract obstruction (two [5%] patients each) among the 42 patients in the nivolumab group and anaemia (eight [19%] patients), hypotension (four [10%] patients), and pain and urinary tract infection (three [7%] patients each) among the 42 patients in the nivolumab plus ipilimumab group. Serious treatment-related adverse events occurred in eight (19%) of 42 patients receiving monotherapy and 11 (26%) of 42 patients receiving combination therapy, and included anaemia, anorexia, dehydration, decreased platelet count, diarrhoea, fatigue, fever, increased creatinine, increased alanine aminotransferase, increased aspartate aminotransferase, hyponatraemia, pain, pleural effusion, and pruritus. There were no treatment-related deaths. INTERPRETATION Nivolumab alone does not warrant further study in an unselected sarcoma population given the limited efficacy. Nivolumab combined with ipilimumab demonstrated promising efficacy in certain sarcoma subtypes, with a manageable safety profile comparable to current available treatment options. The combination therapy met its predefined primary study endpoint; further evaluation of nivolumab plus ipilimumab in a randomised study is warranted. FUNDING Alliance Clinical Trials in Oncology, National Cancer Institute Cancer Therapy Evaluation Program, Bristol-Myers Squibb, Cycle for Survival.

First-in-Human Phase I Study of the Tamoxifen Metabolite Z-Endoxifen in Women With Endocrine-Refractory Metastatic Breast Cancer

Purpose Endoxifen is a tamoxifen metabolite with potent antiestrogenic activity. Patients and Methods We performed a phase I study of oral Z-endoxifen to determine its toxicities, maximum tolerated dose (MTD), pharmacokinetics, and clinical activity. Eligibility included endocrine-refractory, estrogen receptor-positive metastatic breast cancer. An accelerated titration schedule was applied until moderate or dose-limiting toxicity occurred, followed by a 3+3 design and expansion at 40, 80, and 100 mg per day. Tumor DNA from serum (circulating cell free [cf); all patients] and biopsies [160 mg/day and expansion]) was sequenced. Results Of 41 enrolled patients, 38 were evaluable for MTD determination. Prior endocrine regimens during which progression occurred included aromatase inhibitor (n = 36), fulvestrant (n = 21), and tamoxifen (n = 15). Patients received endoxifen once daily at seven dose levels (20 to 160 mg). Dose escalation ceased at 160 mg per day given lack of MTD and endoxifen concentrations > 1,900 ng/mL. Endoxifen clearance was unaffected by CYP2D6 genotype. One patient (60 mg) had cycle 1 dose-limiting toxicity (pulmonary embolus). Overall clinical benefit rate (stable > 6 months [n = 7] or partial response by RECIST criteria [n = 3]) was 26.3% (95% CI, 13.4% to 43.1%) including prior tamoxifen progression (n = 3). cfDNA mutations were observed in 13 patients ( PIK3CA [n = 8], ESR1 [n = 5], TP53 [n = 4], and AKT [n = 1]) with shorter progression-free survival ( v those without cfDNA mutations; median, 61 v 132 days; log-rank P = .046). Clinical benefit was observed in those with ESR1 amplification (tumor; 80 mg/day) and ESR1 mutation (cfDNA; 160 mg/day). Comparing tumor biopsies and cfDNA, some mutations ( PIK3CA, TP53, and AKT) were undetected by cfDNA, whereas cfDNA mutations ( ESR1, TP53, and AKT) were undetected by biopsy. Conclusion In endocrine-refractory metastatic breast cancer, Z-endoxifen provides substantial drug exposure unaffected by CYP2D6 metabolism, acceptable toxicity, and promising antitumor activity.

Combined KIT and CTLA-4 Blockade in Patients with Refractory GIST and Other Advanced Sarcomas: A Phase Ib Study of Dasatinib plus Ipilimumab.

Purpose: A phase Ib study of dasatinib plus ipilimumab in patients with gastrointestinal stromal tumor (GIST) and other sarcomas was performed on the basis of preclinical data demonstrating that combined KIT and CTLA-4 blockade is synergistic. Experimental Design: A standard 3 + 3 design was used to evaluate the safety, efficacy, and immune correlates of treatment. Dose escalation cohorts received ipilimumab 10 or 3 mg/kg every 3 weeks, followed by maintenance every 12 weeks with escalating doses of dasatinib (70 mg daily, 100 mg daily, or 70 mg twice daily). Response was assessed by RECIST 1.1, Choi, and immune-related RECIST criteria (irRC). Results: A total of 28 patients (17 male) were enrolled. Histologic subtypes included GISTs (n = 20) and other sarcomas (n = 8.) Dasatinib 70 mg/day with ipilimumab 10 mg/kg or dasatinib 140 mg/day with ipilimumab 3 mg/kg can be safely administered. Dose-limiting toxicities included grade 3 gastric hemorrhage and anemia. No partial or complete responses were noted by RECIST or irRC. There were 7 of 13 partial responses in the GIST patients by Choi criteria, and 3 of 13 patients each had stable and progressive disease, respectively. Conclusions: Dasatinib and ipilimumab can be safely administered to GIST and sarcoma patients. However, dasatinib was not synergistic with ipilimumab, as there was limited clinical efficacy with the combination. This limited cohort provides prospective data that indoleamine-2,3-dioxygenase (IDO) suppression may potentially correlate with antitumor efficacy in GIST. Given the small cohort, it is only hypothesis generating and additional data would be required. In the era of more modern and effective checkpoint inhibitors, next steps could be consideration of tyrosine kinase inhibitors or IDO inhibitors in combination with anti-PD-1 therapy. Clin Cancer Res; 23(12); 2972–80. ©2016 AACR.