Immunotherapy

Identification of Different Form Tim-3 Proteins by a Unique Set of Tim-3 Monoclonal Antibodies T-cell immunoglobulin and mucin domain-3 (Tim-3) has been suggested to be a critical immune checkpoint target for cancer immunotherapy. However, limited progress with Tim-3 immunotherapy has been achieved over the last decade due to the lack of specific Tim-3 monoclonal antibodies. In this study, we have successfully developed a unique set of Tim-3 antibodies that are able to detect different molecular weights (by Western blot mobility) of Tim-3 proteins ectopically expressed in the same CHO cells. Some of the antibody clones detect only 33 or 55 kDa bands, the rest can recognize both 33 and 55 kDa bands on polyacrylamide gel electrophoresis gel. Antibody clones with 55 kDa specificity uniquely bind to the membrane form of Tim-3 on macrophage, which colocalizes with the CD68, and could be used as a specific marker for tumor-associated macrophage, whereas other clones showed cytoplasmic staining in tumor cells. The membrane form of Tim-3 on tumor-associated macrophages may bear significant roles for clinical application of Tim-3, but less likely for cytoplasmic one. The availability of this unique set of antibodies will be critical for an ultimate understanding of Tim-3 function in tumor microenvironment and potential clinical applications.

An IgG1 Version of the Anti-transferrin Receptor 1 Antibody ch128.1 Shows Significant Antitumor Activity Against Different Xenograft Models of Multiple Myeloma: A Brief Communication The transferrin receptor 1 (TfR1) is a meaningful target for antibody-based cancer therapy given its overexpression on malignant cells and its central role in cancer pathology. We previously developed a mouse/human chimeric IgG3 targeting human TfR1 (ch128.1), which exhibits significant antitumor activity against multiple myeloma (MM) in xenograft models of SCID-Beige mice bearing disseminated ARH-77 or KMS-11 tumors. This activity is observed in early and late disease stages of disseminated KMS-11 tumors and, in this model, the mechanism of antitumor activity is Fc-mediated, involving macrophages. As human IgG1 is the isotype of choice for therapeutic antibodies targeting malignant cells and has several advantages compared with IgG3, including established manufacturability, we now developed an IgG1 version of ch128.1. A single dose of ch128.1/IgG1 shows significant antitumor activity, not only against early and late stages of disseminated KMS-11 tumors (Asian origin) but also against these stages of disseminated disease following injection of human MM cells MM.1S (African American origin) or its variant that is resistant to dexamethasone MM.1R. Treatment with the Fc mutant version of ch128.1/IgG1 (L234A/L235A/P329S) with impaired effector functions fails to confer protection against MM.1S and MM.1R tumors, indicating a crucial role of the Fc fragment in the antitumor activity, similar to its IgG3 counterpart. In fact, we found that ch128.1/IgG1, but not the mutant, elicits antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated phagocytosis in the presence of murine bone marrow–derived macrophages. Our results suggest that ch128.1/IgG1 is a promising therapeutic against human B-cell malignancies such as MM.

Applicability of Routine Targeted Next-generation Sequencing to Estimate Tumor Mutational Burden (TMB) in Patients Treated With Immune Checkpoint Inhibitor Therapy It remains unclear whether targeted next-generation sequencing (tNGS) conveys a reliable estimate of tumor mutational burden (TMB). We sequenced 79 archival samples of immune checkpoint inhibitors (ICPIs) recipients (57% lung cancer, 43% melanoma) using Ion Ampliseq Cancer Hotspot Panel. Employing multiple cutoff values, we verified that TMB by tNGS did not correlate with response or survival following ICPI. We found enrichment of ATM mutations in ICPI-refractory tumors (P=0.01) to correlate with worse survival (4.2 vs. 10 mo, P=0.03). Limited-coverage tNGS delivers an imprecise estimate of patients’ TMB but may aid identification of candidate somatic variants of predictive/prognostic significance.

Blood Immune Cell Biomarkers in Patient With Lung Cancer Undergoing Treatment With Checkpoint Blockade Characterization of host immune cell parameters before and during immunotherapy is expected to identify predictive biomarkers for clinical outcome. We prospectively monitored blood immune cells from 35 patients with advanced non–small cell lung cancer undergoing checkpoint inhibitor monotherapy. The aim was to identify parameters correlating with better/worse outcome. Peripheral blood was serially collected before each infusion at the onset and at cycle 3 and 5 of immunotherapy. A complete leukocyte blood count, the lymphocytic subpopulations and the percentages of both HLA-DRlow monocytes and dendritic cells (DC) were monitored. Disease control was defined as partial/complete response and stable disease on computed tomography scan according to RECIST 1.1. The predictive value of the immune cell parameters investigated was evaluated by patients’ survival analysis. Forty percent of patients showed a clinical response, and the global median overall survival was 7.0 months (95% confidence interval: 3.5–10.5). Patients with an initial neutrophil-to-lymphocyte ratio (NLR) ≥5.2, and/or an amount of HLA-DRlow monocytes ≥11% and/or a total DC level ≤0.4% of leukocytes did rarely respond to PD-1 inhibitor therapy. Otherwise, the immunotherapy-induced decrease of the neutrophil-to-lymphocyte ratio and/or HLA-DRlow monocytes and the increase of total DC frequencies were correlated with improved therapy response and prolonged overall survival. Blood values in the third cycle of immunotherapy did already reflect the effects observed. On the basis of the 3 immune cell parameters identified we created 3 different variants of scores that enable to stratify patients into groups of risk/therapy response. Our results warrant further investigation in larger prospective clinical trials for validation.

Immune-related Neutropenia Following Treatment With Immune Checkpoint Inhibitors The existing data with regard to immune-related neutropenia (irN), a rare (incidence—1%) immune-related adverse event of immune checkpoint inhibitors, are scarce. Eight patients with irN were identified through internal databases of 3 participating Israeli cancer centers. In addition, 11 original articles focusing on the clinical course of 24 patients with irN were selected during the PubMed search. Descriptive analysis of clinical and pathologic factors related to irN was performed (n=32); the effect of these on the irN outcomes was assessed. An algorithm for irN evaluation and treatment was proposed. The median time-to-onset of irN (n=32) was 60 days (range, 10–465 d). Grade 3–5 irN, febrile neutropenia, and irN-related death occurred in 81%, 50%, and 9% of patients, respectively. In all, 56%, 22%, 62%, and 25% of patients received PO corticosteroids, IV corticosteroids, granulocyte colony–stimulating factor (GCSF), and intravenous immunoglobulins (IVIG), respectively, with an improvement/resolution rate of 84%. Odds ratios for irN improvement/resolution were as follows: 1.40 [95% confidence interval (CI), 0.03–68.72], 0.43 (95% CI, 0.04–4.22), 2.60 (95% CI, 0.07–97.24), 0.36 (95% CI, 0.03–4.38), 4.02 (95% CI, 0.16–99.48), 2.01 (95% CI, 0.32–12.70), 1.08 (95% CI, 0.02–49.89), 0.42 (95% CI, 0.06–2.91), and 2.73 (95% CI, 0.42–17.51) for granulocyte hyperplasia, granulocyte/all lineage hypoplasia, granulocyte maturation blockade, lymphocyte infiltration on bone marrow biopsy, IV corticosteroids, PO corticosteroids, cyclosporine, IVIG, and GCSF, respectively (P>0.05 for all factors). IrN recurrence rate following immune checkpoint inhibitors rechallenge was 80%. IrN is a rare, life-threatening, early-onset immune-related adverse event. Differentiating between the central, peripheral, and modified peripheral types allows a better prognosis definition. Corticosteroids and GCSF represent the main treatment approaches; IVIG and cyclosporine should be used as salvage treatment.

Sarcoid-like Reaction Discovered on EBUS-TBNA of Intrathoracic Lymph Nodes During Immunotherapy for Metastatic Melanoma The use of immune checkpoint inhibitors has dramatically improved outcomes for patients with advanced melanoma and other malignancies. Checkpoint inhibitors are associated with a unique set of toxicities collectively known as immune-related adverse events, the incidence of which is rising in parallel with their increasing use in clinical practice. Immune-related adverse events are widely variable in their presentation and can affect virtually any organ system in the body. Sarcoid-like reactions in patients being treated with immune checkpoint inhibitors are rare and are typically multisystemic in nature with isolated pulmonary involvement representing only a small minority of cases reported in the literature. Herein we describe 2 patients who developed progressively enlarging lymphadenopathy while receiving checkpoint inhibitors for metastatic melanoma. Both patients were initially noted to have an excellent clinical response to immunotherapy but their treatment was interrupted pending further investigation as they were suspected to have progressive disease. They were ultimately diagnosed with sarcoid-like reactions after an endobronchial ultrasound-guided lymph node biopsy revealed noncaseating granulomas and were able to resume their immunotherapy without any further interventions or negative effect on their disease course. These 2 cases illustrate the importance of obtaining a tissue diagnosis when imaging reveals enlarging lymph nodes while on immunotherapy for solid malignancies as the differential diagnosis includes benign entities such as sarcoid-like reactions in addition to disease progression. Timely diagnosis through minimally invasive tissue sampling techniques, such as endobronchial ultrasound, can help rule out malignant etiologies of lymphadenopathy and minimize interruptions in treatment.