703 Favorable pre-clinical safety profile of the novel not-alpha IL-2 agonist ANV419 supports first in human clinical development

BackgroundANV419 is a novel interleukin-2 (IL-2)/anti-IL-2 fusion protein with preferential signaling through the IL-2 beta/gamma receptor that induces selective proliferation of CD8 T cells and NK cells in vivo for the treatment of cancer. The safety and pharmacodynamic effects of ANV419 were studied in a 4-week cynomolgus monkey GLP study to support the ongoing PhI dose escalation clinical trial.MethodsANV419 was administered by i.v. injection over 1 min at doses of 0.03, 0.1, 0.3 mg/kg, or vehicle control on days 1 and 15 of the 29-day study. Assessments included body weight, blood pressure, hematology, clinical pathology, serum cytokines, immunophenotyping, histopathology, and pharmacokinetics.ResultsThe pharmacokinetics of ANV419 were characterized by target mediated disposition, with a half-life of approximately 24h at concentrations not affected by target mediated clearance. Dose-dependent increases in WBC were observed after each injection, driven by preferential expansion of CD8 T cells and NK cells over Tregs. NK cells were more sensitive to ANV419 than CD8 T cells reaching maximal proliferation in blood at 0.03 mg/kg vs. 0.3 mg/kg for CD8 T cells. Hematological changes included: transient dose-dependent increase in basophils; elevation in eosinophils, up to 2.2-fold above control animals at > 0.03 mg/kg, remaining within the normal range for cynomolgus monkeys (<1.94 G/L); minor decrease in platelets at day 4 after each injection. There were no relevant treatment-related changes in inflammatory serum cytokines (IL-1b, IL-5, IL-6, IL-8, IFNg, TNFa, GM-CSF). A mild systemic inflammatory response was observed at 0.3 mg/kg evidenced by a transient increase of CRP on days 4 and 19, preceded after the first injection by a slight dose dependent increase in IL-1RA at 4h post injection, and an increase in IL-10 at 24h post treatment at 0.3mg/kg. No significant changes in body weights or blood pressure and no signs of capillary leak were observed during the entire study.A multi-part PhI dose-escalation study of ANV419 has been initiated in cancer patients. In the part A single patient escalation cohort, two patients have been dosed Q2W multiple times with 0.003mg/kg and 0.006mg/kg respectively with the expected PD profile and no DLT observed.ConclusionsConsistent findings, relating to expected effects of ANV419 as a not-alpha IL-2 agonist, demonstrated a favorable tolerability and safety profile at pharmacodynamically relevant doses that strongly support its translational development in cancer patients to identify clinical benefits.

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'First-in-human' phase I dose escalation trial of IL-15N72D/IL-15Rα-Fc superagonist complex (ALT-803) demonstrates immune activation with anti-tumor activity in patients with relapsed hematological malignancy

Blood ◽

10.1182/blood.v126.23.1957.1957 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1957-1957 ◽

Cited By ~ 5

Author(s):

Jeffrey S. Miller ◽

Sarah Cooley ◽

Shernan Holtan ◽

Mukta Arora ◽

Celalettin Ustun ◽

...

Keyword(s):

T Cells ◽

Phase I ◽

Nk Cells ◽

Cd8 T Cells ◽

Dose Escalation ◽

Allogeneic Transplantation ◽

Donor Chimerism ◽

Grade Ii ◽

Anti Tumor Activity ◽

Dose Dependent

Abstract Disease-free survival after allogeneic transplantation for hematological malignancies may be enhanced with post-transplant immunotherapy. IL-15 is a homeostatic cytokine that stimulates both NK cells and CD8+ T cells, but unlike IL-2 it does not stimulate suppressive regulatory T cells. Physiologic IL-15 signaling occurs after trans-presentation with IL-15Rα to IL-15Rβ and IL15Rγ (common chains shared with the IL-2R). ALT-803 (Altor BioSciences) is an IL-15/IL-15Rα-Fc superagonist complex, which includes an activation mutation in IL-15 (N72D), a sushi domain to increase transpresentation, and an IgG1 Fc domain to increase serum half-life and stability. In preclinical mouse models, ALT-803 exhibits potent anti-tumor activity. We therefore performed a 'first-in-human' phase I open label dose escalation trial of ALT-803 in patients with relapsed hematologic malignancy after allogeneic hematopoietic cell transplantation (> 60 days) with no active GVHD and having >10% residual donor chimerism. Sixteen patients (AML, n=10; MDS, n=2; myeloma, n=2; ALL, n=1; NHL, n=1) received escalating doses of intravenous ALT-803 given weekly x 4 doses in 4 cohorts of 1 (n=6), 3 (n=3), 6 (n=4), and 10 (n=3) mcg/kg. Cohort 1 was expanded because of atrial fibrillation (grade 2 toxicity) in an AML subject with a prior history. One unevaluable subject in Cohort 3 who did not receive the minimum 3 doses of ALT-803 was replaced. There was highly reproducible and dose-dependent constitutional symptoms, consistent with immune activation (fevers and tachycardia) that peaked at 5-6 hours after each dose despite pre-medication with acetaminophen & diphenhydramine. Fever ≥103 degrees F was observed in cohorts 3 and 4 in patients receiving 6 and 10 mcg/kg, respectively, but was self-limited with resolution within 12 hours. These side effects correlated with ALT-803 dose-dependent increases in serum IFNγ and IL-6 levels 4 hours after dosing. Grade 2 hypotension (asymptomatic) occurred and responded to saline infusions, resulting in saline bolus prophylaxis in later patients. No patients experienced systemic capillary leak syndrome. Oxygen saturations were normal at all time points except in one patient (6 mcg dose) with an isolated 02 saturation of 88% and 89% five hours after doses 2 and 3, respectively. No dose limiting toxicities were observed. Dose dependent increases in CD8+ T cell and NK proliferation (Ki-67+) were observed at 3 and 5 days after the first dose of ALT-803 (Figure, top) with a slight increase in Ki67+ Treg and CD4+ T cells, but less than NK cells and CD8+ T cells. NK proliferation remained above pre-treatment baseline for 7 days in the 10 mcg/kg cohort, resulting in a sustained increased in absolute NK cells for 28 days (Figure, bottom). Functional studies at the 10 mcg/kg dose showed an increased frequency of NK cells that degranulated against K562 targets and produced IFNγ after IL-12/IL-18 stimulation. Clinical responses occurred in 2 patients with stable disease 1 month after the last dose of ALT-803 and one complete response. This 69 y/o man had recurrent RAEBT (9% blasts) with complex cytogenetics (monosomy 7 with cytogenetic evolution in 5/20 metaphases), relapsing after prior second unrelated donor transplant with 46% donor chimerism at the time of treatment. After 4 doses of ALT-803 at 6 mcg/kg without DLT, he developed a biopsy proven cutaneous grade II GVHD that resolved with topical steroids alone. His follow-up bone marrow biopsy revealed 3% blasts, normal karyotype, 93% donor chimerism with normalization of blood counts (Hgb from 9.6 to 12.5, platelets from 61K to 168K, WBC from 1.7 to 4.2 and ANC from 400 to 1400 from pre-therapy to one month post-therapy). An additional patient had grade II skin GVHD with disease response measures pending. This phase I trial demonstrates that ALT-803 safely induces proliferation of functional NK cell and CD8+ T cells with in vivo cytoxicity. Recent pre-clinical data suggest that subcutaneous dosing of ALT-803 can decrease peak serum concentrations, increase half-life and preferentially distributes to lymphoid tissues potentially optimizing drug delivery. The ongoing trial now utilizes subcutaneous dosing at 6 mcg/kg followed by dose escalation. IL-15N72D /IL-15Rα-Fc superagonist complex is a promising new therapeutic strategy for the treatment of relapse, a major obstacle after allogeneic transplantation for high risk hematologic malignancies. Figure 1. Figure 1. Disclosures Miller: Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau. Jeng:Altor BioScience Corporatoin: Employment. Wong:Altor BioScience Corporation: Employment, Other: stockholder of Altor Bioscience Corporation.

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509 A first-in-human phase 1 study of NL-201 in patients with relapsed or refractory cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.509 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A540-A540

Author(s):

Aung Naing ◽

Margaret Callahan ◽

Brian Costello ◽

Carlo Bifulco ◽

Evan Hall ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Cancer Immunotherapy ◽

Cd8 T Cells ◽

Dose Escalation ◽

Safety Profile ◽

Phase 1 ◽

Response To Treatment ◽

Carcinomatous Meningitis ◽

Link Type

BackgroundNL-201 is a selective and long-acting computationally designed alpha-independent agonist of the IL-2 and IL-15 receptors, which share beta and gamma signaling subunits. NL-201 is being developed as a potent activator of CD8+ T cells and NK cells for cancer immunotherapy. Binding to the beta and gamma subunits selectively stimulates dose-dependent expansion and tumor infiltration of cytotoxic CD8+ T cells and NK cells, thereby enhancing the immune response in the tumor. The absence of binding to the IL-2 alpha subunit reduces the undesirable effects of traditional IL-2 therapies, such as vascular leak syndrome and expansion of immunosuppressive regulatory T cells. As such, NL-201 is designed to promote the desired immunomodulatory anti-tumor effects of IL-2 with an improved safety profile.MethodsNL201-101 is a Phase 1 first-in-human, open-label, dose-escalation, and cohort expansion study consisting of two parts. Part 1 is an adaptive monotherapy dose escalation study in up to 60 adult patients with advanced and/or refractory solid tumors to determine the safety profile and the recommended phase 2 dose (RP2D) and schedule of NL-201. During dose escalation, two different schedules will be evaluated: dosing every 21 days or on days 1 and 8 of each 21-day cycle. Tumor response to treatment will be assessed by Response Evaluation Criteria in Solid Tumours (RECIST) 1.1 and/or RECIST for use in cancer immunotherapy trials (iRECIST). In Part 2, patients with pathologically proven diagnosis of indication-specific cohorts (up to N=30/cohort), who have advanced and/or refractory measurable disease and have failed at least one line of treatment, which may include checkpoint inhibitors, will be enrolled. Key exclusion criteria include history of brain cancer, carcinomatous meningitis, neurologic autoimmune disease, or active central nervous system metastases; patients previously receiving CAR-T or IL-2-based therapies are not eligible. Recruitment of Part 1 began in April 2021, and the trial is actively enrolling. Clinicaltrials.gov identifier: NCT04659629.Trial RegistrationClinicaltrials.gov identifier: NCT04659629.Ethics ApprovalAll relevant documents have been or will be submitted to an Institutional Review Board (IRB)/Independent Ethics Committee (IEC) by the investigator and reviewed and approved by the IRB/IEC before the study is initiated. Site 1001: Belberry HREC, application number 2020–09–925 (Belberry does not provide an approval number); Site 1003: Austin Health HREC, approval number HREC/69340/Austin-2020; Site 2003: MDACC Office of Human Subjects Protection, approval number 2020–0383.

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Determination of Soluble MICA in Serum as a Novel Marker for Tumor Stage and Metastasis.

Blood ◽

10.1182/blood.v104.11.1344.1344 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1344-1344

Author(s):

Helmut R. Salih ◽

Petra Stieber ◽

Andrea Peterfi ◽

Dorothea Nagel ◽

Lothar Kanz ◽

...

Keyword(s):

T Cells ◽

Differential Diagnosis ◽

Nk Cells ◽

Cancer Patients ◽

Tumor Cells ◽

Cd8 T Cells ◽

Control Group ◽

Healthy Individuals ◽

Benign Disorders

Abstract The human NKG2D ligands (NKG2DL) MICA and MICB have been shown to be expressed on tumors of epithelial and hematopoietic origin in vivo. Recently we reported that MICA is shed from the cell surface of tumor cells and is present in sera of tumor patients (J Immunol169:4098 (2002), Blood102:1389 (2003)). Since the strength of an anti-tumor response by NK cells and CD8 T cells is critically depending on NKG2DL expression levels, down-regulation of MICA-expression on tumor cells represents an immune escape mechanism that diminishes anti-tumor reactivity of NKG2D-bearing lymphocytes. However, no data are yet available regarding the correlation of soluble MICA (sMICA) levels with specific tumor entities, aggressiveness of the disease, and hence the potential implementation of sMICA as novel marker in differential diagnosis and prognosis of cancer. In this study, we determined sMICA levels in sera of 512 individuals including 296 patients with various cancers, 154 patients with benign disorders and 62 healthy individuals. Healthy individuals revealed significantly lower sMICA values (median<30pg/mL) than patients with benign diseases (84pg/mL; p=0.005) and cancer patients (161pg/mL; p<0.0001). In addition, sMICA levels differed significantly between cancer patients and patients with benign disorders (p<0.0001) that represent the most relevant control group for differential diagnosis. In cancer patients, while there was no association between sMICA levels and tumor size (p=0.456), cell differentiation (p=0.271), or lymph node involvement (p=0.674), sMICA correlated significantly with cancer stage and metastasis (p=0.015 and p=0.007, respectively). Our data indicate that release of MICA might play a role in late stages of tumor progression by overcoming the confining effect of NK cells and CD8 T cells. Thus, determination of sMICA levels provides valuable information for cancer staging, and sMICA in serum seems to be an indicator for systemic manifestation of malignancy rather than for local tumor extent.

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New Insights into the Mechanism of Action (MoA) of First-in-Class IgG-Based Bcma T-Cell Bispecific Antibody (TCB) for the Treatment of Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v128.22.2096.2096 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2096-2096 ◽

Cited By ~ 7

Author(s):

Laura Moreno ◽

Aintzane Zabaleta ◽

Diego Alignani ◽

Marta Lasa ◽

Patricia Maiso ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

T Cell Activation ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Activation ◽

Rational Combination ◽

Dose Dependent ◽

Tumor Cell Lysis

Abstract Novel agents have improved outcomes in MM, but prognosis after patients relapse remains poor and new drugs with novel MoA are needed. The breakthrough of immuno-oncology has rendered new therapeutic options, and most recently we reported on EM801, a novel BCMA-TCB that showed remarkably efficacy when used as single agent in primary bone marrow (BM) samples from MM patients (Seckinger, Blood 2015;126: abstr 117). Because of its novelty, further knowledge about the MoA of BCMA-TCB is of utmost importance to improve its efficacy by designing rational treatment combinations. In order to optimize the in vitro efficacy of the BCMA-TCB, we started by investigating in primary BM samples from 6 MM patients whether longer treatment periods with BCMA-TCB2 (a BCMA-TCB candidate sharing similar "2+1" structure of EM801 but displaying higher affinity to BCMA) would increase MM cell death. Upon treating samples with BCMA-TCB2 for 48h vs 96h, we noted a 2-fold increment in MM tumor cell lysis at 1nM and 10nM concentrations (Panel A). In parallel, the phenotypic profiles of CD4 and CD8 T cells showed that BCMA-TCB2 induced robust activation (ie. dose-dependent increment in CD69, CD25, HLADR after exposure to 100pM, 1nM and 10nM of BCMA-TCB2), but also led to the natural emergence of the checkpoint inhibitor PD-1 in the surface of activated CD4 and CD8 T cells (Panel B). We then investigated if there was a correlation between the percentage of PD-1 positive CD4 and CD8 T cells and the efficacy of BCMA-TCB2; interestingly, those patients with lower frequencies of PD-1 positive CD4 and CD8 T cells prior to treatment showed the highest rates of MM tumor cell lysis after 48h and 96h of BCMA-TCB2 at 10nM of (r=0.6, P=0.04; Panel C). By contrast, upon measuring the concentration of soluble BCMA and APRIL in the supernatants of primary BM samples from 16 MM patients treated with BCMA-TCB, we found no significant differences between responding (n=11) and non-responding (n=5) patients. Similar results were observed upon comparing the density of BCMA in the surface of MM tumor cells from responding vs non-responding patients (1256 vs 1522 SABC units; P=87). Since the efficacy of BCMA-TCB2 was found to be intrinsically related to the phenotype and activation status of T cells, we then investigated whether we could further harness immune cells by combining BCMA-TCB2 with three drugs representing different types of immunotherapy: lenalidomide (IMIDs), anti-PD1 (checkpoint inhibitors) and daratumumab (mAb). H929 MM cells were co-cultured with human leukocytes (n=5) and challenged to suboptimal concentrations of BCMA-TCB2 (10pM) alone, or in combination with standard doses of lenalidomide (1µM), anti-PD1 (10µg/ml) and daratumumab (10µg/ml) (Panel D). Interestingly, we observed that combining BCMA-TCB2 with lenalidomide or daratumumab significantly increased their anti-MM efficacy by 4-fold and 2.5-fold, respectively. Because lenalidomide and daratumumab share in common that they rely, at least in part, on activated NK cells to eradicate MM cells, we hypothesized whether such robust T cell activation induced by BCMA-TCB2 was leading to co-stimulation of NK cells. First, we demonstrated by analyzing the transcriptomes of T cells prior and after treatment exposure (n=3), that BCMA-TCB2 modulated the transcriptomes of CD4 and CD8 T cells (159 and 141 deregulated genes, respectively), consistent with enhanced activation and T-cell mediated inflammatory response (eg. TNFRS18, STAT1, CCL4). Furthermore, we observed a dose-dependent and significant increment of the CD69 (2-fold), CD25 (2.5-fold) and HLADR (4-fold) activation markers in the surface of NK cells from primary BM samples of 11 MM patients treated with BCMA-TCB2 (Panel E), suggesting a functional crosstalk between activated T cells and NK cells. In conclusion, we showed that the promising pre-clinical activity of the first-in-class IgG-based BCMA-TCB can be further enhanced by longer treatment periods followed by robust T cell activation. The observation that the efficacy of BCMA-TCB is intrinsically related to the activation status of T cells suggests its rational combination with IMIDs as demonstrated here. Most interestingly, potential crosstalk between activated T and NK cells could lead to enhanced function of the later immune subset, and provide a rational combination between BCMA-TCB and anti-CD38 antibodies to eradicate MM cells through highly activated T and NK cells. Figure Figure. Disclosures Strein: EngMab: Employment. Vu:EngMab: Employment. Paiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

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Pharmacokinetic and Pharmacodynamic Study of NKTR-255, a Polymer-Conjugated Human IL-15, in Cynomolgus Monkey

Blood ◽

10.1182/blood-2018-99-115638 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2952-2952

Author(s):

Takahiro Miyazaki ◽

Peiwen Kuo ◽

Mekhala Maiti ◽

Palakshi Obalapur ◽

Murali Addepalli ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cynomolgus Monkey ◽

Memory T Cells ◽

Granzyme B ◽

Employment Equity ◽

Equity Ownership ◽

Dose Dependent

Abstract Introduction IL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey. Methods In vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry. Results NKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis. Conclusions Nektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

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Correlation between driver mutation and immune microenvironment in colorectal cancer patients.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e15513 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e15513-e15513

Author(s):

Xiaochen Cai ◽

Yuezong Bai ◽

Xiaochen Zhao ◽

Longgang Cui ◽

Hui Chen

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

Nk Cells ◽

Cancer Patients ◽

Cd8 T Cells ◽

Braf Mutation ◽

Kras Mutation ◽

Tumor Stroma ◽

Wild Type ◽

Colorectal Cancer Patients

e15513 Background: Immune checkpoint inhibitor (ICI) therapy has made great achievements, but ICI monotherapies show less effectiveness in colorectal cancer patients. Biomarker exploration have carried out from PD-L1 expression, neo-antigen, gene mutation, etc., but no satisfactory results have been obtained. Methods: Patients, Colorectal cancer patients. Methods, Multi-color immunohistochemistry (multi-IHC) was used to evaluate CD8+ T cells, macrophages and natural killer cell (NK cell) in tumors and tumor stroma. The Shapiro-Wilk method was used to test the normality of the data, and the t-test or Mann-Whitney U test was used according to the test results. A two-sided P < 0.05 was considered a significant difference. Results: The study included 72 colorectal cancer patients, including 26 female (36.7%) and 46 male (63.8%), with a median age of 59.5 (50-67.3). There were 6 patients (8.3%) with BRAF mutation, 43 patients (59.7%) with KRAS mutation, and 56 patients (77.8%) with TP53 mutation. The results of immunohistochemistry showed that, BRAF mutation Vs BRAF wild-type or KRAS mutation Vs KRAS wild-type, the number or proportion of CD8+ T cells, macrophages or NK cells in tumor and tumor stroma were not statistically different. For TP53 mutation Vs TP53 wild-type, the number and proportion of CD8+ T cells or macrophages in tumor and tumor stroma were not statistically different. There was no statistical difference in the number and proportion of NK cells in tumor. But, the median number of NK cells in tumor stroma was 345 Vs 129, p = 0.06, and the proportion of NK cells was 5.2% Vs 1.39%, p = 0.02. Conclusions: There is no significant change in the immune microenvironment of colorectal cancer patients with BRAF mutation and KRAS mutation. There are more NK cells in tumor stroma of colorectal cancer patients with TP3 mutation.

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Large-Scale Mass Cytometry Reveals Significant Activation of Innate and Adaptive Immunity in Bone Marrow Tumor Microenvironment of Iberdomide-Treated Myeloma Patients

Blood ◽

10.1182/blood-2021-151542 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 730-730

Author(s):

Oliver Van Oekelen ◽

Michael Amatangelo ◽

Manman Guo ◽

Bhaskar Upadhyaya ◽

Geoffrey Kelly ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Dose Escalation ◽

Research Funding ◽

Large Scale ◽

Post Treatment ◽

Publicly Traded ◽

Heavily Pretreated

Abstract Background: Iberdomide (IBER) is a potent cereblon E3 ligase modulator (CELMoD agent) with direct anti-tumor and immunomodulatory activities in multiple myeloma (MM). An ongoing phase 1b/2a multicenter, open-label, dose-escalation study has reported favorable efficacy and safety of IBER plus low-dose dexamethasone (DEX) in heavily pretreated patients with relapsed/refractory MM (RRMM). While previous studies focused on IMiD/CELMoD agent immune effects in peripheral blood (Amatangelo, ASH 2019), a large-scale characterization of pharmacodynamics in the bone marrow (BM) tumor microenvironment (TME) has not been reported. Here, we present data on BM aspirates (BMAs) of 99 individuals pre-/post-treatment with IBER in the largest study of TME immune dynamics of RRMM patients to date. Methods: A mass cytometry (CyTOF) panel was designed/validated to capture deep and comprehensive immunophenotyping of T, B, and NK cell subpopulations. In all, staining with 37 metal-tagged Abs characterized 110 supervised cell phenotypes. Paired longitudinal assessment was conducted for viably preserved BMAs of IBER±DEX treated patients in dose-escalation of the CC220-MM-001 Ph 1b/2a study pre (SCREEN) and post treatment (cycle 2 day 15, C2D15). For a subset of patients (n=12), samples were available at disease progression (PD) . A training set analysis (n=64, data reported here) collected in North America, was validated against an independent test set (n=35) recruited in Europe. An R-based computational workflow and manual hierarchical gating identified subpopulations of B, T, NK and myeloid cells. In total, more than 5M immune cells were captured (approx. 40K per specimen). Cell populations are expressed as percentage of non-tumor bone marrow mononuclear cells (BMMC, i.e. CD45+CD66b-). P values are by Mann-Whitney U test for unpaired and Wilcoxon test for paired analyses. Results: IBER treatment resulted in profound immune shifts in the TME. B cells decreased (median SCREEN 3.3% vs C2D15 0.7%, p=0.001), with significant reduction of naïve (p=0.001) and regulatory B cells (p<0.001). Decrease occurred in CD4+ T cells (median 15.3% vs 8.9%, p<0.001), whereas CD8+ T cells increased (median 14.9% vs 17.4%). T cell subtypes showed a shift towards cytotoxic effector-memory phenotype (CCR7-CD45RA) (median 67.6% vs 80.9% of CD4+ T cells, p<0.001 and median 78.2% vs 89.2% of CD8+ T cells, p<0.001) with concurrent reduction of naive (CD45RA+CCR7+), central-memory (CD45RA-CCR7+) and EMRA (CD45RA+CCR7-) T cells. The shift towards a cytotoxic TME is confirmed by significant increases of GZMB+, HLA-DR+, ICOS+, Ki-67+, CXCR3+ and CD38+ activated CD8+ T cells (p≤0.001, paired). Conversely, CD8+ T cells expressing inhibitory checkpoints TIGIT and KLRG1 decreased significantly (p≤0.001, paired). Similar changes (with minor deviations) were noted in the CD4+ T cell compartment. NK cells increased (median 4.2% vs 8.7%, p=0.003), with increase of both CD56hi cytokine-producing (median 1.9% vs 4.5%, p<0.001) and CD16+ cytolytic (median 1.8% vs 2.6%, p=0.06) subsets. NKG2A+ and NKG2D+ subsets were increased (p<0.001, paired), as well as subsets expressing markers of activation: GZMB, ICOS, CD38 and PD-1 (p<0.05, paired). TIGIT+ NK cells decreased significantly. NKT cells were also significantly increased (median 1.4% vs 2.2%, p<0.001). Increase of NK and NKT cells expressing the activating receptor NKG2D was limited to patients achieving best response of PR or better. Additional analyses correlating immune phenotypes at baseline/post-treatment with outcomes are ongoing and will be reported at the meeting, as will data on how prior treatment (e.g. with CD38-mAb) shapes the TME at baseline and affects response. Conclusion: Our analysis on a large cohort of RRMM patients provides unique insights into the heterogeneity of the immune TME of heavily pretreated MM patients and how it changes upon treatment with IBER±DEX. We found significant increases of effector T and NK cells in paired analysis, demonstrating innate and adaptive immune enhancement in the MM bone marrow niche as an important mechanism of action. Importantly, these changes were observed throughout dose escalation and in patients previously refractory to lenalidomide/pomalidomide. The presented platform of large-scale immune profiling demonstrates a strategy to design and study rational combinations with (other) immune-enhancing therapies in MM. Figure 1 Figure 1. Disclosures Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gooding: Bristol Myers Squibb: Research Funding. Jagannath: Legend Biotech: Consultancy; Bristol Myers Squibb: Consultancy; Karyopharm Therapeutics: Consultancy; Janssen Pharmaceuticals: Consultancy; Takeda: Consultancy; Sanofi: Consultancy. Pierceall: BMS: Current Employment, Current equity holder in publicly-traded company. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

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Immune Mobilization with IL-2 and Growth Factors: Differential in Vivo Effects of the NKG2D Receptor on CD8+ and CD56+ Effector Cells

Blood ◽

10.1182/blood.v112.11.3491.3491 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3491-3491

Author(s):

Kenneth R Meehan ◽

John M. Hill ◽

Marc Ernstoff ◽

Charles L Sentman

Keyword(s):

T Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

High Dose ◽

Myeloma Cells ◽

Effector Cells ◽

Nkg2d Expression ◽

Dose Dependent ◽

Nkg2d Receptor

Abstract Background: NKG2D, one of four NK cell activating receptors characteristically found on NK cells, has been identified on some CD8+T cells that mediate TCR-independent and non-MHC restricted tumor cell killing. These cytotoxic NKG2D+CD8+ effector cells aggressively kill myeloma cells. Since the NKG2D receptor recognizes ligands expressed on myeloma cells, NKG2D+ CD8+ effector cells provide a unique immunotherapy opportunity. Methods: We designed an immune mobilization trial to mobilize autologous cytotoxic effector cells, with peripheral CD34+ hematopoietic progenitor cells. IL-2 (dose escalation) began on day 0 of mobilization and continued as a daily SQ injection for 11 days. On day 7 of mobilization, GM-CSF (7.5 mcg/kg/d) and G-CSF (5 mcg/kg/d) were initiated for 5 days (days 7–11). Leukapheresis was performed on Day 11. Results: Eleven of 12 patients completed therapy (myeloma, n=11; NHL, n=1). One patient (NHL) progressed during treatment. The MTD of IL-2 was 6×105 IU/m2/d. All patients successfully mobilized and received an autologous transplant with normal engraftment (ANC recovery: day 13 median; range 10–14 days; platelet recovery: day 12 median; range of 0–13 days). When compared to baseline, the immune-mobilized cells demonstrate an increase in CD3+CD8+ T cells (p = 0.01), CD3+CD8+CD56+ T cells (p = 0.01) and CD56+ NK cells (p = 0.002). Cytotoxicity directed against a human myeloma cells increased from 9% (baseline) to 43% (p=0.02). Importantly, immune mobilization resulted in an IL-2 dose-dependent expression of NKG2D on CD8+T cells or CD56+ NK cells. Low dose IL-2 (6 × 105 IU/m2/d) moderately increased NKG2D expression on CD8+ T cells when compared to baseline (p = 0.02). High dose IL-2 (1.5 × 106 IU/m2/d) strongly enhanced NKG2D expression on CD 8+ T cells (p = 0.004). Low dose IL-2 (6 × 105 IU/m2/d) and high dose IL-2 (1.5 × 106 IU/m2/d) increased NKG2D expression on CD3-CD 56+ NK cells, with p values of 0.03 and 0.002, respectively. Preliminary data indicate these effects are sustained in vivo post-transplant. Conclusions: Immune mobilization resulted in an IL-2 dose-dependent expression of NKG2D on CD8+T cells and CD56+ NK cells, thereby altering the cellular components of the mobilized cells. Since the NKG2D receptor recognizes ligands expressed on myeloma cells, NKG2D+ CD8+ effector cells provide a unique immunotherapy opportunity for the treatment of myeloma.

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Infiltration Patterns of Immune Cells in Gastric Cancer and Their Clinical Correlation with Prognosis

10.21203/rs.3.rs-18120/v1 ◽

2020 ◽

Author(s):

XiaoLi Wu ◽

Hongbo Ma ◽

YanYan Li

Keyword(s):

Gastric Cancer ◽

T Cells ◽

Mast Cells ◽

Nk Cells ◽

Cancer Patients ◽

Cd8 T Cells ◽

Immune Cells ◽

Immune Cell ◽

M1 Macrophages ◽

Gastric Cancer Patients

Abstract Abstract: Objective Gastric cancer is a malignant tumour that severely affects the health of patients. This study analyses the correlation between gastric cancer-infiltrating immune cell patterns and clinical prognosis and provides a scientific basis for the development of comprehensive tumour prevention and treatment strategies. Method Transcripts and related clinical data from 9-2019 for gastric cancer were downloaded from the TCGA database. The proportions of 22 kinds of immune cells were calculated by CIBERSORT software, and the correlation of each immune cell component ratio with tumour grade, clinical stage and overall survival (OS) was evaluated. Results A total of 413 gene transcript data sets were obtained from the TCGA database, including 381 for gastric cancer and 32 for normal tissues. The expression of various macrophages in tumour tissues was abundant. The immune cell composition, which included resting dendritic cells (p=0.02), M1 macrophages (p=0.031), resting mast cells (p=0.02), CD8 T cells (p=2.445e-04), M0 macrophages (p=6.353e-04), activated mast cells (p=0.006), neutrophils (p=0.003), resting NK cells (p=0.014), and gamma delta T cells (p=0.033), is related to the pathological grade. As the tumour stage of gastric cancer patients progresses, the proportion of some immune cells, including eosinophils (p=0.013), activated mast cells (p=0.042), neutrophils (p=0.007), and resting NK cells (p=0.036) gradually increases, while the proportion of other immune cells, for example, CD8 T cells (p=0.018), Tregs (p=0.039), M1 macrophages (p=0.018), and activated NK cells (p=0.042) gradually decreases. Higher expression of CD8 T cells suggests a better prognosis. Conclusion The composition of tumour-infiltrating immune cells differed greatly in different pathological grades and stages of gastric cancer. CD8 T cells can be used as a prognostic factor for gastric cancer patients.

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263 Pleiotropic effects of IL-7 in prostate cancer patients receiving Sipuleucel-T vaccination

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0263 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A287-A287

Author(s):

Caroline Duault ◽

Nirasha Ramchurren ◽

Russell Pachynski ◽

Lawrence Fong ◽

Sean Bendall ◽

...

Keyword(s):

Prostate Cancer ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Cancer Patients ◽

T Cell Activation ◽

Cd8 T Cells ◽

Therapeutic Potential ◽

Intracellular Cytokine Staining ◽

Γδ T Cells

BackgroundSipuleucel-T (Provenge) is the first therapeutic vaccination approved by the FDA so far, indicated for advanced metastatic prostate cancer patients. Despite an improvement of the overall survival, the benefits of the therapy are still short-term so increasing the duration of the efficacy is necessary. Specifically, T-cell anergy is one of the challenges that we need to overcome to improve the overall efficacy. IL-7 is known to promote the naive T cell activation and to increase the proliferation and activation of the T cell memory subsets. Therefore, in this phase II clinical trial, we tested the therapeutic potential of a human recombinant glycosylated IL-7 after completion of the Provenge therapy on asymptomatic advanced prostate cancer patients.MethodsTo get a comprehensive analysis of the immune landscape in these patients, we performed CyTOF analysis on PBMC samples obtained at week 1 (baseline) and week 6 after the beginning of the IL-7 therapy. After stimulation with PMA/Ionomycin, we proceeded to surface and intracellular cytokine staining before acquisition on the CyTOF. The data were then analyzed by expert gating on Cytobank.ResultsAt 6 weeks post therapy, our data showed an increase in the number of circulating T lymphocytes in the IL-7 cohort, especially CD8 T cells, in accordance with previous literature. Even though of the frequency of CD4 T cells did not increase, the cells showed greater functionalities, with increased expression of IL-2, TNFα and IL-6 upon stimulation by PMA-Ionomycine. Cytotoxic subsets were also positively affected, with increased expression of IFNγ in CD8 T cells, TNFα in NK cells and IL-2 in γδ T cells. Moreover, PD-1 expression was decreased on CD4, CD8 and γδ T cells while CD137 increased on CD4, CD8 and NK cells. In addition, despite a reduction in the pool of circulating monocytes, we observed higher TNFα expression in these cells.ConclusionsAltogether, our data revealed multiple effects of IL-7 in these patients, highlighting a complex set of in vivo mechanisms. In the future, knowledge of these effects may help in choosing the best agents to use in combination with IL-7 and/or the best patients to benefit from IL-7 as part of their therapeutic approach.Trial RegistrationNCT01881867Ethics ApprovalThe study was last approved by Fred Hutchinson Cancer Research Center Institutional Review Board, IR file 8037, on January 23, 2020

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