Tumor Clearance Research Articles

The Outcomes and Surgical Nuances of Minimally Invasive Parotid Surgery for Pleomorphic Adenoma.

Benign parotid tumors follow an indolent course and present as slow-growing painless swelling in the pre-and-infra-auricular areas. The treatment of choice is surgery. Though the gold standard technique is Superficial Parotidectomy, Extracapsular Dissection (ECD) is an alternative option with the same outcome and decreased complications. This study discusses our experience with extracapsular dissection and the surgical nuances for better results. A retrospective study of histologically confirmed cases of pleomorphic adenoma of the parotid gland, who underwent Extracapsular dissection between September 2019 and March 2023, was done. The demographic details, clinical characteristics, and outcomes were evaluated. There were 33 patients, including 16 females and 17 males, with a mean age of 32.75years. All cases presented as slow-growing painless swelling for a mean duration of 5years. Most of the tumors (94%) were of size between 2 and 4cm, with few tumors more than 4cm. All underwent extracapsular dissection with complete excision. There was only one complication (seroma) and no incidence of facial palsy in our experience with ECD. The goal of a benign parotid surgery is the complete removal of the tumor with minimum complications, which could be achieved with ECD, which has good tumor clearance and lesser rates of complications with good cosmesis. Thus, this minimally invasive parotid surgery could be a worthwhile option in properly selected cases.

DIPG-32. PRECLINICAL TESTING OF THE EFFICACY AND SAFETY OF CRISPR/CAS9 GENE EDITED CAR-T CELL THERAPY FOR DIFFUSE MIDLINE GLIOMA (DMG)

Abstract DMGs are highly aggressive and difficult-to-treat brain tumors in children. Recent advances in Chimeric Antigen Receptor (CAR) expressing T cell therapy suggest application for brain tumors. Recent studies also demonstrate feasibility of GD2, Her2 and B7H3 CAR-T infusions in childhood brain tumors. We identified and developed a novel CAR-T therapy, targeting the CD99 antigen, which is homogenously expressed at high levels on DMG tumor cells. These CAR-T cells showed initial clearance of tumor, but the long-term persistence and anti-tumor efficacy of these CAR-T cells in DIPG xenografts were limited due to fratricide, as T cells also express CD99. To overcome this obstacle, we have further optimized a protocol in which we first knock-out CD99 from the human donor T cells using CRISPR-cas9 gene-editing and subsequently transducing with our CD99 virus in the pure population of CD99KO T cells. Systemic delivery of these gene-edited CAR-T cells showed enhanced in vivo persistence with complete clearance of tumor in DMG mouse models, and no tumor recurrence was seen well-beyond the time frame of expected tumor recurrence after treatment with un-edited CD99 CAR-T cells. In addition, loco-regional delivery of these gene-edited CD99 CAR-T cells when administered directly to the 4th ventricle, even at a low dose, showed similar anti-tumor efficacy with significantly increased animal survival. These data suggest a regional approach analogous to Omaya reservoir administered drug in humans may be a promising alternative approach to limit any toxicity.

IMMU-22. ALLOGENEIC NK CELLS SECRETING IL15 AND RESISTANT TO TGF-β Show Antitumor Activity Against Gliobastoma

Abstract INTRODUCTION High grade brain cancers, like most solid tumors, often relapse and are resistant to current therapies. Limited efficacy to cell therapy may be related to intratumoral heterogeneity and the suppressive tumor microenvironment. Hence, we aimed to modify allogeneic natural killer (NK) cells, which can recognize expressed tumor stress ligands, to resist TGF-β and secrete IL15. METHODS Cord blood-derived NK cells were expanded and modified to express IL15 and a dominant negative receptor (DNR) against TGF-β. NK cell product activity against the GBM cell line U87MG were evaluated in vitro and in vivo. The behavior of these cells were also analyzed ex vivo, to evaluate the impact (including potential toxicity) of increasing the potency of this allogeneic NK cell product. RESULTS While animals injected with NK cells secreting IL15 and expressing DNR successfully cleared tumors 6 days after T cell infusion, we did not observe significant differences in survival between the groups (no treatment control mice vs treated with mock-transduced NK cell vs DNR-transduced NK cells vs IL-15/DNR-transduced NK cells). Therefore, we sought to determine whether cytokine-related toxicity was responsible for death in the setting of tumor clearance. We compared no treatment control mice vs mice treated with mock-transduced or IL-15/DNR-transduced NK cell products. Elevated levels of IL-15 were detected in the plasma of mice treated with IL-15/DNR-transduced NK cells. These elevated IL-15 levels corresponded with NK cell persistence in the brain measured by ddPCR. However, no other cytokines were upregulated. CONCLUSIONS We observed improved tumor clearance when NK cells were both protected against immune suppressive TGF-β and were capable of secreting the stimulatory cytokine IL15. We are currently performing additional experiments to determine whether toxicity from IL15 secretion occurs. To potentially mitigate this toxicity we plan to engineer a suicide gene into these constructs.

MDB-20. IMPROVING THE PERSISTENCE OF CAR T CELLS AGAINST GROUP 3 MEDULLOBLASTOMAS VIA EPIGENETIC REGULATION OF EXHAUSTION

Abstract Medulloblastomas (MB) are the most common malignant pediatric brain tumor with molecular subclass Group 3 (G3) requiring the most intense treatment regimen including surgery, intensive radiation followed by chemotherapy. Despite an overall 40% survival rate, survivors’ quality of life is often impaired due to whole-brain radiation leading to neurocognitive defects. Chimeric antigen receptor (CAR) T cell therapy has emerged clinically as a safe, tumor-targeted immunotherapy with the most success observed against hematological malignancies. Translating CAR T cell therapy to brain tumors has proven challenging due to lack of targetable antigens on the surface of tumor cells and lack of persistence of CAR T cells. We have previously identified that patient samples and PDOX-derived cell lines of G3 MBs consistently over-express B7-H3 (CD267) compared to normal tissue. Herein, we investigated B7-H3 targeted CAR T cell products against G3 MB in vitro and in vivo. Initial results show that B7-H3 CAR T cells are highly effective in clearing human MB tumors in NSG mice, however, 4 out 5 mice experienced late relapses that showed reduced, but still present, B7-H3 antigen expression. We hypothesize that the lack of CAR T cell persistence is ultimately responsible for tumor regrowth. To overcome this limitation, we have identified epigenetic regulators DNMT3A and TET2 that control T cell exhaustion. Using CRISPR/Cas9, we genetically knocked out DNMT3A and TET2 in CAR T cells and investigated the impact on persistence and anti-tumor activity. Our data supports the hypothesis that knock out of DNMT3A and TET2 improves antigen-dependent proliferation and persistence of B7-H3 CAR T cells against G3 MB tumor lines in vitro. DNMT3A KO shows a more sustained effect over TET2. Currently, we are optimizing in vivo experiments to investigate prolonged tumor clearance in both immunocompromised and fully immunocompetent murine models.

AMPLIFY-201, a first-in-human safety and efficacy trial of adjuvant ELI-002 2P immunotherapy for patients with high-relapse risk with KRAS G12D- or G12R-mutated pancreatic and colorectal cancer.

2528 Background: RAS mutations occur in 25% of solid tumors with G12D being the most frequent variant. ELI-002 2P is a vaccine comprised of lymph-node targeted Amphiphile (Amph)-modified G12D and G12R mutant KRAS peptides with an Amph-modified CpG oligonucleotide adjuvant designed to expand polyfunctional mutant KRAS-specific T cells. Preclinical amphiphile data (relative to non-amphiphile controls) showed increased immunogenicity, tumor clearance and survival in mouse models. Methods: This first-in-human multicenter phase I trial assessed safety, immunogenicity and antitumor activity using a novel adjuvant trial design in patients (pts) with minimal residual disease (MRD) following standard locoregional treatment. Eligibility: elevated circulating tumor DNA (ctDNA) and/or serum tumor biomarker elevation (CA19-9/CEA), and KRAS/NRAS mutation. In a 3+3 dose escalation, n=22 received a subcutaneous course of 6 prime and 4 booster injections of fixed dose Amph-peptides (1.4 mg), admixed with escalating Amph-CpG-7909 (Table). Dose escalation based on observed safety in first 28 days. Safety, antitumor activity including biomarker reduction/clearance and relapse free survival using immune Response Evaluation Criteria in Solid Tumors (iRECIST) were assessed. Immunogenicity assessed by direct ex vivo Fluorospot and intracellular cytokine staining of peripheral blood mononuclear cells (T-cell responders = ≥ 2-fold increase over baseline) and by tumor CD3 immunohistochemistry in subset who underwent a biopsy. Results: No dose-limiting toxicities, treatment related SAEs or cytokine release syndrome were observed, and no maximum tolerated dose was identified. Safety: all grade 1, fatigue (19%), headache (19%), and injection site reaction (9.5%). Biomarker reduction was observed in 15/19 (79%) and in 5/5 at highest doses; clearance of MRD was observed in 4/19 (21% - n=2 pancreas, n=2 colorectal). Polyfunctional mKRAS-specific T cell responses observed in 80% of pts (n=12/15), with both CD8+ and CD4+ T cell responses for most, and CD3+ T cells observed on biopsies. The recommended phase 2 dose (RP2D) is 10.0 mg Amph-CpG-7909. Conclusions: ELI-002 2P, evaluated in a novel MRD+ trial, was safe with ctDNA and serum tumor biomarker reduction and clearance and notable immune responses. The RP2D is the start dose for a phase 1/randomized phase 2 study evaluating a new seven peptide formulation for G12 D, R, V, S, A, C, and G13D mutated solid tumors (NCT05726864). Clinical trial information: NCT04853017 . [Table: see text]

Clinical-grade natural killer cells expanded from donor peripheral blood and anti-leukemia effect against myeloid leukemia.

e19018 Background: Natural killer (NK) cells play a crucial role in immune surveillance and tumor clearance. It has been reported that allogeneic NK cell therapy has good safety and curative effect in patients with refractory and relapsed acute myeloid leukemia (AML). However, the clinical application of adoptive NK infusion is limited due to the lower number of human NK cells. We established a feeder-free NK cell expansion system from peripheral blood (PB) and explore the anti-leukemia potential in preclinical and clinical studies. Methods: The killing capacity of ex vivo-expanded NK cells were detected by Flow cytometry. The in vivo expansion, survival and anti-leukemia activity of the expanded NK cells were detected in K562-bearing NSG mice. Six AML patients with hematological/extra medullary relapsed (n = 3) or persistent positive minimal residual disease (n = 3) after consolidation and maintenance therapy were enrolled in Peking University Institute of Hematology from 2021 to 2022. The patients were treated with high-dose cyclophosphamide and fludarabine, then combined with adoptive infusion of donor-derived expanded NK cells. The chimerism, the number and function of NK cells were analyzed, and the safety and clinical efficacy were evaluated. Results: The ex vivo-expanded NK cells exhibit highly anti-leukemia activity. NK cells proliferate in mice and the CD56+ cells to reach the peak time in each organ is 3-7 days and drop to the level before infusion after 14 days. NK cell infusion significantly improve the survival of leukemia mice, and the anti-leukemia effect is NK cell dose-dependent. In the clinical trial, six patients received a total of 7 cases of NK cell infusion, including 3 cases in low-dose group and 4 cases in middle-dose group, among which 1 patient received 2 dose (1 low-dose and 1 middle-dose). In the low-dose group, 1 patient got response (1/3), with the minimal residual disease turned negative. In the middle-dose group, 3 patients responded (3/4), of which 1 patient had the fusion gene CBFB /ABL decreased from 0.99% to 0.13%, 1 case of extramedullary recurrence of AML turned negative, and NK cells could be detected in cerebrospinal fluid, 1 case of hematological recurrence got hematological remission after treatment. The survival time of NK cells in vivo was 7d-30d, with longer time in middle dose group than those in low dose group. The proportion and number of NK cells in responding cases were significantly higher than those in non-responsive group. Conclusions: Clinical grade expanded NK cells has strong anti-leukemia activity in patients with relapse or minimal residual positive AML patients. NK cells can survive for 2 weeks and can enter the central nervous system without obvious adverse reactions. The response rate in middle-dose group is higher than that in low-dose group. However, the efficacy of NK cells still needs to be further confirmed in a large sample size. Clinical trial information: NCT04221971 .

Impact of postoperative residual disease in patients with primary epithelial ovarian cancer in the era of maintenance therapies with targeted agents: A systematic review and meta-analysis.

e17555 Background: To investigate the impact of residual disease after cytoreductive surgery for epithelial ovarian cancer (EOC) on survival outcomes in the era of targeted maintenance approaches, including bevacizumab and poly (ADP-ribose) polymerase (PARP) inhibitor administration. Methods: We searched relevant literature from the MEDLINE, Embase, and Cochrane Library databases to identify randomized controlled trials and prospective clinical trials of primary EOC published between 1 January 2000 and 22 September 2022. To evaluate the impact of postoperative residual tumors on PFS and OS, we constructed a linear regression model for log-transformed median progression-free survival (PFS) and overall survival (OS). Patients with and without first-line maintenance therapy were examined. The protocol was registered in the PROSPERO (CRD42021265810). Results: In total, 97 trials with 41,799 patients were included: 2476 received PARP inhibitors, and 6920 received bevacizumab. The median rate of complete cytoreduction was 36.3% (range, 0–99.4%), regardless of the timing of surgery. Multivariable analysis of the linear regression model of all studies revealed that the median OS increased by 12.97% for every 10% increase in complete cytoreduction rates, independently of the use of systemic maintenance. In subgroup analysis of patients with maintenance therapies, the effect of complete tumor clearance was potentiated, with a median OS increased by 19.13% for every 10% increase in complete cytoreduction rates. The previously described significant effect of total macroscopic tumor clearance of PFS could not be retained when adjusting for maintenance treatment. Conclusions: Total macroscopic tumor clearance at the initial presentation of EOC significantly prolongs OS. Our results establish the importance of complete surgical cytoreduction in the era of novel targeted systemic agents.

Intralesional administration of VAX014 facilitates in situ immunization and potentiates immune checkpoint blockade in immunologically cold tumors

BackgroundImmunologically cold tumors with an ‘immune desert’ phenotype lack tumor-infiltrating lymphocytes (TILs) and are typically impervious to systemic immune checkpoint blockade (ICB). Intratumoral treatment of tumors with immunomodulatory agents can...

Targeting CD47 in lymphoid malignancies with CAR T cells.

2513 Background: Despite major therapeutic advances of chimeric antigen receptor (CAR) T cells directed against CD19 in relapsed and refractory non-Hodgkin lymphoma (NHL), two thirds of patients experience relapse within the first two years. Nearly one third of these relapses are associated with CD19 antigen-loss escape thus leveraging alternate CAR targets may improve outcomes. CD47, an anti-phagocytic “don’t eat me” receptor known to be overexpressed in NHL, is an ideal CAR target as it is potentially indispensable for tumor progression. Methods: CD47-CAR binding domains were engineered either as a camelid single variable heavy chain nanobody (CD47-VHH-CAR) or as the CD47 binding domain of cognate receptor SIRPα (SIRPα-CAR), each combined with a CD28 costimulatory domain and a CD3ζ activation domain with two mutated ITAMs (1XX). In vitro co-culture assays were performed with lymphoid mouse tumor cell lines BM185 and A20. For in vivo experiments, syngeneic BALB/c mice were sublethally irradiated for lymphodepletion then injected intravenously with 1x105 luciferase+ BM185 cells. Two days later, mice were injected with 2x106 CAR T cells, and bioluminescence imaging was performed to monitor tumor progression. For bio-localization experiments, CAR T cells were co-transduced with a separate vector encoding luciferase. Results: CD47-CAR T cell expansion in vitro was hindered due to fratricide, which can be rescued by CD47 CRISPR knockout. Both CD47-VHH-CAR and SIRPa-CAR T cells exhibited potent and specific in vitro lysis of BM185 and A20 cells. CD47-VHH-CAR T cells showed transient anti-leukemia activity, however, survival was not significantly prolonged. T cell bio-localization analysis revealed that, compared to CD19-CAR T cells, CD47-VHH-CAR T cells exhibited prolonged retention in the thorax with minimal bone marrow trafficking, suggesting CD47 antigen-specific pulmonary sequestration, and inferior expansion. SIRPα-CAR T cells engineered to secrete pro-inflammatory cytokine IL-18 significantly improved CAR T cell expansion, resulting in bone marrow trafficking and clearance of BM185 tumor. However, recipients of IL-18-secreting SIRPa-CAR T cells experienced lethal CAR T cell-related toxicity. To improve the safety of CD47-CAR T cells, we engineered an NFAT-responsive vector expressing the CD47-CAR modified with E. coli-derived dihydrofolate reductase drug destabilization domain to limit CD47-CAR expression to T cells exposed to the stabilizing antibiotic trimethoprim and receiving a licensing NFAT signal from a CD19-CAR. Conclusions: In vivo activity of CD47-targeting CAR T cells is limited by thoracic retention and limited expansion, which can be partially overcome by overexpression of IL-18, though enhanced toxicity precludes survival prolongation. Our findings suggest that strategies to spatiotemporally restrict CAR expression or activation will be necessary to achieve safe and efficacious CD47-CAR T cell activity.

T cell CD62L expression following nivolumab therapy is associated with response to rituximab‐nivolumab in treatment naïve follicular lymphoma: the 1st FLOR study

Introduction: Inter-tumoral T cell dysfunction and efficacy of immune checkpoint inhibitors (ICI) in follicular lymphoma (FL) has been well described. However, few studies have examined peripheral blood immunity at diagnosis or the impact of frontline ICI on circulating immunity in FL. We hypothesised that immune dysregulation in peripheral blood would be present in treatment naïve FL and would correlate to response to frontline ICI. Methods: PBMC samples from 34 untreated FL patients receiving rituximab (R) and nivolumab (nivo) in the 1st FLOR trial (Hawkes JCO 2021) were collected at baseline, post 4 cycles of nivo and after 6 months of nivo±R. Immune profile was assessed using a single-tube 29 antibody FACS panel on a spectral flow cytometer and compared to the profile of 12 age matched healthy donors. Results were correlated with centrally determined PET response (Lugano criteria). Results: Compared to healthy donors, FL patients had increased proportions of TRegs (P < 0.0001) with decreased HLA-DR+ (P < 0.01) and increased PD-1+ (P < 0.01) populations. NK cells were increased (P < 0.05) with a 2-fold increase in TIM3 expression (P < 0.01). While total T cells were unchanged, expression of PD-1, TIM3, HLA-DR and 4-1BB were significantly increased in T cells from FL patients. Treatment with nivo significantly increased the populations of TRegs expressing 41BB, LAG-3, TIM3 or PD-L2 (P < 0.05) with a decreased PD-L1 positive population at PET-CT2. Expression of immune checkpoints on T cells was unchanged by therapy. To assess biomarkers of response, patients were stratified into sustained CR (CR achieved by PET-CT4 and maintained for 6 months without evidence of relapse, n = 15) vs. PR/PD (n = 21). At baseline, sustained CR was associated with a trend for increased proportions of Naïve CD4 and CD8 T cells and decreased TRegs and PD-L2 expressing TRegs. Baseline expression of PD-1 on T and NK cells did not correlate with sustained response. Most strikingly, expression of CD62L was significantly downregulated across total CD4 and CD8 T cells and TRegs at PET-CT2 in PR/PD patients (P < 0.05) and maintained at baseline levels in those patients who achieved a sustained CR. At this early timepoint final patient responses had not been established with most patients having either progressive or stable disease, suggesting that dysfunctional downregulation of CD62L in response to nivo may reflect the inability of patient’s T cells to activate and/or migrate to the tumour site and facilitate tumour clearance and long-term treatment responses. Conclusions: Grade 1-3A treatment naïve FL is associated with significant dysregulation of peripheral blood T and NK cells prior to therapy. Early downregulation of CD62L on T cells of patients treated with nivo was associated with the inability to achieve long term complete responses and may indicate aberrant T cell activation and/or function which impacts on long term response to therapy. Encore Abstract - previously submitted to EHA 2023 The research was funded by: Bristol Myers Squibb Keywords: Basic and Translational Science, Diagnostic and Prognostic Biomarkers Conflicts of interests pertinent to the abstract. A. Barraclough Honoraria: Roche, Gilead G. Chong Consultant or advisory role: Bristol Myers Squibb Research funding: Bristol Myers Squibb, Merck Serono, Pharmacyclics, Regeneron, Bayer, Astra Zeneca, Amgen, Hutch Med, Incyte E. A. Hawkes Consultant or advisory role: Roche, Merck Sharpe & Dohme, Astra Zeneca, Gilead, Antigene, Novartis, Regeneron, Janssen, Specialised Therapeutics Research funding: Bristol Myers Squibb, Roche, Merck KgA, Astra Zeneca

A phase I/II trial investigating safety and efficacy of autologous TAC01-HER2 in relapsed or refractory solid tumors.

2519 Background: Despite recent therapeutic developments for patients with advanced, metastatic, unresectable HER2+ solid cancers, significant unmet medical needs still exist, especially in tumors other than breast and gastric. The T cell antigen coupler (TAC) technology is a novel approach to modifying T cells, allowing them to recognize and treat HER2+ solid tumors. The TAC receptor redirects T cells to tumor cells, and upon tumor cell recognition, co-opts the natural T cell receptor (TCR) to yield safer anti-tumor responses versus chimeric antigen receptor (CAR) T cells. In preclinical studies, TAC T cells led to complete tumor clearance in mouse models of human cancer, without any TAC-related toxicities. Methods: In the ongoing clinical trial (NCT04727151), TAC01-HER2 treatment of HER2+ solid tumors is hypothesized to result in safe and effective anti-tumor responses. Subjects undergo leukapheresis, bridging therapy (if needed) while their TAC01-HER2 are engineered, lymphodepletion chemotherapy (LDC), and finally TAC01-HER2 infusion. In phase I dose escalation, the primary objective is to evaluate the safety of TAC01-HER2 at increasing doses of 0.3, 0.8, 3, and 8 x 106 cells/kg in HER2+ solid tumors (1+, 2+ or 3+ as identified by immunohistochemistry) in adult subjects who have progressed after ≥2 lines of systemic therapy. Dose limiting toxicities (DLTs) are assessed up to 28 days from cell infusion. In Phase II, dose expansion groups will further evaluate the safety, efficacy, and pharmacokinetics of the optimal TAC01-HER2 dose in HER2+ breast and other solid tumor types. Results: As of 10 Feb 2023, 18 patients have been treated in Cohorts 1-4 with breast, colorectal, gall bladder, gastroesophageal junction, gastric, esophageal, lung, and ovarian cancers. One DLT event of grade 3 pneumonitis has been reported in one subject in Cohort 4 (8 x 106 cells/kg). No neurotoxicity has been reported. Most subjects treated at Cohorts 3 and 4 experienced Grade ≤2 CRS which resolved with standard therapy. Twelve subjects have reported a total of 21 serious adverse events, 17 unrelated to TAC01-HER2 except for one Grade 3 pneumonitis, one Grade 1 and two Grade 2 CRS. Most adverse events were related to LDC or the underlying malignancy. At Cohort 2 (0.8 x 106 cells/kg), a partial response was observed in a subject with refractory metastatic gastric adenocarcinoma (3+ HER2) at 1st scan, with a 35% reduction in measurable disease and clinical benefit was maintained for 4 months. A 55% disease control rate was observed at Cohorts 2-4 at 1st scan. Conclusions: Dose escalation of TAC01-HER2 is ongoing, with seven subjects treated and three more scheduled in Cohort 4. These results in a heavily pre-treated cancer population show manageable safety and promising clinical activity with a novel T cell therapy that may have broad clinical applicability in HER+ cancers. Clinical trial information: NCT04727151 .

Polymeric nanoparticle gel for intracellular mRNA delivery and immunological reprogramming of tumors

Immuno-oncology therapies have been of great interest with the goal of inducing sustained tumor regression, but clinical results have demonstrated the need for improved and widely applicable methods. An antigen-free method of cancer immunotherapy can stimulate the immune system to recruit lymphocytes and produce immunostimulatory factors without prior knowledge of neoantigens, while local delivery reduces the risk of systemic toxicity. To improve the interactions between tumor cells and cytotoxic lymphocytes, a gene delivery nanoparticle platform was engineered to reprogram the tumor microenvironment (TME) in situ to be more immunostimulatory by inducing tumor-associated antigen-presenting cells (tAPCs) to activate cytotoxic lymphocytes against the tumor. Biodegradable, lipophilic poly (beta-amino ester) (PBAE) nanoparticles were synthesized and used to co-deliver mRNA constructs encoding a signal 2 co-stimulatory molecule (4-1BBL) and a signal 3 immuno-stimulatory cytokine (IL-12), along with a nucleic acid-based immunomodulatory adjuvant. Nanoparticles are combined with a thermoresponsive block copolymer for gelation at the injection site for local NP retention at the tumor. The reprogramming nanoparticle gel synergizes with immune checkpoint blockade (ICB) to induce tumor regression and clearance in addition to resistance to tumor rechallenge at a distant site. In vitro and in vivo studies reveal increases in immunostimulatory cytokine production and recruitment of immune cells as a result of the nanoparticles. Intratumoral injection of nanoparticles encapsulating mRNA encoding immunostimulatory agents and adjuvants via an injectable thermoresponsive gel has great translational potential as an immuno-oncology therapy that can be accessible to a wide range of patients.

A novel polymer-conjugated human IL-15 improves efficacy of CD19-targeted CAR T-cell immunotherapy.

Chimeric antigen receptor (CAR)-modified T-cell therapies targeting CD19 represent a new treatment option for patients with relapsed/refractory (R/R) B-cell malignancies. However, CAR T-cell therapy fails to elicit durable responses in a significant fraction of patients. Limited invivo proliferation and survival of infused CAR T cells are key causes of failure. In a phase 1/2 clinical trial of CD19 CAR T cells for B-cell malignancies (#NCT01865617), low serum interleukin 15 (IL-15) concentration after CAR T-cell infusion was associated with inferior CAR T-cell kinetics. IL-15 supports T-cell proliferation and survival, and therefore, supplementation with IL-15 may enhance CAR T-cell therapy. However, the clinical use of native IL-15 is challenging because of its unfavorable pharmacokinetic (PK) and toxicity. NKTR-255 is a polymer-conjugated IL-15 that engages the entire IL-15 receptor complex (IL-15Rα/IL-2Rβγ) and exhibits reduced clearance, providing sustained pharmacodynamic (PD) responses. We investigated the PK and immune cell PDs in nonhuman primates treated with NKTR-255 and found that NKTR-255 enhanced the invivo proliferation of Tcells and natural killer cells. Invitro, NKTR-255 induced dose-dependent proliferation and accumulation of human CD19 CAR T cells, especially at low target cell abundance. Invivo studies in lymphoma-bearing immunodeficient mice demonstrated enhanced antitumor efficacy of human CD19 CAR T cells. In contrast to mice treated with CAR T cells alone, those that received CAR T cells and NKTR-255 had markedly higher CAR T-cell counts in the blood and marrow that were sustained after tumor clearance, without evidence of persistent proliferation or ongoing activation/exhaustion as assessed by Ki-67 and inhibitory receptorcoexpression. These data support an ongoing phase 1 clinical trial of combined therapy with CD19 CAR T cells and NKTR-255 for R/R B-cell malignancies.

Immunogenetic Metabolomics Reveals Key Enzymes That Modulate CAR T-cell Metabolism and Function.

Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we investigated whether it is possible to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism whereby cancer cells suppress T-cell function by generating a metabolically unfavorable tumor microenvironment (TME). In an in silico screen, we identified ADA and PDK1 as metabolic regulators. We then showed that overexpression (OE) of these genes enhanced the cytolysis of CD19-specific chimeric antigen receptor (CAR) T cells against cognate leukemia cells, and conversely, ADA or PDK1 deficiency dampened this effect. ADA-OE in CAR T cells improved cancer cytolysis under high concentrations of adenosine, the ADA substrate, and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics analysis of these CAR T cells revealed alterations of global gene expression and metabolic signatures in both ADA- and PDK1-engineered CAR T cells. Functional and immunologic analyses demonstrated that ADA-OE increased proliferation and decreased exhaustion in CD19-specific and HER2-specific CAR T cells. ADA-OE improved tumor infiltration and clearance by HER2-specific CAR T cells in an in vivo colorectal cancer model. Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR T cells and reveal potential targets for improving CAR T-cell therapy.

Nationwide Impact of Centralization, Neoadjuvant Therapy, Minimally Invasive Surgery, and Standardized Pathology Reporting on R0 Resection and Overall Survival in Pancreatoduodenectomy for Pancreatic Cancer

BackgroundSurgeons aim for R0 resection in patients with pancreatic cancer to improve overall survival. However, it is unclear whether recent changes in pancreatic cancer care such as centralization, increased use of neoadjuvant therapy, minimally invasive surgery, and standardized pathology reporting have influenced R0 resections and whether R0 resection remains associated with overall survival.MethodsThis nationwide retrospective cohort study included consecutive patients after pancreatoduodenectomy (PD) for pancreatic cancer from the Netherlands Cancer Registry and the Dutch Nationwide Pathology Database (2009–2019). R0 resection was defined as > 1 mm tumor clearance at the pancreatic, posterior, and vascular resection margins. Completeness of pathology reporting was scored on the basis of six elements: histological diagnosis, tumor origin, radicality, tumor size, extent of invasion, and lymph node examination.ResultsAmong 2955 patients after PD for pancreatic cancer, the R0 resection rate was 49%. The R0 resection rate decreased from 68 to 43% (2009–2019, P < 0.001). The extent of resections in high-volume hospitals, minimally invasive surgery, neoadjuvant therapy, and complete pathology reports all significantly increased over time. Only complete pathology reporting was independently associated with lower R0 rates (OR 0.76, 95% CI 0.69–0.83, P < 0.001). Higher hospital volume, neoadjuvant therapy, and minimally invasive surgery were not associated with R0. R0 resection remained independently associated with improved overall survival (HR 0.72, 95% CI 0.66–0.79, P < 0.001), as well as in the 214 patients after neoadjuvant treatment (HR 0.61, 95% CI 0.42–0.87, P = 0.007).ConclusionsThe nationwide rate of R0 resections after PD for pancreatic cancer decreased over time, mostly related to more complete pathology reporting. R0 resection remained associated with overall survival.

Breast Cancer Immunity: It is TIME for the Next Chapter.

Our ability to interrogate the tumor immune microenvironment (TIME) at an ever-increasing granularity has uncovered critical determinants of disease progression. Not only do we now have a better understanding of the immune response in breast cancer, but it is becoming possible to leverage key mechanisms to effectively combat this disease. Almost every component of the immune system plays a role in enabling or inhibiting breast tumor growth. Building on early seminal work showing the involvement of T cells and macrophages in controlling breast cancer progression and metastasis, single-cell genomics and spatial proteomics approaches have recently expanded our view of the TIME. In this article, we provide a detailed description of the immune response against breast cancer and examine its heterogeneity in disease subtypes. We discuss preclinical models that enable dissecting the mechanisms responsible for tumor clearance or immune evasion and draw parallels and distinctions between human disease and murine counterparts. Last, as the cancer immunology field is moving toward the analysis of the TIME at the cellular and spatial levels, we highlight key studies that revealed previously unappreciated complexity in breast cancer using these technologies. Taken together, this article summarizes what is known in breast cancer immunology through the lens of translational research and identifies future directions to improve clinical outcomes.

Precise activation of antiviral memory T cells in solid tumors triggers a multi-cytokine response that ultimately drives tumor clearance.

Abstract The reactivation of CD8 +tissue-resident memory T cells (T RM) triggers a broad, yet local response that mobilizes adaptive and innate immune cells. This immunostimulatory process can be initiated in solid tumors when infiltrating antiviral memory T cells are reactivated. The intratumoral delivery of viral peptides is sufficient to clear poorly immunogenic tumors in mice and synergizes with immune checkpoint therapy. This study investigated the mechanisms required for this anti-tumor response and novel “Peptide Alarm Therapy” that is currently being tested in a Phase I clinical trial. In contrast to expectations, viral peptide presentation by cancer cells was not required for treatment efficacy, and while NK and CD4 T cells are activated and/or recruited to the tumor microenvironment (TME) following antiviral memory T cell activation, depletion of either population did not limit tumor clearance. Interestingly, inflammatory monocytes were greatly increased in both the TME and tumor-draining lymph node (tdLN) following therapy. Moreover, antigen presenting cells (APCs) containing tumor-derived antigen accumulated within the tdLN. TNFα, IFNγ, and IL-2 are cytokines that can signal to APC populations and are produced by activated T RM. When these cytokines were blocked collectively, but not independently, the treatment efficacy of Peptide Alarm Therapy was greatly reduced. Further, when these cytokines were intratumorally injected, they recapitulated some phenomena achieved by antiviral T cell activation. Thus, local reactivation of antiviral memory T cells, which infiltrate tumors and possess known specificities, drives a multi-cytokine response that promotes the immune-stimulation that effectuates tumor clearance. 1R01CA238439-01A1, 1F30CA253992-01

Generation of novel anti-tumor chimeric antigen receptors incorporating downstream T-cell signaling proteins

Abstract Chimeric antigen receptors (CAR) are molecules with an antibody-derived extracellular domain combined intracellularly with T cell receptor (TCR) signaling proteins. Although CAR-T cells have been a breakthrough clinically, several challenges persist: adverse effects from cytokine secretion, CAR-T cell exhaustion, and limited sensitivity in tumor microenvironments with low density of target antigen (i.e. tumor relapse and solid tumors). While many efforts have focused on identifying new cell surface targets, we have modified CAR intracellular domains. This idea arose from our super-resolution microscopy study that revealed that adapter molecules form clusters distinct from the TCR complex and that full activation necessitates passing a signaling threshold. From these findings, novel Chimeric Adapter Proteins (CAPs) were designed to trigger signaling downstream of the TCRζ chain. CAPs replace the TCRζ with ZAP70 intracellularly and are fused to an extracellular targeting domain. Thus, the potency problem associated with CARs would be ameliorated as CAPs would bypass kinetic proofreading steps defining the signaling threshold and the inhibitory regulation of upstream molecules. Indeed, CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo persistence of tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19–28ζ CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28ζ-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.

The efficacy of dermoscopy in defining the surgical margins of cutaneous squamous cell carcinoma: a retrospective study.

To investigate the diagnostic value of dermoscopy in defining the tumor margin of cutaneous squamous cell carcinoma (cSCC) for the appropriate surgical margin. A total of 90 cSCC patients were enrolled in the study. All patients were recruited into two groups: those who preserved intact macroscopic features of neoplasms without or after incisional biopsy and those with uncertain residual tumors after excisional biopsy. A dermoscopy-defined surgical margin of 8mm outward was used according to the tumor boundaries observed with the naked eye and dermoscopy. All excised tumor specimens were divided into serial sections according to the four "3, 6, 9, 12" directions at every 4-mm interval from the dermoscopy-detected tumor margin. Pathological examination was performed at 0mm, 4mm, and 8mm margins to confirm tumor remnants. Retrospective analysis of dermatoscopic results showed inconsistent clinical and dermatoscopic borders in 43 of 90 cases (47.8%). The ability of dermoscopy to detect tumor borders showed no statistical difference between the two groups (p > 0.05). In the unbiopsy or incisional biopsy group, 66.6% of the tumors were resected with a 4-mm margin and 98.3% with an 8-mm margin, with significant differences (p = 0.047). For patients with inconspicuous clinical evidence of residual tumor after excisional biopsy, the tumor clearance rate was 53.3% at 0mm, 93.3% at 4mm, and 100.0% at 8mm. Statistically significant differences were noted between 0mm and 4mm (p = 0.017), as well as between 0mm and 8mm (p = 0.043) but did not differ between 4mm and 8mm (p>0.05). Dermoscopy defined the tumor margin of cSCC better than visual inspection alone. Direct dermoscopic-guided surgery with at least 8-mm expansion was recommended for high-risk cSCC. Dermoscopy also assisted in identifying surgical margins at the healing biopsy site, making 8mm still the recommended expansion range.

Human CD4 cytotoxic T lymphocytes mediate potent tumor control in humanized immune system mice

Efficacy of immune checkpoint inhibitors in cancers can be limited by CD8 T cell dysfunction or HLA-I down-regulation. Tumor control mechanisms independent of CD8/HLA-I axis would overcome these limitations. Here, we report potent CD4 T cell-mediated tumor regression and memory responses in humanized immune system (HIS) mice implanted with HT-29 colorectal tumors. The regressing tumors showed increased CD4 cytotoxic T lymphocyte (CTL) infiltration and enhanced tumor HLA-II expression compared to progressing tumors. The intratumoral CD4 T cell subset associated with tumor regression expressed multiple cytotoxic markers and exhibited clonal expansion. Notably, tumor control was abrogated by depletion of CD4 but not CD8 T cells. CD4 T cells derived from tumor-regressing mice exhibited HLA-II-dependent and tumor-specific killing ex vivo. Taken together, our study demonstrates a critical role of human CD4 CTLs in mediating tumor clearance independent of CD8 T cells and provides a platform to study human anti-tumor immunity in vivo.