Antibody-based Immunotherapy Research Articles

Abstract 6612: Anti-GD2 antibody internalization by neuroblastoma as a mechanism of immunotherapy resistance

Abstract Background: Neuroblastoma, a sympathetic nervous system cancer, accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy including the relatively recent introduction of antibody targeting disialoganglioside (GD2), a common neuroblastoma antigen. The mechanisms underlying therapeutic resistance, specifically to antibody-based therapy, in some patients remains poorly understood. Aims: We hypothesized that differential internalization of anti-GD2 antibody by tumor cells upon binding to surface GD2 may act as a mechanism of escape by decreasing tumor sensitivity to antibody-dependent cellular cytotoxicity (ADCC). We aimed to measure antibody internalization differences across 22 human neuroblastoma cell lines and investigate how it relates to GD2 expression and ADCC sensitivity. Methods: Antibody internalization was measured using IncucyteS3 live cell imaging and anti-GD2 antibody (14G2a) conjugated to a pH-sensitive red fluorescent dye (pHrodo). GD2 surface expression was quantified by flow cytometry and BD QuantiBrite Beads. Neutrophil-mediated ADCC was measured in vitro via one of two methods - DIMSCAN and calcein-labeled tumor cells or IncucyteS3 and GFP-expressing tumor cells. Agents targeting endocytosis (chlorpromazine, cytochalasin-D, methyl-beta-cyclodextrin/MβCD, Pitstop2, and EIPA) were then used to assess inhibition of internalization and subsequently sensitization to ADCC. Results: Internalization of 14G2a-pHrodo over 24 hours varied significantly across cell lines (AUC range: 50-529) with LAN1 demonstrating the highest internalization AUC (529 +/- 40). Internalization rate was not correlated with GD2 surface expression (r=0.15). However, a moderate inverse correlation was found between antibody internalization and sensitivity to neutrophil-mediated ADCC in the 10 cell lines tested (r=-0.57). Antibody internalization by LAN1 was significantly inhibited by treatment with EIPA, chlorpromazine, and MβCD (AUC 1.1, 3.9, 104, respectively), all without significant change in surface GD2 expression. Treatment with MβCD (2mM) additionally was able to sensitize LAN1 to neutrophil-mediated ADCC in a dose-dependent manner. Conclusion: Anti-GD2 antibody internalization varies widely across human neuroblastoma cell lines, is inversely correlated with ADCC sensitivity, and, in a high internalizing cell line, can be effectively modulated by inhibitors of pinocytosis. The finding that membrane cholesterol depletion with MβCD sensitized LAN1 to neutrophil-ADCC suggests that membrane organization of GD2, specifically in relation to cholesterol-rich domains, may play a more significant role in antibody internalization than specific endocytosis processes. Elucidating further the mechanisms underlying differential internalization across cell lines could lead to the development of agents targeting internalization and ultimately augmenting tumor sensitivity to antibody-based immunotherapy. Citation Format: Rachelle J. Tibbetts, Kee Kiat Yeo, Sakunthala Muthugounder, Shahab Asgharzadeh. Anti-GD2 antibody internalization by neuroblastoma as a mechanism of immunotherapy resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6612.

3058 – SURFACE PROTEOMICS REVEALS NOVEL POTENTIAL AML ANTIGENS FOR IMMUNOTHERAPEUTIC TARGETING

Compared to solid tumors, antibody-based immunotherapy is underexploited in acute myeloid leukemia (AML), likely due to the limited availability of targetable surface antigens specifically expressed on AML cells. While targeted immunotherapies hold great promise, their development inexorably depends on the identification of cell surface antigens selectively expressed by cancer cells. The identification of such antigens has been challenging due to the characteristic hydrophobicity of plasma membrane proteins. Another level of complexity is brought by the fact that AML is a heterogeneous disease comprised of different subgroups with associated prognosis ranging from favorable to adverse. In order to identify novel AML antigens to be targeted using immunotherapeutic approaches, we have analysed the surface proteome of a collection of 100 primary human AML specimens, enriched in poor prognosis AML samples. These analyses revealed that AML subgroups have distinct surfaceome signatures. We also confirmed the expression of known AML markers at the surface of specific AML subgroups. Our search for novel AML antigens focused on four poor prognosis AML subgroups: MLL-rearranged, complex karyotype, RUNX1-mutated and normal karyotype with NPM1, DNMT3A and FLT3 (ITD) mutations. We identified proteins selectively expressed at the surface of AML cells from these subgroups. Analysis of the whole cohort of specimens also identified known and novel pan AML antigens. Single cell RNA-sequencing analysis of aforementioned subgroup-specific and pan AML antigens revealed that many of these are expressed by the vast majority of blast populations within primary AML specimens, including most primitive ones. Overall, these results suggest that studying the surface proteome of AML cells has the potential to reveal promising AML antigens to be targeted using immunotherapeutic approaches.

Monoclonal Antibodies in Cancer Therapy.

Monoclonal antibody-based immunotherapy is now considered to be a main component of cancer therapy, alongside surgery, radiation, and chemotherapy. Monoclonal antibodies possess a diverse set of clinically relevant mechanisms of action. In addition, antibodies can directly target tumor cells while simultaneously promoting the induction of long-lasting anti-tumor immune responses. The multifaceted properties of antibodies as a therapeutic platform have led to the development of new cancer treatment strategies that will have major impacts on cancer care. This review focuses on the known mechanisms of action, current clinical applications for the treatment of cancer, and mechanisms of resistance of monoclonal antibody therapy. We further discuss how monoclonal antibody-based strategies have moved towards enhancing anti-tumor immune responses by targeting immune cells instead of tumor antigens as well as some of the current combination therapies.

Chondroitin Sulphate Proteoglycan 4 (NG2/CSPG4) Localization in Low- and High-Grade Gliomas.

Background: Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines. Methods: NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques. Results: NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors. Conclusion: The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.

Polypharmacy as a prognostic factor in older patients with advanced non-small-cell lung cancer treated with anti-PD-1/PD-L1 antibody-based immunotherapy.

Polypharmacy is a common problem among older adults. However, its prevalence and impact on the clinical outcomes of anticancer treatment, such as survival and adverse events, in older patients with advanced cancer have not been well investigated. We retrospectively reviewed data from Japanese patients treated with an immune checkpoint inhibitor (ICI) for advanced or recurrent non-small-cell lung cancer (NSCLC) between 2016 and 2019. Among 157 older (aged ≥ 65years) patients, the prevalence of polypharmacy, defined as ≥ 5 medications, was 59.9% (94/157). The prevalence of potentially inappropriate medication use, according to the screening tool of older people's prescription (STOPP) criteria version 2, was 38.2% (60/157). The median progression-free survival (PFS) in patients with and without polypharmacy was 3.7 and 5.5months, respectively (P = 0.0017). The median overall survival (OS) in patients with and without polypharmacy was 9.5 and 28.1months, respectively (P < 0.001). Multivariate analysis revealed marked associations between polypharmacy and OS, but no significant associations between polypharmacy and PFS. Polypharmacy was not associated with immune-related adverse events but was associated with higher rate of unexpected hospitalizations during ICI treatment (59.6% vs. 31.7%, P < 0.001). Polypharmacy is an independent prognostic factor in older patients with advanced NSCLC treated with ICI. Also, polypharmacy could be utilized as a simple indicator of patients' comorbidities and symptoms or as a predictive marker of unexpected hospitalizations during ICI treatment.

Gut microbiome to predict efficacy and immune-related toxicities in patients with advanced non-small cell lung cancer treated with anti-PD-1/PD-L1 antibody-based immunotherapy.

3095 Background: The gut microbiome (GM) plays an important role in shaping systemic immune responses. Preclinical and clinical data suggest that GM influences anti-PD-1/PD-L1 or -CTLA-4 Antibody (Ab)-mediated anti-cancer responses. Furthermore, there is strong evidence that antibiotics (ATB) worsen clinical outcomes based on multiple retrospective and one prospective studies using immune checkpoint inhibitor (ICI). However, whether GM profiling, at baseline or post-ATB, could represent a biomarker of response in advanced non-small cell lung cancer (NSCLC) during ICI therapy remains unknown. Methods: We prospectively collected baseline (pre-ICI) fecal samples and clinical data Japanese patients (pts) with NSCLC treated with anti-PD-1/PD-L1 Abs in first or second-line therapy. We performed a 16S rRNA V3-V4 sequencing of gene amplicons of fecal microbes. Amplicon sequence variants were generated with dada2 R package. Diversity analysis was performed with phyloseq R. Differential abundance analysis was performed with both LEfSe and DESeq2 methods. Clinical endpoints were progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and immune-related adverse events (irAE). Results: 70 fecal samples were analyzed. Median OS and PFS in all patients were 16.1 and 5.2 months, respectively. 16 pts (23%) were exposed to ATB 1 month prior to ICI initiation. Pts on ATB had lower α-diversity at baseline and underrepresentation of Clostridiales and Ruminococcaceae UCG 13. When analyzing ATB-free pts, lower α-diversity was observed in non-responders. In addition, Ruminococcaceae UCG 13 was enriched in patients with OS &gt; 12 months, favorable ORR, and PFS &gt; 6 months. Clostridiales order was also enriched in patients with OS &gt; 12 months. Compositional GM differences were also observed between the patients who experienced clinically significant (≥grade 2) irAE; Lactobacillaceae and Raoultella were enriched in pts who had no significant irAE. Conclusions: We demonstrated the negative influence of ATB on GM composition and identified differential bacteria repertoire in pts experiencing favorable clinical outcomes or low grade irAE. Our data pave the way to the development of diagnosis tools aimed at identifying gut dysbiosis to predict resistance or irAE during ICI for NSCLC.

Fibrillar form of \u03b1-synuclein-specific scFv antibody inhibits \u03b1-synuclein seeds induced aggregation and toxicity

Synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn). Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area. To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar α-syn, a putative disease-relevant form of α-syn. The purified scFvs showed specific activity towards α-syn fibrils and oligomers in comparison to monomers and recognized intracellular inclusions in human post-mortem brain tissue of Lewy body disease cases, but not aged controls. In vitro studies indicated scFvs inhibit the seeding of α-syn aggregation in a time-dependent manner, decreased α-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble α-syn phosphorylated at Ser-129 (pS129-α-syn). These results suggest that our α-syn fibril-specific scFvs recognize α-syn pathology and can inhibit the aggregation of α-syn in vitro and prevent seeding-dependent toxicity. Therefore, the scFvs described here have considerable potential to be utilized towards immunotherapy in synucleinopathies and may also have applications in ante-mortem imaging modalities.

TLR7/8 Agonist-Loaded Nanoparticles Augment NK Cell-Mediated Antibody-Based Cancer Immunotherapy.

Activated natural killer (NK) cells can kill malignant tumor cells via granule exocytosis and secretion of IFN-γ, a key regulator of the TH1 response. Thus, mobilization of NK cells can augment cancer immunotherapy, particularly when mediated through antibody-dependent cellular cytotoxicity (ADCC). Stimulation of toll-like receptor (TLR)7/8 activity in dendritic cells promotes pro-inflammatory cytokine secretion and costimulatory molecule upregulation, both of which can potentiate NK cell activation. However, currently available TLR7/8 agonists exhibit unfavorable pharmacokinetics, limiting their in vivo efficacy. To enable efficient delivery to antigen-presenting cells, we encapsulated a novel imidazoquinoline-based TLR7/8 agonist in pH-responsive polymeric NPs. Enhanced costimulatory molecule expression on dendritic cells and a stronger pro-inflammatory cytokine response were observed with a NP-encapsulated agonist, compared to that with the soluble form. Treatment with NP-encapsulated agonists resulted in stronger in vivo cytotoxicity and prolonged activation of NK cells compared to that with a soluble agonist. In addition, TLR7/8 agonist-loaded NPs potentiated stronger NK cell degranulation, which resulted in enhanced in vitro and in vivo ADCC mediated by the epidermal growth factor receptor-targeting antibody cetuximab. TLR7/8 agonist-loaded NP treatment significantly enhanced the antitumor efficacy of cetuximab and an anti-HER2/neu antibody in mouse tumor models. Collectively, our data show that a pH-responsive NP-encapsulating TLR7/8 agonist could be used as a potent immunostimulatory adjuvant for antibody-based cancer immunotherapy by promoting NK cell activation.

Neuropilin-1, an immune checkpoint for controlling tumor-specific CD8 T-cell functions whose blockade improves anti-PD-1 immunotherapy

Abstract Neuropilin-1 (Nrp-1) has been defined as a marker of murine CD4+Foxp3+ regulatory T (Treg) cells and a subset of human CD4+ Treg cells. It is also expressed by a subpopulation of CD8+ T cells infiltrating some human solid tumors. However, little is known about its contribution to regulation of tumor-specific CD8 T-cell functions. We show that Nrp-1 defines a subset of CD8+ T cells displaying PD-1high (PD-1hi) status and infiltrating human non-small cell lung cancer (NSCLC). Using a human cytotoxic T lymphocyte (CTL) clone model, we show that the interaction of Nrp-1 with its ligand semaphorin-3A (Sema-3A), secreted by autologous NSCLC cells, inhibits both CTL migration and tumor-specific lytic function. In vivo experiments revealed that this Nrp-1+PD-1hi CD8+ tumor-infiltrating T lymphocyte (TIL) subset is also found in engrafted B16F10 mouse melanoma, and is enriched with tumor-specific T cells exhibiting an exhausted state, with co-expression of Tim-3, LAG-3 and CTLA-4 inhibitory receptors. Functionally, anti-Nrp-1 neutralizing antibodies increase the migratory capacity of Nrp+PD-1hi CD8+ TIL toward autologous cancer cells and enhance their specific cytotoxic activity ex vivo. Remarkably, in vivo immunotherapeutic blockade of Nrp-1 results in control of tumor growth, with a parallel increase in tumor infiltration by CD8+ TIL and enhanced proliferative and cytotoxic potential. This blockade is cumulative with anti-PD-1 treatment in improving tumor regression. Thus, Nrp-1 appears to be a novel immune checkpoint target on CD8+ tumor-specific T cells for combined antibody-based cancer immunotherapies. More recent advances on Nrp-1 expression regulation and functions in CD4+ and CD8+ T cells will be presented.

BRD4 inhibition suppresses PD-L1 expression in triple-negative breast cancer

Programmed death-ligand 1 (PD-L1) expression on the surface of tumour cells can cause tumour immune evasion. Benefits of combining anti-PD-L1 therapy with nab-paclitaxel in patients with advanced triple-negative breast cancer (TNBC) have been reported. However, some patients cannot tolerate the immune-related adverse effects (irAEs) caused by antibody-based immunotherapy. BRD4 is a member of the bromodomain and extra-terminal domain (BET) family. BRD4 inhibition has shown antitumour effects in many tumours, but its role in TNBC has not been definitively concluded. In particular, the immune regulation of BRD4 in TNBC has been rarely studied. In this study, we used JQ1, a BET inhibitor, and small interfering RNAs (siRNAs) targeting BRD4 to explore the influence of BRD4 on PD-L1 expression in TNBC. The results indicated that BRD4 inhibition suppressed PD-L1 expression and the PD-L1 upregulation induced by interferon-γ (IFN-γ). In the in vivo experiments, we found that JQ1 not only reduced the PD-L1 expression level but also changed the proportions of T lymphocyte subsets in the spleens of tumour-bearing mice, which helped to relieve immunosuppression. Briefly, our study reveals that BRD4 regulates PD-L1 expression and may provide a potential method for blocking the programmed death 1 (PD-1)/PD-L1 immune checkpoint in TNBC.

Programmed cell death ligand-1: A dynamic immune checkpoint in cancer therapy.

Antibody-based immunotherapies play a pivotal role in cancer research with efficient achievements in tumor suppression. Tumor survival is assisted by modulation of immune checkpoints to create imbalances between immune cells and cancer cell's environment. The modulation results in T-cell signal inhibition ultimately inert its proliferation and activation against various tumor cells. PD-L1, a 40kDa transmembrane protein of B7 family, binds with PD-1 on the membrane of T cells which results in inhibition of T-cell proliferation and activation. PD-L1/PD-1 pathway has generated novel target sites for antibodies that can block PD-L1/PD-1 interactions. The blockage results in T-cell proliferation and tumor cell suppression. The PD-L1 immune checkpoint strategies' development, expression and regulations, signal inhibitions, and developmental stages of PD-L1/PD-1 antibodies are briefly discussed here in this review. All this information will provide a base for new therapeutic development against PD-L1 and PD-1 immune checkpoint interactions and will make available promising treatment options.

Targets and Antibody Formats for Immunotherapy of Neuroblastoma.

Neuroblastoma (NB) is a malignant embryonal tumor of the sympathetic nervous system that is most commonly diagnosed in the abdomen, often presenting with signs and symptoms of metastatic spread. Three decades ago, high-risk NB metastatic to bone and bone marrow in children was not curable. Today, with multimodality treatment, 50% of these patients will survive, but most suffer from debilitating treatment-related complications. Novel targeted therapies to improve cure rates while minimizing toxicities are urgently needed. Recent molecular discoveries in oncology have spawned the development of an impressive array of targeted therapies for adult cancers, yet the paucity of recurrent somatic mutations or activated oncogenes in pediatric cancers poses a major challenge to the evolving paradigm of personalized medicine. Although low tumor mutational burden is a major hurdle for immune checkpoint inhibitors, an immature or impaired immune system and inhibitory tumor microenvironment can further complicate the prospects for successful immunotherapy. In this regard, despite the poor immunogenic properties of NB, the success of antibody-based immunotherapy and radioimmunotherapy directed at single targets (eg, GD2 and B7-H3) is both encouraging and surprising, given that most solid tumor antibodies that use Fc-dependent mechanisms or radioimmunotargeting have largely failed. Here, we summarize the current information on the immunologic properties of this tumor, its potential immunotherapeutic targets, and novel antibody-based strategies on the horizon.

Probody Therapeutics: An Emerging Class of Therapies Designed to Enhance On-Target Effects with Reduced Off-Tumor Toxicity for Use in Immuno-Oncology.

The deep and durable antitumor effects of antibody-based immunotherapies such as immune checkpoint inhibitors (ICIs) have revolutionized oncology and transformed the therapeutic landscape for many cancers. Several anti-programmed death receptor 1 and anti-programmed death receptor ligand 1 antibodies have been approved for use in advanced solid tumors, including melanoma, non-small cell lung cancer, bladder cancer, and other cancers. ICIs are under development across many tumor types and preliminary results are compelling. However, ICIs have been associated with severe immune-related adverse events (irAEs), including rash, diarrhea, colitis, hypophysitis, hepatotoxicity, and hypothyroidism, which in some cases lead to high morbidity, are potentially life-threatening, and limit the duration of treatment. The incidence of severe irAEs increases further when programmed cell death-1 and programmed cell death ligand-1 inhibitors are combined with anti-CTLA-4 and/or other multidrug regimens. Probody therapeutics, a new class of recombinant, proteolytically activated antibody prodrugs are in early development and are designed to exploit the hallmark of dysregulation of tumor protease activity to deliver their therapeutic effects within the tumor microenvironment (TME) rather than peripheral tissue. TME targeting, rather than systemic targeting, may reduce irAEs in tissues distant from the tumor. Probody therapeutic technology has been applied to multiple antibody formats, including immunotherapies, Probody drug conjugates, and T-cell-redirecting bispecific Probody therapeutics. In preclinical models, Probody therapeutics have consistently maintained anticancer activity with improved safety in animals compared with the non-Probody parent antibody. In the clinical setting, Probody therapeutics may expand or create therapeutic windows for anticancer therapies.

Downregulation of PD-L1 via amide analogues of brefelamide: Alternatives to antibody-based cancer immunotherapy.

The therapeutic blockade of immune checkpoint has emerged as an effective treatment option for a broad range of tumors. However, the objective tumor response is still limited to a small number of cases and tumor types. The full utility of monoclonal antibody (mAb)-based treatment is hindered by several inherent limitations. Thus, there is an urgent requirement to explore alternative modalities targeting the same pathways. In the present study, two amide analogues of brefelamide, TPFS-201 and TPFS-202, were identified as small molecular immune checkpoint inhibitors, as they downregulated PD-L1 expression in tumor cells. PD-L1 was suppressed in cancer cells treated with TPFD compounds at both mRNA and protein levels, as detected by reverse transcription quantitative PCR and flow cytometric analysis, respectively. Reporter assays using a PD-L1 promoter luciferase construct confirmed the transcriptional inhibition of PD-L1 by TPFS compunds. TPFS compound-mediated PD-L1 downregulation in cancer cells consequently restored T cell activity, as identified by the reduction of apoptosis and an increase in interleukin-2 promoter activity in Jurkat T cells, which were co-cultured with TPFS compound-treated A549 cells. TPFS compound-mediated PD-L1 inhibition was partially abolished by the disruption of the putative transcriptional co-activator with PDZ (TAZ)/TEA domain (TEAD)-binding motif in the PD-L1 promoter. The inhibitory effect of TPFS compounds on PD-L1 was markedly inhibited in mouse cell lines, which is consistent with previous research demonstrating that PD-L1 regulation by TAZ is not conserved in mice due to distinct promoter sequences flanking the TAZ/TEAD-binding motif. Together, the data of the current study indicated the potential utility of the brefelamide amide analogues as small molecule immune checkpoint inhibitors, thereby providing therapeutic alternatives, which could be used as monotherapy or in combination with mAbs-based treatment.

Gene-specific treatment approaches in Alzheimer's disease and other tauopathies

The pathological hallmarks of Alzheimer's disease are aggregation and accumulation of amyloid-β and tau proteins. So far most interventional studies have focused on the removal of the toxic protein products, such as antibody-based immunotherapies targeted against amyloid-β and tau proteins; however, the development of gene therapies targeting gene products involved in the disease has opened up new therapeutic strategies to reduce the development of toxic protein aggregates by inhibiting the translation of pathological Alzheimer genes using antisense oligonucleotides (ASO). This has a timely influence on development of the disease. This article gives an overview of new advances in ASO-based treatment strategies for Alzheimer's disease.

The future of cellular immunotherapy for childhood leukemia.

Exciting translational discoveries in recent years have brought realized promise of immunotherapy for children with high-risk leukemias. This review summarizes the current immunotherapeutic landscape with a focus on key clinical trials for patients with acute lymphoblastic leukemia or acute myeloid leukemia. Chemotherapy resistance remains a major barrier to cure in children with high-risk leukemias. Immunotherapy approaches have potential to overcome this resistance given alternative mechanisms of action. Based upon preclinical activity and/or success in adult patients, recent clinical trials have demonstrated safety and efficacy of various mAb, antibody-drug conjugate, bispecific T-cell-engaging antibody, natural killer cell, and chimeric antigen receptor-redirected T-cell immunotherapies for children with acute lymphoblastic leukemia or acute myeloid leukemia. Food and Drug Administration approval of several of these immunotherapies has increased the pediatric leukemia therapeutic portfolio and improved clinical outcomes for previously incurable patients. Several antibody-based or cellular immunotherapy modalities have demonstrated appreciable efficacy in children with relapsed or chemotherapy-refractory leukemia via early-phase clinical trials. Some studies have also identified critical biomarkers of treatment response and resistance that merit further investigation. Continued preclinical and clinical evaluation of novel immunotherapies is imperative to improve cure rates for children with high-risk leukemias.

Machine learning-aided quantification of antibody-based cancer immunotherapy by natural killer cells in microfluidic droplets

Natural killer (NK) cells have emerged as an effective alternative option to T cell-based immunotherapies, particularly against liquid (hematologic) tumors. However, the effectiveness of NK cell therapy has been less than optimal for solid tumors, partly due to the heterogeneity in target interaction leading to variable anti-tumor cytotoxicity. This paper describes a microfluidic droplet-based cytotoxicity assay for quantitative comparison of immunotherapeutic NK-92 cell interaction with various types of target cells. Machine learning algorithms were developed to assess the dynamics of individual effector-target cell pair conjugation and target death in droplets in a semi-automated manner. Our results showed that while short contacts were sufficient to induce potent killing of hematological cancer cells, long-lasting stable conjugation with NK-92 cells was unable to kill HER2+ solid tumor cells (SKOV3, SKBR3) significantly. NK-92 cells that were engineered to express FcγRIII (CD16) mediated antibody-dependent cellular cytotoxicity (ADCC) selectively against HER2+ cells upon addition of Herceptin (trastuzumab). The requirement of CD16, Herceptin and specific pre-incubation temperature served as three inputs to generate a molecular logic function with HER2+ cell death as the output. Mass proteomic analysis of the two effector cell lines suggested differential changes in adhesion, exocytosis, metabolism, transport and activation of upstream regulators and cytotoxicity mediators, which can be utilized to regulate specific functionalities of NK-92 cells in future. These results suggest that this semi-automated single cell assay can reveal the variability and functional potency of NK cells and may be used to optimize immunotherapeutic efficacy for preclinical analyses.

Innovative therapies for malignant brain tumors: the road to a tailored cure.

Background:Immune tolerance, immune escape, neoangiogenesis, phenotypic changes, and glioma stem cells are all responsible for the resistance of malignant brain tumors to current therapies and persistent recurrence. The present study provides a panoramic view of innovative therapies for malignant brain tumors, especially glioblastoma, aimed at achieving a tailored approach.Methods:PubMed/Medline and ClinicalTrials.gov were the main sources of an extensive literature review in which “Regenerative Medicine,” “Cell-Based Therapy,” “Chemotherapy,” “Vaccine,” “Cell Engineering,” “Immunotherapy, Active,” “Immunotherapy, Adoptive,” “Stem Cells,” “Gene Therapy,” “Target Therapy,” “Brain Cancer,” “Glioblastoma,” and “Malignant Brain Tumor” were the search terms. Only articles in English published in the last 5 years were included. A further selection was made according to the quality of the studies and level of evidence.Results:Cell-based and targeted therapies represent the newest frontiers of brain cancer treatment. Active and adoptive immunotherapies, stem cell therapies, and gene therapies represent a tremendous evolution in recent years due to many preclinical and clinical studies. Clinical trials have validated the effectiveness of antibody-based immunotherapies, including an in-depth study of bevacizumab, in combination with standard of care. Preclinical data highlights the role of vaccines, stem cells, and gene therapies to prevent recurrence.Conclusion:Monoclonal antibodies strengthen the first-line therapy for high grade gliomas. Vaccines, engineered cells, stem cells, and gene and targeted therapies are good candidates for second-line treatment of both newly diagnosed and recurrent gliomas. Further data are necessary to validate this tailored approach at the bedside. (www.actabiomedica.it)

Antibody-based immunotherapy targeting cytokines and atherothrombotic cardiovascular diseases

Antibody-based immunotherapy targeting cytokines and atherothrombotic cardiovascular diseases

Treatment with anti CD19 chimeric antigen receptor T cells after antibody-based immunotherapy in adults with acute lymphoblastic leukemia

Purpose of the studyThe prognosis of patients with relapsed/refractory precursor B-acute lymphoblastic leukemia (ALL) is dismal. Antibody-based therapies, such as blinatumomab or inotuzumab ozogamycin (IO) have led to improved outcomes. The impact of prior immunotherapy on chimeric antigen receptor (CAR) T-Cell therapeutic efficacy and toxicity is unknown. MethodsWe describe a case series of ALL patients with prior exposure to blinatumomab or IO, who were treated with anti-CD19 CAR T cells with CD28 co-stimulatory domain (NCT02772198). We then review the literature on CAR-T post antibody-based therapy with either antibodies. ResultsFive adult patients with B-ALL were included. Three had active disease, and two were in morphological complete remission (CR) with minimal residual disease (MRD+). Therapy before CAR-T included blinatumomab (3/5 [60 %]) and IO (3/5 [60 %]), with one patient receiving both. One patient experienced severe cytokine release syndrome and central nervous system toxicity and subsequently died. At 28 days following treatment, two patients achieved CR with MRD negativity, and two had an MRD + CR. Two patients received allogeneic hematopoietic stem cell transplantation. At a median of 10 months (range, 5–26, three out of the four patients are still in CR, and one relapsed. The literature review identified a deficiency on data on the influence of blinatumumab and IO on outcomes post CAR-T therapy. ConclusionsCD19 CAR T-cell therapy after treatment with blinatumomab and/or IO in patients with relapsed/refractory B-ALL is feasible and results in promising response rates in this case series. Future trails should specifically address outcomes in this population.