Monoclonal Antibody-based Immunotherapy Research Articles

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Identification of the functional PD-L1 interface region responsible for PD-1 binding and initiation of PD-1 signaling

The PD-1/PD-L1 checkpoint pathway is important for regulating immune responses and can be targeted by immunomodulatory drugs to treat a variety of immune disorders. However, the precise protein-protein interactions required for the initiation of PD-1/PD-L1 signaling are currently unknown. Previously, we designed a series of first-generation PD-1 targeting peptides based on the native interface region of programmed death ligand 1 (PD-L1) that effectively reduced PD-1/PD-L1 binding. In this work, we further characterized the previously identified lead peptide, MN1.1, to identify key PD-1 binding residues and design an optimized peptide, MN1.4. We show MN1.4 is significantly more stable than MN1.1 in serum and retains the ability to block PD-1/PD-L1 complex formation. We further characterized the immunomodulatory effects of MN1.4 treatment by measuring markers of T cell activation in a co-culture model with ovarian cancer cells and peripheral blood mononuclear cells. We found MN1.4 treatment reduced cytokine secretion and suppressed T cell responses in a similar manner as recombinant PD-L1. Therefore, the PD-L1 interface region used to design MN1.4 appeared sufficient to initiate PD-1 signaling and likely represents the minimum necessary region of PD-L1 required for PD-1 recognition. We propose a peptide agonist for PD-1, such as MN1.4, could have several applications for treating autoimmune disorders caused by PD-1 deficiencies such as type 1 diabetes, inflammatory arthritis, or autoimmune side effects arising from monoclonal antibody-based cancer immunotherapies.

Abstract 1346: Phagocytosis of solid tumors favors macrophage clusters and cooperativity in tumor elimination

Abstract Macrophages are abundant in many solid tumors and are potential effector cells for monoclonal antibody-based immunotherapies because they can in principle phagocytose IgG-opsonized cancer cells. However, it remains unknown whether phagocytic macrophages can overcome the cohesive forces between cancer cells in solid tumors. We show that maximum phagocytosis of B16 melanoma tumors and ‘tumoroids’ requires IgG-opsonization and very deep suppression of the macrophage checkpoint ligand CD47. Under these conditions, phagocytic macrophages cluster or ‘phase separate’, and the elimination exhibits clear cooperativity with respect to macrophage number and phagocytic activity. In vitro disruption of phagocytic clusters specifically implicates Mg2+ and macrophage integrins. Cytokine treatments and other inhibitors further implicate roles for integrins and phagocytic surface receptors. Although tumor-associated macrophages correlate with negative prognoses in many cancers, our findings are among the first to reveal advantages of high macrophage numbers, macrophage clusters, and resulting cooperative effects when combined with opsonizing antibodies and CD47 disruption. Lastly, the challenging B16 tumors are durably eliminated by the approach and induce de novo IgG's that also drive anti-cancer phagocytosis. Citation Format: Dennis E. Discher. Phagocytosis of solid tumors favors macrophage clusters and cooperativity in tumor elimination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1346.

The prophylactic effects of monoclonal antibodies targeting the cell wall Pmt4 protein epitopes of Candida albicans in a murine model of invasive candidiasis.

Candida albicans (C. albicans) is the most prevalent opportunistic human pathogen, accounting for approximately half of all clinical cases of candidemia. Resistance to the existing antifungal drugs is a major challenge in clinical therapy, necessitating the development and identification of novel therapeutic agents and potential treatment strategies. Monoclonal antibody-based immunotherapy represents a promising therapeutic strategy against disseminated candidiasis. Protein mannosyltransferase (Pmt4) encodes mannosyltransferases initiating O-mannosylation of secretory proteins and is essential for cell wall composition and virulence of C. albicans. Therefore, the Pmt4 protein of C. albicans is an attractive target for the discovery of alternative antibody agents against invasive C. albicans infections. In the present study, we found that monoclonal antibodies (mAbs) C12 and C346 specifically targeted the recombinant protein mannosyltransferase 4 (rPmt4p) of C. albicans. These mAbs were produced and secreted by hybridoma cells isolated from the spleen of mice that were initially immunized with the purified rPmt4p to generate IgG antibodies. The mAbs C12 and C346 exhibited high affinity to C. albicans whole cells. Remarkably, these mAbs reduced the fungal burden, alleviated inflammation in the kidneys, and prolonged the survival rate significantly in the murine model of systemic candidiasis. Moreover, they could activate macrophage opsonophagocytic killing and neutrophil killing of C. albicans strain in vitro. These results suggested that anti-rPmt4p mAbs may provide immunotherapeutic interventions against disseminated candidiasis via opsonophagocytosis and opsonic killing activity. Our findings provide evidence for mAbs as a therapeutic option for the treatment of invasive candidiasis.

Human lung-cancer-cell radioresistance investigated through 2D network topology

Radiation therapy (RT) is now considered to be a main component of cancer therapy, alongside surgery, chemotherapy and monoclonal antibody-based immunotherapy. In RT, cancer tissues are exposed to ionizing radiation causing the death of malignant cells and favoring cancer regression. However, the efficiency of RT may be hampered by cell-radioresistance (RR)—that is a feature of tumor cells of withstanding RT. To improve the RT performance, it is decisive developing methods that can help to quantify cell sensitivity to radiation. In acknowledgment of the fact that none of the existing methods to assess RR are based on cell graphs topology, in this work we have examined how 2D cell networks, within a single colony, from different human lung cancer lines (H460, A549 and Calu-1) behave in response to doses of ionizing radiation ranging from 0 to 8 Gy. We measured the structure of resulting cell-graphs using well-assessed networks-analysis metrics, such as the clustering coefficient (cc), the characteristic path length (cpl), and the small world coefficient (SW). Findings of the work illustrate that the clustering characteristics of cell-networks show a marked sensitivity to the dose and cell line. Higher-than-one values of SW coefficient, clue of a discontinuous and inhomogeneous cell spatial layout, are associated to elevated levels of radiation and to a lower radio-resistance of the treated cell line. Results of the work suggest that topology could be used as a quantitative parameter to assess the cell radio-resistance and measure the performance of cancer radiotherapy.

Focused ultrasound effects on the glioblastoma immune microenvironment

Focused ultrasound (FUS) is an attractive method of treating neurological diseases, such as glioblastoma (GBM). An accumulating body of evidence has confirmed the efficacy and safety of using FUS with systematically circulating microbubbles, which operate as acoustic enhancers, to temporarily open the blood-brain barrier for drug delivery. However, there is very limited knowledge of how immune cell respond to mechanical stimuli such as FUS, particularly in the complex immune microenvironments of brain tumors. Here, we report FUS immunomodulation effects may also include the enhanced recruitment and activation of other mononuclear phagocytes in murine models of GBM in tests with or without monoclonal antibody-based passive immunotherapy. Flow cytometry, immunohistochemistry, and in vitro cell co-culture studies were performed for immunoprofiling and immune effector determination. In murine GBM models, our results demonstrated that FUS enhanced antigen presentation behaviors of tumor-associated macrophages without affecting the microglia. FUS also reprogrammed the macrophages locally towards the anti-cancer phenotype. Taken together, our results offer new evidence in FUS immunomodulation on the myeloid compartment of the brains in GBM mouse models.

Label-Free, Rapid and Facile Gold-Nanoparticles-Based Assay as a Potential Spectroscopic Tool for Trastuzumab Quantification.

Monoclonal antibody-based immunotherapy is one of the pillars of cancer treatment. However, for an efficient and personalized approach to the therapy, a quantitative evaluation of the right dose for each patient is required. In this study, we developed a simple, label-free, and rapid approach to quantify Trastuzumab, a humanized IgG1 monoclonal antibody used against human epidermal growth factor receptor 2 (HER2), overexpressed in breast cancer patients, based on localized surface plasmon resonance (LSPR). The central idea of this work was to use gold nanoparticles (AuNPs) as plasmonic scaffolds, decorated with HER2 binders mixed with oligo-ethylene glycol (OEG) molecules, to tune the surface density of the attached macromolecules and to minimize nonspecific binding events. Specifically, we characterized and optimized a self-assembled monolayer of mixed alkylthiols terminated with nitrilotriacetic acid (NTA), and OEG3 as a spacing ligand to achieve both excellent dispersibility and high reliability in protein immobilization. The successful immobilization of histidine-tagged HER2 (His-tagged HER2) on NTA via cobalt (II) chelates was demonstrated, confirming the fully functional attachment of the proteins to the AuNP surface. The proposed design demonstrates the capability of producing a clear readout that enables the transduction of a Trastuzumab/HER2 binding event into optical signals based on the wavelength shifts in LSPR, which allowed for detecting clinically relevant concentrations of Trastuzumab down to 300 ng/mL in the buffer and 2 µg/mL in the diluted serum. This strategy was found to be fast and highly specific to Trastuzumab. These findings make the present platform an auspicious tool for developing affordable bio-nanosensors.

Focused ultrasound immunomodulation on mononuclear phagocytes in the brain

Focused ultrasound (FUS)-mediated blood–brain barrier opening is an attractive method of treating neurological diseases, such as glioblastoma (GBM) and Alzheimer’s disease (AD). As an established drug delivery approach, FUS has also been associated with the stimulation of neuro-inflammation, including the morphological activation of resident microglia. Here we report FUS immunomodulation effects may also include the enhanced recruitment and activation of other mononuclear phagocytes in murine models of GBM (GL261) and AD (APP/PS1dE9) in tests with or without monoclonal antibody-based passive immunotherapy. Immunoprofiling via flow cytometry and immunofluorescent staining were performed in the harvested brains. In the GBM model, our results demonstrated that FUS enhanced antigen presentation behaviors of tumor-associated macrophages without affecting the microglia. FUS also reprogrammed the macrophages locally towards the anti-cancer phenotype. In the AD model, plaque-associated Ly6G+ phagocytes were only present in FUS-treated areas, while microglia activation was found in both antibody-treated groups. Taken together, our results offer new evidence in FUS immunomodulation on the myeloid compartment of the brains in GBM and AD-like mouse models.

Role of Complement in Regulating Inflammation Processes in Renal and Prostate Cancers.

For decades, the complement system, the central pillar of innate immune response, was recognized as a protective mechanism against cancer cells and the manipulation of complement effector functions in cancer setting offered a great opportunity to improve monoclonal antibody-based cancer immunotherapies. Similarly, cellular senescence, the process of cell cycle arrest that allow DNA and tissue repair has been traditionally thought to be able to suppress tumor progression. However, in recent years, extensive research has identified the complement system and cellular senescence as two main inducers of tumour growth in the context of chronic, persistent inflammation named inflammaging. Here, we discuss the data describing the ambivalent role of senescence in cancer with a particular focus on tumors that are strongly dependent on complement activation and can be understood by a new, senescence-related point of view: prostate cancer and renal cell carcinoma.

Perfusable host microfluidic endothelial networks for quantitative assessment of the effects of interstitial flows, macrophage polarization and monoclonal‐based immunotherapy on immune cell infiltration into a tumor spheroid

Monoclonal antibody-based immunotherapy is a promising treatment for several diseases such as cancer and virus infection. However, due to its highly selective target, drug testing using animal models or classical 2D human cell cultures are not sufficient to predict the drug effects on patients. New 3D organotypic models that employ 3D cell culture of human cells within a microfluidic device allow detailed characterization of critical biological interactions. In this project, we describe a new 3D cell culture model that allows the creation of in-vitro human perfusable vasculature containing hollow spaces for the insertion of tumor spheroids. A polydimethylsiloxane device with several vertical holes on top of the gel channel is used to create the host microvascular endothelial (HOME) network (Fig.1A). Endothelial cells and fibroblasts create perfusable vascular networks (Fig. 1B). Tumor spheroids are formed by coculturing MDA-MB-468 tumor cells (T), fibroblasts (F), and non-polarized (M0) or M2 macrophages, and denoted by TFM0 and TFM2 respectively (Fig. 1C). One day after inserting a tumor spheroid in collagen into the HOME networks through the hole, freshly-isolated monocytes are perfused into the vascular networks by luminal flows. We observe that after 2 days, the majority of monocytes remain either within the vasculature or extravasated but stay inside the fibrin gel outside of the hole containing the tumor spheroid (Fig. 1Di). On day 2, interstitial flows from the tumor spheroid cause monocyte migration from the vascular network side into the hole containing the tumor spheroid (figure 1Dii). When a TFM2 spheroid is inserted into the HOME networks, it recruits more monocytes than the TFM0 spheroid (Fig. Ei and Eii). Recruitment % is the percentage of monocytes that leave the blood vessel and migrate into the hole among the total number of monocytes in 3mmx3mm region of interest around the hole. Monocytes are recruited better by the TFM2 spheroid than the TFM0 spheroid (Fig. 1Gi). These results showed that interstitial flows from the tumor spheroid to the vascular networks and macrophage polarization are essential for monocyte recruitment. Multi-specific antibody UniTI102 developed by Elstar Therapeutics (MA, USA) neutralizes chemokine CCL2 to block monocyte recruitment. To characterize the response to the drug UniTI102, we compared devices having a TFM0 spheroid that are untreated (Fig. 1D) or treated with UniTI102 (Fig. 1F) and observed that less monocytes has migrated into the hole in the presence of UniTI102 at 100nM. Quantitative data also showed that less monocytes are recruited when devices are treated with 100nM UniTI102 than 100nM IgG (Fig. 1Gii).

Monoclonal Antibody-Based Immunotherapy and Its Role in the Development of Cardiac Toxicity.

Simple SummaryThe application of immunotherapies to treat cancer patients has significantly improved over the last two decades and extended many patients’ life spans. Monoclonal antibodies are synthetic proteins employed as immunotherapies to treat and manage cancers that require a complete understanding of cancer biology and the host’s immune system. However, activated immune responses, by monoclonal antibodies, can target nonspecific cancer cells, causing frequent immune-related adverse events that can lead to permanent disorders among cancer patients. The immune-related adverse events pose a risk of cardiac toxicity that includes hypertension, heart failure, arrhythmias, and left ventricular dysfunction during and after monoclonal antibody immunotherapy. Moreover, with the mortality rate of 47% attributed to heart disease and cancer, it is imperative to employ reliable, sensitive, and clinically relevant models for efficacy and safety assessment of immune drugs for cancer and the prevention of cardiotoxicities.Immunotherapy is one of the most effective therapeutic options for cancer patients. Five specific classes of immunotherapies, which includes cell-based chimeric antigenic receptor T-cells, checkpoint inhibitors, cancer vaccines, antibody-based targeted therapies, and oncolytic viruses. Immunotherapies can improve survival rates among cancer patients. At the same time, however, they can cause inflammation and promote adverse cardiac immune modulation and cardiac failure among some cancer patients as late as five to ten years following immunotherapy. In this review, we discuss cardiotoxicity associated with immunotherapy. We also propose using human-induced pluripotent stem cell-derived cardiomyocytes/ cardiac-stromal progenitor cells and cardiac organoid cultures as innovative experimental model systems to (1) mimic clinical treatment, resulting in reproducible data, and (2) promote the identification of immunotherapy-induced biomarkers of both early and late cardiotoxicity. Finally, we introduce the integration of omics-derived high-volume data and cardiac biology as a pathway toward the discovery of new and efficient non-toxic immunotherapy.

How Does Complement Affect Hematological Malignancies: From Basic Mechanisms to Clinical Application.

Complement, as a central immune surveillance system, can be activated within seconds upon stimulation, thereby displaying multiple immune effector functions. However, in pathologic scenarios (like in tumor progression), activated complement can both display protective effects to control tumor development and passively promotes the tumor growth. Clinical investigations show that patients with several hematological malignancies often display abnormal level of specific complement components, which in turn modulates complement activation or deregulated cascade. In the past decades, complement-dependent cytotoxicity and complement-dependent cell-mediated phagocytosis were fully approved to display vital roles in monoclonal antibody-based immunotherapies, especially in therapies against hematological malignancies. However, tumor-mediated complement evasion presents a big challenge for such a therapy. This review aims to provide an integrative overview on the roles of the complement in tumor promotion, highlights complement mediated effects on antibody-based immunotherapy against distinct hematological tumors, hopefully provides a theoretical basis for the development of complement-based cancer targeted therapies.

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.

Osteoclast Immunosuppressive Effects in Multiple Myeloma: Role of Programmed Cell Death Ligand 1.

Immunomodulatory drugs and monoclonal antibody-based immunotherapies have significantly improved the prognosis of the patients with multiple myeloma (MM) in the recent years. These new classes of reagents target malignant plasma cells (PCs) and further modulate the immune microenvironment, which prolongs anti-MM responses and may prevent tumor occurrence. Since MM remains an incurable cancer for most patients, there continues to be a need to identify new tumor target molecules and investigate alternative cellular approaches using gene therapeutic strategies and novel treatment mechanisms. Osteoclasts (OCs), as critical multi-nucleated large cells responsible for bone destruction in >80% MM patients, have become an attractive cellular target for the development of novel MM immunotherapies. In MM, OCs are induced and activated by malignant PCs in a reciprocal manner, leading to osteolytic bone disease commonly associated with this malignancy. Significantly, bidirectional interactions between OCs and MM cells create a positive feedback loop to promote MM cell progression, increase angiogenesis, and inhibit immune surveillance via both cell–cell contact and abnormal production of multiple cytokines/chemokines. Most recently, hyper-activated OCs have been associated with activation of programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway, which impairs T cell proliferation and cytotoxicity against MM cells. Importantly, therapeutic anti-CD38 monoclonal antibodies and checkpoint inhibitors can alleviate OC-induced immune suppression. Furthermore, a proliferation-inducing ligand, abundantly secreted by OCs and OC precursors, significantly upregulates PD-L1 expression on MM cells, in addition to directly promoting MM cell proliferation and survival. Coupled with increased PD-L1 expression in other immune-suppressive cells, i.e., myeloid-derived suppressor cells and tumor-associated macrophages, these results strongly suggest that OCs contribute to the immunosuppressive MM BM microenvironment. Based on these findings and ongoing osteoimmunology studies, therapeutic interventions targeting OC number and function are under development to diminish both MM bone disease and related immune suppression. In this review, we discuss the classical and novel roles of OCs in the patho-immunology of MM. We also describe novel therapeutic strategies simultaneously targeting OCs and MM interactions, including PD-1/PD-L1 axis, to overcome the immune-suppressive microenvironment and improve patient outcome.

Monoclonal Antibody: A New Treatment Strategy against Multiple Myeloma

2015 was a groundbreaking year for the multiple myeloma community partly due to the breakthrough approval of the first two monoclonal antibodies in the treatment for patients with relapsed and refractory disease. Despite early disappointments, monoclonal antibodies targeting CD38 (daratumumab) and signaling lymphocytic activation molecule F7 (SLAMF7) (elotuzumab) have become available for patients with multiple myeloma in the same year. Specifically, phase 3 clinical trials of combination therapies incorporating daratumumab or elotuzumab indicate both efficacy and a very favorable toxicity profile. These therapeutic monoclonal antibodies for multiple myeloma can kill target cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent phagocytosis, as well as by direct blockade of signaling cascades. In addition, their immunomodulatory effects may simultaneously inhibit the immunosuppressive bone marrow microenvironment and restore the key function of immune effector cells. In this review, we focus on monoclonal antibodies that have shown clinical efficacy or promising preclinical anti-multiple myeloma activities that warrant further clinical development. We summarize mechanisms that account for the in vitro and in vivo anti-myeloma effects of these monoclonal antibodies, as well as relevant preclinical and clinical results. Monoclonal antibody-based immunotherapies have already and will continue to transform the treatment landscape in multiple myeloma.

Involvement of HMGB1 in Resistance to Tumor Vessel-Targeted, Monoclonal Antibody-Based Immunotherapy.

High mobility group box 1 (HMGB1) is a member of the “danger associated molecular patterns” (DAMPs) than can localize in various compartments of the cell (from the nucleus to the cell surface) and subserve different functions accordingly. HMGB1 is implicated in maintenance of genomic stability, autophagy, immune regulation, and tumor growth. HMGB1-induced autophagy promotes tumor resistance to chemotherapy, as shown in different models of malignancy, for example, osteosarcoma, leukemia, and gastric cancer. To the best of our knowledge, there is virtually no information on the relationships between HMGB1 and resistance to immunotherapy. A recent study from our group has shed new light on this latter issue. We have demonstrated that targeting of tumor-derived endothelial cells with an anti-human CD31 monoclonal antibody in a human neuroblastoma model was unsuccessful due to a complex chain of events involving the participation of HMGB1. These results are discussed in detail since they provide the first evidence for a role of HMGB1 in resistance of tumor cells to monoclonal antibody-based immunotherapy.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

Potential Use of Interleukin-10 Blockade as a Therapeutic Strategy in Human Cutaneous Leishmaniasis.

Interleukin-10 overproduction has been associated with worse prognosis in human cutaneous leishmaniasis, while IFN-γ-dependent responses are associated with parasite killing and host protection. Innovative strategies are needed to overcome therapeutic failure observed in endemic areas. The use of monoclonal antibody-based immunotherapy targeting IL-10 cytokine was evaluated here. Partial IL-10 blockade in Leishmania braziliensis whole soluble antigen-stimulated cells from endemic area CL patients with active or healed lesions and asymptomatic controls was evaluated. Overall decrease in IL-10, IL-4, and TNF-α production was observed in all groups of subjects. Only patients with active lesions still produced some levels of TNF-α after anti-IL-10 stimulation in association with Leishmania antigens. Moreover, this strategy showed limited modulatory effects on IFN-γ-dependent chemokine CXCL10 production. Results suggest the potential immunotherapeutic use of partial IL-10 blockade in localized cutaneous leishmaniasis.

Novel Tumor Antigen-Specific Monoclonal Antibody-Based Immunotherapy to Eradicate Both Differentiated Cancer Cells and Cancer-Initiating Cells in Solid Tumors

A growing body of experimental and clinical evidence strongly suggests that the resistance of cancer-initiating cells (CICs) to conventional therapies represents a major obstacle to the successful treatment of a malignant disease. To overcome this limitation a novel combinatorial tumor antigen (TA)-specific monoclonal antibody (mAb) strategy has been developed. In this strategy TA-specific mAbs are combined with chemotherapeutic agents and/or small molecules that inhibit aberrantly activated signaling pathways in cancer cells and especially in CICs. The in vitro results we have obtained indicate that this strategy is very effective in eradicating both differentiated cancer cells and CICs in several types of malignant disease. If the in vitro results have in vivo relevance, the strategy we have designed may have an impact on the treatment of malignant diseases.

Monoclonal antibody-based immunotherapy of ovarian cancer: Targeting ovarian cancer cells with the B7-H3-specific mAb 376.96

ObjectiveThe high rate of relapse in patients with advanced ovarian cancer likely reflects the chemoresistance of cancer initiating cells (CICs). We evaluated the anti-tumor activity of monoclonal antibody (mAb) 376.96, which recognizes a B7-H3 epitope expressed on ovarian carcinoma cells (OCCs), in combination with the tyrosine kinase inhibitor Sunitinib and chemotherapy on chemosensitive and chemoresistant cells and CICs. MethodsEight ovarian cancer cell lines including platinum- and taxane-resistant cell lines were analyzed by flow cytometry to establish expression of the mAb 376.96-defined-B7-H3-epitope on differentiated ovarian cancer cells and CICs. Samples from 10 ovarian cancer patients were analyzed via immunohistochemistry for mAb 376.96-defined-B7-H3-epitope expression. In vitro studies assessed mAb 376.96 alone and in combination with Sunitinib on the growth of chemosensitive and chemoresistant cell lines and on the content of CICs. ResultsThe mAb-376.96-defined-B7-H3 epitope is expressed on both differentiated cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines and 10 patient derived ovarian cancer tumors. In vitro treatment of chemoresistant cell lines with mAb 376.96 resulted in decreased cell viability. mAb 376.96 enhanced the cytotoxicity of Sunitinib and reduced the content of CICs. ConclusionThe mAb-376.96-defined-B7-H3-epitope was found to be expressed on both differentiated ovarian cancer cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines. mAb 376.96 inhibited the in vitro growth of chemosensitive and chemoresistant OCCs and reduced the content of CICs when used with Sunitinib. Further studies examining B7-H3 as a potential target of mAb-based immunotherapy for this type of malignancy are warranted.