Host Effector Mechanisms Research Articles

Dual Nature of Relationship between Mycobacteria and Cancer.

Although the therapeutic effect of mycobacteria as antitumor agents has been known for decades, recent epidemiological and experimental studies have revealed that mycobacterium-related chronic inflammation may be a possible mechanism of cancer pathogenesis. Mycobacterium tuberculosis and non-tuberculous Mycobacterium avium complex infections have been implicated as potentially contributing to the etiology of lung cancer, whereas Mycobacterium ulcerans has been correlated with skin carcinogenesis. The risk of tumor development with chronic mycobacterial infections is thought to be a result of many host effector mechanisms acting at different stages of oncogenesis. In this paper, we focus on the nature of the relationship between mycobacteria and cancer, describing the clinical significance of mycobacteria-based cancer therapy as well as epidemiological evidence on the contribution of chronic mycobacterial infections to the increased lung cancer risk.

Host-directed therapy to combat mycobacterial infections.

Upon infection, mycobacteria, such as Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM), are recognized by host innate immune cells, triggering a series of intracellular processes that promote mycobacterial killing. Mycobacteria, however, have developed multiple counter‐strategies to persist and survive inside host cells. By manipulating host effector mechanisms, including phagosome maturation, vacuolar escape, autophagy, antigen presentation, and metabolic pathways, pathogenic mycobacteria are able to establish long‐lasting infection. Counteracting these mycobacteria‐induced host modifying mechanisms can be accomplished by host‐directed therapeutic (HDT) strategies. HDTs offer several major advantages compared to conventional antibiotics: (a) HDTs can be effective against both drug‐resistant and drug‐susceptible bacteria, as well as potentially dormant mycobacteria; (b) HDTs are less likely to induce bacterial drug resistance; and (c) HDTs could synergize with, or shorten antibiotic treatment by targeting different pathways. In this review, we will explore host‐pathogen interactions that have been identified for Mtb for which potential HDTs impacting both innate and adaptive immunity are available, and outline those worthy of future research. We will also discuss possibilities to target NTM infection by HDT, although current knowledge regarding host‐pathogen interactions for NTM is limited compared to Mtb. Finally, we speculate that combinatorial HDT strategies can potentially synergize to achieve optimal mycobacterial host immune control.

Immune Response to Tick-Borne Hemoparasites: Host Adaptive Immune Response Mechanisms as Potential Targets for Therapies and Vaccines.

Tick-transmitted pathogens cause infectious diseases in both humans and animals. Different types of adaptive immune mechanisms could be induced in hosts by these microorganisms, triggered either directly by pathogen antigens or indirectly through soluble factors, such as cytokines and/or chemokines, secreted by host cells as response. Adaptive immunity effectors, such as antibody secretion and cytotoxic and/or T helper cell responses, are mainly involved in the late and long-lasting protective immune response. Proteins and/or epitopes derived from pathogens and tick vectors have been isolated and characterized for the immune response induced in different hosts. This review was focused on the interactions between tick-borne pathogenic hemoparasites and different host effector mechanisms of T- and/or B cell-mediated adaptive immunity, describing the efforts to define immunodominant proteins or epitopes for vaccine development and/or immunotherapeutic purposes. A better understanding of these mechanisms of host immunity could lead to the assessment of possible new immunotherapies for these pathogens as well as to the prediction of possible new candidate vaccine antigens.

Hypothesis: Alpha-1-antitrypsin is a promising treatment option for COVID-19

No definitive treatment for COVID-19 exists although promising results have been reported with remdesivir and glucocorticoids. Short of a truly effective preventive or curative vaccine against SARS-CoV-2, it is becoming increasingly clear that multiple pathophysiologic processes seen with COVID-19 as well as SARS-CoV-2 itself should be targeted. Because alpha-1-antitrypsin (AAT) embraces a panoply of biologic activities that may antagonize several pathophysiologic mechanisms induced by SARS-CoV-2, we hypothesize that this naturally occurring molecule is a promising agent to ameliorate COVID-19. We posit at least seven different mechanisms by which AAT may alleviate COVID-19. First, AAT is a serine protease inhibitor (SERPIN) shown to inhibit TMPRSS-2, the host serine protease that cleaves the spike protein of SARS-CoV-2, a necessary preparatory step for the virus to bind its cell surface receptor ACE2 to gain intracellular entry. Second, AAT has anti-viral activity against other RNA viruses HIV and influenza as well as induces autophagy, a known host effector mechanism against MERS-CoV, a related coronavirus that causes the Middle East Respiratory Syndrome. Third, AAT has potent anti-inflammatory properties, in part through inhibiting both nuclear factor-kappa B (NFκB) activation and ADAM17 (also known as tumor necrosis factor-alpha converting enzyme), and thus may dampen the hyper-inflammatory response of COVID-19. Fourth, AAT inhibits neutrophil elastase, a serine protease that helps recruit potentially injurious neutrophils and implicated in acute lung injury. AAT inhibition of ADAM17 also prevents shedding of ACE2 and hence may preserve ACE2 inhibition of bradykinin, reducing the ability of bradykinin to cause a capillary leak in COVID-19. Fifth, AAT inhibits thrombin, and venous thromboembolism and in situ microthrombi and macrothrombi are increasingly implicated in COVID-19. Sixth, AAT inhibition of elastase can antagonize the formation of neutrophil extracellular traps (NETs), a complex extracellular structure comprised of neutrophil-derived DNA, histones, and proteases, and implicated in the immunothrombosis of COVID-19; indeed, AAT has been shown to change the shape and adherence of non-COVID-19-related NETs. Seventh, AAT inhibition of endothelial cell apoptosis may limit the endothelial injury linked to severe COVID-19-associated acute lung injury, multi-organ dysfunction, and pre-eclampsia-like syndrome seen in gravid women. Furthermore, because both NETs formation and the presence of anti-phospholipid antibodies are increased in both COVID-19 and non-COVID pre-eclampsia, it suggests a similar vascular pathogenesis in both disorders. As a final point, AAT has an excellent safety profile when administered to patients with AAT deficiency and is dosed intravenously once weekly but also comes in an inhaled preparation. Thus, AAT is an appealing drug candidate to treat COVID-19 and should be studied.

Serine protease inhibitors rich Coccinia grandis (L.) Voigt leaf extract induces protective immune responses in murine visceral leishmaniasis

Leishmaniasis is a parasite-mediated tropical disease affecting millions of individuals worldwide. The available antileishmanial chemotherapeutic modalities exhibit adverse toxicity, exorbitant price and advent of drug-resistant parasites. Hence, plant-derived products are an alternative preference for the emergence of novel and effective antileishmanial agents that rejuvenate the host immunity with limited toxicity. The present work is complementary to our previous report that revealed the in vitro antileishmanial and immunomodulatory activity of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) rich in serine protease inhibitors. Thus, preliminary objectives of the study were to elucidate the leishmanicidal activity and host effector mechanism in Leishmania donovani infected BALB/c mice treated with Cg-Ex. Oral administration of Cg-Ex significantly reduced the spleen and liver parasite burden at dose-dependently. The parasite elimination was associated with generation of ROS and NO that are interrelated with up-regulation of disease-suppressing Th1 cytokines and down-regulation of disease-promoting Th2 cytokines at both protein and mRNA level. Moreover, Cg-Ex augmented the delayed-type hypersensitivity (DTH) response and serum IgG2a level which are correlated with the diminution of parasite burden with no hepatic and renal toxicity. Additionally, histological analysis of spleen depicted the improvement of structural disorganization of white and red pulp after Cg-Ex treatment. Therefore, our intriguing findings have presented the first indication of in vivo antileishmanial efficacy through activation of pro-inflammatory immune responses of the host by a natural plant leaf extract (Cg-Ex) containing serine protease inhibitors which could have a role as a potential immunomodulator against visceral leishmaniasis.

Neutrophils use superoxide to control bacterial infection at a distance.

Understanding the roles of neutrophils and macrophages in fighting bacterial infections is a critical issue in human pathologies. Although phagocytic killing has been extensively studied, little is known about how bacteria are eliminated extracellularly in live vertebrates. We have recently developed an infection model in the zebrafish embryo in which leukocytes cannot reach the injected bacteria. When Escherichia coli bacteria are injected within the notochord, both neutrophils and macrophages are massively recruited during several days, but do not infiltrate the infected tissue presumably because of its tough collagen sheath. Nevertheless, the bacteria are killed during the first 24 hours, and we report here that neutrophils, but not macrophages are involved in the control of the infection. Using genetic and chemical approaches, we show that even in absence of phagocytosis, the bactericidal action relies on NADPH oxidase-dependent production of superoxide in neutrophils. We thus reveal a host effector mechanism mediated by neutrophils that eliminates bacteria that cannot be reached by phagocytes and that is independent of macrophages, NO synthase or myeloperoxidase.

Neoadjuvant combination immunotherapy with pembrolizumab and high dose IFN-α2b in locally/regionally advanced melanoma.

181 Background: Neoadjuvant pembrolizumab at 200 mg in combination with high dose IFNα2b (HDI) for locally/regionally advanced or recurrent melanoma may improve the clinical outcomes of these high risk patients (pts), and provide access to blood and tumor pre/post pembro-HDI to illuminate the host effector and suppressor immune mechanisms. Methods: Pts were treated with pembro 200 mg IV every 3 weeks (wk) x 2 doses followed by definitive surgery, then every 3 wks for up to one year. HDI (20 MU/m²/d IV x 5 days (d)/wk for 4 wks then 10 MU/m²/d SC every other d TIW for 48 wks) was given concurrently. Tumor samples were obtained at baseline and at definitive surgery (wk 6-8) and serum/PBMC at baseline, 6 wks, 3, 6, 12 months (mo). Results: Twenty evaluable pts (14 male, 6 female, 14 cutaneous primary, 4 unknown, 3 mucosal), age 29-82 were treated. 5 had Stage IIIB (N1b, N2b, M2c), 11 IIIC (N3) and 4 IV (M1a, M1b) melanoma. Over 230 cycles have been delivered to date (median 14). Worst toxicities included grade (Gr) 3: fatigue (8; 40%), ↑CPK (5; 25%), hypophosphatemia (5; 25%), ↑lipase (3; 15%), lymphopenia (3; 15%), hypertension (2; 10%), diarrhea/colitis (1; 5%), arthralgia (1, 5%), syncope (1, 5%), hyponatremia (1, 5%), neutropenia (1; 5%), anemia (1, 5.00%) nausea (2, 10%), flu like symptoms (1, 5%). There were 3 Gr 4 events (CPK, hyperglycemia, lymphopenia). One suspected grade 5 event occurred 6 months after completion of therapy with autopsy evidence of pneumonia and myocarditis. Among 20 evaluable pts, 4 relapsed and 1 died. Median follow-up for pts who have not relapsed is 11 months. The radiologic preoperative response rate (WHO; unconfirmed) was 65%. The pathologic complete response rate (no viable tumor on histologic assessment) was 35%. Conclusions: Neoadjuvant pembro-HDI exhibited promising clinical activity. Longer follow up is underway in order to define the long term benefits and risks. Clinical trial information: NCT02339324.

TLR Specific Immune Responses against Helminth Infections.

Despite marked improvement in the quality of lives across the globe, more than 2 million individuals in socioeconomically disadvantaged environments remain infected by helminth (worm) parasites. Owing to the longevity of the worms and paucity of immunologic controls, these parasites survive for long periods within the bloodstream, lymphatics, and gastrointestinal tract resulting in pathologic conditions such as anemia, cirrhosis, and lymphatic filariasis. Despite infection, an asymptomatic state may be maintained by the host immunoregulatory environment, which involves multiple levels of regulatory cells and cytokines; a breakdown of this regulation is observed in pathological disease. The role of TLR expression and function in relation to intracellular parasites has been documented but limited studies are available for multicellular helminth parasites. In this review, we discuss the unique and shared host effector mechanisms elicited by systemic helminth parasites and their derived products, including the role of TLRs and sphingolipids. Understanding and exploiting the interactions between these parasites and the host regulatory network are likely to highlight new strategies to control both infectious and immunological diseases.

Association of rituximab with graphene oxide confers direct cytotoxicity for CD20-positive lymphoma cells.

Non-Hodgkin lymphoma (NHL) is one of the most common hematologic malignancies among adults for which the chimeric monoclonal anti-CD20 antibody (Ab) rituximab (RTX) is used as first-line therapy. As RTX itself is not directly cytotoxic but relies on host immune effector mechanisms or chemotherapeutic agents to attack target cells, its therapeutic capacity may become limited when host effector mechanisms are compromised. Currently, refractory disease and relapse with NHL are still common, highlighting the need for novel anti-CD20 antibody strategies with superior therapeutic efficacy over current protocols. We hypothesized that making RTX directly cytotoxic might improve the therapeutic efficacy. Graphene oxide (GO) has recently emerged as a highly attractive nanomaterial for biomedical applications; and several studies have reported cytotoxic effect of GO on benign and malignant cells in vitro. Herein, we report that RTX can be stably associated with GO, and that GO-associated RTX (RTX/GO) demonstrates remarkably high avidity for CD20. Binding of GO-associated RTX to CD20-positive lymphoma cells induces CD20 capping and target cell death through an actin dependent mechanism. In vivo, GO-associated RTX, but not free RTX, quickly eliminates high-grade lymphomas in the absence of host effector mechanisms in a xenograft lymphoma mouse model. Our findings represent the first demonstration of using GO-associated antibody as effective cytotoxic therapy for human B cell malignancies in the absence of chemotherapy, and these findings could have important clinical implications.

Leucocyte-derived extracellular trap formation significantly contributes to Haemonchus contortus larval entrapment.

BackgroundPolymorphonuclear neutrophil (PMN) and eosinophil extracellular trap (ETs) formation has recently been described as an important host effector mechanism against invading pathogens. So far, scarce evidence on metazoan-triggered ET formation has been published. We here describe for the first time Haemonchus contortus-triggered ETs being released by bovine PMN and ovine eosinophils in response to ensheathed and exsheathed third stage larvae (L3).MethodsThe visualization of ETs was achieved by SEM analysis. The identification of classical ETs components was performed via fluorescence microscopy analysis. The effect of larval exsheathment and parasite integrity on ET formation was evaluated via Pico Green®- fluorescence intensities. ETs formation under acidic conditions was assessed by using media of different pH ranges. Parasite entrapment was evaluated microscopically after co-culture of PMN and L3. ET inhibition experiments were performed using inhibitors against NADPH oxidase, NE and MPO. Eosinophil-derived ETs were estimated via fluorescence microscopy analysis.ResultsL3 significantly induced PMN-mediated ETs and significant parasite entrapment through ETs structures was rapidly observed after 60 min of PMN and L3 co-culture. Co-localization studies of PMN-derived extracellular DNA with histones (H3), neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite-entrapping structures confirmed the classical characteristics of ETs. Haemonchus contortus-triggered ETs were significantly diminished by NADPH oxidase-, NE- and MPO-inhibition. Interestingly, different forms of ETs, i.e. aggregated (aggETs), spread (sprETs) and diffused (diffETs) ETs, were induced by L3. AggETs and sprETs firmly ensnared larvae in a time dependent manner. Significantly stronger aggETs reactions were detected upon exposure of PMN to ensheathed larvae than to exsheathed ones. Low pH conditions as are present in the abomasum did not block ETosis and led to a moderate decrease of ETs. Eosinophil-ETs were identified extruding DNA via fluorescence staining.ConclusionWe postulate that ETs may limit the establishment of H. contortus within the definitive host by immobilizing the larvae and hampering them from migrating into the site of infection. Consequently, H. contortus-mediated ET formation might have an impact on the outcome of the disease. Finally, besides PMN-triggered ETs, we here present first indications of ETs being released by eosinophils upon H. contortus L3 exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1219-1) contains supplementary material, which is available to authorized users.

NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation

Cryptosporidium parvum causes a zoonotic infection with worldwide distribution. Besides humans, cryptosporidiosis affects a wide range of animals leading to significant economic losses due to severe enteritis in neonatal livestock. Neutrophil extracellular trap (NET) formation has been demonstrated as an important host effector mechanism of PMN acting against several invading pathogens. In the present study, C. parvum-mediated NET formation was investigated in human and bovine PMN in vitro. We here demonstrate that C. parvum sporozoites indeed trigger NET formation in a time-dependent manner. Thereby, the classical characteristics of NETs were demonstrated by co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO). A significant reduction of NET formation was measured following treatments of PMN with NADPH oxidase-, NE- and MPO-inhibitors, confirming the key role of these enzymes in C. parvum-induced NETs. Additionally, sporozoite-triggered NETosis revealed as dependent on intracellular Ca++ concentration and the ERK 1/2 and p38 MAPK-mediated signaling pathway. Moreover, sporozoite-triggered NET formation led to significant parasite entrapment since 15% of the parasites were immobilized in NET structures. Consequently, PMN-pre-exposed sporozoites showed significantly reduced infectivity for epithelial host cells confirming the capability of NETs to prevent active parasite invasion. Besides NETs, we here show that C. parvum significantly up-regulated CXCL8, IL6, TNF-α and of GM-CSF gene transcription upon sporozoite confrontation, indicating a pivotal role of PMN not only in the bovine and human system but most probably in other final hosts for C. parvum.

Potential Function of Granulysin, Other Related Effector Molecules and Lymphocyte Subsets in Patients with TB and HIV/TB Coinfection

Background: Host effector mechanism against Mycobacterium tuberculosis (Mtb) infection is dependent on innate immune response by macrophages and neutrophils and the alterations in balanced adaptive immunity. Coordinated release of cytolytic effector molecules from NK cells and effector T cells and the subsequent granule-associated killing of infected cells have been documented; however, their role in clinical tuberculosis (TB) is still controversy.Objective: To investigate whether circulating granulysin and other effector molecules are associated with the number of NK cells, iNKT cells, Vγ9+Vδ2+ T cells, CD4+ T cells and CD8+ T cells, and such association influences the clinical outcome of the disease in patients with pulmonary TB and HIV/TB coinfection.Methods: Circulating granulysin, perforin, granzyme-B and IFN-γ levels were determined by ELISA. The isoforms of granulysin were analyzed by Western blot analysis. The effector cells were analyzed by flow cytometry.Results: Circulating granulysin and perforin levels in TB patients were lower than healthy controls, whereas the granulysin levels in HIV/TB coinfection were much higher than in any other groups, TB and HIV with or without receiving HAART, which corresponded to the number of CD8+ T cells which kept high, but not with NK cells and other possible cellular sources of granulysin. In addition, the 17kDa, 15kDa and 9kDa isoforms of granulysin were recognized in plasma of HIV/TB coinfection. Increased granulysin and decreased IFN-γ levels in HIV/TB coinfection and TB after completion of anti-TB therapy were observed.Conclusion: The results suggested that the alteration of circulating granulysin has potential function in host immune response against TB and HIV/TB coinfection. This is the first demonstration so far of granulysin in HIV/TB coinfection.

Neoadjuvant ipilimumab in locally/regionally advanced melanoma: Clinical outcome and immune monitoring.

8533 Background: Neoadjuvant ipilimumab (ipi) for stage IIIB-C melanoma may improve the clinical outcomes and provide access to pre/post ipi blood and tumor to gain insight into host effector and suppressor immune response mechanisms. Methods: Patients were treated with ipi (10 mg/kg IV q3weeks x 4doses total) bracketing definitive surgery. Tissue samples were obtained at baseline and at definitive surgery (week ≥ 6) and serum/PBMC collected at baseline, 6 weeks, then at 3, 6, 9, 12 months and/or progression. Flow cytometry was used to monitor the host effector and suppressor immune response in blood and evaluable tumor. Results: Thirty pts (21 male, 9 female), age 40-87 were enrolled (25 cutaneous primary, 1 unknown, 4 mucosal). Six had AJCC stage IIIB (N2b, N2c) and 24 IIIC (N3) melanoma. Ninety-three cycles have been delivered (median 4). Worst toxicities included grade 3 diarrhea/colitis (5 patients; 17%), hepatic enzyme elevations (2; 7%), rash (2; 7%), lipase (1; 3%), all manageable. Median follow up is 14 months: among 29 evaluable pts 15 (52%) continue disease free. Median PFS is 15.5 months, 95% CI = (8.1,-). The probability of 6 and 12 month PFS is 82.4% (95% CI=0.63, 0.92) and 53% (95% CI=0.31, 0.70) respectively. Peripherally, a significant increase in frequency of circulating T-regs (CD4+CD25hi+ Foxp3+; p=0.02 CD4+CD25hi+CD39+; p=0.001) from baseline to 6 weeks was observed. Greater increases in T-regs were associated with improved PFS (p=0.045; HR=0.54). Significant decreases in circulating MDSCs, were observed in monocytic HLA-DR+/low/CD14+ MDSC subtype (p<0.0001). Spontaneous in vivo cross presentation was observed resulting in Th1 CD4 and CD8 antigen specific T-cell immunity (gp-100, MART-1, NY-ESO-1 peptides) with increase in frequency after ipi. Activated TIL in tumor increased after ipi (CD3+/CD4+/CD69+;p=0.06 and CD3+/CD8+/CD69+) with significant induction/potentiation of T-cell memory (CD8+/CD45RO+/TNF-α+;p=0.03). Conclusions: Neoadjuvant ipi exhibits promising clinical activity and significantly modulates the host effector and suppressor immune response. Full analysis of this completed trial and its correlates will be presented. Support: BMS, P30CA047904 and P50CA121973.

Expression of either Lethal Toxin or Edema Toxin by Bacillus anthracis Is Sufficient for Virulence in a Rabbit Model of Inhalational Anthrax

The development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics against Bacillus anthracis. We investigated the roles of B. anthracis capsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability of B. anthracis to traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA- mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA- mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.

Exhaustion of Cytotoxic Effector Systems May Limit Monoclonal Antibody-Based Immunotherapy in Cancer Patients

The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.

Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated approximately 80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.

The effectors responsible for gastrointestinal nematode parasites, Trichinella spiralis, expulsion in rats

Helper T (Th2) cells type 2 have a central role in host protective responses to gastrointestinal nematode parasite, Trichinella spiralis infection, but the actual effector mechanisms involved in parasite expulsion are still uncertain. Recent evidences suggest that mast cell recruitment and activation may associate with parasite elimination from host intestines in mice. On the other hand, IgE production may induce defensive responses to primary infection with the helminth in rats. The differences of host effector mechanisms to the same experimental infections might disturb our understanding of the host protective mechanisms to gastrointestinal nematode parasite infection. In order to redefine these differences, we investigated in detail the relationship between intestinal immune responses and worm expulsion following T. spiralis infection among several rat strains including mutants. As a result, there were significant correlations of parasite expulsion with mast cell hyperplasia in addition to serum IgE level. Moreover, mast cell-deficient and dysfunction rats showed delayed worm elimination from their gut. Therefore, the present study suggests that mast cells should also be one of the prominent effector cells involved in T. spiralis parasite expulsion in rats as well as mice.

Follicular NHL: From Antibodies and Vaccines to Graft-versus-Lymphoma Effects

Monoclonal antibody therapy with rituximab in combination with standard chemotherapy has improved the survival of patients with advanced-stage follicular lymphoma (FL). A series of next-generation anti-CD20 antibodies may be less immunogenic and have even greater antitumor activity through augmented interactions with host effector mechanisms responsible for tumor cell kill. Additional approaches with patient-specific immunoglobulin idiotype vaccines; novel monoclonal antibodies binding to biologically active cell-surface antigen are also demonstrating early clinical activity. Antibodies targeting radioisotopes, toxins or drugs are also slowly entering clinical trials and practice. Last, allogeneic stem cell transplantation following reduced-intensity conditioning provides graft-versus-tumor immune responses that may be able to control FL and allow this risky but potentially curative treatment option to older patents or those with comorbidities.

Mechanistic Evaluation of Siplizumab (MEDI-507) Activity on Normal and Malignant T-Lymphocytes.

Siplizumab is a humanized monoclonal antibody that selectively binds human CD2 (huCD2), which is expressed on virtually all cells of the T- or NK-cell lineage. Also, known as MEDI-507, siplizumab is being developed as a potential new treatment for lymphoid malignancies involving T- or NK-cells. Our present study was designed to increase further the mechanistic understanding of siplizumab activity. Preclinical studies demonstrated that siplizumab mediated efficient lysis of malignant T-cells in a concentration-dependent and effector-to-target cell ratio-dependent manner. ADCC contributed prominently to tumor cell clearance whereas complement-mediated killing (CDC) did not. Siplizumab treatment alone was sufficient to trigger limited internalization of its target antigen (CD2) from the surface of tumor cells. Moreover, the efficiency of CD2 internalization was increased when such studies were carried out in the presence of an activated monocyte cell line (THP-1) bearing Fcg receptors and incapable of mediating ADCC, and suggested that CD2 clearance from the cell surface can be distinguished from host effector mechanisms. CD2 has been long known to confer both inhibitory and stimulatory signals and we reasoned that target internalization might disrupt survival signals independent of host effector activity. Consistent with this concept, siplizumab treatment in the absence of effector cells transduced apoptotic signals as indicated by multiple standard markers of tumor cell apoptosis , including activation of caspase 3 activity and an increase in tumor cells with a sub-G1 DNA content. In addition, the use of transgenic mouse models demonstrated that intravenous administration of siplizumab caused a rapid (≤ 24 hours) but transient reduction in the number of peripheral huCD2-positive T-cells. The number of T-cells gradually recovered to normal levels within 30 days of treatment. In contrast, siplizumab treatment did not alter the number of CD19-positive B-cells. These results indicate that siplizumab can utilize host effector mechanisms (ADCC) to facilitate malignant T-cell elimination. Our present findings also suggest that siplizumab can mediate changes in CD2 expression or function that result in apoptosis of CD2-positive tumor cells. In light of this potent anti-tumor activity, two Phase I dose escalation trials (MI-CP099 and MI-CP107) are ongoing to assess the maximum tolerated dose and safety of siplizumab in patients with CD2-positive T- and NK-cell malignancies.

Fcγ Receptors Play a Dominant Role in Protective Tumor Immunity against a Virus-Encoded Tumor-Specific Antigen in a Murine Model of Experimental Pulmonary Metastases

Simian virus 40 (SV40) large tumor antigen (Tag) represents a virus-encoded tumor-specific antigen expressed in many types of human cancers and a potential immunologic target for antitumor responses. Fc receptors are important mediators in the regulation and execution of host effector mechanisms against conditions including infectious diseases, autoimmunity, and cancer. By examining tumor protection in SV40 Tag-immunized wild-type BALB/c mice using an experimental pulmonary metastasis model, we attempted to address whether engagement of the immunoglobulin G Fc receptors (FcgammaRs) on effector cells is necessary to mediate antitumor responses. All immunized BALB/c FcgammaR-/- knockout mice developed anti-SV40 Tag antibody responses prior to experimental challenge with a tumorigenic cell line expressing SV40 Tag. However, all mice deficient in the activating FcgammaRI (CD64) and FcgammaRIII (CD16) were unable to mount protective immunologic responses against tumor challenge and developed tumor lung foci. In contrast, mice lacking the inhibitory receptor FcgammaRII (CD32) demonstrated resistance to tumorigenesis. These results underscore the importance of effector cell populations expressing FcgammaRI/III within this murine tumor model system, and along with the production of a specific humoral immune response, antibody-dependent cell-mediated cytotoxicity (ADCC) may be a functioning mechanism of tumor clearance. Additionally, these data demonstrate the potential utility of ADCC as a viable approach for targeting vaccination strategies that promote FcgammaRI/III scavenging pathways against cancer.