Effect of ibrutinib on CCR7 expression and functionality in chronic lymphocytic leukemia and its implication for the activity of CAP-100, a novel therapeutic anti-CCR7 antibody.

CAP-100 is a novel therapeutic antibody directed against the ligand binding site of human CCR7. This chemokine receptor is overexpressed in chronic lymphocytic leukemia (CLL) and orchestrates the homing of CLL cells into the lymph node. Previous studies, on a very limited number of samples, hypothesized that the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib might induce loss of surface CCR7 levels in CLL cells. CAP-100 will be evaluated in clinical trials as a therapy for relapse/refractory CLL patients, who have received at least two systemic therapies (NCT04704323). As nowadays many relapse/refractory CLL patients will have received ibrutinib as a prior therapy, we aimed to investigate in a large cohort of CLL patients the impact of this BTKi on CCR7 expression and functionality as well as on the therapeutic activity of CAP-100. Our data confirm that ibrutinib moderately down-regulates the very high expression of CCR7 in CLL cells but has no apparent effect on CCR7-induced chemotaxis. Moreover, CLL cells are perfectly targetable by CAP-100 which led to a complete inhibition of CCR7-mediated migration and induced strong target cell killing through antibody-dependent cell-mediated cytotoxicity, irrespective of previous or contemporary ibrutinib administration. Together, these results validate the therapeutic utility of CAP-100 as a next-line single-agent therapy for CLL patients who failed to ibrutinib and confirm that CAP-100 and ibrutinib have complementary non-overlapping mechanisms of action, potentially allowing for combination therapy.

Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-cancer Agents.

The transferrin receptor 1 (TfR1), also known as cluster of differentiation 71 (CD71), is a type II transmembrane glycoprotein that binds transferrin (Tf) and performs a critical role in cellular iron uptake through the interaction with iron-bound Tf. Iron is required for multiple cellular processes and is essential for DNA synthesis and, thus, cellular proliferation. Due to its central role in cancer cell pathology, malignant cells often overexpress TfR1 and this increased expression can be associated with poor prognosis in different types of cancer. The elevated levels of TfR1 expression on malignant cells, together with its extracellular accessibility, ability to internalize, and central role in cancer cell pathology make this receptor an attractive target for antibody-mediated therapy. The TfR1 can be targeted by antibodies for cancer therapy in two distinct ways: (1) indirectly through the use of antibodies conjugated to anti-cancer agents that are internalized by receptor-mediated endocytosis or (2) directly through the use of antibodies that disrupt the function of the receptor and/or induce Fc effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). Although TfR1 has been used extensively as a target for antibody-mediated cancer therapy over the years, interest continues to increase for both targeting the receptor for delivery purposes and for its use as direct anti-cancer agents. This review focuses on the developments in the use of antibodies targeting TfR1 as direct anti-tumor agents.

Drug Use is Associated with Anti-CD4 IgG-mediated CD4+ T Cell Death and Poor CD4+ T Cell Recovery in Viral-suppressive HIV-infected Individuals Under Antiretroviral Therapy.

BACKGROUND: The role and mechanism of drug use or abuse in Antiretroviral Therapy (ART)-treated HIV disease are not completely known. METHODS: To investigate the impact of drug use on HIV pathogenesis without confounding by HIV replication and ART adherence, we first analyzed the data from our clinical database in 103 HIV+ subjects with viral-suppressed ART treatment by a multiple regression test. RESULTS: We found that HIV+ drug users had lower CD4+ T cell counts but higher CD8+ T cell counts compared to HIV+ non-drug users, and both drug use and nadir CD4+ T cell counts was independently associated with CD4+ T cell recovery after controlling for sex and age. Next, we enrolled individuals from four study groups, HIV-negative and HIV+ subjects without any substance use, HIV-negative and HIV+ subjects with current illicit drug use (either non-injection cocaine or cannabis). All HIV+ subjects were viral-suppressed with ART treatment (≥ 2 years). Notably, HIV+ drug users had increased plasma anti-CD4 IgG levels compared to the other three study groups which were inversely correlated with decreased CD4+ T cell counts only in HIV+ drug users. There was a significant increase in CD4+ T cell recovery following ART in HIV+ non-drug users but not in HIV+ drug users. Anti-CD4 IgGs purified from plasma of HIV+ drug users induced CD4+ T cell death in vitro through Antibody-Dependent Cytotoxicity (ADCC). CONCLUSION: These results suggest that drug use prevents immune reconstitution in HIV-infected individuals despite long-term ART treatment and viral suppression.

A Novel, Fully Human Anti-fucosyl-GM1 Antibody Demonstrates Potent In Vitro and In Vivo Antitumor Activity in Preclinical Models of Small Cell Lung Cancer.

Purpose: The ganglioside fucosyl-GM1 (FucGM1) is a tumor-associated antigen expressed in a large percentage of human small cell lung cancer (SCLC) tumors, but absent in most normal adult tissues, making it a promising target in immuno-oncology. This study was undertaken to evaluate the preclinical efficacy of BMS-986012, a novel, nonfucosylated, fully human IgG1 antibody that binds specifically to FucGM1.Experimental Design: The antitumor activity of BMS-986012 was evaluated in in vitro assays using SCLC cells and in mouse xenograft and syngeneic tumor models, with and without chemotherapeutic agents and checkpoint inhibitors.Results: BMS-986012 showed a high binding affinity for FcγRIIIa (CD16), which resulted in enhanced antibody-dependent cellular cytotoxicity (ADCC) against FucGM1-expressing tumor cell lines. BMS-986012-mediated tumor cell killing was also observed in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) assays. In several mouse SCLC models, BMS-986012 demonstrated efficacy and was well tolerated. In the DMS79 xenograft model, tumor regression was achieved with BMS-986012 doses of 0.3 mg/kg and greater; antitumor activity was enhanced when BMS-986012 was combined with standard-of-care cisplatin or etoposide. In a syngeneic model, tumors derived from a genetically engineered model of SCLC were treated with BMS-986012 or anti-FucGM1 with a mouse IgG2a Fc and their responses evaluated; when BMS-986012 was combined with anti-PD-1 or anti-CD137 antibody, therapeutic responses significantly improved.Conclusions: Single-agent BMS-986012 demonstrated robust antitumor activity, with the addition of chemotherapeutic or immunomodulatory agents further inhibiting SCLC growth in the same models. These preclinical data supported evaluation of BMS-986012 in a phase I clinical trial of patients with relapsed, refractory SCLC. Clin Cancer Res; 24(20); 5178-89. ©2018 AACR.

Anti-PD-L1/TGFßR2 (M7824) fusion protein induces immunogenic modulation of human urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis.

BACKGROUND: Avelumab has recently been approved by the Food and Drug Administration for the therapy of Merkel cell carcinoma and urothelial carcinoma. M7824 is a novel first-in-class bifunctional fusion protein comprising a monoclonal antibody against programmed death-ligand 1 (PD-L1, avelumab), fused to the extracellular domain of human transforming growth factor beta (TGFß) receptor 2, which functions as a TGFß "trap." Advanced urothelial tumors have been shown to express TGFß, which possesses immunosuppressive properties that promote cancer progression and metastasis. The rationale for a combined molecule is to block the PD-1/PD-L1 interaction between tumor cells and immune cell infiltrate and simultaneously reduce or eliminate TGFß from the tumor microenvironment. In this study, we explored the effect of M7824 on invasive urothelial carcinoma cell lines. METHODS: Human urothelial (transitional cell) carcinoma cell lines HTB-4, HTB-1, and HTB-5 were treated with M7824, M7824mut (M7824 that is mutated in the anti-PD-L1 portion of the molecule and thus does not bind PD-L1), anti-PD-L1 (avelumab), or IgG1 isotype control monoclonal antibody, and were assessed for gene expression, cell-surface phenotype, and sensitivity to lysis by TRAIL, antigen-specific cytotoxic T lymphocytes and natural killer cells. RESULTS: M7824 retains the ability to mediate antibody-dependent cellular cytotoxicity of tumor cells, although in some cases to a lesser extent than anti-PD-L1. However, compared to anti-PD-L1, M7824 increases (A) gene expression of molecules involved in T-cell trafficking in the tumor (e.g., CXCL11), (B) TRAIL-mediated tumor cell lysis, and (C) antigen-specific CD8+ T-cell-mediated lysis of tumor cells. CONCLUSIONS: These studies demonstrate the immunomodulatory properties of M7824 on both tumor cell phenotype and immune-mediated lysis. Compared to anti-PD-L1 or M7824mut, M7824 induces immunogenic modulation of urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis. These findings show the relevance of the dual blockade of PD-L1 and TGFß in urothelial carcinoma cell lines and thus support the rationale for future clinical studies of M7824 in patients with urothelial cancer.

Antitumor activity of an anti-CD98 antibody.

CD98 is expressed on several tissue types and specifically upregulated on fast-cycling cells undergoing clonal expansion. Various solid (e.g., nonsmall cell lung carcinoma) as well as hematological malignancies (e.g., acute myeloid leukemia) overexpress CD98. We have identified a CD98-specific mouse monoclonal antibody that exhibits potent preclinical antitumor activity against established lymphoma tumor xenografts. Additionally, the humanized antibody designated IGN523 demonstrated robust tumor growth inhibition in leukemic cell-line derived xenograft models and was as efficacious as standard of care carboplatin in patient-derived nonsmall lung cancer xenografts. In vitro studies revealed that IGN523 elicited strong ADCC activity, induced lysosomal membrane permeabilization and inhibited essential amino acid transport function, ultimately resulting in caspase-3 and -7-mediated apoptosis of tumor cells. IGN523 is currently being evaluated in a Phase I clinical trial for acute myeloid leukemia (NCT02040506). Furthermore, preclinical data support the therapeutic potential of IGN523 in solid tumors.

Preclinical Evidence for the Therapeutic Potential of CD38-Targeted Immuno-Chemotherapy in Multiple Myeloma Patients Refractory to Lenalidomide and Bortezomib.

PURPOSE: Novel therapeutic agents have significantly improved the survival of patients with multiple myeloma. Nonetheless, the prognosis of patients with multiple myeloma who become refractory to the novel agents lenalidomide and bortezomib is very poor, indicating the urgent need for new therapeutic options for these patients. The human CD38 monoclonal antibody daratumumab is being evaluated as a novel therapy for multiple myeloma. Prompted with the encouraging results of ongoing clinical phase I/II trials, we now addressed the potential value of daratumumab alone or in combination with lenalidomide or bortezomib for the treatment of lenalidomide- and bortezomib-refractory patients. EXPERIMENTAL DESIGN: In ex vivo assays, mainly evaluating antibody-dependent cell-mediated cytotoxicity, and in an in vivo xenograft mouse model, we evaluated daratumumab alone or in combination with lenalidomide or bortezomib as a potential therapy for lenalidomide- and bortezomib-refractory multiple myeloma patients. RESULTS: Daratumumab induced significant lysis of lenalidomide/bortezomib-resistant multiple myeloma cell lines and of primary multiple myeloma cells in the bone marrow mononuclear cells derived from lenalidomide- and/or bortezomib-refractory patients. In these assays, lenalidomide but not bortezomib, synergistically enhanced daratumumab-mediated multiple myeloma lysis through activation of natural killer cells. Finally, in an in vivo xenograft model, only the combination of daratumumab with lenalidomide effectively reduced the tumorigenic growth of primary multiple myeloma cells from a lenalidomide- and bortezomib-refractory patient. CONCLUSIONS: Our results provide the first preclinical evidence for the benefit of daratumumab plus lenalidomide combination for lenalidomide- and bortezomib-refractory patients.

Target-directed development and preclinical characterization of the proposed biosimilar rituximab GP2013.

Biosimilar development involves a target-directed iterative process to ensure a similar product to the originator. Here we report the preclinical development of the proposed biosimilar rituximab (GP2013). Post-translational modifications and bioactivities of GP2013 versus originator rituximab were engineered and monitored to ensure similar pharmacological profiles. Antibody-dependent cellular cytotoxicity (ADCC) was used to illustrate how different glycosylation patterns and structure-function relationships were controlled during process development. Pharmacological comparability between GP2013 and originator rituximab were confirmed in preclinical studies using clinical scale drug product. Similar in vitro ADCC potency was demonstrated when compared in a dose-response manner against two lymphoma cell lines using freshly purified human natural killer (NK) cells. In vivo efficacy was demonstrated in two well characterized mouse xenograft models, testing at sensitive sub-therapeutic dose levels. Pharmacokinetics and pharmacodynamics (CD20 cell depletion) were likewise comparable in cynomolgus monkeys. This preclinical comparability exercise confirms that GP2013 and originator rituximab are pharmacologically similar.

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study.

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.

A mechanistic rationale for combining alemtuzumab and rituximab in the treatment of ALL.

B-lineage acute lymphoblastic leukemia (ALL) may express CD52 and CD20. Alemtuzumab (ALM) and rituximab (RTX) are therapeutic antibodies directed against CD52 and CD20, respectively, but showed limited activity against ALL in clinical trials. The mechanisms for the impaired responses remained unclear. We studied expression of CD52 and CD20 on ALL cells and found that most cases coexpressed CD52 and CD20. However, distinct CD52-negative (CD52(-)) subpopulations were detected in most cases as the result of defective glycophosphatidyl-inositol anchoring. Although ALM efficiently eradicated CD52-positive (CD52(+)) cells in NOD/scid mice engrafted with primary human ALL, CD52(-) subclones escaped therapy. In the same model, RTX showed limited activity resulting from occurrence of CD20 down-modulation. However, CD52(-) cells concurrently lacked the glycophosphatidyl-inositol-anchored complement regulators CD55 and CD59 and showed increased susceptibility to RTX-mediated complement-dependent cytotoxicity in vitro. At the same time, ALM was shown to inhibit down-modulation of CD20 in response to RTX by depleting the trogocytic capacity of phagocytic cells. Probably because of these complementary mechanisms, combined administration of ALM and RTX induced complete responses in vivo. Based on these data, we propose a mechanistic rationale for combined application of RTX and ALM in ALL.

Novel anti-CD20 antibody TGLA with enhanced antibody-dependent cell-mediated cytotoxicity mediates potent anti-lymphoma activity.

Rituximab is the first anti-cancer antibody approved by the FDA for the treatment of B-cell lymphoma. However, its efficacy remains variable and often modest. Some patients are initially unresponsive to rituximab or later develop resistance to it, and require alternative therapies. Rituximab activity has been thought to involve antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and apoptosis. Present studies suggest that the patients unresponsive to rituximab may be helped with other CD20 antibodies with enhanced activities. In this study, we characterized a novel anti-CD20 chimeric antibody, TGLA, which binds to various B-cell lines specially and shares an epitope with rituximab. TGLA shows equal activities with rituximab, such as CDC, cell growth arrest and so on. Interestingly, TGLA also shows significant ADCC activity. Immunotherapeutic studies further show that TGLA is far more effective in delaying tumor growth than rituximab. These findings suggest that the ADCC-enhanced anti-CD20 antibody TGLA might be an alternative therapeutic agent for B-cell lymphoma.

P-selectin glycoprotein ligand-1 as a potential target for humoral immunotherapy of multiple myeloma.

Monoclonal antibodies (mAbs), successfully adopted in the treatment of several haematological malignancies, have proved almost ineffective in multiple myeloma (MM), because of the lack of an appropriate antigen for targeting and killing MM cells. Here, we demonstrate that PSGL1, the major ligand of P-Selectin, a marker of plasmacytic differentiation expressed at high levels on normal and neoplastic plasma cells, may represent a novel target for mAb-mediated MM immunotherapy. The primary effectors of mAb-induced cell-death, complement-mediated lysis (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), were investigated using U266B1 and LP1 cell-lines as models. Along with immunological mechanisms, the induction of apoptosis by PSGL1 cross-linking was assessed. The anti-PSGL1 murine mAb KPL1 induced death of MM cells in a dose- and time-dependent fashion and mediated a significant amount of ADCC. KPL1 alone mediated C1q deposition on target cells but proved unable to induce CDC due to inhibition of the lytic activity of complement by membrane complement regulators (mCRP) expressed on the cell surface. Consistently, CDC was induced by KPL1 upon mCRP blockage. Our results suggest a role for PSGL1 in MM humoral immunotherapy and support further in vivo studies assessing the effects of anti-PSGL1 mAbs on MM growth and interaction with the bone marrow microenvironment.

Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation.

Anti-CD20 monoclonal antibodies (mAbs) are classified into type I (rituximab-like) or type II (tositumomab-like) based on their ability to redistribute CD20 molecules in the plasma membrane and activate various effector functions. To compare type I and II mAbs directly in vivo and maximize Fc effector function, we selected and engineered mAbs with the same mouse IgG(2)a isotype and assessed their B-cell depleting activity in human CD20 transgenic mice. Despite being the same isotype, having similar affinity, opsonizing activity for phagocytosis, and in vivo half-life, the type II mAb tositumomab (B1) provided substantially longer depletion of B cells from the peripheral blood compared with the type I mAb rituximab (Rit m2a), and 1F5. This difference was also evident within the secondary lymphoid organs, in particular, the spleen. Failure to engage complement did not explain the efficacy of the type II reagents because type I mAbs mutated in the Fc domain (K322A) to prevent C1q binding still did not display equivalent efficacy. These results give support for the use of type II CD20 mAbs in human B-cell diseases.

Preclinical evaluation of MORAb-003, a humanized monoclonal antibody antagonizing folate receptor-alpha.

The highly restricted distribution of human folate receptor-alpha (FRalpha) in normal tissues and its high expression in some tumors, along with its putative role in tumor cell transformation, make this antigen a suitable target for antigen-specific, monoclonal antibody-based immunotherapy for oncology indications. We have developed a therapeutic humanized monoclonal antibody with high affinity for FRalpha, named MORAb-003, which was derived from the optimization of the LK26 antibody using a whole cell genetic evolution platform. Here we show that MORAb-003 possesses novel, growth-inhibitory functions on cells overexpressing FRalpha. In addition, MORAb-003 elicited robust antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vitro, and inhibited growth of human ovarian tumor xenografts in nude mice. Because of its multimodal activity in vitro and its safe toxicology profile in non-human primates, MORAb-003 development has recently been advanced to clinical trials involving ovarian cancer patients.

Modulation of natural killer cell activity, antibody-dependent cellular cytotoxicity, and antibody-dependent complement-mediated cytotoxicity by andrographolide in normal and Ehrlich ascites carcinoma-bearing mice.

Modulation of immune responses is highly relevant in tumor cell destruction. The present study is focused on the effect of Andrographis paniculata extract (APE) and its isolated compound andrographolide (ANDLE) on cell-mediated immune responses in normal and tumor-bearing control animals. Treatment with APE and ANDLE significantly enhanced natural killer cell activity in normal (APE, 46.82% cell lysis; ANDLE, 40.79% cell lysis) and tumor-bearing animals (APE, 48.66% cell lysis; ANDLE, 42.19% cell lysis) on the fifth day, and it was observed earlier than in tumor-bearing control animals (12.89% cell lysis on day 9). Antibody-dependent cellular cytotoxicity was also increased in APE (45.17% cell lysis on day 11) as well as ANDLE (39.92% cell lysis on day 11)-treated normal and tumor-bearing animals (APE, 47.39% cell lysis; ANDLE, 41.48% cell lysis on day 11) compared to untreated tumor-bearing control animals (maximum of 11.76% cell lysis on day 17). An early enhancement of antibody-dependent complement-mediated cytotoxicity was also observed by the administration of APE and ANDLE in normal as well as tumor-bearing animals. APE and ANDLE administration could significantly enhance the mitogen-induced proliferation of splenocyte, thymocyte, and bone marrow cells. Moreover, treatment of APE and ANDLE significantly elevated the production of interleukin-2 and interferon-gamma in normal and Ehrlich ascites carcinoma-bearing animals.

Evaluation of passively transferred, nonneutralizing antibody-dependent cellular cytotoxicity-mediating IgG in protection of neonatal rhesus macaques against oral SIVmac251 challenge.

Previously, Ab-dependent cellular cytotoxicity (ADCC) was significantly correlated with reduced acute viremia upon intrarectal SIVmac251 challenge of immunized rhesus macaques. To directly assess ADCC protective efficacy, six neonatal macaques were infused s.c. with immune IgG (220 mg/kg) purified from the immunized animals and positive for ADCC and Ab-dependent cell-mediated viral inhibition (ADCVI) activities. Six neonates received control IgG. The neonates were challenged twice orally with 10(5) 50% inhibiting tissue culture-infective dose of SIVmac251 2 days post-IgG infusion. At challenge, plasma of neonates that received immune IgG did not neutralize SIVmac251 but had geometric mean ADCC titers of 48,130 and 232,850 against SIVmac251 -infected and gp120-coated targets, respectively. Peak ADCVI activity varied from 62 to 81%. ADCC activity declined with the 2-wk IgG half-life but was boosted at wk 4, together with de novo ADCC-mediating Abs in controls, by postchallenge viremia. ADCVI activity was similarly induced. No protection, assessed by viral burdens, CD4 counts, and time to euthanasia was observed. Possible factors contributing to the discrepancy between the previous correlation and lack of protection here include: the high oral challenge dose compared with the 400-fold lower intrarectal dose; the challenge route with regard to viral dissemination and distribution of infused IgG; insufficient NK effector activity and/or poor functionality in newborns; insufficient immune IgG; and the possibility that the previous correlation of ADCC with protection was augmented by cellular immune responses also present at challenge. Future studies should explore additional challenge routes in juvenile macaques using higher amounts of potent IgG preparations.

Augmentation of natural killer cell and antibody-dependent cellular cytotoxicity in BALB/c mice by sulforaphane, a naturally occurring isothiocyanate from broccoli through enhanced production of cytokines IL-2 and IFN-gamma.

Effect of sulforaphane on cell-mediated immune (CMI) response was studied in normal as well as Ehrlich ascites tumor-bearing BALB/c mice. Administration of sulforaphane significantly enhanced natural killer (NK) cell activity in both normal as well as tumor-bearing animals, and the activity was observed earlier than in tumor-bearing control animals. Antibody-dependent cellular cytotoxicity (ADCC) also was enhanced significantly in both normal as well as tumor-bearing animals after sulforaphane administration compared with untreated control tumor-bearing animals. An early antibody-dependent complement-mediated cytotoxicity (ACC) also was observed in sulforaphane-treated normal and tumor-bearing animals. Administration of sulforaphane significantly enhanced the production of Interleukin-2 and Interferon-gamma in normal as well as tumor-bearing animals. In addition, sulforaphane significantly enhanced the proliferation of splenocytes, bone marrow cells, and thymocytes by stimulating the mitogenic potential of various mitogens such as concanavalin A, phytohaemagglutinin, poke weed mitogen, and lipopolysaccharide.

Effect of abrin on cell-mediated immune responses in mice.

Effect of abrin isolated from Abrus precatorius on the cellular immune responses was studied in normal as well as tumor-bearing animals. Administration of abrin was found to enhance the proliferation of splenocytes and thymocytes (lymphocytes in general) in responses to mitogens. Natural killer cell activity was enhanced significantly by abrin in both the normal (49.8% cell lysis on day 9) and the tumor-bearing group (51.7% cell lysis on day 9), and it was found to be earlier than the control. Antibody dependent cellular cytotoxicity was enhanced in the abrin treated tumor-bearing group on the ninth day (44% cell lysis). An early antibody dependent complement mediated cytotoxicity was observed in the abrin treated group on day 15 (27.6% cell lysis). Results of our present study suggest the immunomodulatory property of abrin.

Antiviral activity against CMV-infected fibroblasts in pediatric patients transplanted with CD34(+)-selected allografts from alternative donors.

Human cytomegalovirus (HCMV) remains a cause of serious infectious complications after allogeneic transplantation of hematopoietic stem cells, especially in recipients of T-cell-depleted grafts. Here we investigated the antiviral activity of natural killer (NK) cells from healthy donors (n = 8) as well as of mononuclear cells (MNC) from transplanted pediatric patients (n = 11) who had received CD34(+) selected (and thus T-cell-depleted) stem cells from unrelated and mismatched related donors. Allogeneic human fibroblasts infected with HCMV laboratory strain AD169 for 5 days were used as targets in a 2-h cytotoxicity assay. Downregulation of human leukocyte antigen class I and upregulation of the adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3) were observed after infection. In this experimental setting, NK cells from healthy donors exerted no specific lysis. However, antibody-dependent cellular cytotoxicity (ADCC) mediated by human anti-CMV IgG (cytoglobin) as well as stimulation with low-dose interleukin-(IL)-2 or IL-15 enhanced lysis markedly. MNC from two thirds of the patients (7/11) were capable of lysing infected targets without stimulation. Here also, lytic activity was significantly increased by IL-2 or IL-15, used in combination with ADCC. In contrast, 4/11 patients exerted no lysis. The observed antiviral activity may contribute to the low incidence of CMV DNAemia (29% at day 100, detected by polymerase chain reaction) in the whole group of our patients who have been transplanted with CD34(+)-selected allografts since 1995. Furthermore, our data suggest a potential benefit of using low-dose IL-2 or IL-15, also combined with anti-CMV immunoglobulinG, for immune modulation in CMV disease.