Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus.

INTRODUCTION: The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. METHODS: T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5'- and 3'-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays. RESULTS: Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells. CONCLUSION: Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.

Overexpression of SIRT6 alleviates apoptosis and enhances cell viability and monoclonal antibody expression in CHO-K1 cells.

BACKGROUND: Chinese hamster ovary (CHO) cells are the most predominantly utilized host for the production of monoclonal antibodies (mAbs) and other complex glycoproteins. A major challenge in the process of CHO cell culture is the occurrence of cell death following different stressful conditions, which hinders the production yield. Engineering genes involved in pathways related to cell death is a remarkable strategy to delay apoptosis, improve cell viability and enhance productivity. SIRT6 is a stress-responsive protein that regulates DNA repair, maintains genome integrity, and is critical for longevity and cell survival in organisms. METHODS AND RESULTS: In this study, SIRT6 was stably overexpressed in CHO-K1 cells and the impact of its expression on apoptosis related gene expression profile, viability, apoptosis, and mAb productivity was investigated. While a significant increase was observed in Bcl-2 mRNA level, caspase-3 and Bax mRNA levels were decreased in the SIRT6 engineered cells compared to the parental CHO-K1 cells. Moreover, improved cell viability and decreased rate of apoptotic progression was observed in a SIRT6-derived clone in comparision to the CHO-K1 cells during 5 days of batch culture. anti-CD52 IgG1 mAb titers were improved up to 1.7- and 2.8-fold in SIRT6-derived clone during transient and stable expression, respectively. CONCLUSIONS: This study indicates the positive effects of SIRT6 overexpression on cell viability and anti-CD52 IgG1 mAb expression in CHO-K1 cells. Further studies are needed to examine the potential of SIRT6-engineered host cells for the production of recombinant biotherapeutics in industrial settings.

Alemtuzumab-induced immune phenotype and repertoire changes: implications for secondary autoimmunity.

Alemtuzumab is a monoclonal antibody that causes rapid depletion of CD52-expressing immune cells. It has proven to be highly efficacious in active relapsing-remitting multiple sclerosis; however, the high risk of secondary autoimmune disorders has greatly complicated its use. Thus, deeper insight into the pathophysiology of secondary autoimmunity and potential biomarkers is urgently needed. The most critical time points in the decision-making process for alemtuzumab therapy are before or at Month 12, where the ability to identify secondary autoimmunity risk would be instrumental. Therefore, we investigated components of blood and CSF of up to 106 multiple sclerosis patients before and after alemtuzumab treatment focusing on those critical time points. Consistent with previous reports, deep flow cytometric immune-cell profiling (n = 30) demonstrated major effects on adaptive rather than innate immunity, which favoured regulatory immune cell subsets within the repopulation. The longitudinally studied CSF compartment (n = 18) mainly mirrored the immunological effects observed in the periphery. Alemtuzumab-induced changes including increased numbers of naïve CD4+ T cells and B cells as well as a clonal renewal of CD4+ T- and B-cell repertoires were partly reminiscent of haematopoietic stem cell transplantation; in contrast, thymopoiesis was reduced and clonal renewal of T-cell repertoires after alemtuzumab was incomplete. Stratification for secondary autoimmunity did not show clear immununological cellular or proteomic traits or signatures associated with secondary autoimmunity. However, a restricted T-cell repertoire with hyperexpanded T-cell clones at baseline, which persisted and demonstrated further expansion at Month 12 by homeostatic proliferation, identified patients developing secondary autoimmune disorders (n = 7 without secondary autoimmunity versus n = 5 with secondary autoimmunity). Those processes were followed by an expansion of memory B-cell clones irrespective of persistence, which we detected shortly after the diagnosis of secondary autoimmune disease. In conclusion, our data demonstrate that (i) peripheral immunological alterations following alemtuzumab are mirrored by longitudinal changes in the CSF; (ii) incomplete T-cell repertoire renewal and reduced thymopoiesis contribute to a proautoimmune state after alemtuzumab; (iii) proteomics and surface immunological phenotyping do not identify patients at risk for secondary autoimmune disorders; (iv) homeostatic proliferation with disparate dynamics of clonal T- and B-cell expansions are associated with secondary autoimmunity; and (v) hyperexpanded T-cell clones at baseline and Month 12 may be used as a biomarker for the risk of alemtuzumab-induced autoimmunity.

Anti-CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems.

Anti-CD52 antibody (anti-CD52-Ab) leads to a rapid depletion of T and B cells, followed by reconstitution of immune cells with tolerogenic characteristics. However, very little is known about its effect on innate immune cells. In this study, experimental autoimmune encephalomyelitis mice were administered murine anti-CD52-Ab to investigate its effect on dendritic cells and monocytes/macrophages in the periphery lymphoid organs and the central nervous system (CNS). Our data show that blood and splenic innate immune cells exhibited significantly increased expression of MHC-II and costimulatory molecules, which was associated with increased capacity of activating antigen-specific T cells, at first day but not three weeks after five daily treatment with anti-CD52-Ab in comparison with controls. In contrast to the periphery, microglia and infiltrating macrophages in the CNS exhibited reduced expression levels of MHC-II and costimulatory molecules after antibody treatment at both time-points investigated when compared to controls. Furthermore, the transit response of peripheral innate immune cells to anti-CD52-Ab treatment was also observed in the lymphocyte-deficient SCID mice, suggesting the changes are not a direct consequence of the mass depletion of lymphocytes in the periphery. Our study demonstrates a dynamic and tissue-specific modulation of the innate immune cells in their phenotype and function following the antibody treatment. The findings of differential modulation of the microglia and infiltrating macrophages in the CNS in comparison with the innate immune cells in the peripheral organs support the CNS-specific beneficial effect of alemtuzumab treatment on inhibiting neuroinflammation in multiple sclerosis patients.

Anti-CD52 blocks EAE independent of PD-1 signals and promotes repopulation dominated by double-negative T cells and newly generated T and B cells.

Lymphocyte depletion using anti-CD52 antibody effectively reduces relapses of multiple sclerosis (MS). To begin to understand what mechanisms might control this outcome, we examined the effect of a murine-CD52-specific mAb on the depletion and repopulation of immune cells in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We tested whether the tolerance-promoting receptor programmed cell death protein-1 (PD-1) is required for disease remission post anti-CD52, and found that PD-1-deficient mice with a more severe EAE were nevertheless effectively treated with anti-CD52. Anti-CD52 increased the proportions of newly generated T cells and double-negative (DN) T cells while reducing newly generated B cells; the latter effect being associated with a higher expression of CD52 by these cells. In the longer term, anti-CD52 caused substantial increases in the proportion of newly generated lymphocytes and DN T cells in mice with EAE. Thus, the rapid repopulation of lymphocytes from central lymphoid organs post anti-CD52 may limit further disease. Furthermore, these data identify DN T cells, a subset with immunoregulatory potential, as a significant hyperrepopulating subset following CD52-mediated depletion.

Preclinical pharmacology and toxicology evaluation of an anti-CD52 monoclonal antibody produced by perfusion fermentation process.

Anti-cluster of differentiation 52 (CD52) monoclonal antibody (mAb) has been employed in the treatment of chronic lymphoblastic leukemia and multiple sclerosis. Previously we developed a perfusion process to produce the biosimilar mAb named "Mab-TH." A series of quality assessments was conducted in the fields of structural identification, purity analysis, and activity measurement. After these quality researches, this report laid emphasis on preclinical pharmacology and toxicology evaluation. Mab-TH was characterized in biological, pharmacological, and toxicological properties in comparison with the original drug, alemtuzumab. Binding activity and immune-dependent toxicity as in vitro activity were evaluated. Severe immunodeficient mice transplanted with a human leukemia cell line were also used as an in vivo pharmacological model and a 4-week repeated dosing study in cynomolgus monkeys was conducted to evaluate the safety differences. Our results demonstrated that Mab-TH, the anti-CD52 antibody generated by a perfusion process, had high similarity in in vitro and in vivo activities compared with alemtuzumab in relevant preclinical models. The results supported it as a biosimilar candidate for clinical evaluation.

CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T cell function.

CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.

Mutations in PIGA cause a CD52-/GPI-anchor-deficient phenotype complicating alemtuzumab treatment in T-cell prolymphocytic leukemia.

OBJECTIVE: Infusional alemtuzumab followed by consolidating allogeneic hematopoietic stem cell transplantation in eligible patients is considered a standard of care in T-cell prolymphocytic leukemia (T-PLL). Antibody selection against CD52 has been associated with the development of CD52-negative leukemic T cells at time of relapse. Clinical implications and molecular mechanisms underlying this phenotypic switch are unknown. METHODS: We performed flow cytometry and real-time-PCR for CD52-expression and next generation sequencing for PIGA mutational analyses. RESULTS: We identified loss of CD52 expression after alemtuzumab treatment in two of 21 T-PLL patients resulting from loss of GPI-anchor expression caused by inactivating mutations of the PIGA gene. One patient with relapsed T-PLL exhibited a single PIGA mutation, causing a CD52-negative escape variant of the initial leukemic cell clone, preventing alemtuzumab-retreatment. The second patient with continued complete remission after alemtuzumab treatment harbored three different PIGA mutations that affected either the non-neoplastic T cell or the mononuclear cell compartment and resulted in symptomatic paroxysmal nocturnal hemoglobinuria. Next generation sequencing of T-PLL cells collected before the initiation of treatment revealed PIGA wild-type sequence reads in all 16 patients with samples available for testing. CONCLUSION: These data indicate that PIGA mutations were acquired during or after completion of alemtuzumab treatment.

Effects of guluronic acid (G2013) on gene expression of TLR2, TLR4, MyD88, TNF-α and CD52 in multiple sclerosis under in vitro conditions.

Context: Multiple sclerosis (MS) is an autoimmune and chronic inflammatory disease of CNS. The α-L-guluronic acid (G2013) as novel NSAID with immunomodulatory effects has shown its positive effects in various investigations.Objective: Present research aimed to study the potency of G2013 on gene expression of TLR2, TLR4, MyD88, TNF-α and CD52 in PBMCs of MS patients under in vitro conditions. Materials and methods: 24 blood samples from MS patients and healthy controls were considered for RT-PCR and flow cytometry techniques under two different doses of G2013.Results: Our research indicated that this drug could significantly decrease the gene expression of TLR2, TLR4 and TNF-α compared to untreated group. Conclusion: Data demonstrated that the guluronic acid is able to modify the expression levels of TLR2, TLR4 and TNF-α genes to less than the pathogenic boarder line level, which it might be recommended for reducing the pathological process in multiple sclerosis.

Alemtuzumab depletion failure can occur in multiple sclerosis.

Alemtuzumab is a lymphocyte-depleting antibody and one of the most effective treatments for relapsing multiple sclerosis. However, it also causes loss of immune-tolerance leading to secondary autoimmunity and marked anti-drug antibody responses. Although these anti-drug responses have been reported to be of no significance, we hypothesized that they will affect the depleting capacity and treatment response in some individuals. This was found following analysis of the regulatory submission of the pivotal phase III trials, which was obtained from the European Medicines Agency. At the population level there was lack of influence of 'ever-positive' alemtuzumab-specific antibody responses on lymphocyte depletion, clinical efficacy and adverse effects during the 2-year trial. This was not surprising as no one before the first infusion, and only 0·6% of people before the second-infusion, had pre-infusion, neutralizing antibodies (NAbs). However, at the individual level, NAbs led to poor lymphocyte depletion. Importantly, it was evident that 31% of people had NAbs and 75% had binding antibodies at the end of treatment-cycle 2, which suggests that problems may occur in people requiring additional alemtuzumab cycles. In addition, we also identified individuals, following 'post-marketing' alemtuzumab use, whose lymphocyte level was never effectively depleted after the first infusion cycle. Hence, although alemtuzumab depletes lymphocytes in most individuals, some people fail to deplete/deplete poorly, probably due to biological-response variation and NAbs, and this may lead to treatment failure. Monitoring depletion following infusion and assessment of the neutralizing response before re-infusion may help inform the decision to retreat or switch therapy to limit treatment failure.

CD4+ CD52lo T-cell expression contributes to the development of systemic lupus erythematosus.

The cell-surface glycoprotein CD52 is widely expressed in lymphocytes. CD4+CD52hi T cells are functioning suppressor CD4+T cells. We investigated the role of the immune regulation of CD4+CD52 T cells in systemic lupus erythematosus (SLE). CD4+CD52lo T cells were increased in SLE patients, in positive correlation with SLEDAI, anti-ds-DNA antibody, and IgG concentration. Circulating follicular helper-like T cells (Tfh-like cells) were also increased in SLE, in positive correlation with CD4+CD52lo T cells. Chemokine receptor 8 (CCR8) expression in CD4+CD52lo T cells was increased. In vitro experiments using CD4 T cells of SLE patients showed that thymus and activation-regulated chemokine (TARC), a ligand of CCR8, contributed to the development of CD4+CD52hi T cells into CD4+CD52lo T cells. Our findings suggest that CD4+CD52lo T-cell upregulation is involved in the production of pathogens by autoantibodies, and TARC may contribute to the development of SLE through an aberrant induction of CD4+CD52lo T cells.

Anti-CD52 antibody treatment depletes B cell aggregates in the central nervous system in a mouse model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) for which several new treatment options were recently introduced. Among them is the monoclonal anti-CD52 antibody alemtuzumab that depletes mainly B cells and T cells in the immune periphery. Considering the ongoing controversy about the involvement of B cells and in particular the formation of B cell aggregates in the brains of progressive MS patients, an in-depth understanding of the effects of anti-CD52 antibody treatment on the B cell compartment in the CNS itself is desirable. METHODS: We used myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 (B6) mice as B cell-dependent model of MS. Mice were treated intraperitoneally either at the peak of EAE or at 60 days after onset with 200 µg murine anti-CD52 vs. IgG2a isotype control antibody for five consecutive days. Disease was subsequently monitored for 10 days. The antigen-specific B cell/antibody response was measured by ELISPOT and ELISA. Effects on CNS infiltration and B cell aggregation were determined by immunohistochemistry. Neurodegeneration was evaluated by Luxol Fast Blue, SMI-32, and Olig2/APC staining as well as by electron microscopy and phosphorylated heavy neurofilament serum ELISA. RESULTS: Treatment with anti-CD52 antibody attenuated EAE only when administered at the peak of disease. While there was no effect on the production of MP4-specific IgG, the treatment almost completely depleted CNS infiltrates and B cell aggregates even when given as late as 60 days after onset. On the ultrastructural level, we observed significantly less axonal damage in the spinal cord and cerebellum in chronic EAE after anti-CD52 treatment. CONCLUSION: Anti-CD52 treatment abrogated B cell infiltration and disrupted existing B cell aggregates in the CNS.

Anti-mouse CD52 Treatment Ameliorates Colitis through Suppressing Th1/17 Mediated Inflammation and Promoting Tregs Differentiation in IL-10 Deficient Mice.

Recent studies suggested that excessive T helper (Th)1/17 cells concomitant with regulatory T cell deficiency might play important roles in Crohn's disease. Anti-cluster of differentiation 52 (CD52) monoclonal antibody (mAb), which aims on CD52 antigen on mature immunocytes, has both T cell depletion and immunosuppressive activities. In this study, we evaluated the therapeutic effects and possible mechanisms of anti-CD52 treatment on interleukin-10 (IL-10) deficient mouse. Anti-mouse CD52 mAb was administered to C3H/HeJBir.IL-10-/- (C3H.IL-10-/-) mice intraperitoneally 20 µg per week for 2 weeks. The disease activity index, body weight, the histological grading of colitis, and levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-17 and IL-6 in colon were quantified after treatment. In addition, CD25, Forkhead box P3 (Foxp3) and transforming growth factor (TGF)-ß gene as well as the percentage of CD25+Foxp3+ T cells in colon were also measured. The severity of colitis in IL-10-/- mice was significantly decreased by the treatment, with improvement of colon histological grade. The treatment also decreased the TNF-α, IFN-γ, IL-17 and IL-6 levels in colon. Furthermore, the treatment up-regulated the mRNA expression of CD25, Foxp3 and TGF-ß gene as well as the percentage of CD25+Foxp3+ T cells in colon lamina propria mononuclear cells (LPMCs) of IL-10-/- mice. Our data might indicate that anti-CD52 treatment could ameliorate the colitis of C3H.IL-10-/- mice and it might be related to the suppression of Th1/17 related inflammation and the promotion of regulatory T cell differentiation. Thus, our data reveals that anti-CD52 treatment may hold potential for clinical applications for Crohn's disease treatment.

[Anti-mouse CD(52) antibody ameliorates colitis through suppressing Th1/17 mediated inflammation and promoting regulatory T cell response in interleukin-10 deficient mice].

In this study, we evaluated the therapeutic effects and possible mechanisms of anti-CD(5)2 treatment on interleukin-10 (IL-10) deficient mice. Anti-mouse CD(52) monoclonal antibody was administrated to C3H. IL-10(-/-)mice. The disease activity index, histological grading of colitis, serum Th1/17 related cytokines, percentage of CD(25+)Foxp3(+) T cells in colon as well as CD(25), Foxp3 gene expression were measured. Our data suggested that anti-CD(52) treatment inhibited colitis in C3H.IL-10(-/-)mice and it might be related to the suppression of Th1/17 related inflammation and the promotion of regulatory T cell differentiation.

Alemtuzumab improves quality-of-life outcomes compared with subcutaneous interferon beta-1a in patients with active relapsing-remitting multiple sclerosis.

BACKGROUND: Alemtuzumab was superior on clinical and magnetic resonance imaging (MRI) outcomes versus subcutaneous interferon beta-1a in phase 3 trials in patients with relapsing-remitting multiple sclerosis. OBJECTIVE: To examine quality-of-life (QoL) outcomes in the alemtuzumab phase 3 trials. METHODS: Patients who were treatment naive (Comparison of Alemtuzumab and Rebif® Efficacy in Multiple Sclerosis I [CARE-MS I]) or had an inadequate response to prior therapy (CARE-MS II) received annual courses of alemtuzumab 12 mg/day at baseline (5 days) and Month 12 (3 days) or subcutaneous interferon beta-1a 44 µg three times/week. QoL was measured every 6 or 12 months using Functional Assessment of Multiple Sclerosis (FAMS), European Quality of Life-5 Dimensions (EQ-5D) and its visual analog scale (EQ-VAS), and 36-Item Short-Form Survey (SF-36). RESULTS: Statistically significant improvements from baseline with alemtuzumab were observed on all three QoL instruments at the earliest post-baseline assessment and sustained through Year 2. Statistically significant greater QoL improvements over subcutaneous interferon beta-1a were seen at all time points in CARE-MS II with FAMS, EQ-VAS and SF-36 physical component summary, and in CARE-MS I with FAMS. CONCLUSION: Patients treated with alemtuzumab had improvements in physical, mental, and emotional QoL regardless of treatment history. Improvements were significantly greater with alemtuzumab versus subcutaneous interferon beta-1a on both disease-specific and general measures of QoL.

The immunological function of CD52 and its targeting in organ transplantation.

INTRODUCTION: CD52 (Campath-1 antigen), a glycoprotein of 12 amino acids anchored to glycosylphosphatidylinositol, is widely expressed on the cell surface of immune cells, such as mature lymphocytes, natural killer cells (NK), eosinophils, neutrophils, monocytes/macrophages, and dendritic cells (DCs). The anti-CD52 mAb, alemtuzumab, was used widely in clinics for the treatment of patients such as organ transplantation. In the present manuscript, we will briefly summarize the immunological function of CD52 and discuss the application of anti-CD52 mAb in transplantation settings. FINDINGS: We reviewed studies published until July 2016 to explore the role of CD52 in immune cell function and its implication in organ transplantation. We showed that ligation of cell surface CD52 molecules may offer costimulatory signals for T-cell activation and proliferation. However, soluble CD52 molecules will interact with the inhibitory sialic acid-binding immunoglobulin-like lectin 10 (Siglec10) to significantly inhibit T cell proliferation and activation. Although the physiological and pathological significances of CD52 molecules are still poorly understood, the anti-CD52 mAb, alemtuzumab, was used widely for the treatment of patients with chronic lymphocytic leukemia, autoimmune diseases as well as cell and organ transplantation in clinics. CONCLUSION: Studies clearly showed that CD52 can modulate T-cell activation either by its intracellular signal pathways or by the interaction of soluble CD52 and Siglec-10 expressing on T cells. However, the regulatory functions of CD52 on other immune cell subpopulations in organ transplantation require to be studied in the near future.

Ex vivo expansion of natural killer cells from human peripheral blood mononuclear cells co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies.

BACKGROUND AIMS: This study developed a new method to expand CD3(-)CD56(+) natural killer (NK) cells from human peripheral blood mononuclear cells (PBMCs) without feeder cells for clinical trials. METHODS: PBMCs from healthy subjects were co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies and cultured for 14 days in newly developed NKGM-1 medium containing autologous plasma and interleukin-2. Expanded NK cells were examined for cell number, phenotype, in vitro and in vivo cytotoxicity and interferon (IFN)-γ secretion. We also evaluated the proliferative ability of NK cells after cryopreservation. A patient with advanced pancreatic cancer was treated with autologous-expanded NK cells through the use of this method in combination with chemotherapy. RESULTS: Expanded NK cells expressed higher levels of activating molecules compared with resting NK cells and exhibited potent cytotoxicity against K562 cells and IFN-γ secretion by cytokine stimulation. Significant anti-tumor activity was observed in immunodeficient mice injected with the human pancreatic cancer cell line BxPC-3. Large-scale cultures generated a median 5.7 × 10(9) NK cells from 20 mL of peripheral blood (n = 38) after 14 days of culture and 8.4 × 10(9) NK cells after 18 days of culture through the use of a cryopreservation procedure. The number of NK cells and cytotoxic activity in the peripheral blood of the patient with pancreatic cancer greatly increased, and successful clinical responses were observed after multiple infusions of expanded NK cells. CONCLUSIONS: These data demonstrate that this simple and safe methodology for the ex vivo expansion of NK cells can be used for cancer immunotherapy.

Histopathologic spectrum of hypersensitivity reactions associated with anti-CD52 therapy (alemtuzumab).

BACKGROUND: Alemtuzumab is a humanized monoclonal antibody directed against CD52, a cell surface antigen on B and T lymphocytes, and used to treat B-cell chronic lymphocytic leukemia and cutaneous T-cell lymphoma. Skin rash is a common adverse reaction following treatment with alemtuzumab. However, the clinicopathologic features and immunologic basis for the reaction have not been previously reported. METHODS: Our hospital's electronic pathology database was searched for cases with documentation of 'alemtuzumab' or 'anti-CD52' in the clinical history provided by either the ordering physician or the pathologist. Clinical and histopathologic review of the cases was performed. RESULTS: Five patients with cutaneous T-cell lymphoma (CTCL) or chronic lymphocytic leukemia (CLL) were treated with alemtuzumab, and developed pruritic, erythematous papules and plaques. Histopathology of the skin lesions revealed subacute spongiotic dermatitis with multifocal parakeratosis, endothelial activation and perivascular lymphocytic infiltrate. Eosinophils were not a prominent feature. CONCLUSIONS: We describe the clinicopathologic features of a novel hypersensitivity reaction to alemtuzumb, and hypothesize it may be due to an immunologic response precipitated by the persistence of resident memory T-cells (TRM ) in the skin. Our findings raise awareness for a novel reaction pattern and guide the histopathologic interpretation of lesions which may clinically mimic residual or recurrent cutaneous lymphoproliferative disorders.

Anti-mouse CD52 monoclonal antibody ameliorates intestinal epithelial barrier function in interleukin-10 knockout mice with spontaneous chronic colitis.

Intestinal inflammation causes tight junction changes and death of epithelial cells, and plays an important role in the development of Crohn's disease (CD). CD52 monoclonal antibody (CD52 mAb) directly targets the cell surface CD52 and is effective in depleting mature lymphocytes by cytolytic effects in vivo, leading to long-lasting changes in adaptive immunity. The aim of this study was to investigate the therapeutic effect of CD52 mAb on epithelial barrier function in animal models of IBD. Interleukin-10 knockout mice (IL-10(-/-) ) of 16 weeks with established colitis were treated with CD52 mAb once a week for 2 weeks. Severity of colitis, CD4(+) lymphocytes and cytokines in the lamina propria, epithelial expression of tight junction proteins, morphology of tight junctions, tumour necrosis factor-α (TNF-α)/TNF receptor 2 (TNFR2) mRNA expression, myosin light chain kinase (MLCK) expression and activity, as well as epithelial apoptosis in proximal colon were measured at the end of the experiment. CD52 mAb treatment effectively attenuated colitis associated with decreased lamina propria CD4(+) lymphocytes and interferon-γ/IL-17 responses in colonic mucosa in IL-10(-/-) mice. After CD52 mAb treatment, attenuation of colonic permeability, increased epithelial expression and correct localization of tight junction proteins (occludin and zona occludens protein-1), as well as ameliorated tight junction morphology were observed in IL-10(-/-) mice. CD52 mAb treatment also effectively suppressed the epithelial apoptosis, mucosa TNF-α mRNA expression, epithelial expression of long MLCK, TNFR2 and phosphorylation of MLC. Our results indicated that anti-CD52 therapy may inhibit TNF-α/TNFR2-mediated epithelial apoptosis and MLCK-dependent tight junction permeability by depleting activated T cells in the gut mucosa.

Alemtuzumab: a new therapy for active relapsing-remitting multiple sclerosis.

Alemtuzumab is a humanized monoclonal antibody directed against CD52 to deplete circulating T and B lymphocytes; lymphocyte depletion is followed by a distinctive pattern of T- and B-cell repopulation, changing the balance of the immune system. This review reports the efficacy and safety findings of the phase 2 CAMMS223 trial and the phase 3 CARE-MS I and II trials investigating alemtuzumab for the treatment of active relapsing-remitting MS. Alemtuzumab, administered intravenously, was shown to improve relapse rate versus subcutaneous interferon beta-1a in patients who were treatment-naive (CAMMS223 and CARE-MS I) or had relapsed on prior therapy (CARE-MS II), and to reduce sustained accumulation of disability (CAMMS223 and CARE-MS II). Important adverse events were infusion-associated reactions, serious infections and autoimmune events. A safety monitoring program allowed for early detection and management of autoimmune events. Recommendations for the monitoring of adverse events are made. Alemtuzumab's mechanism of action, pharmacodynamics and opportunities for future research are discussed.