Complement-dependent Lysis Research Articles

IGN523: A Therapeutic Anti-CD98 Antibody With Multiple Mechanisms Of Action Demonstrates Anti-Tumor Efficacy

Abstract Background Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults resulting in >10,000 deaths per year in the US. Limited treatment options and poor clinical response to current standard of care (SOC) necessitate development of more efficacious therapeutics. CD98 is a heterodimeric cell surface protein that is implicated in the pathogenesis of many hematological and solid malignancies and its elevated expression is correlated with poor prognosis and patient outcome. Previous studies demonstrated that CD98 functions both in integrin signaling and amino acid transport processes, which support the proliferation, anchorage-independence, invasion, and metastasis of tumor cells. Here we demonstrate that IGN523, a humanized monoclonal antibody targeting CD98, possesses multiple mechanisms of action (MOAs) and elicits potent anti-tumor activity against a variety of leukemic xenograft models. Methods CD98 expression was assessed on CD34+/CD33- progenitors and more mature CD34+/CD33+ precursors from AML patient bone marrow samples by flow cytometry. Anti-tumor efficacy of IGN523 treatment was evaluated across 11 lymphoma or leukemic xenograft models, including 5 AML, established in NOD/SCID, CB17, or NSG mice. Animals were treated with either IGN523 alone or in combination with SOC anti-tumor agents and dose-responses were performed in selected models. IGN523’s ability to elicit NK-cell mediated antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent tumor cell lysis was determined. Additional in vitro MOA studies investigated IGN523’s effect upon lysosomal and mitochondrial membrane permeabilization, amino acid transport, tumor cell proliferation and apoptosis. Results Flow cytometry data demonstrate that CD98 is differentially expressed on CD34+/CD33- and CD34+/CD33+ AML bone marrow cells compared to healthy donors. In AML patients, CD98 expression was significantly increased on the CD34+/CD33+ cells versus the CD34+/CD33- population. Leukemic tumors were established in mice (100-200mm3 starting volumes) prior to weekly treatment with IGN523 dosed at 15 or 30 mg/kg. IGN523 elicited robust and significant tumor growth inhibition (TGI; p<0.01) in the majority of models including Ramos (87% TGI), HL-60 (70% TGI), KG-1 (53% TGI) and OCI-AML-3 (74% TGI). A well-defined IGN523 dose response (0.1 – 30 mg/kg) was also observed. IGN523 was at least as efficacious as SOC anti-tumor agents (e.g. cytarabine). In vitro data established that IGN523 elicits strong ADCC activity, induces lysosomal membrane permeabilization, and inhibits essential amino acid transport function. Moreover, IGN523 impedes cell proliferation/survival signaling cascades through reduction of PRAS40 and p70s6K phosphorylation, ultimately resulting in caspase-mediated apoptosis. Conclusion The combination of increased CD98 expression on AML cells, pre-clinical efficacy of IGN523 in xenograft models, and identification of diverse MOAs provide strong scientific rationale for therapeutic use in patients. Clinical trials are therefore warranted to evaluate the efficacy and safety of IGN523 in AML. Disclosures: Hayes: Igenica, Inc.: Employment. Chinn:Igenica, Inc.: Employment. Cantor:Igenica, Inc.: Consultancy. Velilla:Igenica, Inc.: Employment. Ginsberg:Igenica, Inc.: Consultancy. van der Horst:Igenica, Inc.: Employment.

T-Cell Engineering For “off-The-shelf” Adoptive Immunotherapy

Adoptive T-cell therapies, where exogenous expression of a chimeric antigen receptor (CAR) confers cancer recognition, have shown significant promise in initial clinical trials. However, present adoptive immunotherapy Methods are limited by the need for manipulation of autologous patient T-cells. To permit such an approach in an allogeneic context, Transcription Activator-Like Effector Nucleases (TALENTM) have been used to simultaneously inactivate the endogenous T cell receptor and CD52, a cellular target for a lymphodepleting treatment. This approach reduces the risk of GVHD while permitting proliferation and activity of the introduced T lymphocytes in the presence of the immunosuppressive drug alemtuzumab. Electroporation of primary T cells with mRNA coding for the appropriate TALENTM result in double knock-out (dKO) frequencies of up to 70%. Furthermore, functional characterization demonstrates that the dKO cells are resistant to complement dependent lysis or in vivo depletion by alemtuzumab, and show no apparent potential for TCR-mediated activation. Finally, endowing the dKO cells with a CD19 CAR supports their capacity to kill CD19+ tumor targets as efficiently as unedited T-cells both in vitro and in vivo. Disclosures:Poirot:CELLECTIS THERAPEUTICS: Employment. Schiffer-Mannioui:CELLECTIS THERAPEUTICS: Employment. Philip:UCL Cancer Institute, London, United Kingdom: Employment. Derniame:CELLECTIS THERAPEUTICS: Employment. Gouble:CELLECTIS THERAPEUTICS: Employment. Chion-Sotinel:CELLECTIS THERAPEUTICS: Employment. Le Clerre:CELLECTIS THERAPEUTICS: Employment. Lemaire:CELLECTIS THERAPEUTICS: Employment. Grosse:CELLECTIS THERAPEUTICS: Employment. Cheung:UCL Cancer Institute, London, United Kingdom: Employment. Arnould:CELLECTIS THERAPEUTICS: Employment. Smith:CELLECTIS THERAPEUTICS: Employment. Pule:UCL Cancer Institute, London, United Kingdom: Employment. Scharenberg:CELLECTIS THERAPEUTICS: Employment.

Myeloid cells as effector cells for monoclonal antibody therapy of cancer

Monoclonal antibodies (mAbs) have become an important addition to chemo- and/or radiotherapy for the treatment of cancer. They have multiple effector functions that can lead to eradication of tumor, including induction of apoptosis, growth inhibition, and initiation of complement-dependent lysis. Furthermore, mAbs can recruit immune effector cells. Traditionally, natural killer cells have been considered as the main effector cell population in mAb-mediated tumor killing. Myeloid cells have potent cytotoxic ability, as well. Monocytes and macrophages have been shown to induce antibody-dependent cytotoxicity and phagocytosis of tumor cells in the presence of IgG anti-tumor mAb. Furthermore, neutrophils are the most abundant population of circulating white blood cells, and as such may constitute a formidable source of effector cells. However, when targeting neutrophils for tumor therapy, antibodies of the IgA subclass may be more effective. This article focuses on enlisting myeloid effector cells for mAb-based immunotherapy of cancer. Additionally, methods to study mAb-dependent phagocytosis of tumor cells by macrophages are compared.

Oncolytic and Immunotherapeutic Vaccinia Induces Antibody-Mediated Complement-Dependent Cancer Cell Lysis in Humans

Oncolytic viruses cause direct cytolysis and cancer-specific immunity in preclinical models. The goal of this study was to demonstrate induction of functional anticancer immunity that can lyse target cancer cells in humans. Pexa-Vec (pexastimogene devacirepvec; JX-594) is a targeted oncolytic and immunotherapeutic vaccinia virus engineered to express human granulocyte-macrophage colony-stimulating factor (GM-CSF). Pexa-Vec demonstrated replication, GM-CSF expression, and tumor responses in previous phase 1 trials. We now evaluated whether Pexa-Vec induced functional anticancer immunity both in the rabbit VX2 tumor model and in patients with diverse solid tumor types in phase 1. Antibody-mediated complement-dependent cancer cell cytotoxicity (CDC) was induced by intravenous Pexa-Vec in rabbits; transfer of serum from Pexa-Vec-treated animals to tumor-bearing animals resulted in tumor necrosis and improved survival. In patients with diverse tumor types treated on a phase 1 trial, CDC developed within 4 to 8 weeks in most patients; normal cells were resistant to the cytotoxic effects. T lymphocyte activation in patients was evidenced by antibody class switching. We determined that patients with the longest survival duration had the highest CDC activity, and identified candidate target tumor cell antigens. Thus, we demonstrated that Pexa-Vec induced polyclonal antibody-mediated CDC against multiple tumor antigens both in rabbits and in patients with diverse solid tumor types.

A bit of antithymocyte globulin can take you a long way!

The success of allogeneic hematopoietic SCT (allo-SCT) owes much to the improvements in immunosuppressive regimens that prevent GVHD. For the past 25 years, the mainstay of immunosuppression has been the calcineurin antagonists CSA and tacrolimus. However, their immunosuppressive effects can be unpredictable. The so-called ‘T-cell-depleting’ antibodies such as antilymphocyte or antithymocyte globulins (ATGs) have been used for decades. The general common belief is that ATG efficacy relies on its potent capacity to deplete T lymphocytes of the graft. However, an ever-growing body of more recent data suggested that ATG affects not only the T lymphocytes, but also other key cells involved in the immune reaction. Indeed, the immunosuppressive activity of ATG has been thought to result primarily from the depletion of peripheral T lymphocytes from the circulating pool through complement-dependent lysis or activation-associated apoptosis. Other potential mechanisms of action include the modulation of surface adhesion molecules or chemokine receptor expression. In addition to their T-cell-depleting properties, ATGs can modulate the immune response by affecting or interfering with the function of different immune effectors such as B lymphocytes, regulatory T lymphocytes, natural killer-T lymphocytes and DCs.1

Abstract 331: Epithelial-mesenchymal transition confers resistance to complement-dependent cytotoxicity in lung cancer cells

Abstract Recent studies have implicated Epithelial-mesenchymal transition (EMT) in conferring several clinically relevant properties to cancer cells, in addition to initiating tumor metastasis. These include stem-cell like properties, resistance to targeted therapies and ability to evade immune surveillance. Pathway analysis of gene expression changes in TGF-β induced EMT identified the complement cascade as one of the significantly enriched pathway. Further analysis of the genes in the complement pathway revealed EMT-induced expression of complement inhibitors and decrease in expression of proteins essential for complement activity, suggesting resistance to complement dependent cytotoxicity (CDC) during EMT. We found lung cancer cells that undergo EMT showed marked decrease in C3 deposition and concomitant resistance to apoptosis by CDC as measured by 7-Aminoactinomycin D staining. Flow cytometric and RT-PCR analysis showed a significant increase in the CD59 expression on the surface of cells undergoing EMT, which is a potent inhibitor of formation of membrane attack complex that mediates complement dependent cell lysis. Furthermore, CD59 inhibition by siRNA mediated knock-down overcame the EMT-induced resistance to anti-EGFR antibody (Cetuximab) mediated CDC. The increase in CD59 was abrogated on disruption of TGF-β signaling as revealed by TGF- β Receptor kinase inhibitor and is dependent on Smad3 signaling. Chromatin immunoprecipitation analysis revealed increased occupancy of Smad3 on CD59 promoter, suggesting a direct regulation by Smad3. These results demonstrate that TGF-β-induced EMT confers another novel immune evasive mechanism to tumor cells that facilitates tumor progression and CD59 as a potential therapeutic target for enhancing the efficacy of anti-EGFR Antibody therapies and against tumor metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 331. doi:1538-7445.AM2012-331

Constitutive Production of Catalytic Antibodies to a Staphylococcus aureus Virulence Factor and Effect of Infection

Antibodies that recognize microbial B lymphocyte superantigenic epitopes are produced constitutively with no requirement for adaptive immune maturation. We report cleavage of the Staphylococcus aureus virulence factor extracellular fibrinogen-binding protein (Efb) by catalytic antibodies produced with no exposure to the bacterium and reduction of the catalytic antibody activity following infection. IgG catalytic antibodies that specifically hydrolyzed Efb via a nucleophilic catalytic mechanism were found in the blood of healthy humans and aseptic mice free of S. aureus infection. IgG hydrolyzed peptide bonds on the C-terminal side of basic amino acids, including a bond located within the C3b-binding domain of Efb. Efb digested with the IgG lost its ability to bind C3b and inhibit complement-dependent antibody-mediated red blood cell lysis. In addition to catalysis, the IgG expressed saturable Efb binding activity. IgG from S. aureus-infected mice displayed reduced Efb cleaving activity and increased Efb binding activity compared with uninfected controls, suggesting differing effects of the infection on the antibody subsets responsible for the two activities. IgG from children hospitalized for S. aureus infection also displayed reduced Efb cleavage compared with healthy children. These data suggest a potential defense function for constitutively produced catalytic antibodies to a putative superantigenic site of Efb, but an adaptive catalytic response appears to be proscribed.

Complement-Dependent Lysis of Influenza A Virus-Infected Cells by Broadly Cross-Reactive Human Monoclonal Antibodies

Complement-Dependent Lysis of Influenza A Virus-Infected Cells by Broadly Cross-Reactive Human Monoclonal Antibodies

In Vivo T Cell Depletion Using Rabbit Derived ATG Leads to An Increased EBV-PTLD Risk Due to An Induced Imbalance Between B and T Cell Recovery Which Is Not Seen After Horse Derived ATG or Alemtuzumab

Abstract 1979Control of replication of endogenous viruses like CMV and EBV is fully dependent on CMV or EBV specific T cells after allogeneic stem cell transplantation (alloSCT). In the absence of specific CD8 T cell control, proliferation of EBV infected B cells can lead to post transplantation lymphoproliferative disease (PTLD). In an initial cohort of patients treated with horse derived anti thymocyte globulin (h-ATG), no early PTLD was observed. However, due to unavailability in Europe, h-ATG had to be replaced by rabbit derived ATG (r-ATG), leading to an unacceptable high incidence of EBV-PTLD (26% during first 3 months after alloSCT). Replacement of r-ATG by alemtuzumab (ALT) significantly reduced the incidence of EBV-PTLD (3 months incidence of EBV-PTLD 2%). To determine the immunological basis of these findings we performed a detailed analysis of immune reconstitution in these three cohorts of transplanted patients. The first cohort (41 patients) received h-ATG (Lymphoglobulin) 10 mg/kg/day for 4 days. The second cohort (19 patients) received r-ATG (Thymoglobulin) 2.0 or 3.5 mg/kg/day for 4 days and the third cohort (60 patients) received ALT, 15 mg/day for 2 days. All grafts consisted of PBSC to which 20 mg of ALT was added for in vitro T cell depletion. All patients received a fludarabin and busulphan based conditioning regimen. No standard post transplantation immunosuppressive treatment was given.In the r-ATG cohort, early EBV-PTLD occurred after a median of 7 weeks (range 4–12 weeks) post alloSCT. Three r-ATG treated patients died while high levels of circulating EBV-DNA were present (> log 4.0 copies/mL). Incidence of CMV disease was not significantly different in the three cohorts (5%, 6% and 0%, respectively). In contrast to the other 2 cohorts, immune reconstitution in the r-ATG cohort was characterized by an imbalance between recovery of B cells and CD8 T cells. Already 3 weeks after alloSCT, the majority (67%) of r-ATG patients showed a more rapid reconstitution of B cells than CD8 T cells, leading to B cells outnumbering CD8 T cells. This was seen in only a small minority of patients after h-ATG and ALT (17% and 6%, respectively, p<0.01 versus r-ATG).Because rapid recovery of T cells in the alemtuzumab patients was frequently found in the presence of circulating ALT (mean concentration 0.43 μg/mL and 0.12 μg/mL after 3 and 6 weeks, respectively), the phenotype of circulating CD4 and CD8 T cells at 6 weeks after ALT was analyzed. The majority of circulating CD8 and CD4 T cells lacked CD52 expression (56% (range 0–99%) and 81% (range 0–93%), respectively). Using tetramer staining, cytotoxicity assays and analysis of cytokine production, we demonstrated the presence of functional CD52 negative as well as CD52 positive CMV and EBV specific CD8 T cells. Based on FLAER negativity, it was demonstrated that the CD52 negative T cells are GPI anchor deficient, representing a PNH-like clone escaping ALT induced cell lysis. Because almost half of the circulating CD8 T cells were CD52 positive, we examined expression of CD52 and the in-vitro sensitivity to ALT-mediated complement-dependent cell lysis (CDC) of B cells, CD4 and CD8 T cells of healthy donors. The highest CD52 expression was observed on B cells (mean fluorescence intensity (MFI) 120), resulting in 95% lysis after incubation with ALT and complement. Differential expression of CD52 was observed on CD4 and CD8 T cells, MFI 120 and 101 respectively, resulting in relative protection of CD52 positive CD8 compared to CD4 T cells against ALT-mediated CDC (52% and 90% lysis).We conclude that the high incidence of EBV-PTLD after in-vivo T cell depletion with r-ATG is caused by an induced imbalance between B and T cell recovery, which is not seen after h-ATG or ALT. In-vivo T cell depletion with ALT is associated with a relatively low risk of EBV disease because of efficient B cell depletion and persistent EBV immunity due to the relative insusceptibility for ALT of CD8 T cells and the development of functional CD52 negative escape variants of CD4 and CD8 T cells. Disclosures:Off Label Use: Alemtuzumab and Anti Thymocyte Globulin used for in vivo T cell depletion prior to allogeneic stem cell transplantation.

Complement-Dependent Lysis of Influenza A Virus-Infected Cells by Broadly Cross-Reactive Human Monoclonal Antibodies

We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics.

A novel anti-GD2 monoclonal antibody (mAb), hu14.18K322A, in children with refractory or recurrent neuroblastoma: Early-phase evaluation.

9523 Background: Immunotherapy, including combination anti-GD2 mAb ch14.18 and cytokines, improves the outcome of patients with advanced neuroblastoma. Dose escalation of antibodies targeting GD2 antigen on neuroblastoma cells has been limited by toxicities which may be partially mediated by complement activation. Hu14.18K332A is a humanized anti-GD2 mAb with a single point mutation (K322A) reducing complement-dependent lysis. We report the preliminary results of a phase I trial to determine the maximum tolerated dose (MTD) and toxicity profile of hu14.18K322A. Methods: Cohorts of 3-6 eligible patients with refractory or recurrent neuroblastoma received a 4-hour daily IV infusion of escalating doses of hu14.18K322A (2, 4, 6, 10, 20, 40, 50, and 60 mg/m2/dose) for 4 consecutive days every 28 days (1 course). Patients were allowed to receive up to 4 courses in the absence of progressive disease or unacceptable toxicity. Results: A total of 32 patients (22 males, median age 6.5 years) received a total of 71 courses. Patients received a median of 2 courses. Dose-limiting grade 3 toxicities occurred in 2 of 29 evaluable patients and were characterized by cough and asthenia. The most common grade 3 or 4 toxicities were pain (65% of patients), fever (19%) and hyponatremia (13%). Pain was well managed and predominately occurred on the first day of course 1. Grade 1 or 2 ocular abnormalities were noted in 13 patients and appear to be dose related. No objective responses by RECIST have been observed; however, 9 patients completed the maximum of 4 courses allowed by protocol. Conclusions: Hu14.18K322A is well tolerated at the doses tested which exceed the dosages of similar GD2 mAbs used in other trials. MTD has not been defined and the study continues to accrue patients at higher doses.

Enhancing the action of rituximab by adding fresh frozen plasma for the treatment of fludarabine refractory chronic lymphocytic leukemia

Complement deficiencies have been identified in many chronic lymphocytic leukemia (CLL) patients. Supplying fresh frozen plasma (FFP)-derived complement can enhance complement-dependent cell lysis by the rituximab. The objective of our study was to evaluate the clinical efficacy and safety of the treatment by adding FFP to rituximab in fludarabine refractory CLL patients. Twenty-two patients were treated with two units of FFP followed with rituximab, 375 mg/m(2), as a single agent, repeated every 1-2 weeks. Patients received a median of four courses of the combined FFP and rituximab treatment (range: 2-6). Sixteen patients (72.7%) responded to treatment and seven (31.8%) achieved a complete remission. Three (13.6%) of which had no evidence of minimal residual disease after treatment. Patients with high expression of ZAP-70 or CD38, unmutated immunoglobulin heavy chain variable region, mutated p53, or adverse cytogenetic features, achieved response to treatment at rates that appeared similar to those who did not have such characteristics. With a median follow-up of 12 (4-19) months, the median overall survival and progression free survival have not been achieved. Toxicity was minimal, and the treatment was well tolerated. Our data suggest that the adding FFP to rituximab is an effective nonmyelotoxic regimen for the treatment of fludarabine refractory CLL patients.

Ofatumumab for newly diagnosed and relapsed/refractory chronic lymphocytic leukemia

Monoclonal antibodies have become an increasingly utilized treatment option for many hematological malignancies, including chronic lymphocytic leukemia (CLL). Ofatumumab is a second-generation fully human anti-CD20 monoclonal antibody that binds to the small extracellular loop of CD20, thereby producing complement-dependent cell lysis and antibody-mediated cell cytotoxicity in cells expressing CD20. Ofatumumab has shown efficacy in the treatment for relapsed or refractory CLL. This success has resulted in the recent US FDA approval of ofatumumab for the treatment of CLL in patients who are refractory to fludarabine and alemtuzumab-based regimens. Major side effects of ofatumumab include infusion reactions, neutropenia and increased risk for infection. This article provides an overview of the current data supporting the use of ofatumumab for CLL and projects the future role of ofatumumab as monotherapy and combination therapy.

Low Incidence of Post-Transplant EBV-Related Disease After Alemtuzumab-Mediated T Cell Depletion Is Explained by the Differential Susceptibility to Alemtuzumab of B Cells and Protective CD8 and CD4 T Cells

AbstractAbstract 2330In vivo T cell depletion with anti-thymocyte globulin (ATG) or alemtuzumab (anti-CD52) before reduced intensity allogeneic stem cell transplantation (alloSCT) in combination with in vitro T cell depletion with alemtuzumab reduces the risk of GVHD. Detectable levels of circulating antibodies are present up to several months after the alloSCT, leading to a delayed immune reconstitution which is associated with an increased incidence of opportunistic infections and early relapses. Prior to 2007, combined in vitro (Alemtuzumab 20 mg added “to the bag”) and in vivo T cell depletion with horse-derived ATG (h-ATG) resulted in good engraftment without GVHD in the absence of GVHD prophylaxis after reduced intensity alloSCT using conditioning with fludarabine and busulphan. Due to the unavailability of h-ATG, rabbit-derived ATG (r-ATG) 10–14 mg/kg was introduced in the conditioning regimen in 2007. Strikingly, in this cohort of patients, early EBV reactivation and EBV-associated post-transplantation lymphoproliferative disease (PTLD) was observed in 10 out of 18 patients at a median time of 6 weeks after alloSCT (range 5 to 11 weeks) in the absence of GVHD or immunosuppressive treatment. Analysis of T and B cell recovery early after transplantation revealed preferential depletion of T cells as compared to B cells, thereby allowing unrestricted proliferation of EBV infected B cells. Due to this unacceptable high incidence of EBV-related complications, in the conditioning regimen r-ATG was replaced by low dose alemtuzumab (15 mg i.v. day -4 and -3) in 2008. In this cohort of 60 patients, only 2 patients experienced transient EBV reactivation during the first 3 months after alloSCT and one patient developed an EBV-associated lymphoma 4 weeks after alloSCT. To investigate the mechanisms underlying the low incidence of EBV reactivation using alemtuzumab for T cell depletion, we studied the in vivo and in vitro effects of alemtuzumab on different lymphocyte subsets. First, lineage-specific reconstitution was studied in 20 patients from the alemtuzumab cohort with known CD52 negative diseases (11 AML and 9 multiple myeloma) to exclude the confounding effect of antibody absorption by malignant cells. Whereas at 3 weeks after alloSCT detectable numbers of circulating NK cells and T cells were observed (medians 71 (range 6–378), and 12 (range 1–1164)E6/L, respectively), no circulating B cells could be detected (median 0, range 0–1 E6/L). At 6 weeks after alloSCT, NK and T cell numbers further increased (medians 212 (52-813), and 130 (range 25–1509)E6/L, respectively), whereas B cell numbers still remained low in the majority of patients (median 15, range 0–813E6/L). In all patients, T cells were detectable before the appearance of circulating B cells. Furthermore, the expression of CD52 and the sensitivity to alemtuzumab-mediated complement-dependent cell lysis (CDC) of B cells, T cells and NK cells was measured in vitro. The highest CD52 expression was observed on B cells (mean fluorescence intensity (MFI) 120), resulting in 95% lysis after incubation with 10ug/mL alemtuzumab and rabbit complement. NK cells showed a significantly lower CD52 expression (MFI 41), which was also reflected by a lower susceptibility to alemtuzumab-mediated CDC (62% lysis). Interestingly, differential expression of CD52 was observed on CD4 and CD8 T cells (MFI 120 and 101, respectively). Cytotoxicity analysis revealed relative protection of CD8 compared to CD4 T cells against alemtuzumab-mediated CDC, resulting in 52% and 90% lysis, respectively. Based on these results, we investigated in detail the presence and phenotype of the CD4 and CD8 subsets and EBV-specific CD8 T cells using tetramer staining at 6 weeks after alloSCT. In accordance with the in-vitro expression and susceptibility data, circulating CD52+ CD8 T cells including EBV-specific T cells were detectable. Interestingly, the majority of circulating CD4 T cells (64-93%, n=4) lacked CD52 expression, explaining their capacity to persist in the presence of alemtuzumab. We conclude that in vivo and in vitro T cell depletion with alemtuzumab is associated with a relatively low risk of EBV-associated PTLD because of efficient B cell depletion and persistent EBV immunity allowed by the relative insusceptibility for alemtuzumab of CD8 T cells and the development of CD52 negative escape variants of CD4 T cells. Disclosures:No relevant conflicts of interest to declare.

Complement-dependent lysis of mAb-opsonized B cells can increase resistance of naïve B cells to subsequent attack by mAb and complement

Complement-dependent lysis of mAb-opsonized B cells can increase resistance of naïve B cells to subsequent attack by mAb and complement

Anti-GD 2 with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia

Monoclonal antibodies against GD 2 ganglioside, such as ch14.18, the human–mouse chimeric antibody, have been shown to be effective for the treatment of neuroblastoma. However, treatment is associated with generalized, relatively opiate-resistant pain. We investigated if a point mutation in ch14.18 antibody (hu14.18K332A) to limit complement-dependent cytotoxicity (CDC) would ameliorate the pain behavior, while preserving antibody-dependent cellular cytotoxicity (ADCC). In vitro, CDC and ADCC were measured using europium-TDA assay. In vivo, allodynia was evaluated by measuring thresholds to von Frey filaments applied to the hindpaws after injection of either ch14.18 or hu14.18K332 into wild type rats or rats with deficient complement factor 6. Other rats were pretreated with complement factor C5a receptor antagonist and tested following ch14.18 injection. The mutation reduces the antibody’s ability to activate complement, while maintaining its ADCC capabilities. Injection of hu14.18K322 (1 or 3 mg/kg) produced faster resolving allodynia than that engendered by ch14.18 (1 mg/kg). Injection of ch14.18 (1 mg/kg) into rats with C6 complement deficiency further reduced antibody-induced allodynia, while pre-treatment with complement factor C5a receptor antagonist completely abolished ch14.18-induced allodynia. These findings showed that mutant hu14.18 K322 elicited less allodynia than ch14.18 and that ch14.18-elicited allodynia is due to activation of the complement cascade: in part, to formation of membrane attack complex, but more importantly to release of complement factor C5a. Development of immunotherapeutic agents with decreased complement-dependent lysis while maintaining cellular cytotoxicity may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of therapeutic antibodies.

Ofatumumab, a human anti-CD20 monoclonal antibody

Importance of the field: Since relapse of chronic lymphocytic leukemia (CLL), and refractoriness to treatment following relapse, is inevitable after initial treatment, development of novel treatment strategies is necessary.Areas covered in this review: CD20 as a therapeutic target for CLL, successes and limitations of current anti-CD20 monoclonal antibody (mAb) therapy, and implications of preclinical and clinical developments of novel alternatives, including ofatumumab, that may enhance anti-CD20 mAb therapy are reviewed. The literature reviewed encompasses papers and congress abstracts from the past 13 years.What the reader will gain: While rituximab combined with chemotherapy has considerably improved outcomes for some but not all CLL patients, single-agent use is limited in relapsed/refractory CLL. Novel anti-CD20 mAbs in development, such as ofatumumab, may bypass some limitations by virtue of alternative epitope binding and modified effector functions. Ofatumumab induces significant complement-dependent cell lysis in vitro, particularly in cells with low CD20 expression. The FDA recently approved ofatumumab for treatment of CLL refractory to fludarabine and alemtuzumab.Take home message: CD20-targetting chemoimmunotherapeutic options have advanced treatment of CLL. Results for single-agent ofatumumab and other new CD20 mAbs, in different lines of therapy and in combination with chemotherapy, will guide optimal use of these alternative therapies for B-cell malignancies.

Hepatitis B virus sensitizes hepatocytes to complement-dependent cytotoxicity through downregulating CD59

Hepatitis B virus (HBV) infection afflicts over 350 million people worldwide and is a leading cause of hepatitis, cirrhosis and hepatocellular carcinoma. HBV replicates noncytopathically in hepatocytes, and most of the hepatic injury is caused by the immune response to the virus. While most studies focused on the adaptive immune response, the role of the innate immune response, especially the complement activation, in HBV infection remains obscure. To identify proteins that are involved in the pathogenesis of HBV infection, we carried out gene microarray analysis to compare the gene expression profile of HBV transgenic BALB/c mice with that of control mice. CD59 mRNA, which encodes an important complement regulatory protein (CRP) expressed on cell surface, was found to be significantly downregulated in HBV transgenic liver, a result that was further confirmed by RT-PCR and real-time PCR. To explore the relationship between CD59 and HBV infection, we examined the effect of HBV on CD59 expression and complement-dependent cytolysis in two hepatocyte cell lines. We found that HBV could significantly downregulate CD59 expression and sensitize cells to complement-dependent lysis. Blocking CD59 function using a CD59-specific antibody greatly diminished the HBV effect. Similar CD59 downregulation was also observed in the livers of patients with chronic HBV infection. These results demonstrate that HBV can sensitize hepatocytes to complement-dependent cytotoxicity (CDC) through downregulating CD59, which may lead to the activation of complement system and cause liver inflammation.

Experimental verification of the model of complement-dependent immune lysis of liposomes

The quantitative dependences of the immune complement-dependent lysis of a monodisperse suspension of 200-nm liposomes whose membrane was sensitized by monovalent hapten (2,4-DNP-epsilon-caproyl-DPPE) or a polyvalent antigen (LPS from F. tularensis) on the initial concentration of specific antibodies (IgG) to hapten and the antigen have been investigated. The quantity of antibodies binding sites on the surface of liposome was evaluated. The difference between the complement-dependent immune lysis of poly- and monodisperse suspensions of liposomes was shown. The experimental results are well described by the direct binding model.

Clinical improvement with a novel CD20 mAb, ofatumumab, in fludarabine-refractory chronic lymphocytic leukemia (CLL) also refractory to alemtuzumab or with bulky lymphadenopathy

7043 Background: Patients (pts) with CLL refractory tofludarabine and alemtuzumab (double-refractory, DR) or refractory to fludarabine with bulky (&gt;5 cm) lymphadenopathy (bulky fludarabine-refractory, BFR) have a poor prognosis. Ofatumumab is a human mAb specific for a distinctive small-loop epitope of CD20 that appears more potent than rituximab in eliciting complement-dependent lysis of B cells in vitro. We report, for the first time, results from the planned interim analysis of the clinical benefit observed in pts with DR or BFR CLL treated with ofatumumab in an international pivotal clinical study. Methods: Pts with DR or BFR CLL received 8 weekly then 4 monthly ofatumumab infusions (Dose 1, 300 mg; Doses 2–12, 2,000 mg). Primary endpoint was overall response rate (ORR; 1996 NCI-WG criteria), as assessed by an Independent Review Committee, over 24 wks. Results: Of 138 treated pts (DR: N = 59; BFR: N = 79; median age 64 and 62 yrs, respectively), 63% had Rai stage III/IV disease at screening. Pts had a median of 5 prior therapies. ORR (99% CI) was 58% (40, 74%) in the DR and 47% (32, 62%) in the BFR groups, and median overall survival (95% CI) was 13.7 mo (9.4, NR) and 15.4 mo (10.2, 20.2), respectively. Resolution of disease symptoms (maintained for ≥2 mo) were observed in a large proportion of pts (Table), including in pts considered nonresponders by NCI-WG criteria. Improvements in hematologic values were also observed in some pts with abnormal baseline values, particularly for platelet counts. Pts with thrombocytopenia at baseline (n = 73) experienced sustained increases in median platelet counts from 65 × 109/L to over 100 × 109/L by Wk 8; a similar pattern of rapid improvement was observed in Hgb values. Conclusions: Ofatumumab as single-agent achieves high ORR, and improves disease symptoms and hematologic parameters in heavily pretreated pts with DR and BFR disease who lack standard treatment options. [Table: see text] [Table: see text]