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Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein, Ad35K++, which is capable of transiently removing CD46 from the cell surface. We also produced a peptide inhibitor of CD59 (rILYd4). In this study, we tested Ad35K++ and rILYd4 in combination with daratumumab and isatuximab in MM cells as well as in cells from two other B-cell malignancies. We showed that Ad35K++ and rILYd4 increased CDC triggered by daratumumab and isatuximab. The combination of both inhibitors had an additive effect in vitro in primary MM cells as well as in vivo in a mouse xenograft model of MM. Daratumumab and isatuximab treatment of MM lines (without Ad35K++ or rILYd4) resulted in the upregulation of CD46/CD59 and/or survival of CD46high/CD59high MM cells that escaped the second round of daratumumab and isatuximab treatment. The escape in the second treatment cycle was prevented by the pretreatment of cells with Ad35K++. Overall, our data demonstrate that Ad35K++ and rILYd4 are efficient co-therapeutics of daratumumab and isatuximab, specifically in multi-cycle treatment regimens, and could be used to improve treatment of multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein, Ad35K++, which is capable of transiently removing CD46 from the cell surface. We also produced a peptide inhibitor of CD59 (rILYd4). In this study, we tested Ad35K++ and rILYd4 in combination with daratumumab and isatuximab in MM cells as well as in cells from two other B-cell malignancies. We showed that Ad35K++ and rILYd4 increased CDC triggered by daratumumab and isatuximab. The combination of both inhibitors had an additive effect in vitro in primary MM cells as well as in vivo in a mouse xenograft model of MM. Daratumumab and isatuximab treatment of MM lines (without Ad35K++ or rILYd4) resulted in the upregulation of CD46/CD59 and/or survival of CD46high/CD59high MM cells that escaped the second round of daratumumab and isatuximab treatment. The escape in the second treatment cycle was prevented by the pretreatment of cells with Ad35K++. Overall, our data demonstrate that Ad35K++ and rILYd4 are efficient co-therapeutics of daratumumab and isatuximab, specifically in multi-cycle treatment regimens, and could be used to improve treatment of multiple myeloma. [ABSTRACT FROM AUTHOR]

ABSTRACTThe highly pathogenic avian influenza A (H5N6) virus has caused sporadic human infections with a high case fatality rate. Due to the continuous evolution of this virus subtype and its ability to transmit to humans, there is an urgent need to develop effective antiviral therapeutics. In this study, a murine monoclonal antibody 9F4 was shown to display broad binding affinity against H5Nx viruses. Furthermore, 9F4 can neutralize H5N6 pseudotyped particles and prevent entry into host cells. Additionally, ADCC/ADCP deficient L234A, L235A (LALA) and CDC deficient K322A mutants were generated and displayed comparable binding affinity and neutralizing activity as wild type 9F4 (9F4-WT). Notably, 9F4-WT, 9F4-LALA and 9F4-K322A exhibit in vivo protective efficacies against H5N6 infections in that they were able to reduce viral loads in mice. However, only 9F4-WT and 9F4-K322A but not 9F4-LALA were able to reduce viral pathogenesis in H5N6 challenged mice. Furthermore, depletion of phagocytic cells in mice lungs nullifies 9F4-WT's protection against H5N6 infections, suggesting a crucial role of the host's immune cells in 9F4 antiviral activity. Collectively, these findings reveal the importance of ADCC/ADCP function for 9F4-WT protection against HPAIV H5N6 and demonstrate the potential of 9F4 to confer protection against the reassortant H5-subtype HPAIVs.

Rhesus macaques are a common non-human primate model used in the evaluation of human monoclonal antibodies, molecules whose effector functions depend on a conserved N-linked glycan in the Fc region. This carbohydrate is a target of glycoengineering efforts aimed at altering antibody effector function by modulating the affinity of Fcγ receptors. For example, a reduction in the overall core fucose content is one such strategy that can increase antibody-mediated cellular cytotoxicity by increasing Fc-FcγRIIIa affinity. While the position of the Fc glycan is conserved in macaques, differences in the frequency of glycoforms and the use of an alternate monosaccharide in sialylated glycan species add a degree of uncertainty to the testing of glycoengineered human antibodies in rhesus macaques. Using a panel of 16 human IgG1 glycovariants, we measured the affinities of macaque FcγRs for differing glycoforms via surface plasmon resonance. Our results suggest that macaques are a tractable species in which to test the effects of antibody glycoengineering. [ABSTRACT FROM AUTHOR]

• Generation of bi-specific nanobodies with specificity towards human properdin and EGFR. • Bi-specific nanobodies induce complement activation on targeted human cancer cells. • Bi-specific nanobodies induce complement dependent lysis of baby hamster kidney cells. • Complement regulators protect human cancer cells against nanobody-mediated lysis. The complement system represents a powerful part of the innate immune system capable of removing pathogens and damaged host cells. Nevertheless, only a subset of therapeutic antibodies are capable of inducing complement dependent cytotoxicity, which has fuelled the search for new strategies to potentiate complement activation. Properdin (FP) functions as a positive complement regulator by stabilizing the alternative pathway C3 convertase. Here, we explore a novel strategy for direct activation of the alternative pathway of complement using bi-specific single domain antibodies (nanobodies) that recruit endogenous FP to a cell surface. As a proof-of-principle, we generated bi-specific nanobodies with specificity toward FP and the validated cancer antigen epidermal growth factor receptor (EGFR) and tested their ability to activate complement onto cancer cell lines expressing EGFR. Treatment led to recruitment of FP, complement activation and significant deposition of C3 fragments on the cells in a manner sensitive to the geometry of FP recruitment. The bi-specific nanobodies induced complement dependent lysis of baby hamster kidney cells expressing human EGFR but were unable to lyse human tumour cells due to the presence of complement regulators. Our results confirm that FP can function as a surface bound focal point for initiation of complement activation independent of prior C3b deposition. However, recruitment of FP by bi-specific nanobodies appears insufficient for overcoming the inhibitory action of the negative complement regulators overexpressed by many human tumour cell lines. Our data provide general information on the efficacy of properdin as an initiator of complement but suggest that properdin recruitment on its own may have limited utility as a platform for potent complement activation on regulated cell surfaces. [ABSTRACT FROM AUTHOR]

Interaction of therapeutic antibodies with complement component C1q are frequently part of pharmaceutical characterization and production process comparability studies. Assays currently used to assess this interaction and/or the activation of the complement cascade are often cumbersome, time consuming or imprecise. We here report the further development, successful qualification and suitability evaluation of an SPR-based C1q binding assay for the characterization of IgG-C1q interactions. We evaluate different IgG subtypes and well-described mutants of IgG1. The assay offers a suitable alternative for extended characterization of interactions of IgG1 and IgG3 with complement component C1q. [ABSTRACT FROM AUTHOR]

We aimed to show the cross-reactivity that may occur between immunoglobulin (Ig) M antibodies that form against Cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV) and human leukocyte antigens (HLA). Complement-dependent cytotoxicity (CDC) cross-reactivity between serum samples of 57 patients with IgM positive CMV and/or EBV infections and T and B cells from 15 healthy donors were evaluated. Dithiothreitol was used to distinguish cross-reactivity caused by IgM antibodies from IgG. The cross-reactivity ratio between pathogenic IgM antibodies with T cell of the 12th donor, and B cell of the 3rd, 4th, and 8th donors was significantly higher (p = 0.011, <0.001, <0.001 and 0.013, respectively). The ratio of B cell CDC cross-reactivity of all donors (26.4%) was higher than the ratio of T cell CDC cross-reactivity (5.2%) (p < 0.001). The ratio of T cell CDC cross-reactivity of sera containing both anti-CMV IgM and anti-EBV IgM antibodies was significantly higher than those of sera containing only anti-CMV IgM or only anti-EBV IgM antibodies (p = 0.002 and p < 0.001, respectively). There was no difference between B cell CDC cross-reactivity rates according to the presence of anti-CMV and/or anti-EBV IgM antibodies. Cross-reactivity may occur between anti-CMV and anti-EBV IgM antibodies with HLA molecules. Thus, in graft recipients, pathogenic IgMs can also act as de novo anti-HLA antibodies and aggravate the rejection process. • Cross-reactive anti-HLA antibodies can arise via exposure to CMV or EBV infections. • Non-sensitized, anti-HLA antibodies in graft recipients may be due to previous CMV and EBV infections. • Anti-CMV IgM and anti-EBV IgM antibodies can predict allo-HLA cross-reactivity. • Cross-reactivity of the anti-viral IgM antibody with allo-HLA may be a potential factor for inducing post-transplantation graft rejection. [ABSTRACT FROM AUTHOR]

Two index cases of living-related donor renal allografts patients developed C4d-negative rejection. Both cases had negative cytotoxic crossmatch and negative flow crossmatch before transplantation. The serum creatinine levels were tabulated. Both cases experienced augmented anti-T cell therapy (intravenous methyl prednisolone) at the time of rejection, which failed to improve renal function. Meantime, our HLA lab identified circulating anti-class I and/or II HLA antibodies towards donor mismatched antigens by Luminex multiplex bead array. Additional therapy included high-dose IVIg and plasma exchange. The renal function improved significantly. Furthermore, the donor-specific antibody strength decreased after combined plasmapheresis and IVIG therapy. These cases highlight the importance of donor-specific antibody detection by sensitive solid phase assays in the context of C4d-negative ABMR.

Bee venom (BV)—a complex mixture of peptides and toxic proteins including phospholipase A2 and melittin—promotes blood clotting. In this study, we investigated the anti-atopic properties of BV and the mechanism associated with its regulation of the complement system. BV treatment upregulated the mRNA and protein levels of CD55 in THP-1 cells. Further experiments revealed that the phosphorylation of ERK was associated with upregulation of CD55. A complement-dependent cytotoxicity assay and a bacteria-killing assay showed that BV inactivated the complement system through the induction of CD55. The serum levels of C3 convertase (C3C) and Membrane attack complex (MAC) increased, while CD55 decreased in mice with AD-like lesions from DNCB treatment. However, the levels were inverted when the AD-like mice were treated with BV using subcutaneous injection, and we observed that the AD symptoms were alleviated. BV is often used to treat AD but its mechanism has not been elucidated. Here, we suggest that BV alleviates AD through the inactivation of the complement system, especially by the induction of CD55.

publisher‐imprint‐name Springer volume‐issue‐count 1 issue‐article‐count 46 issue‐toc‐levels 0 issue‐pricelist‐year 2016 issue‐copyright‐holder The Author(s) issue‐copyright‐year 2016 article‐contains‐esm No article‐numbering‐style Unnumbered article‐registration‐date‐year 2016 article‐registration‐date‐month 10 article‐registration‐date‐day 26 article‐toc‐levels 0 toc‐levels 0 volume‐type Regular journal‐product ArchiveJournal numbering‐style Unnumbered article‐grants‐type OpenChoice metadata‐grant OpenAccess abstract‐grant OpenAccess bodypdf‐grant OpenAccess bodyhtml‐grant OpenAccess bibliography‐grant OpenAccess esm‐grant OpenAccess online‐first false pdf‐file‐reference BodyRef/PDF/40169_2016_Article_125.pdf target‐type OnlinePDF issue‐type Regular article‐type OriginalPaper journal‐subject‐primary Medicine & Public Health journal‐subject‐secondary Medicine/Public Health, general journal‐subject‐collection SC11 --> Background: Only eight women out of one hundred diagnosed with ovarian epithelial cancers, which progressed to the clinical stage IV, survive 10 years. First line therapies: surgery, chemotherapy, and radiation therapy inflict very serious iatrogenic consequences. Passive immunotherapy of ovarian cancers offers only low efficacy. Prophylactic and therapeutic vaccines for ovarian cancers are not available. Interestingly, prophylactic vaccines for Hepatitis B Viruses (HBV) are very effective. Specific aim: The specific aim of this work was to design, synthesize, and administer biomolecules, which would engage prophylactic, vaccination‐induced immunity for HBV towards killing of ovarian cancer cells with high specificity and efficacy. Patients: Tissue biopsies, ascites, and blood were acquired from the patients, whose identities were entirely concealed in accordance with the Declaration of Helsinki, pursuant to the Institutional Review Board approval, and with the Patients' informed consent. Methods and results: By biomolecular engineering, we have created a novel family of biomolecules: antibody × vaccine engineered constructs (AVEC: anti‐HER‐2 × HBsAg). We have collected the blood from the volunteers, and measured the titers of anti‐HBV antibodies resulting from the FDA approved and CDC scheduled HBV vaccinations. We have acquired tumor biopsies, ascites, and blood from patients suffering from the advanced ovarian cancers. We have established cultures of HER‐2 over‐expressing epithelial ovarian cancers: OV‐90, TOC‐112D, SKOV‐3, as well as human ovary surface epithelial (HOSE) and human artery endothelial (HAE) cells. Treatment of the HER‐2+ ovarian cancer cells with AVEC: anti‐HER‐2 × HBsAg, accompanied by administration of blood drawn from patients with high titers of the anti‐HBV antibodies, resulted in much higher therapeutic efficacy as compared to treatment with the naked anti‐HER‐2 antibodies alone and/or with the relevant isotype antibodies. This treatment had practically no effect upon the HOSE and HAE cells. Discussion: Herein, we report attaining the great improvement in eradication efficacy of ovarian epithelial cancer cells' by engaging prophylactic immunity against HBV; thus creating a novel paradigm for immunotherapy of ovarian cancer. We have accomplished that by designing, synthesis, and administration of AVEC. Therefore, the HBV vaccination acquired immunity mounts immune response against the vaccine, but AVEC redirect, accelerate, and amplify this immune response of all the elements of the native and adaptive immune system against ovarian cancer. Our novel paradigm of immunotherapy is currently streamlined to clinical trials also of other cancers, while also engaging prophylactic and acquired immunity. Conclusion: Novel antibody‐vaccine engineered constructs (AVEC) create the solid foundation for redirected, accelerated, and amplified prophylactic, HBV vaccination‐induced immunity immunotherapy (RAAVIIT) of ovarian cancers. [ABSTRACT FROM AUTHOR]

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system and is mediated by complement-dependent cytotoxicity (CDC) of NMO-specific immunoglobulin G (IgG) antibodies (NMO-IgG). Glycyrrhizic acid (GA) has numerous pharmacological effects including inhibition of the complement pathway. We aimed to study the influence of GA on NMO-IgG-induced CDC. NMO-IgG samples from 7 patients with NMO, together with human complement, induced CDC in an aquaporin 4 M23-overexpressing glial cell line, an in vitro NMO model. GA attenuated NMO-IgG-induced CDC in a dose-dependent manner. The mechanism of the GA-related CDC inhibition was sequentially dissected and found to involve inhibition of C1q binding to NMO-IgG. Consequently, GA attenuates NMO-IgG-induced CDC and may be a promising novel therapeutic agent against NMO. [ABSTRACT FROM AUTHOR]

Identifying and monitoring donor-directed anti-human leukocyte antigen antibodies are a rapidly evolving area of solid organ transplantation. Donor-specific antibodies dictate pre-transplant donor choice and donor-recipient matching and underlie much acute and chronic allograft rejection and loss. The evolution of available technology has driven this progress. Early, labor-intensive, whole-cell assays based on complement-dependent cytotoxicity suffered from poor sensitivity and specificity, technical challenges and lack of precision. Sequential improvement in assay performance included anti-human immunoglobulin-enhanced, complement-dependent cytotoxicity techniques followed by cell-based flow cytometry. However, variable specificity and sensitivity inherent in cell-based testing continued to limit flow cytometry. The introduction of solid-phase assays led to a second revolution in histocompatibility testing with the use of purified antigens bound to artificial surfaces rather than whole cells. These techniques augmented sensitivity and specificity to detect even low-titer antibodies to previously undetected antigens. Identification of complement-activating antibodies is being introduced, but current technology is in the developmental stage. While the detection of alloantibodies has improved dramatically, our comprehension of their importance remains imperfect. Variability in methodology and a lack of standardization limits the clinical application of these tests. In spite of the hurdles that remain, antibody-mediated rejection has become a key target to improve graft survival. [ABSTRACT FROM AUTHOR]

Although rituximab, a chimeric monoclonal antibody that specifically binds to CD20, has significantly improved the prognosis for diffuse large B cell lymphoma (DLBCL), one-third of DLBCL patients demonstrate resistance to rituximab or relapse after rituximab treatment. Thus, a novel approach to rituximab-based treatment is likely to be required to improve the efficacy of DLBCL treatment. As complement dependent cytotoxicity (CDC) is a key mechanism mediating rituximab's tumoricidal activity, rituximab binding to CD20 on tumor cells is a critical factor for effective rituximab-based treatments against DLBCL. We found that gemcitabine (GEM), but not lenalidomide (LEN) or azacitidine (AZA), can upregulate CD20 expression in TK and KML-1 cells, two human DLBCL cell lines. Treatment of TK and KML-1 cells with GEM enhanced CD20 expression at both the mRNA and protein levels. CD20 upregulation by GEM treatment was accompanied by increased rituximab binding to CD20. In TK cells, GEM treatment synergistically increased rituximab-mediated CDC activity in a dose-dependent manner. In KML cells, GEM treatment also induced upregulation of complement regulatory proteins, possibly leading to resistance to CDC. Treatment with LEN, a drug that did not upregulate CD20, did not enhance rituximab-mediated CDC activity. GEM treatment activated nuclear factor-kappa B (NF- kB) signaling in these cells. Furthermore, a specific inhibitor to NF- kB suppressed GEM-induced CD20 upregulation, indicating that GEM-induced NF- kB activation is closely associated with CD20 upregulation. These results suggest that when used in combination, GEM might enhance the antitumor efficacy of rituximab against DLBCL due to its unique ability to upregulate CD20. [ABSTRACT FROM AUTHOR]

Two index cases of living-related donor renal allografts patients developed C4d-negative rejection. Both cases had negative cytotoxic crossmatch and negative flow crossmatch before transplantation. The serum creatinine levels were tabulated. Both cases experienced augmented anti-T cell therapy (intravenous methyl prednisolone) at the time of rejection, which failed to improve renal function. Meantime, our HLA lab identified circulating anti-class I and/or II HLA antibodies towards donor mismatched antigens by Luminex multiplex bead array. Additional therapy included high-dose IVIg and plasma exchange. The renal function improved significantly. Furthermore, the donor-specific antibody strength decreased after combined plasmapheresis and IVIG therapy. These cases highlight the importance of donor-specific antibody detection by sensitive solid phase assays in the context of C4d-negative ABMR. [ABSTRACT FROM AUTHOR]

Complement dependent cytotoxicity (CDC) is an important mechanism of action for monoclonal antibodies (mAbs) used in the treatment of chronic lymphocytic leukemia (CLL). We hypothesized that alemtuzumab (ALM) mediated CDC would be increased by the addition of ofatumumab (OFA). CLL cells from 21 previously untreated patients with progressive disease were tested in vitro for mAb binding, complement activation and CDC. The subpopulation of CDC resistant CLL cells was examined for levels of C3b and C5b-9 binding, and expression of complement regulatory proteins. OFA significantly increased complement activation and CDC in ALM-treated CLL cells, suggesting that combining ALM and OFA could improve clinical outcome in patients with CLL. Approximately 10% of CLL cells were resistant to CDC because of lower levels of complement activation or decreased cytotoxicity of activated complement. Improvement of clinical responses will require determining the mechanisms of CDC resistance and developing methods to overcome this problem. [ABSTRACT FROM AUTHOR]

Abstract: Releasing drug molecules at their targets with high spatial and temporal accuracy could aid numerous clinical applications which require low systemic damage and low side effects. Nano-carriers of drugs are an attractive solution for such task, allowing specific accumulation in tumors and gradual release of their payload. Here, we utilize gold nanospheres conjugated to Rituximab, an anti-CD20 monoclonal antibody-based drug, for carrying and releasing the drug upon irradiation of specifically tailored femtosecond laser pulses. The released anti-CD20 molecules retain their functionality and ability of triggering the complement-dependent cytotoxicity. This effect comes in addition to cell necrosis caused by the plasmonic nanometric shock waves emanating from the nanospheres and rupturing the plasma membranes. Main advantages of the presented technique include high spatial and temporal resolution, low toxicity and high repeatability and consistency due to the morphological stability of the nanospheres. [Copyright &y& Elsevier]

Rituximab, an anti-CD20 monoclonal antibody used to treat B cell lymphoproliferative disorders and autoimmune diseases, kills cells through complement dependent cytotoxicity, antibody-dependent cellular toxicity and apoptosis. A mechanism of resistance to rituximab is upregulation of the complement regulatory proteins, CD59 and CD55. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic disorder caused by PIGA mutations that lead to a loss of all glycosylphospatidylinositol (GPI)-anchored proteins including, CD55 and CD59. We compared the cytotoxic activity of rituximab against a PNH B cell line, LD  -  , and the isogenic cell line LD  -  PIGA  +  in which GPI-anchor expression was restored by stable transfection of PIGA. The PNH cell line was more sensitive to rituximab-mediated killing than the LD  -  PIGA  +  cells. Biochemical disruption of GPI anchors with phosphatidylinositol specific phospholipase C (PIPLC), a phospholipase that cleaves GPI-anchored proteins, also increased rituximab-mediated killing. Thus, genetic and biochemical interruption of GPI anchor proteins augments sensitivity to rituximab. [ABSTRACT FROM AUTHOR]

Recombinant monoclonal antibodies (mAbs) have become an important category of biological therapeutics. mAbs share the same structures and biological functions as endogenous IgG molecules. One function is complement-dependent cytotoxicity (CDC) initiation by binding of C1q. Traditionally, ELISA methods have been utilized to measure C1q binding. A new robust capture method was established in this study to measure the binding affinity of C1q to antibodies by surface plasmon resonance (SPR). The utility of this method was demonstrated by determination of the difference in IgG subclass specificity of C1q binding. [ABSTRACT FROM AUTHOR]

The highly pathogenic avian influenza A (H5N6) virus has caused sporadic human infections with a high case fatality rate. Due to the continuous evolution of this virus subtype and its ability to transmit to humans, there is an urgent need to develop effective antiviral therapeutics. In this study, a murine monoclonal antibody 9F4 was shown to display broad binding affinity against H5Nx viruses. Furthermore, 9F4 can neutralize H5N6 pseudotyped particles and prevent entry into host cells. Additionally, ADCC/ADCP deficient L234A, L235A (LALA) and CDC deficient K322A mutants were generated and displayed comparable binding affinity and neutralizing activity as wild type 9F4 (9F4-WT). Notably, 9F4-WT, 9F4-LALA and 9F4-K322A exhibit in vivo protective efficacies against H5N6 infections in that they were able to reduce viral loads in mice. However, only 9F4-WT and 9F4-K322A but not 9F4-LALA were able to reduce viral pathogenesis in H5N6 challenged mice. Furthermore, depletion of phagocytic cells in mice lungs nullifies 9F4-WT's protection against H5N6 infections, suggesting a crucial role of the host's immune cells in 9F4 antiviral activity. Collectively, these findings reveal the importance of ADCC/ADCP function for 9F4-WT protection against HPAIV H5N6 and demonstrate the potential of 9F4 to confer protection against the reassortant H5-subtype HPAIVs.

Bee venom (BV)—a complex mixture of peptides and toxic proteins including phospholipase A2 and melittin—promotes blood clotting. In this study, we investigated the anti-atopic properties of BV and the mechanism associated with its regulation of the complement system. BV treatment upregulated the mRNA and protein levels of CD55 in THP-1 cells. Further experiments revealed that the phosphorylation of ERK was associated with upregulation of CD55. A complement-dependent cytotoxicity assay and a bacteria-killing assay showed that BV inactivated the complement system through the induction of CD55. The serum levels of C3 convertase (C3C) and Membrane attack complex (MAC) increased, while CD55 decreased in mice with AD-like lesions from DNCB treatment. However, the levels were inverted when the AD-like mice were treated with BV using subcutaneous injection, and we observed that the AD symptoms were alleviated. BV is often used to treat AD but its mechanism has not been elucidated. Here, we suggest that BV alleviates AD through the inactivation of the complement system, especially by the induction of CD55. [ABSTRACT FROM AUTHOR]

GD2-directed immunotherapies improve survival of high-risk neuroblastoma (NB) patients (pts). Treatment with chimeric anti-GD2 antibodies (Ab), such as ch14.18, can induce development of human anti-chimeric Ab (HACA). Here, we report HACA effects on ch14.18/CHO pharmacokinetics, pharmacodynamics and pain intensity in pts treated by long-term infusion (LTI) of ch14.18/CHO combined with IL-2. 124 pts received up to 5 cycles of ch14.18/CHO 10 days (d) infusion (10 mg/m2/d; d8–18) combined with s.c. IL-2 (6 × 106 IU/m2/d; d1–5, d8–12). HACA, treatment toxicity, ch14.18/CHO levels, Ab-dependent cellular- (ADCC) and complement-dependent cytotoxicity (CDC) were assessed using respective validated assays. HACA-negative pts showed a steadily decreased pain in cycle 1 (74% pts without morphine by d5 of LTI) with further decrease in subsequent cycles. Ch14.18/CHO peak concentrations of 11.26 ± 0.50 µg/mL found in cycle 1 were further elevated in subsequent cycles and resulted in robust GD2-specific CDC and ADCC. Development of HACA (21% of pts) resulted in strong reduction of ch14.18/CHO levels, abrogated CDC and ADCC. Surprisingly, no difference in pain toxicity between HACA-positive and -negative pts was found. In conclusion, ch14.18/CHO LTI combined with IL-2 results in strong activation of Ab effector functions. Importantly, HACA response abrogated CDC but did not affect pain intensity indicating CDC-independent pain induction. [ABSTRACT FROM AUTHOR]

Chronic inflammation has been linked to the initiation of carcinogenesis, as well as the advancement of established tumors. The polarization of the tumor inflammatory microenvironment can contribute to either the control, or the progression of the disease. The emerging participation of members of the complement cascade in several hallmarks of cancer, renders it a potential target for anti-tumor treatment. Moreover, the presence of complement regulatory proteins (CRPs) in most types of tumor cells is known to impede anti-tumor therapies. This review focuses on our current knowledge of complement's potential involvement in shaping the inflammatory tumor microenvironment and its role on the regulation of angiogenesis and hypoxia. Furthermore, we discuss approaches using complement-based therapies as an adjuvant in tumor immunotherapy.

TNFα played a dominant role in the development and progression of rheumatoid arthritis (RA). Clinical trials proved the efficacies of anti-TNFα agents for curing RA. However, most researchers were concentrating on their abilities of neutralizing TNFα, the potencies of different anti-TNFα agents varied a lot due to the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). For better understanding and differentiating the potentiality of various candidate anti-TNF reagents at the stage of new drug research and development, present study established a cell model expressing the transmembrane TNFα for usage in in vitro ADCC or CDC assay, meanwhile, the assay protocol described here could provide guidelines for screening macromolecular antibody drugs. A stable cell subline bearing transmembrane TNFα was first established by conventional transfection method, the expression of transmembrane TNFα was approved by flow cytometer, and the performance of the stable subline in ADCC and CDC assay was evaluated, using human peripheral blood mononuclear cells as effector cells, and Adalimumab as the anti-TNFα reagent. The stable cell subline demonstrated high level of surface expression of transmembrane TNFα, and Adalimumab exerted both ADCC and CDC effects on this cell model. In conclusion, the stable cell line we established in present research could be used in ADCC or CDC assay for screening antibody drugs, which would provide in-depth understanding of the potencies of candidate antibody drugs in addition to the traditional TNFα neutralizing assay.

Rituximab has been revolutionized and validated CD20 targeting monoclonal antibody. Although, it is widely used for lymphoma therapy and many patients have been benefited. However significant numbers of patients are refractory or developed resistance to current therapies due to low level of CD20 expression and/or availability on cells surface. Thus development of novel anti-CD20 mAbs with great cell killing ability and enhance CD20 levels on cell surface can potentially exploit lymphoma therapy. In this scenario, we are summarizing the recently developed mAbs against CD20 and compounds that have ability to induce CD20 expression at significant level. We also are providing information regarding combination strategy for use of radiation and anti-CD20 mAbs in vitro . However, it will need to be determined by rigorous at pre-clinical and clinic testing. We hope this review will be beneficial for current research in the area of immunotherapy or radio-immunotherapy.

The Fc variant of IgG2, designated as IgG2σ, was engineered with V234A/G237A /P238S/H268A/V309L/A330S/P331S substitutions to eliminate affinity for Fcγ receptors and C1q complement protein and consequently, immune effector functions. IgG2σ was compared to other previously well-characterized Fc 'muted' variants, including aglycosylated IgG1, IgG2m4 (H268Q/V309L/A330S/P331S, changes to IgG4), and IgG4 ProAlaAla (S228P/L234A/L235A) in its capacity to bind FcγRs and activate various immune-stimulatory responses. In contrast to the previously characterized muted Fc variants, which retain selective FcγR binding and effector functions, IgG2σ shows no detectable binding to the Fcγ receptors in affinity and avidity measurements, nor any detectable antibody-dependent cytotoxicity, phagocytosis, complement activity, or Fc-mediated cytokine release. Moreover, IgG2σ shows minimal immunogenic potential by T-cell epitope analysis. The circulating half-life of IgG2σ in monkeys is extended relative to IgG1 and IgG2, in spite of similar in vitro binding to recombinant FcRn. The three-dimensional structure of the Fc, needed for assessing the basis for the absence of effector function, was compared with that of IgG2 revealing a number of conformational differences near the hinge region of the CH2 domain that result from the amino acid substitutions. Modeling reveals that at least one of the key interactions with FcγRs is disrupted by a conformational change that reorients P329 to a position that prevents it from interacting with conserved W90 and W113 residues of the FcγRs. Inspection of the structure also indicated significant changes to the conformations of D270 and P329 in the CH2 domain that could negatively impact C1q binding. Thus, structural perturbations of the Fc provide a rationale for the loss of function. In toto, these properties of IgG2σ suggest that it is a superior alternative to previously described IgG variants of minimal effector function, for future therapeutic applications of non-immunostimulatory mAb and Fc-fusion platforms., (Copyright © 2013 Elsevier Inc. All rights reserved.)