Antibody-dependent Complement-mediated Cytotoxicity Research Articles

Abstract 1335: Dissecting the mechanisms of anti-tumor protection mediated by an antibody targeting the transferrin receptor 1

Abstract The transferrin receptor 1 (TfR1/CD71) is a type II transmembrane homodimeric protein involved in iron uptake and regulation of cell growth. The elevated levels of TfR1 expression on malignant cells, together with its extracellular accessibility, make TfR1 an attractive target for antibody therapy. We developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which exhibits direct cytotoxic activity against certain human malignant B cells in vitro. This anti-tumor activity is due to the induction of TfR1 degradation and lethal iron deprivation. Importantly, ch128.1 also shows extraordinary anti-tumor activity in two xenograft models of the incurable B-cell malignancy multiple myeloma in immunosuppressed mice (SCID-Beige) resulting in up to 100% survival using a single dose. Interesting, this anti-tumor activity occurs even against malignant cell lines that show limited or no sensitivity to ch128.1 in vitro. The mechanism of in vivo protection of ch128.1 is currently unknown. In order to explore the possible role of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) in the anti-tumor mechanism of ch128.1 and to delineate the residues involved in these effector functions, we developed three constructs with mutations in the ch128.1 heavy chain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. We now show that ch128.1 is capable of inducing ADCC and CDC against malignant B cells in vitro suggesting the potential contribution of these antibody effector functions to its effects in vivo. Additionally, we show that the triple mutant consistently displayed significant ADCC reduction in vitro, while the other mutants showed a weaker impairment, suggesting that all three residues are relevant contributors to ADCC. Lack of CDC in the P331S mutant confirmed the critical role of this residue, while the other residues show a lesser role in CDC in vitro. These results provide, for the first time, insights into the human IgG3 effector functions in the context of TfR1 targeting of malignant cells. Intriguingly, we have recently observed that the strong in vivo protection of ch128.1 against disseminated malignant B cells is abolished when the triple ch128.1 mutant (L234A/L235A/P331S) is used as a therapeutic at the same dose as that of ch128.1. These results point to a functional role for the antibody Fc fragment in anti-tumor protection mediated by ch128.1. Further studies aimed to provide a better understanding of the in vivo activity exhibited by ch128.1 are in progress. This antibody may be potentially used in the therapy of incurable human B-cell malignancies, such as multiple myeloma and certain non-Hodgkin lymphomas. Citation Format: Lai Sum Leoh, Tracy R. Daniels-Wells, Otoniel Martinez-Maza, Manuel L. Penichet. Dissecting the mechanisms of anti-tumor protection mediated by an antibody targeting the transferrin receptor 1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1335. doi:10.1158/1538-7445.AM2015-1335

Insights into the effector functions of human IgG3 in the context of an antibody targeting transferrin receptor 1.

The transferrin receptor 1 (TfR1) is involved in cellular iron uptake and regulation of cell proliferation. The increased expression of TfR1 observed in malignant cells, compared to normal cells, together with its extracellular accessibility, make this receptor an attractive target for antibody-mediated cancer therapy. We have developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which shows anti-tumor activity against certain malignant B cells in vitro through TfR1 degradation and iron deprivation, and in vivo through a mechanism yet to be defined. To further explore potential mechanisms of action of ch128.1, we examined its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). We now report that ch128.1 is capable of mediating ADCC and CDC against malignant B cells, which is consistent with its ability to bind FcγRI, FcγRIIIa, and the complement component C1q. To delineate the residues involved in these effector functions, we developed a panel of three constructs with mutations in the lower hinge region and CH2 domain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. The triple mutant consistently displayed a significant reduction in ADCC, while the L234A/L235A mutant exhibited less reduction in ADCC, and the P331S mutant did not show reduced ADCC. However, all three mutants exhibited impaired binding to FcγRI and FcγRIIIa. These results suggest that all three residues contribute to ADCC, although to different degrees. The P331S mutant showed drastically decreased C1q binding and abolished CDC, confirming the critical role of this residue in complement activation, while the other residues play a less important role in CDC. Our study provides insights into the effector functions of human IgG3 in the context of an antibody targeting TfR1.

Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8

Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy.

Quantitative analysis of drug-induced complement-mediated cytotoxic effect on single tumor cells using atomic force microscopy and fluorescence microscopy.

In the antibody-based targeted therapies of B-cell lymphomas, complement-mediated cytotoxicity (CMC) is an important mechanism. CMC is activated after the binding of drugs (monoclonal antibodies) to tumor cells. The activation of CMC ultimately leads to the lysis of tumor cells. However, it remains poorly understood how CMC alters the morphology and mechanics of single tumor cells at the nanoscale. In recent years, nanoscopic observations of cellular behaviors with the use of atomic force microscopy (AFM) have contributed much to the field of cell biology. In this work, by combining AFM with fluorescence microscopy, the detailed changes in cellular ultra-microstructures and mechanical properties during the process of CMC were quantitatively investigated on single tumor cells. AFM imaging distinctly showed that the CMC effect could lead to the formation of nano holes on the tumor cells. Quantitative analysis of AFM images indicated that cell surface became lower and rougher after the CMC process. The cellular mechanics measurements showed that during the process of CMC cells firstly softened and finally stiffened, which was validated by dynamically monitoring the mechanical changes of single living cells during CMC. The experimental results provide novel insights into the antibody-dependent CMC.

Different IVIG glycoforms affect in vitro inhibition of anti-ganglioside antibody-mediated complement deposition.

Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.

Prevention of rejection of allogeneic endothelial cells in a biohybrid lung by silencing HLA-class I expression

Variability in Human Leukocyte Antigens (HLA) remains a hurdle to the application of allogeneic cellular products. Due to insufficient autologous endothelial cell harvesting for the biohybrid lung, allogeneic human cord blood derived endothelial cells (HCBEC) were used for the endothelialization of poly-4-methyl-1-pentene (PMP) gas exchange membranes. Therefore, HLA class I expression was silenced stably in HCBECs to prevent rejection. The capacity of HLA class I-silenced HCBEC to abrogate allogeneic immune responses, their functional properties and suitability for endothelialization of PMP membranes were investigated. Delivery of β2-microglobulin (β2m)-specific shRNAs reduced β2m mRNA levels by up to 90% and caused a knockdown of HLA class I expression by up to 85%. HLA-silenced HCBEC abrogated T-cell responses and escaped antibody-mediated complement-dependent cytotoxicity. The EC phenotype and cytokine secretion profiles between HLA-expressing or -silenced HCBEC remained unaltered. EC specific activation (e.g. ICAM) and thrombogenic markers (e.g. thrombomodulin) remained unaffected by HLA-silencing, but their expression was upregulated by TNFα-stimulation. Furthermore, HLA-silenced HCBECs showed high proliferation rates and built an EC monolayer onto PMP membranes. This study represents a new therapeutic concept in the field of cell and organ transplantation and may bring the bioartificial lung as an alternative to lung transplantation closer to reality.

Characterization of melanocyte-specific autoantibodies in vitiligo-prone Smyth line chickens. (BA2P.128)

Abstract Autoimmune vitiligo is characterized by the loss of pigment-producing melanocytes (MC) in the skin. The autoimmune vitiligo-prone Smyth line (SL) of chicken together with the parental Brown line (BL) and vitiligo-resistant Light Brown Leghorn (LBL) chickens constitutes an excellent animal model for this disorder. Although cell-mediated immunity has long been known to contribute to the etiopathology of SL vitiligo (SLV) the role of humoral immunity is less clear. We recently completed a time-course study examining levels of IgG specific to MC lysate in SL and control plasma. Results showed elevated (P<0.05) MC-lysate specific IgG levels in SL chickens 1-2 weeks prior to and throughout SLV development. To evaluate the binding characteristics of MC-specific IgG, primary MC isolated from growing feathers were incubated with IgG purified from the serum of SLV (birds with SLV) and control chickens. IgG binding was evaluated by indirect immunofluorescent staining and flow cytometry. Regardless of the source of MC (SL, BL, LBL), significant binding was only observed with SLV-IgG (mean fluorescence intensity increased approximately 100%). This effect was not observed when MC were incubated with IgG purified from egg yolk of SLV hens. Further characterization of autoantibodies from SLV chickens may provide insight into their role in the loss of MC in SLV. Studies evaluating such mechanisms as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity are underway.

Antibodies with Higher Bactericidal Activity Induced by a Neisseria gonorrhoeae Rmp Deletion Mutant Strain

Neisseria gonorrhoeae (N. gonorrhoeae) outer membrane protein reduction modifiable protein (Rmp) has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261–460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

Role of Crosslinking for Agonistic CD40 Monoclonal Antibodies as Immune Therapy of Cancer

Agonists of the TNF superfamily of receptors hold promise as novel therapy for cancer. Recent data on agonistic anti-murine TNF receptors (TNFR) such as CD40 suggest that the specific engagement of Fc-receptor (FcR) is required for optimal antitumor effects, prompting calls to engineer anti-human CD40 and other TNFR mAb accordingly. CP-870,893 is a fully human anti-CD40 mAb, selected in part because it is an IgG2 which is presumed to have poor reactivity with FcR; however, CP-870,893 has been evaluated in multiple clinical trials with beneficial activity in patients with melanoma, pancreatic and other cancers. Here, we confirmed that the activity of anti-murine CD40 mAb was dependent on FcγRIIB engagement, was decreased significantly in FcγRIIB (-/-) mice, and upon Fc-crosslinking anti-mouse CD40 mAb enhanced the activation of antigen presenting cells. In contrast, the CP-870,893-mediated activation of human B cells was not enhanced with anti-IgG-crosslinking nor abrogated when used as an F(ab)'2 reagent. Crosslinking of CP-870,893 using the CD32-expressing K562 cells yielded an Fc-dependent modest increase in the expression of some activation markers relative to that of the soluble CP-870,893 mAb. Classic Fc-dependent functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) were minimal for CP-870,893 as compared to the IgG1 anti-CD20 mAb rituximab, which mediated both ADCC and CMC in parallel assays. Anti-mouse CD40 mAb competed for the CD40 ligand binding site, but CP-870,893 did not. Thus, Fc-crosslinking is not an essential requirement for agonistic anti-human CD40 mAb, whose potency is more dependent on the CD40 epitope recognized and the strength of the signal achieved.

Genetically engineered blood pharming: generation of HLA-universal platelets derived from CD34+ progenitor cells.

Blood pharming is a recently designed concept to enable in vitro production of blood cells that are safe, effective and readily available. This approach represents an alternative to blood donation and may contribute to overcome the shortage of blood products. However, the high variability of the human leukocyte antigen (HLA) loci remains a major hurdle to the application of off-the-shelf blood products. Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA class I antigens constitutes a relevant clinical problem. Thus, it would be desirable to generate PLT units devoid of HLA antigens. To reduce the immunogenicity of cell-based therapeutics, we have permanently reduced HLA class I expression using an RNA interference strategy. Furthermore, we demonstrated that the generation of HLA class I-silenced (HLA-universal) PLTs from CD34+ progenitor cells using an shRNA targeting β2-microglobulin transcripts is feasible. CD34+ progenitor cells derived from G-CSF mobilised donors were transduced with a lentiviral vector encoding for the β2-microglobulin-specific shRNA and differentiated into PLTs using a liquid culture system. The functionality of HLA-silenced PLTs and their ability to escape HLA antibody-mediated cytotoxicity were evaluated in vitro and in vivo. Platelet activation in response to ADP and thrombin were assessed in vitro. The immune-evasion capability of HLA-universal megakaryocytes (MKs) and PLTs was tested in lymphocytotoxicity assays using anti-HLA antibodies. To assess the functionality of HLA-universal PLTs in vivo, HLA-silenced MKs were infused into NOD/SCID/IL-2Rγc(-/-) mice with or without anti-HLA antibodies. PLT generation was evaluated by flow cytometry using anti-CD42a and CD61 antibodies. HLA-universal PLTs demonstrated to be functionally similar to blood-derived PLTs. Lymphocytotoxicity assays showed that HLA-silencing efficiently protects MKs against HLA antibody-mediated complement-dependent cytotoxicity. 80-90% of HLA-expressing MKs, but only 3% of HLA-silenced MKs were lysed. In vivo, both HLA-expressing and HLA-silenced MKs showed human PLT production (up to 0.5% within the PLT population) when anti-HLA antibodies were absent. However, in presence of anti-HLA antibodies HLA-expressing MKs were rapidly cleared from the circulation of mice, while HLA-silenced MKs escaped HLA antibody-mediated cytotoxicity and human PLT production was detectable up to 11 days. Our studies show that HLA-silenced PLTs are functional and efficiently protected against HLA antibody-mediated cytotoxicity. In this chapter, we provide a review of our most recent findings in the use of CD34+ progenitor cells for the production of HLA-universal PLTs and their potential clinical applications. Provision of HLA-universal PLT units may become an important component in the management of patients with PLT transfusion refractoriness.

HLA-Universal Platelet Transfusions Prevent Platelet Refractoriness in a Mouse Model

Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA class I antigens constitutes a significant clinical problem. Thus, it would be desirable to have PLT units lacking HLA antigens on the cell surface. Previously, we showed that the generation of functional HLA class I-silenced (HLA-universal) PLTs from CD34(+) cells, using a short hairpin RNA (shRNA) to target β2-microglobulin (β2m) transcripts, is feasible. Here, we assessed the capacity of HLA-silenced PLTs to escape HLA antibody-mediated cytotoxicity in vitro and in vivo. Generation of megakaryocytes (MKs) and PLTs was performed by thrombopoietin-mediated differentiation of HLA-silenced CD34(+) cells within 10 days. Lymphocytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) reporter assays using anti-HLA antibodies and a mouse model for PLT refractoriness were used to assess the immune-evasion capability of HLA-universal MKs and PLTs. To mimic PLT refractoriness in vivo, NOD/SCID/IL-2Rγc(-/-) mice were injected with specific anti-HLA antibodies followed by the infusion of 1 × 10(6) HLA-universal MKs. In vivo PLT generation was evaluated by flow cytometry using anti-CD42a and CD61 antibodies. Cells expressing a nonspecific shRNA were used as control. Lymphocytotoxicity and ADCC reporter assays showed that HLA silencing protects MKs against HLA antibody-mediated complement-dependent and cell-mediated cytotoxicity. In lymphocytotoxicity assays, 80-90% of HLA-expressing MKs but only 3% of HLA-silenced MKs were lysed. In the circulation of mice, HLA-expressing and HLA-silenced MKs showed PLT production in the absence of anti-HLA antibodies, with human PLT frequencies of up to 0.5% within the PLT population. However, in presence of anti-HLA antibodies HLA-expressing MKs were rapidly cleared from the circulation of mice, whereas HLA-silenced MKs escaped HLA antibody-mediated cytotoxicity and produced PLTs that were detectable up to 11 days. Our data show that HLA-silenced PLTs are efficiently protected against HLA antibody-mediated cytotoxicity and prevent PLT refractoriness in vivo. Provision of HLA-silenced PLTs may become an important component in the management of patients refractory to PLT transfusion.

Construction of a recombinant Neisseria gonorrhoeae strain with rmp gene deletion and study on the bactericidal activities of its antibodies

Objective To study the role of the outer membrane protein Rmp of Neisseria gonor- rhoeae strain in immunosuppression and the strategy of eliminating it. Methods The rmp gene of Neisseria gonorrhoeae strain was amplified by PCR and inserted into pMD19-T vector. The recombinant vector pMD19 Armp ：： Kan containing Kan and the 5＇-and 3＇-flanking regions of rmp （ Armp ：： Kan） was constructed by replacing 200 nucleotide residues of pMD19-rmp with kanamycin resistance gene Kan and transformed into Neisseria gonorrhoeae WHO-A strain. PCR and Western blot assay were used to screen and identify the re- combinant mutant strains that could not express Rmp. Mice were immunized with mutant strains and bacteri- cidal activities of the immune sera were detected by antibody-mediated complement-dependent cytotoxicity assay. Results The mutant strains that could not encode Rmp were successfully constructed. Antibodies in- duced by mutant strains showed stronger bactericidal activity against Neisseria gonorrhoeae in comparison with those induced by wild strains. Conclusion The recombinant Neisseria gonorrhoeae strain with rmp gene de- letion might eliminate the immunosuppressive effects of Rmp expressed in wild gonococcal strains, which provides a reference for further development of novel live attenuated whole-cell vaccines of Neisseria gonor- rhoeo, e. Key words: Neisseria gonorrhoeae; Homologous recombination; rmp gene deletion mutants; Live attenuated vaccine

Fibronectin from alpha 1,3-galactosyltransferase knockout pigs is a xenoantigen

BackgroundAntibody-mediated rejection continues to be an obstacle for xenotransplantation despite development of α1,3-galactosyltransferase knockout (GTKO) pigs. Fibronectin (Fn) from GTKO pigs was identified as a xenoantigen in baboons. N-glycolylneuraminic acid (Neu5Gc), similar to galactose α1,3-galactose, is an antigenic carbohydrate found in pigs. We evaluated human antibody reactivity and performed initial antigenic epitope characterization of Fn from GTKO pigs. Materials and methodsGTKO pig aortic endothelial cells (AEC) were isolated and assessed for antibody-mediated complement-dependent cytotoxicity (CDC). Human and GTKO pig Fn were purified and analyzed using immunoblots. GTKO pig and human AEC absorbed human sera were assessed for CDC and anti-GTKO pig Fn antibodies. GTKO pig proteins were assessed for Neu5Gc. Immunoaffinity-purified human IgG anti-GTKO pig (hIgG-GTKOp) Fn using a GTKO pig Fn column were evaluated for cross-reactivity with other proteins. ResultsGTKO pig AEC had greater human antibody binding, complement deposition and CDC compared with allogeneic human AEC. Human sera absorbed with GTKO pig AEC resulted in diminished anti-GTKO pig Fn antibody. Neu5Gc was identified on GTKO pig Fn and other proteins. The hIgG-GTKOp Fn cross-reacted with multiple GTKO pig proteins and was enriched with anti-Neu5Gc antibody. ConclusionsRemoval of antigenic epitopes from GTKO pig AEC would improve xenograft compatibility. GTKO pig Fn has antigenic epitopes, one identified as Neu5Gc, which may be responsible for pathology and cross-reactivity of hIgG-GTKOp Fn. Genetic knockout of Neu5Gc appears necessary to address significance and identification of non-Neu5Gc GTKO pig Fn antigenic epitopes.

Anti-Thy-1 Antibody-mediated Complement-dependent Cytotoxicity is Regulated by the Distribution of Antigen, Antibody and Membrane Complement Regulatory Proteins in Rats.

Some therapeutic antibodies as anticancer agents exert their effects through the host immune system, but the factors that predict their cytotoxicity, including complement-dependent cytotoxicity (CDC), are unclear. In the present study, we attempted to elucidate some of these factors in a preclinical model. CDC-related mesangiolysis caused by administration of the anti-Thy-1.1 antibody can be studied in the rat anti-Thy-1 glomerulonephritis model, so the model was used in this study. Three animals each were sacrificed at 0.5, 1, 8, 24 and 48 hours after i.v. administration of the anti-Thy-1.1 antibody at 1mg/kg. The distribution of the Thy-1.1 antigen and 2 membrane complement regulatory proteins (mCRPs), Crry and CD55, in three non-treated animals and the distribution of the injected antibody and C3 in the model was studied by immunohistochemistry. In the mesangial cells of the kidney, both expression of the antigen and distribution of the antibody with C3 deposition were observed with weak expression of mCRPs. There was also antigen and antibody distribution in the medullary cells of the adrenal gland and in the lymphocytes of the thymus but no C3 deposition, which was thought to be related to high expression of mCRPs. The antigen was observed in several other organs and tissues without distribution of the antibody. Cell death was only observed in the mesangial cells. These results clearly demonstrate that activation of CDC is regulated by several factors, such as distribution of the target molecule, antibody distribution and the balance among the molecules of the CDC cascade and mCRPs.

Double knockout pigs deficient in N‐glycolylneuraminic acid and Galactose α‐1,3‐Galactose reduce the humoral barrier to xenotransplantation

Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. This technology will accelerate the development of pigs for xenotransplantation research.

Rituximab therapy for refractory autoimmune thrombocytopenia in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SlE) and autoimmune polyendocrinopathy are characterized by autoantibodies against a variety of target organs. Traditional treatment strategies for immune cytopenias include global immunosuppression that targets both the humoral and cell-mediated arms of the immune system. B-cells play an important role in the pathogenesis of many autoimmune diseases. Targeted depletion of B-lymphocytes is an alternative treatment approach for autoimmune disorders. rituximab—a chimeric, human Igg1 K monoclonal antibody—is specific for the CD20 antigen and can selectively deplete B-cells via antibody-dependent cell-mediated cytotoxicity, complement-mediated cytotoxicity, and inhibition of cell proliferation with direct induction of B-cell apoptosis. Several studies have shown the efficacy and safety of rituximab in the treatment of autoimmune disorders [1-5].

Modulation of cytotoxic T lymphocyte, natural killer cell, antibody-dependent cellular cytotoxicity, and antibody-dependent complement-mediated cytotoxicity by Vernonia cinerea L. and vernolide-A in BALB/c mice via enhanced production of cytokines IL-2 and IFN-γ

Effect of Vernonia cinerea L. and vernolide-A on cell-mediated immune (CMI) response was studied in normal as well as tumor-bearing BALB/c mice. Administration of V. cinerea and vernolide-A significantly enhanced natural killer (NK) cell activity in both normal as well as tumor-bearing animals, and the activity was observed earlier than in tumor-bearing control animals. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were also enhanced significantly in both normal as well as tumor-bearing animals after V. cinerea and vernolide-A administration compared with untreated control tumor-bearing animals. Extract and vernolide-A showed a significant increase in cytotoxic T lymphocyte (CTL) production in both the in vivo and in vitro models. The level of cytokines such as interleukin (IL)-2 and interferon (IFN)-γ were also enhanced by the treatment of V. cinerea and vernolide-A in both normal as well as tumor-bearing animals. This study demonstrated that V. cinerea extract and vernolide-A stimulate the CTL, NK cell, ADCC, and ADCC through enhanced secretion of IL-2 and IFN-γ.

Ofatumumab demonstrates activity against rituximab‐sensitive and ‐resistant cell lines, lymphoma xenografts and primary tumour cells from patients with B‐cell lymphoma

Ofatumumab is a new monoclonal antibody (mAb) targeting a novel membrane-proximal epitope on CD20. To better define ofatumumab's activity, we conducted pre-clinical studies in rituximab-sensitive cell lines (RSCL), rituximab-resistant cell lines (RRCL), ofatumumab-exposed cell lines (OECLs), primary lymphoma cells, and a lymphoma xenograft model. RRCL and OECL were generated by repeated exposure of sensitive cells to escalating doses of rituximab or ofatumumab ± human serum. Antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) assays were performed to assess cellular sensitivity to rituximab or ofatumumab. Ofatumumab elicited a higher rate of CMC in RSCL, RRCL and primary tumour cells. The chronic exposure of lymphoma cells to ofatumumab resulted in rituximab resistance but less ofatumumab resistance. In an in vivo severe combined immunodeficiency mouse model of human lymphoma, ofatumumab prolonged median survival compared to rituximab. While rituximab CMC diminished with CD20 down-regulation in RRCL passages, ofatumumab activity in vitro diminished to a lesser degree. Our data suggest that ofatumumab is more potent than rituximab in rituximab-sensitive or rituximab-resistant models and has the potential to decrease the development of biological resistance in patients with repeated exposure to anti-CD20 mAbs.

Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn’s neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.

Augmentation of humoral and cell mediated immune responses by Thujone

Thujone, a naturally occurring monoterpene, was found to enhance the total WBC count, bone marrow cellularity, number of α-esterase positive cells, number of plaque forming cells in spleen and circulating antibody titer in Balb/c mice (1mg/kg body weight, intraperitoneally for 5days). Thujone treatment enhanced proliferation of splenocytes and thymocytes, both in the presence and absence of specific mitogens. Administration of Thujone was found to stimulate the cell-mediated immunological response in normal and tumor bearing Balb/c mice. A significant enhancement in natural killer (NK) cell mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement mediated cytotoxicity (ACC) in both normal as well as tumor-bearing animals was observed after the administration of Thujone. Production of cytokines such as IL-2 and IFN-γ was significantly enhanced by the administration of Thujone. The stimulatory effect of Thujone on cytotoxic T lymphocyte (CTL) generation was determined by Winn's neutralization assay using CTL sensitive EL4 thymoma cells. Thujone treatment showed a significant increase in CTL production in both the in vivo and in vitro models, as indicated by a significant increase in the life span of tumor bearing animals. All these results indicate that administration of Thujone could enhance the immune response of mice. There was a significant reduction in solid tumor development, mediated by the presence of alert immune responses during Thujone administration.