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Multivalent antibody-recruiting glycopolymers (MARGs) composed of hyaluronic acid (HA) grafted with multiple copies of dinitrophenol (DNP) were developed for targeted cancer immunotherapy. Structure-activity studies demonstrated that the MARGs were able to specifically recognize CD44-positive cancer cells and displayed remarkable antibody-recruiting capacities and tumor cell killing activities dependent on the introduced multivalent effect and the length of PEG linker. One of the MARGs, HA-[PEG3 -DNP]8 , showed the best capacity for clustering anti-DNP antibodies onto CD44-positive cancer cells and displayed potent in vitro anti-cancer activity by triggering complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, we found that HA-[PEG3 -DNP]8 significantly inhibited the xenograft tumor growth of Babl/c nude mice bearing triple negative breast cancer cells, while it did not cause detectable histological cytotoxicity. Given the easy access of this type of natural glycopolymer and the practical synthesis approach, these MARGs provide promising immunotherapeutics for cancer immunotherapy.

C1q-engagement with IgG and IgM type antibodies is the initiating step of classical complement-mediated immunity. The tumor shed antigen CA125 has been reported to have immunosuppressive effects on host tumor responses as well as commercially approved and experimental monoclonal antibody (mAb)-based therapeutic agents. To better understand this effect, molecular and cellular studies were carried out testing the ability of CA125 to perturb the classical complement pathway. Here, we show that patient-derived CA125 inhibits IgG1, IgG3, and IgM-mediated complement-dependent cytotoxicity (CDC) by perturbing antibody-Fc interaction with the C1q complement-initiating protein only in those mAbs that are directly bound by CA125. This mechanism was found to impact naturally generated IgM antibodies as well as experimental and clinically approved mAbs, such as farletuzumab and rituximab, respectively. These data support a role for CA125 in humoral immune suppression and as a potential mechanism by which tumors may possibly avoid host immune responses.

Background Cyclooxygenase-2 (COX-2) is an inducible enzyme with catalytic activity for biosynthesis of prostaglandins which are the key mediators of inflammation. COX-2 is also the therapeutic target for widely used non-steroidal anti-inflammatory drugs (NSAIDs). However, the involvement of COX-2 in xenotransplantation (eg, pig-to-non-human primate) remains poorly recognized. Methods We investigated the mechanisms that regulate COX-2 expression and the effects of COX-2 on porcine aortic endothelial cell (PAEC) viability using in vitro pig-to-primate xenotransplantation model and in vivo pig-to-mouse cellular transplant model. Regulation of COX-2 expression was assessed by real-time quantitative polymerase chain reaction (qPCR) and Western blotting. The effects of inhibition or downregulation of COX-2 on PAEC viability were assessed by propidium iodide (PI)-Annexin V staining and Cell Counting Kit-8 assay. Results Human serum triggered robust COX-2 expression in PAECs in a dose- and time-dependent manner. Induction of COX-2 expression by human serum was partially through activation of both canonical and non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κb) signaling and increasing intracellular calcium. Cytokines like tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), IL-17, were able to induce COX-2 expression. Selective inhibition of COX-2 by celecoxib dramatically decreased PAEC death in vitro and in vivo as defined by propidium iodide (PI)-Annexin V staining. Consistently, downregulation of COX-2 expression by NF-κb inhibitors or calcium chelator BAPTA decreased human serum-induced PAEC death as well. Silencing of COX-2 expression by small interfering RNA (siRNA) protected PAEC viability when transplanted under kidney capsule of C57BL/6 mice. Conclusions Taken together, our data suggest that COX-2 is highly induced in PAECs by xenogenic serum and associated with human antibody-mediated complement-dependent cytotoxicity. COX-2 might be a potential therapeutic target to improve xenotransplantation.

Summary Background Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Controversy exists, however, over whether major histocompatibility complex (MHC)‐mismatched MSCs are recognised by the recipient immune system and targeted for death by a cytotoxic antibody response. Objectives To determine if cytotoxic anti‐MHC antibodies generated in vivo following MHC‐mismatched MSC injections are capable of initiating complement‐dependent cytotoxicity of MSCs. Study design Experimental controlled study. Methods Antisera previously collected at Days 0, 7, 14 and 21 post‐injection from 4 horses injected with donor MHC‐mismatched equine leucocyte antigen (ELA)‐A2 haplotype MSCs and one control horse injected with donor MHC‐matched ELA‐A2 MSCs were utilised in this study. Antisera were incubated with ELA‐A2 MSCs before adding complement in microcytotoxicity assays and cell death was analysed via eosin dye exclusion. ELA‐A2 peripheral blood leucocytes (PBLs) were used in the assays as a positive control. Results Antisera from all 4 horses injected with MHC‐mismatched MSCs contained antibodies that caused the death of ELA‐A2 haplotype MSCs in the microcytotoxicity assays. In 2 of the 4 horses, antibodies were present as early as Day 7 post‐injection. MSC death was consistently equivalent to that of ELA‐A2 haplotype PBL death at all time points and antisera dilutions. Antisera from the control horse that was injected with MHC‐matched MSCs did not contain cytotoxic ELA‐A2 antibodies at any of the time points examined. Main limitations This study examined MSC death in vitro only and utilized antisera from a small number of horses. Conclusions The cytotoxic antibody response induced in recipient horses following injection with donor MHC‐mismatched MSCs is capable of killing donor MSCs in vitro. These results suggest that the use of allogeneic MHC‐mismatched MSCs must be cautioned against, not only for potential adverse events, but also for reduced therapeutic efficacy due to targeted MSC death.

Background The criterion (gold) standard to detect anti-human leukocyte antigen (HLA) antibodies is the complement-dependent cytotoxicity (CDC) assay. Recently, more sensitive methods have been used for the same purpose. Methods This study analyzed 70 serum samples of patients with end-stage renal disease using CDC, CDC with the addition of anti-human globulin (CDC-AHG), CDC with the addition of dithiothreitol (CDC-DTT), and the recent solid-phase immunoassay (SPI; Labscreen PRA) to detect anti-HLA antibodies. Results Mean percent panel reactive antibodies (PRA) detected by SPI was 37.5% (±34.2) higher than the values detected by the other methods. Comparative analyses revealed significant difference between CDC and CDC-AHG, and between CDC and SPI (P < 0.0001), but not between CDC-AHG and SPI (P = 0.8026). Conclusion Although the CDC-AHG method is “old,” its performance to detect anti-HLA antibodies in the samples analyzed was comparable to the SPI in the evaluation of percent class I PRA.

Our previous studies showed that anti-β2M monoclonal antibodies (mAbs) at high doses have direct apoptotic effects on myeloma cells, suggesting that anti-β2M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the ability of the mAbs at much lower concentrations to indirectly kill myeloma cells by utilizing immune effector cells or molecules. Our results showed that anti-β2M mAbs effectively lysed MM cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which were correlated with and dependent on the surface expression of β2M on MM cells. The presence of MM bone marrow stromal cells or addition of IL-6 did not attenuate anti-β2M mAb-induced ADCC and CDC activities against MM cells. Furthermore, anti-β2M mAbs only showed limited cytotoxicity toward normal B cells and nontumorous mesenchymal stem cells, indicating that the ADCC and CDC activities of the anti-β2M mAbs were more prone to the tumor cells. Lenalidomide potentiated in vitro ADCC activity against MM cells and in vivo tumor inhibition capacity induced by the anti-β2M mAbs by enhancing the activity of NK cells. These results support clinical development of anti-β2M mAbs, both as a monotherapy and in combination with lenalidomide, to improve MM patient outcome.

In this study we have evaluated an alternative 96-well format flow cytometry based (FCtox) method which enable simultaneous detection of cytotoxicity and human leukocyte antigen (HLA) antibody binding. Comparable results were obtained in side-by-side comparisons with conventional complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM) in terms of sensitivity and specificity. There was 91 and 93% agreement between results obtained by FCtox and CDC for T and B cells, respectively. In addition, comparable results were obtained with FCtox IgG and FCXM IgG for both T and B cells. Furthermore, compared with a recently developed and highly sensitive Luminex based C1q assay we obtained close to 90% method agreement with the FCtox assay. Our alternative cytotoxicity and IgG binding assay which exhibit low intra-and inter-assay variation will improve the workflow and speed up the pre-transplant testing and also allow continuous monitoring of assay performance and proper quality assurance.

Compound (IIIb) exhibits inhibitory effects against neuromyelitis optica (NMO)-IgG/CDC and may be a potential therapeutic candidate for NMO treatment.

Basnet NB, Ide K, Tahara H, Tanaka Y, Ohdan H. Deficiency of N-glycolylneuraminic acid and Galα1-3Galβ1-4GlcNAc epitopes in xenogeneic cells attenuates cytotoxicity of human natural antibodies. Xenotransplantation 2010; 17: 440–448. © 2010 John Wiley & Sons A/S. Abstract: Background: A number of carbohydrate structures apart from Galα1-3Galβ1-4GlcNAc (Gal) have been implicated as potential xenoantigens. Epitopes of another carbohydrate structure, namely N-glycolylneuraminic acid (NeuGc), are widely expressed on the surfaces of endothelial cells of all mammals, except humans, and are likely targets of anti-non-Gal antibodies (Abs). The purpose of this study is to assess whether deficiency of NeuGc and Gal epitopes in xenogeneic cells attenuates the cytotoxicity of naturally occurring antibodies in human sera. Methods: We generated mice deficient in both α1,3-galactosyltransferase (GalT) and cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Flow cytometric and immunohistochemical studies confirmed the complete absence of Gal and NeuGc expression in hematopoietic cells and tissue sections of the heart, lungs, liver, kidney, and pancreas in GalT−/−CMAH−/− double knockout (DKO) mice. The thymocytes obtained from wild-type (WT), GalT−/−, CMAH−/−, and DKO mice were used to capture xenoreactive Abs present in human sera from healthy volunteers of each blood group. The titers and complement-dependent cytotoxicity (CDC) of these Abs were determined by flow cytometry and the 51Cr release assay, respectively. Results: In all sera, IgM and IgG Abs bound to the thymocytes of WT, GalT−/−, CMAH−/−, and DKO mice. The average values of IgM Abs against thymocytes of WT and CMAH−/− mice were similar, but were statistically higher than those against thymocytes of GalT−/− and DKO mice. The average value of IgG Abs against WT mouse thymocytes was similar to that against GalT−/− mouse thymocytes, but was significantly higher than that against thymocytes of CMAH−/− and DKO mice. Remarkable CDC of human sera was observed against the thymocytes of WT and CMAH−/− mice, whereas thymocytes of GalT−/− and DKO mice were more resistant to lysis. CDC of human sera against CMAH−/− mouse thymocytes was significantly lower than that against WT mouse thymocytes. In addition, CDC against DKO mouse thymocytes, which appeared undetectable even at lesser serum dilutions, was significantly lower than that against GalT−/− mouse thymocytes. Conclusions: A DKO mice strain lacking both Gal and NeuGc antigens in several tissues has been generated. Comparing CDC of human sera against mouse thymocytes, the DKO strain shows a statistically significant but a small additional reduction in CDC compared to that already achieved in GalT−/− mice.

Complement-dependent serum-mediated cytotoxicity (CDC) was measured in a 51-chromium release assay against autologous tumor cells from 7 non-invasive and 9 invasive transitional-cell tumors of the urinary bladder. CDC was demonstrated against tumor cells from invasive tumors. Heat-inactivation of autologous sera lead to complete loss of cytotoxicity. There were no differences in CDC of autologous sera from patients and allogenic sera from controls. The cytotoxic response seems to be strongly dependent on the target cell. CDC was significantly reduced by use of an allogenic C 2 deficient serum. Direct immunofluorescence did not reveal any tumor cell associated immunoglobulins of IgG or IgM classes. Indirect immunofluorescence with autologous heat-inactivated sera demonstrated in most cases both IgG and IgM attachment to the tumor cells, but there were no obvious relations between indirect immunofluorescence and CDC. Absorption of allogenic sera to trypsin- or neuraminidase-treated erythrocytes did not affect CDC of these sera against invasive tumor targets. The results indicate a complement-dependent cytotoxicity against target cells from invasive bladder tumors. Complement seems to be activated through the classical pathway, but the possible role of naturally-occurring antibodies against invasive tumor targets is not clarified.

Complement-dependent cytotoxicity (CDC) was measured in a 51-Cr release assay against tumor cells from 13 non-invasive and 7 invasive transitional-cell tumors of the urinary bladder. CDC was compared between mechanically dispersed tumor cells and neuraminidase-treated tumor cells. Neuraminidase treatment of bladder tumor cells enhanced their susceptibility to complement-dependent cytolysis. There were no differences in CDC between autologous and allogenic sera. Mechanically dispersed tumor cells showed no significant differences in susceptibility when non-invasive and invasive tumor cells were compared, whereas significant differences in CDC were seen when neuraminidase-treated non-invasive and invasive tumor cells were used as targets. A C2 deficient serum showed significantly reduced cytotoxicity suggesting that the CDC reaction requires classical complement activation. A hypogammaglobulinemic serum showed stronger CDC compared to autologous and other allogenic sera and upon dilution of autologous sera and hypogammaglobulinemic serum CDC declined parallelly.

Two well characterized Eighth International Workshop sera, 8w1090 and 8w112 , described to have anti-DR3 and anti-DR5 activity, respectively, were tested by standard and antiglobulin augmented (AHG) complement dependent cytotoxicity (CDC) assays with 21 target cells. We demonstrate that both sera, in fact, have only one antibody (most likely anti-MT2), but have different CYNAP patterns. These observations are discussed with regard to a more complete analysis of antisera that detect gene products of the HLA-DR region.

In an attempt to study the gene products of the HLA complex, a monoclonal antibody, named HU-30, was produced by immunizing BALB/c mice with a cultured human B lymphoblastoid cell line, Shi-C3 (Aw24, Aw31, Bw51, Bw52, DR2, DR blank, MT1, MT2, MB3). HU-30 belonged to the IgG2 subclass and was active in complement dependent cytotoxicity. When the serological specificity was evaluated with a panel of 15 cultured human lymphoblastoid cell lines, it was found that HU-30 detected a polymorphic determinant, common to HLA-DR1 and 2, with much stronger cytotoxic activity against HLA-DR2 positive B cell lines. When HU-30 was tested against a panel of B cells from 84 healthy donors at a dilution of 2(-13), it gave positive reactions only with cells typed as HLA-DR2. Furthermore, sequential coprecipitation studies indicated that the HU-30 determinant was borne on the molecules carrying the HLA-DR determinants. Thus, HU-30 appears to be of great value as a tissue typing reagent monospecific for HLA-DR2.

The expression of H-Y antigen on preimplantation mouse embryos has been studied by complement dependent cytotoxicity. Embryos at the 8-cell stage were exposed to anti-H-Y and complement and then scored for cell death. Overall, half of the treated embryos were killed, retarded, or contained dead cells. Of those embryos which were not affected by anti-H-Y, 92% were female. In control experiments, neither normal serum plus complement nor complement alone had any specific effect on male embryos. It is concluded, therefore, that the H-Y antigen is present on preimplantation mouse embryos.

Eight human sera from healthy individuals with no history of immunization with human transplantation antigens have demonstrated complement dependent cytotoxicity to cells of some patients with active acute leukemia. The antigen(s) detected by these sera are absent from normal and remission lymphocytes and appear most often in ALL patients with a high peripheral lymphoblast count. Some B and T lymphoblastoid cell lines carry the antigen(s) as evidenced by their ability to react with and absorb the antileukemia activity. The data support the existence of at least two overlapping specificities detected by these antileukemia sera. The leukemia antigen(s) show no strong correlation with any known HLA antigen. These observations provide evidence for a human leukemia blast associated antigen or set of antigens which may be immunogenic in man. Their relationship to normal HLA antigens of loci other than A, B or C is unknown.

Purified HL-A antigen bearing cell membrane components were prepared from human lymphoid cells by papain digestion and physicochemical fractionation. Rabbits immunized with these components produced antisera which had high cytotoxic titers against human lymphoid cells and other human tissue culture cells. The cytotoxicity was inhibited only by those membrane fractions which contained HL-A activity. Absorption and cytotoxicity inhibition studies showed that the antisera possessed antibodies against antigens shared by humans and monkeys, antigens common to different human tissues, lymphoid antigens and allotypic antigens. To determine if the heterologous antisera reacted with immunoglobulin-like receptor sites on the lymphoid cell surface, purified human immunoglobulins were tested for their ability to inhibit the cytotoxic antisera. The evidence obtained from complement and noncomplement dependent systems indicated that the surface components recognized by the antisera did not include immunoglobulins. Addition of IgM to the complement dependent cytotoxicity assay, however, did interfere with the reaction by binding complement.

Complement-dependent cytotoxic antibodies in syngeneic serum from rats carrying transplanted aminoazo dye-induced hepatomas (D23 and D33) and from rats immunized with irradiated tumor cell or homogenates were studied by a short-term 51Cr release assay. The tumor-bearer sera (TBS) were subjected to chromatography on unsolubilized protein A and Sepharose 4B. The cytolytic activity of D23 TBS was recovered in the IgM molecular weight region, whereas no such activity was obtained in the IgG fraction. As judged from immunodiffusion experiments, the IgM molecular weight fraction did not contain any aggregated or complexed IgG. Moreover, in immunofluorescence tests against viable hepatoma cells, using a specific anti-rat IgG conjugate, the TBS were negative. Cross-testing of D23 and D33 TBS against the two hepatomas and cross-absorption of the sera with tumor plasma membranes revealed no tumor specificity in these cytotoxicity reactions. Furthermore, neither fetal nor early postnatal liver cells could absorb out the activity. Absorptions with adult liver plasma membranes, however, abrogated the cytotoxic activity, and even more effective in this respect were homogenates from kidney and small intestine, thus indicating that the cytotoxic antibodies are directed against 'normal' adult antigen(s) present also in tissues other than liver.

The recent introduction of new technologies such as Luminex has provided alternative methods to the Complement Dependent Cytotoxicity (CDC) test for HLA specific antibody detection. In this study we compared the results obtained with CDC to those obtained using a Luminex method with the aim of evaluating the impact of this new technology on antibody screening policies in our transplant setting.A total of 1,421 sera, acquired from patients on the waiting list for a kidney transplant or following transplantation, were tested by both methodologies. CDC was performed using a whole lymphocyte population comprising a panel of 52 cells. The percentage panel reactive antibodies (PRA) and antibody specificity were evaluated using Lambda Scan Analysis software. For the Luminex method sera screening and identification of antibody specificity were carried out using the LABScreen Mixed and LABScreen PRA respectively. The overall concordance between the results obtained using the CDC and the Luminex methods was 85%. HLA antibody specificity was confirmed in 96% of the sera which tested positive using the Luminex system and serum positivity corresponded with a previous sensitisation event in these individuals. Using the Luminex method 18% of patients on the waiting list were considered and managed as sensitised as compared to 7% when testing with CDC alone. The Luminex method was able to detect a number of antibody specificities significantly more frequently than the CDC method and in addition the CDC method failed to detect some of the antibody specificities detected by the Luminex system. Based on this comparison study we have incorporated the Luminex methodology into our screening strategy.

The objective of this study was to evaluate the specificities of HLA class I (-A,-B) and class II (-DR,-DQ) antibodies (Ab) detected in the sera of alloimmunized patients waiting for a subsequent renal transplantation. The study group consisted of 62 dialysis patients (42 men and 20 women, mean age: 43 +/- 18 yr) on waiting list for a subsequent kidney transplant (52 for a second and 10 for a third transplant) at S. Martino Hospital Transplant Centre in Genoa/Italy, who were enrolled from 2002 to 2004 for HLA antibody screening. Complement dependent cytotoxicity (CDC) technique was used firstly to select anti-HLA class I sensitized patients; indeed sera from 50 individuals out of 62 (80.6%) were found to display persistent HLA class I PRA (panel reactive antibody) values >4% (range: 20-100). ELISA technique was subsequently adopted to analyze HLA class I Ab positive sera for the presence also of HLA class II Ab and to characterize class I and class II Ab specificities. Anti-class I immunized patients were divided in three groups according to the type of class I Ab specificities, that were classified as private, public, and multispecific. The first group included 35 patients (70% of the total number of positive patients) showing only antibodies directed against private HLA class I specificities, represented in 33 cases by those expressed by graft donors (first or second transplant). In this group anti-class I PRA% values ranged from 20% to 60%. HLA class II Ab, with an heterogeneous specificity pattern (private, public or multispecific), were present in 25 (78.1%) out of the 32 patients, whose sera were also available for this analysis. The second group comprised 12 patients (24%) who displayed antibodies directed against class I public epitopes belonging to CREGs (Cross reactive Groups) or an association of anti-private and anti-public antibodies. In this group PRA values ranged from 25% to 90%. Five patients (46.7%) were positive for HLA class II Ab, whose specificity pattern appeared also heterogeneous (private or multispecific). The third group was represented by three patients (6%) displaying multispecific antibodies with PRA values > or = 90%. No multispecific class II Ab were found in this group, where only two patients had class II Ab showing anti-private or anti-private plus public specificities. Globally, 74% of anti-class I Ab positive patients, having at least one HLA class II antigen mismatch, appeared also positive for class II Ab. These results indicate that: (i) a large proportion of patients, waiting for a kidney retransplantation, display in their sera alloantibodies specific for graft mismatched HLA class I (80.6%) and class II antigens (54.2); (ii) the immunogenic determinants, mainly involved in HLA class I and II specific Ab production, were, in a significant rate, private specificities of mismatched HLA antigens (70% for class I and 59.4% for class II), and in a lesser percentage by public (CREG) epitopes (24% for class I and 34.3% for class II). In a few patients only no HLA class I and class II Ab specificities could be determined, as they displayed multispecific antibodies (6% for class I and 6.2% for class II). These findings may have important implications to improve donor-recipient matching in dialysis recipients waiting for a subsequent renal transplantation.

Background: Tumor cells can escape complement-dependent cytotoxicity (CDC) by expressing complement restriction factors (CRFs), CD46, CD55 and CD59. CRF-expression in non-neoplastic mucosa of the head and neck was compared with biopsies of the head and neck squamous cell carcinoma (HNSCC) and cell lines derived from oral squamous cell carcinomas (OSCC). Methods: Normal mucosa and HNSCC tumor tissue (poor, moderate, or well differentiated) specimens were immunostained with anti-CRF monoclonal antibodies. Immunostaining of the OSCC cell lines (SCC12 and SCC71) was examined under laser scan fluorescence microscopy. Results: CD46, CD55 and CD59 were highly expressed in HNSCC cells including T1/T2N0M0 stages. The CRF expression was much lower or absent in non-neoplastic squamous epithelia or in the submucosa of both normal and tumor tissues. Conclusions: Enhanced staining of tumor tissues at stages T1/T2 indicates that the CRFs are overexpressed by primary tumors before metastasis to either lymph nodes or organs (N0M0 stage) suggesting that CRFs are formed early during tumorigenesis.

Metastases from renal cell carcinomas (RCC) are resistant to radiation and chemotherapy but are relatively immunogenic. We have investigated the possibility to eliminate human RCC micrometastases using MAb G250. G250 penetrates human micrometastases completely in a spheroid model and induces complement deposition rapidly on the outmost cell layers. However, complement dependent cytotoxicity (CDC) was barely detected using either 51 chromium release assays or confocal microscopy, due to relatively low expression of the G250 antigen and the effect of membrane bound complement regulatory proteins. Addition of blocking anti-CD59 MAbs enhanced formation of C5b-9 and consequently complement mediated lysis (13%). Complement assisted cellular cytotoxicity (CACC) was not detectable, although the iC3b ligand and CR3 receptor were present on respectively target and effector cells. Addition of soluble -glucan induced the killing of MAb and iC3b opsonized spheroids by effector cells (6 –21%). Despite a lower affinity for G250 antigen, a bispecific anti-G250*anti-CD55 MAb enhanced cell killing in spheroids comparable to the parental G250 MAb. Our results suggest that complement-activating G250 in combination with anti-mCRP MAbs is able to kill human RCC cells in micrometastasis in vitro. For CACC the presence of CR3-priming -glucan seems to be obligatory. In vivo, bi-MAb may be more effective as therapeutic agent due to its increased C5a generating properties. © 2004 Wiley-Liss, Inc.

We generated fully human mAbs (HmAbs) to carcinoembryonic antigen (CEA) using the KM mouse, which carries a human chromosome 14 fragment containing the entire Ig H chain loci and human kappa L chain segments in the mouse genome. Forty-six hybridoma clones producing HmAbs to CEA were thus obtained by fusing the P3-U1 mouse myeloma cells with splenocytes of the KM mice immunized with CEA. Among them, 22 clones produced HmAbs that reacted with CEA but not with 3 other CEA-related cell adhesion molecule (CEACAM) family members, CEACAM1, CEACAM6 and CEACAM8. In 12 HmAbs examined, 8 were IgG4, 2 were IgG3, 1 was IgG2, and the other was IgG1. The affinity constants for CEA of these HmAbs were comparable to those of the previously prepared mouse anti-CEA mAbs (MmAbs). BIAcore analyses revealed that 1 and 2 of the 22 HmAbs react with 2 epitopes defined by MmAbs on the domain N and the domain A1 or B1 of CEA, respectively. In the presence of human complement in vitro, 2 HmAbs tested showed substantial cytotoxicity, namely, 50-65%, against CEA-expressing tumor cells. With human lymphokine-activated killer cells in vitro, 3 HmAbs tested exhibited 40-65% Ab-dependent cell-mediated cytotoxicity against the tumor cells. Moreover, one of the HmAbs induced a significant inhibition of tumor growth when administered to mice xenografted with the CEA-expressing cells. Considering their lack of immunogenicity to humans, these CEA-specific HmAbs may be useful for immunotherapeutic approaches as well as for immunodiagnosis.

Rituximab (Mabthera) and alemtuzumab (Campath(R), Mabcampath(R)) are non-conjugated IgG1 therapeutic monoclonal antibodies directed against the CD20 and CD52 surface antigens respectively. They are presently used in the therapy of indolent B-cell non-Hodgkin's lymphoma (B-NHL) and of B-cell chronic lymphocytic leukaemia, and are thought to act mainly through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Here we have analysed the capacity of these two monoclonal antibodies to lyse cell lines of acquired immunodeficiency syndrome (AIDS)-related B-NHL through either complement activation or antibody-dependent cytotoxicity. Rituximab strongly activated both CDC and ADCC against CD20-positive AIDS-NHL cells lines, inducing up to 60-98% and 20% specific lysis respectively. In contrast, alemtuzumab was a poor activator of CDC, even in the AIDS-NHL cell lines expressing high amounts of CD52, leading to a lysis of only 1-30%, whereas it was at least as strong as rituximab in inducing ADCC of the same lines (up to 30% specific lysis). Altogether, these data offer a first in vitro rationale supporting the therapeutic use of rituximab for CD20-positive AIDS-NHL.

The hamster to rat xenograft combination is considered to be a “difficult” concordant combination. Although the rat lacks preformed anti-hamster antibodies, there is rapid production of antibodies upon exposure to hamster antigens. When vascularized hamster xenografts are rejected in the rat, anti-hamster antibodies are invariably detected in both the serum and in the rejected tissue. In allogeneic and other concordant xenogeneic (rat to mouse) combinations, rapamycin has been shown to markedly augment the survival of skin grafts in ALS-treated, bone marrow injected mice. We investigated the ability of rapamycin to prolong the survival of skin grafts and block antibody production in the hamster to rat combination. Outbred Golden Syrian hamsters served as skin and marrow donors and Lewis rats were recipients. ALS was administered on days -1, 0, and +2 relative to skin grafting. Rapamycin was administered at 3 mg/kg on alternate days from day +1 through day +13 (seven doses). Donor specific bone marrow was infused IV on day +4 at a dose of either 100 times 106 or 250 times 106 nucleated cells. Antibody titers were determined serially by using a complement dependent cytotoxicity assay. The administration of rapamycin alone (MST 8.5 days) and ALS alone (MST 10 days) prolonged graft survival slightly but significantly compared with untreated controls (MST 7 days). The addition of BM had no additional effect on graft survival beyond that achieved with ALS alone. Rapamycin markedly prolonged skin graft survival when added to ALS (MST 16 days), ALS+BM100 (MST 19 days), or ALS+BM250 (MST 20.5 days). Rats receiving ALS or ALS+BM produced anti-hamster antibodies in a slightly delayed fashion and at a slightly lower titer when compared to controls. Treatment with rapamycin alone substantially diminished but did not completely eliminate anti-hamster antibody production. Rats receiving rapamycin with ALS or ALS+BM failed to produce anti-hamster antibodies entirely, although they all eventually rejected the hamster skin grafts. Rapamycin, when used in combination with ALS or ALS plus BM is able to prolong skin graft survival and completely abolish antibody production in the hamster to rat combination. The eventual rejection of hamster skin grafts in this circumstance occurs in the absence of anti-hamster antibodies.

Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

Two kinds of human monoclonal antibodies, CLN-IgG and SLN-IgG, were obtained from human x human hybridomas, termed CLNH11 and SLNF10, respectively. These antibodies recognized different molecules which were expressed simultaneously on the cell surface of tumor cells. When both antibodies were added into culture medium in the presence of complement, a remarkable synergistic cytotoxic-effect against glioblastoma cells was observed.

Human lymphoid cells (NC-37) persistently infected with either measles virus (Schwarz and TYCSA strains) or subacute sclerosing panencephalitis (SSPE) virus (Halle and Mantooth strains) were destroyed in the presence of complement by anti-measles sera as well as by sera from SSPE patients. The cytotoxic activity was demonstrated in both IgG and IgM fractions of measles convalescent sera, but only in IgG fraction of SSPE sera. Measles convalescent sera completely lost the cytotoxic activity to all the cell lines, when absorbed with any one of the cell lines, indicating that the viral surface antigens of these cell lines infected with measles or SSPE virus are identical. On the other hand, the cytotoxic activity of SSPE sera could not be readily absorbed with these cells. Thus, the affinity of SSPE sera for the viral surface antigens might be lower than that of measles convalescent sera.

SUMMARY The expression of H-2 antigenic specificities on Meth-A and meth-A-vaccinia were compared. Their ability to inhibit complement dependent cytotoxicity of 51Cr-labelled normal lymphoid target cells was tested by anti H-2 sera of restricted specificity. Normal lymphocytes positive or negative for a particular specificity, were used as controls. With this assay the expression of H-2-like specificities of foreign haplotypes on Meth-A cells passaged together with vaccinia virus was confirmed. In addition, using an immunofluorescence technique the expression of some foreign H-2 antigens on non-infected Meth-A tumour cells was shown. Anti-vaccinia virus sera were used to compare the reactivity of Meth-A and Meth-A-vaccinia. Unexpectedly, anti-vaccinia sera stained non-infected Meth-A and other tumour cells of different H-2 origin and absorption of these sera with normal Meth-A left little activity behind when testing against Meth-A-vaccinia. Finally, the role that the foreign H-2 antigenic specificities present in Meth-A after vaccinia virus infection may have in the growth of these tumour cells in syngeneic recipients was studied. A powerful inhibitory effect was observed and regressor mice were found to be resistant to a challenge of non-infected meth-A. In contrast, vaccinia virus was irrelevant to the rate of growth of P815 Y in DBA/2 mice. These results are discussed in relation to the origin of the foreign H-2 antigens on tumour cells after virus infection (derepression) and the role that this new antigenicity could have in immunopotentiation.

Summary. A patient with agranulocytosis and myeloid marrow hypoplasia following a second exposure to propylthiouracil (PTU) was studied for antibodies against mature blood cells and bone marrow precursor cells. During the acute phase of the agranulocytosis, significant growth inhibition of the myeloid committed progenitor cells (CFU-GM) was found following incubation with complement, indicating the presence of in-vivo cell bound cytotoxic antibodies. Using immunofluorescence and complement dependent cytotoxicity techniques it was demonstrated that acute phase and recovery phase sera contained circulating antibodies, reactive not only with differentiated granulocytes and monocytes but also with myeloid and erythroid (BFU-E/CFU-E) progenitor cells. Complement dependent lysis of the progenitor cells was facilitated by preincubation with PTU. These results indicate that the agranulocytosis was mediated by a PTU dependent antibody that affected both mature blood cells and bone precursor cells.

Bone marrow cells were transferred into 700 RX-irradiated syngeneic mice, and reganeration of the thymus was demonstrated by determining the total amount of DNA in the thymus. The amount of DNA in the regenerated thymus 9 days after X-irradiation paralleled the number of cells injected, the range being 0 to 106 cells. These results indicate that regeneration during this period reflects the proliferation of thymocyte precursors existing in bone marrow cells, therefore such can be used as an indicator to determine the number of thymocyte precursors. Thymocyte precursors were separated from spleen colony forming cells and soft agar colony forming cells by one G velocity sedimentation. Thymocyte precursors (more than 4.3 mm/hour sedimentation rate) sedimented faster than did spleen colony forming cells (peak at about 3.0 mm/hour) and Thy-1 antieen was already evidenced on the cell surface as demonstrated by complement dependent cytotoxicity. Soft agar colony forming cells also sedimented faster, suggesting that cells destined to become one type of cell are larger than multi-potential stem cells.

Complement dependent cytotoxicity to malignant melanoma tumor cells was demonstrated in incubations that included rheumatoid factor (RF) positive plasma and normal plasma. Cytotoxicity was not demonstrated to cells from a number of normal tissues. The phenomenon involved coating of tumor cells with immunoglobulins and complement fixation. The activity in RF positive plasma was present in high titer an may not be RF. The activity in normal plasma was present in low titer, was found in plasma from eight out of 11 healthy subjects and may be a naturally occurring antibody. This previously undescribed humoral cytotoxicity system may participate in tumor host interactions, especially after therapy, when the majority of patients become RF seropositive.

With the aid of an assay measuring complement dependent cytotoxicity mediated by syngeneic antibodies, we performed a serological analysis of surface antigens of a polyoma-virus-induced murine tumor (SEYF-a). In vivo propagated SEYF-a ascites tumor cells expressed a specific membrane antigen in addition to various other cross-reacting antigens. Among these we could identify at least four separate specificities. Two of these were present on Mu LV-induced lymphoma cells, the first on Moloney-virus-induced YAC cells and the second on Gross-virus-induced GHA cells. The third cross-reacting antigen was detected on EL-4 cells. At least one additional specificity was present on two methylcholanthreneinduced murine sarcomas. Normal syngeneic lymphoid cells were insensitive to cytotoxicity mediated by the anti-tumor antisera. Quantitative and perhaps also qualitative differences between the antigenic expression of in vivo propagated and cultured SEYF-a cells were indicated. These studies show that hyperimmune sera produced in syngeneic mice against transplanted tumors may contain a considerable number of antibody specificities, only some of which are specific for the tumor. Furthermore the results also suggest that polyoma-virus-induced tumors may possess individually distinct antigenic specificities, over and above the known cross-reacting TSTA or TSSA type antigen. Analyse Serologique Des Antigenes Specifiques Et A Reactions Croisees De La Membrane Des Cellules D'une Tumeur Murine Induite Par Le Virus Polyome Nous avons effectue une analyse serologique des antigenes de la surface des cellules d'une tumeur murine induite par le virus polyome (SEYF-a) en mesurant la cytotoxicite des anticorps syngeniques liee au complement. Les cellules de la tumeur ascitique SEYF-a propagees in vitro expriment un antigene membranaire specifique ainsi que divers autres antigenes a reactions croisees. Parmi ces derniers, nous avons pu identifier au moins quatre specificites distinctes. Deux de ces antigenes etaient presents sur des cellules de lymphome induit par le MuLV, le premier sur des cellules YAC induites par le virus de Moloney et le second sur des cellules GHA induites par le virus de Gross. Le troisieme a ete detecte sur les cellules EL-4; il y en avait au moins un quatrieme sur des sarcomes murins induits par le methylcholanthrene. Les cellules lymphoides syngeniques normales sont insensibles a la cytotoxicite mediee par les antiserums antitumoraux. Des differences d'expression antigenique, non seulement quantitatives mais peut-ětre aussi qualitatives, sont apparues entre les cellules SEYF-a propagees in vitro et les měmes cellules en culture. Ces etudes montrent que les serums hyperimmuns produits chez des souris syngeniques contre les tumeurs transplantees peuvent contenir un nombre considerable de specificites dont quelques-unes seulement sont specifiques pour la tumeur. De surcroit, les resultats conduisent a penser que les tumeurs induites par le virus polyome peuvent posseder des specificites antigeniques individuellement uniques en plus des antigenes du type TSTA ou TSSA, dont on connait les reactions croisees.