Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions.

Monoclonal antibody-based therapies are increasingly being used to treat cancer. Some mediate their therapeutic effects through modifying the function of immune cells globally, while others bind directly to tumor cells and can recruit immune effector cells through their Fc regions. As new direct-binding agents are developed, having the ability to test their Fc-mediated functions in a high-throughput manner is important for selecting antibodies with immune effector properties. Here, using monoclonal anti-CD20 antibody (rituximab) as an example and the CD20+ Raji cell line as tumor target, we describe flow cytometry-based assays for determining an antibody's capacity for mediating antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). These assays are sensitive, reliable, affordable and avoid the use of radioactivity.

Genetically-engineered pigs as sources for clinical red blood cell transfusion: What pathobiological barriers need to be overcome?

An alternative to human red blood cells (RBCs) for clinical transfusion would be advantageous, particularly in situations of massive acute blood loss (where availability and compatibility are limited) or chronic hematologic diseases requiring frequent transfusions (resulting in alloimmunization). Ideally, any alternative must be neither immunogenic nor pathogenic, but readily available, inexpensive, and physiologically effective. Pig RBCs (pRBCs) provide a promising alternative due to their several similarities with human RBCs, and our increasing ability to genetically-modify pigs to reduce cellular immunogenicity. We briefly summarize the history of xenotransfusion, the progress that has been made in recent years, and the remaining barriers. These barriers include prevention of (i) human natural antibody binding to pRBCs, (ii) their phagocytosis by macrophages, and (iii) the T cell adaptive immune response (in the absence of exogenous immunosuppressive therapy). Although techniques of genetic engineering have advanced in recent years, novel methods to introduce human transgenes into pRBCs (which do not have nuclei) will need to be developed before clinical trials can be initiated.

Targeting Immunity in End-Stage Renal Disease.

BACKGROUND: Despite the stable incidence of end-stage renal disease (ESRD), it continues to be associated with an unacceptably high cardiovascular risk. SUMMARY: ESRD is characterized by enhanced oxidative stress and severe inflammation, which boost cardiovascular risk, thus increasing cardiovascular-associated mortality rate. While substantial effort has been made in the technological innovation of dialytic techniques, few significant advances have been made to reduce inflammation in patients with ESRD. Indeed, this contrasts with the extensive scientific breakthroughs made in the basic field of science in targeting inflammation. There is thus a pressing need for clinical trials to test the effect of reducing inflammation in patients with ESRD. Here, we will revisit the negative effect of ESRD on inflammation and explore the impact of enhanced inflammation on cardiovascular outcomes and survival in patients with ESRD. Finally, we will discuss the need for clinical trials that target inflammation in ESRD, as well as weigh potential disadvantages and offer novel innovative approaches. Key Message: We will try to understand why the issue of inflammation has not been successfully addressed thus far in patients with ESRD, while at the same time weighing the potential disadvantages and offering novel innovative approaches for targeting inflammation in patients with ESRD.

Comparative Assessment of a Single Dose and a 2-dose Vaccination Series of a Quadrivalent Meningococcal CRM-conjugate Vaccine (MenACWY-CRM) in Children 2-10 Years of Age.

BACKGROUND: We compared the immunogenicity, safety and 1-year antibody persistence of a single-dose and a 2-dose series of a licensed meningococcal ACWY-CRM conjugate vaccine (MenACWY-CRM) in 2- to 10-year-old children. METHODS: In this phase III, multicenter, observer-blind study, children aged 2-5 years (n = 359) and 6-10 years (n = 356) were randomized 1:1 to receive 2 doses of MenACWY-CRM (ACWY2) or 1 dose of placebo followed by 1 dose of MenACWY-CRM (ACWY1), 2 months apart. Immunogenicity was measured using serum bactericidal activity with human complement (hSBA). Primary outcomes were to assess the immunologic noninferiority and superiority of ACWY2 versus ACWY1. RESULTS: One-month after the second dose, the hSBA seroresponse in ACWY2 was noninferior to ACWY1 for all 4 serogroups, in both age cohorts, and was superior for serogroups C and Y in the 2- to 5-year-old age cohort and for serogroup Y in the 6- to 10-year-old age cohort. Overall, 90%-99% of subjects in ACWY2 and 65%-96% in ACWY1 had hSBA titers ≥ 8; geometric mean titers were 1.8- to 6.4-fold higher in ACWY2 than ACWY1 across serogroups. At 1 year postvaccination, geometric mean titers declined, and the differences between ACWY2 and ACWY1 remained significant for serogroups A and C in the 2- to 5-year-old age cohort and for serogroups C and Y in the 6- to 10-year-old age cohort. The safety profile of MenACWY-CRM was similar in both groups. CONCLUSIONS: The single dose and 2-dose MenACWY-CRM series were immunogenic and well tolerated. Although antibody responses were greater after 2 doses, especially in the 2- to 5-year-old age cohort, this difference was less pronounced at 1 year postvaccination.

Systematic comparison of four cell- and Luminex-based methods for assessment of complement-activating HLA antibodies.

BACKGROUND: Efforts to increase the specificity and sensitivity of human leukocyte antigen (HLA) antibody detection assays recently led to the establishment of two novel Luminex bead-based assays to detect complement-activating antibodies by the assessment of complement products C1q or C4d. Here, we present a systematic comparison of the four methods, complement-dependent lymphocytotoxicity (CDC) and C1q-, C4d-, and IgG-Luminex, to assess or predict the complement-binding capability of HLA IgG antibodies. METHODS: Forty-five sera of highly immunized patients have been assessed by in-house modified C1q- and C4d-Luminex assays and compared with standard CDC and IgG-Luminex. RESULTS: Antibody specificities assigned by the C1q- and C4d-Luminex assay revealed an excellent concordance of 94% and 97% for HLA class I and II, respectively. Complement-fixing HLA class II antibodies were found less frequently among IgG antibodies compared with class I. Both C1q- and C4d-Luminex detected, on average, three times more specificities than CDC. Although we found a high correlation of mean fluorescence intensity values between C1q- and C4d-Luminex assays, IgG mean fluorescence intensity was not a suitable surrogate marker for the prediction of complement binding. CONCLUSIONS: C1q- and C4d-Luminex assays are characterized by an increased sensitivity and specificity compared with CDC, the current standard in detecting complement-fixing HLA antibodies. Pretransplantation risk assessment for transplantation but also posttransplantation monitoring are important applications for both assays to improve overall allograft survival.

Therapeutic assessment of SEED: a new engineered antibody platform designed to generate mono- and bispecific antibodies.

The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands therapeutic applications of natural antibodies. This new protein engineered platform is based on exchanging structurally related sequences of immunoglobulin within the conserved CH3 domains. Alternating sequences from human IgA and IgG in the SEED CH3 domains generate two asymmetric but complementary domains, designated AG and GA. The SEED design allows efficient generation of AG/GA heterodimers, while disfavoring homodimerization of AG and GA SEED CH3 domains. Using a clinically validated antibody (C225), we tested whether Fab derivatives constructed on the SEED platform retain desirable therapeutic antibody features such as in vitro and in vivo stability, favorable pharmacokinetics, ligand binding and effector functions including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. In addition, we tested SEED with combinations of binder domains (scFv, VHH, Fab). Mono- and bivalent Fab-SEED fusions retain full binding affinity, have excellent biochemical and biophysical stability, and retain desirable antibody-like characteristics conferred by Fc domains. Furthermore, SEED is compatible with different combinations of Fab, scFv and VHH domains. Our assessment shows that the new SEED platform expands therapeutic applications of natural antibodies by generating heterodimeric Fc-analog proteins.

Assessment of vaccine potential of the Neisseria-specific protein NMB0938.

The availability of complete genome sequence of Neisseria meningitidis serogroup B strain MC58 and reverse vaccinology has allowed the discovery of several novel antigens. Here, we have explored the potential of N. meningitidis lipoprotein NMB0938 as a vaccine candidate, based on investigation of gene sequence conservation and the antibody response elicited after immunization in mice. This antigen was previously identified by a genome-based approach as an outer membrane lipoprotein unique to the Neisseria genus. The nmb0938 gene was present in all 37 Neisseria isolates analyzed in this study. Based on amino acid sequence identity, 16 unique sequences were identified which clustered into three variants with identities ranging from 92 to 99%, with one cluster represented by the Neisseria lactamica strains. Recombinant protein NMB0938 (rNMB0938) was expressed in Escherichia coli and purified after solubilization of the insoluble fraction. Antisera produced in mice against purified rNMB0938 reacted with a range of meningococcal strains in whole-cell ELISA and western blotting. Using flow cytometry, it was also shown that anti-rNMB0938 antibodies bound to the surface of the homologous meningococcal strain and activated complement deposition. Moreover, antibodies against rNMB0938 elicited complement-mediated killing of meningococcal strains from both sequence variants and conferred passive protection against meningococcal bacteremia in infant rats. According to our results, NMB0938 represents a promising candidate to be included in a vaccine to prevent meningococcal disease.

Quantitation of HLA proteins incorporated by human immunodeficiency virus type 1 and assessment of neutralizing activity of anti-HLA antibodies.

Human anti-human leukocyte antigen (HLA) antibodies were assessed for neutralizing activity against human immunodeficiency virus type 1 (HIV-1) carrying HLA alleles with matching specificity. Multiparous women carrying anti-HLA antibodies were identified. Plasma samples from those women were confirmed as having antibodies that specifically bound to HLA proteins expressed on the peripheral blood mononuclear cells (PBMCs) of their husbands. A primary HIV-1 isolate was cultured in the husband's PBMCs so that the virus carried matching HLA alleles. To determine the HIV-1-neutralizing activity of anti-HLA antibodies, the infectivity of the virus for GHOST cells (which express green fluorescent protein after HIV infection) was investigated in the presence of a plasma sample positive for the respective anti-HLA antibody. A neutralization assay was also performed using purified immunoglobulin G (IgG) from two plasma samples, and two plasma samples were investigated in the presence of complement. The prerequisite for anti-HLA antibody-mediated neutralization is incorporation of HLA proteins by HIV-1. Therefore, the extent of incorporation of HLA proteins by the primary HIV-1 isolate was estimated. The ratios of HLA class I protein to HIV-1 capsid (p24) protein cultured in the PBMCs of two healthy individuals were 0.017 and 0.054. These ratios suggested that the HIV-1 strain used in the assay incorporated more HLA proteins than gp160 trimers. Anti-HLA antibody-positive plasma was found to contain antibodies that specifically reacted to HIV-1 carrying cognate HLA alleles. However, incubation of HIV-1 with anti-HLA antibody- positive plasma or purified IgG did not show a reduction in viral infectivity. HIV-1-neutralizing activity was also not detected in the presence of complement. This study shows that HIV-1 primary isolates cultured in PBMCs contain significant amounts of HLA proteins. However, the binding of antibodies to those HLA proteins does not mediate a reduction in viral infectivity.

Host defence against disseminated Candida albicans infection and implications for antifungal immunotherapy.

The different manifestations of Candida albicans infection are dictated by an underlying defect in the immune response of the host. Protective immunity to disseminated candidiasis, the manifestation of C. albicans infection discussed in this review, has traditionally been ascribed to innate immunity with emphasis on the role of granulocytes. Lately, however, immunological studies have learned that host defence against disseminated candidiasis is based on a complex interplay between innate and cell-mediated immunity. Despite the availability of new antifungal agents, mortality associated with disseminated C. albicans infection remains high. Immunotherapy that augments host defence is an important strategic option in the battle against disseminated candidiasis. Here, the authors review the chronological events in the pathogenesis of disseminated candidiasis that aid in predicting the impact of existing immunotherapy and the development of future immunomodulating strategies.

Utility of intravenous immune globulin in kidney transplantation: efficacy, safety, and cost implications.

Intravenous immunoglobulin preparations (IVIG) are known to be effective in the treatment of various autoimmune and inflammatory disorders into their immunomodulatory, immunoregulatory, and anti-inflammatory properties. Recently, IVIG has been utilized in the management of highly sensitized patients awaiting renal transplantation. The mechanisms of suppression of panel reactive antibodies (PRA) in patients awaiting transplantation are currently under investigation and appear to be related to anti-idiotypic antibodies present in IVIG preparations. In this review, the various immunomodulatory mechanisms attributable to IVIG and their efficacy in reducing PRAs will be described. In addition, the use of IVIG in solid organ transplant recipients will be reviewed. The adverse events, safety considerations, and economic impact of IVIG protocols for patients awaiting solid organ transplantation will be discussed.

Functional assessment in vitro of human-complement-dependent antibody-induced cytotoxicity of neoplastic cells.

The complement system is one potential cytotoxic effector mechanism that might be effective in immunotherapy of cancer using monoclonal antibodies (mAb) directed against tumor antigens. In order to evaluate the treatment outcome from trials using mAb in cancer patients, assessment of complement-dependent cytotoxicity (CDC) may therefore be of interest. Here we describe the elaboration of a CDC assay in vitro using a rat hepatoma cell line, H4-II-E, as target cells sensitised with mAb F12, directed against the tumor-associated ganglioside antigen fucosyl-GMI. Sensitised cells were incubated with various concentrations of fresh serum as complement source for 48 h and cytotoxicity was then assessed by the tetrazolium bromide (MTT) test. A large variation in CDC efficacy was observed between individual serum donors. No differences in CDC could be seen between healthy donors and cancer patients. The CDC showed a strong correlation to the serum concentrations of complement factor C4, supporting the validity of the assay. Our results suggest that there may be significant variations in complement function within and between individuals that might influence the outcome of clinical mAb therapy. The H4/F12 CDC assay described here, together with measurement of individual complement factors. such as C4, should be further validated in cancer patients at various disease stages and phases of treatment.

An enzyme-release assay for the assessment of the lytic activities of complement or antimicrobial peptides on extracellular Toxoplasma gondii.

A method is described which allows the evaluation of the membrane lytic activity of either complement or antimicrobial peptides against the extracellular stage of the human protozoan parasite Toxoplasma gondii. The assay is based on lacZ transgenic parasites, determining the activity of released cytoplasmic beta-galactosidase into the culture supernatant upon membrane disintegration. This method was used to evaluate the lytic activities of (i) complement which is a natural defense mechanism in infected hosts against extracellular parasites, and (ii) antimicrobial peptides which have not been evaluated against T. gondii before. The results show that the assay provides a simple and convenient way to assess the membrane lytic activity of such compounds and that T. gondii, like other protozoan parasites, is vulnerable to the membrane-lytic effect of antimicrobial peptides.

Levels of complement components, immunoglobulins and acute phase proteins in plasma during aging in Nigeria.

Plasma samples from Nigerians aged 6-95 years were examined for their content of complement components (C3, C4, factor B-Bf), immuloglobins (IgG, IgA, IgM IgD) and acute phase proteins (transferrin, albumin, C-reactive protein--CRP, alpha-2-macroglobulin). Albumin, was estimated colorimetrically and the other components by the single radial immunodiffusion techniques. No significant age-related changes in mean values of the four immunobulins and the four acute phase proteins could be demonstrated. Also, the mean values for C3 and Bf did not change significantly with age but C4 values rose significantly with increasing age (r -0.232: P < 0.01).

Use of highly encapsulated Streptococcus pneumoniae strains in a flow-cytometric assay for assessment of the phagocytic capacity of serotype-specific antibodies.

A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 microliter per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.

[Methods of in vitro assessment of the degree of complement activation by the classical pathway]. / Metodika opredeleniia in vitro stepeni aktivirovaniia komplementa po klassicheskomu puti.

A sensitive a simple method is proposed for assessing the complement activation degree by the monospecific classical route. The alternative pathway of the complement activation proposed by Adachi et al. in 1990 is taken account of. The specific features of the proposed method are 1) use of commercial dry complement of guinea pigs with the regulatory protein defect needed for the alternative pathway of complement activation; 2) absence of EGTA in GVB and presence of Ca2+; 3) use of sensitized sheep red blood cells as the target cells. These conditions prevent the alternative pathway of complement activation and permit only the classical pathway. Effects of some drugs on the classical pathway of complement activation by the new method and on Adachi's alternative method are investigated. Estradurin and fosfestrol affect the complement activation by both pathways.

Shark complement: an assessment.

The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA clone encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish (Triakis scyllia) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C8n and C9n, and is activated by immune complexes in the presence of Ca++ and Mg++ ions. The ACP is antibody independent, requiring Mg++ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C3 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q-like rather than like mannan-binding protein. N-terminal amino acid sequences of the alpha and beta chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4 gamma chain, shows little similarity to human C4 gamma chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and alpha 2-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.

[Assessment of total complement activity of blood plasma (serum) using reader for immunoenzyme analysis]. / Otsenka obshchei komplementarnoi aktivnosti plazmy (syvorotki) krovi na ridere dlia immunofermentnogo analiza.

A method for instrumental assessment of the total complementary activity of human blood plasma (serum) by the classical and alternative activation is proposed. The test is carried out by the micromethod in round-bottom 96-well plates for immunological tests. The results are read by automated analyzer for enzyme immunoassay recording at 405 nm wavelength.

Cell cycle assessment of adoptive transferred lymphokine activated killer cells.

The use of lymphokine activated killer (LAK) cells for adoptive transfer therapy has been reported from number of clinical trials. To our knowledge, however, there has been no report concerning the cell cycle progression of LAK cells. Thus, for the present study we have attempted to examine the LAK cell cycle either before or after transfer. In vitro and in vivo analyses of LAK cells labeled with bromodeoxyuridine (BrdU) were carried out using two-parameter flow cytometries of their nuclear staining using fluorescein isothiocyanate(FITC)-conjugated anti-BrdU antibody and propidium iodide (PI). The in vitro growth of BrdU-positive cells showed the cells to divide once in the S phase, continue to the G2-M phase and return to the G0-G1 phase with a similar pattern after 48 or 72 h culture. They formed a definite subpopulation and were of phenotypes, thy-1.2 (+), Lyt-1.1 (-), Lyt-2.1 (+), L3T4 (-) and AGM1 (+). The percentage of BrdU-positive cells decreased significantly (P < 0.05) when treated with complement plus anti-thy-1.2, anti-Lyt-2.1 or anti-AGM1 anti-bodies, demonstrating BrdU-labeled LAK cells to have the same phenotype as control LAK cells. As in vitro, the in vivo cell cycle of LAK cells 48 and 72 h after transfer had a similar pattern, and the LAK cells also continued on to the S phase after the first division. The in vivo growth of the LAK cells treated with interleukin-2 (IL-2) was promoted 24 and 48 h after transfer. In conclusion, the present study showed LAK cells to be capable of dividing following transfer and to keep their own phenotypic characteristics; also, that treatment with IL-2 may promote their division. Adoptive transfer therapy seems to be a viable therapeutic method.

Assessment of complement activation in vivo.

Hemolytic assays that measure the functional integrity of the complement system and the quantitation of individual components by immunochemical techniques have been widely used in the past for the assessment of in vivo complement activation. However, the complement system comprises a large number of interacting serum proteins which are subject to independent synthetic and catabolic processes. The fact that complement proteins are rapidly metabolized under in vivo conditions adds to the complexity of complement analysis. Assays that are based on monoclonal antibodies with specificities for activation-dependent neoepitopes now allow the direct determination of complement fragments in plasma. These methods are superior to the quantitation of native proteins. Several parameters that differentially affect the generation or the catabolism of individual complement activation products still have to be taken into account when elevated plasma levels of complement fragments suggest in vivo complement activation. These factors include the binding to complement fragment receptors, the degradation by serum proteases and renal or hepatic clearance. An accurate estimation of complement activation in vivo requires the simultaneous determination of both the native components and the activation products.