Presence Of Normal Human Serum Research Articles

Abstract 1237: IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma

Abstract Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. CD38 is an attractive therapeutic target for MM due to its high expression on plasma cells, but it is also expressed on multiple subsets of normal immune cells. Approved therapies including anti-CD38 monoclonal antibodies such as daratumumab are available, however resistance inevitably develops, and toxicities such as reduced immune cell counts put patients at increased risk for infections. To address this and other issues associated with current therapies, we are developing IGM-2644, an engineered high affinity, high avidity bispecific anti-CD38xCD3 IgM T cell engager (TCE) antibody for the treatment of plasma cell disorders. IGM-2644 kills CD38-expressing tumor cells through complement-dependent cytotoxicity (CDC) and T cell-dependent cellular cytotoxicity (TDCC). Live cell imaging of tumor cells in the presence of normal human serum as a source of complement has shown that IGM-2644, compared to daratumumab, has exhibited superior CDC with faster kinetics and higher maximal killing, as well as increased activity on tumor cell lines that express low levels of CD38. IGM-2644 also has displayed enhanced cellular dependent cytotoxicity over daratumumab across tumor cell lines with a range of CD38 expression levels in vitro. In ex vivo assays with MM patient bone marrow mononuclear cells (BMMC), IGM-2644 has shown superior tumor cell depletion compared to daratumumab even with low T cell to tumor cell ratios. Furthermore, IGM-2644 can deplete tumor cells in BMMC from daratumumab-refractory MM patients. Similarly, using in vivo humanized xenograft tumor mouse models with various levels of CD38 expression, greater anti-tumor activity with IGM-2644 over daratumumab was observed. When compared to daratumumab, IGM-2644 demonstrated lower immune cell fratricide in an in vivo xenograft tumor mouse model, suggesting a potentially differentiated safety profile. In summary, IGM-2644 is a bispecific anti-CD38xCD3 IgM TCE antibody that demonstrates superior tumor cell killing when compared to daratumumab in vitro and especially in xenograft tumor models in vivo with low CD38 expression while having low immune cell depletion, potentially reducing the risk of infections. The safety and efficacy of IGM-2644 has been initially studied in a phase 1 clinical trial for relapsed/refractory (r/r) MM. Applicability of IGM-2644 for the treatment of other plasma cell disorders is currently being explored, including autoimmune diseases. Citation Format: Kevin C. Hart, Daniel Santos, Keyu Li, Rui Yun, Min Chai, Andras Kabai, Gene Li, Poonam Yakkundi, Roel P. Funke, Yinghui Guan, Thomas Manley, Liqin Liu, Angus M. Sinclair, Albert F. Candia, Bruce Keyt, Maya F. Kotturi. IGM-2644, a CD38xCD3 bispecific IgM T cell engager, shows enhanced anti-tumor activity compared to daratumumab in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1237.

Functional characterization of the gonococcal polyphosphate pseudo-capsule.

Neisseria gonorrhoeae is an exclusively human pathogen able to evade the host immune system through multiple mechanisms. Gonococci accumulate a large portion of phosphate moieties as polyphosphate (polyP) on the exterior of the cell. Although its polyanionic nature has suggested that it may form a protective shield on the cell surface, its role remains controversial. Taking advantage of a recombinant His-tagged polyP-binding protein, the presence of a polyP pseudo-capsule in gonococcus was demonstrated. Interestingly, the polyP pseudo-capsule was found to be present in specific strains only. To investigate its putative role in host immune evasion mechanisms, such as resistance to serum bactericidal activity, antimicrobial peptides and phagocytosis, the enzymes involved in polyP metabolism were genetically deleted, generating mutants with altered polyP external content. The mutants with lower polyP content on their surface compared to the wild-type strains, became sensitive to complement-mediated killing in presence of normal human serum. Conversely, naturally serum sensitive strains that did not display a significant polyP pseudo-capsule became resistant to complement in the presence of exogenous polyP. The presence of polyP pseudo-capsule was also critical in the protection from antibacterial activity of cationic antimicrobial peptide, such as cathelicidin LL-37. Results showed that the minimum bactericidal concentration was lower in strains lacking polyP than in those harboring the pseudo-capsule. Data referring to phagocytic killing resistance, assessed by using neutrophil-like cells, showed a significant decrease in viability of mutants lacking polyP on their cell surface in comparison to the wild-type strain. The addition of exogenous polyP overturned the killing phenotype of sensitive strains suggesting that gonococcus could exploit environmental polyP to survive to complement-mediated, cathelicidin and intracellular killing. Taken together, data presented here indicate an essential role of the polyP pseudo-capsule in the gonococcal pathogenesis, opening new perspective on gonococcal biology and more effective treatments.

The type VI secretion system in Acinetobacter baumannii clinical isolates and its roles in antimicrobial resistance acquisition

Acinetobacter baumannii is a successful pathogen that can acquire various antibiotic resistance in a short time. However, little is known about how it can evolve from an antibiotic sensitive to a resistant phenotype. In this study, we investigated the roles of the type VI secretion system (T6SS) in the acquisition of antibiotic resistance of A. baumannii. T6SS gene cluster was found to be present in 51 of 77 A. baumannii clinical isolates, of which, it was found in 62% (8/13) of the multiple drug resistant (MDR) isolates, 90% (36/40) of the extensively drug-resistant (XDR) isolates and 26% (6/23) of the antibiotic sensitive isolates. There is a close relationship between the antimicrobial resistance and the presence of T6SS. Besides, T6SS + isolates showed lower biofilm formation activity and higher survival ability in the presence of normal human serum than T6SS- isolates. A. baumannii A152 with complete T6SS can outcompete E.coli effectively and can acquire the antibiotic resistance plasmids released by E.coli. In contrast, the T6SS core gene mutant A152Δhcp showed significantly decreased ability to acquire antimicrobial resistance plasmids from the prey bacteria. These results suggest that T6SS mediated bacterial competition plays important roles in the antimicrobial resistance of A. baumannii, which points out a new direction for us to study the antimicrobial resistance of A. baumannii.

Virulence Genotype and Correlation of Clinical Severeness with Presence of the Type VI Secretion System in Klebsiella pneumoniae Isolates Causing Bloodstream Infections.

BackgroundKlebsiella pneumoniae (K. pneumoniae) causes bloodstream infection (BSI), which is responsible for a high rate of morbidity and mortality among different populations. In mainland China, data on the correlation and features of the type VI secretion system (T6SS) gene cluster in K. pneumoniae is currently scarce. As a result, we conducted a prospective investigation to determine the involvement of the T6SS in K. pneumoniae pathogenicity and antibiotic resistance.MethodsIn this prospective analysis, we enrolled 119 individuals who had been diagnosed with K. pneumoniae bloodstream infection between July 2019 and January 2021 and acquired demographic and clinical data from their medical records. The virulence genes rmpA, rmpA2, aerobactin, iroB, hcp, vgrG, and icmF were tested for K1 and K2, antimicrobial susceptibility. Five T6SS-positive and five T6SS-negative isolates were chosen for the competition, serum resistance, and biofilm formation experiments to further gain insights regarding the microbiological properties of T6SS-positive K. pneumoniae isolates.ResultsAmong 119 isolates obtained from patients with BSIs, 20 (16.8%) were T6SS positive K. pneumoniae. T6SS positive strains had four virulence genes and a greater K1 capsular serotypes rate than T6SS negative bacteria. Among hvKP isolates, the T6SS positive rate was substantially greater than the T6SS negative rate (P = 0.001). T6SS-positive K. pneumoniae strains had a lower rate of antimicrobial resistance in comparison to T6SS-negative bacteria. T6SS-positive isolates may be more competitive with Escherichia coli than T6SS-negative isolates. T6SS-positive isolates, on the other hand, did not show stronger biofilm-forming activity or a higher survival rate in the presence of normal human serum in comparison to T6SS-negative isolates.ConclusionT6SS-positive K. pneumoniae was common in people who had BSIs. In T6SS‐containing K. pneumoniae, the system may play a major role in bacterial competition.

Phenotypic Changes Associated With In Vivo Evolution of Colistin Resistance in ST11 Carbapenem-Resistant Klebsiella pneumoniae.

Colistin is one of the few antibiotics that exhibit bactericidal effect on carbapenemase-producing Klebsiella pneumoniae strains. In recent years, however, colistin resistance is increasingly being reported among clinical carbapenem-resistant K. pneumoniae strains worldwide, posing serious challenge to treatment of infections caused by these organisms. In this study, we investigated one colistin-susceptible (YJH4) and one colistin-resistant (YJH15) K. pneumoniae strain, which were collected from a patient before and after colistin treatment, respectively. We characterized the effects of mgrB inactivation-induced colistin resistance on the physiological fitness and virulence in ST11 carbapenem-resistant K. pneumoniae both in vitro and in vivo. The colistin-resistant strain YJH15 was found to exhibit increased fitness and biofilm formation potential in vitro, and increased survival rate in the presence of normal human serum. Interestingly, YJH15 exhibited reduced virulence in the mouse infection model but enhanced virulence in Galleria mellonella infection model when compared to the colistin-susceptible parental strain YJH4. Infection with YJH15 was also found to result in lower expression level of inflammatory cytokine IL-1β in blood and significantly decreased bacterial loads in heart, liver, spleen, lung, kidney and blood. These results demonstrated that mgrB inactivation-induced colistin resistance has significant effects on multiple fitness and virulence-associated traits in K. pneumoniae.

New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source.

Transplantation of xenogenic porcine chondrocytes could represent a future strategy for the treatment of human articular cartilage defects. Major obstacles are humoral and cellular rejection processes triggered by xenogenic epitopes like α-1,3-Gal and Neu5Gc. Besides knockout (KO) of genes responsible for the biosynthesis of respective epitopes (GGTA1 and CMAH), transgenic expression of human complement inhibitors and anti-apoptotic as well as anti-inflammatory factors (CD46, CD55, CD59, TNFAIP3 and HMOX1) could synergistically prevent hyperacute xenograft rejection. Therefore, chondrocytes from different strains of single- or multi-genetically modified pigs were characterized concerning their protection from xenogeneic complement activation. Articular chondrocytes were isolated from the knee joints of WT, GalTKO, GalT/CMAH-KO, human CD59/CD55//CD46/TNFAIP3/HMOX1-transgenic (TG), GalTKO/TG and GalT/CMAHKO/TG pigs. The tissue-specific effectiveness of the genetic modifications was tested on gene, protein and epitope expression level or by functional assays. After exposure to 20% and 40% normal human serum (NHS), deposition of C3b/iC3b/C3c and formation of the terminal complement complex (TCC, C5b-9) was quantified by specific cell ELISAs, and generation of the anaphylatoxin C5a by ELISA. Chondrocyte lysis was analyzed by Trypan Blue Exclusion Assay. In all respective KO variants, the absence of α -1,3-Gal and Neu5Gc epitope was verified by FACS analysis. In chondrocytes derived from TG animals, expression of CD55 and CD59 could be confirmed on gene and protein level, TNFAIP3 on gene expression level as well as by functional assays and CD46 only on gene expression level whereas transgenic HMOX1 expression was not evident. Complement activation in the presence of NHS indicated mainly effective although incomplete protection against C3b/iC3b/C3c deposition, C5a-generation and C5b-9 formation being lowest in single GalTKO. Chondrocyte viability under exposure to NHS was significantly improved even by single GalTKO and completely preserved by all other variants including TG chondrocytes without KO of xenoepitopes.

A Factor H-Fc fusion protein competes with serum FH for binding to Staphylococcus aureus and boosts complement-mediated opsonization

Abstract Staphylococcus aureus is a significant human pathogen that causes a multitude of superficial and invasive infections worldwide. As antibiotic resistance is increasing, novel therapies are in dire need. As a master of immune evasion, S. aureus produces several virulence factors to persist in the host. A primary target of staphylococcal immune evasion is the Complement system, a component of innate immunity central to controlling bacterial infections. We have previously shown that S. aureus binds Complement regulator Factor H (FH) via surface protein SdrE to inhibit Complement. To address the need for novel therapeutics and take advantage of the SdrE:FH interaction, we tested the binding and downstream effect of a fusion protein comprised of the SdrE-interacting domain of FH coupled with IgG1 Fc using a S. aureus clinical isolate known to bind FH. S. aureus bound significantly more FH-Fc than Fc-control proteins indicating a greater affinity for SdrE compared to S. aureus Fc-binding proteins Protein A and Sbi. In the presence of normal human serum, FH-Fc competitively bound to S. aureus and reduced S. aureus recruitment of serum FH compared to Fc-control. When examining the effect of FH-Fc on complement-mediated opsonization of S. aureus, treatment with FH-Fc resulted in a slight increase in C3-fragment deposition and a significant increase in C5a generation compared to control. This may be attributed to the cumulative effect of serum FH displacement plus a boost in classical pathway activation via additional Fc presence afforded by SdrE:FH-Fc binding. Taken together, these data support FH-Fc as a potential anti-staphylococcal therapeutic. Future studies will explore how FH-Fc affects S. aureus survival when challenged with phagocytes.

Phenotypical profile and global transcriptomic profile of Hypervirulent Klebsiella pneumoniae due to carbapenemase-encoding plasmid acquisition

BackgroundPlasmids play an vital role in driving the rapid global spread of antimicrobial resistance and adaptation to changing ambient conditions. It has been suggested that the presence of plasmids can pose tremendous impacts on the host physiology. However, little is known regarding the contributions of carbapenemase-encoding plasmid carriage on the physiology and pathogenicity of hypervirulent K. pneumoniae (hvKP).ResultsHere we performed a transcriptomic analysis of hvKP with or without carbapenemase-encoding plasmid p24835-NDM5. The results had shown 683 genes with differential expression (false discovery rate, ≤0.001; > 2-fold change), of which 107 were up-regulated and 576 were down-regulated. Gene groups with functions relating to carbohydrate metabolism and multidrug efflux system were increased in genes with increased expression, and those relating to capsule biosynthesis and virulence factors were increased in the genes with decreased expression. In agreement with these changes, survival rate of TfpNDM-hvKP in the presence of normal human serum decreased, and competitive index (CI values) indicated significant fitness defects in the plasmid-carrying hvKP strain when co-cultured with its plasmid-free isogenic ancestor and the ATCC control. Moreover, the p24835-NDM5-containing hvKP strain retained its high neutrophil-mediated phagocytosis and murine lethality.ConclusionThese data indicate that hvKP responds to carbapenemase-encoding plasmid by altering the expression of genes involved in carbohydrate metabolism, antibiotic resistance, capsule biosynthesis and virulence expression. Apart from antibiotic resistance selective advantages, carbapenemase-encoding plasmid carriage may also lead to virulence change or adaption to specific habitats in hvKP strain.

Microbiological features and clinical impact of the type VI secretion system (T6SS) in Acinetobacter baumannii isolates causing bacteremia

ABSTRACTWe investigated the genetic background and microbiological features of T6SS-positive Acinetobacter baumannii isolates and clinical impact of the T6SS in patients with A. baumannii bacteremia. One hundred and 62 A. baumannii isolates from patients with bacteremia in 2 tertiary-care hospitals in Korea were included in this study. Approximately one-third (51/162, 31.5%) of the A. baumannii clinical isolates possessed the hcp gene, and the hcp-positive isolates were found in several genotypes in multilocus sequence typing. The expression and secretion of Hcp protein varied among the clinical isolates. A. baumannii isolates with detectable Hcp secretion (T6SS+) could better outcompete Escherichia coli compared with T6SS- isolates, including hcp-negative and inactivated hcp-positive isolates. In addition, T6SS+ isolates showed higher biofilm-forming activity and better survival in the presence of normal human serum than the T6SS– isolates. T6SS+ isolates were more frequently detected in patients with catheter-related bloodstream infection, haematopoietic stem cell transplant recipients, and patients receiving immunosuppressive agents. However, T6SS was not a prognostic factor for mortality. Our results suggest that the T6SS of A. baumannii is associated with virulence and contributes to infections in immunocompromised patients and those with implanted medical devices.

Loss of hypermucoviscosity and increased fitness cost in colistin-resistant Klebsiella pneumoniae sequence type 23 strains.

In this study, we investigated the effects of colistin resistance on virulence and fitness in hypermucoviscous (HV) Klebsiella pneumoniae sequence type 23 (ST23) strains. Colistin-resistant mutants were developed from three colistin-susceptible HV K. pneumoniae ST23 strains. The lipid A structures of strains were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Changes in HV were investigated using the string test, and extracellular polysaccharide production was quantified. The expression levels of the phoQ, pmrD, pmrB, pbgP, magA, and p-rmpA2 genes, serum resistance, and biofilm-forming activity were determined. The fitness of colistin-resistant mutants compared to that of the parental strains was examined by determining the competitive index (CI). The colistin-resistant mutants exhibited reduced HV, which was accompanied by decreased formation of capsular polysaccharides (CPS) and reduced expression of genes (magA and p-rmpA2). While there was enhanced expression of pmrD and pbgP in all colistin-resistant derivatives, there were differences in the expression levels of phoQ and pmrB between strains. MALDI-TOF analysis detected the addition of aminoarabinose or palmitate to the lipid A moiety of lipopolysaccharide in the colistin-resistant derivatives. In addition, survival rates in the presence of normal human serum were decreased in the mutant strains, and CI values (0.01 to 0.19) indicated significant fitness defects in the colistin-resistant derivatives compared to the respective parental strains. In hypervirulent HV K. pneumoniae strains, the acquisition of colistin resistance was accompanied by reduced CPS production, impaired virulence, and a significant fitness cost.

Hepatitis C Virus Infection Upregulates CD55 Expression on the Hepatocyte Surface and Promotes Association with Virus Particles

CD55 limits excessive complement activation on the host cell surface by accelerating the decay of C3 convertases. In this study, we observed that hepatitis C virus (HCV) infection of hepatocytes or HCV core protein expression in transfected hepatocytes upregulated CD55 expression at the mRNA and protein levels. Further analysis suggested that the HCV core protein or full-length (FL) genome enhanced CD55 promoter activity in a luciferase-based assay, which was further augmented in the presence of interleukin-6. Mutation of the CREB or SP-1 binding site on the CD55 promoter impaired HCV core protein-mediated upregulation of CD55. HCV-infected or core protein-transfected Huh7.5 cells displayed greater viability in the presence of CD81 and CD55 antibodies and complement. Biochemical analysis revealed that CD55 was associated with cell culture-grown HCV after purification by sucrose density gradient ultracentrifugation. Consistent with this, a polyclonal antibody to CD55 captured cell culture-grown HCV. Blocking antibodies against CD55 or virus envelope glycoproteins in the presence of normal human serum as a source of complement inhibited HCV infection. The inhibition was enhanced in the presence of both the antibodies and serum complement. Collectively, these results suggest that HCV induces and associates with a negative regulator of the complement pathway, a likely mechanism for immune evasion.

Interference of antimodified C-reactive protein autoantibodies from lupus nephritis in the biofunctions of modified C-reactive protein

Autoantibodies against modified C-reactive protein (mCRP) are frequently found in patients with lupus nephritis and are associated with disease activity, suggesting their pathogenic role in lupus nephritis. The aim of this study was to investigate the influence of anti-mCRP autoantibodies from patients with lupus nephritis on the biofunctions of mCRP. mCRP plays important roles in the clearance of apoptotic cells and immune complex-mediated injuries via binding to C1q and factor H and acting as an opsonin. In this study, we confirmed that mCRP could bind to C1q and factor H, and the binding between mCRP and C1q was mainly via the collagen-like region of C1q by enzyme-linked immunosorbent assay and surface plasmon resonance. mCRP could significantly enhance the phagocytosis of late apoptotic cells in the presence of normal human serum. Autoantibodies against mCRP, purified from immunoglobulin G fractions of 3 patients with lupus nephritis by affinity chromatography, could significantly inhibit the binding between mCRP and C1q or factor H and reduce the clearance of late apoptotic cells enhanced by mCRP. Our observations suggest that anti-mCRP autoantibodies from patients with lupus nephritis might be pathogenic in systemic lupus erythematosus and lupus nephritis through interfering with the biofunctions of mCRP.

Investigations into human serum sensitivity expressed by stocks of Trypanosoma brucei evansi

Trypanosoma bruceievansi, a widely distributed species of trypanosome infecting different livestock species in many countries in Africa, Asia and South America, has recently been reported as a pathogen causing a case of human trypanosomiasis in India. To date, there is little information regarding the natural resistance of animal-infective stocks of T. b. evansi to normal human serum (NHS). In this study, we investigated the degree of sensitivity to NHS of 15 stocks of T. b. evansi from different geographical origins and found that 10 of the stocks were completely susceptible to the action of NHS; parasites disappeared from the blood of infected mice within a few hours and the mice remained free from infection for more than 1 month. The remaining five stocks were partially resistant to NHS; although parasites initially disappeared from the circulation more than 50% of the mice showed relapse infection 10–18 days later. Studies on one stock, T. b. evansi STIB 810, showed that the changes in parasitaemia in the infected mice were correlated with the amount of NHS inoculated (correlation factor −0.584 and P=0.001). When this stock was passaged 25 times in mice in the presence of NHS it was found that the trypanosomes’ serum resistance increased compared with the parent stock from which they were derived; 40% of the passaged parasites survived after in vitro incubation with 50% NHS for 7h, while only 1% of individual trypanosomes of the parent stock survived under the same conditions. These findings show, to our knowledge for the first time, that human serum sensitivity varies amongst stocks of T. b. evansi, that some of them naturally display resistance to NHS and that, furthermore, T. b. evansi serum resistance can be increased by sub-passage in the presence of NHS.

Daratumumab, a Novel Potent Human Anti-CD38 Monoclonal Antibody, Induces Significant Killing of Human Multiple Myeloma Cells: Therapeutic Implication.

Abstract 608CD38 is a type II transmembrane glycoprotein highly expressed in many hematological malignancies including multiple myeloma (MM), representing a promising target for monoclonal antibody (mAb)-based targeted immunotherapy. We here investigated the mechanisms of anti-MM activity of daratumumab, a novel anti-CD38 mAb currently in clinical development, to treat human MM. Daratumumab was generated in human Ig transgenic HuMab-mice® and was selected for its excellent binding characteristics. It strongly reacts against a panel of CD38-expressing MM cell lines (>94%, 17/18) that are sensitive or resistant to current treatments for MM: median fluorescence intensity (MFI) observed was 378-20764 and 15-140 for daratuzumab and isotype control Ab, respectively. U266 cells, which lack CD38 mRNA, did not bind daratuzumab. Significantly, daratuzumab induced NK-mediated antibody-dependent cellular cytotoxicity (ADCC) against CD38-expressing MM cells but not CD38-negative U266 cells, confirming its specific targeting. Daratuzumab-mediated MM cell lysis was dose-dependent, with maximum lysis of the CD38-expressing MM cell lines (n=17) at concentrations of 0.01-0.1μg/ml. We also determined potential effects of daratumumab on NK and peripheral blood mononuclear cells (PBMC) effector cells, since these cells express CD38, albeit at lower levels than MM cells. Overnight pretreatment of PBMCs or purified NK cells from 2 normal donors with daratumumab (2 μg/ml) did not significantly alter daratumumab-induced ADCC against MM1S target cells. The bone marrow (BM) microenvironment has been shown to protect MM cells against apoptosis and stimulate their growth; we therefore next asked whether daratumumab effectively induced ADCC against MM cells in the presence of BM stromal cells (BMSCs). Daratumumab induced a similar, dose-dependent, ADCC of dexamethasone-sensitive MM1S as well as dexamethasone-resistant MM1R cells both in the presence and absence of BMSCs, suggesting that daratumumab could indeed effectively induce ADCC in the BM microenvironment. Significantly, our findings translated to patient cells, since daratumumab induced ADCC of both allogeneic (n=2) and autologous (n=9) patient MM cells. We next turned our attention to complement-mediated cytotoxicity (CDC). In calcein AM-labeling assays in the presence of normal human serum, daratumumab induced dose-dependent CDC of LP-1 and XG-1 MM cells expressing low levels of CD59 and CD55 both in the absence or presence of BMSCs. Effective daratumumab-induced CDC of CD138-purified MM cells was seen for 6 of 9 patients. Daratumumab-induced CDC was not affected by the expression of complement inhibitor proteins in this cohort of patients. Finally, daratumumab induced apoptosis in Ramos and Daudi cells when crosslinked by anti-IgG antibody, and it induced cell death in CD138-purified patient MM cells, as assessed by annexin V/PI staining. Taken together, our results indicate that daratumumab is able to effectively kill patient MM tumor cells via several cytotoxic mechanisms, supporting clinical development of daratumumab to improve patient outcome in MM. Disclosures:de Weers:Genmab: Employment. Bakker:Genmab: Employment. Vink:Genmab: Employment. Jacobs:Genmab: Employment. Oomen:Genmab: Employment. Bleeker:Genmab: Employment. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. van de Winkel:Genmab: Employment. Parren:Genmab: Employment. Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Responses to Rituximab Vary Among Follicular Lymphoma B Cells of Different Maturation Stages

The chimeric anti-CD20 monoclonal antibody rituximab has been used for the treatment of non-Hodgkin's lymphomas with varying responses. Rituximab has been demonstrated to act by direct complement-dependent cytotoxity (CDC) and by inducing apoptosis, complement-, and antibody-dependent cellular cytotoxity. In the present study, we determined whether rituximab's effector mechanisms differed between two human follicular lymphoma cell lines that originate from different maturation stages of B cell germinal centre (GC) development. The tested HF-1 and HF-4b lymphoma cells represent GC centrocytes and centroblasts, respectively. Both cell lines responded to rituximab treatment by undergoing apoptosis yet the HF-1 cells were more sensitive. A major difference was seen in the proliferation response as only the proliferation of HF-1 cells was inhibited by rituximab. In the presence of normal human serum (NHS) rituximab almost completely inhibited DNA synthesis and induced necrosis of both cell lines because of CDC. Our results show that the CD20-positive HF-1 and HF-4b cells respond differentially to rituximab-induced apoptosis and inhibition of proliferation but similarly to complement-mediated killing. The increased sensitivity of the HF-1 cell line to apoptosis and inhibition of proliferation may reflect a tendency of centrocytic cells for negative selection and a role for CD20 in this process.

The Tick Salivary Protein Salp15 Inhibits the Killing of Serum-Sensitive Borrelia burgdorferi Sensu Lato Isolates

Borrelia burgdorferi, the agent of Lyme disease, is transmitted by ticks. During transmission from the tick to the host, spirochetes are delivered with tick saliva, which contains the salivary protein Salp15. Salp15 has been shown to protect spirochetes against B. burgdorferi-specific antibodies. We now show that Salp15 from both Ixodes ricinus and Ixodes scapularis protects serum-sensitive isolates of Borrelia burgdorferi sensu lato against complement-mediated killing. I. ricinus Salp15 showed strong protective effects compared to those of I. scapularis Salp15. Deposition of terminal C5b to C9 (one molecule each of C5b, C6, C7, and C8 and one or more molecules of C9) complement complexes, part of the membrane attack complex, on the surface of B. burgdorferi was inhibited in the presence of Salp15. In the presence of normal human serum, serum-sensitive Borrelia burgdorferi requires protection against complement-mediated killing, which is provided, at least in part, by the binding to the tick salivary protein Salp15.

Selective blockade of inhibitory Fcgamma receptor enables human dendritic cell maturation with IL-12p70 production and immunity to antibody-coated tumor cells.

The final differentiation or maturation of dendritic cells (DCs) in response to environmental stimuli influences their ability to both initiate immunity and determine the quality of the response to antigens. Circulating immune complexes and cell-bound immunoglobulins present in normal human sera represent a potential stimulus for inadvertent DC activation in the steady state and during autoimmunity. Here, we show that selective blockade of the inhibitory Fcgamma receptor (FcgammaR) FcgammaRIIb with recently developed monoclonal antibodies leads to maturation of human monocyte-derived DCs, which depends on the presence of IgG in normal human plasma. Plasma, in the presence of an FcgammaRIIb blockade, caused the DCs to up-regulate the expression of costimulatory molecules and to produce the inflammatory mediator IL-12p70. FcgammaRIIb blockade of DCs loaded with tumor cells led to increased tumor-specific T cell immunity without the need for exogenous stimuli other than human plasma. Therefore, the activation status of DCs in the presence of normal human serum depends on the balance between activating and inhibitory FcgammaRs and can be enhanced by new antibodies that react selectively with FcgammaRIIb. These data suggest an approach for modifying this balance to enhance immunity to immune complexes and antibody-coated tumor cells and to silence DC activation by immune complexes in autoimmune states.

The Capsular Polysaccharide ofBurkholderia pseudomalleiContributes to Survival in Serum by Reducing Complement Factor C3b Deposition

Burkholderia pseudomallei produces an extracellular polysaccharide capsule -3)-2-O-acetyl-6-deoxy-beta-D-manno-heptopyranose-(1- which has been shown to be an essential virulence determinant. The addition of purified capsule was shown to increase the virulence of a capsule mutant strain in the Syrian hamster model of acute melioidosis. An increase in the number of wild-type B. pseudomallei cells in the blood was seen by 48 h, while the number of capsule mutant cells in the blood declined by 48 h. Capsule expression was shown to be induced in the presence of serum using a lux reporter fusion to the capsule gene wcbB. The addition of purified B. pseudomallei capsule to serum bactericidal assays increased the survival of B. pseudomallei SLR5, a serum-sensitive strain, by 1,000-fold in normal human serum. Capsule production by B. pseudomallei contributed to reduced activation of the complement cascade by reducing the levels of complement factor C3b deposition. An increase in phagocytosis of the capsule mutant compared to the wild type was observed in the presence of normal human serum. These results suggest that the production of this capsule contributes to resistance to phagocytosis by reducing C3b deposition on the surface of the bacterium, thereby contributing to the persistence of bacteria in the blood of the infected host. Continued studies to characterize this capsule are essential for understanding the pathogenesis of B. pseudomallei infections and the development of preventive strategies for treatment of this disease.

Dysregulation of IL-2/IL-2R system alters proliferation of early activated CD4+ T cell subset in patients with end-stage renal failure

Although CD4+ T cells are preactivated in patients with end-stage renal failure (ESRF), these patients present an impairment of T cell immune response, which is partly responsible for the higher incidence of infection in this population. The aim of the present study was to analyze the mechanisms underlying the altered function of activated CD4+ T cells in patients with ESRF. Thirty patients undergoing chronic hemodialysis (HD) and 20 patients with ESRF were compared with 15 sex- and age-matched controls. CD4+ T cell early activation (CD69, CD25), interleukin-2 (IL-2)/IL-2 receptor (IL-2R) system, and proliferation capacity of CD69+/CD4+ T cells were assessed ex vivo after blood draw sampling, in culture conditions and after phytohemagglutinin (PHA) stimulation. Although the CD4+ T cell count was lower in chronic HD patients than in predialysis patients and controls (p = 0.007), CD4+ T cells showed a pre-activation state as demonstrated by higher percentage of CD69+/CD4+ T cells and CD25+/CD4+ T cells in chronic HD patients compared with the other groups ex vivo. Furthermore, CD69+/CD4+ T cells from chronic HD patients spontaneously released more IL-2 (22 +/- 6 pg/ml) than those from pre-dialysis patients (12 +/- 4 pg/ml, p = 0.005) and controls (5 +/- 3 pg/ml, p = 0.001). However, after PHA stimulation, CD69+/CD4+ T cells from chronic HD patients expressed lower cell surface CD25 density, and were unable to show further activation. Indeed, these cells produced less IL-2 and released more soluble IL-2R, and correlatively with IL-2 production, they showed lower proliferation capacity compared with predialysis patients (p = 0.001) and controls (p < 0.001). They also displayed decreased responsiveness to exogenous human recombinant IL-2. The restoration of the PHA stimulation index of CD69+/CD4+ T cells from chronic HD patients in the presence of normal human serum as well as the decreased stimulation index of CD69+/CD4+ T cells from control subjects incubated with HD serum, strongly suggest that uremic toxins and mediators induced by HD affect the IL-2/IL-2R pathway. These findings demonstrate the presence, in chronic HD patients, and to lesser extent, in predialysis patients, of abnormally high proportion of spontaneously preactivated CD4+ T cells whose proliferation and further activation are blunted due to dysregulation of the IL-2/IL-2R system.

Treatment with an Anti-C3b/iC3b Monoclonal Antibody as an Adjuvant to Rituximab Enhances the Killing of Non-Hodgkin's Lymphoma and Chronic Lymphocytic Leukemia Cells.

Complement-dependent cytotoxicity (CDC) is a key mechanism of Rituximab® (RTX) action in killing Non-Hodgkin's Lymphoma (NHL) cells both in vitro and in vivo. Here we present studies of a mouse/human chimeric monoclonal antibody (Mab), ETI-210, specific for human complement C3 cleavage products C3b and iC3b, in enhancing RTX- mediated killing in vitro and ex vivo. Raji (NHL), primary NHL, and CLL cells were treated with RTX and ETI-210 in the presence of normal human serum (NHS) as a source of complement. Cell death was determined by propidium iodide staining or reduction of Alamar Blue®. After 1 hour of treatment, the killing of Raji cells by RTX plus ETI-210 plus NHS was observed to be 30–70% greater than that of RTX alone plus NHS by either detection method. The enhancing effect of ETI-210 was also seen in longer-term treatment (1 – 2 days). The killing by RTX plus ETI-210 was much lower in the absence of NHS than in the presence of NHS. ETI-210 was also tested ex vivo against primary cells from 12 NHL and 9 CLL patients. In 5 NHL samples, ETI-210 significantly enhanced RTX-mediated killing; in the remaining samples RTX alone already caused killing above 90%. The Mab also increased RTX-mediated killing in 4 out of 9 CLL samples. A pilot study examining the safety of administrating ETI-210 (5 and 30 mg/kg) with or without RTX was performed in cynomolgus monkeys. There were no cardiovascular changes. Clinical chemistry (potassium, glucose, sodium), liver function (alkaline phosphatase, ALT, albumin, total protein), kidney function (BUN, creatine, serum electrolytes) and hematological parameters (CBC profile, hemoglobin, MCV, MCH, MCHC) were not changed significantly. There was a transient reduction in CH50 values in the 30mg/kg ETI-210 group, which was independent of RTX administration. Serum concentrations of C3 and C4 were not affected. Because, ETI-210, a chimeric anti-C3b/iC3b Mab, was able to substantially enhance RTX-mediated killing of lymphoma cells via complement-mediated lysis and did not cause toxicity in monkeys, ETI-210 is being developed as a therapeutic agent for NHL and CLL treatment.