Human AB Research Articles

Metal dependent hydrolysis of β-casein by sIgA antibodies from human milk

We present the first evidence that electrophoretically and immunologically homogeneous sIgAs purified from milk of healthy human mothers by chromatography on Protein A-Sepharose and FPLC gel filtration contain intrinsically bound metal ions (Ca > Mg ≥ Al > Fe approximately Zn ≥ Ni ≥ Cu ≥ Mn), the removal of which by a dialysis against ethylenediamine tetraacetic acid (EDTA) leads to a significant decrease in the β-casein-hydrolyzing activity of these antibodies (Abs). An affinity chromatography of total sIgAs on benzamidine-Sepharose interacting with canonical serine proteases separates a small metalloprotease sIgA fraction (6.8 ± 2.4%) from the main part of these Abs with a serine protease-like β-casein-hydrolyzing activity. The relative activity of this metalloprotease sIgA fraction containing intrinsically bound metal ions increases ∼1.2-1.9-fold after addition of external metal ions (Mg(2+) > Fe(2+) > Cu(2+) ≥ Ca(2+) ≥ Mn(2+)) but decreases by 85 ± 7% after the removal of the intrinsically bound metals. The metalloprotease sIgA fraction free of intrinsic metal ions demonstrates a high β-casein-hydrolyzing activity in the presence of individual external metal ions (Fe(2+) > Ca(2+) > Co(2+) ≥ Ni(2+)) and especially several combinations of metals: Co(2+) + Ca(2+) < Mg(2+) + Ca(2+) < Ca(2+) + Zn(2+) < Fe(2+) + Zn(2+) < Fe(2+) + Co(2+) < Fe(2+) + Ca(2+). The patterns of hydrolysis of a 22-mer oligopeptide corresponding to one of sIgA-dependent specific cleavage sites in β-casein depend significantly on the metal used. Metal-dependent sIgAs demonstrate an extreme diversity in their affinity for casein-Sepharose and chelating Sepharose, and interact with Sepharoses bearing immobilized monoclonal mouse IgGs against λ- and κ-type light chains of human Abs. Possible ways of the production of metalloprotease abzymes (Abz) by human immune system are discussed.

Natural Killer Cell Killing of Acute Myelogenous Leukemia and Acute Lymphoblastic Leukemia Blasts by Killer Cell Immunoglobulin-Like Receptor–Negative Natural Killer Cells after NKG2A and LIR-1 Blockade

Although the study of natural killer (NK) cell alloreactivity has been dominated by studies of killer cell immunoglobulin-like receptors (KIRs), we hypothesized that NKG2A and LIR-1, present on 53% +/- 13% and 36% +/- 18% of normal NK cells, respectively, play roles in the NK cell killing of primary leukemia targets. KIR(-) cells, which compose nearly half of the circulating NK cell population, exhibit tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both acute myelogenous leukemia and acute lymphoblastic leukemia targets were rendered susceptible to lysis by fresh resting KIR(-) NK cells when inhibitory receptor-major histocompatibility class I interactions were blocked by pan-HLA antibodies, demonstrating that these cells are functionally competent. Blockade of a single inhibitory receptor resulted in slightly increased killing, whereas combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR(-) NK cells, demonstrating that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR(-) NK cells is mediated by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemias.

Curvature of Synthetic and Natural Surfaces Is an Important Target Feature in Classical Pathway Complement Activation

The binding of Abs to microbial surfaces followed by complement activation constitutes an important line of defense against infections. In this study, we have investigated the relationship between complement activation and the binding of human IgM Abs to surfaces with different curvatures. IgM Abs to dextran were shown to activate complement potently on dextran-coated particles having a diameter around 250 nm, whereas larger (600 nm) particles were less potent activators. This selectivity regarding particle dimension was also found for complement activation by colloidal substances of microbial origin. Peptidoglycan (PGN) is the major chemical component in the cell wall of Gram-positive bacteria. Fragments of purified PGN with sizes of approximately 100 nm promoted complement activation effectively through the classical pathway. By contrast, larger or smaller fragments of PGN did not activate complement strongly. A careful analysis of PGN fragments released during planctonic growth of Staphylococcus aureus showed that these include curvatures that would permit strong IgM-mediated complement activation, whereas the curvature of intact cells would be less effective for such activation. Consistently, we found that the suspended PGN fragments were strong activators of complement through the classical pathway. We suggest that these fragments act as decoy targets for complement activation, providing protection for S. aureus against the host immune response to infection.

Human IgG2 Antibodies against Epidermal Growth Factor Receptor Effectively Trigger Antibody-Dependent Cellular Cytotoxicity but, in Contrast to IgG1, Only by Cells of Myeloid Lineage

Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcgammaRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcgammaRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcgammaRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R-directed immunotherapy.

Anaplasma phagocytophilum propagation is enhanced in human complement-containing medium

Complement was originally discovered as a component of normal plasma that augments opsonisation of bacteria by antibodies with subsequent killing of the pathogen. Complement plays a crucial role in innate and adaptive immune responses to many blood-borne pathogens [1,2], and some pathogens subvert complement function to survive in the blood. Its role inA. phagocytophilum infection has not been explored. We previously showed reduced phagocytosis by neutrophils infected by A. phagocytophilum. Reduced phagocytosis and opsonophagocytic receptor loss could allow A. phagocytophilum to avoid killing by either the neutrophil or by complement activation. Such changes could result from down-regulated expression or shedding of opsonophagocytic receptors on neutrophil surfaces, including various Fc receptors and complement receptors that bind opsonising ligands such as IgG, IgA, IgE, or complement components. This hypothesis was previously tested in our laboratory by assaying for the expression of these receptors after 24 h of A. phagocytophilum infection in human neutrophils. A. phagocytophilum-infected neutrophils had significantly reduced levels of surface-exposed receptors C1qRp, FcRI, FcRII and FcRIII 24 h after infection. The expression of CR1 was slightly reduced, and there was very low or no expression of FcRII and FcRI in all cells [3]. The fact that A. phagocytophilum alters reactions involving components of the complement system raises a more important question: how does it survive in the presence of complement factors during bloodstream infection,where other bacteria are usually effectively removed? Because other bloodstream bacterial pathogens, such as Borrelia burgdorferi [4] and Streptococcus pneumoniae [5], have evolved towards mechanisms that preclude complement-mediated opsonisation, assembly and lysis, it suggests thatA. phagocytophilum is also likely to have evolved towards such mechanisms. We therefore hypothesised that A. phagocytophilum evades complement-mediated degradation to ensure survival. A. phagocytophilum growth in human AB serum (NHS) with known CH50 lytic activity was compared with its growth in other serum supplements. HL-60 cells were used directly or after acclimation to 5% heat-inactivated (HI) NHS for 2–4 weeks. Experimental conditions included infected HL-60 cells in fetal bovine serum (FBS), HI-FBS, NHS and HI-NHS. Experiments were performed at 2.0 · 10 cells ⁄mL starting at concentrations with differing initial infection rates. Infections were allowed to propagate for 5 days or until 100% infection was achieved, and bacterial load was quantified by qPCR using the DDCt method or by using a standard curve of infected cells, and by light microscopy. Without adaptation to NHS, A. phagocytophilum grew equally well in HI-NHS as in NHS, but neither condition supported growth as well as FBS (not shown). Once adapted to NHS, A. phagocytophilum grew more rapidly (maximum infection by day 5 vs. day 6–8 for other sera) than with HI-NHS, HI-FBS or FBS (Fig. 1), as quantitated by the DDCt method and in a separate experiment by the infected cell standard curve. Compared with Corresponding author and reprint requests: Diana G. Scorpio DVM, MPH, DACLAM, Department of Molecular and Comparative Pathobiology, Johns Hopkins Medical Institutions, Ross Research Bldg 459 Baltimore, MD 21205, USA. Tel.: 410-955-3273 E-mail: dscorpio@jhmi.edu

Ex Vivo Expansion of Highly Purified Human NK Cells..

Abstract 2157Poster Board II-134 ObjectTo optimize the expansion of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after Ex vivo expansion. MethodsNK cells were isolated from PBMNC by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II(Miltenyi Biotec, Germany), then they were cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were fed with fresh media and cytokines every 3 days, and were evaluated for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-γ secretion at the end of the culture period. ResultsIn group IL2+IL15 and IL2+IL15+IL12, cells were expanded 50.46±4.31 and 52.35±6.72 fold, respectively, much more higher than others(P<0.01), but no significant difference between them (P>0.05). And the purity of CD3−CD56+NK cells was over 94% in all groups except the control. The expressions of perforin and granzyme B mRNA of expanded NK cells cultured with cytokines was significantly higher than the starting population(P<0.01), although IL2+IL15+IL12 group was slightly higher than that of IL2+IL15 group, without significant difference (P>0.05). There was great increase in IFN-γ levels in the supernatants of NK cells culture in the presence of cytokines; IL2+IL15+IL12 group and IL2+IL12 group was significantly higher than others(P<0.01). ConclusionHigh purity NK cells could be efficiently expanded in culture with IL2+IL15, and its biological functions were enhanced in this condition. Disclosures:No relevant conflicts of interest to declare.

TNF-α-converting enzyme (TACE/ADAM17)-dependent loss of CD30 induced by proteasome inhibition through reactive oxygen species

Combinations with proteasome inhibitors are currently being investigated to improve the therapy of hematological malignancies. We previously found that proteasome inhibition by bortezomib failed to sensitize anti-CD30 antibody (Ab)-based lymphoma cell killing. In this study, we demonstrate in L540 Hodgkin's lymphoma cells that proteasome inhibition not only communicates apoptosis but also more rapidly causes a loss of CD30 antigen from cell membrane and a simultaneous release of soluble CD30, a targeting competitor. This shedding was catalyzed by the tumor necrosis factor (TNF)-alpha-converting enzyme (TACE, ADAM17) and blocked by the ADAM17-selective inhibitor, Ro32-7315. In parallel with CD30 shedding, bortezomib caused the generation of reactive oxygen species (ROS). As apoptosis and shedding were inhibited by the radical scavenger, N-acetyl-L-cysteine, ROS might have a pivotal function in both effects. In contrast, the pan-caspase inhibitor, zVAD-fmk, blocked bortezomib-induced apoptosis but not CD30 shedding, and Ro32-7315 blocked shedding but allowed apoptosis. This suggests independent terminal signaling pathways that are conflicting in Ab-based immunotherapy. Consequently, shedding inhibition substantially improved the synergistic antitumor efficacy of the human anti-CD30 Ab, MDX-060, and bortezomib. As proteasome inhibition also stimulated loss of TNF receptors, interleukin-6 receptor and syndecan-1 in different leukemia and lymphoma cell lines, we concluded that proteasome inhibition might impede targeted therapy against antigens susceptible to shedding.

Genoproteomic Mining of Urothelial Cancer Suggests γ-Glutamyl Hydrolase and Diazepam-Binding Inhibitor as Putative Urinary Markers of Outcome after Chemotherapy

Urinary biomarkers for the detection of bladder cancer have been developed, but no similar markers exist for prediction of clinical outcomes after receiving chemotherapy. Here we evaluate an approach that combines genomic, proteomic, and therapeutic outcome datasets to identify novel putative urinary biomarkers of clinical outcome after neoadjuvant methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC). Using this method, we identified gamma-glutamyl hydrolase (GGH), emmprin, survivin, and diazepam-binding inhibitor (DBI). Interestingly, GGH is a protein associated with methotrexate resistance, whereas emmprin, survivin, and DBI had been previously identified as predictors of outcome after platinum-containing chemotherapeutic regimens when assessed on tumor tissue. Using disease-free survival as a marker for clinical outcome, we evaluated the ability of GGH, emmprin, survivin, and DBI expression in tumor tissue to stratify 27 patients treated with neoadjuvant MVAC. DBI (P = 0.046) but not GGH (P = 0.190), emmprin (P = 0.066), or survivin (P = 0.393) successfully stratified patients. When GGH was used with DBI the significance of stratification improved (P = 0.024), whereas the addition of survivin or emmprin to this latter two-gene model reduced its significance (P = 0.036 and P = 0.040, respectively). Although these predictive results were obtained on tumor tissues, the presence of GGH and DBI in urine serves as a rationale for developing them as urinary markers of clinical outcomes for patients treated with neoadjuvant MVAC.

Potent High-Affinity Antibodies for Treatment and Prophylaxis of Respiratory Syncytial Virus Derived from B Cells of Infected Patients

Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million. Several of the newly cloned Abs bind to the RSV G protein central conserved motif with very high affinity (K(d) 1-24 pM). Two of the Abs were characterized in detail and compared with palivizumab, a humanized mAb against the RSV F protein. Relative to palivizumab, the anti-G Abs showed improved viral neutralization potency in vitro and enhanced reduction of infectious virus in a prophylaxis mouse model. Furthermore, in a mouse model for postinfection treatment, both anti-G Abs were significantly more effective than palivizumab at reducing viral load. The combination of activity in mouse models for both prophylaxis and treatment makes these high-affinity human-derived Abs promising candidates for human clinical testing.

Study on amplification of bone marrow mesenchymal stem cells with serum from different sources in vitro

Objective To investigate whether autologous serum could replace fetal calf serum for the expansion of human human bone marrow mesenchymal stem cells(BMSCs)in vitro and establish a protocol of culturing BMSCs.Methods BMSCs were separated from human bone marrow by modified ficoll density gradient centrifugation combined with adherence culture method and respectively cultured by DMEM/F12 with 10%umbilical cord blood serum,10%fetal calf serum,human AB blood serum,and autologous serum in vitro. The morphology and its growth character of the BMSCs were studied with the phase-contrast microscope.Differentiation potency through condition medium directional induction were observed. The expression of cell surface antigens was detected by flow cytometer,immunocytochemistry. Results There were no significant differences between four groups in morphological changes. BMSCs were induced to differentiate into neuron like cells by growth factor. The induced cells expressed NSE,NF,GFAP after induction. Conclusion High purity and active BMSCs are cultured by autoserum,which grow prosperously and maintain its biological characters for at least 5 passages. Key words: BMSCs; Autologous serum; Differentiation

Inhibition of Erythrocyte Invasion andPlasmodium falciparumMerozoite Surface Protein 1 Processing by Human Immunoglobulin G1 (IgG1) and IgG3 Antibodies

Antigen-specific antibodies (Abs) to the 19-kDa carboxy-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) play an important role in protective immunity to malaria. Mouse monoclonal Abs (MAbs) 12.10 and 12.8 recognizing MSP1(19) can inhibit red cell invasion by interfering with MSP1 processing on the merozoite surface. We show here that this ability is dependent on the intact Ab since Fab and F(ab')(2) fragments derived from MAb 12.10, although capable of binding MSP1 with high affinity and competing with the intact antibody for binding to MSP1, were unable to inhibit erythrocyte invasion or MSP1 processing. The DNA sequences of the variable (V) regions of both MAbs 12.8 and 12.10 were obtained, and partial amino acid sequences of the same regions were confirmed by mass spectrometry. Human chimeric Abs constructed by using these sequences, which combine the original mouse V regions with human gamma1 and gamma3 constant regions, retain the ability to bind to both parasites and recombinant MSP1(19), and both chimeric human immunoglobulin G1s (IgG1s) were at least as good at inhibiting erythrocyte invasion as the parental murine MAbs 12.8 and 12.10. Furthermore, the human chimeric Abs of the IgG1 class (but not the corresponding human IgG3), induced significant NADPH-mediated oxidative bursts and degranulation from human neutrophils. These chimeric human Abs will enable investigators to examine the role of human Fcgamma receptors in immunity to malaria using a transgenic parasite and mouse model and may prove useful in humans for neutralizing parasites as an adjunct to antimalarial drug therapy.

The human neonatal B cell response to respiratory syncytial virus uses a biased antibody variable gene repertoire that lacks somatic mutations

The human Ab repertoire exhibits restrictions during fetal life characterized by biases of variable gene segment usage and lack of junctional diversity. We tested the hypotheses that Ab repertoire restriction persists in the early postnatal period and contributes to the observed poor quality of specific Ab responses made by neonates to viruses and vaccines. We analyzed the molecular determinants of B cell responses in humans to respiratory syncytial virus (RSV). Analysis of the variable gene segment usage of adult RSV-specific B cells revealed a repertoire profile in these cells similar to that seen in randomly selected B cells, which was VH3-dominant. Four gene segments (VH3–23, VH3–30, VH3–33 and VH4–04) accounted for almost half of the VH genes used. In contrast, very young infant RSV-specific antibodies exhibited a biased repertoire characterized by comparable use of the VH1, VH3, and VH4 families, and less common use of the four immunodominant gene segments. Infants and children older than three months used an antibody repertoire similar to that of adults. Mutational analysis revealed that the antibody variable genes of infants under three months of age also possessed significantly fewer somatic mutations in both framework and complementarity-determining region (CDR) regions than those of adults, even in a child with recurrent RSV infection. These data suggest that neonates use a biased antibody gene repertoire that is less VH3-focused and that possesses a dramatically lower frequency of somatic mutations. These biased features of the RSV-specific repertoire likely contribute to the poor functional Ab response in very young infants.

Selective Culture of Mesodermal Progenitor Cells

We have recently identified mesodermal progenitor cells (MPCs) isolated from adult human bone marrow. These cells show unusual phenotypes, having putative embryonic markers and aldehyde dehydrogenase (ALDH) activity. Interestingly, these resting cells, which have been selected by culturing them in the presence of adult human serum, can easily be induced to differentiate into mature mesenchymal stromal cells (MSCs) after substituting the adult human serum for fetal bovine serum (FBS) or human cord serum. MPC-derived MSCs are, in turn, able to differentiate toward osteoblasts, chondrocytes, and adipocytes. Furthermore, MPCs are able to differentiate into endothelial cells. MPCs have been proven to be strongly adherent to plastic culture bottles and to be trypsin-resistant. In the present article, we show a simple and inexpensive method to isolate highly selected mesodermal progenitors from bone marrow or cord blood. The optimization of standard culture conditions (using commercial human AB sera and appropriate concentrations for cell seeding in plastics) allows a pure population of MPCs to be obtained even after a short culture period. We believe that this simple, repeatable, and standardized method will facilitate studies on MPCs.

Drug‐induced Lupus Associated With Infliximab and Adalimumab in an Adolescent With Crohn Disease

Drug‐induced Lupus Associated With Infliximab and Adalimumab in an Adolescent With Crohn Disease

Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope

Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3(B)-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.

Antibodies as Crypts of Antiinfective and Antitumor Peptides

Antibodies (Abs), often associated with antimicrobial and antitumor agents, have emerged as an important class of novel drugs for antigen-driven therapeutic purposes in diverse clinical settings, including oncology and infectious diseases. Abs commonly give rise in the treated host to anti-Ab responses, which may induce adverse reactions and limit their therapeutic efficacy. Their modular domain architecture has been exploited to generate alternative reduced formats (Fabs, scFvs, dAbs, minibodies, multibodies), essentially devoid of the Fc region. The presence of complementarity determining regions (CDRs) ensures the maintenance of selective binding to antigens and supports their use for biotechnological and therapeutic applications. Paradigmatic Abs mimicking the wide-spectrum antimicrobial activity of a yeast killer toxin (killer Abs) have revealed the existence of a family of Abs exerting a direct in vitro and/or in vivo microbicidal activity. Based on the variable sequence of an antiidiotypic recombinant killer Ab, CDR-related peptides have been synthesized, engineered by alanine-scanning and selected according to antimicrobial, antiviral and immunomodulatory properties. Irrespective of the native Ab specificity, synthetic CDRs from unrelated murine and human monoclonal Abs, have shown to display differential in vitro, in vivo and/or ex vivo antifungal (Candida albicans), antiviral (HIV-1) and antitumor (melanoma cells) activities. Alanine substitution of single residues of synthetic CDR peptides resulted in further differential increased/unaltered/decreased biological activity. The intriguing potential of Abs as source of antiinfective and antitumor therapeutics will be discussed, in light of recent advances in peptide design, stability and delivery.

Phase I study of an anti-CD30 Fc engineered humanized monoclonal antibody in Hodgkin lymphoma (HL) or anaplastic large cell lymphoma (ALCL) patients: Safety, pharmacokinetics (PK), immunogenicity, and efficacy

8531 Background: XmAb2513 is a novel 2nd-generation humanized monoclonal antibody (mAb) directed against CD30 (a cell surface antigen expressed on Reed-Sternberg cells of HL and ALCL), with an Fc region engineered to have increased binding affinity to Fcγ receptors (FcγRs) leading to improved FcγR-dependent effector cell functions. In vitro, XmAb2513 was more potent and more efficacious than 1st-generation anti-CD30 mAbs (e.g., SGN-30 &amp; MDX-060). In cynomolgus monkeys (cynos), XmAb2513 exposure was dose-proportional with half-lives ranging from 12 - 17d in repeat dose studies, which suggested biweekly (QOW) dosing in man. A Phase 1 study has been initiated to examine the safety and efficacy of XmAb2513 in patients (pts) with relapsed HL and ALCL. Methods: Phase 1 open-label study with QOW dosing of XmAb2513; dose-escalation study design with doses of 0.3, 1, 3, 6, 9, and 12 mg/kg via IV-infusion to establish the maximum tolerated dose (MTD). Pts receive up to 4 cycles (8 infusions) with sample collections at regular intervals for safety, PK, soluble CD30 (sCD30) and detection of immunogenicity [i.e., human Abs to humanized Abs (HAHA)]. Tumor response is assessed every 2 cycles. Results: 13 HL pts have been enrolled in the 1st 4 dose levels (0.3 to 6 mg/kg). XmAb2513 has been well tolerated. The MTD has not been achieved. 1 pt with an extensive treatment history and chronic immunosuppression died due to fungal pneumonia after a single 3 mg/kg dose. The event was considered unrelated to XmAb2513. PK parameters were computed for the 1st dose alone and for the entire course of XmAb2513 treatment. PK parameters appeared non-linear across the dose range from 0.3 - 3 mg/kg. Half-life appeared to increase after multiple infusions and ranged from 5.3 - 30.2 days in the 1 and 3 mg/kg cohorts. Immunogenicity (HAHA) results have been negative. Tumor reduction has been observed in 1 pt in the 1 mg/kg cohort and 2 in the 3 mg/kg cohort. Conclusions: The MTD of XmAb2513 has not yet been reached due to its tolerability and lack of immunogenicity. PK results are on track with simulations based on PK results from cynos. XmAb2513 has demonstrated encouraging biologic activity in patients with refractory HL. [Table: see text]

716. Selection of Human AB Serum for Ex Vivo Expansion of T Cells Displaying an Early Differentiation Phenotype

716. Selection of Human AB Serum for Ex Vivo Expansion of T Cells Displaying an Early Differentiation Phenotype

Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis.

Airway Smooth Muscle Cells Synthesize Hyaluronan Cable Structures Independent of Inter-α-inhibitor Heavy Chain Attachment

The covalent association of inter-alpha-inhibitor-derived heavy chains (HCs) with hyaluronan was first described in synovial fluid from arthritic patients and later described as a structural and functional component of hyaluronan "cable" structures produced by many different cells and stimuli. HC transfer has been shown to be mediated by the protein product of TSG-6 (tumor necrosis factor-stimulated gene 6). Considering the accumulation of hyaluronan in airways following asthmatic attacks and the subsequent infiltration of leukocytes, we sought to characterize HC substitution of hyaluronan "cables" in primary mouse airway smooth muscle cells (MASM) and primary human airway smooth muscle cells (HASM). We found that cells derived from mice lacking TSG-6 had no defect in hyaluronan production or hyaluronan-mediated leukocyte adhesion when treated with the viral mimic poly(I,C). Functional hyaluronan cables were induced by cycloheximide in the confirmed absence of protein synthesis, with or without simultaneous treatment with poly(I,C). We characterized the species specificity of the antibody other investigators used to describe the HC-hyaluronan complex of hyaluronan cables and found minimal affinity to bovine-derived HCs in contrast to HCs from mouse and human sera. Thus, we cultured MASM and HASM cells in serum from these three sources and analyzed hyaluronan extracts for HCs and other hyaluronan-binding proteins, using parallel cumulus cell-oocyte complex (COC) extracts as positive controls. We conclude that, if hyaluronan cables derived from MASM and HASM cells are substituted with HCs, the amount of substitution is significantly below the limit of detection when compared with COC extracts of similar hyaluronan mass.