anti-Fc Antibody Research Articles

Orchestrating NK and T cells via tri-specific nano-antibodies for synergistic antitumor immunity

The functions of natural killer (NK) and T cells in innate and adaptive immunity, as well as their functions in tumor eradication, are complementary and intertwined. Here we show that utilization of multi-specific antibodies or nano-antibodies capable of simultaneously targeting both NK and T cells could be a valuable approach in cancer immunotherapy. Here, we introduce a tri-specific Nano-Antibody (Tri-NAb), generated by immobilizing three types of monoclonal antibodies (mAbs), using an optimized albumin/polyester composite nanoparticle conjugated with anti-Fc antibody. This Tri-NAb, targeting PDL1, 4-1BB, and NKG2A (or TIGIT) simultaneously, effectively binds to NK and CD8+ T cells, triggering their activation and proliferation, while facilitating their interaction with tumor cells, thereby inducing efficient tumor killing. Importantly, the antitumor efficacy of Tri-NAb is validated in multiple models, including patient-derived tumor organoids and humanized mice, highlighting the translational potential of NK and T cell co-targeting.

Developing selection strategy for CHO-K1 cell line that secretes scfv-Fc fusion antibodies using ClonePix2

In the pharmaceutical industry, biopharmaceuticals (biologics) are gaining market share. There has been a dramatic increase in the sale and market penetration of monoclonal antibodies in particular. Typically, therapeutic antibodies are produced using high-expression, clonal, or recombinant CHO cell lines. CHO cells dominate the market as a commercial production host due to their ease of use, built-in regulatory records, and security profiles. While traditional limiting-dilution and cloning-ring regulations are frequently used to select mammalian cell lines that produce high levels of proteins, they have a number of drawbacks. ClonePix2 is a fully automated, single cell-based clone selector that significantly increases the likelihood of rapidly selecting high-production clones with high monoclonality. Scfv-Fc recombinant antibody structures with a variety of therapeutic advantages have gained prominence in recent years. Single cell cloning of CHO cells expressing the scfv-Fc fusion protein, which differs from the classical immunoglobulin structure, was performed in situ using the ClonePix2 device using FITC-tagged anti-Fc and anti-H+L antibodies. The fluorescent intensity parameters of the resulting cell clones were analyzed. Additionally, ELISA was used to determine the production capacities of the best clones. As a result, it was established that anti-Fc antibody recognizes the scfv-Fc fusion protein in a semi-solid environment, enabling the identification of higher production clones.

Neutrophil Activation Through High-Affinity Fey Receptor Using a Monomeric Antibody With Unique Properties

The high-affinity, type I Ig Fc receptor (Fc gamma RI) for human IgG1, human IgG3, murine IgG2a, and murine IgG3 is highly expressed on monocytes, neutrophils (PMN) in certain disease states, and phagocytes treated with interferon-gamma (IFN-gamma). We studied the activation of the human PMN oxidative burst and stimulated fluid pinocytosis induced by three monoclonal antibodies (MoAbs) directed against Fc gamma RI (CD64) to study the role of this receptor in Fc-mediated cellular activation. All three MoAbs were capable of triggering PMN activation from IFN-gamma-treated PMN when cross-linked with goat antimurine Ig reagents. However, MoAb 197 alone demonstrated a concentration-dependent activation of the oxidative burst without the use of a second cross-linking antibody. The oxidative burst and stimulated fluid pinocytosis responses induced by monomeric MoAb 197 could be elicited only after the IFN-gamma induction of approximately 8,000 Fc gamma RI receptor equivalents and did not occur in freshly isolated or control-cultured PMN. Competitive blocking of Fc binding of MoAb 197 by human IgG or purified Fc fragments inhibited cellular activation. We believe the ability of MoAb 197 to activate these oxidative burst and fluid pinocytic responses was because of its murine IgG2a subclass, which allowed it to function as a trivalent anti-Fc gamma RI antibody binding through the combination of its two FAB regions and the Fc domain. This study demonstrates that the cross-linking of CD64 can activate PMN oxidative and endocytic responses and supports a role for Fc gamma RI in the human neutrophil inflammatory response.

Identification of lectin counter-receptors on cell membranes by proximity labeling.

Lectin–glycan interactions play important roles in many biological systems, but the nature of glycoprotein counter-receptors expressed on cell membranes is often poorly understood. To help overcome this problem, we developed a method based on proximity labeling technology. Using a peroxidase-coupled lectin, addition of H2O2 and tyramide-biotin substrates leads to generation of short-range biotin radicals that biotinylate proteins in the immediate vicinity of the bound lectin, which can subsequently be identified. As a proof-of-principle, sialoadhesin-horseradish peroxidase-human IgG1 Fc recombinant protein constructs were precomplexed with anti-Fc antibodies, bound to human erythrocytes and reacted with H2O2 and tyramide-SS-biotin. The erythrocyte membrane protein with strongest biotinylation was identified as glycophorin A, in agreement with early studies using lectin overlay and reglycosylation approaches. As a further test of the method, the plant lectin MAL II was conjugated with horseradish peroxidase and used in proximity labeling of human erythrocytes. Glycophorin A was again selectively labeled, which is consistent with previous reports that MAL II has high affinity for glycophorin. This method could be applied to other lectins to identify their membrane counter-receptors.

Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors

Drozitumab is an agonistic therapeutic monoclonal antibody (mAb) against the pro-apoptotic death receptor 5 (DR5). In vitro cell killing assays using drozitumab have traditionally required cross-linking with anti-Fc antibody to amplify the pro-apoptotic signal, although drozitumab shows activity in in vivo tumor models without artificial cross-linking. Recently it has been shown that FcγR expressing cells play an important role in the activity of drozitumab by mediating cross-linking in vivo (Wilson et al., 2011). To provide a more biologically relevant alternative to cross-linking with anti-Fc antibody in in vitro bioassays, methods for cross-linking with soluble FcγR extracellular domain (ECD) were developed in this work.FcγR cross-linking methods developed in this work were assessed in solution, bead-bound, and plate-bound assay formats, as well as a cell-based assay format. The assays showed reproducible drozitumab dose-response curves in the concentration range of 5–20,000ng/mL and had acceptable precision and accuracy. The assays are also able to detect degradative changes in drozitumab samples subjected to thermal stress. The data suggest that FcγR cross-linking of drozitumab is a viable alternative to anti-Fc cross-linking of drozitumab to measure effector mediated apoptosis of drozitumab in vitro.

Notch Ligand Binding Assay Using Flow Cytometry.

Notch signaling is an evolutionary conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most important aspects of regulation is the binding of different Notch ligands to each Notch receptor (NOTCH1-NOTCH4). Canonical ligands Delta or Serrate (in Drosophila), and Delta-like (DLL1 and DLL4) or Jagged (JAG1 and JAG2) (in mammals), are transmembrane glycoproteins. Ligands expressed on one cell bind to Notch receptors on an adjacent cell to induce Notch signaling. Glycosylation of Notch receptor extracellular domain by O-fucose and O-GlcNAc glycans is well established as critical regulators for Notch signaling strength (Stanley and Okajima, 2010; Haltom and Jafar-Nejad, 2015; Sawaguchi et al., 2017). In order to characterize Notch ligand binding to Notch receptors in isolated cells, we utilize Notch ligand extracellular domains tagged at the C-terminus by a human Fc domain, and determine binding of fluorescent anti-Fc antibody by flow cytometry.

Humanized Anti-PR1/HLA-A2 T Cell Receptor-like Monoclonal Antibody, h8F4, Has Multiple Effector Mechanisms

Previously we developed a T cell receptor (TCR) -like murine IgG2a monoclonal antibody, m8F4, which binds to the PR1 peptide/HLA-A2 complex, and targets both HLA-A2+ leukemia cell lines and primary HLA-A2+ patient blasts of various AML subtypes. We reported that m8F4 mediated complement dependent cytotoxicity (CDC) of AML in vitro and actively depleted AML in vivoin AML patient-derived xenograft (PDX) models. Due to the therapeutic potential of 8F4, we humanized 8F4 for clinical development. Here we report the specificity of this humanized IgG1κ 8F4 (h8F4) to PR1/HLA-A2 and document the activity of h8F4 against AML.Both ELISA and surface plasmon resonance (SPR) were used to study h8F4 specificity to recombinant monomer, consisting of PR1 peptide, HLA-A2 and β2 microglobulin. Like mouse 8F4, human 8F4 bound PR1/HLA-A2 monomer with high affinity (KD= 6.5 nM), while no binding was detected to control monomers pp65/HLA-A2 and WT1/HLA-A2. Moreover, FACS of peptide-pulsed T2 cells showed that h8F4 bound PR1- pulsed T2, but did not bind T2 cells loaded with other HLA-A2-restricted peptides, including MART1, pp65, WT1 and gp100, thus confirming h8F4 specificity to PR1/HLA-A2. To test whether h8F4 retains the in vivoanti-leukemia activity seen with m8F4, we used an NSG mouse model. Similar to m8F4, h8F4 treatment reduced leukemia growth in NSG mice with established disease in 3 out of 4 AML PDX models tested.To determine the mechanism of action (MOA) of humanized 8F4, we first tested h8F4 activity in the presence of complement because we had previously demonstrated the primary MOA of m8F4 to be CDC. However, unlike m8F4, even when tested at high concentrations up to 20 µg/ml, h8F4 did not mediate CDC of HLA-A2 transduced U937, HL60, or PR1-loaded T2 cells. To further examine h8F4 MOA, we tested antibody dependent cellular cytotoxicity (ADCC) of h8F4 using an lactase dehydrogenase (LDH) assay with pooled human PBMC as effectors. H8F4 induced ADCC of U937-A2 at E:T ratio 50 (EC50=0.95 µg/ml), thus indicating improved ADCC anti-AML activity of h8F4. Introduction of alanine substitutions in positions 234-235 (AAh8F4), which disrupted the binding of h8F4 to Fc receptor (FcR), abolished ADCC activity against U937-A2 in vitro. In contrast, AA8F4 still retained partial in vivo activity against the same target cells in the NSG mouse model, suggesting that ADCC is important, but not the exclusive mechanism of h8F4 in vivo. To investigate whether h8F4 had direct activity against AML, as possible alternative MOA, we measured apoptosis of target cells by h8F4 with and without crosslinking anti-Fc antibody fragments (CLA). Indeed, we found, by using annexin V and propidium iodide staining, that a 24 hours incubation with h8F4 (1µg/ml) in the presence of CLA induced apoptosis of 55.5 ± 3.5% of U937-A2 cells, as compared to 23.5 ± 2.1% in CLA-only control (p < 0.05). Moreover, caspase 3 activation was observed as early as 10 minutes after adding h8F4+CLA, and was time- and h8F4 concentration- dependent.Our data demonstrate that specificity of humanized anti-PR1/HLA-A2 TCR-like antibody, h8F4, is preserved and that h8F4 has a similar affinity compared with m8F4. In addition to inducing apoptosis, h8F4 mediates greater ADCC compared with m8F4, but lacks CDC activity in vitro. Together, our data is consistent with multiple mechanisms of action in vivo, including ADCC. Our results support further testing of h8F4 for patients with AML. DisclosuresSergeeva:Astellas Pharma: Patents & Royalties. Molldrem:Astellas Pharma: Patents & Royalties.

Preparation and characterization of novel IgG affinity resin coupling anti-Fc camelid single-domain antibodies.

This work aimed to evaluate novel affinity resin used to purify immunoglobulin G (IgG) with a variable domain of the heavy chain of the heavy-chain antibody (VHH) as an affinity ligand. The VHH, isolated from a naïve camelid single-domain phage display library, exhibits not only affinity to the fragment crystallizable (Fc) region of IgG but also high thermal stability. This anti-Fc VHH (AFV) was expressed as a soluble protein in Escherichia coli and purified using a simple heat treatment procedure. The effects of pH and NaCl concentrations on the capacity of AFV resin were also investigated. Results showed a robust property of the AFV resin. It could bind IgGs at various pH conditions (from 6.0 to 9.0) and NaCl concentrations. The static binding capacities of AFV resin ranged from 3.40±0.53mg/ml to 15.04±0.37mg/ml measured using rabbit, mouse, and human IgGs. The bound IgGs can be efficiently eluted at pH 5.0, which is conducive to acid-sensitive IgGs and prevents the aggregation of IgGs. After 10 purification cycles or a 7-day period of storage at 37°C, recovery did not decrease. These findings suggested that VHHs from non-immunized library could also be robust and functional reagent as an affinity purification ligand.

Dasatinib Is Effective in the Treatment of Mice Models with Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is an autoimmune disease in which anti-platelet antibody (APA) is produced. APA-coated platelets are captured and phagocytized by macrophages in the spleen. Recent study revealed that spleen tyrosine kinase (Syk) inhibitor is effective in the treatment of ITP, because Syk phosphorylation is the key step of the phagocytosis by macrophages. Activated Fc receptor signal transduction is initiated by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) tyrosine residues by SRC family kinases. Recruitment of Syk to dually phosphorylated ITAMs triggers the activation of Syk. To prove the hypothesis that the inhibition of SRC family kinase induces the decreased phosphorylation of Syk, resulting in decreased phagocytosis by macrophages, a SRC family kinase inhibitor, dasatinib, was used in the experiments.Methods, Results and Discussion:In vitro study; Murine macrophage cell line, RAW, was incubated with APA coated murine platelets for 30 minutes. Phagocytosis by RAW was significantly decreased with dasatinib (100nM, p<0.01), indicating SRC family kinase activity is required for efficient phagocytosis. Phosphorylated Syk was decreased in RAW, incubated with anti-Fc receptor antibody (rat IgG) and anti-rat IgG antibody with dasatinib (100nM), shown in the Western blot analysis (Figure 1). These results suggest that Syk phosphorylation is the key step in phagocytosis.In vivo study; (1) Three hours before APA intra-peritoneal injection, dasatinib (2.5mg/kg) was oral-administrated. Six hours after APA injection, platelet counts were measured. The platelet counts were 366 ± 164 x109/L with dasatinib (n=4, mean ± SD) and 114 ± 51 x109/L without dasatinib (n=4)(P=0.026)(Figure 2). (2) Osmotic pump, filled with APA, were inserted in murine intra-peritoneal cavity and dasatinib (2.5mg/kg) was oral-administered once daily for 7 days. The platelet counts were 499 ± 98 x109/L with dasatinib (n=4, mean ± SD) and 82 ± 131 x109/L without dasatinib (n=4) at day 7 (p<0.0022) (Figure 3). These results strongly suggest that dasatinib inhibit the phagocytosis in vivo.Conclusion: Dasatinib inhibits phosphorylation of Syk, inducing decreased phagocytosis of APA-coated platelets via decreased SRC family kinase activity. These findings reveal that SRC family kinase controls the efficiency of phagocytosis in part through the regulation of Syk function. Dasatinib might be effective in the treatment of ITP. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recapitulating cell-cell adhesion using N-cadherin biologically tethered to substrates.

Intercellular adhesion modulated by cadherin molecules plays an important role in diverse cellular functions including tissue morphogenesis, regeneration, and pathogenesis. However, it is a challenging task to decipher the effects of cell-cell adhesion in vitro because of difficulty in controlling the extent and numbers of cell-cell contacts. In this study, we hypothesize that tethering recombinant extracellular domains of neural cadherin with a C-terminal immunoglobulin Fc domain (N-Cad-Fc) to a substrate with an immobilized anti-Fc antibody (Fc-antibody) and a bifunctional polymer, which is reactive to both protein and substrate, would allow us to recapitulate cell-cell adhesion, independent of the number of cells plated on the substrate. To examine this hypothesis, we first immobilized Fc-antibody to a polyacrylamide hydrogel and a methacrylate-substituted glass using poly(amino-2-hydroxyethyl-co-2-methacryloxyethyl aspartamide)-g-poly(ethylene glycol)-N-hydroxysuccinimide ester (PHMAA-g-PEGNHS) and then incubated the gel in medium containing defined concentrations of the recombinant N-Cad-Fc. The resulting N-Cad-conjugated substrate enabled us to modulate adhesion of bone marrow stromal cells to the gel surface by varying the surface density of N-Cad-Fc. In contrast, direct chemical conjugation of N-Cad-Fc to the gel surface did not support cell adhesion. Additionally, the glass substrate biologically tethered with N-Cad-Fc promoted neuronal adhesion significantly more than substrates coated with poly-l-lysine. We suggest that this novel biological tethering method could be broadly applicable for modifying substrates with a variety of classical cadherins to enable the systematic study of the effects of cadherin-modulated cell-cell adhesion on cellular activities.

High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy

The discovery of monoclonal antibodies (mAbs) that bind to a particular molecular target is now regarded a routine exercise. However, the successful development of mAbs that (1) express well, (2) elicit a desirable biological effect upon binding, and (3) remain soluble and display low viscosity at high concentrations is often far more challenging. Therefore, high throughput screening assays that assess self-association and aggregation early in the selection process are likely to yield mAbs with superior biophysical properties. Here, we report an improved version of affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) that is capable of screening large panels of antibodies for their propensity to self-associate. AC-SINS is based on concentrating mAbs from dilute solutions around gold nanoparticles pre-coated with polyclonal capture (e.g., anti-Fc) antibodies. Interactions between immobilized mAbs lead to reduced inter-particle distances and increased plasmon wavelengths (wavelengths of maximum absorbance), which can be readily measured by optical means. This method is attractive because it is compatible with dilute and unpurified mAb solutions that are typical during early antibody discovery. In addition, we have improved multiple aspects of this assay for increased throughput and reproducibility. A data set comprising over 400 mAbs suggests that our modified assay yields self-interaction measurements that are well-correlated with other lower throughput assays such as cross-interaction chromatography. We expect that the simplicity and throughput of our improved AC-SINS method will lead to improved selection of mAbs with excellent biophysical properties during early antibody discovery.

Visualization of Subunit-Specific Delivery of Glutamate Receptors to Postsynaptic Membrane during Hippocampal Long-Term Potentiation

An increase in the number of AMPA-type glutamate receptors (AMPARs) is critical for long-term potentiation (LTP), synaptic plasticity regarded as a basal mechanism of learning and memory. However, when and how each type of AMPAR reaches the postsynaptic membrane remain unclear. We have developed experimental methods to form postsynaptic-like membrane (PSLM) on a glass surface to precisely visualize the location and movement of receptors. We observed fluorescence-labeled AMPAR subunits (GluA1-3) around PSLM with total internal reflection fluorescence microscopy. The increases of GluA1, 2, and 3 in PSLM showed different time courses after LTP induction. GluA1 increased first, and was exocytosed to the periphery of PSLM soon after LTP induction. GluA2 and GluA3 initially decreased, and then increased. Exocytosis of GluA2 and GluA3 occurred primarily in non-PSLM, and later than exocytosis of GluA1. Thus, GluA1-3 appear to increase in the postsynaptic membrane through distinct pathways during LTP.

Transfer of HBV Genomes Using Low Doses of Adenovirus Vectors Leads to Persistent Infection in Immune Competent Mice

Studies of mechanisms responsible for the persistence of hepatitis B virus (HBV) infection have been hindered by a lack of appropriate animal models. HBV genomes can be delivered to livers of mice using hydrodynamic injection or high doses of an adenoviral vector; these lead to clearance of HBV. We found that infection of immunocompetent mice with low doses of an adenoviral vector resulted in persistent HBV infection; the mice neither underwent seroconversion to production of antibodies against HBV nor developed a strong HBV-specific effector T-cell response. As in patients with chronic HBV infection, DNA vaccination failed to generate T cells that cleared infection. This model of persistent HBV infection could be used to study the pathogenesis of chronic HBV infection and develop new therapeutic strategies.

Monoclonal antibodies against ROR1 induce apoptosis of chronic lymphocytic leukemia (CLL) cells

ROR1 is a receptor tyrosine kinase (RTK) recently identified to be overexpressed at the gene and protein levels in chronic lymphocytic leukemia (CLL). Monoclonal antibodies (MAbs) against RTKs have been successfully applied for therapy of solid tumors. We generated five MAbs against the Ig (n = 1), cysteine-rich (CRD) (n = 2) and kringle (KNG) (n = 2) domains, respectively, of the extracellular part of ROR1. All CLL patients (n = 20) expressed ROR1 on the surface of the leukemic cells. A significantly higher frequency of ROR1 expression was found in patients with progressive versus non-progressive disease, and in those with unmutated versus mutated IgVH genes. All five MAbs alone induced apoptosis in the absence of complement or added effector cells (Annexin-V and MTT, as well as cleavage of poly-(ADP ribose)-polymerase, caspase-8 and caspase-9) of CLL cells but not of normal B cells. Most effective were MAbs against CRD and KNG, significantly superior to rituximab (P < 0.005). Cross-linking of anti-ROR1 MAbs using the F(ab')(2) fragments of anti-Fc antibodies significantly augmented apoptosis. Two of the MAbs induced complement-dependent cytotoxicity (CDC) similar to that of rituximab and one anti-ROR1 MAb (KNG) (IgG1) showed killing activity by antibody-dependent cellular cytotoxicity. The identified ROR1 epitopes may provide a basis for generating human ROR1 MAbs for therapy.

FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons

Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A-Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.

Complement Activation and Cytotoxicity In CLL Cells Treated with Alemtuzumab and Ofatumumab: Evidence for Multiple Mechanisms of Resistance to Complement Dependent Cytotoxicity

AbstractAbstract 3575Unconjugated monoclonal antibodies (MoAb) have proven value in the therapy of CLL, and alemtuzumab (ALEM) has efficacy in patients with purine analog refractory/p53 defective disease. Although the mechanisms of action of ALEM in vivo are not fully defined, complement dependant cytotoxicity (CDC) appears to play an important role. ALEM CDC resistance in a subpopulation of CLL cells in vivo could thus contribute to treatment failure. We hypothesized that the CD20 targeted complement activating human MoAb ofatumumab (OFA) would increase complement activation (as manifested by C3b deposition) as well as CDC in CLL cells treated with ALEM in vitro. Methods:We have completed experiments on 4 of 15 specimens collected from patients with progressive untreated CLL. CLL cells were isolated from blood by density gradient centrifugation and negative selection with magnetic beads. CD52 and CD20 expression were measured with fluorescent beads (Quantum MESF, Bangs Laboratories, IN). CLL cells were treated at a concentration of 2 × 106/ml with 10 mcg/ml of ALEM (Genzyme) and/or OFA (G.S.K.) in AIM V medium (Invitrogen CA) and 10% standard normal human serum (NHS) (Sigma, MO) as a source of complement for 1 hour at 37C. Absolute viable cell counts were assayed with counting beads (TruCOUNT, BD, CA) and propidium iodide (PI) staining (Sigma) using a FACSCalibur (BD) and CellQuest Pro software (BD). CDC was determined by comparing counts to CLL cells treated with NHS alone. Membrane levels of CD59, bound MoAb, and deposited C3b on the CLL cells membranes were measured by flow cytometry using using the anti-CD59 antibody FITC-CD59 (BD), mouse anti-human Fc antibody FITC-HB43, and the anti-C3b antibody FITC-7C12. To study ALEM CDC resistant cells, CLL cells were first treated with ALEM and NHS as detailed above. Surviving viable cells were isolated using density gradient centrifugation and then retreated. C5 deficient serum (Sigma) was used in experiments to determine MoAb binding and C3b deposition in the entire CLL cell population. Results:Binding of either ALEM or OFA to the CLL cells in NHS resulted in activation of complement and deposition of large amounts of membrane C3b on the cells. However, ALEM induced higher levels of C3b deposition (delta MFI 220) and caused more CDC (73% vs. 18%) compared to OFA. This correlated well with the higher levels of expression of CD52 vs. CD20 expression in CLL cells (143 ×103, range 131 ×103 – 159 ×103 vs. mean 20 ×103 molecules/cell, range 10 ×103 – 28 ×103). Studies with the anti-Fc antibody showed that only a small subpopulation of CLL cells had weak MoAb binding. Addition of OFA to ALEM increased complement-mediated C3b deposition. In three patients with high initial ALEM CDC, addition of OFA did not appreciably increase CDC (81% to 87%). However, in one patient with cells that were less sensitive to ALEM CDC, addition of OFA did appreciably increase CDC (51% to 86%). There was no relationship between levels of CD59 expression and CDC. All four CLL specimens studied contained a subpopulation of cells that were resistant to CDC (mean 13%, range 9–18%) despite robust C3b deposition. Conclusions:These data suggests that there are multiple mechanisms of resistance to MoAb-mediated CDC including: 1. Low MoAb binding because of low target antigen expression; 2. Insufficient C3b deposition after MoAb ligation and; 3. Intrinsic resistance in some cells in cells in which high levels of C3b deposition were clearly demonstrable. OFA can increase MoAb binding, C3b deposition, and CDC in CLL cells treated with ALEM. However, addition of OFA does not overcome the intrinsic resistance of a small subpopulation of CLL cells to CDC. Future studies will be needed to examine these resistance mechanisms and to develop interventions to disrupt them. In addition, membrane C3b could be a target for complement dependent cellular cytotoxity in these CDC resistant cells. Disclosures:Taylor:G.S.K.: Research Funding; Genmab: Research Funding. Zent:Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

The immunogenic part of infliximab is the F(ab′)2, but measuring antibodies to the intact infliximab molecule is more clinically useful

ObjectivesTo localise the immunogenic part of infliximab and evaluate the clinical usefulness of measuring antibodies against infliximab fragments.DesignObservational study.SettingsA specialised inflammatory bowel disease (IBD) centre in a tertiary hospital.InterventionsSerum was...

Type-I IFN Signaling Suppresses an Excessive IFN-γ Response and Thus Prevents Lung Damage and Chronic Inflammation During Pneumocystis (PC) Clearance in CD4 T Cell-Competent Mice

Immune-reconstitution after highly active antiretroviral therapy (HAART) is often incomplete, and some HIV-infected individuals fail to regenerate type-I interferon (IFN)-producing pDCs. We recently demonstrated that during Pneumocystis (PC) infection in CD4 T cell-competent mice the absence of type-I IFN signaling results in chronic pulmonary inflammation and fibrosis despite clearance. Because the mechanisms involved are poorly understood, we further characterized the role of type-I IFN signaling in immune responses to PC. We show that type-I IFN signaling around day 7 postinfection is critical to the outcome of inflammation. Microarray analysis of pulmonary CD11c(+) cells revealed that at day 7 post infection, wild-type cells up-regulated type-I IFN-responsive genes as well as SOCS1, which is a critical negative-regulator of type-I IFN and IFN-gamma signaling. This was associated with an eosinophilic lung inflammation, PC clearance, and complete restitution. However, pulmonary CD11c(+) cells from IFNAR(-/-) mice demonstrated increased tumor necrosis factor (TNF)-alpha production and lacked SOCS1-induction at day 7. This was followed by a transient lymphocytic and IFN-gamma response before switching to a chronic eosinophilic inflammation of the lung. Early neutralization of TNF-alpha did not prevent chronic inflammation in IFNAR(-/-) mice, but treatment with an anti-IFN-gamma antibody did. We propose that during PC lung infection type-I IFNs induce SOCS1-associated regulatory mechanisms, which prevent excessive IFN-gamma-mediated responses that cause chronic lung damage. Therefore, partial immune-reconstitution in AIDS, attributable to reduced type-I IFN actions, might disrupt regulatory aspects of inflammation, causing unexplained chronic pulmonary complications as seen in some patients during HAART.

Immunoliposomes Incorporated with Humanized Monoclonal Antibody, Milatuzumab, Induce Cell Death in CLL by Retention of the CD74 Receptor On the Surface of B Cells.

Abstract 721Chronic lymphocytic leukemia (CLL) is a progressive disease for which most patients require treatment. Therapy with monoclonal antibodies (mAbs) such as rituximab (anti-CD20) and alemtuzumab (anti-CD52) have improved the outcomes of patients with CLL; however, patients display varied levels of response to these agents and many develop resistance. We describe here a humanized mAb, milatuzumab (Immunomedics, Inc.), against CD74. The CD74 antigen has several ligands, including macrophage inhibitory factor, MIF, whose binding promotes internalization and nuclear localization with concomitant activation of NF-κB. Milatuzumab is currently in clinical trials in multiple myeloma and CLL; however, the mechanism by which this antibody induces apoptosis is still unknown. We demonstrate surface expression of CD74 on CLL and circulating normal CD19+ B cells, but no expression on T cells. Milatuzumab-induced cytotoxicity occurs very rapidly, with 70% cell survival at 4 h and 45% at 24 h, which is superior to that observed with the CD20 antibody rituximab. No toxicity was detected in NK and T cells treated with milatuzumab. The mode of cell death induced by milatuzumab was found to be independent of caspase activation, with no detectable caspase-3 processing, PARP cleavage, or inhibition of death with the pan-caspase inhibitor, BocD-fmk. In vitro, the addition of a-Fc crosslinking antibody is necessary for milatuzumab-induced cell death. However, previous reports noted milatuzumab does not mediate antibody-dependent cellular cytotoxicity (ADCC) in lymphoma cell lines and this was verified in CLL using peripheral blood mononuclear cells, NK cells, and granulocytes as effector cells. Thus, milatuzumab-mediated death appears to be mediated directly through interaction with CD74 and effects on signal transduction. We further investigated the role of anti-Fc crosslinking antibody in mediating milatuzumab-induced cell death, and found that upon milatuzumab treatment in the presence of anti-Fc, CLL cells aggregated in culture. Furthermore, the mean fluorescent intensity (MFI) of CD74 on the cell surface increased, which did not occur following treatment with anti-Fc alone, milatuzumab alone, or anti-Fc along with other anti-B-cell mAbs such as rituximab. This suggests that milatuzumab, when crosslinked, promotes the maintenance of CD74 on the cell surface and thereby prevents receptor internalization and subsequent signaling. However, association with Fc receptors on other cells in the microenvironment may not be sufficient to mediate this effect, as indicated by the lack of ADCC. Furthermore, in vitro co-culture of NK cells in the presence of milatuzumab did not induce the same cellular aggregation and elevated CD74 surface expression as seen with the anti-Fc antibody. These results indicate that milatuzumab treatment in vivo may not be as efficient as suggested by the in vitro studies without a sufficient method to sequester CD74 on the cell surface. We tested this hypothesis by incorporating milatuzumab into liposomes (Mila-ILP). Our results show that this conjugate mediates the same receptor aggregation on the cell surface as milatuzumab combined with anti-Fc crosslinking antibody. This effect was not evident with incorporation of irrelevant immunoglobulin into liposomes (IgG-ILP). Furthermore, Mila-ILP induced the same degree of cell death as observed previously with in vitro antibody crosslinking. Therefore, the incorporation of milatuzumab into liposomes induces cell death independent of other cell types in the microenvironment. Together, these data describe a mechanism of milatuzumab-induced cytotoxicity in CLL, and encourage the clinical application of milatuzumab-immunoliposome-based therapy in CLL and other CD74-positive lymphoid malignancies. Disclosures:Goldenberg:Immunomedics: Employment, Equity Ownership.

CD8+ T Cells Engineered to Express a ROR1-Specific Chimeric Antigen Receptor Specifically Recognize ROR1 Positive B Cell Tumors.

Abstract 930The orphan tyrosine kinase receptor ROR1 was previously identified as a highly expressed gene by expression profiling of B cell chronic lymphocytic leukemia (B-CLL), [Klein et al. J Exp Med 2001], and has subsequently been shown to be expressed on mantle cell lymphoma (MCL) and a subset of B cell acute lymphoblastic leukemias (B-ALL). ROR1 encodes a 105 kDa protein that contains Ig-like, cysteine rich, kringle, tyrosine kinase and proline rich domains and is expressed during embryonic development but is absent on normal adult tissues including non-malignant B cells. The function of ROR1 in normal and malignant cells is not known, although secreted Wnt proteins have been proposed as candidate ligands. Analysis of ROR1 protein expression using specific polyclonal antibodies revealed uniform, stable, and restricted cell surface expression on B-CLL, suggesting this molecule is a candidate for targeted immunotherapy of B cell malignancies [Baskar et al. Clin Cancer Res 2008]. We constructed a lentiviral vector that encodes a chimeric antigen receptor (CAR) consisting of single chain variable (scFV) fragments of the heavy and light chains of a murine monoclonal antibody specific for ROR1, linked to an IgG4 Fc domain, the T cell receptor CD3 zeta chain and a CD28 costimulatory domain. The specificity and function of the ROR1 CAR was compared with a similarly designed CAR specific for the CD20 molecule, which is expressed on both malignant and normal B cells, and is being targeted with gene-modified T cells in clinical trials. Primary human CD8+ T cells were transduced with the ROR1 and CD20-specific CARs respectively, and T cells expressing high levels of the receptors were sort-purified using an anti-Fc antibody. T cells that expressed either the ROR1-specific CAR or the CD20-specific CAR efficiently lysed primary B-CLL samples (5/5) obtained from patients with advanced disease, and also lysed a MCL cell line (JeKo-1), and a ROR1+ B-ALL cell line (BALL-1). ROR1-specific T cells did not recognize the myeloid leukemia cell line K562, but efficiently lysed K562 cells that had been transfected to express ROR1, confirming the specific recognition of ROR1 on target cells. Consistent with the expression pattern of the target molecules, T cells that expressed the CD20-specific CAR also efficiently lysed normal primary and EBV-transformed B cells, but T cells that expressed the ROR1-specific CAR did not recognize nonmalignant or EBV-transformed B cells. Activation of normal B cells by engagement of the B cell receptor or activation through CD40 induced B cell proliferation and upregulation of the CD80 and CD86 costimulatory molecules, but did not result in ROR1 surface expression by flow cytometry or recognition by T cells that expressed the ROR1-specific CAR. These results suggest that targeting ROR1 with gene-modified T cells may have advantages over targeting B cell-lineage restricted molecules such as CD19 and CD20 that are expressed on normal mature B cells. Studies to determine whether ROR1 is expressed during a stage of normal B cell development are in progress. ROR1 is highly conserved in non-human primates and this model may be suitable to determine potential toxicities of adoptive immunotherapy with ROR1-specific CAR expressing T cells. Disclosures:No relevant conflicts of interest to declare.