Irrelevant mAb Research Articles

Correction: Treatment of mice with a ligand binding blocking anti-CD28 monoclonal antibody improves healing after myocardial infarction.

[This corrects the article DOI: 10.1371/journal.pone.0227734.].

PD-1 blockade later but not at post-transplant attenuates chronic graft-versus-host disease

Abstract PD-1 and its ligand PD-L1 are widely appreciated as targets of immune checkpoint blockade. We sought to investigate the role of the PD-1 pathway in the context of chronic graft-versus-host disease (cGVHD). To induce cGVHD, mice are conditioned with Cytoxan and radiation prior to MHC disparate bone marrow (BM) + T cells (70k) and develop multi-organ symptoms with bronchiolitis obliterans lung manifestations. CGHVD mice have high survival rates past 50 days, with <15% weight loss over this period. Anti-PD-1 mAb given to mice with established cGVHD resulted in disease attenuation assessed by significantly improved pulmonary function tests compared to irrelevant mAb controls. PD-1 blockade significantly reduced germinal center (GC)-promoting GC B and T follicular helper (Tfh) cell and increased GC-suppressing T-follicular regulatory (Tfr) cell frequencies. However, in studies with mice given five percent higher T cell dose, resulting in T cell rather than GC-mediated disease, anti-PD-1 mAb dramatically worsened cGVHD severity. To assess GC cell-specific effects, recipients were given PD-1-deficient cells in graft compartments. Mice receiving PD-1 KO T cells predictably exhibited marked escalation of cGVHD severity and lethality, while mice receiving PD-1 KO B cells showed diminished disease, with significant improvement in pulmonary function. Co-culture of isolated B, Tfh and Tfr cells from NP-OVA immunized mice to determine effects of individual GC cell populations on Ig class switching demonstrated significant increase in Tfr suppressive capacity with addition of anti-PD-1 mAb. Together, these data provide evidence for PD-1 support of the GC reaction and identifies the PD-1/PD-L1 axis as a potential target for cGVHD therapy.

Treatment of mice with a ligand binding blocking anti-CD28 monoclonal antibody improves healing after myocardial infarction.

Both conventional and regulatory CD4+ T-cells rely on costimulatory signals mediated by cell surface receptors including CD28 for full activation. We showed previously that stimulation of CD4+ Foxp3+ regulatory T-cells by superagonistic anti-CD28 monoclonal antibodies (mAb) improves myocardial healing after experimental myocardial infarction (MI). However, the effect of ligand binding blocking anti-CD28 monoclonal antibodies has not yet been tested in this context. We hypothesize that ligand blocking anti-CD28 mAb treatment might favorably impact on healing after MI by limiting the activation of conventional CD4+ T-cells. Therefore, we studied the therapeutic effect of the recently characterized mAb E18 which blocks ligand binding to CD28 in a mouse permanent coronary ligation model. E18 or an irrelevant control mAb was applied once on day two after myocardial infarction to wildtype mice. Echocardiography was performed on day 7 after MI. E18 treatment improved the survival and reduced the incidence of left ventricular ruptures after experimental myocardial infarction. Accordingly, although we found no difference in infarct size, there was significantly less left ventricular dilation after E18 treatment in surviving animals as determined by echocardiography at day 7 after MI. In sham operated control mice neither antibody had an impact on body weight, survival, and echocardiographic parameters. Mechanistically, compared to control immunoglobulin, E18 treatment reduced the number of CD4+ T-cells and monocytes/macrophages within the infarct and periinfarct zone on day 5. This was accompanied by an upregulation of arginase which is a marker for alternatively differentiated macrophages. The data indicate that CD28-dependent costimulation of CD4+ T-cells impairs myocardial healing and anti-CD28 antibody treatment constitutes a potentially clinically translatable approach to improve the outcome early after MI.

Complement Inhibition as Potential Novel Therapy for Antibody-Mediated Rejection after Lung Transplantation in Murine Antibody-Mediated Rejection Model

Antibody-mediated rejection (AMR) can induce acute or chronic graft failure in organ transplantations. There are few reports regarding AMR in lung transplant, and no reports of effective therapy for AMR after lung transplant. This study aimed to verify the efficacy of anti-complement 5(anti-C5), which is reported to prevent AMR in kidney transplant, in an AMR-enhanced mouse model after lung transplant. As allograft model, BALB/c (Major histocompatibility complex; MHC H2b) and C57BL/6 (MHC H2b) were used for donor and recipient in order to detect and enhance AMR. Skin transplants were performed to pre-sensitize, and subsequently, orthotopic left lung transplants were performed 14 days after skin transplant. The mice were treated with anti-C5 (40mg/kg/day) (n=5), or cyclosporine (CyA, 10mg/kg/day) /methylprednisolone (mPSL, 1.6mg/kg/day) (n=5), or anti-C5/CyA/mPSL (n=5), or with isotype-matched irrelevant control mAb (n=5) subcutaneously until sacrifice. The mice were sacrificed 2 days after lung transplant and evaluated for histopathology, including acute rejection score, C4d immunostaining, and donor-specific antibody (DSA) in serum. The mice with anti-C5 showed significantly lower C5 in serum compared with the mice with isotype control (1472.50±110.44 vs. 2081.87±75.17 pg/ml, p=0.002). The mice treated with anti-C5 and anti-C5/CyA/mPSL showed significantly lower rejection A score (0.63±0.23; p=0.002, 0.59±0.22; p=0.001) compared with the mice with isotype control (2.58±0.35). All groups showed C4d deposition on vascular endothelium and DSA in serum. Anti-C5 effectively prevented AMR against transplanted allogeneic lung in the pre-sensitized murine model, in which AMR was strongly augmented.

Monoclonal Antibodies Against Human Papillomavirus E6 and E7 Oncoproteins Inhibit Tumor Growth in Experimental Cervical Cancer

Nearly all cases of cervical cancer are initiated by persistent infection with high-risk strains of human papillomavirus (hr-HPV). When hr-HPV integrates into the host genome, the constitutive expression of oncogenic HPV proteins E6 and E7 function to disrupt p53 and retinoblastoma regulation of cell cycle, respectively, to favor malignant transformation. HPV E6 and E7 are oncogenes found in over 99% of cervical cancer, they are also expressed in pre-neoplastic stages making these viral oncoproteins attractive therapeutic targets. Monoclonal antibodies (mAbs) represent a novel potential approach against the actions of hr-HPV E6 and E7 oncoproteins.In this report, we describe the utilization of anti-HPV E6 and HPV E7 mAbs in an experimental murine model of human cervical cancer tumors. We used differential dosing strategies of mAbs C1P5 (anti-HPV 16 E6) and TVG701Y (anti-HPV E7) administered via intraperitoneal or intratumoral injections. We compared mAbs to the action of chemotherapeutic agent Cisplatin and demonstrated the capacity of mAbs to significantly inhibit tumor growth. Furthermore, we investigated the contribution of the immune system and found increased complement deposition in both C1P5 and TVG701Y treated tumors compared to irrelevant mAb therapy. Taken together, the results suggest that anti-HPV E6 and E7 mAbs exert inhibition of tumor growth in a viral-specific manner and stimulate an immune response that could be exploited for an additional treatment options for patients.

The Hemagglutinin A Stem Antibody MEDI8852 Prevents and Controls Disease and Limits Transmission of Pandemic Influenza Viruses.

MEDI8852 is a novel monoclonal antibody (mAb) that neutralizes both group I and group II influenza A viruses (IAVs) in vitro. We evaluated whether MEDI8852 was effective for prophylaxis and therapy against representative group I (H5N1) and group II (H7N9) pandemic IAVs in mice and ferrets and could be used to block transmission of influenza H1N1pdm09 in ferrets, compared to an irrelevant control mAb R347 and oseltamivir. MEDI8852 was administered to mice and ferrets by intraperitoneal injection at varying doses, 24 hours prior to intranasal infection with H5N1 and H7N9 viruses for prophylaxis, and 24, 48, and 72 hours post-infection for treatment. A comparison with oseltamivir alone and combination of MEDI8852 and oseltamivir was included in some studies. Survival, weight loss, and viral titers were assessed over a 14-day study period. For the transmission study, naive respiratory contact ferrets received MEDI8852 or R347 prior to exposure to ferrets infected with an H1N1pdm09 virus. MEDI8852 was effective for prophylaxis and treatment of H7N9 and H5N1 infection in mice, with a clear dose-dependent response and treatment with MEDI8852 24, 48, or 72 hours postinfection was superior to oseltamivir for H5N1. MEDI8852 alone was effective treatment for lethal H5N1 infection in ferrets compared to oseltamivir and R347, and MEDI8852 plus oseltamivir was better than oseltamivir alone. MEDI8852 or oseltamivir alone early in infection was equally effective for H7N9 infection in ferrets while the combination yielded similar protection when treatment was delayed. MEDI8852 was able to protect naive ferrets from airborne transmission of H1N1pdm09. MEDI8852, alone or with oseltamivir, shows promise for prophylaxis or therapy of group I and II IAVs with pandemic potential. Additionally, MEDI8852 blocked influenza transmission in ferrets, a unique finding among influenza-specific mAbs.

BAMBI, a new molecular target in autoimmunity

Abstract We have previous demonstrated that BAMBI (BMP and Activin Membrane Bound Inhibitor) negatively regulates the functional differentiation of CD4 T cells into tolerogenic regulatory T-cells (Tregs) and positively the differentiation into pro-inflammatory TH17 cells. Accordingly, mice deficient in BAMBI are resistant against the development of collagen type II-induced arthritis (CIA). To analyze the therapeutic effects of BAMBI inhibition in normal mice in the development of autoimmunity, we developed new inhibitory mouse anti-mouse BAMBI mAbs. Treatment of B10RIII wild type (WT) mice with a high dose of a mouse IgG1 anti-mouse BAMBI mAb (clone B101.37; 2 mg/mice/week), but not with a low dose of B101.37 (0.3 mg/mice/week) or a high dose of an irrelevant IgG1 mAb (2 mg/mice/week), inhibited the development of CIA as efficiently as observed in B10RIII.BAMBI-KO mice. We further explore the therapeutic potential of B101.37 in experimental models of psoriasis (Ps) and psoriasis arthritis (PsA). A single ip injection of mannan from Saccharomyces cerevisiae induces an acute inflammation resembling human Ps and PsA in B10RIII.WT but with a reduced severity in B10RIII.BAMBI-KO mice. Again, treatment of WT mice with B101.37, but not with an irrelevant IgG1 antibody, protects the animals against mannan-induced PsA and Ps by a Treg- and TGFβ-dependent mechanism. B101.37 also exhibits a therapeutic activity against mannan-induced PsA and Ps once the disease has been established. Similar results have been observed in a model of Ps induced after the topic administration of the TLR-7 agonist imiquimod. Altogether, our results demonstrate the beneficial effects of BAMBI inhibitors in the treatment of autoimmune skin and joint diseases.

Mechanism of quinine-dependent monoclonal antibody binding to platelet glycoprotein IIb/IIIa

AbstractDrug-dependent antibodies (DDAbs) that cause acute thrombocytopenia upon drug exposure are nonreactive in the absence of the drug but bind tightly to a platelet membrane glycoprotein, usually αIIb/β3 integrin (GPIIb/IIIa) when the drug is present. How a drug promotes binding of antibody to its target is unknown and is difficult to study with human DDAbs, which are poly-specific and in limited supply. We addressed this question using quinine-dependent murine monoclonal antibodies (mAbs), which, in vitro and in vivo, closely mimic antibodies that cause thrombocytopenia in patients sensitive to quinine. Using surface plasmon resonance (SPR) analysis, we found that quinine binds with very high affinity (KD ≈10−9 mol/L) to these mAbs at a molar ratio of ≈2:1 but does not bind detectably to an irrelevant mAb. Also using SPR analysis, GPIIb/IIIa was found to bind monovalently to immobilized mAb with low affinity in the absence of quinine and with fivefold greater affinity (KD ≈2.2 × 10−6) when quinine was present. Measurements of quinine-dependent binding of intact mAb and fragment antigen-binding (Fab) fragments to platelets showed that affinity is increased 10 000- to 100 000-fold by bivalent interaction between antibody and its target. Together, the findings indicate that the first step in drug-dependent binding of a DDAb is the interaction of the drug with antibody, rather than with antigen, as has been widely thought, where it induces structural changes that enhance the affinity/specificity of antibody for its target epitope. Bivalent binding may be essential for a DDAb to cause thrombocytopenia.

Monoclonal antibodies to heat shock protein 60 induce a protective immune response against experimental Paracoccidioides lutzii

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America. PCM is primarily caused by Paracoccidioides brasiliensis and less frequently by the recently described, closely related species Paracoccidioides lutzii. Current treatment requires protracted administration of systemic antibiotics and relapses may frequently occur despite months of initial therapy. Hence, there is a need for innovative approaches to treatment. In the present study we analyzed the impact of two monoclonal antibodies (mAbs) generated against Heat Shock 60 (Hsp60) from Histoplasma capsulatum on the interactions of P. lutzii with macrophages and on the experimental P. lutzii infection. We demonstrated that the Hsp60-binding mAbs labeled P. lutzii yeast cells and enhanced their phagocytosis by macrophage cells. Treatment of mice with the mAbs to Hsp60 before infection reduced the pulmonary fungal burden as compared to mice treated with irrelevant mAb. Hence, mAbs raised to H. capsulatum Hsp60 are protective against P. lutzii, including mAb 7B6 which was non-protective against H. capsulatum, suggesting differences in their capacity to bind to these fungi and to be recognized by macrophages. These findings indicate that mAbs raised to one dimorphic fungus may be therapeutic against additional dimorphic fungi, but also suggests that biological differences in diseases may influence whether a mAb is beneficial or harmful.

Tissue Factor On The Herpes Simplex Virus Type 1 Surface Enhances Infection In Vivo

Tissue factor (TF) is a multifunctional transmembrane receptor. As the pivotal initiator of the coagulant response to vascular damage, it accelerates factor (F) VIIa-dependent generation of FXa. However, TF also functions in FVIIa/FXa-dependent cell signaling via protease activated receptors (PARs), and has consequently been implicated in a wide variety of physiological and pathological conditions. Our previous work adds to this repertoire by revealing that TF, originating from the host cell membrane, is on the surface of enveloped viruses. Using herpes simplex virus type 1 (HSV1) as a model for enveloped viruses, we recently produced TF +/- HSV1 variants, which showed that viral TF enhances infection of cultured cells through a mechanism involving FXa/FVIIa-mediated activation of PAR-2. In the current project we extended these studies in vivo, hypothesizing that TF on the HSV1 surface would enhance infection by influencing the activation of coagulation proteases and/or their role in cell signaling. As before, HSV1 NS strain (with restored glycoprotein C) was propagated in TF- or genetically engineered TF+ human melanoma A7 cells, then purified and quantified. HSV1 TF+ or HSV1 TF- virus (5 x 105 virus plaque forming units of similar particle number) was injected slowly in 100 µL into the tail vein of female, eight-week old balb/c mice. On the third day, organs and blood were harvested. Live virus and total HSV1 genome were evaluated by plaque assays and rtPCR, respectively. Strikingly, production of live HSV1 TF+ (n=13) in the lung, heart, spinal cord, liver and brain was greater by an order of magnitude compared to HSV1 TF- (n=13), which was not detected in some tissues. The presence of TF on HSV1 furthermore increased virus titers in spleen and blood by several fold. Viral replication in organs was corroborated by rtPCR. The procoagulant and signaling functions of TF can be dissected using epitope-specific monoclonal antibodies (mAbs) selective for human TF. These mAbs only recognize HSV1 surface TF and not the endogenous mouse TF. These mAbs or an irrelevant mAb (TIB115, n=8) were administered by intraperitoneal injection 4 hours pre-inoculation (1 mg/mouse). When TF-dependent coagulation and the related effects on cell signaling due to attenuated protease activation was blocked by a potent combination of three mAbs to non-overlapping epitopes (5G9/6B4/9C3; 0.33 mg/mouse each, n=10), reduced levels of live HSV1 TF+ were seen in lung, heart, liver, brain and blood, with concordantly reduced viral genome levels measured by rtPCR. Similar results were obtained with mAb 5G9 alone (n=6), which selective blocks FXa generation. Interestingly, titers of HSV1 TF+ in blood and spleen were not affected by mAB 10H10 (n=6) that has no effect on coagulation, but is an effective direct inhibitor of TF signaling. Despite normal blood titers, mAb 10H10 (n=6) reduced virus titers in heart and brain, but not in other organs. These data demonstrate that virus surface TF is important for HSV1 infection in vivo and indicate that distinct functions of TF contribute to organ-specific tropism. Disclosures:No relevant conflicts of interest to declare.

CD44v6 Is Required For In Vivo Tumorigenesis Of Human AML and MM Cells: Role Of Microenvironmental Signals and Therapeutic Implications

BackgroundTargeting the interactions between tumor cells and their microenvironment is an exciting new frontier in cancer therapy. The biology of acute myeloid leukemia (AML) and multiple myeloma (MM) is characterized by addiction to specific signals uniquely provided within the bone marrow (BM), where tumor cells preferentially home and locally thrive. The hyaluronate receptor CD44 was shown to be required for retroviral-induced leukemogeneis in syngeneic mouse models. Conversely, CD44 mAbs interfere with human leukemia initiation in immunocompromised mice by inhibiting leukemia stem cell homing to the bone marrow (BM). The therapeutic potential of CD44 mAbs is also under clinical investigation in humans. Much less is known on the role of the differently spliced CD44 variant isoforms. The expression of exon 6 (CD44v6) conveys additional properties to standard CD44, like binding to osteopontin and cooperation with different tyrosine kinase receptors (RTKs), like VEGF receptor type II and c-Met. Interestingly, CD44v6 is the most abundantly expressed CD44 isoform in both AML and MM, where it correlates with a bad prognosis. Since CD44v6 expression is much more tumor-restricted than CD44, targeting this isoform may have a better efficacy/toxicity profile than targeting the standard molecule. AimTo preclinically validate CD44v6 as a therapeutic target in AML and MM ResultsBy FACS analysis and RT-qPCR, we established CD44v6 over expression in a relevant fraction of leukemic blasts from AML pts (15/25, 60%) with preference for the M4-5 FAB subtypes, and in the majority of malignant plasmacells from MM pts (13/15, 87%). CD44v6 was also over expressed on THP-1, Kasumi and U937 human AML cells, and on MM.1S, XG-6 and XG-7 MM cells. To address the specific role of CD44v6 in BM homing, we pretreated MM1.S cells with either a CD44 mAb (SFF-2) or a CD44v6 mAb (VFF-18) and infused them i.v. in NSG mice. Unexpectedly, while SFF-2 almost completely inhibited early (18hrs) homing to the BM compared with an irrelevant mAb, VFF-18 had no effect. To rule out confounding variables associated with specific mAb clones, we silenced CD44v6 expression in MM1.S cells by lentiviral-mediated shRNA transduction and confirmed no difference in BM homing compared with control LV-transduced cells. Longer follow-ups (4-6 weeks) however revealed that, despite unaltered rates of in vitro proliferation, CD44v6-silenced MM1.S cells were severely hampered in their tumorigenic capacity in vivo (P<0.001). These results were confirmed by using THP-1 cells (P<0.001) and primary leukemic blasts (P<0.01). Hypothesizing that CD44v6 may be crucial for in vivo tumorigenesis by cooperating with RTKs, we set-up a co-culture system with BM-derived mesenchymal stromal cells (MSCs), which are producers of VEGF and the c-Met ligand HGF. MSCs protected a wide range of tumor cells, including primary leukemic blasts, from spontaneous apoptosis (P<0.05) and from apoptosis induced by Ara-C or daunorubicin (P<0.01), or bortezomib in the case of MM cells (P<0.001). Comparable results were obtained by using MSC supernatants, hinting to a causative soluble factor, which was neither VEGF nor HGF, as demonstrated by inhibition experiments with bevacizumab and crizotinib, respectively. Noteworthy, MSCs or their supernatants prompted a significant up-regulation of CD44v6 expression levels (P<0.01). Most importantly, preventing CD44v6 up-regulation on tumor cells by shRNA silencing restored their sensitivity to spontaneous and drug-induced apoptosis (P<0.01). ConclusionsThese results clearly indicate that CD44v6 is dispensable for BM homing, but responsible for AML and MM addiction to microenvironmental signals. Combining CD44v6 targeting with cytotoxic chemotherapy might interfere with this vicious circle and result in higher and/or more durable response rates. Disclosures:No relevant conflicts of interest to declare.

ImmunoPET Imaging of Renal Cell Carcinoma with 124I- and 89Zr-Labeled Anti-CAIX Monoclonal Antibody cG250 in Mice

Monoclonal antibody (mAb) cG250 recognizes carbonic anhydrase IX (CAIX), overexpressed on clear cell renal cell carcinoma (ccRCC). (124)I-cG250 is currently under clinical investigation for the detection of ccRCC. However, the (124)I label is rapidly excreted from the tumor cells after internalization of the radiolabeled mAb. We hypothesized that labeling cG250 with the residualizing positron emitter (89)Zr would lead to higher tumor uptake and more sensitive detection of ccRCC lesions. Nude mice with CAIX-expressing ccRCC xenografts (SK-RC-52 or NU-12) were i.v. injected with (89)Zr-cG250 or (124)I-cG250. To determine specificity of (89)Zr-cG250 uptake in ccRCC, one control group was i.v. injected with (89)Zr-MOPC21 (irrelevant mAb). PET images were acquired using a small animal PET camera and the biodistribution of the radiolabeled mAb was determined. The ccRCC xenografts were clearly visualized after injection of (89)Zr-cG250 and (124)I-cG250. Tumor uptake of (89)Zr-cG250 was significantly higher compared with (124)I-cG250 in the NU-12 tumor model (114.7% ± 25.2% injected dose per gram (%ID/g) vs. 38.2 ± 18.3%ID/g, p=0.029), but in the SK-RC-52 the difference in tumor uptake was not significant (48.7 ± 15.2%ID/g vs. 32.0 ± 22.9%ID/g, p=0.26). SK-RC-52 tumors were not visualized with (89)Zr-MOPC21 (tumor uptake 3.0%ID/g). Intraperitoneal SK-RC-52 lesions as small as 7 mm(3) were visualized with (89)Zr-cG250 PET. ImmunoPET imaging with cG250 visualized s.c. and i.p. ccRCC lesions in murine models. This confirms the potential of cG250 immunoPET in the diagnosis and (re)staging of ccRCC. PET imaging of ccRCC tumors with (89)Zr-cG250 could be more sensitive than (124)I-cG250-PET.

Strain-related differences in antibody-mediated changes in gene expression are associated with differences in capsule and location of binding

We recently established that antibody (Ab)-binding can induce gene expression changes in a serotype A strain (H99) of the pathogenic yeast, Cryptococcus neoformans. That study showed that monoclonal antibodies (mAbs) differing in epitope specificity and protective efficacy elicited differences in gene expression. Because many mAbs bind to serotypes A and D strains differently, we now investigate the binding of one mAb to two strains representing these serotypes. Cells of the serotype A strain H99 and the serotype D strain 24067 were incubated with near saturating concentrations of the IgG1 capsule-binding mAb 18B7 or MOPC, an irrelevant mAb matched control. Comparative immunofluorescence analysis of mAb 18B7 binding revealed that it bound closer to the cell wall in H99 than 24067, where it was associated with decreased or increased cell diameter, respectively. A comparison of encapsulated cell compressibility showed that strain 24067 was more compressible than that of strain H99. RNA was extracted and used for gene expression analysis using the C. neoformans JEC21 genomic microarray. After 1h incubation with mAb 18B7, there were just 2 gene expression changes observed with strain 24067 or strain JEC21, unlike the 43 seen with strain H99. After 4h incubation with mAb 18B7, there were 14 and 140 gene expression changes observed with strain 24067 and JEC21, respectively. Thus, C. neoformans strains differ both in the response and the time of response to mAb binding and these differences may reflect differences in the location of Ab binding, Ab-mediated changes in cell diameter and compressibility of the capsular polysaccharide.

ROR1 Isoforms Are Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) - a Survival Factor for CLL Cells

Abstract 1778 Background:Phosphorylation of receptor tyrosine kinases (RTK) plays an important role for many aspects of cell life, as cell-cycle progression, differentiation, apoptosis, intercellular communication and cell survival. RTKs as e.g. EGFR, HER2/neu, VEGFR, IGFR etc. are important structures contributing to the malignant phenotype of many tumors. Targeting RTKs by monoclonal antibodies (MAb) or small inhibitory molecules has been successful for the treatment of various cancers. ROR is one of the twenty RTK families and consists of two members, ROR1 and ROR2. ROR1 and ROR2 have important functions during embryogenesis. ROR1 is uniquely expressed in CLL cells compared to normal tissues. ROR1 siRNA transfection of CLL cells induced specific apoptosis of the leukemic cells. Aims:To study ROR1 protein variants in CLL, ROR1 phosphorylation and relation to clinical activity of the disease. Methods:Phosphorylation of primary human CLL cells from patients with non-progressive and progressive disease, and a series of cell lines were studied applying immunoprecipitation (IP) of the ROR1 molecule, using anti ROR1 and irrelevant MAbs by Western blotting as well as in intracytoplasmic staining of the cells by flowcytometry, using an anti-phospho ROR1 (pROR1) MAb (a MAb specifically recognizing a phospho-peptide of the cytoplasmic TK domain of ROR1) and semi quantification of the staining intensity as well as by p-tyrosine and p-serine MAbs. Results:IP of the ROR1 molecule of fresh leukemic cells using ROR1 specific antibodies and subsequent Western blot with anti ROR1 MAbs, showed various protein bands. Two bands had the size of 105 and 130 KDa probably representing unglycosylated or partially glycosylated ROR1 and the fully glycosylated glycoform respectively. One of the two bands dominated in individual patients. A band of 260 KDa could also be detected in a large number of patients probably representing dimerized monomers. Finally a band of 64 KDa could also be noted which may represent an intracellular part of ROR1, as has been described in fetal and adult human CNS, leukemia and lymphoma cell lines (Reddy UR et al, Oncogene. 1996; 13:1555–9). The 64, 105 and 130 KDa bands were constitutively phosphorylated in all patients both at tyrosine and serine residues. The intensity of phosphorylation of the 130 KDa band was significantly higher in patients with progressive disease vs. non-progressive disease (p=0.0001). There was no relation between the pattern of the different ROR1 protein bands and disease activity.Using 9 cell lines of different hematologic malignancies including CLL, similar protein bands and phosphorylation status as for fresh CLL cells were noted.We have also produced specific mouse monoclonal antibodies against defined epitopes of the extracellular parts of ROR1. One of those MAbs (anti-KNG, IgG1) induced a high degree of specific apoptosis of the CLL cells (ASH Annual Meeting Abstracts, Nov 2010; 116: 916) and could also be shown to induce a specific dephosphorylation of the cytoplasmic TK domain which was noted already after 20 min and gradually increased up to 4h. No effects were noted using irrelevant MAbs or healthy donor lymphocytes. Conclusion:Our results showed that the ROR1 molecule in CLL cells are expressed in various protein variants probably representing different glycosylation patterns (105–130 KDa), dimerized monomers (260 KDa) and a truncated protein variant (64 KDa). ROR1 was constitutively phosphorylated (64, 105 and 130 KDa) both at serine and tyrosine residues. Treatment of CLL cells in vitro with a ROR1 specific antibody induced specific apoptosis as well as rapid dephosphorylation of ROR1. Collectively the data suggest that phosphorylated ROR1 might be an important structure for the growth potential of CLL cells and an interesting structure to target in a therapeutic intervention. Disclosures:Mellstedt:Kancera AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hojjat-Farsangi:Kancera AB: Equity Ownership. Rabbani:Kancera AB: Equity Ownership.

Binding of Anti-Membrane-Proximal gp41 Monoclonal Antibodies to CD4-Liganded and -Unliganded Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus Virions

The broadly neutralizing monoclonal antibodies (MAbs) 4E10, 2F5, and Z13e1 target membrane-proximal external region (MPER) epitopes of HIV-1 gp41 in a manner that remains controversial. The requirements for initial lipid bilayer binding and/or CD4 ligation have been proposed. To further investigate these issues, we probed for binding of these MAbs to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions with protein A-conjugated gold (PAG) nanoparticles using negative-stain electron microscopy. We found moderate levels of PAG associated with unliganded HIV-1 and SIV virions incubated with the three MAbs. Significantly higher levels of PAG were associated with CD4-liganded HIV-1 (epitope-positive) but not SIV (epitope-negative) virions. A chimeric SIV virion displaying the HIV-1 4E10 epitope also showed significantly higher PAG association after CD4 ligation and incubation with 4E10. MAbs accumulated rapidly on CD4-liganded virions and slowly on unliganded virions, although both reached similar levels in time. Anti-MPER epitope-specific binding was stable to washout. Virions incubated with an irrelevant MAb or CD4-only (no MAb) showed negligible PAG association, as did a vesicle-rich fraction devoid of virions. Preincubation with Fab 4E10 inhibited both specific and nonspecific 4E10 IgG binding. Our data provide evidence for moderate association of anti-MPER MAbs to viral surfaces but not lipid vesicles, even in the absence of cognate epitopes. Significantly greater MAb interaction occurs in epitope-positive virions following long incubation or CD4 ligation. These findings are consistent with a two-stage binding model where these anti-MPER MAbs bind first to the viral lipid bilayer and then to the MPER epitopes following spontaneous or induced exposure.

Toward Developing a Universal Treatment for Fungal Disease Using Radioimmunotherapy Targeting Common Fungal Antigens

Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall. MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls. (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing. Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Combination immunotherapy of MAb B6.1 with fluconazole augments therapeutic effect to disseminated candidiasis

We recently reported that IgM MAb B6.1, specific for β-1, 2-mannotriose on the cell wall of Candida albicans, is therapeutic to disseminated candidiasis due to C. albicans. In the current study, we examined if MAbB6.1 enhances therapeutic effect of fluconazole (FLC) to the disseminated disease. To assess the combination effect, determination by the kidneys-colony forming unit and survival times were used. Results showed that the therapeutic effect of FLC on mice with disseminated candidiasis was dose-dependent, but a FLC dose at 0.8 mg/kg body weight of mice was ineffective. To determine combination effect, mice treated intraperitoneally with a combination of FLC plus MAb B6.1 at 1 h post-infection - a condition of developing partial therapeutic activity - enhanced survival times beyond the effect by only antibody (p < 0.05). The resulting MST (mean survival times) value from the combination-received mice was almost the same as MST value from 3.2 mg FLC dose-given animals (p < 0.05). Another combination of 1.6 mg FLC dose and B6.1 reduced severity of the disseminated disease at almost the same rate as combination efficacy of 0.8 mg FLC dose plus B6.1. This data indicates that B6.1 acts in concert with FLC and that this combination therapy augments protection, which suggests a possibility of reducing FLC dose. The augmentation response was specific because an irrelevant IgM MAb S9 was not effective to the disseminated disease. Thus, our present studies demonstrate that this combination immunotherapy may be a way of solving the problem of limited antifungal drug choices caused by drug-resistant C. albicans.

MicroSPECT/CT Imaging of Human Leukemia Engraftment In NOD-Scid Mice Using [111In]-Labeled 7G3 Anti-CD123 Antibodies

AbstractAbstract 968Radioimmunotherapy (RIT) of acute myelogenous leukemia (AML) is a promising strategy that could improve the long-term outcome of patients. A limitation of current therapies is their inability to eradicate the leukemic stem cell (LSC), which is responsible for causing and propagating the disease and the failure to achieve long-term survival. Monoclonal antibody (mAb) 7G3 recognizes CD123 (IL-3R α) in the absence of CD131 (IL-3R β) a LSC characteristic but not of normal hematopoietic stem cells. Our laboratory is developing 111In-labeled 7G3 modified with nuclear localization sequence (NLS) peptides for Auger electron RIT of AML aimed at the LSC. These electrons have only nanometer-micrometer range but are lethal when released inside the cell nucleus. Our objective was to examine the ability of 111In-NLS-7G3 to localize at sites of AML engraftment in NOD-scid mice taking advantage of the concurrent gamma emissions of 111In for microSPECT/CT imaging. These properties were examined in NOD-scid mice engrafted with CD123+/CD131- AML cell lines which avoids the challenges of engrafting primary AML specimens including their variability in engraftment efficiency and heterogeneity in CD123 expression. Alternatively, mice bearing s.c. CD123-transfected Raji (Raji-CD123) tumors were studied. 7G3 was reacted with sulfo-SMCC to introduce maleimide groups for conjugation to NLS peptides (CGYGPKKKRKVGG) then directly derivatized with DTPA for complexing 111In. The pharmacokinetics (PK) of elimination from the blood of 111In-NLS-7G3 intact IgG2a and F(ab')2 fragments were studied in NOD-scid and Balb/c mice. The effect of pre-administration of a 10-fold excess of unlabeled BM4 IgG2a or IgG1 irrelevant mAbs on the PK was assessed. Imaging and biodistribution studies were performed at 48 to 72 h p.i. of 111In-NLS-7G3 IgG2a and F(ab')2 in AML-engrafted mice. Sites of engraftment visualized on the images were analyzed for AML by flow cytometry. PK studies revealed an unusually rapid elimination of 111In-NLS-7G3 IgG2a but not F(ab')2 in NOD-scid compared to Balb/c mice with blood radioactivity levels at 18 h 4-fold lower (3.9 ± 0.9 vs. 17.0 ± 4.4% i.d./ml, respectively). This rapid elimination prevented uptake into Raji-CD123 xenografts and did not allow their visualization by microSPECT/CT. The elimination of 111In-NLS-7G3 F(ab')2 was not significantly different in NOD-scid or Balb/c mice (0.60 ± 0.17 vs. 0.87 ± 0.06 %i.d./ml, respectively at 72 h p.i.). Pre-administration of unlabeled BM4 improved the retention of 111In-NLS-7G3 in the blood by 3.2-fold and allowed for high uptake (14.7 ± 2.8 % i.d./g) and imaging of tumors. Xenografts also had high uptake (7.2 ± 3.8 % i.d./g) and imaged with 111In-NLS-7G3 F(ab')2. AML-3, -4 and -5 cells engrafted into bone marrow (BM) and NOD-scid mice assessed by the proportion of CD45+ cells (12%, 0.5% and 63%, respectively). CD45+ cells in the spleen for AML-5 engraftment was 83%. Uptake of 111In-NLS-7G3 F(ab')2 in the spleen and BM of AML-3 engrafted mice were 5-fold and 1.5-fold higher than 111In-NLS-BM4 control F(ab')2. The spleen was visualized by microSPECT/CT in engrafted mice receiving 111In-NLS-7G3 F(ab')2 but not with 111In-NLS-BM4 F(ab')2. In AML-3 engrafted mice imaged with 111In-NLS-7G3 IgG2a, multiple intense foci of radioactivity were noted in the spine, pelvis and femur, representing leukemia in the marrow. Extramedullary (EM) disease was also imaged in the brain and lymph nodes. Uptake in the femur was 2-fold higher in engrafted mice compared to non-engrafted mice. In AML-5 engrafted mice, outgrowths of AML at the femoral heads was imaged with 111In-NLS-7G3 IgG2a following pre-administration of an excess of unlabeled BM4. No visualization of these AML growths was noted for mice receiving 111In-NLS-BM4. We conclude that microSPECT/CT allowed sensitive detection of the specific uptake of 111In-NLS-7G3 and its F(ab')2 fragments in AML engrafted mice. PK and Raji-CD123 xenograft microSPECT/CT studies identified a tumor delivery barrier. A robust AML engraftment model was established with cells displaying the CD123+/CD131- phenotype. These results are promising for preclinical evaluation of 111In-NLS-7G3 for Auger electron RIT of AML and suggest that imaging is a useful tool to assess engraftment in NOD-scid mice as well as for evaluating response to 111In-NLS-7G3. Preliminary results of studies to examine 111In-NLS-7G3 for RIT of AML will also be presented. Disclosures:No relevant conflicts of interest to declare.

Efficacy of combination immunotherapy of IgM MAb B6.1 and amphotericin B against disseminated candidiasis

We previously reported that the IgM MAb B6.1, specific for β-1,2-mannotriose on the surface of the cell wall of Candida albicans, prevents mice from disseminated candidiasis. The preventive activity of the antibody is mediated by enhanced phagocytosis that caused killing of the fungus and involvement of the complement system. In the present study, MAb B6.1 was tested if the antibody enhances therapeutic efficacy of amphotericin B (Amp B) in a murine model of disseminated candidiasis due to C. albicans. Determination by the kidneys-cfu (colony forming unit) and survival times was used to assess treatment. Mice treated intraperitoneally with MAb B6.1 at 1 h post-infection with C. albicans (5 × 10 5 yeast cells/mouse) developed fewer 28% cfu and prolonged survival rates than negative controls, whereas administration of B6.1 to mice at 2 h post-infection was ineffective. Therapeutic effect of Amp B on mice with the disseminated disease was dose-dependent, but dosage of Amp B (0.5 mg/kg body weight of mice) was not effective. A combination of MAb B6.1given at 1 h post-infection plus Amp B (0.5 mg dose) enhanced survival times beyond the effect due to only antibody ( P < 0.05). The MST (mean survival times) value resulted from the combination therapy-received mice was as almost the same as MST value from 2 mg dose of Amp B-given animals ( P < 0.05). In case mice given a combination of Amp B (0.5 mg dose) plus MAb B6.1 at 2 h post-infection — a condition of developing no therapeutic efficacy, the combination also reduced disease severity. All these data indicate that MAb B6.1 acts in concert with Amp B, the antibody enhances therapeutic efficacy of Amp B, and this combination therapy augments protection which implies a possibility of reducing Amp B dose. The augmentation of the response appeared to be specific because an irrelevant IgM carbohydrate-specific MAb was not protective. In conclusion, these studies show that Amp B combined with MAb B6.1 may be an option of solving the problem of limited antifungal drug choices due to drug-resistant C. albicans

Pro-prion Binds Filamin A, Facilitating Its Interaction with Integrin β1, and Contributes to Melanomagenesis

Filamin A (FLNA) is an integrator of cell mechanics and signaling. The spreading and migration observed in FLNA sufficient A7 melanoma cells but not in the parental FLNA deficient M2 cells have been attributed to FLNA. In A7 and M2 cells, the normal prion (PrP) exists as pro-PrP, retaining its glycosylphosphatidyl-inositol (GPI) anchor peptide signal sequence (GPI-PSS). The GPI-PSS of PrP has a FLNA binding motif and binds FLNA. Reducing PrP expression in A7 cells alters the spatial distribution of FLNA and organization of actin and diminishes cell spreading and migration. Integrin β1 also binds FLNA. In A7 cells, FLNA, PrP, and integrin β1 exist as two independent, yet functionally linked, complexes; they are FLNA with PrP or FLNA with integrin β1. Reducing PrP expression in A7 cells decreases the amount of integrin β1 bound to FLNA. A PrP GPI-PSS synthetic peptide that crosses the cell membrane inhibits A7 cell spreading and migration. Thus, in A7 cells FLNA does not act alone; the binding of pro-PrP enhances association between FLNA and integrin β1, which then promotes cell spreading and migration. Pro-PrP is detected in melanoma in situ but not in melanocyte. Invasive melanoma has more pro-PrP. The binding of pro-PrP to FLNA, therefore, contributes to melanomagenesis.