BL-01, an Fc-bearing, tetravalent CD20 × CD5 bispecific antibody, redirects multiple immune cells to kill tumors in vitro and in vivo.

BACKGROUND AIMS: The authors describe here a novel therapeutic strategy combining a bispecific antibody (bsAb) with cytokine-induced killer (CIK) cells. METHODS: The authors have designed, produced and purified a novel tetravalent IgG1-like CD20 × CD5 bsAb called BL-01. The bsAb is composed of a fused heavy chain and two free light chains that pair correctly to the heavy chain sequences thanks to complementary mutations in the monoclonal antibody 2 CH1/CL sequences. RESULTS: The authors show that BL-01 can bind specifically to CD20 and CD5 with an affinity of 4-6 nM, demonstrating correct pairing of two light chains to the fused heavy chain. The CD20 × CD5 BL-01 bsAb has a functional human IgG1 Fc and can induce up to 65% complement-dependent cytotoxicity of a CD20+ lymphoma cell line in the presence of human complement, similar to anti-CD20 rituximab. The bsAb also induces significant natural killer cell activation and antibody-dependent cytotoxicity of up to 25% as well as up to 65% phagocytosis by human macrophages in the presence of CD20+ tumor cells. The BL-01 bsAb binds to CD20 and CD5 simultaneously and can redirect CIK cells in vitro to kill CD20+ targets, increasing the cytotoxicity of CIK cells by about 3-fold. The authors finally show that the CD20 × CD5 BL-01 bsAb synergizes with CIK cells in vivo in controlling tumor growth and prolonging survival of nonobese diabetic/severe combined immunodeficiency mice inoculated with a patient-derived, aggressive diffuse large B-cell lymphoma xenograft. CONCLUSIONS: The authors suggest that the efficacy of bsAb in vivo is due to the combined activation of innate immunity by Fc and redirection of CIK cells to kill the tumor target.

A Nano-Emulsion Platform Functionalized with a Fully Human scFv-Fc Antibody for Atheroma Targeting: Towards a Theranostic Approach to Atherosclerosis.

Atherosclerosis is at the onset of the cardiovascular diseases that are among the leading causes of death worldwide. Currently, high-risk plaques, also called vulnerable atheromatous plaques, remain often undiagnosed until the occurrence of severe complications, such as stroke or myocardial infarction. Molecular imaging agents that target high-risk atheromatous lesions could greatly improve the diagnosis of atherosclerosis by identifying sites of high disease activity. Moreover, a "theranostic approach" that combines molecular imaging agents (for diagnosis) and therapeutic molecules would be of great value for the local management of atheromatous plaques. The aim of this study was to develop and characterize an innovative theranostic tool for atherosclerosis. We engineered oil-in-water nano-emulsions (NEs) loaded with superparamagnetic iron oxide (SPIO) nanoparticles for magnetic resonance imaging (MRI) purposes. Dynamic MRI showed that NE-SPIO nanoparticles decorated with a polyethylene glycol (PEG) layer reduced their liver uptake and extended their half-life. Next, the NE-SPIO-PEG formulation was functionalized with a fully human scFv-Fc antibody (P3) recognizing galectin 3, an atherosclerosis biomarker. The P3-functionalized formulation targeted atheromatous plaques, as demonstrated in an immunohistochemistry analyses of mouse aorta and human artery sections and in an Apoe-/- mouse model of atherosclerosis. Moreover, the formulation was loaded with SPIO nanoparticles and/or alpha-tocopherol to be used as a theranostic tool for atherosclerosis imaging (SPIO) and for delivery of drugs that reduce oxidation (here, alpha-tocopherol) in atheromatous plaques. This study paves the way to non-invasive targeted imaging of atherosclerosis and synergistic therapeutic applications.

Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies.

We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-hinge domains of mAb2 are fused through a peptidic linker to the N terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDR-CR3 or -MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR(+) leukemic cells by human CD5(+) cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDR-CR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia.

Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

In Vivo Human Single-Chain Fragment Variable Phage Display-Assisted Identification of Galectin-3 as a New Biomarker of Atherosclerosis.

Background Atherosclerosis is a complex pathology in which dysfunctional endothelium, activated leucocytes, macrophages, and lipid-laden foam cells are implicated, and in which plaque disruption is driven by many putative actors. This study aimed to identify accurate targetable biomarkers using new in vivo approaches to propose tools for improved diagnosis and treatment. Methods and Results Human scFv (single-chain fragment variable) selected by in vivo phage display in a rabbit model of atherosclerosis was reformatted as scFv fused to the scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G format) antibodies. Their reactivity was tested using flow cytometry and immunoassays, and aorta sections from animal models and human carotid and coronary artery specimens. A pool of atherosclerotic proteins from human endarterectomies was co-immunoprecipitated with the selected scFv-Fc followed by mass spectrometry for target identification. Near-infrared fluorescence imaging was performed in Apoe-/- mice after injection of an Alexa Fluor 647-labeled scFv-Fc-2c antibody produced in a baculovirus system with 2 additional cysteine residues (ie, 2c) for future coupling to nano-objects for theranostic applications. One scFv-Fc clone (P3) displayed the highest cross-reactivity against atherosclerotic lesion sections (rabbit, mouse, and human) and was chosen for translational development. Mass spectrometry identified galectin-3, a ß-galactoside-binding lectin, as the leader target. ELISA and immunofluorescence assays with a commercial anti-galectin-3 antibody confirmed this specificity. P3 scFv-Fc-2c specifically targeted atherosclerotic plaques in the Apoe-/- mouse model. Conclusions These results provide evidence that the P3 antibody holds great promise for molecular imaging of atherosclerosis and other inflammatory pathologies involving macrophages. Recently, galectin-3 was proposed as a high-value biomarker for the assessment of coronary and carotid atherosclerosis.

Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival.

Anti­Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets in ovarian carcinoma. Conversely, the role of the three AMH type I receptors (AMHRIs), namely activin receptor­like kinase (ALK)2, ALK3 and ALK6, in ovarian cancer remains to be clarified. To determine the respective roles of these three AMHRIs, the present study used four ovarian cancer cell lines (COV434­AMHRII, SKOV3­AMHRII, OVCAR8, KGN) and primary cells isolated from tumor ascites from patients with ovarian cancer. The results demonstrated that ALK2 and ALK3 may be the two main AMHRIs involved in AMH signaling at physiological endogenous and supraphysiological exogenous AMH concentrations, respectively. Supraphysiological AMH concentrations (25 nM recombinant AMH) were associated with apoptosis in all four cell lines and decreased clonogenic survival in COV434­AMHRII and SKOV3­AMHRII cells. These biological effects were induced via ALK3 recruitment by AMHRII, as ALK3­AMHRII dimerization was favored at increasing AMH concentrations. By contrast, ALK2 was associated with AMHRII at physiological endogenous concentrations of AMH (10 pM). Based on these results, tetravalent IgG1­like bispecific antibodies (BsAbs) against AMHRII and ALK2, and against AMHRII and ALK3 were designed and evaluated. In vivo, COV434­AMHRII tumor cell xenograft growth was significantly reduced in all BsAb­treated groups compared with that in the vehicle group (P=0.018 for BsAb 12G4­3D7; P=0.001 for all other BsAbs). However, the growth of COV434­AMHRII tumor cell xenografts was slower in mice treated with the anti­AMRII­ALK2 BsAb 12G4­2F9 compared with that in animals that received a control BsAb that targeted AMHRII and CD5 (P=0.048). These results provide new insights into type I receptor specificity in AMH signaling pathways and may lead to an innovative therapeutic approach to modulate AMH signaling using anti­AMHRII/anti­AMHRI BsAbs.

A recombinant human monoclonal anti-R7V antibody as a potential therapy for HIV infected patients in failure of HAART.

The construction of a recombinant antibody directed against the cellular epitope R7V acquired by HIV during the viral budding has been realized. The c-DNAs encoding the variable regions of the anti-R7V antibody have been cloned from B lymphocytes of a non-progressor patient. Two transfer vectors containing complete coding sequences for heavy and light chains of this antibody were constructed and a recombinant baculovirus was generated by a double recombination between baculovirus DNA and the two transfer vectors. Insect cells infected with this baculovirus produced a complete human anti-R7V immunoglobulin. This recombinant antibody, specific to the R7V peptide, recognizes and neutralizes all clades of HIV1 including resistant viruses, opening new perspectives in anti-HIV therapy.

Data on atherosclerosis specific antibody conjugation to nanoemulsions.

This article present data related to the publication entitled "Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging" (Prévot et al., 2017) [1]. Herein we describe the engineering in the baculovirus-insect cell system and purification processes of the human scFv-Fc TEG4-2C antibody, specific of platelets within the atheroma plaque. For molecular targeting purpose, atheroma specific antibody was conjugated to nanoemulsions (NEs) using a heterobifunctional linker (DSPE-PEG-maleimide). Atheroma labelling was assayed by immunochemistry on arterial sections from rabbits.

Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging.

PURPOSE: For early atherosclerosis imaging, magnetic oil-in-water nanoemulsion (NE) decorated with atheroma specific monoclonal antibody was designed for Magnetic Particle Imaging (MPI) and Magnetic Resonance Imaging (MRI). MPI is an emerging technique based on direct mapping of superparamagnetic nanoparticles which may advantageously complement MRI. METHODS: NE oily droplets were loaded with superparamagnetic iron oxide nanoparticles of 7, 11 and 18nm and biofunctionalized with atheroma specific scFv-Fc TEG4-2C antibody. RESULTS: Inclusion of nanoparticles inside NE did not change the hydrodynamic diameter of the oil droplets, close to 180nm, nor the polydispersity. The droplets were negatively charged (ζ=-30mV). In vitro MPI signal was assessed by Magnetic Particle Spectroscopy (MPS). NE displayed MRI and MPS signals confirming its potential as new contrast agent. NE MPS signal increase with NPs size close to the gold standard (Resovist). In MRI, NE displayed R2* transversal relaxivity of 45.45, 96.04 and 218.81mM-1s-1 for 7, 11 and 18nm respectively. NE selectively bind atheroma plaque both in vitro and ex vivo in animal models of atherosclerosis. CONCLUSION: Magnetic NE showed reasonable MRI/MPS signals and a significant labelling of the atheroma plaque. These preliminary results support that NE platform could selectively image atherosclerosis.

Determination and characterization of bovine interleukin-17 cDNA.

Interleukin-17 (IL-17) is a proinflammatory cytokine produced by activated memory T cells, and it appears to play an upstream role in T cell-triggered inflammation by stimulating stromal cells to secrete other cytokines. We hypothesize that IL-17 plays a role in the recruitment of neutrophils in the bovine mammary gland during infection or immune-mediated inflammation. The rapid amplification of cDNA ends (RACE) method was used to obtain a cDNA of bovine IL-17 (BoIL-17) containing a 462-bp open reading frame (ORF) encoding a protein of 153 amino acids (aa) with a molecular mass of 17.2 kDa, a 23-residue NH(2)-terminal signal peptide, a single potential N-linked glycosylation site, and 6 cysteine residues. BoIL-17 protein shared 73.5% identity with the human protein and 67% with the mouse and rat proteins. Sf9 insect cells were transfected with BoIL-17 cDNA, and supernatant was tested for biologic activity on a primary culture of bovine mammary epithelial cells (MECs). mRNA synthesis of IL-6, IL-8, and growth-related oncogene alpha (Groalpha) was induced, suggesting a functional role for IL-17 in mammary immunity.

Molecular cloning and expression of a human hST8Sia VI (alpha2,8-sialyltransferase) responsible for the synthesis of the diSia motif on O-glycosylproteins.

Based on BLAST analysis of the human and mouse genome databases using the human CMP sialic acid; alpha2,8-sialyltransferase cDNA (hST8Sia I; EC 2.4.99.8), a putative sialyltransferase gene, was identified on human chromosome 10. The genomic organization was found to be similar to that of hST8Sia I and hST8Sia V. Transcriptional expression analysis showed that the newly identified gene was constitutively expressed at low levels in various human tissues and cell lines. We have isolated a full-length cDNA clone from the breast cancer cell line MCF-7 that encoded a type II membrane protein of 398 amino acid residues with the conserved motifs of sialyltransferases. We have established a mammary cell line (MDA-MB-231) stably transfected with the full-length hST8Sia VI and the analysis of sialylated carbohydrate structures expressed at the cell surface clearly indicated the disappearance of Neu5Acalpha2-3-sialylated structures. The transient expression of a truncated soluble form of the enzyme in either COS-7 cells or insect Sf-9 cells led to the production of an active enzyme in which substrate specificity was determined. Detailed substrate specificity analysis of the hST8Sia VI recombinant enzyme in vitro, revealed that this enzyme required the trisaccharide Neu5Acalpha2-3Galbeta1-3GalNAc (where Neu5Ac is N-acetylneuraminic acid and GalNAc is N-acetylgalactosamine) to generate diSia (disialic acid) motifs specifically on O-glycans.

Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines.

Equine luteinizing hormone (eLH) and chorionic gonadotropin (eCG) are composed of identical alpha and beta polypeptide chains, but eCG subunits are much more heavily glycosylated and sialylated. Consequently, eCG exhibits a much longer half-life than eLH in blood. Recombinant eLH/CG, expressed in Sf9 and Mimic insect cells, were compared with one another and to the natural hormones eCG and eLH. Mimic cells are stably-transformed Sf9 cells, expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex N-carbohydrate chains. Recombinant eLH/CG expressed in Mimic cells exhibited a higher apparent molecular weight (MW) than that expressed in Sf9 cells, suggesting that its N-glycosylation was, as expected, more complete. Nevertheless, the two recombinant eLH/CG exhibited lower MW than natural eCG from pregnant mare plasma. The two eLH/CG produced in Sf9 and Mimic cells were found to be active in in vitro LH and FSH bioassays, with potencies similar to those of eCG. By contrast, they exhibited no significant in vivo bioactivity, neither in the specific follicle-stimulating hormone (FSH) assay nor in the specific eCG assay. Although recombinant eLH/CG produced in Mimic cells bears more elaborate carbohydrate chains than recombinant eLH/CG from Sf9 cells, it exhibits no significant in vivo bioactivity, probably because of insufficient terminal sialylation of its carbohydrate chains, leading to its rapid removal from blood.

The α1,6-fucosyltransferase gene (fut8) from the Sf9 lepidopteran insect cell line: insights into fut8 evolution.

The core alpha1,6-fucosyltransferase (FUT8) catalyzes the transfer of a fucosyl moiety from GDP-fucose to the innermost asparagine-linked N-acetylglucosamine residue of glycoproteins. In mammals, this glycosylation has an important function in many fundamental biological processes and although no essential role has been demonstrated yet in all animals, FUT8 amino acid (aa) sequence and FUT8 activity are very well conserved throughout the animal kingdom. We have cloned the cDNA and the complete gene encoding the FUT8 in the Sf9 (Spodoptera frugiperda) lepidopteran cell line. As in most animal genomes, fut8 is a single-copy gene organized in different exons. The open reading frame contains 12 exons, a characteristic that seems to be shared by all lepidopteran fut8 genes. We chose to study the gene structure as a way to characterize the evolutionary relationships of the fut8 genes in metazoans. Analysis of the intron-exon organization in 56 fut8 orthologs allowed us to propose a model for fut8 evolution in metazoans. The presence of a highly variable number of exons in metazoan fut8 genes suggests a complex evolutionary history with many intron gain and loss events, particularly in arthropods, but not in chordata. Moreover, despite the high conservation of lepidoptera FUT8 sequences also in vertebrates and hymenoptera, the exon-intron organization of hymenoptera fut8 genes is order-specific with no shared exons. This feature suggests that the observed intron losses and gains may be linked to evolutionary innovations, such as the appearance of new orders.

Functional analysis of evolutionary conserved clustering of bZIP binding sites in the baculovirus homologous regions (hrs) suggests a cooperativity between host and viral transcription factors.

The genome of the Autographa californica Multinucleocapsid Polyhedrosis Virus (AcMNPV) contains nine interspersed homologous regions (hrs) that function as potent enhancer sequences when linked in cis to either viral or heterologous RNA polymerase II-dependent promoters. Their activity is strongly increased by the binding of the major immediate early viral transregulator IE1 on 28-mer palindromic sites present in hrs. We show that hrs of AcMNPV additionally carry, in the interpalindromic sequences, a large number of cAMP response elements (CRE) and TPA response elements (TRE), known to bind ubiquitous cellular transcription factors of the bZIP family. Moreover, these clusters of CRE and TRE motifs are concentrated in hrs. Analysis of the 25 baculovirus genomes sequenced so far reveals that these motifs are evolutionary conserved in Lepidoptera NPVs, suggesting a functional role in the hr enhancer function. Consistently, EMSA experiments indicate that CRE and on a lesser extent TRE sites specifically bind insect host factors. Moreover, reporter assays reveal that these CRE sites have an additive stimulatory effect on RNAPol II-dependent transcription in Sf9 cells and are potentially able to synergize with the IE1-binding palindrome.

Establishment of cell lines from the wasp Hyposoter didymator (Hym., Ichneumonidae) containing the symbiotic polydnavirus H. didymator ichnovirus.

Cell lines derived from polydnavirus-associated wasps should constitute a valuable tool for investigations of polydnavirus replication, but none is yet available. In this work, we describe the first cell lines, named Hd-AA, -AD, -BBA and -K, to have been established from the ichneumonid wasp Hyposoter didymator, associated with the polydnavirus H. didymator ichnovirus (HdIV). Southern blot analysis indicated that the viral DNA was present in all four cell lines and co-localized with high molecular mass DNA, probably the wasp chromosomes. Northern blot analysis of mRNAs extracted from the AA cell line showed transcription of some HdIV-encoded genes, although at low level. The effects of ecdysone treatment, HdIV re-infection and 42 degrees C heat-shock were analysed in the AA cell line. No effect was detected at the DNA (virus replication) or RNA (gene expression) levels, which may be due to the limitation of the present available tools.

Effect of the 5' non-translated region on self-assembly of hepatitis C virus genotype 1a structural proteins produced in insect cells.

The effect of the 5' non-translated region (5'NTR) on hepatitis C virus (HCV) morphogenesis in insect cells is investigated in this study. Expression in baculovirus-infected cells of a sequence encoding the C and E1 structural proteins under the control of the very late promoter P10 (AcSLP10-C-E1) led to the synthesis of C and C-E1 complexes, essentially found in dense reticular material associated with the ER and sedimenting at a density of 1.24-1.26 g ml(-1). Addition of the 5'NTR upstream of the C-E1 sequence (AcSLP10-5'NTR-E1) prevents translation from the initiating codon, probably because of the presence of five AUG codons in this sequence. When cells were co-infected with these two viruses, virus-like particles (VLPs) were found in the cytoplasm. The size and shape of these VLPs were variable. Concomitantly, a shift in the sedimentation profile from 1.24-1.26 to 1.15-1.18 g ml(-1) was observed, suggesting an association of C/E1 with the ER membrane. A unique vector was then constructed bearing a mutated 5'NTR (mutation of the five AUGs) and the sequence encoding all of the structural proteins and part of NS2 (5'NTRm-C-E1-E2-p7-NS2Delta). Translation of structural proteins was restored and electron microscopic observation of a cytoplasmic extract showed the presence of icosahedral particles with a density of 1.15-1.18 g ml(-1).

Persistent expression of a newly characterized Hyposoter didymator polydnavirus gene in long-term infected lepidopteran cell lines.

An Hyposoter didymator ichnovirus (HdIV) gene was stably maintained and efficiently transcribed in lepidopteran cell lines more than 3 years after HdIV infection. This K-gene had two introns and the fully spliced cDNA, named K19, comprised a short open reading frame and a long 3'-untranslated region with 13 imperfectly repeated sequences (44 to 102 nt). Transcripts related to the K-gene were detected in several long-term infected cell lines (Sf9, Spodoptera littoralis haemocytes, Trichoplusia ni). Conversely, no transcripts related to seven other viral cDNAs were detected, suggesting that the K-related DNA is selectively retained in long-term infected Sf9 cells. The function of the K-gene product and its association with stably transformed insect cell lines remains to be investigated.