Co-engaging CD47 and CD19 with a bispecific antibody abrogates B-cell receptor/CD19 association leading to impaired B-cell proliferation

CD19 is a B cell-specific receptor that regulates the threshold of B cell receptor (BCR)-mediated cell proliferation. A CD47xCD19 bispecific antibody (biAb) was generated to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.     

Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector

Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34(+) cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34(+) cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha(v) integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34(+) cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34(+) cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34(+) cells expressing alpha(v) integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34(+) cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34(+) cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34(+) c-Kit(+) cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34(+) c-Kit(+) cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.     

Improved gene delivery into neuroglial cells using a fiber-modified adenovirus vector

One impediment to treating neuronal diseases is finding ways to introduce genes into specific neuroglial cell types. Here we describe the strategy for efficient gene delivery via transferrin receptor using an adenovirus bearing a peptide mimic for transferrin. The attachment of the peptide consisted of 12 amino acids on the C-terminus of adenovirus fiber protein significantly improved entry and expression of a beta-galactosidase transgene into neuroglial cells such as astrocytes, and Schwann cells. The entry of re-targeted viruses into cells depends on the attached peptide and the transferrin receptor. Furthermore, transferrin did not affect gene delivery by the engineered adenovirus, suggesting that the effectiveness of therapeutic agents targeted to the receptor would not be diminished by competition with the abundant endogenous transferrin present in the plasma. Therefore, such transduction systems hold promise for efficient delivering gene to neuroglial cells in gene therapy protocols.     

Modulation of adenovirus vector tropism via incorporation of polypeptide ligands into the fiber protein

The efficacy of adenovirus (Ad)-based gene therapy might be significantly improved if viral vectors capable of tissue-specific gene delivery could be developed. Previous attempts to genetically modify the tropism of Ad vectors have been only partially successful, largely due to the limited repertoire of ligands that can be incorporated into the Ad capsid. Early studies identified stringent size limitations imposed by the structure of the Ad fiber protein on ligands incorporated into its carboxy terminus and thus limited the range of potential ligand candidates to short peptides. We have previously identified the HI loop of the fiber knob domain as a preferred site for the incorporation of targeting ligands and hypothesized that the structural properties of this loop would allow for the insertion of a wide variety of ligands, including large polypeptide molecules. In the present study we have tested this hypothesis by deriving a family of Ad vectors whose fibers contain polypeptide inserts of incrementally increasing lengths. By assessing the levels of productivity and infectivity and the receptor specificities of the resultant viruses, we show that polypeptide sequences exceeding by 50% the size of the knob domain can be incorporated into the fiber with only marginal negative consequences on these key properties of the vectors. Our study has also revealed a negative correlation between the size of the ligand used for vector modification and the infectivity and yield of the resultant virus, thereby predicting the limits beyond which further enlargement of the fiber knob would not be compatible with the virion's integrity.     

Suppression of normal and malignant kit signaling by a bispecific antibody linking kit with CD300a

Through its receptor Kit (CD117), stem cell factor (SCF) critically regulates human mast cell (MC) differentiation, survival, priming, and activation. The dominance of SCF in setting these parameters compels stringent contra-regulation to maintain a balanced MC phenotype. We have synthesized a library of bispecific Ab fragments to examine the effect of linking Kit with CD300a. In this study, we report that CD300a exerts a strong inhibitory effect on Kit-mediated SCF-induced signaling, consequently impairing MC differentiation, survival, and activation in vitro. This effect derives from Kit-mediated tyrosine phosphorylation of CD300a and recruitment of the SHIP-1 but not of SH2-containing protein phosphatase 1. CD300a inhibits the constitutive activation of the human leukemic HMC-1 cells but not their survival. Finally, CD300a abrogates the allergic reaction induced by SCF in a murine model of cutaneous anaphylaxis. Our findings highlight CD300a as a novel regulator of Kit in human MC and suggest roles for this receptor as a suppressor of Kit signaling in MC-related disorders.     

Enhancement of antibody responses in DNA vaccination using a vector encoding a universal T-helper cell epitope

DNA vaccination appears as a very promising approach to raise protective antibodies against a variety of proteins from pathogens or tumor cells, but is often hindered by the low immunogenicity of the genetic vectors used for the immunizations. To enhance the humoral response through improvement of the antigenic presentation of newly synthesized proteins upon vaccination, we engineered a plasmid coding for a low immunogenic protein (an scFv, i.e. the single-chain Fragment variable of a well-characterized antibody) fused to a small-size universal T-helper cell epitope derived from tetanus toxin, whose efficiency in classical protein-based immunization protocols has already been demonstrated. We found that immunization of C57Bl/6 mice using this vector greatly enhanced the production not only of specific antibodies recognizing essentially conformational epitopes on the undenatured scFv protein but also of antibodies against linear epitopes on the denatured protein. Since this T-epitope is known to be accommodated by several haplotypes of H-2 molecules in mice, as well as by various class II MHC molecules in humans, the results reported here allow us to conclude that this method could be of general interest for future applications of genetic immunization, including DNA-based vaccinations in humans.     

Enhancement of Natural Killer cell cytotoxicity by a CD18 integrin-activating antibody

Natural Killer (NK) cells kill certain tumor cells and virus infected cells in an antigen-independent manner. Members of CD18 integrins such as CD11a, CD11b, and CD11c are expressed in all NK cells. CD18-blocking mAbs inhibit the killing activity of NK cells implying an essential role of these integrins in NK cell cytotoxicity. In this report we show that the pan CD18-activating mAb, 240Q, augments cytotoxicity of resting NK cells. Since activation of either CD11a or CD11c alone fails to augment the NK cell activity, we postulate that a functional synergy of the individual CD18 integrins is responsible for the observed stimulatory effect of pan CD18 activation on NK cell cytotoxicity.     

Enhancement of the antibody response to flavivirus B-cell epitopes by using homologous or heterologous T-cell epitopes.

We have been investigating the T-helper (Th)-cell response to the flavivirus envelope (E) glycoprotein. In our studies with Murray Valley encephalitis (MVE) virus, we previously identified synthetic peptides capable of priming Th lymphocytes for an in vitro antivirus proliferative response (J. H. Mathews, J. E. Allan, J. T. Roehrig, J. R. Brubaker, and A. R. Hunt, J. Virol. 65:5141-5148, 1991). We have now characterized in vivo Th-cell priming activity of one of these peptides (MVE 17, amino acids 356 to 376) and an analogous peptide derived from the E-glycoprotein sequence of the dengue (DEN) 2, Jamaica strain (DEN 17, amino acids 352 to 368). This DEN peptide also primed the Th-cell compartment in BALB/c mice, as measured by in vitro proliferation and interleukin production. The failure of some MVE and DEN virus synthetic peptides to elicit an antibody response in BALB/c mice could be overcome if a Th-cell epitope-containing peptide was included in the immunization mixture. A more detailed analysis of the structural interactions between Th-cell and B-cell epitope donor peptides revealed that the peptides must be linked to observe the enhanced antibody response. Blockage or deletion of the free cysteine residue on either peptide abrogated the antibody response. The most efficient T-B-cell epitope interaction occurred when the peptides were colinearly synthesized. These Th-cell-stimulating peptides were also functional with the heterologous B-cell epitope-containing peptides. The Th-cell epitope on DEN 17 was more potent than the Th-cell epitope on MVE 17.     

Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein.

The adenovirus fiber protein is responsible for attachment of the virion to unidentified cell surface receptors. There are at least two distinct adenovirus fiber receptors which interact with the group B (Ad3) and group C (Ad5) adenoviruses. We have previously shown by using expressed adenovirus fiber proteins that it is possible to change the specificity of the fiber protein by exchanging the head domain with another serotype which recognizes a different receptor (S. C. Stevenson et al., J. Virol. 69:2850-2857, 1995). A chimeric fiber cDNA containing the Ad3 fiber head domain fused to the Ad5 fiber tail and shaft was incorporated into the genome of an adenovirus vector with E1 and E3 deleted encoding beta-galactosidase to generate Av9LacZ4, an adenovirus particle which contains a chimeric fiber protein. Western blot analysis of the chimeric fiber vector confirmed expression of the chimeric fiber protein and its association with the adenovirus capsid. Transduction experiments with fiber protein competitors demonstrated the altered receptor tropism of the chimeric fiber vector compared to that of the parental Av1LacZ4 vector. Transduction of a panel of human cell lines with the chimeric and parental vectors provided evidence for a different cellular distribution of the Ad5 and Ad3 receptors. Three cell lines (THP-1, MRC-5, and FaDu) were more efficiently transduced by the vector containing the Ad3 fiber head than by the Ad5 fiber vector. In contrast, human coronary artery endothelial cells were transduced more readily with the vector containing the Ad5 fiber than with the chimeric fiber vector. HeLa and human umbilical vein endothelial cells were transduced at equivalent levels compared with human diploid fibroblasts, which were refractory to transduction with both vectors. These results provide evidence for the differential expression of the Ad5 and Ad3 receptors on human cell lines derived from clinically relevant target tissues. Furthermore, we show that exchange of the fiber head domain is a viable approach to the production of adenovirus vectors with cell-type-selective transduction properties. It may be possible to extend this approach to the use of ligands for a range of different cellular receptors in order to target gene transfer to specific cell types at the level of transduction.     

Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab'gamma)2 antibody

We have developed a system for the targeted delivery of adeno-associated virus (AAV) vectors. Targeting is achieved via a bispecific F(ab')2 antibody that mediates a novel interaction between the AAV vector and a specific cell surface receptor expressed on human megakaryocytes. Targeted AAV vectors were able to transduce megakaryocyte cell lines, DAMI and MO7e, which were nonpermissive for normal AAV infection, 70-fold above background and at levels equivalent to permissive K562 cells. Transduction was shown to occur through the specific interaction of the AAV vector-bispecific F(ab')2 complex and cell-associated targeting receptor. Importantly, targeting appeared both selective and restrictive as the endogenous tropism of the AAV vector was significantly reduced. Binding and internalization through the alternative receptor did not alter subsequent steps (escape from endosomes, migration to nucleus, or uncoating) required to successfully transduce target cells. These results demonstrate that AAV vectors can be targeted to a specific cell population and that transduction can be achieved by circumventing the normal virus receptor.     

Stable transfer of a mouse dihydrofolate reductase gene into a deficient cell line using human adenovirus vector

A plasmid containing the mouse dihydrofolate reductase (dhfr) gene was rescued in a human adenovirus in early region 3. Analysis of the insert in the recombinant virus revealed that the dhfr sequences were intact in the viral genome, whereas a part of the ampicillin gene in the plasmid sequences was deleted. The recombinant virus could successfully express this gene in a deficient cell line. A permanent dhfr+ cell line was established by stable transfer of the gene using the recombinant virus.     

Functional and selective targeting of adenovirus to high-affinity Fcgamma receptor I-positive cells by using a bispecific hybrid adapter

Adenovirus (Ad) efficiently delivers its DNA genome into a variety of cells and tissues, provided that these cells express appropriate receptors, including the coxsackie-adenovirus receptor (CAR), which binds to the terminal knob domain of the viral capsid protein fiber. To render CAR-negative cells susceptible to Ad infection, we have produced a bispecific hybrid adapter protein consisting of the amino-terminal extracellular domain of the human CAR protein (CARex) and the Fc region of the human immunoglobulin G1 protein, comprising the hinge and the CH2 and CH3 regions. CARex-Fc was purified from COS7 cell supernatants and mixed with Ad particles, thus blocking Ad infection of CAR-positive but Fc receptor-negative cells. The functionality of the CARex domain was further confirmed by successful immunization of mice with CARex-Fc followed by selection of a monoclonal anti-human CAR antibody (E1-1), which blocked Ad infection of CAR-positive cells. When mixed with Ad expressing eGFP, CARex-Fc mediated an up to 250-fold increase of transgene expression in CAR-negative human monocytic cell lines expressing the high-affinity Fcgamma receptor I (CD64) but not in cells expressing the low-affinity Fcgamma receptor II (CD32) or III (CD16). These results open new perspectives for Ad-mediated cancer cell vaccination, including the treatment of acute myeloid leukemia.     

Gene delivery into neuronal and glial cells by using a replication-deficient adenovirus vector: prospects for neurological gene therapy

We have used a recombinant adenovirus vector (E1−) expressing β-galactosidase to explore a novel mechanism with which to transfer genes into cells of the central nervous system (CNS). The replication-deficient adenovirus vector expressing β-galactosidase (RAd35) was propagated on a permissive helper cell line (293 cells). High level protein expression from the human cytomegalovirus immediate early promoter (hCMV IE) was obtained in a target cell population of RAd35 infected cultured neuronal and glial cell lines. Light microscopy showed that over 50% of the glial cells studied expressed β-galactosidase. Following retinoic acid treatment, RAd35 infected cell lines ND7/23, NG108 and NTera2, showed β-galactosidase expression in up to 90% of the cells. In addition, these cells showed morphological evidence of differentiation into neurons. This pattern of β-galactosidase expression was also observed in primary rat cerebella granule neuron cultures. In vivo studies were performed in Balb/c mice following direct intracranial injections of RAd35 into the brain. Cell sections showed a localised staining in the brain at the site of injection of the virus. Non-replicating adenovirus vectors are therefore highly efficient systems for delivering a transgene into brain cells. However, their broad cell tropism may limit their applications for genetic disorders in which a specific cell type is to be targeted for gene therapy. To address this problem, we have constructed adenovirus vectors which contain specific neuronal promoters and are currently assessing in vitro expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Construction of a diabody (small recombinant bispecific antibody) using a refolding system

Diabodies are the recombinant bispecific antibodies (BsAbs), constructed from heterogeneous single-chain antibodies. Usually, diabodies have been prepared from bacterial periplasmic fraction using a co-expression vector (i.e. genes encoding two chains were tandemly located under the same promoter). Some diabodies, however, cannot be expressed as a soluble material owing to inclusion body formation, which limits the utilization of diabodies in various fields. Here we report an improved method for the construction of diabodies using a refolding system. As a model, a bispecific diabody binding to adenocarcinoma-associated antigen MUC1 and to CD3 on T cells was studied. One chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). The second was composed of a VL specific for MUC1 linked to a VH specific for CD3. The two hetero scFvs were independently obtained from intracellular insoluble fractions of Escherichia coli, purified, mixed stoichiometrically (at an equivalent molar ratio of 1:1) and refolded. The refolded two hetero scFv has a hetero-dimeric structure, with complete specificity for both target cells [i.e. MUC1 positive cells and CD3 positive lymphokine-activated killer cells with a T cell phenotype (T-LAK)]. Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach approximately 98% at an effector:target ratio (E:T ratio) of 10, almost identical with that with anti-MUC1xanti-CD3 chemically synthesized BsAbs (c-BsAbs). This is the first report of the construction of a diabody using a refolding system.     

Formation of a bispecific antibody by the use of leucine zippers.

A new method is described for the production of bispecific F(ab')2 heterodimers using leucine zippers. Two heterodimer-forming "zipper" peptides derived from the Fos and Jun proteins were respectively linked to the Fab' portions of two different mAb by gene fusion. The antibodies used were 145-2C11, which binds to murine CD3, and anti-Tac, which binds to the p55 chain of the human IL-2R. Anti-Tac Fab'-Jun and anti-CD3 Fab'-Fos were expressed individually as F(ab'-zipper)2 homodimers in the mouse myeloma cell line Sp2/0. When these homodimers were reduced at the hinge region to form monomers and then reoxidized together, the resulting end products were mostly F(ab'-zipper)2 heterodimers. Bispecific anti-CD3 x anti-Tac F(ab'-zipper)2 heterodimers produced by this method were shown to be highly effective in recruiting cytotoxic T cells to lyse IL-2R-bearing HuT-102 cells in vitro.     

Serum-free recombinant production of adenovirus using a hollow fiber capillary system

A novel method for the production of adenoviral vectors on a scale sufficient to support most research applications and early phase clinical trials is presented. This method utilizes serum-free cell culture medium and a hollow fiber cell culture apparatus. Significantly less time and space are required than in conventional methods, and the resulting adenovirus is collected in a much smaller volume, simplifying the purification steps. The protocol described is a reproducible, convenient, biologically safe, and environmentally sound method for the production of adenoviral vectors for laboratory use and has the potential to scale-up the adenovirus production for clinical use.     

Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate

G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.     

Preparation of a Tat-related transporter peptide for carrying the adenovirus vector into cells

We synthesized a Tat-related peptide acetyl-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, Ac-Tat(48-60)-Gly-Cys-NH(2), having high intracellular permeability, and conjugated this peptide to adenovirus vector to enhance gene transfer efficiency of adenovirus vector into cells. The peptide was prepared by the solid-phase peptide synthesis method and a bifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide ester was used to conjugate the peptide to adenovirus vector containing luciferase gene. The novel conjugate of adenovirus vector and Ac-Tat(48-60)-Gly-Cys-NH(2) peptide exhibited excellent gene transfer efficacy in B16BL6 cells.