Ectodomain of coxsackievirus and adenovirus receptor genetically fused to epidermal growth factor mediates adenovirus targeting to epidermal growth factor receptor-positive cells

Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.     

Targeting avian leukosis virus subgroup A vectors by using a TVA-VEGF bridge protein

Previously, we have demonstrated that bridge proteins comprised of avian leukosis virus (ALV) receptors fused to epidermal growth factor (EGF) can be used to selectively target retroviral vectors with ALV envelope proteins to cells expressing EGF receptors. To determine whether another type of ligand incorporated into an ALV receptor-containing bridge protein can also function to target retroviral infection, the TVA-VEGF110 bridge protein was generated. TVA-VEGF110 consists of the extracellular domain of the TVA receptor for ALV subgroup A (ALV-A), fused via a proline-rich linker peptide to a 110-amino-acid form of vascular endothelial growth factor (VEGF). This bridge protein bound specifically to its cell surface receptor, VEGFR-2, and efficiently mediated the entry of an ALV-A vector into cells. These studies indicate that ALV receptor-ligand bridge proteins may be generally useful tools for retroviral targeting approaches.     

Identification of key residues in subgroup A avian leukosis virus envelope determining receptor binding affinity and infectivity of cells expressing chicken or quail Tva receptor

To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells ( approximately 200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.     

Production and characterization of a soluble, active form of Tva, the subgroup A avian sarcoma and leukosis virus receptor

The receptor for the subgroup A avian sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of avian cells by ASLV(A) with a 90% inhibitory concentration of approximately 25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva.     

Mutational analysis of the subgroup A avian sarcoma and leukosis virus putative fusion peptide domain

Short hydrophobic regions referred to as fusion peptide domains (FPDs) at or near the amino terminus of the membrane-anchoring subunit of viral glycoproteins are believed to insert into the host membrane during the initial stage of enveloped viral entry. Avian sarcoma and leukosis viruses (ASLV) are unusual among retroviruses in that the region in the envelope glycoprotein (EnvA) proposed to be the FPD is internal and contains a centrally located proline residue. To begin analyzing the function of this region of EnvA, 20 substitution mutations were introduced into the putative FPD. The mutant envelope glycoproteins were evaluated for effects on virion incorporation, receptor binding, and infection. Interestingly, most of the single-substitution mutations had little effect on any of these processes. In contrast, a bulky hydrophobic substitution for the central proline reduced viral titers 15-fold without affecting virion incorporation or receptor binding, whereas substitution of glycine for the proline had only a nominal effect on EnvA function. Similar to other viral FPDs, the putative ASLV FPD has been modeled as an amphipathic helix where most of the bulky hydrophobic residues form a patch on one face of the helix. A series of alanine insertion mutations designed to interrupt the hydrophobic patch on the helix had differential effects on infectivity, and the results of that analysis together with the results observed with the substitution mutations suggest no correlation between maintenance of the hydrophobic patch and glycoprotein function.     

Acutely transforming avian leukosis virus subgroup J strain 966: defective genome encodes a 72-kilodalton Gag-Myc fusion protein

Avian leukosis virus subgroup J (ALV-J), the most recent member of the avian retroviruses, is predominantly associated with myeloid leukosis in meat-type chickens. We have previously demonstrated that the acutely transforming virus strain 966, isolated from an ALV-J-induced tumor, transformed peripheral blood monocyte and bone marrow cells in vitro and induced rapid-onset tumors, suggesting transduction of oncogenes (L. N. Payne, A. M. Gillespie, and K. Howes, Avian Dis. 37:438-450, 1993). In order to understand the molecular basis for the rapid transformation and tumor induction, we have determined the complete genomic structure of the provirus of the 966 strain. The sequence of the 966 provirus clone revealed that its genome is closely related to that of HPRS-103 but is defective, with the entire pol and parts of the gag and env genes replaced by a 1,491-bp sequence representing exons 2 and 3 of the c-myc gene. LSTC-IAH30, a stable cell line derived from turkey monocyte cultures transformed by the 966 strain of ALV-J, expressed a 72-kDa Gag-Myc fusion protein. The identification of the myc gene in 966 virus as well as in several other ALV-J-induced tumors suggested that the induction of myeloid tumors by this new subgroup of ALV occurs through mechanisms involving the activation of the c-myc oncogene.     

Evolutionary pressure of a receptor competitor selects different subgroup a avian leukosis virus escape variants with altered receptor interactions

A complex interaction between the retroviral envelope glycoproteins and a specific cell surface protein initiates viral entry into cells. The avian leukosis-sarcoma virus (ALV) group of retroviruses provides a useful experimental system for studying the retroviral entry process and the evolution of receptor usage. In this study, we demonstrate that evolutionary pressure on subgroup A ALV [ALV(A)] entry exerted by the presence of a competitive inhibitor, a soluble form of the ALV(A) Tva receptor linked to a mouse immunoglobulin G tag (quail sTva-mIgG), can select different populations of escape variants. This escape population contained three abundant ALV(A) variant viruses, all with mutations in the surface glycoprotein hypervariable regions: a previously identified variant containing the Y142N mutation in the hr1 region; a new variant with two mutations, W141G in hr1 and K261E in vr3; and another new variant with two mutations, W145R in hr1 and K261E. The W141G K261E and W145R K261E viruses escape primarily by lowering their binding affinities for the quail Tva receptor competitive inhibitor while retaining wild-type levels of binding affinity for the chicken Tva receptor. A secondary phenotype of the new variants was an alteration in receptor interference patterns from that of wild-type ALV(A), indicating that the mutant glycoproteins are possibly interacting with other cellular proteins. One result of these altered interactions was that the variants caused a transient period of cytotoxicity. We could also directly demonstrate that the W141G K261E variant glycoproteins bound significant levels of a soluble form of the Tvb(S3) ALV receptor in a binding assay. Alterations in the normally extreme specificity of the ALV(A) glycoproteins for Tva may represent an evolutionary first step toward expanding viral receptor usage in response to inefficient viral entry.     

The protein tyrosine phosphatase TCPTP suppresses the tumorigenicity of glioblastoma cells expressing a mutant epidermal growth factor receptor

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and GBMs frequently contain amplifications or mutations of the EGFR gene. The most common mutation results in a truncated receptor tyrosine kinase known as Delta EGFR that signals constitutively and promotes GBM growth. Here, we report that the 45-kDa variant of the protein tyrosine phosphatase TCPTP (TC45) can recognize Delta EGFR as a cellular substrate. TC45 dephosphorylated Delta EGFR in U87MG glioblastoma cells and inhibited mitogen-activated protein kinase ERK2 and phosphatidylinositol 3-kinase signaling. In contrast, the substrate-trapping TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, suppressed the activation of ERK2 but not phosphatidylinositol 3-kinase. TC45 inhibited the proliferation and anchorage-independent growth of Delta EGFR cells but TC45-D182A only inhibited cellular proliferation. Notably, neither TC45 nor TC45-D182A inhibited the proliferation of U87MG cells that did not express Delta EGFR. Delta EGFR activity was necessary for the activation of ERK2, and pharmacological inhibition of ERK2 inhibited the proliferation of Delta EGFR-expressing U87MG cells. Expression of either TC45 or TC45-D182A also suppressed the growth of Delta EGFR-expressing U87MG cells in vivo and prolonged the survival of mice implanted intracerebrally with these tumor cells. These results indicate that TC45 can inhibit the Delta EGFR-mediated activation of ERK2 and suppress the tumorigenicity of Delta EGFR-expressing glioblastoma cells in vivo.     

Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.     

Identification and characterization of the viral interaction determinant of the subgroup A avian leukosis virus receptor.

The cellular receptor for subgroup A avian leukosis viruses (ALV-A) has a small, 83-amino-acid extracellular domain containing a motif that is related in sequence to the ligand binding repeats of the low-density lipoprotein receptor. Extensive mutagenesis of the ALV-A receptor has identified two acidic amino acids (Asp-46 and Glu-47) and an adjacent aromatic amino acid (Trp-48) in the carboxy-terminal portion of this low-density lipoprotein receptor-related motif that are crucial for efficient viral entry. In addition, a 19-amino-acid peptide derived from this region efficiently and specifically blocked subgroup A viral infection when oxidized to form a disulfide bond previously predicted to form in the native receptor (C. Bélanger, K. Zingler, and J. A. T. Young, J. Virol. 69:1019-1024, 1995). Thus, the charged and aromatic amino acid determinants that are required for viral infection appear to lie on a small loop region of the ALV-A receptor. Previously, a single aromatic and one or more charged residues on the CD4 receptor for human and simian immunodeficiency viruses, and the MCAT receptor for ecotropic murine leukemia viruses, were shown to be important for viral entry. These results suggest that different retroviruses may recognize related determinants on structurally divergent cellular receptors.     

Extracellular heat shock protein 70 mediates heat stress-induced epidermal growth factor receptor transactivation in A431 carcinoma cells

The initial steps of heat stress in A431 cells were previously characterized by ligand-independent EGFR transactivation via an unknown mechanism and concomitant secretion of Hsp70. In this work we demonstrate that the depletion of Hsp70 from the conditioned medium of heated cells abolishes EGFR transactivation indicating that secreted Hsp70 is essential for EGFR transactivation during heat shock. This notion is supported by the findings that purified Hsp70 can induce EGFR transactivation and the activation of EGFR-dependent signaling pathways. Both heat stress and pure Hsp70 stimulate activation of TLR2/4 and their association with EGFR. These results suggest that the secreted Hsp70 mediates the cross-communication of TLR and EGFR signaling systems in A431 cells.     

Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor

BACKGROUND: The analysis of the subcellular distribution of proteins is essential for the understanding of processes such as signal transduction. In most cases, the parallel analysis of multiple components requires fixation and immunofluorescence labeling. Therefore, one has to ascertain that the fixation procedure preserves the in vivo protein distribution. Fusion proteins with the green fluorescent protein (GFP) are ideal tools for this purpose. However, one must consider specific aspects of the fluorophore formation or degradation, i.e. reactions that may interfere with the detection of GFP fusion proteins. METHODS: Fusion proteins of the epidermal growth factor receptor (EGFR) with GFP as well as free, soluble GFP stably or transiently expressed in adherent cultured cells served as test cases for comparing the distribution in vivo with that after fixation by conventional epifluorescence and laser scanning microscopy. Indirect immunofluorescence was employed to compare the distributions of the GFP signal and of the GFP polypeptide in the fusion protein. RESULTS: Paraformaldehyde (PFA) fixation with subsequent mounting in the antifading agent Mowiol, but not in Tris- or HEPES buffered saline, led to a partial redistribution of the EGFR from the plasma membrane to the perinuclear region. The redistribution was confirmed with the GFP and EGFR immunofluorescence. The in vivo distribution in Mowiol mounted cells was preserved if cells were treated with a combined PFA/methanol fixation procedure, which also retained the fluorescence of soluble GFP. The anti-GFP antiserum was negative for the N-terminal fusion protein. CONCLUSIONS: The combined PFA/methanol protocol is universally applicable for the fixation of transmembrane and soluble cytoplasmic proteins and preserves the fluorescence of GFP.     

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.Key words: EGFR, antibody synergy, functional screening, epitope binning, antibody combinations     

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.     

Isolation and identification of a subgroup A avian leukosis virus from imported meat-type grand-parent chickens

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%–90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for ALV of subgroup A isolated from imported breeders.     

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.     

Transcytosis of epidermal growth factor. The epidermal growth factor receptor mediates uptake but not transcytosis

Madin-Darby canine kidney (MDCK) cells polarize and generate distinct apical and basolateral membrane domains when grown on permeable filter supports. Under these conditions, they transcytose fluid-phase markers. Recently, receptor-mediated transcytosis of epidermal growth factor (EGF) across MDCK cells has been reported (Maratos-Flier, E., Kao, C.-Y. Y., Verdin, E. M., and King, G. L. (1987) J. Cell Biol. 105, 1595-1601). We examined the role of the EGF receptor in this process. Transcytosis of EGF occurred only in the basolateral-to-apical direction, was time-dependent, and inhibited by the addition of unlabeled EGF in a concentration-dependent manner. In contrast to previous work, we found that only about 5% of basolaterally bound EGF was transported to the apical chamber. The half-time of transport was 90 min. A mutant cell line of MDCK, MDCKII-RCAr, was used to study the expression of the EGF receptor. Cell surface glycoproteins of these mutant cells can be efficiently labeled with [3H]galactose by exogalactosylation. The EGF receptor was found to be expressed only on the basolateral surface. Addition of EGF to the basolateral medium resulted in rapid internalization and degradation of the receptor. Testing directly for transcytosis of basolateral glycoproteins, we detected several proteins transported across the cell. The EGF receptor, however, was not among this group of proteins. Taking these results together, we suggest the following model. Internalization of EGF on the basolateral surface is mediated by the EGF receptor. EGF dissociates from the receptor in an endocytic compartment. A fraction of the EGF is then diverted nonselectively to the transcytotic pathway, as found for other fluid-phase markers previously (Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J., and Simons, K. (1989) J. Cell Biol. 109, 3243-3258.     

Induction of immunogenic apoptosis by blockade of epidermal growth factor receptor activation with a specific antibody

Despite promising results in the use of anti-epidermal growth factor receptor (EGFR) Abs for cancer therapy, several issues remain to be addressed. An increasing emphasis is being placed on immune effector mechanisms. It has become clear for other Abs directed to tumor targets that their effects involve the adaptive immunity, mainly by the contribution of Fc region-mediated mechanisms. Given the relevance of EGFR signaling for tumor biology, we wonder whether the oncogene inhibition could contribute to Ab-induced vaccine effect. In a mouse model in which 7A7 (an anti-murine EGFR Ab) and AG1478 (an EGFR-tyrosine kinase inhibitor) displayed potent antimetastatic activities, depletion experiments revealed that only in the case of the Ab, the effect was dependent on CD4(+) and CD8(+) T cells. Correspondingly, 7A7 administration elicited a remarkable tumor-specific CTL response in hosts. Importantly, experiments using 7A7 F(ab')(2) suggested that in vivo Ab-mediated EGFR blockade may play an important role in the linkage with adaptive immunity. Addressing the possible mechanism involved in this effect, we found quantitative and qualitative differences between 7A7 and AG1478-induced apoptosis. EGFR blocking by 7A7 not only prompted a higher proapoptotic effect on tumor metastases compared with AG1478, but also was able to induce apoptosis with immunogenic potential in an Fc-independent manner. As expected, 7A7 but not AG1478 stimulated exposure of danger signals on tumor cells. Subcutaneous injection of 7A7-treated tumor cells induced an antitumor immune response. This is the first report, to our knowledge, of a tumor-specific CTL response generated by Ab-mediated EGFR inhibition, suggesting an important contribution of immunogenic apoptosis to this effect.     

A soluble form of a receptor for subgroup A avian leukosis and sarcoma viruses (ALSV-A) blocks infection and binds directly to ALSV-A.

A receptor that confers susceptibility to infection by subgroup A avian leukosis and sarcoma viruses (ALSV-A) has been described (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993). A soluble form of the receptor was generated to determine whether this protein interacts directly with virus particles in the absence of other cell surface factors. The soluble protein comprised the extracellular region of the ALSV-A receptor fused to an antibody epitope tag and six histidine residues. Preincubating this protein with virus led to an efficient block to infection of avian cells by ALSV-A but had no effect on infection by ALSV-B, ALSV-C, or ALSV-D. Furthermore, an antibody directed against the introduced epitope tag immunoprecipitated ALSV-A particles bound to the soluble receptor. In contrast, other ALSV subgroups were not immunoprecipitated by this procedure. These data demonstrate that the cloned receptor interacts directly with ALSV-A and discriminates between different ALSV subgroups at the level of virus binding.     

Epidermal growth factor receptor. Characterization of a monoclonal antibody specific for the receptor of A431 cells

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.