Effect of intravenous bisphosphonates on release of basic fibroblast growth factor in serum of patients with cancer-associated hypercalcemia

Basic fibroblast growth factor (bFGF) is a potent tumor angiogenesis factor and normal constituent of bone extracellular matrix which does not normally circulate in serum of nonpregnant adult humans. We examined the effects of acute administration of intravenous bisphosphonates on release of bFGF in human serum. Twenty seven men and women (mean age, 64 yr) with cancer-associated hypercalcemia, the majority of whom had osseous metastases, were treated once with an intravenous bisphosphonate. Nearly all twelve patients with elevated baseline serum bFGF ranging from 5-27 pg/mL showed significant decreases in serum bFGF (2-7 days) after iv bisphosphonate treatment. The mathematical product of the patients' initial serum bFGF and intial serum calcium concentration, the 'Ca x bFGF product', was significantly negatively (r = -0.91, P < 0.001) correlated with the acute change in serum bFGF level. No consistent relationship was observed between serum bFGF and serum parathyroid hormone related peptide (PTHrP) levels in the hypercalcemic cancer patients. In a subset of patients with non-hematological malignancies and low baseline serum bFGF, acute changes in serum bFGF were significantly negatively (r = -0.66, P < 0.01) correlated with acute change in serum calcium concentration. These results indicate that release of bFGF in serum of patients with cancer-associated hypercalcemia likely depends predominantly on increased bone resorption. Acute change in low serum levels of bFGF in patients with cancer-associated hypercalcemia treated with intravenous bisphosphonates may be physiologically inversely regulated by acute change in the serum calcium concentration.     

Basic fibroblast growth factor in the chick embryo: immunolocalization to striated muscle cells and their precursors

The identification of acidic and basic fibroblast growth factors (FGFs) in a number of embryonic tissue extracts has implicated these growth factors in the regulation of a variety of embryonic events including angiogenesis, eye development, and muscle differentiation. Lack of information concerning the cellular distribution of the growth factor within these tissues has made it extremely difficult to assign developmental roles to FGF. We have localized bFGF in the developing chick embryo using immunohistochemical techniques and our monospecific polyclonal rabbit anti-human bFGF IgG. The spatial pattern for bFGF localization was highly specific. The anti-human bFGF antibodies recognized striated muscle cells and their precursors in 2-6-d chick embryos. Myocardium, somite myotome, and limb bud muscle all stain positively for bFGF. In addition, the anti-human bFGF antibodies localized specifically to the cell, rather than to the extracellular matrix or nucleus of myotubes. The localization of bFGF demonstrated here provides further support for the hypothesis (Clegg et al., 1987; Seed et al., 1988) that this growth factor is involved in muscle development.     

IgG anti-IgE autoantibodies in visceral leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.     

Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA.

The expression of human basic fibroblast growth factor (bFGF) cDNA in mouse BALB/c 3T3 clone A31 cells induced morphological transformation. These transformed cells grew well and reached more than a sixfold-higher saturation density than parental A31 cells even in serum-free medium. They were able to form colonies in soft agar. The phenotypic alteration in the transformed cells was reversed by the addition of anti-human bFGF antibodies to the medium. These results suggest that the cellular transformation mediated by bFGF is caused by autocrine stimulation with secreted bFGF molecules.     

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTβR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti-LTβR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a full-length anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.Key words: bispecific antibodies, single-chain Fv, immunoglobulins, antibody therapeutics, protein stability, pharmacokinetics, protein engineering, tumor inhibition, cancer treatment     

Early Detection of NSCLC with scFv Selected against IgM Autoantibody

Survival of patients with lung cancer could be significantly prolonged should the disease be diagnosed early. Growing evidence indicates that the immune response in the form of autoantibodies to developing cancer is present before clinical presentation. We used a phage-displayed antibody library to select for recombinant scFvs that specifically bind to lung cancer-associated IgM autoantibodies. We selected for scFv recombinant antibodies reactive with circulating IgM autoantibodies found in the serum of patients with early stage lung adenocarcinoma but not matched controls. Discriminatory performance of 6 selected scFvs was validated in an independent set of serum from stage 1 adenocarcinoma and matching control groups using two independent novel methods developed for this application. The panel of 6 selected scFvs predicted cancer based on seroreactivity value with sensitivity of 0.8 and specificity of 0.87. Receiver Operative Characteristic curve (ROC) for combined 6 scFv has an AUC of 0.88 (95%CI, 0.76–1.0) as determined by fluorometric microvolume assay technology (FMAT) The ROC curve generated using a homogeneous bridging Mesa Scale Discovery (MSD) assay had an AUC of 0.72 (95% CI, 0.59–0.85). The panel of all 6 antibodies demonstrated better discriminative power than any single scFv alone. The scFv panel also demonstrated the association between a high score - based on seroreactivity - with poor survival. Selected scFvs were able to recognize lung cancer associated IgM autoantibodies in patient serum as early as 21 months before the clinical presentation of disease. The panel of antibodies discovered represents a potential unique non-invasive molecular tool to detect an immune response specific to lung adenocarcinoma at an early stage of disease.     

Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates

The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)--based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galbeta1-3GalNAcalpha- and Galbeta1-3GalNAcbeta-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcalpha-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 x 10(-8) M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcbeta and GalNAcbeta1-3GalNAcbeta ligands was found in human serum.     

Purification of basic fibroblast growth factor from rat brain: identification of a Mr 22,000 immunoreactive form

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several anti-human placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF.     

Proteomics-based identification of HSP60 as a tumor-associated antigen in early stage breast cancer and ductal carcinoma in situ

The detection of autoantibodies in cancer patients has been shown to constitute an excellent tool for early diagnosis. Because breast cancer still lacks early diagnostic markers, we investigated novel tumor-associated antigens and related autoantibodies in sera from patients with early stage breast cancer compared to autoimmune disease, other cancers, and healthy volunteers, using a proteomics-based approach. Among the 26 protein antigens specifically recognized by early stage breast cancer sera, we focused on Heat Shock Protein 60 (HSP60). Using ELISA, we investigated the frequency of autoantibodies directed against this protein in the sera of 240 individuals, comprising patients with either ductal carcinoma in situ (DCIS) ( n = 49) or early stage breast cancer ( n = 58), other cancers ( n = 20), autoimmune disease ( n = 20), and healthy subjects ( n = 93). Autoantibodies directed against HSP60 were present in 16/49 (31%) early stage breast cancer and 18/58 (32.6%) DCIS patients, compared to 4/93 (4.3%) healthy subjects. In particular, autoantibodies were present in 11/23 patients (47.8%) with high-grade DCIS, compared to 5/26 (19.2%) with low-grade DCIS. HSP60 mRNA levels were significantly higher in primary breast cancer compared to healthy breast tissues. Using immunohistochemistry, we found that HSP60 expression gradually increases from normal through DCIS to invasive tissues. Our results indicate that HSP60 autoantibodies may be of interest in terms of clinical utility for the early diagnosis of breast cancer and more particularly in DCIS. Moreover, HSP60 overexpression during the first steps of breast carcinogenesis may be functionally correlated to tumor growth and/or progression.     

Antigen-specific IgG antibodies in stage IV long-time survival breast cancer patients

BACKGROUND: Profiling the immune responses in patients with cancer is expected to facilitate the design of diagnostic tests and therapeutic vaccines. Such studies usually require the parental antigens. We attempted to profile the immune responses in patients with breast cancer using a peptide phage display selection strategy, which identifies antibody specificities whether or not the antigens are known. MATERIALS AND METHODS: A panel of random peptide phage libraries was panned on serum IgG antibodies from breast cancer patients with stage IV, seeking for disease specific IgG epitopes. ELISA, immunoscreening, and Western blotting techniques were the main approaches used. RESULTS: Phage-displayed peptides were specifically enriched for binding to IgG antibodies from patients with breast cancer. Several peptides have been identified, in particular the SQRIPARIHHFPTSI peptide, which was recognized by IgG antibodies from breast cancer patients, but not from normals (p < 0.0004). In patients who responded to the selected peptides, in particular the SQRIPARIHHFPTSI peptide, antibodies against a 66 kDa cellular protein were found. Interestingly, three out of six patients with the strongest immunoreactivity are still alive, with a mean survival time from first recurrence until now of 2553 days. In contrast, all the nonresponders (n = 10) are deceased. The mean survival time of these patients was 784 days, whereas the mean survival time of the three deceased responders was 1050 days (p < 0.02). CONCLUSIONS: The data provide the first example in which panning of peptide phage display libraries on patient IgG antibodies results in the isolation of breast cancer specific IgG epitopes, some of which correlate with patient survival time. Thus, the identified B-cell epitopes should be of great interest in vaccine development.     

Single variable domain-IgG fusion. A novel recombinant approach to Fc domain-containing bispecific antibodies

Both laboratory and early clinical studies to date have demonstrated that bispecific antibodies (BsAb) may have potentially significant application in cancer therapy. The clinical development of BsAb as therapeutics has been hampered, however, by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods has been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. Here we describe a novel recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a single variable domain antibody to the N terminus of the light chain of a functional IgG antibody of different specificity. A model BsAb was constructed using a single variable domain antibody to mouse platelet-derived growth factor receptor alpha and a conventional IgG antibody to mouse vascular endothelial growth factor receptor 2. The BsAb was expressed in mammalian cells and purified to homogeneity by one-step protein A affinity chromatography. Furthermore, the BsAb retains the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. This design and expression of Fc domain-containing, IgG-like BsAb should be applicable to the construction of similar BsAb from antibodies recognizing any pair of antigens.     

FCgammaRII (CD32)-dependent induction of interferon-alpha by serum from patients with lupus erythematosus

Interferon-alpha (IFN-alpha) is detected in the serum of 70-80% of patients with systemic lupus erythematosus (SLE). Furthermore, soluble factors in SLE serum can induce peripheral blood mononuclear cells (PBMC) to produce IFN-alpha. The purpose of this work was to investigate the mechanism of this IFN-alpha induction. In eleven of fifteen SLE serum samples, an IFN-alpha inducing activity was detected, whereas serum from healthy controls, patients with other autoimmune disease and patients with viral infections were ineffective under the same conditions. After gel filtration of the serum, the inducing activity was found in the same fraction as IgG. The IFN-alpha inducing activity was inhibited by native monoclonal antibodies to the receptors for the Fc portion of IgG: FcgammaRIIA/C and FcgammaRIIB subclasses (CD32) and by their F(ab)'2 fragments. Purified Fc fragments of human IgG were also effective in abolishing the IFN-alpha-inducing activity. Since no anti-CD32 autoantibodies were found in SLE serum, this IFN-alpha-inducing activity may be due to immune complex antibodies. Such results may allow better understand the origin of endogenous IFN-alpha, which has a deleterious effect on the course of this autoimmune disease. The inhibition of this function by the CD32 antibody could lead to new therapeutic approach in SLE.     

Modulation of human rheumatoid factor-specific lymphocyte responses with a cross-reactive anti-idiotype bearing the internal image of antigen

Rabbit anti-idiotypic antibodies to human rheumatoid factor (RF) autoantibodies were isolated by affinity chromatography on rabbit anti-human IgG Fc Sepharose 4B. The anti-idiotypic antibodies bore the "internal image" of the antigen, human IgG. They reacted specifically with multiple human monoclonal and polyclonal IgM-RF, independent of any particular light or heavy chain amino acid sequence. The anti-idiotypes did not react with IgM or IgG proteins lacking RF activity. The present experiments determined the potential of the "internal image" antibodies to modulate in vitro lymphocyte functions. The addition of anti-idiotypic antibody to peripheral blood mononuclear cell cultures from patients with rheumatoid arthritis elicited lymphocyte proliferation, but not RF synthesis. The antibody did not induce the proliferation of lymphocytes from a normal individual. Moreover, the anti-idiotype specifically suppressed IgM-RF secretory responses when preincubated with B cells before co-culture with autologous pokeweed mitogen-activated T cells. The data show that the anti-idiotypic antibodies with the "internal image" of antigen are capable of interacting with B cell receptors in an antigen-restricted manner, and possess specific immunomodulatory properties.     

甲状腺特异性抗体与乳腺癌相关性研究的系统评价

目的：评价血清中甲状腺特异性抗体的存在对乳腺癌患病风险的影响,为评估乳腺癌的预后及制定治疗方案提供理论依据。方法：计算机检索Medline（1950～2012）、EMBASE（1949-2012）、Pubmed（1946-2012）、Current Contents Connect（1998-2012）和Google Scholar（1992-2012）等英文数据库。收集关于甲状腺特异性抗体与乳腺癌（BreastCancer,Bc）相关性分析的横断面研究或队列研究。按Cochrane系统评价方法,评价所纳入研究的文献质量,有效数据采用RevMan5．2软件进行系统评价。结果：最终纳入6项研究,共计6945例患者。系统评价结果显示：乳腺癌的风险会随血清中甲状腺特异性抗体（包括甲状腺过氧化物酶抗体anti-TP0和甲状腺球蛋白抗体anti—TG）的存在而增加（anti—TP00R2．51．95％CI：1．94—3．25;anti—TG2．67,95％CI：1．65—4．33）。结论：乳腺癌的风险会随血清中甲状腺特异性抗体的存在而增加,甲状腺特异性抗体可能为乳腺癌预后的评估以及治疗原则的制定提供理论基础。     

Cancer-associated antigens and antigen arrays in serological diagnostics of malignant tumors

The appearance of antibodies to cancer-associated antigens in biological fluids (particularly, in blood sera) of cancer patients is now a well-established fact, and their detection by immunochemical methods is a promising approach to diagnostics of malignant neoplasms. In this review, we consider some immunobiological aspects of the most extensively studied cancer-associated B-cell antigens, various applications of autoantibodies as cancer biomarkers, and prospects for the use of antigen arrays for improving diagnostic sensitivity.     

Humoral Response against Small Heat Shock Proteins in Parkinson’s Disease

Introduction

In the light of evidence for the increased heat shock proteins (HSP) expression in neurodegenerative disorders, the presence of the adaptive humoral response of the immune system can be expected. The aim of the study was to check whether Parkinson’s disease (PD) has the ability to elicit immune response against small heat shock proteins.

Methods

IgG and IgM autoantibodies against alpha B-crystallin were assessed in 26 PD patients 26 healthy subjects. For the assessment of anti-HSP IgG autoantibodies serum samples from 31 parkinsonian patients and 31 healthy control subjects were collected. Serum samples from PD patients and healthy control subjects were collected twice, at baseline and after mean of 13 months follow up.

Results

Both IgM and IgG autoantibodies against alpha ß-crystallin in PD patients were significantly higher compared to healthy controls (p<0.05). We also found statistically significant increase in antibodies titers against alpha ß-crystallin over the time of 13 months, both for IgG (p = 0.021) and for IgM (p<0.0001). Additionally, PD patients presented higher levels of anti-HSP IgG autoantibodies than healthy controls (p = 0.02).

Conclusions

Increase of IgG and IgM autoantibodies against alpha B-crystallin in PD patients over time may suggest their involvement in the disease pathogenesis and progression. Further studies are required to confirm the role of this antibody as a biomarker of the disease progression.     

A genetic variant of immunoglobulin γ2 is strongly associated with immunity to mucin 1 in patients with breast cancer

High levels of antibodies to mucin 1 (MUC1), a membrane-bound glycoprotein that is overexpressed in adenocarcinomas, are associated with good prognosis in patients with breast cancer. The aim of the present investigation was to determine whether GM and KM allotypes—genetic markers of IgG heavy chains and κ-type light chains, respectively—contribute to the magnitude of natural antibody responsiveness to MUC1 in patients with breast cancer. A total of 153 Caucasian subjects with breast cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. Anti-MUC1 IgG antibody levels in subjects who were carriers of the immunoglobulin γ2 allele GM 23 were significantly higher than in those who were noncarriers (P = 0.003). These results could potentially divide the population into high or low responders to MUC1, which has important implications for MUC1-based immunotherapeutic interventions in breast cancer.     

A monoclonal antibody to the desmosomal glycoprotein desmoglein I binds the same polypeptide as human autoantibodies in pemphigus foliaceus

Recent studies have demonstrated that antibodies from about half of patients with pemphigus foliaceus (PF) bind to a 160 kd polypeptide ("PF antigen") in sodium dodecyl sulfate (SDS) extracts of normal human epidermis. Desmoglein (DG) I, a glycoprotein enriched in desmosomal cores, is approximately the same m.w. as PF antigen. To demonstrate that PF autoantibodies bind to DG I, we used a monoclonal IgG antibody (MmDGI-1) that was raised against bovine muzzle desmosomal cores, and that specifically binds DG I. Double immunofluorescence labeling was performed on the same section of normal human skin with PF antibodies, detected by fluorescein-conjugated goat anti-human IgG, and MmDGI-1, detected by rhodamine-conjugated goat anti-mouse IgG. The pattern of reactivity with both antibodies was identical. Immunoblotting studies on proteins extracted from normal human epidermis and separated by SDS-polyacrylamide gel electrophoresis demonstrated that PF antibodies and MmDGI-1 bound co-migrating polypeptide bands of approximate m.w. 160,000. To confirm that these were identical polypeptides, we performed immunoblots of these epidermal extracts that were separated by two-dimensional gel electrophoreses (isoelectric focusing followed by SDS-PAGE). PF antibodies and MmDGI-1 bound identical spots with pI approximately 5.4 to 5.7 and m.w. approximately 160,000. These studies demonstrate that autoantibodies from certain patients with PF, a disorder of cell adhesion, bind to DG I, a desmosomal core glycoprotein.     

Probing the normal and autoimmune B cell repertoire with Epstein-Barr virus. Frequency of B cells producing monoreactive high affinity autoantibodies in patients with Hashimoto's disease and systemic lupus erythematosus

The frequency of cell precursors producing Ig of different classes and Ag-binding activities were determined, using EBV-infection and limiting dilution assays, in healthy subjects and patients with autoimmune disease. A large proportion of circulating B cells from healthy subjects were committed to the production of IgM antibodies that were polyreactive and bound a variety of self- and exogenous Ag, i.e., IgG Fc fragment, ssDNA, thyroglobulin, thyroid microsomal Ag, insulin, and tetanus toxoid. Similar frequencies of these polyreactive antibody-producing cells were found in patients with Hashimoto's disease and SLE. In contrast, significantly higher frequencies of cell precursors producing monoreactive IgG autoantibodies to thyroid Ag (thyroglobulin and thyroid microsomal Ag) and ssDNA were found in Hashimoto's disease and SLE patients, respectively. Calculation of the Kd revealed that monoclonal polyreactive antibodies were in general low affinity (Kd, 10(-3) to 10(-7) mol/liter), whereas monoclonal monoreactive autoantibodies were high affinity (Kd, 10(-9) to 10(-11) mol/liter). The detected frequency and high affinity of the monoreactive autoantibodies in Hashimoto's disease and SLE patients were comparable to those of anti-tetanus toxoid and anti-insulin IgG mAb produced by B cell clones from vaccinated healthy subjects and insulin-treated patients with insulin-dependent diabetes mellitus, respectively. These findings support the hypothesis that the autoimmune B cell repertoire in patients with organ-specific and systemic autoimmunity is shaped by Ag-driven responses rather than merely reflecting a polyclonal B cell activation.     

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTβR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti- LTβR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a full-length anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.