Di-diabody: a novel tetravalent bispecific antibody molecule by design

The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods have been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. These recombinant antibody molecules possess dual antigen-binding capability with, in most cases, monovalency to each of their target antigens. Here, we describe an efficient approach for the production of a novel tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a bispecific/divalent diabody to, via the hinge region, the N-terminus of the CH(3) domain of an IgG. The novel BsAb, which we termed "di-diabody", represents a tetravalent diabody dimer resulting from dimerization between the hinge region and the CH(3) domains. A di-diabody was constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor, expressed both in a single Escherichia coli host and in mammalian cells, and purified to homogeneity by a one-step affinity chromatography. Compared to the bispecific/divalent diabody, the tetravalent di-diabody binds more efficiently to both of its target antigens and is more efficacious in blocking ligand binding to the receptors. The di-diabody retained good antigen-binding activity after incubation at 37 degrees C in mouse serum for 72 h, demonstrating good product stability. Finally, expression of the di-diabody in mammalian cells yielded higher level of production and better antibody activity. This design and expression for BsAb fragments should be applicable to any pair of antigen specificities.     

A new format of bispecific antibody: highly efficient heterodimerization, expression and tumor cell lysis

Bispecific antibodies (BsAbs) have been considered as potential therapeutics for cancer. A major obstacle in the development of BsAb has been the difficulty in producing a heterodimer with two different arms and in sufficient quantity for clinical application by the traditional methods. We describe a new format of BsAb that consists of two single-chain variable fragment of antibodies (scFvs), one for human epidermal growth factor receptor 2 (HER2)/neu and the other for CD16, heterodimerized by a "knobs-into-holes" device from the CH3 domains of the human IgG1 Fc fragment. The two chains were functionally expressed in CHO cells and assembled into heterodimers with dual antigen-binding specificity. Compared with other types of engineered BsAbs expressed in mammalian cells, the yield of this BsAb was relatively high (12-14 mg/l). In vitro experiments demonstrated that the BsAb was able to recruit human peripheral blood mononuclear cells (PBMC) to kill SK-BR-3 cells more effectively than the commercial anti-HER2/neu antibody Herceptin (Roche, Shanghai). This new format of BsAb possesses properties that support its potential as a new antitumor agent.     

Fab-scFv fusion protein: an efficient approach to production of bispecific antibody fragments

The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. Here, we describe an efficient approach for the production of a novel bispecific antibody fragment by genetically fusing a single-chain Fv (scFv) to the C-terminus of either the light chain or the heavy chain of a Fab fragment of different antigen-binding specificity. The bispecific Fab-scFv fragments were expressed in a single Escherichia coli host and purified to homogeneity by a one-step affinity chromatography. Two different versions of the bispecific Fab-scFv fragments were constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor. These bispecific antibody fragments not only retained the antigen-binding capacity of each of the parent antibodies, but also are capable of binding to both targets simultaneously as demonstrated by a cross-linking ELISA. Further, the bispecific antibodies were comparable to their parent antibodies in their potency in blocking ligand binding to the receptors and in inhibiting ligand-induced biological activities. This design for BsAb fragments should be applicable to any pair of antigen specificities.     

Mechanisms by which accessory cells contribute in growth of resting T lymphocytes initiated by OKT3 antibody

This study was undertaken to determine how accessory cells (AC) participate in growth of normal resting T cells initiated by anti-T3 monoclonal antibodies. Highly purified peripheral blood resting T cells were obtained by sequentially using three procedures (adherence to plastic surface, adherence to nylon wool columns and treatment with four monoclonal antibodies against antigens on AC and activated T cells plus complement). The assays for T cell growth were carried out at low cell density (10(4) cells/well) and with T cell populations where we could not detect cells bearing OKM1 and Ia antigens. Soluble OKT3 antibody, concanavalin A, recombinant interleukin 2 (IL 2) or purified interleukin 1 (IL 1) alone did not induce proliferation of purified resting T cells. Recombinant IL2 together with soluble OKT3 antibody stimulated significant growth whereas purified IL1 and two distinct preparations derived from AC containing IL 1 activity did not. Nevertheless, purified IL 1 amplified the proliferation of T cells induced by soluble OKT3 antibody in the presence of a small number of irradiated AC (3%). Phorbol myristate acetate (PMA) together with soluble OKT3 antibody activated purified resting T cells to proliferate, but PMA alone had little growth-promoting activity only. Soluble OKT3 antibody did not by itself induce a detectable number of resting T cells to express receptors for IL2 as determined by direct immunofluorescence staining and FACS analysis with monoclonal anti-IL2 receptor antibody. Cloned IL2 or purified IL1 alone did not induce resting normal T cells to express receptors for IL2 either. In contrast, T cells exposed to both soluble OKT3 antibody and IL2 exhibited IL2 receptors. PMA alone stimulated some resting T cells to express IL2 receptors and this response was significantly increased when the drug was used together with soluble OKT3 antibody. Studies were performed with unfractionated mononuclear cells from a donor whose cells respond to OKT3 (IgG2) but not to Leu 4 (IgG1) anti-T3 antibodies. Recombinant IL 2 but not purified IL 1 corrected the defective response to Leu 4 antibody. Finally, OKT3 antibody linked to beads, but not in soluble form, and purified IL1 replaced AC in growth of purified resting T cells. Based on these data I conclude the following: (a) AC participate in growth of resting normal T cells initiated by anti-T3 antibodies through their Fc receptors in two ways, namely, by providing a matrix to favour cross-linking of the T3 complex and simultaneously by secreting IL1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bispecific anti-human red blood Rhesus-D antigen x anti Fc gamma RI targeted antibody-dependent cell-mediated cytotoxicity and phagocytosis by mononuclear leucocytes.

The Fc receptor mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis induced by bispecific antibody (BsAb) to the high-affinity Fc receptor for IgG (Fc gamma RI) and to human red blood group antigen RhD were studied in vitro, using human mononuclear leucocytes as effector cells. The results were compared with those obtained by using a human monoclonal IgG1 anti-RhD used alone and a reference human polyclonal anti-RhD antibody. The effect of non-specific human IgG on FcR-mediated functions by mononuclear leucocytes was checked. The results demonstrate that BsAb presents a high resistance of Fc-mediated function to blockade by non-specific human IgG compared with that of both polyclonal and monoclonal anti-RhD antibodies. These results further encourage possible clinical application of bispecific antibody in passive immunotherapy.     

Two distinct monoclonal antibodies raised against mouse beta nerve growth factor. Generation of bi-specific anti-nerve growth factor anti-horseradish peroxidase antibodies for use in a homogeneous enzyme immunoassay

Two hybridomas producing monoclonal antibodies against mouse beta nerve growth factor (NGF) were obtained from the fusion of hyperimmune splenocytes from rats immunized with polymerized beta-NGF and Sp2/0.Ag mouse myeloma cells. The monoclonal antibodies coded IgG 24 and 30 produced and secreted by the hybrid cells are both of the IgG2a subclass. Both monoclonal antibodies are capable of recognizing native NGF coated on microassay plates as well as the denatured factor on Western blots. However, only IgG 30 has been found to block NGF-induced process outgrowth from the rat pheochromocytoma cell line (PC12) as well as NGF-induced increase in choline acetyltransferase activity in rat primary septal cell cultures. In addition, only IgG 30 was able to detect immunocytochemically NGF-immunoreactive sites in fixed tissue. And, finally, IgG 24 could not compete for IgG 30 binding to immobilized native NGF. Consequently, it appears that these antibodies are recognizing different epitopes on the NGF molecule. Neither monoclonal antibody displayed any crossreactivity with serum albumin, aprotinin, epidermal growth factor or insulin. A hybrid-hybridoma producing bi-specific anti-NGF anti-horseradish peroxidase (HRP) monoclonal antibodies was generated from the fusion of an azaguanine resistant anti-HRP hybridoma, coded RAP2.Ag and the anti-NGF IgG 30 hybridoma treated with emetine. The potential merits of using these bi-specific antibodies in combination with their mono-specific anti-NGF parent in a homogeneous sandwich immunoassay for the quantitation of NGF are discussed.     

Murine Bispecific Antibody 1A10 Directed to Human Transferrin Receptor and a 42-kDa Tumor-Associated Glycoprotein

Previously, we observed that bispecific antibodies (“antigen forks”) that bound to certain pairs of different tumor surface antigens could inhibit cell growth. The chemically linked heteroconjugate of MAb 454A12 (murine IgG1 recognizing human transferrin receptor) and 317G5 (murine IgG1 recognizing a 42-kDa tumor-associated glycoprotein) was particularly inhibitory toward human colorectal cancer cell lines, and the iron-chelating agent deferoxamine was found to augment inhibition of tumor cell growth by this antigen fork. Further experiments revealed that an antigen fork constructed by linking Fab′ fragments instead of whole antibodies retained activity, which led us to construct a fork-secreting hybrid hybridoma. Hybridoma 454A12 was fused with hybridoma 34F2 (murine IgG1 with the same specificity as 317G5). Hybrid hybridomas whose supernatants blocked binding of both 454A12 and 34F2 probes were further tested for the ability to block growth of SW948 human colorectal cancer cells in an MTT growth assay, and were chosen for subcloning. Ascites produced by clone 1A10 was purified by affinity and cation exchange chromatography. Purified 1A10 bispecific antibody showed growth inhibitory activity comparable to that of a chemically linked heteroconjugate of its parental antibodies 34F2 and 454A12. Adding deferoxamine greatly enhanced the inhibitory activity of 1A10 and effectively prevented regrowth of tumor cellsin vitro.By heterologously crosslinking two antigens that are coexpressed on many tumor cells, this bispecific antibody is able to inhibit tumor growth with enhanced selectivity.     

CD98 regulates the phosphorylation of HER2 and a bispecific anti-HER2/CD98 antibody inhibits the growth signal of human breast cancer cells

Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.     

Phenotype, cytotoxic, and helper functions of T cells from varicella zoster virus stimulated cultures of human lymphocytes

Blood mononuclear cells (MNC) were stimulated with VZV in the form of live cell-associated virus, glutaraldehyde-fixed VZV-infected fibroblasts or an extracted VZV antigen. After 7 days of culture with live virus, IL2 receptors (IL2R) were found on CD4 and CD8 cells while cultures stimulated with fixed or extracted antigen had IL2R only on CD4 cells. Clones derived by limiting dilution from these cultures were tested for specificity by cytotoxicity to autologous VZV-superinfected B lymphoblasts, and by proliferation in the presence of antigen and antigen presenting cells. The CD8+ clones were not VZV specific in tests of cytotoxicity or proliferation. 50% of the CD4+ clones with specificity for VZV also proliferated in cultures stimulated with individual VZV glycoproteins gp I, II or III. Included amongst these were clones cytotoxic for lymphoblast targets prepared with live VZV. Most of the VZV-specific T cell clones which failed to lyse targets prepared with live VZV lysed targets prepared with heat killed VZV. Target cells were susceptible to lysis after VZV antigens were no longer demonstrable on the cell surface by immunofluorescence and monoclonal antibodies to VZV glycoproteins failed to block cytotoxicity. 5 of 8 VZV-specific CD4+ clones provided antigen-specific help to B cells for IgG antibody production. The data are consistent with the view that CD4+ T cells respond to processed VZV antigen fragments, and that these fragments include epitopes present on gp I, gp II and gp III. Additionally, some the cloned progeny of VZV specific T cells were bifunctional (expressing cytotoxicity and help for B cells) in tissue culture.     

Infection enhancement of influenza A H1 subtype viruses in macrophage-like P388D1 cells by cross-reactive antibodies

The contribution of cross-reactive hemagglutination inhibition (HI) antibodies to infection enhancement of influenza A H1 subtype NWS virus and two antigenic drift strains was investigated in a macrophage-like cell line P388D1. When P388D1 cells, previously treated with neuraminidase (NA) to remove the viral receptors, were infected with NWS virus exposed to rabbit antiviral immunoglobulin (IgG) showing various levels of cross-HI titers, virus yields were enhanced in the presence of a subneutralizing antibody, depending on their cross-HI titers. By flow cytometric analysis using a fluorescein isothiocyanate (FITC)-labeled NWS virus, the efficiency of attachment of virus-rabbit IgG complexes to Fc receptors on NA-treated cells showed close correlation with its cross-HI titer. These data suggest that cross-reactive HI antibodies could contribute to infection enhancement through the formation of potent infectious immune complexes with drift strains to mediate virus infection via Fc receptor uptake. Two monoclonal antibodies (mAB) in mouse IgG subclasses IgG1 and IgG2a showing strain-specific or cross-reactive HI activity were tested for their infection enhancement characteristics. A strain-specific mAB enhanced infection of homologous NWS virus, but not that of two other drift strains in either antibody dilution. In contrast, a cross-reactive mAB caused infection enhancement of all three virus strains in the presence of the subneutralizing antibody. This indicates that cross-reactivity, but not the IgG subclass, acts as an enhancing factor to this phenomenon. The antibody, with the same specificity as cross-reactive mAB, was detected semiquantitatively by competitive enzyme-linked immunosorbent assay (ELISA) with results almost consistent with cross-HI titers of polyclonal rabbit antiviral IgGs. These data suggest that the antibody detected by this assay might be one of the potent antibodies governing cross-HI activity as a whole antibody and causing infection enhancement of drift strains.     

EDTA dependent pseudothrombocytopenia caused by antibodies against the cytoadhesive receptor of platelet gpIIB-IIIA.

AIMS--To clarify the mechanisms involved in the development of EDTA dependent pseudothrombocytopenia, particularly the platelet receptors. METHODS--Platelets were measured in 33 patients with pseudothrombocytopenia, using different anticoagulants to collect blood samples (direct test). The results were compared with the counts obtained by adding patients'' serum or immunoglobulins to normal blood samples (indirect test). The role of platelet function was explored using ASA, PGE1, and apyrase as platelet inhibitors. The contribution of platelet receptor/s was investigated using antigens to gpIb-IX and gpIIb-IIIa monoclonal antibodies. Immunoglobulin class was estimated by the ability of IgG, IgA, and IgM antibodies to prevent platelet clumping. RESULTS--Agglutinating antibodies were IgA in 40%, IgG in 30%, and IgM in 10% of patients studied. Both patients'' serum and immunoglobulins induced platelet clumping in normal samples anticoagulated with EDTA (indirect test). This was prevented by incubation of blood samples at 37 degrees C and almost completely inhibited by the platelet inhibitors ASA, PGE1, and apyrase. Pseudothrombocytopenia was also entirely prevented by an antigen to gpIIb-IIIa monoclonal antibody that recognises fibrinogen and the von Willebrand factor binding site. Pseudothrombocytopenia was almost completely abolished after the addition of RGD peptide, the recognition sequence of cytoadhesive proteins. CONCLUSIONS--These findings suggest that EDTA dependent pseudothrombocytopenia is caused by agglutinating antibodies that recognise cytoadhesive receptors on platelet gpIIb-IIIa and that an efficient platelet metabolism is required.     

Naturally occurring human IgG antibodies to intracellular and cytoskeletal components of human platelets.

Immunoblotting of platelets that have been subjected to SDS-PAGE has revealed that sera from normal individuals contain IgG which binds to many platelet components. This binding was seen with autologous and heterologous platelets using serum of males and of nulliparous females who had not received blood transfusions. Although binding patterns of different sera were not identical, almost all sera caused IgG binding to platelet components of 87-90 kD, 140 kD (identified as vinculin) and 220-240 kD (tentatively identified as talin and actin-binding protein). Purified IgG showed the same binding pattern as whole serum and F(ab')2 fragments retained their ability to bind to many components. The titre of IgG binding in serum was 1:50-1600 while that of alloantibodies to the PlA1 antigen was 1:3200. IgG binding components were not secreted when platelets were stimulated and were rarely associated with isolated membranes, but were located either in platelet cytoplasm or cytoskeletons. IgG binding was decreased by absorbing sera with lysed platelets or isolated cytoskeletons, but only slightly with intact platelets. Microaffinity purification of IgG which formed a major band on immunoblots showed that it was antibody with specificity for vinculin or its degradation products. These findings suggest that normal sera contain naturally occurring IgG antibodies with specificity for intracellular platelet antigens and that in some cases their titre approaches that of antibodies of pathological significance.     

Complexes of soluble HLA antigens and anti-HLA autoantibodies in human sera: Possible role in maintenance of self-tolerance

The MHC plays an essential role in regulating the process of self-nonself discrimination. T cells recognize nonself antigens only in the context of self-MHC gene products. It is not clear, however, whether B cells are also endowed with immune receptors for self-MHC antigens. We have explored the existence of antibodies against self-MHC antigens (HLA) in the human by analyzing the specificity of anti-HLA antibodies developed by a population of 727 dialysis patients who had been monitored monthly over a period of 3-78 months. Anti-HLA autoantibodies were identified by serum screening in 119 patients. Twenty-five of these 119 patients had not been exposed to alloantigens before, indicating that the production of anti-HLA autoantibodies is not necessarily stimulated by allogeneic HLA antigens. Cross-matching of sera with autologous lymphocytes, confirmed the autoreactive nature of these anti-HLA antibodies which were of IgM or of IgG isotype in approximately equal numbers of patients. The fine specificities of anti-HLA autoantibodies was ascertained in studies which showed that antibodies can be adsorbed, and the eluted, from the membranes of target cells carrying then relevant HLA antigen. Since the antibodies characterized in our studies may be functionally active in vivo we examined the possibility that their level is controlled by anti-idiotypic antibodies or by soluble HLA antigens. We found that the titer of anti-HLA autoantibodies increased significantly if soluble HLA antigens were depleted from the serum. Our data suggest that circulating HLA antigens form immune complexes with anti-HLA autoantibodies and contribute to the maintenance of self-tolerance by inhibiting antibody binding to membrane HLA antigens. The finding that the immune repertoire includes B cells with receptors for self-MHC opens new perspectives for the study of network perturbations in autoimmune diseases.     

A peptide mimic blocks the cross‐reaction of anti‐DNA antibodies with glomerular antigens

Anti‐DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross‐reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti‐DNA antibodies to self‐antigens is isotype‐dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti‐DNA isotypes [derived from a single murine immunoglobulin (Ig)G3 monoclonal antibody, PL9‐11] that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti‐DNA IgG isotypes to glomerular antigens. The PL9‐11 panel of IgG anti‐DNA antibodies (IgG1, IgG2a, IgG2b and IgG3) was used for screening a 12‐mer phage display library. Binding affinity was determined by surface plasmon resonance. Enzyme‐linked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or ‘ALW’) which binds to all PL9‐11 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL9‐11 anti‐DNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti‐DNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal anti‐DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti‐DNA antibody binding to renal tissue.     

Does mitogen-induced antibody production by normal blood cells mimic spontaneous production in lupus?

Blood cells from patients with systemic lupus erythematosus (SLE) showed a raised level of spontaneous IgG production that included antibodies to DNA and to common environmental antigens (influenza virus haemagglutinin, adenovirus hexon and mannan from Candida albicans). In contrast, no IgG antibody was produced against an antigen not normally encountered in the UK (egg antigen from Schistosoma mansoni) or a self-antigen not generally associated with SLE (thyroglobulin). IgM production was raised to a lesser extent and only antibodies to DNA were detected. When normal cells were stimulated with pokeweed mitogen or S. aureus organisms, the specificity pattern of IgG production was similar to that described above for SLE with the major exception of the absence of IgG anti-DNA. IgM antibodies to DNA and all the other antigens were detected, but the specificity of the IgM ELISA assays for the protein antigens needs further clarification. The activity of IgM anti-DNA relative to total IgM was far greater in the SLE system. These results provide further evidence that a response to self-antigen is required for production of pathogenic IgG autoantibodies in SLE.     

抗人红细胞膜抗原非凝集型单克隆抗体的研制及特性鉴定

目的:制备抗人红细胞膜抗原的非凝集型单克隆抗体(mAb)并鉴定其特性。方法:应用杂交瘤技术,以人O型红细胞膜抗原免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,用聚凝胺试管法筛选识别红细胞表面共同抗原的抗体,玻片凝集实验剔除凝集型抗体(完全抗体),再将分泌非凝集型mAb(不完全抗体)的杂交瘤细胞株用有限稀释法克隆化3次。对杂交瘤细胞的稳定性和mAb的特性进行鉴定。结果:获得1株可稳定分泌mAb的杂交瘤细胞2E8。mAb2E8为IgG1类,可特异性地识别红细胞膜上的H抗原,没有种属交叉血凝反应。杂交瘤细胞的培养上清与人的A、B、AB和O型红细胞均能产生强凝集,凝集效价为1∶1024,腹水mAb的凝集效价达到1∶64×106。mAb的亲和力用凝集试验检测,出现血凝的时间为7s,3min以内凝块>1mm。结论:成功地制备了针对红细胞膜H抗原的非凝集性mAb,此mAb的凝集效价、相对亲和力及特异性均较良好,为构建双特异性抗体奠定了基础。     

Designing of a bispecific antibody against SARS-CoV-2 spike glycoprotein targeting human entry receptors DPP4 and ACE2

COVID-19 originated in Wuhan city, China, in 2019 erupted a global pandemic that had put down nearly 3 million lives and hampered the socio-economic conditions of all nations. Despite the available treatments, this disease is not being controlled totally and spreading swiftly. The deadly virus commences infection by hACE2 receptor and its co-receptors (DPP4) engagement with the viral spike protein in the lung alveolar epithelial cells, indicating a primary therapeutic target. The current research attempts to design an in-silico Bispecific antibody (BsAb) against viral spike glycoprotein and DPP4 receptors. Regdanvimab and Begelomab were identified to block the D614G mutated spike glycoprotein of SARS-CoV-2 and host DPP4 receptor, respectively. The designed BsAb was modified by using KIH (Knobs into Holes) and CrossMAb techniques to prevent heavy chain and light chain mispairings. Following the modifications, the site-specific molecular docking studies were performed, revealing a relatively higher binding affinity of BsAb with spike glycoprotein and DPP4 co-receptor than control BsAb. Also, for blocking the primary entry receptor, hACE2, an anti-viral peptide was linked to the Fc region of BsAb that blocks the hACE2 receptor by linker cleavage inside the infected host. Thus, the designed BsAb and anti-viral peptide therapy could be a promising triumvirate way to obstruct the viral entry by blocking the receptor engagement.     

The combination therapy with EpCAM/CD3 BsAb and MUC-1/CD3 BsAb elicited antitumor immunity by T-cell adoptive immunotherapy in lung cancer

Lung cancer remains a global challenge due to high morbidity and mortality rates and poor response to treatment, and there are still no effective strategies to solve it. The bispecific antibody (BsAb) is a novel antibody, which can target two different antigens and mediate specific killing effects by selectively redirecting effector cells to the target cells. In this study, we combined two BsAbs to achieve a dual-target therapy strategy of EpCAM⁺ and MUC-1⁺ with high affinity and specificity. The results showed that the combination of two BsAbs against EpCAM and MUC-1 could inhibit the growth of lung cancer more effectively in cell lines and primary tumors. The superior antitumor effect of two BsAbs could be attributable to enhanced CTL and increased production of type I IFNs. At the same time, the combination of EpCAM/CD3 BsAb and MUC-1/CD3 BsAb significantly regulated T population in the TDLNs. Therefore, we have found a potential immunotherapeutic strategy, which was the combination therapy with EpCAM/CD3 BsAb and MUC-1/CD3 BsAb for the treatment of non-small cell lung cancer.     

Stimulation of stromal cell growth by normal rat urothelial cell-derived epidermal growth factor

Primary cultured adult rat urothelial (RU) cells caused increased thymidine incorporation in rat bladder stromal (RS) cells in a coculture system. The concentrated conditioned medium derived from RU cell culture (CM-RU) also stimulated the growth of RS cells, and induced anchorage independent growth of NRK-49F cells. Since these activities were heat and acid resistant, we investigated whether epidermal growth factor (EGF) and/or transforming growth factors were the humoral factor(s) responsible. The immunodiffusion analysis of CM-RU gave a positive precipitin line with rabbit anti-rat EGF IgG but not with rabbit anti-rat transforming growth factor-alpha antibodies. The anti-rat EGF IgG inhibited CM-RU-stimulated thymidine incorporation into RS cells, whereas normal rabbit IgG did not. By immunofluorescent technique using rabbit anti-rat EGF antibodies, immunoreactive EGF was demonstrated in RU cells and the urothelial of cryoinjury-induced reparative hyperplasia. Immunofluorescent technique also demonstrated the presence of EGF receptors on the cell membrane of RU and RS cells, basal cells of normal rat urothelium, and cells of whole epithelial layers of reparative hyperplasia. These data strongly suggest that EGF or an EGF-like substance produced by RU cells and released into medium stimulates the growth of RS cells possibly mediated by EGF receptors.     

Acquired antagonistic activity of a bispecific diabody directed against two different epitopes on vascular endothelial growth factor receptor 2

Bispecific antibody (BsAb) technology has been successfully used as a means to construct novel antibody (Ab) molecules with increased avidity for binding, by combining two Ab or their fragments directed against different epitopes within the same antigen. Using two single chain antibodies (scFv) isolated from a phage display library, we have constructed a bispecific diabody directed against two different epitopes on the extracellular domain (ECD) of human vascular endothelial growth factor receptor 2 (VEGFR2), the kinase-insert domain-containing receptor (KDR). Neither of the parent scFv blocks KDR/VEGF interactions or inhibits VEGF-induced receptor activation. The diabody binds to KDR with an affinity that is 1.5- to 3-fold higher than its parent scFv, mainly due to a much slower dissociation rate (k_off), which is approximately 17- to 26-fold slower than that of the individual scFv. In addition, the diabody binds simultaneously to, and thus cross-links, the two epitopes on the receptor(s). It is rather unexpected that the diabody effectively blocked KDR/VEGF interactions, and inhibited both VEGF-induced activation of the receptor and mitogenesis of human endothelial cells. Taken together, our results suggest that the diabody is most likely to exert its effect through steric hindrance and/or causing major conformational changes of the receptor. This is the first report on the construction of a bispecific diabody with acquired novel antagonistic activity.