A general strategy for generating intact,full-length IgG antibodies that penetrate into the cytosol of living cells

Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were co-expressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira®) and bevacizumab (Avastin®), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.     

抗EGFRvⅢ噬菌体抗体库的构建和筛选

目的:应用噬菌体展示技术筛选针对表皮生长因子受体突变体Ш(epidermal growth factorreceptor variant typeⅢ,EGFRvIII)的单链抗体(single chain Fv,scFv)。方法:利用原核表达纯化的人EGFRvIIIex蛋白和高表达EGFRvIIIex的小鼠成纤维细胞系NIH3T3免疫小鼠,扩增VH和VL片段并拼装成scFv基因,连接至噬菌粒pCANTAB5E,电击转化Hpd3cells,构建噬菌体单链抗体库,并进行3轮富集筛选。在第4轮筛选时,采用了降低抗原浓度的方法。然后将筛选得到的阳性克隆测序分析,转化E.coliHB2151,IPTG诱导可溶性scFv的表达。结果:构建了库容为7.9×107的噬菌体单链抗体库。经过第4轮低浓度抗原筛选,得到了较高亲和力的克隆。取单个阳性克隆测序分析结果表明,该抗EGFRvIII scFv基因序列长807bp,编码268个氨基酸。IPTG诱导后表达的可溶性scFv可分别与纯化的EGFRvⅢex抗原以及细胞表面的EGFRvⅢex结合。结论:利用噬菌体抗体库筛选得到了高亲和力的抗EGFRvⅢ scFv,为开发针对EGFRvⅢ...     

Generation by phage display and characterization of drug-target complex-specific antibodies for pharmacokinetic analysis of biotherapeutics

Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format.

Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.     

Isolation and characterization of an active variable domain from a homogeneous rabbit antibody light chain.

The variable domain (VL) of allotype b4 light chains of rabbit IgG was isolated from both nonimmune heterogeneous IgG and a homogeneous antibody directed against type III pneumococcal polysaccharide. Light chains were first isolated and then cleaved under mild acidic conditions between residues 109 and 110. Reduction with dithiothreitol in guanidine hydrochloride cleaved both intradomain disulfide bridges as well as the interdomain disulfide bridge joining the variable and constant domain. The sulfhydryl groups were protected after reduction by p-chloromercuribenzoate. VL was isolated from this mixture of variable and constant domains by affinity chromatography, utilizing sheep antibodies directed against a peptide including residues 110--211 from nonimmune IgG light chain. The isolated VL domain was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and automated Edman degradation. VL from a homogeneous antibody was treated with dithiothreitol to remove p-chloromercuribenzoate, reoxidized, and recombined with homologous heavy chain. The binding of this recombinant to type III pneumococcal polysaccharide was identical with that of the light-chain--heavy-chain recombinant.     

Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies

The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long‐lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use.     

Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells

Bispecific IgG production in single host cells has been a much sought-after goal to support the clinical development of these complex molecules. Current routes to single cell production of bispecific IgG include engineering heavy chains for heterodimerization and redesign of Fab arms for selective pairing of cognate heavy and light chains. Here, we describe novel designs to facilitate selective Fab arm assembly in conjunction with previously described knobs-into-holes mutations for preferential heavy chain heterodimerization. The top Fab designs for selective pairing, namely variants v10 and v11, support near quantitative assembly of bispecific IgG in single cells for multiple different antibody pairs as judged by high-resolution mass spectrometry. Single-cell and in vitro-assembled bispecific IgG have comparable physical, in vitro biological and in vivo pharmacokinetics properties. Efficient single-cell production of bispecific IgG was demonstrated for human IgG₁, IgG₂ and IgG₄ thereby allowing the heavy chain isotype to be tailored for specific therapeutic applications. Additionally, a reverse chimeric bispecific IgG_2a with humanized variable domains and mouse constant domains was generated for preclinical proof-of-concept studies in mice. Efficient production of a bispecific IgG in stably transfected mammalian (CHO) cells was shown. Individual clones with stable titer and bispecific IgG composition for >120 days were readily identified. Such long-term cell line stability is needed for commercial manufacture of bispecific IgG. The single-cell bispecific IgG designs developed here may be broadly applicable to biotechnology research, including screening bispecific IgG panels, and to support clinical development.     

单域抗体的研究和应用进展

传统IgG抗体分子一般由轻链和重链组成,轻链包含1个可变区(VL)和1个恒定区(CL),重链包含1个可变区(VH)和3个恒定区(CH1,CH2,CH3)。单域抗体(Single domain antibody,sdAb),是指缺失抗体轻链而只有重链可变区的一类抗体,因其分子量小,也被称为纳米抗体(Nanobody)。20世纪90年代,单域抗体最早在骆驼科动物中被发现,之后在护士鲨、大星鲨和鳐鱼等软骨鱼纲动物中也发现了类似的抗体。单域抗体虽然结构简单,但仍然可以达到与传统抗体相当甚至更高的与特异抗原结合的亲和力。相比于传统抗体,单域抗体具有分子量小、稳定性强、易于重组表达等优点。近年来在生物学基础研究和医学临床应用方面均备受关注并被广泛应用。文中将从单域抗体的结构特征、理化性质、筛选方法及其在生物医学领域的重要应用进展进行综述。     

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.     

Studies on the influence of different designs of eukaryotic vectors on the expression of recombinant IgA

Production and application of therapeutic monoclonal antibodies are second only to vaccines in the world pharmaceutical market. The most common therapeutic antibodies are monoclonal antibodies (mAbs) of the IgG isotype that are produced in eukaryotic CHO cells. In recent years, there has been a considerable interest in developing treatment medications based on IgA antibodies, which can have a wide range of effector functions on human mucous membranes. To study the expression level of immunoglobulin A (IgA) in mammal cells, we designed a set of bipromoter (CMV and EF1α) vectors. The vectors contain gene fragments that encode the heavy chain variable domain (VH) and the light chain variable domain (VL) of the human monoclonal antibody FI6v3 against the hemagglutinin of influenza virus A. They also contain gene fragments that encode the light chain (kappa type) constant domain and the heavy chain constant domain of the human antibody IgA1. The expression vectors differed in the orientation of the promoters and the presence or absence of introns. Two variants of the full-length light and heavy chains were cloned into a eukaryotic expression vector in head-to-head and head-to-tail orientations. The resulting plasmids were transfected into CHO-DG44 and HEK-293T cells. The antibody expression level for the stable transfection of CHO-DG44 and HEK-293T cell cultures was determined by ELISA. The results of the experiments showed that the expression of FI6v3-IgA1 antibodies significantly increased when eukaryotic cells were transfected with the plasmid pBiPr-ABIgA1FI6-Iht in which the heavy chain of IgA1 contains introns and the promoters are arranged head-to-tail.     

Recombinant shark natural antibodies to thyroglobulin

As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.     

Structural and functional characterization of the trifunctional antibody catumaxomab

The Triomab® family of trifunctional, bispecific antibodies that maintain an IgG-like shape are novel tumor targeting agents. These chimeras consist of two half antibodies, each with one light and one heavy chain, that originate from parental mouse IgG2a and rat IgG2b isotypes. This combination allows cost-effective biopharmaceutical manufacturing at an industrial scale since this specific mouse/rat isotype combination favors matching of corresponding antibody halves during production by means of quadroma technology. Whereas every Triomab® family member is composed of an anti-CD3 rat IgG2b half antibody for T cell recognition, the antigen binding site presented by the mouse IgG2a isotype is exchangeable. Several Triomab® antibodies have been generated that bind to tumor-associated antigens, e.g., EpCAM (catumaxomab), HER2/neu (ertumaxomab), CD20 (FBTA05), gangliosides GD2/GD3 (Ektomun®), on appropriate tumor target cells associated with carcinomas, lymphomas or melanomas. Catumaxomab (Removab®) was launched in Europe for treatment of malignant ascites in April 2009. Here, we report the structural and functional characterization this product. Mass spectrometry revealed an intact mass of 150511 Dalton (Da) and 23717 Da, 24716 Da, 51957 Da and 52019 Da of the reduced and alkylated rat light chain, mouse light chain, rat heavy chain, mouse heavy chain chains, respectively. The observed masses were in agreement with the expected masses based on the amino acid sequence obtained from cDNA sequencing. The glycosylation profile was similar to other human IgG consisting of biantennary oligosaccharides with different numbers of terminal galactose. CD spectroscopy showed mainly β-sheets secondary structure that is typical for IgG antibodies. Binding measurement revealed the unique trifunctional features of catumaxomab. Other analytical tools were used to evaluate characteristics of catumaxomab preparations, including the presence of isoforms and aggregates.     

Improvement of antigen binding ability of human antibodies by light chain shifting

Human HB4C5 hybridoma cells produce a lung cancer-specific IgM human monoclonal antibody (mAb). HB4C5 human mAb cross-reacts with Candida cytochrome c (Cyt c) and carboxypeptidase (Cpase). Concanavalin A (ConA)-resistant variants of HB4C5 cells loss the original light chain followed by expression of various new light chains at a high incidence (light chain shifting) (Tachibana et al., 1996). HTD8 cells, one of the ConA-resistant variant subclones of HB4C5 cells, undergo the active light chain shifting and produce various sublines, each of which stably secretes new mAb consisting of a new light chain and a HB4C5 heavy chain. The new mAb exhibits altered antigen binding ability from that of the original antibody. We could expect that HTD8 cells can be used as ‘a light chain stem cell line’ to improve antigen binding ability and specificity of established human mAbs. A BD9D12 IgG human mAb recognizes lung cancer cells and cross-reacts with cytokeratin 8. Introduction of the heavy chain gene of BD9D12 mAb into HTD8 cells resulted in establishment of various sublines which secreted various kinds of hybrid antibodies consisting of different light chains derived from HTD8 subclones which underwent light chain shifting and a common IgG heavy chain derived from BD9D12. These hybrid antibodies exhibited different or improved reactivities to Cyt, Cpase, cytokeratin 8 and various cancer cells from those of parental mAb, demonstrating that light chain shifting can be applied to improve the affinity and specificity of human mAb. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparing domain interactions within antibody Fabs with kappa and lambda light chains

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.     

Humanization of anti-human insulin receptor antibody for drug targeting across the human blood-brain barrier

A murine monoclonal antibody (MAb) to the human insulin receptor (HIR) has been engineered for use as a brain drug delivery system for transport across the human blood-brain barrier (BBB). The HIRMAb was humanized by complementarity determining region (CDR) grafting on the framework regions (FR) of the human B43 IgG heavy chain and the human REI kappa light chain. A problem encountered in the humanization process was the poor secretion of the CDR-grafted HIRMAb by myeloma cells. This problem was solved by the production of human/mouse hybrids of the engineered heavy chain variable region (VH), which led to the replacement of five amino acids in the FR3 of the VH with original murine amino acids. No replacement of FR amino acids in the light chain variable region (VL) was required. The affinity of the humanized HIRMAb for the HIR was decreased 27% relative to the murine HIRMAb. The humanized HIRMAb avidly bound to the HIR of isolated human brain capillaries, which are used as an in vitro model system of the human BBB. The HIRMAb cross reacts with the HIR of Old World primates such as the Rhesus monkey. The humanized HIRMAb was radiolabeled with 125-iodine, and injected intravenously into an adult, anesthetized Rhesus monkey. Brain scanning showed the humanized HIRMAb was rapidly transported into all parts of the primate brain after intravenous administration. The humanized HIRMAb may be used as a brain drug and gene delivery system for the targeting of large molecule therapeutics across the BBB in humans.     

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5′-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.     

Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG1 or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-chain recombinant antibodies in eukaryotic cells.     

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant‐ and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell‐penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell‐specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb‐DNA or –protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.     

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.     

Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)–variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH–VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.     

Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)–variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH–VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.