A new model for intermediate molecular weight recombinant bispecific and trispecific antibodies by efficient heterodimerization of single chain variable domains through fusion to a Fab-chain

Due to their specificity and versatility in use, bispecific antibodies (BsAbs) are promising therapeutic tools in tomorrow's medicine, provided sufficient BsAb can be produced. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. Recombinant BsAb can be made by fusing single chain variable fragments (scFv) to a heterodimerization domain. We compare the efficiency of the isolated CL and CH1 constant domains with complete Fab chains to drive heterodimerization of BsAbs in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete Fab chains were used, secretion of a heterodimerized bispecific antibody was successful. Since the Fab chain encodes a binding specificity on its own, bispecific (BsAb) or trispecific (TsAb) antibodies can be made by C-terminal fusion of scFv molecules to the L or Fd Fab chains. This gave rise to disulphide stabilized Fab-scFv BsAb (Bibody)or Fab-(scFv)2 TsAb (Tribody) of intermediate molecular size. Heterodimerization of the L and Fd-containing fusion proteins was very efficient, and up to 90% of all secreted antibody fragments was in the desired heterodimerized format. All building blocks remained functional in the fusion product, and the bispecific character of the molecules as well as the immunological functionality was demonstrated.     

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.     

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.     

Simultaneous blockade of both the epidermal growth factor receptor and the insulin-like growth factor receptor signaling pathways in cancer cells with a fully human recombinant bispecific antibody

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. Here we constructed and produced two fully human recombinant bispecific antibodies (BsAb) that target both EGFR and IGFR, using two neutralizing human antibodies originally isolated from a phage display library. The BsAb not only retained the antigen binding capacity of each of the parent antibodies, but also were capable of binding to both targets simultaneously as demonstrated by a cross-linking enzyme-linked immunosorbent assay. Furthermore, the BsAb effectively blocked both ligands, EGF and IGF, from binding to their respective receptors, and inhibited tumor cell proliferation as potently as a combination of both the parent antibodies. More importantly, the BsAb were able to completely block activation of several major signal transduction molecules, including Akt and p44/p42 MAP kinases, by both EGF and IGF, whereas each individual parent antibody was only effective in inhibiting those signal molecules activated by the relevant single growth factor. The BsAb molecules retained good antigen binding activity after incubation with mouse serum at 37 degrees C for up to 6 days. Taken together, our results underscore the benefits of simultaneous targeting multiple growth factor receptor pathways for more efficacious cancer treatment. This report describes the first time use of a recombinant BsAb for targeting two tumor-associated molecules on either a single or adjacent tumor cells for enhanced antitumor activity.     

The effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody

In recent years a variety of recombinant methods have been developed for efficient production of bispecific antibodies (BsAb) in various formats. Bispecific diabody (bDAb), a 55-60 kDa molecule comprising two non-covalently associated cross-over single chain Fv (scFv) polypeptides, represents one of the most promising as well the most straightforward approaches to BsAb production. Here we constructed a bDAb, using two human scFv, 11F8 and A12, directed against the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR), respectively, as the building blocks. A total of 8 scFv and diabody constructs were prepared comprising the same two variable heavy (V(H)) and variable light (V(L)) chain domains but arranged in different orientations. V(H)/V(L) orientation, i.e., V(H)-linker-V(L) or V(L)-linker-V(H), showed significant effects on the expression and antigen-binding activity of scFv and monospecific diabody of both 11F8 and A12. Further, only 2 out of the 4 possible V(H)/V(L) orientations/arrangements in bDAb construction yielded active products that retain binding activity to both EGFR and IGFR. Both active bDAb preparations retained their original antigen-binding activity after incubation at 37 degrees C in mouse serum for up to 7 days, indicating excellent stability of the constructs. Taken together, our results underscore the importance of identifying/selecting optimal V(H)/V(L) orientation/arrangement for efficient production of active bDAb.     

Integrating molecular detection and response to create self-signalling antibodies

Monoclonal antibodies are reproducible, specific, and cost-effective molecular probes; use outside the laboratory is, however, restricted by technical limitations. Addressing these constraints, the first self-signalling antibodies are now described, where specific antigen binding causes release of bound reporter from bispecific antibodies (BsAb) to generate a detectable signal. The report examines the concept that two different antibody binding sites in close proximity can promote interaction between molecules recognised by these sites, generating a signal by molecular crowding. Signal strength is found to increase with increasing homogeneity for a BsAb reactive with multimeric surfactant antigen; signal response is linear for a BsAb reactive with univalent small analyte deoxypyridinoline. Self-signalling is consistent with intramolecular steric hindrance. This is the first report detailing integration of two different functions, molecular detection and signal response, into BsAbs and with detection of large and small analytes, has generic application to antibody-based systems.     

双特异性抗体构建在肿瘤临床治疗中的应用

双特异性抗体(Bispecific antibody,BsAb)是具有两个不同抗原结合位点的抗体,可分为含Fc段和不含Fc段的BsAb,不同结构的BsAb具有不同的特点和应用领域。相比于传统的单克隆抗体,BsAb的灵敏度和特异性更高。更重要的是,BsAb具有募集免疫细胞、双重阻断信号通路等功能,在免疫诊断和治疗中扮演重要角色。随着全球环境的恶化以及人们生活习惯的不规律,肿瘤的发病率越来越高,成为仅次于心脑血管疾病的全球第二大致死疾病,全球每年有1200万新发癌症病例。肿瘤的治疗手段包括手术切除、放化疗和靶向治疗等。肿瘤免疫疗法是近几年新兴的治疗方法,其通过激发自身免疫系统的能力来清除肿瘤细胞。传统的单抗药物虽在肿瘤靶向治疗和免疫治疗中取得了一定的疗效,但肿瘤具有高度的异质性和可塑性,常常引发肿瘤耐药性的出现。双特异性抗体能够同时靶向多个靶点,目前已用于肿瘤的临床治疗,并取得了一定的治疗效果。文中就双特异性抗体在肿瘤临床治疗中的研究进展和应用作一综述。     

A comparative study of the bispecific monoclonal antibody,blinatumomab expression in CHO cells and E. coli

Abstract

The “bispecifics” market improved over the past decade due to the development of many technological platforms including bispecific T cell engagers (BiTEs). The approval of blinatumomab, the most advanced bispecific T-cell engager (BiTE) in clinical trials, can be a significant milestone in the development of bispecific antibodies. Both Chinese hamster ovary (CHO) cells and E. coli strain are considered as the most widely used hosts for the large-scale production of therapeutic monoclonal antibodies. Since both of the economic and qualitative aspects of protein production are important in industry, selection of a suitable protein expression system is very critical. The BsAb gene was cloned into the expression vectors FC550A-1, pcDNA3.1 (+), and PET22b and 6?×?His-tagged BsAb then purified on a Ni-NTA chromatography column. Both SDS–PAGE and Western blotting analysis of the purified protein demonstrated that blinatumomab was successfully expressed as a 55?kDa in both expression systems. The antigen-binding properties of blinatumomab were compared in the mammalian system versus Escherichia coli. The results showed that the purified antibody from a mammalian expression system has better binding activity than the one from E. coli host.     

A Novel Bispecific Antibody against Human CD3 and Ephrin Receptor A10 for Breast Cancer Therapy

Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3) was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (≤10⁻¹ μg/mL) and a lower effector to target (E/T) cell ratio (E/T = 2.5). Dimeric BsAb (EphA10/CD3) also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3), which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors.     

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla^TM). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA’) fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA’, was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA’-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.     

Improving target cell specificity using a novel monovalent bispecific IgG design

Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, “DuetMab,” for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the C_H1-C_L interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.     

Nanocell targeting using engineered bispecific antibodies

There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or EDV^TMnanocell) to the epidermal growth factor receptor (EGFR). EDV^TMnanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDV^TMnanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDV^TMnanocells actively targeted with tandem scFv BsAb in vivo to MDA-MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively targeted EDV^TMnanocells. BsAbs therefore provide a functional means to deliver EDV^TMnanocells to target cells.     

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla^TM). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA’) fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA’, was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA’-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.     

Chemically generated IgG2 bispecific antibodies through disulfide bridging

Bispecific antibodies (BsAbs) are designed to engage two antigens simultaneously, thus, effectively expanding the ability of antibody-based therapeutics to target multiple pathways within the same cell, engage two separate soluble antigens, bind the same antigen with distinct paratopes, or crosslink two different cell types. Many recombinant BsAb formats have emerged, however, expression and purification of such constructs can often be challenging. To this end, we have developed a chemical strategy for generating BsAbs using native IgG2 architecture. Full-length antibodies can be conjugated via disulfide bridging with linkers bearing orthogonal groups to produce BsAbs. We report that an αHER2/EGFR BsAb was successfully generated by this approach and retained the ability to bind both antigens with no significant loss of potency.     

Improving target cell specificity using a novel monovalent bispecific IgG design

Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, “DuetMab,” for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the C_H1-C_L interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.     

A bispecific antibody targeting HER2 and PD-L1 inhibits tumor growth with superior efficacy

Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2⁺ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2⁺ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.     

Purified anti-CD3 × anti-HER2 bispecific antibody potentiates cytokine-induced killer cells of poor spontaneous cytotoxicity against breast cancer cells

Background

Chemical crosslinking is the most straightforward method to produce bispecific antibodies (BsAb) for arming ex vivo activated cytotoxic T lymphocytes. However, heterogeneous polymers are produced by chemical crosslinking. Currently, it is not known under what circumstances or to what extent further purification is needed.

Results

In this study, we purified Traut’s Reagent-Sulfo-SMCC crosslinked anti-CD3?×?anti-HER2 by size-exclusion column chromatography and compared the capacity of the crude and the purified forms of the BsAb in enhancing cytokine-induced killer (CIK) cell-mediated cytotoxicity in vitro. We found that the purified BsAb assisted CIK cells more efficiently than the crude form only when the spontaneous cytotoxicity of the CIK cells was relatively low; otherwise, the two forms performed almost identically.

Conclusions

For the CIK cells of low spontaneous cytotoxicity, purified BsAb is a more powerful substitute for crude BsAb in enhancing their killing efficacy. However, that purification of BsAb is not necessary for robust CIK cells. This phenomenon also corroborates that CIK-mediated cytotoxicity is highly dependent on cell contact.

     

An efficient route to the production of an IgG-like bispecific antibody

Production of IgG-form bispecific antibody (BsAb-IgG) by co-expressing two antibodies in transfected cells is often inefficient owing to the unwanted pairing between the component heavy and light chains. We have developed an efficient method for the production of a novel IgG-like BsAb by using the natural dimerization mechanism between IgG heavy and light chains. Two single-chain Fv (scFv) of different specificity are fused to the constant domain of human kappa chain (C(L)) and the first constant domain of human heavy chain (C(H1)), to form two polypeptides, (scFv)(1)-C(L) and (scFv)(2)-C(H1)-C(H2)-C(H3), respectively. Co-expression of the two polypeptides in mammalian cells results in the formation of a covalently linked IgG-like hetero-tetramer, Bs(scFv)(4)-IgG, with dual specificity. Our approach yields a homogeneous bispecific IgG-like antibody product with each molecule containing four antigen binding sites, two for each of its target antigens. A Bs(scFv)(4)-IgG was prepared using two scFv antibodies each directed against a different epitope of a vascular endothelial growth factor receptor, the kinase insert domain-containing receptor (KDR). The Bs(scFv)(4)-IgG is capable of simultaneously binding to the two epitopes on the receptor. Further, the Bs(scFv)(4)-IgG also retains the antigen-binding efficacy and biological activity of its component antibodies.     

Nanocell targeting using engineered bispecific antibodies

There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or EDV^TMnanocell) to the epidermal growth factor receptor (EGFR). EDV^TMnanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDV^TMnanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDV^TMnanocells actively targeted with tandem scFv BsAb in vivo to MDA-MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively targeted EDV^TMnanocells. BsAbs therefore provide a functional means to deliver EDV^TMnanocells to target cells.     

Aberrant bispecific antibody pharmacokinetics linked to liver sinusoidal endothelium clearance mechanism in cynomolgus monkeys

Bispecific antibodies (BsAbs) can affect multiple disease pathways, thus these types of constructs potentially provide promising approaches to improve efficacy in complex disease indications. The specific and non-specific clearance mechanisms/biology that affect monoclonal antibody (mAb) pharmacokinetics are likely involved in the disposition of BsAbs. Despite these similarities, there are a paucity of studies on the in vivo biology that influences the biodistribution and pharmacokinetics of BsAbs. The present case study evaluated the in vivo disposition of 2 IgG-fusion BsAb formats deemed IgG-ECD (extracellular domain) and IgG-scFv (single-chain Fv) in cynomolgus monkeys. These BsAb molecules displayed inferior in vivo pharmacokinetic properties, including a rapid clearance (> 0.5 mL/hr/kg) and short half-life relative to their mAb counterparts. The current work evaluated factors in vivo that result in the aberrant clearance of these BsAb constructs. Results showed the rapid clearance of the BsAbs that was not attributable to target binding, reduced neonatal Fc receptor (FcRn) interactions or poor molecular/biochemical properties. Evaluation of the cellular distribution of the constructs suggested that the major clearance mechanism was linked to binding/association with liver sinusoidal endothelial cells (LSECs) versus liver macrophages. The role of LSECs in facilitating the clearance of the IgG-ECD and IgG-scFv BsAb constructs described in these studies was consistent with the minimal influence of clodronate-mediated macrophage depletion on the pharmacokinetics of the constructs in cynomolgus monkeys The findings in this report are an important demonstration that the elucidation of clearance mechanisms for some IgG-ECD and IgG-scFv BsAb molecules can be unique and complicated, and may require increased attention due to the proliferation of these more complex mAb-like structures.