Dissection of the combining site in a humanized anti-Tac antibody.

The genetically engineered "humanized" anti-Tac antibody (HAT) has been shown to bind the p55 chain of the human IL-2R with an affinity close to that of the murine anti-Tac. Although the HAT molecule contains all six mouse CDR, it was not known which, and to what extent, each of the CDR contributes to Ag binding. These questions were addressed by constructing a series of variant HAT antibodies, each substituting a single HAT CDR with a heterologous CDR. The association constants of the variant HAT antibodies to p55 were determined by competitive binding analysis. We find that CDR 1 and 3 of the H chain and CDR 3 of the L chain are essential for maintaining binding. The remaining three CDR appear to be involved to a lesser degree.     

Distinct reactivities of four monoclonal antibodies with human interleukin 2 receptor

Two new murine monoclonal IgG1 antibodies, H-31 and H-A26, were characterized in comparison with two previously obtained monoclonal antibodies against human interleukin 2 (IL-2) receptor (IL-2 R), anti-Tac and HIEI. In immunofluorescence assays with various human hematopoietic cells, H-31 and H-A26 antibodies both reacted with only IL-2 R-positive cells, and they precipitated IL-2 R molecules, glycoproteins with molecular weights of 60K and 53K daltons (gp60/gp53), from human T-cell leukemia virus type I (HTLV-I)-carrying MT-2 cells, as demonstrated by sequential immunoprecipitation after absorption of IL-2 R with anti-Tac. Antibody-binding competition assays showed that H-31 and anti-Tac, and H-A26 and HIEI, respectively, competed reciprocally in binding to the cells, and that anti-Tac also inhibited the binding of HIEI but not vice versa. H-31, like anti-Tac, strongly inhibited the IL-2-dependent proliferation of normal activated T-cells, absorption of IL-2 and direct binding of IL-2 to the cells, while H-A26, like HIEI, inhibited those processes only weakly. The spectra of reactivities of these antibodies with various simian cell lines derived by HTLV-I infection were different, as revealed by immunofluorescence studies. Human IL-2 R was shown to express a unique antigenic determinant, detected with HIEI, that was not detectable in IL-2 R molecules of Old and New World monkeys, and also to express determinants common to simian IL-2 R molecules. These observations indicate that H-31 and H-A26 recognize human IL-2 R molecules and that the antigenic sites on the IL-2 R molecule defined by H-31, H-A26, anti-Tac, and HIEI are different.     

Immunological properties of a single-chain fragment of the anti-idiotypic antibody ACA125

Vaccination with anti-idiotypic antibodies has been described as a promising concept for treatment of several malignant diseases. The murine monoclonal anti-idiotypic antibody ACA125 imitates a specific epitope of the tumor-associated antigen CA125 expressed by 80% of ovarian carcinomas. In the first clinical trial it could be shown that mAb ACA125 is able to elicit anti-anti-idiotypic antibodies (Ab3) with anti-CA125 specificity in patients with advanced ovarian cancer. In order to improve the capabilities of anti-idiotype vaccines we generated a genetically engineered single-chain fragment (scFv) ACA125 composed of heavy- and light-chain variable regions connected by a flexible linker. The antigenicity of scFv ACA125 was demonstrated by immunizing rats i.p. with scFv or complete mAb in complete/incomplete Freund's adjuvants (CFA/IFA) or precipitated by aluminium hydroxide. Negative control groups included applications of irrelevant mouse IgG or adjuvants alone. Anti-anti-idiotypic antibodies (Ab3) directed against the mAb ACA125 as well as specific anti-CA125 antibodies (Ab1′) could be detected in all animals treated with scFv in CFA/IFA. Nevertheless, antibody titers were lower than when the complete mAb ACA125 was used. Suprisingly, an increase of specificity could not be observed in scFv-immunized animals, which had been expected because of the lack of heavy- and light-chain constant regions that could raise rather unspecific anti-isotypic and anti-allotypic rat anti-(mouse Ig) antibodies (RAMA). In contrast, the RAMA responses detected in these rats were even stronger than those following immunization with complete mAb ACA125. In conclusion, the anti-idiotypic scFv ACA125 alone cannot improve the immunogenic features of the corresponding mAb, but provides a useful tool for the further development of genetic vaccines. Received: 20 January 2000 / Accepted: 24 April 2000     

Human monoclonal anti-idiotypic antibodies. I. Establishment of immortalized cell lines from a tumor patient treated with mouse monoclonal antibodies

Patients who undergo immunotherapy with a murine anti-colon carcinoma mAb (mAb17-1A) generate high titers of anti-idiotype and anti-isotype antibodies. Specifically selected anti-idiotypic antibodies that elicit in vivo a humoral and a cellular immune response against the nominal Ag can be used as surrogate Ag for immunization. We established from the B lymphocytes of a treated patient a series of EBV-transformed cell lines. Three weeks after immortalization, the cells were selected for production of antibodies (Ab2) against the Fab fragment of the murine mAb17-1A. The selected cells were cloned and screened by ELISA for specific anti-mAb17-1A idiotypic antibodies. Thirty-six out of 89 clones were anti-idiotypes. Cell culture supernatants and the purified Ig derived from 10 clones completely inhibited the specific binding of radiolabeled mAb17-1A to HT-29 colon carcinoma cells thus resembling Ab2-gamma anti-idiotypes. These cell lines which grow now in culture for 18 mo, continuously secrete IgG,K anti-Ab1-idiotype mAb. Human anti-idiotypic mAb might be candidates for vaccines when the nominal Ag itself is not available or cannot be used as such.     

Induction of effective immunity to Moloney murine sarcoma virus using monoclonal anti-idiotypic antibody as immunogen

We have isolated an anti-idiotypic mAb (RS1.1.3), which recognizes an idiotope present on several IgM mAb specific for Moloney murine leukemia virus (M-MuLV)-determined cell surface Ag. The binding of RS1.1.3 to idiotypic antibody could be inhibited by specific Ag. Intraperitoneal immunization of mice with purified RS1.1.3 antibody-induced effective immunity against Moloney murine sarcoma virus challenge. A single injection of RS1.1.3 7 days before virus challenge resulted in a 27% reduction in tumor load compared to non-immune control mice challenged with the same dose of virus, whereas multiple injections of RS1.1.3 before virus challenge resulted in a 75% reduction in tumor load. The protective effect of anti-idiotype immunization appeared to be T dependent, because immunization of athymic mice had no effect on their susceptibility to tumor virus challenge. Administration of the anti-idiotypic antibody after virus inoculation caused an increase in tumor load of nearly 50% compared to non-immune controls. BALB/c mice immunized with RS1.1.3 developed anti-anti-idiotypic antibodies, as well as M-MuLV Ag-specific antibodies. Analysis of sera from RS1.1.3-immune mice subsequently challenged with Moloney murine sarcoma virus indicated an inverse relationship between tumor load and M-MuLV-specific serum IgG titers induced by the RS1.1.3 immunization. These results indicate that anti-idiotypic mAb may be used as immunogen to induce Ag-specific antibody responses, and to cause effective immunity to a retro-virus-induced tumor.     

Anti-idiotype-induced, lipopolysaccharide-specific antibody response to Pseudomonas aeruginosa

Antibody directed to the O-specific polysaccharide (Ps) side chain of Pseudomonas aeruginosa LPS provides immunotype-specific protection against infection by virtue of enhancing opsonophagocytosis. We have developed a syngeneic anti-idiotypic antibody (mAb2) directed to a functionally active monoclonal immunotype 1 Ps-antibody (mAb1). The mAb2 performed as a molecular mimic of Ps as evidenced by 1) blocking of mAb1/mAb2 interaction by Ps, 2) blocking of mAb1/Ps binding by mAb2, 3) cross-species binding of mAb2 to human Ps antibodies from individuals immunized with the same immunotype 1 Ps, and 4) induction of anti-LPS antibody by immunization with mAb2 in syngeneic mice. Our studies thus show that an anti-idiotypic antibody may functionally mimic the O-polysaccharide of P. aeruginosa LPS, and bind to cross-reactive Id present in human Ps antibodies. We have further shown that this anti-idiotypic antibody induces anti-LPS antibody when used as an Ag in syngeneic mice, suggesting that this approach may eventually be used to successfully immunize humans.     

Antibody response against a possible arthritogenic epitope on human type II collagen induced by anti-idiotypic antibody

We reported the presence of three distinct epitopes commonly present on murine and human type II collagen (CII), observed using mAb. To investigate the possible involvement of these epitopes in collagen-induced arthritis, we raised rabbit anti-idiotypic antibodies that may bear the internal image of these epitopes. Anti-idiotypic antibodies developed against three anti-CII mAb designated as 1-5, 2-14, and 2-15 were demonstrated to recognize idiotype expressed on Ag-binding site (paratope) of their related mAb. Anti-CII antibody response specific for a given epitope could be induced in DBA/1J mice upon immunization with anti-idiotypic antibodies coupled to keyhole limpet hemocyanin. Anti-idiotypic antibody to 1-5 antibody in particular could stimulate all DBA/1J mice for production of anti-CII antibody possessing Ag specificity and idiotype similar to those of 1-5 antibody. Although the mice immunized with anti-1-5 antibody alone did not develop arthritis, they did show a much more enhanced antibody response against a given epitope than did control mice non-treated with anti-idiotypic antibody upon the subsequent immunization with human CII. Some of the mice immunized with anti-1-5 antibody and challenged with human CII developed arthritis, whereas the control mice did not. These findings strongly suggest that a common epitope recognized by 1-5 antibody might be involved in the induction of arthritis.     

Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman’s Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.     

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200?d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.     

Treatment with GM-CSF and IL-2 in patients with metastatic colorectal carcinoma induced high serum levels of neopterin and sIL-2R,an indicator of immune suppression

Cytokines may enhance the effect of therapeutic monoclonal antibodies (mAb). Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) have been shown to increase ADCC levels. GM-CSF may augment the induction of an idiotypic network response (anti-tumour immunity). The clinical anti-tumour effect of a combination of mouse mAb17-1A-1A [anti-colorectal carcinoma (CRC)], and GM-CSF was, however, not enhanced by the addition of IL-2. In the present study, some immune functions considered to be involved in mAb-mediated tumour cell killing were analysed in patients receiving GM-CSF and GM-CSF/IL-2 respectively together with the mAb17-1A-1A. Ten patients received mAb17-1A and GM-CSF, and ten patients mAb17-1A with GM-CSF and IL-2. During a 10- day cytokine treatment period, a significantly higher increase in white blood cell counts was noted in the GM-CSF/IL-2 treatment group as compared to GM-CSF-treated patients. In the GM-CSF/IL-2 group, significantly higher serum concentrations of neopterin and soluble IL-2 receptor (sIL-2R) respectively were induced as compared to GM-CSF-treated patients. However, the ADCC of peripheral blood mononuclear cells (PBMC) against a CRC cell line was significantly higher in the GM-CSF group than in the GM-CSF/IL-2 group. The frequencies of patients developing human anti-mouse antibodies (HAMA) and anti-idiotypic antibodies were the same in both groups, while serum concentrations were significantly lower in the GM-CSF/IL-2 group as compared to the GM-CSF group. GM-CSF/IL-2 therapy seems to induce an immune suppressive stage compared to GM-CSF alone affecting cytotoxic mononuclear cells and B cells, which might be mediated through the neopterin metabolic pathway or other inducible immune suppressive factors such as reactive oxygen and nitrogen intermediates.     

Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72

The use of anti-idiotypic antibodies as immunogens represents one potential approach to active specific immunotherapy of cancer. Two panels of syngeneic monoclonal anti-idiotypic antibodies were generated. One panel was directed against mAb CC49 and the other to mAb COL-1. mAb CC49 recognizes the pancarcinoma antigen (Ag), tumor-associated glycoprotein-72 (TAG-72), and mAb COL-1 recognizes carcinoembryonic antigen (CEA). Seven anti-idiotypic (AI) antibodies (Ab2) designated AI49-1–7 were generated that recognize the variable region of mAb CC49. These mAb were shown to inhibit the interaction of mAb CC49 (Ab1) with TAG-72 (Ag). Five anti-idiotypic antibodies designated CAI-1–5 were also generated to the anti-CEA mAb, COL-1 (Ab1). These Ab2 were shown to inhibit the interaction between COL-1 (Ab1) and CEA (Ag). Immunization of mice, rats, and rabbits with Ab2 directed against CC49 or COL-1 could not elicit specific Ab3 humoral immune responses, i.e., antibody selectively reactive with their respective target antigens. However, immunization of mice with the CC49 anti-idiotypic antibody (Ab2), designated AI49-3, could induce a delayed-type hypersensitivity response (DTH) specific for tumor cells that express TAG-72. Similarly, immunization of mice with an anti-idiotypic antibody directed against COL-1, designated CAI-1, could induce specific DTH cell-mediated immune responses to murine tumor cells that express human CEA on their surface. These results thus demonstrate that while some anti-idiotype mAb may not be potent immunogens in eliciting Ab3 humoral responses, they are capable of eliciting specific cellular immune responses against human carcinoma-associated antigens. This type of mAb may ultimately be useful in active immunotherapy protocols for human carcinoma.Some of the studies described in this paper were in partial fulfillment of requirements for the completion of Dr. Irvine's dissertation at the George Washington University     

Whole-molecule antibody engineering: generation of a high-affinity anti-IL-6 antibody with extended pharmacokinetics

The differentiation of therapeutic monoclonal antibodies in an increasingly competitive landscape requires optimization of clinical efficacy combined with increased patient convenience. We describe here the generation of MEDI5117, a human anti-interleukin (IL)-6 antibody generated by variable domain engineering, to achieve subpicomolar affinity for IL-6, combined with Fc (fragment crystallizable) engineering to enhance pharmacokinetic half-life. MEDI5117 was shown to be highly potent in disease-relevant cellular assays. The pharmacokinetics of MEDI5117 were evaluated and compared to those of its progenitor, CAT6001, in a single-dose study in cynomolgus monkeys. The antibodies were administered, either subcutaneously or intravenously, as a single dose of 5 mg/kg. The half-life of MEDI5117 was extended by approximately 3-fold, and clearance was reduced by approximately 4-fold when compared to CAT6001. MEDI5117 therefore represents a potential ‘next-generation’ antibody; future studies are planned to determine the potential for affinity-driven efficacy and/or less frequent administration.     

Suppression of anti-mouse immunoglobulin antibodies in subhuman primates receiving murine monoclonal antibodies against T cell antigens

Murine monoclonal antibodies stimulate the production of human anti-mouse immunoglobulin antibodies (AMIA) when administered to patients. This limits their long-term usefulness as therapeutic and diagnostic agents. We report the use of three maneuvers to suppress AMIA against T cell-specific monoclonal antibodies in cynomolgus monkeys. Twelve monkeys received daily i.v. infusions of 1 mg each of anti-Leu-2a, -3a, and -5 on days 1 through 10. One group (control) received no suppressive regimen. The second group received cyclosporine, 12.5 mg/kg daily on days -7 to +14. The third group (PI) were passively immunized with 0.4 ml of hyperimmune monkey AMIA serum on days -7, -1, 2, 4, 6, and 8. The fourth group (TLI) received 1700 rad fractionated total lymphoid irradiation ending on day -1. The animals treated with TLI were markedly delayed in the onset of AMIA, which was suppressed to less than 1% of the control group. The AMIA specific for the constant region of animals receiving PI was also suppressed to one-third of control. The majority of the AMIA in all the animals was anti-idiotypic and wholly anti-idiotypic in the TLI animals.     

Formation of a bispecific antibody by the use of leucine zippers.

A new method is described for the production of bispecific F(ab')2 heterodimers using leucine zippers. Two heterodimer-forming "zipper" peptides derived from the Fos and Jun proteins were respectively linked to the Fab' portions of two different mAb by gene fusion. The antibodies used were 145-2C11, which binds to murine CD3, and anti-Tac, which binds to the p55 chain of the human IL-2R. Anti-Tac Fab'-Jun and anti-CD3 Fab'-Fos were expressed individually as F(ab'-zipper)2 homodimers in the mouse myeloma cell line Sp2/0. When these homodimers were reduced at the hinge region to form monomers and then reoxidized together, the resulting end products were mostly F(ab'-zipper)2 heterodimers. Bispecific anti-CD3 x anti-Tac F(ab'-zipper)2 heterodimers produced by this method were shown to be highly effective in recruiting cytotoxic T cells to lyse IL-2R-bearing HuT-102 cells in vitro.     

Flow cytometric analysis on reactivity of human T lymphocyte-specific and cytokine-receptor-specific antibodies with peripheral blood mononuclear cells of chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and squirrel monkey (Saimiri sciureus)

Abstract: There are relatively few monoclonal antibodies (mAb) that have been characterized for their applicability in studies on the immune system of various nonhuman primates. In the present study, we identified a large number of mAb that can be used in future immunological studies in three different nonhuman primates, i.e., chimpanzees, rhesus macaques, and squirrel monkeys. The reactivity of 161 anti-human mAb to T-cell antigens and cytokine receptors were tested on peripheral blood mononuclear cells (PBMC) from the three primate species by flow cytometric analysis. A total of 105 (65%), 73 (45%), and 68 (42%) antibodies reacted with PBMC from chimpanzees, rhesus macaques, and squirrel monkeys, respectively. Out of the 161 mAb, 38 reacted with all three species and 112 reacted with one or two of the species. No specific reaction was observed with mAb to receptors to GM-CSF, 4–1BB, FLT3, FLX2, common β-chain, IL-1 (type I receptor), and IL-8.     

Correlations between pharmacokinetics of IgG antibodies in primates vs. FcRn-transgenic mice reveal a rodent model with predictive capabilities

Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.     

Activation of a functional idiotype network response by monoclonal antibody specific for a virus (M-MuLV)-induced tumor antigen

BALB/c mice were injected with IgM mAb specific for Moloney murine leukemia virus (M-MuLV)-determined cell surface Ag in an attempt to inhibit Moloney sarcoma growth. The monoclonal IgM significantly inhibited sarcoma growth when given to the mice after inoculation with Moloney murine sarcoma/leukemia virus, and also potentiated the in vivo antibody response specific for M-MuLV Ag. These responses were significantly greater than the primary response to the virus alone in age- and sex-matched control mice, and were also seen in mice which were injected with the IgM antibody only and not with virus, suggesting that an Ag-independent mechanism may be involved. The M-MuLV-specific serum antibody responses induced by the monoclonal IgM, with or without prior virus inoculation, were predominantly of the IgG1 isotype, with some IgG2a; no other isotypes were found to have titers significantly higher than in the normal response to virus alone. M-MuLV-specific IgG1 was detected only in mice injected with monoclonal IgM, and not in the response to virus alone. The same sera also had high titers of anti-idiotypic antibodies, (Ab2), as well as anti-anti-idiotypic antibodies (Ab3). It appears, therefore, that passive immunization with M-MuLV-specific IgM mAb activates an idiotypic network, which results in both Ab2 and Ab3 responses; the M-MuLV-specific response may be considered a subset of Ab3.     

A strategy for risk mitigation of antibodies with fast clearance

A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4–61.3 mL/day/kg), with 15 (29%) having clearance > 10 mL/day/kg. Alteration in the interaction with the recycling FcRn receptor did not account for the faster than expected clearance observed for the antibodies; off-target binding was presumed to account for the fast clearance. We developed an assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies having increased risk for fast clearance. This assay can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.     

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.