In Vitro Binding Correlated To In Vivo Properties

In vitro cell-binding experiments employ highly simplified model systems lacking the physiological complexities of living organisms. This distinction underscores a critical uncertainty that trends observed in binding to cultured cells expressing the target antigen alone may or may not necessarily translate into the same trend with respect to targeting, efficacy, or other in vivo behaviors. In some regards, in vitro models are best-case scenarios in which the chances are stacked in favor of successful binding because of an inherent lack of physical barriers between the Ab and its receptor. In certain cases, however, specific in vivo factors or conditions that are absent in vitro may also cause discrepancies between the two measurements. To demonstrate these concepts, we will cite examples of Ab studies that relate in vitro binding to in vivo PK, in vivo targeting, and in vivo PD.

In Vitro Binding Correlated to In Vivo Pharmacokinetics

Correlations between in vitro antigen binding and in vivo PK must be carefully interpreted because binding events do not exclusively determine Ab PK. In

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addition to the effects of antigen binding, the valency, shape, size, isoelectric pH (pI), and blood pool concentration are all factors that govern the speed, magnitude, and depth (penetration) of tissue uptake and clearance of a given Ab (37). In some cases, however, differences in in vitro binding affinities can be correlated to differences in PK profiles in vivo assuming that Ab valency, shape, size, and pI are similar and that antigen density remains constant. Both the binding affinity of a given Ab for its antigen and the expression levels of the target antigen can heavily influence PK; however, nontarget specific binding interactions between Fc-binding domains of a given Ab and endogenous Fc receptors can also play an important role.

Target Antigen Binding Correlated to Antibody Pharmacokinetics The exploration of cluster of differentiation (CD)11a as a target for psoriasis treatment illustrates how target antigen expression can affect Ab PK. Efalizumab (anti-CD11a, Raptiva®) has been approved for treatment of moderate to severe psoriasis, a chronic skin disease involving T cells, and its PK and PD have been reviewed by Joshi et al. (38). This Ab binds the subunit CD11a of the human integrin lymphocyte function-associated antigen-1, and interferes with the T-cell infiltration, activation, migration to the skin, and reactivation process, all hallmarks of the disease. Coffey et al. used an in vitro human T-cell model to study cellular uptake and clearance of the anti-CD11a Ab (39). Flow cytometric analysis was used to examine the cell surface and intracellular expression of CD11a following in vitro incubation of anti-CD11a with human T cells, demonstrating downmodulation of both surface and intracellular CD11a expression over time. Addition of a secondary cross-linking Ab revealed CD11a internalization in vitro, and it was rationalized that a similar phenomenon helps mediate the in vivo clearance of anti-CD11a (39). In vivo experiments were conducted by intravenously administering mice with M17, a rat anti-mouse CD11a mAb, and muM17 (40). Similar flow cytometric analysis of muM17 binding to CD11a on CD3+, CD4+, and CD8+ T cells and cellular clearance in vivo was performed. The results were consistent with the in vitro results, suggesting that anti-CD11a clearance in vivo was driven by receptor-mediated internalization and lysoso-mal degradation by cells expressing CD11a (40).

Fcg Receptor Binding Correlated to Antibody Pharmacokinetics

Nearly every cell type of the immune system expresses FcgRs, including B lymphocytes, dendritic cells, macrophages, natural killer (NK) cells, neutrophils, mast cells, platelets, Langerhans cells, eosinophils, mesangial cells, and endothelial cells (41). Three different FcgRs exist: FcgRI (CD64), which demonstrates high affinity for monomeric IgG (Kd & 10~9 M), and two low-affinity receptors, FcgRII (CD32) and FcgRIII (CD16), which bind to monomeric IgG with a dissociation constant (Kd) of approximately 10~6 M but have high avidity for IgG-opsonized particles and IgG immune complexes (42). FcgRs are known to play a critical role in linking IgG Ab-mediated immune response with cellular effector functions. In addition, Ab PK may also be impacted through FcgR-mediated elimination.

The correlation of FcgR binding affinity to mAb PK is complicated and dependent on many factors. Target expression is a major determinant of Ab PK; however, when comparing the PK between a wild-type (WT) and an Fcg mutant Ab

(having equal antigen expression levels), Fcg affinity becomes an important parameter. Some Abs bind to soluble targets to form soluble immune complexes that can promote binding to the low-affinity FcgRII or FcgRIII. Once the soluble immune complex has been engaged by FcgRs, the complexes are internalized by hepatocytes including Kupffer cells and sinusoidal endothelial cells. A change in binding affinity to the FcgR will noticeably impact PK if the target antigen concentration is high relative to the mAb of interest when the contribution of the target-mediated clearance to the total clearance is significant. The binding of Ab to antigen is a dynamic process, and Fcg influences not only the PK of the mAb but also the Ab-antigen complex. As such, an Fcg-driven decrease in clearance of the Ab-antigen complex will result in a concomitant decrease in Ab clearance. For example, anti-IgE mAb (IgE, a soluble target with high endogenous concentration) with reduced FcgR binding affinity demonstrated slower clearance compared with its WT counterpart in monkeys. However, another undisclosed mAb against a different target (soluble target with low endogenous concentration) with reduced FcgR binding affinity had similar clearance compared with its WT counterpart in mice (Internal data, Genentech, Inc., unpublished data).

In contrast, Ab binding to cell membrane-bound targets can result in ADCC-driven cellular depletion through binding to the FcgRs on NK cells or other immune cells; the cell membrane-bound Ab is also destroyed in this process. Again, changes in binding affinity to FcgRs will significantly impact Ab PK only if the target expression level is high. For example, murine mAbs against OX40L (OX40L, cell-bound targets with low expression level) with reduced FcgR binding affinity have similar clearance when compared with their WT counterparts in mice (Internal data, Genentech, Inc., unpublished data). Gillies et al. reported an inverse correlation between the affinity of the interleukin-2 (IL-2) fusion protein for FcgRI and its half-life in Balb/C mice (43). The target for the IL-2 fusion protein is the IL-2 receptor expressed on cancer cells. The amount of IgG1-IL-2 mutant, which has reduced affinity to mouse FcgRI, remaining in the circulation at all time points, was significantly higher than that in the original IgG1-IL2. Campath-IH is a humanized mAb that reacts with CD52 antigen present on human lymphoid and myeloid cells. The blood concentration for the Fc mutants with a greatly reduced capacity to interact with FcgR was approximately two- to threefold higher than that of Campath-IH WT; this effect is due to both decreased clearance and a decrease in normal tissue uptake. In particular, the mutants showed significantly less spleen, liver, and bone uptake, with an overall trend toward lower normal tissue to blood ratios but with higher tumor uptake (44).

In the case of membrane-bound target antigens that undergo rapid inter-nalization upon mAb binding, differences in binding affinity to FcgR have only a minor impact on mAb PK. For example, keliximab and clenoliximab are monkey/human chimeric antihuman CD4 Abs with similar binding affinities to human CD4. Keliximab shows strong binding to FcgRs in vitro and can deplete CD4+ T cells. Clenoliximab has an approximately 10- to 100-fold lower affinity to FcgRs compared with keliximab and cannot deplete CD4+ T cells (45). Once an anti-CD4 mAb binds to CD4+ T cells, it will either be internalized or the anti-CD4-coated T cell will be cleared through an FcgR-mediated clearance pathway (e.g., phagocytosis). Since the internalization pathway appears much faster than FcgR-mediated clearance pathway and because the internalization pathway is not dependent on the Fcg affinity, the PK profiles of keliximab and clenoliximab should be similar. Accordingly, these two mAbs showed similar PK profiles in

24 Boswell et al.

transgenic mice bearing human CD4; however, the PD profiles (CD4+ T cells) were different (45). Binding of the anti-CD4 mAb to cell surface CD4 resulted in rapid internalization of the complex, and this internalization clearance pathway dominated the total clearance of the mAb. As such, although Fcg does play a substantial role for anti-CD4 mAb PD, the binding affinity to FcgRs has only a minor impact on anti-CD4 mAb PK due to rapid receptor-mediated internal-ization. It should be noted, however, that receptor saturation may be an additional contributing factor to similarity in PK between keliximab and clenoliximab. In addition, internalization induced by Ab binding also plays a critical role in the PK of another anti-CD4 Ab, TRX1 (see section "In Vitro Binding Correlated to In Vivo Pharmacodynamics").

In addition, the relative contribution of the FcgR-mediated clearance pathway to the total clearance of mAb compared with other factors also has impact on the correlation of FcgR binding affinity to mAb PK. For instance, fucosylation may impact FcgR binding affinity, as the removal of the core fucose from the biantennary complex-type oligosaccharides attached to Fc regions results in dramatically enhanced ADCC of Abs via improved Fcg binding. Core-fucosylated and nonfucosylated antihuman CD20 IgG1 from Chinese hamster ovary (CHO) cells with similar binding affinity to FcRn demonstrated the same PK in mice, although the nonfucosylated molecule has a threefold increased binding affinity to FcgRs compared with the core-fucosylated one (46). Other relevant factors include the size and, more importantly, the complexity (i.e., number of antigen and Ab molecules in a complex) of the immune complex (47). Multivalent immune complexes tend to be cleared more rapidly than simpler immune complexes because of increased Fc receptor-driven elimination resulting from multivalent Fc interactions (47). In summary, a number of factors must be considered for the effects of FcgR binding affinity on the mAb PK, including antigen location (i.e., membrane vs. soluble), antigen size, antigen expression level, internalization rate, and the relative contribution of the FcgR-mediated clearance pathway to the total clearance of mAb compared with other factors.

FcRn Binding Correlated to Antibody Pharmacokinetics

Recent reports have demonstrated that the FcRn receptor is a prime determinant of the disposition of IgG Abs (48-50). FcRn, which protects IgG from catabolism and contributes to the long plasma half-life of IgG, was first postulated by Brambell in 1964 (51) and cloned in the late 1980s (52,53). FcRn is a heterodimer consisting of a P2m light chain and a major histocompatibility (MHC) class I-like heavy chain. The receptor is widely expressed in cells and tissues. Several studies have shown that IgG clearance in P2m knockout mice (48,49) and FcRn-heavy chain knockout mice (54) is increased 10- to 15-fold, with no changes in the elimination of other Ig. The FcRn receptor binds to IgG in a pH-dependent manner, binding to IgG within acidic endosomes (pH * 6.0) and releasing IgG in the plasma (pH * 7.4) or interstitial (pH * 7.0) compartments. Any unbound IgG proceeds to the lysosome and undergoes proteolysis, therefore, IgG clearance is dependent on its affinity to FcRn receptors. The shorter half-life of the IgG3 isotype was attributed to its lower binding affinity to the FcRn receptor (50,55). Murine mAbs generally have much shorter serum half-lives (few days) than human mAbs (few weeks) due to their low binding affinity to the human FcRn receptor. It is also reported that human FcRn binds human, rabbit, and guinea pig IgG but not rat, mouse, sheep, and bovine IgG; however, mouse FcRn binds IgG from all of these species (56).

Interestingly, human IgG1 has been shown to have an eightfold higher affinity to murine FcRn than that for human FcRn (57-59), indicating a potential limitation in using mice as preclinical models for human IgG1 PK evaluation. In this context, Vaccaro et al. confirmed that an engineered human IgG1 had disparate properties in murine and human systems (60). Engineered IgG with higher affinity to human FcRn receptor had two- to threefold longer half-lives in some instances compared with WT in human FcRn-expressing mice and monkeys (57,61). Hinton et al. found that the half-life of an IgG1 FcRn mutant with increased binding affinity to human FcRn at pH 6.0 was about 2.5-fold longer than that of the WT Ab in monkey (Table 3) (61). However, Dall'Acqua et al. reported that an increase in binding affinity for FcRn at both pH 6.0 and pH 7.4 in mice was paralleled by a decrease in serum concentration of such variants (65). They proposed that a higher affinity to FcRn at pH 7.4 adversely affects release into the serum and offsets the benefit of the enhanced binding at pH 6.0. Furthermore, unlike previously reported PK studies with Abs having the same or similar FcRn mutations, Datta-Mannan et al. did not observe a direct relationship between increased binding affinity to FcRn and improved PK properties for their Abs in the limited number of mice and monkeys tested (62,63). In Table 3, the published PK properties of humanized IgG1 variants with differential binding properties to FcRn are summarized. In general, a number of factors may impact the influence of FcRn binding affinity on mAb PK: differences in the absolute IgG-FcRn affinity at pH 6.0 and pH 7.4, the kinetics of IgG/FcRn interaction at pH 6.0 versus pH 7.4, and the relative contribution of the FcRn-mediated clearance pathway to the total clearance of mAb compared with other factors. Another excellent example of FcRn binding correlated to PK in primates was recently published (64).

Albumin Binding Correlated to Antibody Pharmacokinetics

Albumin is the most abundant (50 mg/mL) plasma protein and has a half-life of about 19 days in humans, similar to a typical IgG1 (67). Albumin binding (AB) can be an effective strategy to improve the PK properties of otherwise shortlived molecules such as small molecules (68), peptides (69), and fragments of IgG (e.g., Fab) (70-72). Dennis et al. demonstrated that the clearance of an Fab fragment can be dramatically decreased through association with albumin (71). They developed one high-affinity AB peptide and fused it to an anti-tissue factor Fab D3H44. The clearance of AB-Fab (AB-Fab D3H44-L) decreased approximately 58-fold, and the half-life increased approximately 40-fold to 32.4 hours compared with 0.8 hours for Fab D3H44 in rabbits (71). Building on Dennis's work, Nguyen et al. found that the PK of an anti-human epidermal growth factor receptor 2 (HER2) AB-Fab 4D5 could be modulated as a function of affinity for albumin (72). There was an inverse correlation between affinity for albumin (ranging from 0.04 to 2.5 pM) and clearance (ranging over * 50-fold in rats and * 20-fold in rabbits) for AB-Fab variants (72). Later, Dennis et al. also showed that AB-Fab 4D5 rapidly targeted tumors, achieved tumor concentrations comparable to that of IgG, and quickly achieved higher tumor to normal tissue ratios compared with IgG (70). They also found that AB-Fab 4D5 did not accumulate in the kidney, suggesting that association with albumin leads to an altered route of clearance and metabolism (70). Overall, the increased half-life of Fab through association with albumin is consistent with the longer half-life of

TABLE 3 Humanized lgG1 Variants with Differential Binding Properties to the Neonatal Crystallizable Fragment Receptor: Relationship to Pharmacokinetics
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