Monoclonal antibodies represent one of the fastest-growing classes of new therapeutic and diagnostic agents, with nearly 2 dozen FDA-approved antibodies for the treatment of cancer or inflammatory and autoimmune disorders now available. Most native therapeutic antibodies provide modest response rates against solid tumors, and strategies to improve potency typically involve the conjugation of a drug, toxin, or radioisotope to the antibody. However, this strategy does not markedly improve the therapeutic index because the slow clearance of monoclonal antibodies results in increased normal-tissue toxicity, typically to the bone marrow and secondarily to the kidneys, lungs or liver. The ability to accelerate the clearance and reduce the plasma half-life of IgG antibodies (several weeks in humans) after their infusion should improve their therapeutic indices and the resultant therapeutic and diagnostic efficacy.
The MHC-1, like FcRn, binds IgG and albumin at distinct sites and protects both proteins from catabolism (30
). However, this protective capacity of FcRn is saturable. It has been shown that the plasma half-life of IgG is inversely proportional to its serum concentration and that the plasma half-life of radiolabeled γ globulin is shorter in patients with monoclonal gammopathy (31
). Because it is FDA approved and large amounts are readily available, we used human polyclonal IgG to pharmacologically inhibit the murine FcRn, which is promiscuous and can bind human IgG with high affinity (33
Biodistribution studies in mice revealed enhanced blood clearance and a higher liver uptake of the radiolabeled antibody following high-dose IgG therapy. An enhancement in liver uptake was expected because it was previously reported that FcRn is expressed in hepatic endothelium and the hepatocytes (5
) and that blockade of FcRn should result in degradation of the radiolabeled IgG and radiometal accumulation in these organs. Despite an estimated doubling of the radiation dose to the liver, no significant hepatic pathology was observed in IgG-treated animals. A possible explanation for this observation is the relative radioresistance of the liver (35
). Our previous data in mice and nonhuman primates, in which high doses of 225
Ac-HuM195 resulted in severe renal toxicity but no hepatic toxicity, are consistent with this finding (29
The biodistribution and whole-body clearance data in mice indicate that high-dose IgG therapy markedly increased the blood clearance of the radiometal-labeled antibody, which was followed by gradual elimination of the radioactivity from the liver and thus enhanced whole-body clearance. For whole-body clearance and postmortem biodistribution studies, radiometals (111In or 225Ac) were used to label antibodies instead of radioiodine because iodine can be rapidly released from tissues following endocytosis and catabolism of radioiodinated antibodies and may therefore lead to an overestimation of the normal-organ and whole-body clearance. For the same reason, although enhancement in liver uptake of radioactivity was seen following high-dose IgG administration in biodistribution experiments, no liver uptake was seen in PET images, which used 124I as a radiolabel. For PET studies, in which enhanced tumor-to-blood contrast is desirable, 124I provides a quantitative measure of the blood clearance and tumor targeting. We showed the versatility of this approach in altering the pharmacokinetics of IgG antibodies by testing 3 different radiolabeled antibodies, each of which has been used in patients with respective tumor types. 225Ac-HuM195, an antibody construct currently in human clinical trials for patients with advanced myeloid malignancies, does not have a suitable mouse tumor model. Therefore, biodistribution and imaging studies in tumor-bearing mice were performed with radiolabeled cG250 or A33 antibodies because of the availability of reliable mouse tumor xenograft models.
The plasma clearance of 124
I-A33 in humans, although resulting in favorable image contrast, was not as pronounced as that seen in mice that received the same dose (1 g/kg) of polyclonal human IgG. The blunted effect in humans may be explained not only by biologic differences, but also by the 8-fold higher affinity of human IgG for mouse FcRn than for human FcRn (37
). Therefore, higher doses of IgG may be required in humans to achieve the effects seen in mice. Petkova, Roopenian, and colleagues have addressed this issue by generating mice that express human FcRn (38
), which should serve as a good surrogate for preclinical evaluation of diagnostic and therapeutic human IgG antibodies. The pharmacokinetic behavior of human IgG is also expected to differ in mouse and human systems. Moreover, the routes of administration of high-dose IgG were also different in mouse and human studies (i.p. and i.v., respectively). Plasma IgG concentrations of approximately 10 and 35 mg/ml in mice and humans, respectively, should suffice to functionally inhibit the ability of FcRn to protect IgG from catabolism (26
). Based on basal plasma IgG concentration and its volume of distribution, 2 daily 1 g/kg doses (the recommended therapeutic dose for autoimmune disorders) should result in a peak plasma IgG concentration of 40 mg/ml (39
) and may be sufficient to saturate the FcRn in humans.
Previous studies have tried to mutate key amino acid residues in the IgG molecule that are critical for FcRn binding in order to generate antibodies with altered binding to FcRn (18
). Although enhanced blood clearance of fragments with low affinity for FcRn was seen, their tumor uptake was also compromised (18
). An important advantage of the high-dose IgG therapy described here, besides obviating the need for engineering each antibody, is that it allows for timing of the IgG administration to clear excess circulating antibody after tumor targeting has occurred. This is crucial, because the time required for optimal tumor targeting varies with each antigen-antibody-tumor system. For example, optimal tumor targeting in mice with A33 and cG250 was seen at 4 and 24 hours after injection, respectively, and therefore the IgG was administered to mice after 6 or 24 hours, respectively.
Another strategy, developed by Vaccaro et al., uses administration of engineered IgG antibodies with high affinity and reduced pH-dependent binding to FcRn that are not readily released at the cell surface during exocytosis (40
). As a consequence, low doses of engineered antibodies were required relative to wild-type polyclonal IgG to enhance blood clearance of endogenous antibodies. A relatively longer duration of action of this therapeutic via its inability to dissociate at the cell surface may be useful in autoimmune disorders, in which sustained degradation of endogenous pathogenic autoantibodies is desirable. However, for imaging or therapy with conjugated antibodies, in which a transient FcRn blockade to sufficiently accelerate the blood clearance of injected diagnostic or therapeutic antibody is desirable, an optimally timed high-dose IgG therapy is preferable.
In summary, we have shown that it is possible to effectively control the blood half-lives and therefore the therapeutic index of targeted IgG antibodies via pharmacological modulation of their interaction with the protective FcRn. The described approach resulted in enhanced tumor contrast and reduction of normal-tissue toxicity for tumor imaging and therapy.