In this study, we assessed dose-dependent and dose-independent uptake of the 89Zr-labelled anti-CD44 antibody RG7356 in normal tissues to identify specific, target-mediated uptake on immuno-PET in a dose escalation phase 1 study.
Both dose-dependent and dose-independent uptake were observed, reflecting specific as well as non-specific uptake of RG7356. For tissues without antigen expression, a linear increase in antibody concentrations can be expected for increasing antibody doses, driven by perfusion and blood volume of the tissue. However, our results suggest a mechanism that extracts antibody from the blood pool to tissues, in addition to the non-specific uptake mechanisms (Fig. 3c). Therefore, tissue-to-blood AUC ratios were used to evaluate dose-dependent uptake of RG7356 for the following tissues: liver, spleen, bone marrow, kidney, lung and brain. For the brain, a constant low tissue-to-blood AUC ratio was observed for all dose cohorts. Assuming that RG7356 does not cross the blood–brain barrier, this value is determined by the blood volume fraction of the brain. For the spleen, liver, bone marrow, kidney and lung, dose-dependent uptake of 89Zr-RG7356 was observed, indicating target antigen-mediated specific uptake in these tissues. A very similar pattern of dose-dependent uptake in the spleen, liver and bone marrow has been reported previously in the preclinical study with 89Zr-RG7356 in cynomolgus monkeys, indicating that such preclinical immuno-PET studies can be predictive with respect to normal tissue uptake in human [9].
Target antigen expression in these tissues is a plausible explanation for dose-dependent uptake, as protein expression of CD44 has been reported for normal bone marrow, spleen, lung, kidney and liver (bile ducts) [16, 17]. Although dose-dependent uptake in tissues was observed, a constant tissue-to-blood AUC ratio was reached at 450 mg for all tissues, indicating target antigen saturation.
In addition, dose-independent uptake of the tracer in the liver, spleen, bone marrow, kidney and lung was observed, indicating non-specific uptake. For the liver, based on a 30% blood volume fraction [18], a liver-to-blood AUC ratio of 0.3 would be expected. However, we observed a liver-to-blood AUC ratio of 0.85 ± 0.08 for the 675 mg dose cohort. The difference between the tissue-to-blood AUC ratio and blood volume fraction represents an additional accumulation mechanism in the liver, for example, the large endothelial fenestrae or antibody catabolism. Stability of 89Zr-labelled antibodies, with minimal release of 89Zr, has been demonstrated in many in vitro and in vivo preclinical as well as clinical studies [11, 19, 20]. There are no experimental data supporting accumulation of free 89Zr in normal tissues, except for the observation that free 89Zr, arising after internalisation and intracellular catabolism of the conjugate, may accumulate in bone tissue (not bone marrow) [19]. However, in our study, we did not observe 89Zr accumulation in the bone (Fig. 2).
Although dose-dependent, as well as dose-independent, uptake in normal tissues was found in this imaging study, there were no safety concerns in the corresponding phase I dose escalation study, with treatment doses up to 1500 mg biweekly/2250 mg weekly. The overall safety profile of RG7356 was acceptable. Dose-limiting toxicities included febrile neutropenia and aseptic meningitis [10]. However, this phase I study was terminated at an early stage due to the lack of evidence of a clinical and/or pharmacodynamic (PD) dose–response relationship with RG7356.
We observed tumour uptake in all patients receiving ≥ 450 mg, with an extremely high tumour blood volume fraction estimated for the 675 mg cohort. Although this might suggest target-mediated specific tumour uptake, another study design would have been more informative on this point [21]. This requires measurement of the same tumour lesion after administration of different antibody doses to exclude differences in tumour characteristics, for example, blood volume fraction. Learning from the present study, we recently demonstrated target-mediated specific tumour uptake in a PET imaging study with an anti-HER3 mAb in which the 89Zr-labelled antibody was administered twice (with a variable dose of unlabelled antibody) to a single patient [21]. Although biopsies taken after the immuno-PET could have provided additional confirmation with immunohistochemistry, this was not included in the study design due to the fragile patient population.
No focal tumour uptake was visualized in the lowest dose cohorts (1–200 mg). This observation cannot be explained by the level of CD44 expression or the percentage of CD44-positive tumour cells (Table 1). A probable explanation why tumour visualization is hampered for the lowest dose cohorts is that dose-dependent uptake in normal tissues leads to lower visual tumour contrast, assuming similar binding constants and accessibilities of the target antigen in both normal tissues and tumour. However, in the higher dose cohorts, differences between binding constant became apparent where the dose-dependent tracer uptake in normal tissues does not significantly contribute to the imaging signal anymore (Fig. 1); target antigen-mediated tracer uptake will result in sufficient visual contrast to allow identification and quantification of tumour targeting.
In this study, PET imaging with the novel anti-CD44 monoclonal antibody RG7356 confirmed tumour uptake for patients receiving ≥ 450 mg. However, dose-dependent uptake of RG7356 in normal tissues indicates target antigen expression, limiting the use of RG7356 for targeting toxic payloads to the tumour like in antibody–drug conjugate (ADC) approaches.
This exploratory imaging study demonstrates how immuno-PET with a 89Zr-labelled mAb can be used as a general method during phase I dose escalation studies to evaluate the therapeutic potential of an antibody. Evaluation of dose-dependent and dose-independent normal tissue uptake with immuno-PET reflects specific and non-specific uptake. Antibody quantification obtained by molecular imaging provides an additional, non-invasive method to study in vivo mAb biodistribution, besides traditional PK obtained by blood sampling. Especially for a candidate mAb with a potential future as ADC, the resulting information (prevention of potential toxicity/additional development costs) may justify the patient burden and cost for additional scans in a limited number of patients in a phase I setting.
For a mAb which continues in further stages of drug development, in vivo measurements of antibody concentrations in tissue and tumour can be of value for PK/PD modelling/dose optimization and response prediction to guide individualized treatment.