The quantitative nature of PET facilitates the accurate measurement of tracer concentration within a lesion and such measurements correlate well with those obtained through standard biodistribution studies (19
). Monoclonal antibodies, combined with flow cytometry, have long been used to quantitatively measure the expression of cell surface proteins. This has led us, and others, to hypothesize that antibody-based radiotracers, coupled with PET, can be used to measure antigen expression in vivo
. In addition, significant data in the literature suggests that smaller antibody fragments, affibodies, or engineered antibody fragments are poised to be more effective than intact mAbs as PET radiotracers due to their faster blood clearance and higher tumor:background ratios (32
At the most basic level, response to mAb-based therapies requires that the target protein be expressed on the surface of tumor cells, and that the therapeutic mAb effectively target and accumulate to sufficient levels within the tumor. The ability to monitor each of these variables has the potential to guide patient selection and treatment plans. In the setting of HER2-positive breast cancer, response to trastuzumab positively correlates with the level of HER2 expression. High-level overexpression in biopsied tumor tissue, as measured by IHC or FISH, is the critical criteria for treatment eligibility. Thus we and others (34
) have speculated that a molecular imaging based approach to evaluate HER2 expression across a patient's entire tumor burden could provide a more complete analysis of HER2 expression, potentially providing a better prediction of initial response to trastuzumab-based therapy or even obviating the need for invasive biopsy procedures. The data we present here demonstrates that tumor uptake of the C6.5db is dependent upon antigen density on the surface of the tumor cells and suggest that C6.5db-based radiotracers may be useful for evaluating the levels of HER2 expression on tumor cells in vivo
, and by extension predicting initial response to trastuzumab therapy. Data from Cai et al (35
) suggests that this approach may be applicable to target antigens beyond HER2. Uptake of [64
Cu-DOTA]-cetuximab correlated with the level of EGFR expression across a number of tumor models, suggesting that immunoPET-based determination of antigen density could be applied to a broader range of target antigens.
Biological properties of the target antigen, the strategy employed to radiolabel the tracer, and the intended imaging application are all critical components in radiotracer design. ImmunoPET images obtained with a residualizing radionuclide, such as the 64
Cu or 89
Zr used to label cetuximab by Cai (35
) and Aerts (36
), depict the cumulative antibody bound to, and internalized by the cells over the course of the experiment. This is ideal for the purpose of lesion detection but potentially suboptimal for monitoring antigen levels. When targeting rapidly internalizing antigens, such as EGFR or HER2, the long half-life of intact mAbs coupled with residualizing radionuclides would be predicted to obscure internalization rates, and thereby provide an inaccurate estimate of level of antigen expression. The radiohalogen 124
I has a physical half-life that pairs appropriately with the biological half-lives of mAb-based tracers (33
). However, it has been speculated that 124
I is inappropriate for labeling of mAbs because internalization and degradation leads to rapid loss of the iodine from cells resulting in both insufficient tumor:normal tissue contrast for imaging (37
) and unwanted uptake by tissues, such as the thyroid, that express the Na/I symporter. Engineered antibodies such as the C6.5db have the potential to function as effective radiotracers, in part, because their rapid systemic clearance leads to positive tumor-to-blood ratios early after administration (19
). Our results demonstrate that sufficient uptake of 124
I-C6.5db is achieved to afford PET detection of tumors as small as 50 mg at 48 h p.i. in our preclinical model. In addition, we argue that 124
I-C6.5db, and by extension other 124
I-mAbs, provide a representation of the antibody bound to the tumor cell surface at the time of imaging, thus decreasing the impact of internalization rates on tumor signal and potentially providing a more quantitative approach to measuring either inherent differences in antigen expression between tumors or changes in antigen expression within a tumor in response to therapy. However, the positive impact of the rapid clearance of C6.5db is balanced by its negative effect on the limiting time available for the antibody to accumulate to high levels in the tumor (38
). This rapid clearance could in principle be exploited to enable the use of residualizing isotopes with similar quantitative results. Although thyroid uptake was not quantified in this study we have demonstrated in previous work that the use of 124
I in conjunction with a partially residualizing labeling strategy (e.g. SHPP) does not dramatically alter the performance of the C6.5db as a radiotracer and decreases thyroid uptake (19
). This suggests that such a labeling strategy could be used in conjunction with thyroid blocking to reduce thyroid exposure in patients and still provide quantitative analysis of HER2 levels.
Accumulation of antibody-based therapeutics to sufficient levels within a tumor is essential for therapeutic efficacy. The decrease in overall tumor uptake seen with the C6.5db upon trastuzumab treatment implies that trastuzumab is effectively targeting tumor in our preclinical models to induce this effect. It is intriguing to speculate that molecular imaging with agents such as the C6.5db, when used in the clinical setting, could potentially shed light on whether and how trastuzumab is targeting lesions in a patient. In addition, significant effort is ongoing in the preclinical setting to understand both how physical properties of mAbs (e.g. intrinsic affinity, molecular size, PK) dictate tumor targeting and how those properties can be modified to improve antibody targeting (for review see (39
). Imaging strategies that can function as companion diagnostics during the development process have the potential to aid in translation of new therapeutic antibodies during translation into the clinic.
Antibody-based cancer therapeutics can induce anti-tumor effects through a number of mechanisms of action including inhibiting signal transduction and/or focusing the anti-tumor effects of the immune system (40
). Since its initial approval more than a decade ago trastuzumab has become standard-of-care for HER2-postive breast cancer. Despite this fact, trastuzumab's mechanism of action has yet to be definitively identified. It most likely functions through multiple processes including antibody-dependent cellular cytotoxicity (ADCC), inhibiting HER2 shedding, and blocking signaling (for review see (41
)). Although somewhat controversial (42
) and its relevance to the clinical setting not yet fully demonstrated (43
), trastuzumab-induced down-regulation of HER2 has also been reported in both in vitro
cell culture (46
) and xenograft models (48
). Our IHC and FACS results are in alignment with clinical findings, in that trastuzumab treatment failed to induce detectable levels of HER2 down-regulation in our model systems. Consistent with our findings, McLarty et al report that trastuzumab treatment (4 mg/kg) of athymic mice bearing MDA-MB361 xenografts followed by SPECT imaging (3 d p.i. of trastuzumab) with 111
In-diethylenetriaminepenta-acetic acid-pertuzumab (111
In-DTPA-pertuzumab) showed a significant decrease in the tumor uptake of 111
In-DTPA-pertuzumab, despite no apparent decrease in HER2 levels by IHC (32
). Interestingly, chronic treatment (3 weeks) induced a significant decrease in HER2 levels by IHC, and was associated with loss of HER2-positive tumor cells. In our studies, chronic treatment of mice bearing SK-OV-3 or BT-474 xenografts failed to induce an obvious change in HER2 expression (data not shown), similar to the situation seen in the clinic (43
). It is worthy of note in this context that when McLarty et al compared trastuzumab-induced changes in HER2 density between SKBR-3 (high HER2 expression) and MDA-MB361 (moderate HER2 expression) BrCa cells in culture, they found that the effects of trastuzumab on HER2 density was more profound in MDA-MB361 cells than SKBR-3 cells (32
). One possible explanation for the apparent differences in HER2 down-regulation seen in these studies may be cell line-dependent variability in receptor down-regulation.
Despite an inability to detect down-regulation of HER2 by FACS and IHC, our results are consistent with those of other groups (42
) and suggest that in vivo
targeting of 124
I-C6.5db is perturbed as an early response to trastuzumab-based therapy. This is particularly true in the context of the results obtained with 111
In-DTPA-pertuzumab and our data demonstrating that the C6.5db binds to HER2 near the epitope bound by pertuzumab. The mechanism by which trastuzumab treatment inhibits the targeting of both pertuzumab and C6.5db-based PET radiotracers is unclear. The epitopes for C6.5db and pertuzumab are located in domain II of the HER2 extracellular domain, distinct from the domain IV epitope bound by trastuzumab (51
). This, coupled with the inability of trastuzumab to compete with C6.5db for HER2 binding, suggested that the therapeutic levels of trastuzumab circulating in the animals, or by extension patients, should not compete for HER2 binding and therefore should not have resulted in the decreased in vivo
targeting, nor the time-dependent decrease in binding to cells treated with trastuzumab in culture. In light of trastuzumab's complicated mechanism of action, it is interesting to speculate that trastuzumab treatment results in a physical change to the receptor, such as altered clustering or dimerization patterns. Data from Kani et al (52
) demonstrating that binding of antibodies to HER2 alters its partitioning in the membrane, particularly with regards to localization with HER3, coupled with recent data from Junttila et al (53
) demonstrating that trastuzumab inhibits ligand-independent signaling through the HER2/HER3 heterodimer supports this hypothesis. The manner in which this would result in decreased binding of C6.5db is not yet clear. Altered packing of the receptor may result in steric inhibition of C6.5db binding to domain II. Alternatively, binding of trastuzumab to HER2 may prevent cross-linking of two HER2 molecules by C6.5db, forcing monovalent association of the radiotracer. Monovalent binding of the C6.5db is predicted to lower its functional affinity 40-fold, decrease its cell surface residence from 5 hr to 5 min, and significantly lower tumor uptake in vivo
). Efforts to more fully address the basis for the decreased uptake and determine whether this decreased binding can function as a measure of therapeutic response are underway.
Beyond treatment with trastuzumab, agents such as the hsp-90 inhibitors 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-demethoxygeldanamycin (17-DMAG) have been reported to induce rapid and transient degradation of HER2 as part of their proposed mechanisms of action and this down-regulation has been imaged with trastuzumab-based radiotracers (20
). The ability to monitor the efficacy of this type of agent in a robust manner, with dedicated radiotracers such as the C6.5db, has the potential to improve the development and clinical outcome associated with its use. Smith–Jones (20
) demonstrated that 68
can detect a 50% decrease in HER2 expression in BT-474 xenografts treated with Hsp-90 inhibitors and that the change in HER2 density can be detected before subsequent tumor inhibition is apparent by FDG-based imaging (57
). Interestingly, and consistent with our inability to observe down-regulation of HER2 upon trastuzumab treatment, IHC was unable to detect less than a 70% reduction in HER2 expression in BT-474 BrCa xenografts in athymic mice treated with the heat shock protein-90 (Hsp-90) inhibitor 17-demethoxygeldanamycin (17-DMAG) (55
). Thus, differences in trastuzumab-based HER2 down-regulation seen between preclinical and clinical studies may be due, at least in part, to the inability of IHC to sensitively detect those changes.
In conclusion, we hypothesize that molecular imaging with antibody-based radiotracers has the potential to make a positive impact in both guiding the development and use of targeted therapies that inhibit either the activity or expression of cell-surface proteins. The targeting properties of engineered antibody fragments, such as the C6.5db, are well suited for PET imaging and can provide specific information regarding the expression and modulation of targets in a non-invasive manner, regardless of their location. One important future goal is to test C6.5db in transgenic mouse models that express HER2 antigen on normal tissues and shed those antigens into the bloodstream, similar to the clinical setting (58
). The development of trastuzumab has revolutionized the treatment of both early and advanced stage HER2-positive BrCa, but acquired resistance to treatment is frequently encountered in advanced disease, and in a small proportion of early stage patients after adjuvant therapy. It is interesting to speculate that molecular imaging, as with the C6.5db or similar antibody-based agents, may serve as an effective method to monitor patients for initial response as well as for development of resistance to trastuzumab.
Statement of Translational Relevance
Strategies to both predict and monitor patient response are critical for the effective development and clinical implementation of targeted therapies. Molecular imaging strategies, such as positron emission tomography (PET) are well suited to this role. We have previously described an antibody-based PET radiotracer, C6.5 diabody (C6.5db), which selectively binds to HER2. Here we demonstrate that imaging with the C6.5db has the potential to quantify HER2 levels in vivo thus predicting response to trastuzumab therapy. We also provide data to suggest that C6.5db-based PET imaging may be an effective strategy for monitoring patient response to trastuzumab or other HER2-directed therapies.