This study describes a method for the analysis and quantification of ADCC killing by trastuzumab using an xCELLigence instrument and a PanToxiLux assay. Similar results were observed using the two different methods to measure ADCC activity. These data suggest that granzyme B and upstream caspases are involved in the killing of the tumor cells. The advantage of the xCELLigence system is it measures the rate of killing, is mostly reproducible, and can be standardized with the main disadvantage residing in the equipment cost. The advantage of the PanToxiLux assay is it is less expensive if not purchasing a flow cytometer, it can be done rapidly, and it measures specific enzyme activity. In addition, frozen down labeled target cells can also be used for this assay (data not included). The disadvantage is that not all effector cells will use these enzymes as a mechanism of action and the standardization may be difficult.
Four breast cancer cell lines were used in the studies to analyze ADCC killing with multiple MNC preparations and a control U937 cell line. Our data show that a low, physiologic dose of (0.1ug/ml) of trastuzumab has little, if any, effect on CI values for any of the cell lines when compared with the untreated target cells. Even when the trastuzumab was increased to 20 ug/ml, there was no change in the CI values over time for BT474 clone 5 and SKBR3v cells. It was previously demonstrated that BT474 clone 5 is resistant to trastuzumab based on in vitro studies but still overexpresses HER2 and is still sensitive to trastuzumab under in vivo conditions.17,18
These cells were used as targets to minimize the intracellular cell signaling component of trastuzumab killing.18
It should be noted that the parental BT474 cell line is sensitive to killing by trastuzumab as measured by cell counting, flow cytometry, animal studies and xCELLigence profiles.16-18
The SKBR3 cells used in this study have been defined as a variant since these cells no longer overexpress HER2 at similar levels to BT474. Several investigators have measured SKBR3 for HER2 expression levels and found them to be similar to BT474.The cell line described here, SKBR3v, expresses only 20% of the HER2 levels observed in BT474 cells based on flow cytometry measurements. These cell lines remained stable based on flow cytometry expression of HER2 and therefore were considered good targets for representing high and low expression of HER2.
In this study, the ADCC activity is dependent on the E:T ratios (), is reproducible over multiple samples from the same individual against two targets and does not display changes over time. In order to be clinically meaningful, there needs to be significant variation for the ADCC activity among individuals which indeed was observed. Furthermore, it is clear that the expression of HER2 affects the ability of the MNCs to kill in the presence of trastuzumab. The range and median or mean values of MNCs for ADCC activity overlap between normal individuals and breast cancer patients who are HER2 positive and will be treated with trastuzumab. This range is very broad and we hope our clinical study comparing trastuzumab response to ADCC activity will be correlated in our future studies. Our attempts to use cryopreserved MNCs indicated that the ADCC activity is greatly diminished compared with the fresh MNCs. The loss of ADCC activity following cryopreservation correlated with the loss of NK cells during the cryopreservation process, which seems to be a likely reason for decrease in ADCC activity.
Since this a biological assay, it is important to have a reproducible positive control for ADCC killing. Our use of U937 cells provides a reproducible effector cell control that acts differently in the two targets. In the BT474 clone 5, the addition of U937 cells alone has minimal effect, but when trastuzumab is added, there is a significant decrease in cell index. In SKBR3v, one observes a relative decrease in cell index when U937 cells are added alone but there is little further decrease in the presence of trastuzumab. It also should be noted that this cell line can be frozen and thawed immediately prior to use with similar results.
These studies suggest that the major immune cell subtype contributing to the ADCC effect are NK cells. This is based on measuring ADCC activity following MNC fractionation with Miltenyi immunoaffinity beads and by image analysis. This is in agreement with other investigators24
that have demonstrated that the Fcγ receptor is critical for the trastuzumab to interact with the effector cell and bring the effector cell into a “kiss of death” relationship with the target. The Costa group initiated a neoadjuvant study on women with primary breast cancer being treated with trastuzumab given 4 weeks prior to surgery.11
Comparison of responding vs. non-responding patients, indicated no change in serum or tissue levels of trastuzumab, no change in HER2 expression in the tumors, no change in blood vessel diameter, and no change in proliferation as measured by Ki-67 staining. They did demonstrate a relationship of response to an increase in infiltrating leukocytes and a higher ADCC activity. Varchetta et al. have further shown that the ADCC activity for trastuzumab was related to number of NK cells present.25
Additional studies are needed to compare the relationship of trastuzumab response in breast cancer patients with the assay described in this study. It would be of great value to determine if patients with low ADCC activity (lower tertile) do not respond as well to trastuzumab as those patients with a high degree of ADCC activity (upper tertile) based on this assay. The patients with low activity might then be candidates for other treatment such as lapatinib, a tyrosine kinase inhibitor, or T-DM1, a trastuzumab convalantly bound to maytansine which is a microtubule- disrupting drug. These compounds target the HER2 positive cells but are not thought to use the immune system to kill tumor cells. Another question of importance is whether this ADCC activity is affected by chemotherapy or other treatments (surgery, etc) the patient is undergoing. If the patient displays depressed ADCC due to chemotherapy, then it may be clinically beneficial to wait until the patient’s activity is high before treating with trastuzumab. Although these studies were limited to trastuzumab only, similar studies could be performed with any of the other monoclonal antibody treatments since any monoclonal antibody therapy has a high likelihood of displaying a similar mechanism of action.