Targeted therapy is a novel approach in the treatment and control of cancers, and has the potential to enhance the therapeutic efficacy, minimize undesirable side effects and help achieve a better clinical outcome in patients. With respect to B cell malignancies, others and we have demonstrated uniform and restricted expression of ROR1 in CLL and MCL cells, and suggested its potential as a therapeutic target.9-11,36
The present study describes the development, characterization and validation of four mouse mAbs against hROR1. Subsequently, we focused on one of the two mAbs (2A2-IgG) that selectively bound ROR1-positive primary CLL and MCL cells as well as MCL cell lines and revealed the overall strongest reactivity with hROR1 and mROR1. Nonetheless, 2A2-IgG alone or after conversion to chimeric mouse/human IgG1 (data not shown) and three chimeric rabbit/human IgG1 against ROR130
failed to induce significant direct cytotoxicity, CDC or ADCC in primary CLL cells or ROR1-positive MCL cell lines. Such unarmed mAb-mediated effector functions have generally been demonstrated in cells with high target antigen density (e.g., CDC activity of rituximab on human B cells with > 40,000 CD20 molecules/cell).37
Our previous study estimated the ROR1 density on CLL cells to be between 2,500 and 7,000 molecules/cell,9
which may be below the threshold needed for unarmed mAbs to mediate effector functions.38,39
It is also possible that the effector functions of unarmed mAbs against ROR1 are influenced by epitope location.40
Knowing that cell surface ROR1 mediated internalization of bound antibody in primary CLL cells and MCL cell lines, the use of a ROR1-immunotoxin was sought to potentially overcome this limitation. The induction of profound apoptosis by mAb 2A2-derived ROR1-immunotoxin BT-1 in dsFv-PE38 format in ROR1-positive MCL cell lines, but not in ROR1-negative B cell lines validated that ROR1 can function as a therapeutic target. The specific cytotoxicity induced by BT-1 was confirmed by three independent measurements: PS exposure to cell surface, change in mitochondrial membrane potential and activation of caspase-3, events that have been well documented in apoptotic cell death. The maximum effect was seen after approximately 3 d and this is in agreement with other studies using PE38-based immunotoxins.16
The dose-response curves depicted differences in BT-1 sensitivity among different MCL cell lines, and this could reflect cell specific intrinsic factors as regulators of apoptosis.
A number of parameters determine the efficacy of immunotoxins. First, the affinity of a mAb or a mAb fragment (e.g., dsFv) to its target antigen largely determines the stability of the antibody/antigen complex on the cell surface. A head-to-head comparison of the observed KD
values of bivalent 2A2-IgG (0.42 nM) and 2A2-derived monovalent BT-1 (64.9 nM) binding to immobilized Fc-ROR1 revealed an approximately 150-fold difference in BT-1 binding. Assuming that similar binding might occur with cell surface ROR1 and given that the half-life of the antigen/antibody interaction equals ln2/koff
, the estimated cell surface retention of BT-1 would be 19 sec compared with 34 min for 2A2-IgG. Although the dose-response curves with BT-1 showed robust killing of ROR1-positive MCL lines at low concentration (with EC50
values of 16 pM to 16 nM), there is likely room for improvement. Other PE38-based immunotoxins that target different antigens on malignant B cells (CD19 and CD22) or mesothelioma (mesothelin) showed higher potency presumably due to higher affinity, i.e., lower dissociation rate constants, for the respective antigens, resulting in longer cell surface retention.41
Currently, we are looking into different antibody engineering strategies to improve the affinity or avidity of BT-1. Alternatively, a new panel of chimeric rabbit/human mAbs to ROR1 that was selected by phage display in the monovalent Fab format for high affinity binding to ROR1 and reveals dissociation rate constants up to 120-fold lower than BT-1,30
could be employed for the next generation of ROR1-immunotoxins.
Second, the target antigen density on the cell surface could influence immunotoxin efficacy. Our data showed an approximately 10-fold difference in EC50
values among ROR1-positive MCL cell lines (0.016 nM for Rec-1, 0.16 nM for HBL-2, 16 nM for JeKo-1 and 16 nM for Mino) and overall this paralleled ROR1 expression levels among these cell lines. A correlation between target antigen density and apoptosis induction was also reported for CD22- and FCRL1-immunotoxins.16,42
However, preliminary results indicate that Rec-1 cells expressed the lowest level of the anti-apoptotic protein BCL2 compared with the other MCL cell lines we tested. It is conceivable that BCL2 levels in addition to the level of other anti-apoptotic proteins (e.g., MCL1, survivin, X1AP) can influence the efficacy of BT-1 as has been reported for other immunotoxins.43,44
Third, our epitope mapping studies indicated that 2A2-IgG (the parent antibody of BT-1) bound to the membrane distal Ig-domain of ROR1. Immunotoxins generated from antibodies recognizing different epitopes on the same antigen showed significant differences in their potency in vitro.45,46
An immunotoxin binding to a membrane proximal epitope on the target antigen, CD2, induced higher cytotoxicity than those binding to distal epitopes.47
Therefore, it may be worthwhile to generate ROR1-immunotoxins based on mAbs recognizing different epitopes and compare their abilities to kill ROR1-expressing cells.
Fourth, the rate of internalization of surface bound immunotoxin could affect its efficacy. A recent study compared the efficacy of two immunotoxins, RFB4 (Fv)-PE38 also known as BL22 (anti-CD22) and FMC63 (Fv)-PE38 (anti-CD19) on lymphoma cell lines. Despite lower cell surface expression of CD22, rapid internalization of large amounts of BL22 made CD22 a better target than CD19 present in high density.48
There was clear evidence for BT-1-mediated apoptosis in ROR1-positive cell lines within 24 h, but how much BT-1 was actually internalized is unknown. Further studies on the membrane dynamics of ROR1, intracellular trafficking of BT-1 and its degradation rate are required to better understand and improve the efficacy of next generation ROR1-immunotoxins.
Despite the profound cytotoxicity induced by BT-1 in vitro, the surface plasmon resonance data indicated relatively fast dissociation of the monovalent BT-1 compared with its parent antibody, and it is likely that this could be a limitation in exploring its in vivo activity. We are currently working on generating different formats of ROR1 immunotoxins with a goal to enhance the affinity/avidity, and minimize potential immunogenicity. The efficacy of improved immunotoxins can be tested in vivo using MCL cell lines as xenografts in nude mice. The cytotoxicity studies described here were based on MCL cell lines and, unlike primary CLL and MCL cells, they do not undergo spontaneous apoptosis in vitro. A more clinically relevant system for evaluating the ability of ROR1-immunotoxins to kill primary CLL and MCL cells may be a recently developed mouse model with adoptively transferred human primary CLL cells that retain ROR1 expression in vivo.49
In summary, as a proof of concept, we have demonstrated that a ROR1-immunotoxin can induce apoptosis resulting in robust killing of ROR1-expressing MCL cell lines in vitro. Owing to its exquisite specificity, BT-1 could function as a leukemia/lymphoma specific therapeutic agent in vivo, and unlike unarmed mAbs, would not depend on the effector machinery such as complement proteins, NK cells, macrophages or T cells as these may not be optimally available in cancer patients. BT-1 provides a platform for next generation ROR1-immunotoxins with defined modifications in both antibody and toxin portion designed to enhance affinity and efficacy, as well as minimize immunogenicity.50,51
In addition, combination strategies with small molecule inhibitors of anti-apoptotic proteins could help to mitigate the opposing effects and boost the efficacy of BT-1 and next generation ROR1-immunotoxins.