In our studies, the CiTE antibody demonstrated a high efficiency to initiate T cell immune responses. It was able to activate T cells polyclonally and thus irrespective of their MHC:antigen specificity, as measured by the general upregulation of CD69 and CD25. This implies that a much larger effector T cell pool can be addressed than in a physiological immune response, where only T cells carrying the specific TCR are engaged.76,277 Since in AML after chemotherapy or HSCT T cell numbers are often reduced and low lymphocyte counts are associated with a poor prognosis, polyclonal activation and proliferation of remaining T cells might be particularly beneficial.278-280 Furthermore, CiTE-mediated T cell activation results in the release of IFN-γ and IL-2, thereby promoting a proinflammatory microenvironment and potentially leading to the attraction of other
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immune cells to the tumor site. In vitro, the CiTE was able to specifically induce cytotoxic lysis of AML cells at very low concentrations with calculated EC50 values between 2.3 pM and 132.5 pM. This is comparable to published values for BiTE® antibodies.70,75,76 Taking blinatumomab as reference, in a clinical application this might translate into effective therapeutic doses of around 15 µg/m2/day, which is significantly lower than conventional mABs.63,281,282 Notably, there is a considerable difference between EC50 values, which were determined to be in the picomolar range, and the calculated dissociation constants of the separate tumor binding scFvs in the nanomolar range. This highlights the high biologic activity of the CiTE and suggests that a few molecules at the tumor site are already sufficient to induce T cell effector functions. In contrast, as example for conventional IgG antibodies rituximab was described to exhibit a 100,000-fold higher EC50 value than the corresponding CD19xCD3 BiTE®, indicating that Fc-induced tumor cell depletion through NK cells and macrophages is less potent than cytotoxic lysis by T cells.70 An additional component that enhances CiTE-mediated T cell cytotoxicity is its ability to induce serial lysis of cancer cells, as demonstrated in assays with patient-derived AML cells. Similar to other T cell engagers, this is attributed to the low binding affinity of the CD3ε binding module compared to the tumor-targeting arm, allowing T cell migration between tumor cells.69,86
It is important to mention that the CiTE antibody is able to induce cytotoxic lysis of target cells with varying target antigen densities. For the BiTE® AMG 330 it has been described that the kinetics of induced lysis correlate with CD33 surface levels, however, elimination of different AML cell lines with different CD33 antigen densities was still achieved.75,221 Similar results were obtained for the CiTE antibody. This is especially relevant since individuals reveal a large inter- and intra-patient heterogeneity regarding their CD33 expression.79 In addition, the supposed source of relapse in many patients is a population of chemoresistant LSCs that expresses CD33 but at lower levels.79 By depleting AML cells irrespective of absolute CD33 density, the CiTE might provide a promising strategy to eliminate remaining LSCs and thus prevent reoccurring disease outgrowth.79,213 First evidence for the efficient eradication of MRD was provided by the in vivo
xenograft experiments that were performed as part of this study. By injecting MOLM-13:PD-L1 cells and obtaining 1-3% engraftment in the bone marrow, we were able to provide a model that resembled an MRD-positive state with ˂5% myeloblasts in the bone marrow.265 As the CiTE antibody was able to completely eradicate AML cells in our setting, we propose that it might also be a highly promising therapeutic to eliminate MRD cells in humans. However, future studies will have to elucidate safety and efficacy in preclinical models before transferring the CiTE format into
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the clinics. From the current perspective similar patient groups that are addressed with GO might benefit from a CiTE therapy.232 These include adult patients at relapse or elderly patients that cannot be treated with conventional therapies in particular, as GO monotherapy turned out to be advantageous in a randomized trial (EORTC-GIMEMA AML-19) with patients aged 62 years or older.232,283 Further, GO was efficient in newly diagnosed patients in combination with chemotherapy (e.g. studies ALFA-0701, MRC AML-15), wherefore accordingly a combination of CiTE and chemotherapeutic agents appears reasonable.231,232,284 However, due to the large heterogeneity within the disease we do not expect all patients to respond equally to CiTE administration.79 This was already suggested by our ex vivo studies on primary AML patient samples, in which variable efficiency of AML depletion and differential upregulation of PD-1 on T cells as well as PD-L1 on AML cells were observed. Moreover, the release of proinflammatory cytokines by healthy donor T cells in response to CiTE-mediated activation indicated differences. The implementation of biomarker screenings might help to identify patients that benefit from CiTE therapy. These could include the quantification of CD33 expression and the determination of the responsiveness to PD-1/PD-L1 blockade, for instance by assessing the transcriptional IPRES signature.154,155