In this study, we show for the first time that ADCC can be promoted by blocking inhibitory self-recognition receptors on human NK cells. This mechanism was shown in an appropriate autologous human system, providing physiologic “checkpoints”. This approach offers a new direction in the development and improvement of new and existing therapeutic Ab treatments of cancer. A broad assortment of NK cell inhibitory and activating receptors create important immune response checkpoints by surveying self-cells that express different ligands (39
). These checkpoints are exquisitely regulated in part by the defined inherited repertoire of inhibitory KIRs distributed on different NK cell subpopulations within an individual; this basis provides a mechanism by which NK cells sense changes in self-molecule expression or expression of stress-activating ligands while protecting healthy cells. Based on our results, disrupting interactions that lead to inhibitory self-recognition can efficiently improve ADCC for the elimination of Ab-coated malignant cells.
CD16 is the predominant Fc receptor on NK cells and has been shown to be involved in the capacity of NK cells to mediate ADCC (2
). In our cell line model, NK-92.26.5 cells were transduced with one of the two variants of CD16 (determined by the aa 158 polymorphism valine (V) or phenylalanine (F)) (). CD16.NK-92.26.5 effector cells were treated with DX9 to block KIR3DL1 inhibitory receptor and used to mediate ADCC against rituximab-coated 721.221-B*5101 (B51) target cells. Lysis of the target cells was improved when DX9 was used in combination with rituximab, compared with rituximab alone, indicating that our approach can improve ADCC ().
The results show that NK cells that do not express CD16 predictably do not mediate Ab-dependent cytotoxicity. However, CD16-expressing NK cells efficiently mediate ADCC against cells bearing relevant target Ags, CD20+
, using rituximab or trastuzumab, respectively (). Moreover, in determining how the impact of CD16 polymorphisms on the degree of response correlates with Fc binding affinity (1
), an enhanced cytotoxicity was seen with the V158 variant compared with F158 at any given Ab concentration ().
To validate our hypothesis that RISER will augment Ab-dependent NK cell-mediated cytotoxicity we combined the two complementary NK cell-activating mechanisms of CD16 engagement and blockade of inhibitory self-recognition (). The Ab isotypes of mouse origin that we used in our study show low affinity binding to human CD16 (64
). We initially used F(ab′)2
fragments of the DX9 Ab in our cytotoxicity experiments. This process was performed in anticipation of the potential problem of cross-linking two NK cells and a possible competition with the Ab that mediates ADCC. In our CD16.NK-92 cells system, an intact mouse IgG1 Ab (DX9) was used with no such effect, as described in Results
. In our model, the blockade of KIR3DL1 on NK-92.26.5 cells was sufficient to significantly activate lysis toward 721.221-B*5101 (B51) target cells (on which the KIR3DL1 cognate ligand HLA-B B*5101 is the only expressed class I MHC molecule). We found that although blocking of KIR3DL1-HLA-Bw4 interaction by the DX9 Ab or mediating ADCC by rituximab were separately effective in triggering NK cell cytotoxicity alone, the combination of these two approaches yielded the most efficient cytotoxicity level. Our results indicate that blocking KIR-HLA interactions could overcome the effects of CD16 Fc receptor low affinity in rituximab-mediated ADCC.
An individual NK cell population contains a diverse repertoire of NK cells, each expressing one of a varied spectrum of inhibitory receptors that recognize class I MHC self-molecules and thereby achieve tolerance. Therefore, it is predictable, that in a biologic setting, the blockade of any single receptor is unlikely to have a substantial overall impact on inhibitory self-recognition. Thus, broad targeting of inhibitory NK cell receptors might be required to effectively block NK cell recognition, allowing a nonrestricted enhanced antitumor response in patients. The NK-92.26.5 studies provided a useful starting point for obtaining proof-of-concept and elucidating key principles that must be fulfilled in order for inhibitory self-recognition to cooperate with CD16 signaling to efficiently promote ADCC. Nonetheless, an autologous setting using primary NK cells and controlled by realistic checkpoints was needed.
A unique donor gave us the opportunity to test how KIR blockade affected NK-mediated lysis in a human autologous in vitro system (). Ab blockage of KIR2DL1, which was expressed on virtually all NK cells from this subject, did not allow cytotoxicity of either 721.221 or autologous target cells that expressed HLA-C ligands for KIR2DL1. However, Ab blockage of KIR2DL1 did augment rituximab-mediated ADCC of these target cells. This property offers potential advantages for clinical translation because cytotoxicity will not be directed to healthy cells, but more specifically toward Ab-coated tumor cells. When we tested DX9 to block KIR3DL1 (expressed on 13% of the donor NK cells) in , neither basal NK activity nor ADCC were augmented (), suggesting that the inhibitory receptors on a more substantial proportion of the NK cell population must be blocked to effectively improve NK cell cytotoxicity.
More donors were characterized for varying expression patterns of inhibitory receptors and CD16 polymorphism status (), and these cells were tested for the impact of blocking inhibitory receptors on ADCC. Because the interference with self-recognition through a single inhibitory receptor had variable and generally modest effects on ADCC promotion, we applied a combination of NK cell-binding Abs to interfere with multiple inhibitory receptors. One challenge for this set of experiments was that most anti-KIR Abs bind to polymorphic extracellular domain determinants that are shared among several activating and inhibitory receptors of a given KIR family (e.g., KIR2D). However, we found that a mixture of mAb molecules with broad coverage of a large number of NK cell receptors, most of which are inhibitory, had a dramatic influence on the capacity of donor PBMC or NK cells to lyse autologous B cell targets in the presence of rituximab, but not in its absence (). The results of blocking autologous recognition by pan anti-MHC class I Ab were similar to those obtained when autologous self-inhibition was blocked with the NK cell receptor combination of Abs, suggesting that we successfully achieved maximal inhibition of MHC class I-regulated self-recognition, even though some activating signals were targeted (). Due to the variability among our donors and among NK cell subpopulations within each donor, attempts to identify a critical subset of KIR receptors required to achieve these effects have not yielded consistent results, suggesting that optimal ADCC promotion by interfering with inhibitory self-recognition requires the blocking of multiple inhibitory receptors. A combination of Abs targeting NKG2A/CD94 and multiple KIRs generally yielded the most consistent promotion of cytotoxicity.
Other laboratories have used strategies to modify mAb structure to better interact with the immune system and more efficiently mediate ADCC (65
). These approaches have included manipulation of the mAb Fc region. Examples include introducing mutations within the Fc domain of mAb to selectively tune the affinity for Fcγ
RIII and other Fcγ
) and modifying Fc glycosylation by the Ab-producing cell line (67
). Importantly, the improvement in rituximab-mediated ADCC when combined with the RISER combination of Abs () was also seen using an anti-CD19 Ab with a mutated Fc domain that mediates enhanced ADCC (CD19 Ab) obtained from Xencor (). Accordingly, the blockade of inhibitory self-recognition further augments ADCC promoted by optimized CD16 signaling. Thus, if the current generation of high affinity ADCC promoting anti-CD20 Abs possesses improved antitumor activity in lymphoma, additional gains may be anticipated by combining our approach.
Interestingly, in some of our donors, the relatively low in vitro rituximab-promoted cytotoxicity of autologous cells was not consistent with the ubiquitous and rapid systemic B cell depletion that follows rituximab therapy. It must be considered that we have used short-term assay with purified NK cells. However, additional mechanisms such as macrophage- and neutrophil-mediated damage of Ab-coated cells, complement fixation, and signaling perturbation of the B cells probably contributed to rituximab-initiated B cell depletion in vivo. We believe, nonetheless, that more efficient ADCC promotion is still a valuable benefit, particularly because CD16 polymorphism status can dictate clinical response to rituximab therapy in lymphoma. Increased ADCC thus offers the possibility of improving treatment outcomes, particularly in individuals who carry the low response polymorphism forms of CD16 (F/F and V/F). This accomplishment can be furthered by altering target cell sensitivity to effector cell lysis, by modulating the affinity of the Ag-binding domains for the tumor target, and by improving the affinity of the Ab Fc domain for activating cellular Fc receptors (65
). Our results suggest that lowering the threshold for NK cell activation through the blockade of inhibitory cellular receptors can complement attempts to create more powerful activating Abs that signal through CD16. This novel approach offers a clear pathway for clinical development of a new way to improve Ab therapy.