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Multispecific antibodies set the rules of engagement for NK cell-mediated tumor control

June 5, 2019

Many bispecific T-cell engager antibodies have been developed and tested in recent years, but challenges with off-target toxicities and lack of efficacy against solid tumors remain. Instead of targeting T cells for antitumor immunity, Gauthier et al. designed and developed trifunctional NK-cell engagers (NKCEs) with the goal of reducing off-target cytotoxicity and enabling control of both hematological and solid malignancies. The results were recently published in Cell.

The researchers began by analyzing the expression of activating receptors on human intratumoral NK cells. They found that the natural cytotoxicity receptor NKp46 was frequently expressed on NK cells in many types of human solid cancers, suggesting that it may be a good candidate for therapeutic targeting.

To target NKp46 in patients, Gauthier et al. generated a library of 14 monoclonal, humanized IgG1 anti-NKp46 antibodies that bound with various affinities to different parts of the human NKp46 extracellular domain. Next, the team created bispecific NKCEs by combining NKp46 antigen-binding fragments (Fabs) with a single-chain variable fragment (scFv) targeting a tumor antigen (e.g. CD19) and a monomeric “silent” Fc that is unable to bind to CD16 (a.k.a. FcγRIIIA, an NK cell activating receptor that promotes antibody-dependent cellular cytotoxicity [ADCC]). When targeting CD19+ human Daudi B cell lymphoma cells in vitro, the bispecific NKCEs induced NK cell-mediated tumor lysis more effectively than the anti-CD19 antibody, indicating that engagement of NKp46 was more effective than ADCC in this setting. In addition, NKCEs that bound NKp46 led to more efficient lysis of tumor cells in vitro than NKCEs that bound NKG2D (another activating NK cell receptor).

Based on these experiments, the researchers selected one particular anti-NKp46 antibody candidate, which bound NKp46 at the junction between its two extracellular domains, for further development due to its superior ability to induce NK cell activation. The NKCE format was then optimized for production, purification, and pharmacological properties. The format that included Fab-based antigen-binding sites, a silent Fc fragment, a long terminal half-life of 11 days, and good manufacturability was selected for further evaluation.

For in vivo studies, Gauthier et al. generated bispecific NKCEs targeting murine NKp46 and human CD20, and tested them in a solid tumor model of human Raji B cell lymphoma in immunodeficient SCID mice. The NKCEs slowed tumor growth and controlled tumors in 50% of mice in an NK cell-dependent manner. Using an RNA-scope-based assay, the researchers showed that the NKCE treatment not only induced tumor clearance, but also increased the NK cell count within the tumors, either via increased infiltration or intratumoral proliferation.

Aiming to further improve antitumor efficacy, the team developed trifunctional NKCEs that could simultaneously target a tumor antigen and co-engage NKp46 and CD16 in order to promote ADCC. The trifunctional NKCEs were engineered to bind CD16 at either wild-type or enhanced levels. The NKCEs targeted human B cell lymphoma either via CD19 or CD20, or human lung carcinoma via EGFR. Both Fc-competent NKCEs were more effective at NK cell activation and tumor cell lysis than Fc-silent NKCEs due to the co-engagement of CD16 and NKp46. The NKCEs with enhanced CD16 binding had superior performance in NK cell activation and tumor cell killing compared with other NKCE formats and compared with the corresponding, clinically used tumor-targeting antibodies (anti-CD20 rituximab, anti-CD20 obinutuzumab, and anti-EGFR cetuximab). Trifunctional NKCEs were also more potent than a mixture of bispecific molecules that engaged NKp46 and CD16 separately, suggesting synergistic binding effects.

The researchers then evaluated the safety of NKCEs. In vitro, NKCE-induced tumor cell killing was tumor antigen-specific. In addition, Fc-competent trifunctional NKCEs did not induce fratricidal killing of NK cells. Testing with human PBMCs showed no elevated cytokine release. Thus, Fc-competent trifunctional NKCEs efficiently mediated tumor cell lysis by NK cells without off-target toxicities.

Having analyzed the trifunctional NKCEs in vitro, Gauthier et al. proceeded to in vivo studies. In a solid tumor model of subcutaneously injected Raji B lymphoma cells, trifunctional NKCEs targeting CD20 slowed tumor growth more effectively than obinutuzumab. In mice with intravenously injected Raji tumor cells, trifunctional NKCEs improved survival compared with obinutuzumab at all doses. 100% cures were achieved with NKCEs at the highest dose tested whereas only 60% cures were mediated by obinutuzumab at the same dose. These in vivo results provide the rationale for clinical development of trifunctional NKCEs.

by Anna Scherer

Meet the Researcher

This week, Laurent Gauthier, first author on the paper, answers our questions.

Laurent Gauthier, first author

What prompted you to tackle this research question?
We were interested in NK cells because these cells are naturally equipped to discriminate stressed cells, like virus-infected cells or cancer cells, from healthy cells and kill them. NK cells express several activating and inhibiting receptors; the NK cell killing activity is controlled by a balance of many positive or negative signals delivered by the target cells. One possible angle to address the problem of NK cell activation to fight cancer was to develop antibodies targeting and blocking the inhibitory receptors expressed on NK cells like KIRs or NKG2A. This approach led to the development at Innate Pharma of two clinical grade antibodies: lirilumab targeting KIRs and monalizumab targeting NKG2A. A second possible angle was to develop multifunctional molecules targeting both an activating receptor on NK cells and an antigen on cancer cells. Unfortunately, when we started to work on that idea in the early 2000s, the technical options to generate multifunctional antibodies were pretty limited and we had to wait for the emergence of recombinant antibody technology and engineering to concretize the idea.
NK cells express several activating receptors that can be targeted to induce NK cell-mediated antitumor immunity, such as CD16 (FcγRIIIA), NKG2D, and the natural cytotoxicity receptors NKp30, NKp44, and NKp46. We focused on NKp46 because this activating receptor is expressed on all NK cells and NKp46 expression is not downmodulated on tumor infiltrating NK cells.

What was the most surprising finding of this study for you?

One of the most surprising findings of this study was the encouraging activating potential of NKp46. Indeed, even though circulating NK cells express higher levels of CD16 than of NKp46, we observed in vitro that NK cell engager molecules (NKCEs) targeting NKp46 were at least equivalent or more effective to mediate anti-tumor activity than the regular IgG1 mAb targeting the same antigens, suggesting that engaging NKp46 was a more efficient approach than inducing ADCC in this experimental setting. Another surprising finding was the synergy of NK cell activation observed with trifunctional NKCEs co-engaging NKp46 and CD16 on NK cells, showing that trifunctional molecules were more potent than a mixture of the bispecific reagents activating NKp46 and CD16 separately.


Gauthier L., Morel A., Anceriz N., Rossi B., Blanchard-Alvarez A., Grondin G.,Trichard S., Cesari C., Sapet M., Bosco F., Rispaud-Blanc H., Guillot F., Cornen S., Roussel A., Amigues B., Habif G., Caraguel F., Arrufat S., Remark R., Romagné F., Morel Y., Narni-Mancinelli E., Vivier E. Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity. Cell. 2019 May 22.

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