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A tetraspecific NK cell engager armed with IL-2v targets B-NHL

November 27, 2024

To capitalize on the antitumor potential of NK cells for the treatment of B cell non-Hodgkin lymphoma (B-NHL), Demaria et al. developed a clinical-grade tetraspecific NK cell engager (NKCE) that targets NKp46 and CD16a on NK cells and CD20 on tumor cells, and armed it with an IL-2 variant (CD20-NKCE-IL2v). The mechanism and antitumor effects of this novel CD20-NKCE-IL2v molecule were recently evaluated in vitro and in vivo, and the results were published in Science Immunology.

To begin, Demaria et al. developed a fully human CD20-NKCE-IL2v consisting of a CD20 antibody fragment, an IgG1 Fc portion (to target CD16a), an NKp46 antibody fragment, and an IL-2 variant engineered to eliminate CD25 (IL-2Rα) binding and reduce Treg activation. In short-term cytotoxicity assays of NK cells and CD20+ RAJI B-NHL, CD20-NKCE-IL2v induced on-target cytotoxic activity, dependent on simultaneous engagement of CD16a and NKp46. The inclusion of the IL-2v component improved tumor control in longer-term assays. Target cell killing was observed across a broad range of CD20 expression levels, and occurred regardless of MHC-I expression. Within NK cells, CD20-NKCE-IL2v induced IL-2R signaling, STAT5 phosphorylation, and proliferation, regardless of antigen-specific targeting. scRNAseq revealed that CD20-NKCE-IL2v induced changes across all NK cell subsets (NK1, NK2, and NK3), and led to the emergence of another NK cell cluster that resembled cytokine-induced memory-like (CIML) NK cells by 4 hours, though they appeared to diminish by 24 hours. These cells showed enhanced activation and cytokine and chemokine expression. Changes in NK cell subsets could also be observed over time; NK cells stimulated for 4 hours showed upregulation of IFNγ, IFN-regulated genes, and genes related to inflammation, while NK cells stimulated for 24 hours were marked by genes associated with cell cycle regulation, proliferation, cytoskeletal components, proinflammatory mediators, and negative regulators of cytokine signaling.

Comparing the efficacy of CD20-NKCE-IL2v to other existing therapeutics, Demaria et al. showed that in 4-day in vitro killing assays of RAJI B-NHL cells, CD20-NKCE-IL2v-stimulated NK cells outperformed the anti-CD20 antibodies rituximab and obinutuzumab, and (at higher doses) a CD20-TCE that is a biosimilar of epcoritamab. These results were confirmed in assays using healthy PBMCs, where autologous B cells were more effectively depleted. Further, at comparable doses, CD20-NKCE-IL2v induced lower levels of pro-inflammatory cytokines, suggesting a better safety profile.

Next, Demaria et al. evaluated the efficacy of CD20-NKCE-IL2v in mice using a surrogate molecule engineered to target murine NKp46 to overcome a lack of cross-reactivity. In a

disseminated B cell lymphoma xenograft model in SCID mice, which are immunocompromised, but maintain the NK cell compartment, treatment with the surrogate CD20-NKCE-IL2v improved survival. Similar effects were observed in a more resistant subcutaneous RAJI model, where treatment also controlled tumor growth. In a syngeneic mouse model intravenously engrafted with B16F10 tumor cells engineered to express human CD20, a single dose of CD20-NKCE-IL2v controlled tumor growth in the lungs, even at doses low enough to avoid temporary weight loss. A time-course analysis showed that NK cells expanded in the blood and spleen in a dose-dependent manner, with increased markers of proliferation, which normalized by day 13. NK cells in the blood and spleen were selectively activated. Higher doses of CD20-NKCE-IL2v also induced increases in CD8+ T cells, affecting mainly effector and central memory subsets, but did not increase their activation. Only the highest tested dose of CD20-NKCE-IL2v induced detectable cytokine levels in the blood. In an evaluation of NK cell trafficking, CD20-NKCE-IL2v induced NK cell accumulation in tumors, suggesting a capacity to redirect NK cells from the periphery to the TME.

Moving towards clinical translation, the researchers evaluated CD20-NKCE-IL2v in non-human primates, and found that i.v. administration of the drug induced strong dose-dependent depletion of B cells within 3 days, with 3 weekly injections yielding prolonged responses; B cells recovered to 50% at around 3-4 weeks after the last dose. As in mice, NK cells were found to increase in the blood in a dose-dependent manner, with evidence of enhanced proliferation. Only the highest dose increased numbers of cytotoxic T cells in the blood, though some doses did increase markers of proliferation in effector memory cytotoxic T cells. IHC analysis showed that these results were mirrored in lymphoid organs. Further, cytokine response profiles in treated NHPs were benign, and treatment was well tolerated, suggesting a strong safety profile.

Turning their attention towards relapsed/refractory B-NHL, Damaria et al. evaluated patient samples and found that r/r B-NHL cells maintained CD20 expression, and that patient PBMCs maintained normal NK cell levels, though the NK cells did show slightly reduced CD16 and IL-2Rβ compared to NK cells from healthy donors. In assays using samples from patients who were in the leukemic phase of their disease, CD20-NKCE-IL2v still stimulated NK cell proliferation and depleted autologous CD20+ target cells. These effects were stronger compared to treatment with CD20-TCE, further underscoring the potential of CD20-NKCE-IL2v in a challenging clinical setting.

Finally, Demaria et al. investigated the specific contributions of the IL-2v to the efficacy of CD20-NKCE-IL2v, and found that it contributed to the upregulation of expression of activating receptors (NKp30, DNAM-1, and NKG2D) on the surface of NK cells, regardless of CD20 antigen targeting. Cytotoxicity assays showed that this enhanced the agnostic activity of CD20-NKCE-IL2v-treated NK cells, enabling lysis of B16F10-huMICA cells, which lack CD20 expression, but express the NKG2D ligand MICA. These results could be reversed with an NKG2D-blocking antibody, were not induced by conventional therapeutic antibodies, and did not impede the recognition and destruction of CD20+ target cells. Similar observations were made in assays with CD20- human cells, where both CD20-NKCE-IL2v and a control IC-NKCE-IL2v controlled the growth of RAJI cells, though the effects of the CD20-NKCE-IL2v molecule were stronger. In vivo testing yielded similar results.

Overall, Demaria et al. showed that the tetraspecific CD20-NKCE-IL2v boosts NK cell proliferation, cytotoxicity, surface expression of activating receptors, and tumor homing, ultimately supporting antitumor activity, even against tumor cells with low or no CD20 and tumor cells expressing MHC-I. Compared to comparable treatments, including clinically approved CD20-targeting antibodies and T cell engagers, CD20-NKCE-IL2 showed stronger antitumor efficacy with reduced toxicity, even in r/r B-NHL models, and was well tolerated in NHPs, suggesting strong potential for clinical applications.

Write-up and image by Lauren Hitchings

Meet the researcher

This week, first author Olivier Demaria and lead author Eric Vivier answered our questions.

From left to right: Olivier Demaria and Eric Vivier

What was the most surprising finding of this study for you?
 IPH6501 is the leading asset of a new generation of NK cell engagers that incorporate a cytokine component. It includes an IL-2 variant designed to not only stimulate NK cell activation, but also to promote their proliferation. In preclinical studies, IPH6501 demonstrated the ability to stimulate NK cell proliferation both in vitro, with human NK cells, and in vivo, in mouse and non-human primate models. A key finding of our study was that IPH6501 also triggers their recruitment of NK cells from the periphery to the tumor site. This discovery has significant implications. Given the well documented negative effects of the tumor microenvironment on infiltrating immune cells, a therapy that amplifies and mobilizes NK cells from outside this immunosuppressive milieu may have a higher potential for antitumor activity. Furthermore, this observation that NK cells in the bloodstream are recruited to the tumor site following treatment supports the rationale for using blood-derived NK cells in preclinical ex vivo assays with patient samples to anticipate in vivo efficacy.

What is the outlook?
The first NK cell engagers targeting NKp46 have entered clinical trials to treat patients. Following promising data in patients, including those obtained in relapsed or refractory acute myeloid leukemia (AML) (NCT05086315), the front runner asset in this class of molecule, SAR443579/IPH6101, received Fast Track Designation from the FDA. It is now progressing to a phase 2 trial, underscoring its potential and supporting the development of other molecules of this type. IPH6501, as a first in class NK cell engager targeting NKp46 and incorporating a cytokine component, was specifically developed to address the critical need for effective therapeutic options in patients with B-NHL. Currently in the dose-escalation phase in relapsed/refractory B-NHL patients (NCT06088654), we are looking forward to analysing the first clinical data on IPH6501, focusing not only on its efficacy, but also on its pharmacodynamics to confirm whether this new molecular platform achieves in patients the biological activity it was designed to deliver.

What was the coolest thing you’ve learned (about) recently outside of work?
We both enjoy climbing with our kids. After an intense week of work, there's nothing more relaxing than getting outdoors and climbing together. The combination of spending time in nature with loved ones while engaging in a physical activity is one of the most refreshing things you can do.

"A big thank you to all the people from Innate Pharma who contributed to the project." Olivier Demaria
References:

Demaria O, Habif G, Vetizou M, Gauthier L, Remark R, Chiossone L, Vagne C, Rebuffet L, Courtois R, Denis C, Le Floch F, Muller M, Girard-Madoux M, Augier S, Lopez J, Carrette B, Maguer A, Vallier JB, Grondin G, Baron W, Galluso J, Yessaad N, Giordano M, Simon L, Chanuc F, Alvarez AB, Perrot I, Bonnafous C, Represa A, Rossi B, Morel A, Morel Y, Paturel C, Vivier E. A tetraspecific engager armed with a non-alpha IL-2 variant harnesses natural killer cells against B cell non-Hodgkin lymphoma. Sci Immunol. 2024 Nov 15.

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