Franceline Guillot

Abstract CD123, the alpha chain of the IL-3 receptor, is an attractive target for acute myeloid leukemia (AML) treatment. However, cytotoxic antibodies or T cell engagers targeting CD123 had insufficient efficacy or safety in clinical trials. We show that expression of CD64, the high-affinity receptor for human IgG, on AML blasts confers resistance to anti-CD123 antibody-dependent cell cytotoxicity (ADCC) in vitro. We engineer a trifunctional natural killer cell engager (NKCE) that targets CD123 on AML blasts and NKp46 and CD16a on NK cells (CD123-NKCE). CD123-NKCE has potent antitumor activity against primary AML blasts regardless of CD64 expression and induces NK cell activation and cytokine secretion only in the presence of AML cells. Its antitumor activity in a mouse CD123 + tumor model exceeds that of the benchmark ADCC-enhanced antibody. In nonhuman primates, it had prolonged pharmacodynamic effects, depleting CD123 + cells for more than 10 days with no signs of toxicity and very low inflammatory cytokine induction over a large dose range. These results support clinical development of CD123-NKCE.

<h3>Background</h3> There is a clear need for targeted therapies to treat acute myeloid leukemia (AML), the most common acute leukemia in adults. CD123 (IL-3 receptor alpha chain) is an attractive target for AML treatment.¹ However, cytotoxic antibody targeting CD123 proved insufficiently effective in a combination setting in phase II/III clinical trials.² T-cell engagers targeting CD123 displayed some clinical efficacy but were often associated with cytokine release syndrome and neurotoxicity.³ Interest in the use of NK cells for therapeutic interventions has increased in recent years, as a potential safer alternative to T cells. Several NK-cell activating receptors, such as CD16a, NKG2D, and the natural cytotoxicity receptors NKp30 and NKp46, can be targeted to induce antitumor immunity. We previously reported the development of trifunctional NK-cell engagers (NKCEs) targeting a tumor antigen on cancer cells and co-engaging NKp46 and CD16a on NK cells.⁴ <h3>Methods</h3> We report here the design, characterization and preclinical development of a novel trifunctional NK cell engager (NKCE) targeting CD123 on AML cells and engaging the activating receptors NKp46 and CD16a on NK cells. The CD123 NKCE therapeutic molecule was engineered with humanized antibodies targeting NKp46⁴ and CD123.⁵ We compared CD123-NKCE and a cytotoxic ADCC-enhanced antibody (Ab) targeting CD123, in terms of antitumor activity in vitro, ex vivo and in vivo. Pharmacokinetic, pharmacodynamic and safety profile of CD123-NKCE were evaluated in non-human primate (NHP) studies. <h3>Results</h3> The expression of the high affinity Fc gamma receptor CD64 on patient-derived AML cells inhibited the ADCC of the Ab targeting CD123 in vitro and ex vivo, but not the antitumor activity of CD123-NKCE. CD123-NKCE had potent antitumor activity against primary AML blasts and AML cell lines, promoted strong NK-cell activation and induced cytokine secretion only in the presence of AML target cells. Its antitumor activity in mouse model was greater than that of the comparator antibody. Moreover, CD123-NKCE had strong and prolonged pharmacodynamic effects in NHP when used at very low doses, was well-tolerated up to high 3 mg/kg dose and triggered only minor cytokine release. <h3>Conclusions</h3> The data for activity, safety, pharmacokinetics, and pharmacodynamics provided here demonstrate the superiority of CD123-NKCE over comparator cytotoxic antibody, in terms of antitumor activity in vitro, ex vivo, in vivo, and its favorable safety profile, as compared to T-cell therapies. These results constitute proof-of-principle for the efficacy of CD123-NKCE for controlling AML tumors in vivo, and provide consistent support for their clinical development. <h3>References</h3> Ehninger A, Kramer M, Rollig C, <i>et al</i>. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. <i>Blood Cancer J</i> 2014;<b>4</b>:e218. Montesinos P, Gail J Roboz GJ, <i>et al</i>. Safety and efficacy of talacotuzumab plus decitabine or decitabine alone in patients with acute myeloid leukemia not eligible for chemotherapy: results from a multicenter, randomized, phase 2/3 study. <i>Leukemia</i> 2021;<b>35</b>(1):62–74. Uy GL, Aldoss I, Foster MC, <i>et al</i>. Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia. <i>Blood</i> 2021;<b>137</b>(6):751–762. Gauthier L, Morel A, Anceriz N, <i>et al</i>. Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity. <i>Cell</i> 2019;<b>177</b>(7):1701–13. Jin L, Lee EM, Ramshaw HS, <i>et al</i>. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. <i>Cell Stem Cell</i> 2009;<b>5</b>:31–42.

Introduction: KIR3DL2, a killer immunoglobulin-like receptor normally expressed by a subset of natural killer (NK) cells and a minority of CD4+ and CD8+ T lymphocytes, is aberrantly expressed in cutaneous T-cell lymphomas (CTCL), particularly in Sézary Syndrome (SS)1. IPH4102, a monoclonal antibody directed against KIR3DL2, demonstrated in-vitro antitumor activity and has shown beneficial clinical activity in a phase 1 dose-escalation plus expansion cohort study in relapsed advanced CTCL patients (NCT02593045)2,3. Methods: Our cohort included a total of 91 peripheral TCL (PTCL) patients. NK receptors expression was assessed by immunohistochemistry (IHC) on frozen biopsies (n = 49), and by flow-cytometry (n = 42) on peripheral blood samples (n = 12) and lymph node/tumor tissue (n = 30). Ex-vivo antibody dependent cell cytotoxicity (ADCC) assays are currently being performed on sorted primary KIR3DL2-positive PTCL cells with IPH4102. Finally, IPH4102 and gemcitabine plus oxaliplatin (GemOx), a combination chemotherapy regimen commonly used in relapsed PTCL, was studied in-vitro. Results: Overall, irrespective of the staining technique, KIR3DL2 expression was evidenced in 41/91 (45%) of PTCL patients. More precisely, KIR3DL2 was expressed on 7/20 (35%) PTCL-not otherwise specified (NOS), 9/25 (36%) angioimmunoblastic TCL (AITL), 8/14 (57%) anaplastic large-cell lymphomas (ALCL), 5/11 (45%) enteropathy-associated TCL (EATL), 5/11 (45%) NK/T-cell lymphomas, 0/2 hepatosplenic TCL and 7/8 (88%) T-cell large granular lymphocyte leukemias (T-LGL). By IHC, within all PTCL categories, > 5% of lymphoid cells were KIR3DL2-positive in 22 out of 49 cases (45 %). High expression (> 50% KIR3DL2-positive lymphoid cells) was found in 11 (22 %) patients ( figure 1A ). In addition, by flow cytometry, KIR3DL2 was expressed on tumor cells compared to isotype control in 18/42 PTCL (43%). Incubation of T-cell lymphoma cell lines with GemOx enhances baseline KIR3DL2 expression. In-vitro, IPH4102 ADCC against KIR3DL2-positive tumor T-cell lines is increased by GemOx ( figure 1B ). Keywords: monoclonal antibodies (MoAb); peripheral T-cell lymphomas (PTCL). Disclosures: Cheminant, M: Research Funding: yes. Bruneau, J: Research Funding: yes. Peri, V: Employment Leadership Position: yes. Guillot, F: Employment Leadership Position: yes. Paturel, C: Employment Leadership Position: yes. Sicard, H: Employment Leadership Position: yes. Bonnafous, C: Employment Leadership Position: yes; Stock Ownership: yes. Hermine, O: Research Funding: yes.