Background
Shedding of CD16, a potent activating receptor on natural killer (NK) cells that mediates antibody dependent cellular cytotoxicity (ADCC), limits the efficacy of NK cell immunotherapies that depend on this mechanism. Blocking A Disintegrin And Metalloprotease 17 (ADAM17), the enzyme that clips CD16 upon NK cell activation, may overcome this limitation. To achieve this, we improved upon a previously described Tri-specific Killer Engager (TriKE) platform. The original TriKE, which showed strong preclinical efficacy, consists of a nanobody arm that engages CD16, an interleukin (IL)-15 moiety that can drive expansion of NK cells, and a nanobody arm binding B7H3, a pan-cancer antigen on various solid tumors. Here, we incorporated a Poly-Antigen Cytokine-receptor Complex (PACC), consisting of interleukin-15 receptor alpha (IL-15R) linked to an ADAM17-blocking arm, to noncovalently bind IL-15 in the TriKE molecule and enhance CD16-mediated NK cell-directed anti-tumor responses against B7H3-expressing solid tumors (figure 1).
Methods
Formation of the TriKE-PACC complex was validated using ELISA and flow cytometry-based binding assays. Phorbol 12-myristate 13-acetate (PMA) stimulation of NK cells treated with TriKE-PACC evaluated the ability of the anti-ADAM17 PACC arm to block CD16 shedding. To investigate prolonged ADCC capacity, an ovarian cancer mouse xenograft model, examined the efficacy of TriKE-PACC in enhancing NK cell-mediated anti-tumor response.
Results
Formation of TriKE-PACC complex was confirmed, indicating incorporation of this ‘backpack’ is stable and retained through manufacturing. Upon PMA stimulation of untreated cells, CD16 and CD62L (another ADAM17 substrate vital for NK cell trafficking into tumor site), were potently shed from the NK cell surface. This effect was reversed by use of an ADAM17-blocking antibody or our novel TriKE-PACC (figure 2) In the in vivo model, TriKE-PACC treatment resulted in better tumor control, demonstrating enhanced anti-tumor activity compared to TriKE or tumor alone controls (figure 3). Survival of mice in this study is still being tracked.
Conclusions
ADAM17-blocking in a TriKE-PACC complex can overcome CD16 shedding, and be directed towards B7H3-expressing solid tumors, leading to better tumor control in a long-term in vivo model. Evaluations of TriKE-PACC efficacy are currently ongoing in other settings of CD16 down-regulation, including ovarian cancer patient ascites, mesothelioma cancer patient pleural fluid samples, and cryopreserved NK cell products. This innovative approach holds great promise for enhancing the efficacy of NK cell immunotherapies, potentially transforming the treatment landscape for various solid tumors where prolonged and sustained effector functions are especially critical.
Ethics Approval
Mouse studies were carried out in accordance with guidelines from Institutional Animal Care and Use Committee (IACUC) at the University of Minnesota. The ID of the IACUC protocol for this project is 2207A40255.
Abstract 1071 Figure 1
Graphical abstract. To abrogate CD16 shedding from NK cells by ADAM17, a single chain variable fragment of an ADAM17 inhibitor, MEDI3622, can be engineered onto an existing B7H3-targeting TriKE molecule via an IL-15Ra ectodomain
Abstract 1071 Figure 2
ADAM17 TriKE-PACC can retain CD16 and CD62L on NK cells after strong activation. NK cells were incubated with different drugs for 30 minutes prior to PMA stimulation. Following stimulation, NK cells were assessed for CD16 and CD62L expression using flow cytometry
Abstract 1071 Figure 3
ADAM17 TriKE-PACC controls tumor burden in Ovcar8 mouse xenograft model. Immunocompromised mice were engrafted with Ovcar8 cells four days prior to NK cell administration intraperitoneally. Treatment groups are dosed twice per week with either the TriKE or the TriKE-PACC. BLIs are conducted every week to track tumor burden