Site-specific 68Ga-labeled nanobody for PET imaging of CD70 expression in preclinical tumor models.

BACKGROUND: CD70-CD27 is a costimulatory ligand-receptor pair in the tumor necrosis factor receptor family. With only limited expression in normal tissues, CD70 is constitutively expressed in a variety of solid tumors and hematologic malignancies, facilitating immunosuppression through CD27 signaling in the tumor microenvironment by enhanced survival of regulatory T cells, induction of T cell apoptosis, and T cell exhaustion. Consequently, CD70 is an increasingly recognized target for developing antibody-based therapies, but its expression patterns vary among different tumor types in spatial distribution, magnitude of expression and percentage of positive cells. In that regard, individual confirmation of CD70 expression at screening and during treatment could enhance the successful implementation of anti-CD70 therapies. Here, we developed a gallium-68 (68Ga) radiolabeled single-domain antibody-fragment targeting CD70 for in vivo positron emission tomography (PET) imaging. RESULTS: An anti-CD70 VHH construct containing a C-direct-tag with a free thiol was developed to enable site-specific conjugation to a NOTA bifunctional chelator for 68Ga radiolabeling. [68Ga]Ga-NOTA-anti-CD70 VHH was obtained in good radiochemical yield of 30.4 ± 1.7% and high radiochemical purity (> 94%). The radiolabeled VHH showed excellent in vitro and in vivo stability. Specific binding of [68Ga]Ga-NOTA-anti-CD70 VHH was observed on CD70high 786-O cells, showing significantly higher cell-associated activity when compared to the blocking condition (p < 0.0001) and CD70low NCl-H1975 cells (p < 0.0001). PET imaging showed specific radiotracer accumulation in CD70 expressing human tumor xenografts, which was efficiently blocked by prior injection of unlabeled anti-CD70 VHH (p = 0.0029). In addition, radiotracer uptake in CD70high tumors was significantly higher when compared with CD70low tumors (p < 0.0001). The distribution of the radioactivity in the tumors using autoradiography was spatially matched with immunohistochemistry analysis of CD70 expression. CONCLUSION: [68Ga]Ga-NOTA-anti-CD70 VHH showed excellent in vivo targeting of CD70 in human cancer xenografts. PET imaging using this radioimmunoconjugate holds promise as a non-invasive method to identify and longitudinally follow-up patients who will benefit most from anti-CD70 therapies.

Complement inhibition prevents glial nodal membrane injury in a GM1 antibody-mediated mouse model.

The involvement of the complement pathway in Guillain-Barré syndrome pathogenesis has been demonstrated in both patient biosamples and animal models. One proposed mechanism is that anti-ganglioside antibodies mediate neural membrane injury through the activation of complement and the formation of membrane attack complex pores, thereby allowing the uncontrolled influx of ions, including calcium, intracellularly. Calcium influx activates the calcium-dependent protease calpain, leading to the cleavage of neural cytoskeletal and transmembrane proteins and contributing to subsequent functional failure. Complement inhibition has been demonstrated to provide effective protection from injury in anti-ganglioside antibody-mediated mouse models of axonal variants of Guillain-Barré syndrome; however, the role of complement in the pathogenesis of demyelinating variants has yet to be established. Thus, it is currently unknown whether complement inhibition would be an effective therapeutic for Guillain-Barré syndrome patients with injuries to the Schwann cell membrane. To address this, we recently developed a mouse model whereby the Schwann cell membrane was selectively targeted with an anti-GM1 antibody resulting in significant disruption to the axo-glial junction and cytoplasmic paranodal loops, presenting as conduction block. Herein, we utilize this Schwann cell nodal membrane injury model to determine the relevance of inhibiting complement activation. We addressed the early complement component C2 as the therapeutic target within the complement cascade by using the anti-C2 humanized monoclonal antibody, ARGX-117. This anti-C2 antibody blocks the formation of C3 convertase, specifically inhibiting the classical and lectin complement pathways and preventing the production of downstream harmful anaphylatoxins (C3a and C5a) and membrane attack complexes. Here, we demonstrate that C2 inhibition significantly attenuates injury to paranodal proteins at the node of Ranvier and improves respiratory function in ex vivo and in vivo Schwann cell nodal membrane injury models. In parallel studies, C2 inhibition also protects axonal integrity in our well-established model of acute motor axonal neuropathy mediated by both mouse and human anti-GM1 antibodies. These data demonstrate that complement inhibition prevents injury in a Schwann cell nodal membrane injury model, which is representative of neuropathies associated with anti-GM1 antibodies, including Guillain-Barré syndrome and multifocal motor neuropathy. This outcome suggests that both the motor axonal and demyelinating variants of Guillain-Barré syndrome should be included in future complement inhibition clinical trials.

Cusatuzumab for treatment of CD70-positive relapsed or refractory cutaneous T-cell lymphoma.

BACKGROUND: The clinical benefit of cusatuzumab, a CD70-directed monoclonal antibody with enhanced effector functions, was investigated in patients with relapsed/refractory (R/R) cutaneous T-cell lymphoma (CTCL). METHODS: In this cohort expansion of the ARGX-110-1201 study, 27 patients with R/R CTCL received cusatuzumab at 1 (n = 11) or 5 mg/kg (n = 16) once every 3 weeks to investigate its safety, dose, and exploratory efficacy. The pharmacokinetics, immunogenicity, CD70 expression, and CD70/CD27 biology were also assessed. RESULTS: The most common adverse events included infusion-related reactions, pyrexia, and asthenia. Eighteen serious adverse events (grade 1-3) were reported in 11 patients; 1 of these (vasculitis) was considered drug-related. For 8 of the 11 patients receiving 1 mg/kg, anti-drug antibodies (ADAs) affected the minimal concentration, and this resulted in undetectable cusatuzumab concentrations at the end of treatment and, in some cases, a loss of response. This effect was greatly reduced in the patients receiving 5 mg/kg. The overall response rate was 23%; this included 1 complete response and 5 partial responses (PRs) in 26 of the 27 evaluable patients. In addition, 9 patients achieved stable disease. The mean duration on cusatuzumab was 5.2 months, and the median duration was 2.5 months. Patients with Sézary syndrome (SS) achieved a 60% PR rate with a dosage of 5 mg/kg and a 33% PR rate with a dosage of 1 mg/kg; this resulted in an overall response rate of 50% for patients with SS at both doses. CONCLUSIONS: Cusatuzumab was well tolerated, and antitumor activity was observed at both 1 and 5 mg/kg in highly pretreated patients with R/R CTCL. The observed dose-dependent effect on exposure supports the use of 5 mg/kg for future development.

An open-label, nonrandomized, phase Ib feasibility study of cusatuzumab in patients with nasopharyngeal carcinoma.

CD70 is expressed in up to 80% of nasopharyngeal carcinoma (NPC) cases. Cusatuzumab is a humanized anti-CD70 monoclonal antibody, with dual action mechanisms: induction of cytotoxicity against CD70+ tumor cells and reduction in CD70-CD27 signaling mediated immune evasion. The aim of this study was to assess the safety, pharmacokinetic profile, immunogenicity, pharmacodynamic profile, and preliminary activity of cusatuzumab in advanced NPC. Eleven patients were enrolled: one patient was assigned to arm A (adjuvant cusatuzumab monotherapy after curative chemoradiation), nine patients to arm B (cusatuzumab monotherapy; noncurative setting), and one patient to arm C (cusatuzumab + chemotherapy; noncurative setting); irrespective of tumoral CD70 expression. Both patients in arms A and C completed the study. All patients in arm B discontinued at an early stage. Five patients experienced grade greater than or equal to 3 nondrug related treatment-emergent adverse events, most commonly fatigue and pneumonia (18%). An infusion-related reaction was observed in two of 11 patients. Laboratory results showed no trend over time. Seven patients were eligible for response evaluation. No objective response to cusatuzumab was observed with stable disease being the best response. The current study indicates that the safety profile of cusatuzumab (with or without concurrent chemotherapy) is manageable in patients with advanced NPC, which is consistent with known safety profile. Limited activity of cusatuzumab in advanced NPC was observed. Combination therapies of cusatuzumab and other types of therapy should be explored for the improvement of activity in NPC and other CD70-expressing malignancies.

Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents.

Acute myeloid leukemia (AML) is driven by leukemia stem cells (LSCs) that resist conventional chemotherapy and are the major cause of relapse1,2. Hypomethylating agents (HMAs) are the standard of care in the treatment of older or unfit patients with AML, but responses are modest and not durable3-5. Here we demonstrate that LSCs upregulate the tumor necrosis factor family ligand CD70 in response to HMA treatment resulting in increased CD70/CD27 signaling. Blocking CD70/CD27 signaling and targeting CD70-expressing LSCs with cusatuzumab, a human αCD70 monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity activity, eliminated LSCs in vitro and in xenotransplantation experiments. Based on these preclinical results, we performed a phase 1/2 trial in previously untreated older patients with AML with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine ( NCT03030612 ). We report results from the phase 1 dose escalation part of the clinical trial. Hematological responses in the 12 patients enrolled included 8 complete remission, 2 complete remission with incomplete blood count recovery and 2 partial remission with 4 patients achieving minimal residual disease negativity by flow cytometry at <10-3. Median time to response was 3.3 months. Median progression-free survival was not reached yet at the time of the data cutoff. No dose-limiting toxicities were reported and the maximum tolerated dose of cusatuzumab was not reached. Importantly, cusatuzumab treatment substantially reduced LSCs and triggered gene signatures related to myeloid differentiation and apoptosis.