CD74 Antibody Conjugated to an MCL1 Inhibitor (S227928) Combines Synergistically with Venetoclax to Enhance Killing of Acute Myeloid Leukemia

Background: BCL2 pro-survival proteins (BCL2, MCL1, BCL-XL) function as key regulators of oncogenic survival. BH3-mimetic drugs bind to pro-survival proteins (e.g. venetoclax to BCL2), enhancing BH3-only proteins to induce BAX/BAK dependent apoptosis. MCL1 is overexpressed in acute myeloid leukemia (AML) and increased expression has been observed in leukemias resistant to venetoclax-based therapy. Co-targeting BCL2 and MCL1 with selective BH3-mimetics has synergistic activity in diverse AML cases, including TP53-mutated disease (Thijssen, Blood, 2021). Clinical development of MCL1 inhibitors has been hampered by treatment-associated rises in serum troponin (Yuda, Commun Med, 2023). To overcome this, a novel antibody drug conjugate (S227928; Servier-Novartis) has been designed, comprising a monoclonal antibody directed at human CD74, bridged by a linker to an MCL1 selective BH3-mimetic. CD74 was chosen for its high expression on AML cells and low expression on critical organs, including cardiomyocytes. In this study, we highlight the activity of S227928 in combination with venetoclax in models of AML. Aims: To assess the pre-clinical efficacy and CD74 target selectivity of S227928 alone and in combination with venetoclax in AML. Methods: CD74 expression was determined by APC labelled anti-human CD74 (Clone REA1103;Miltenyi). CRISPR-CAS9 gene editing was performed to generate CD74-/- AML cell lines. Drug sensitivity assays were evaluated by flow cytometry. Patient derived xenografts were performed in NSG-SG3 mice. S227928, isotype-MCL1i and unconjugated anti-CD74 were from Servier/Novartis. Results: Expression of CD74 was highest on EOL-1>MV4;11, MOLM13, OCI-AML3>>K562 cells. Pro-apoptotic S227928 activity by 72 h was highest in EOL-1 cells (LC50<10 nM) and least in K562 cells (LC50~10 µM). Combined venetoclax and S227928 was synergistic in AML cell lines, including those with TP53 deleted by CRISPR. Cell death was significantly reduced in BAX/BAK or CD74 deficient cells, confirming a mitochondrial apoptosis mechanism specific to CD74 expressing cells. In patient samples, CD74 expression was highest in B-lymphoid (CD20+) and monocytic blasts (CD64+ or CD14+) > leukemic stem and progenitor cells (CD34+ and/or CD117+) > myeloid blasts (CD33+) >> T lymphoid cells (CD3+). Single-agent S227928 activity in primary AML was observed in 3/14 cases (LC50<100 nM). In contrast, S227928 + venetoclax was active in 9/14 primary AML cases, comparable to non-conjugated MCL1 inhibitor + venetoclax (activity in 9/14) and greater than either unconjugated anti-CD74 + venetoclax (activity in 3/14) or an isotype-conjugated MCL1 inhibitor control (activity in 0/14). Concordant with CD74 expression being highest in monocytoid leukemias, S227928 + venetoclax appeared most potent against monocytic lineage AML. Expression of CD74 on non-leukemic CD34+ progenitors was significantly lower than expression in primary AML samples. Concordantly, viability of normal CD34+ cells was not significantly impacted by either S227928 alone or in combination with venetoclax, in contrast to the cytotoxic impact of cytarabine or daunorubicin on CD34+ cell viability. To assess activity of S227928 + venetoclax in vivo, mice were transplanted with AML cells expressing CD74, CD14 and CD64 and harboring biallelic TP53 V173M and V143M variants. Cohorts were treated with 1) vehicle, 2) S227928 (30 mg/kg IV q 14 days), 3) isotype-MCL1i q 14 days + venetoclax 50 mg/kg days 1-14 (week 1-2) and weekdays (week 3-8), 4) unconjugated anti-CD74 q 14 days + venetoclax, 5) S227928 (10 mg/kg q 14 days) + venetoclax or 6) S227928 (30 mg/kg q 14 days) + venetoclax for 8 weeks. Analysis of bone marrow 2 weeks after commencing treatment revealed blast reduction greatest for the S227928/veneteclax combination arms. Overall survival was longest for S227928/venetoclax combination therapy (median 78 and 96 days), compared to vehicle (median 41 days), S227928 (median 49 days), isotype-MCL1i + venetoclax (median 59 days) or unconjugated anti-CD74 + venetoclax (median 49 days). Conclusion: S227928 is a novel CD74 ADC that combines synergistically with venetoclax to enhance killing in pre-clinical models of AML. Importantly, this combination is active in TP53 mutated and monocytoid AML, sub-groups associated with venetoclax failure. These findings support the clinical development of S227928 alone and in combination in patients with AML.