High Frequency of CD74 Expression in B-Cell Non-Hodgkin's Lymphoma (NHL) and Targeting with SΤRO-001, a Novel Anti-CD74 Antibody Drug Conjugate (ADC) with Potent I n Vitro Cytotoxicty and In Vivo Anti-Tumor Activity

Introduction: STRO-001 is a novel CD74-targeting ADC comprised of an anti-CD74 human IgG1 antibody (SP7219) genetically incorporating the non-natural amino acid para-azidomethyl-L-phenylalanine (pAMF) with a non-cleavable dibenzocyclooctyne (DBCO)-maytansinoid linker-warhead. Highly efficient and precise site-specific conjugation enabled by cell-free antibody synthesis technology produced a well-defined homogeneous ADC with a drug-antibody ratio (DAR) of 2. Conjugation sites were selected based on highest stability both in vitro and in vivo . The aim of this study was to investigate the expression of CD74 in NHL and explore the therapeutic potential of STRO-001 in B-cell NHL cell lines and xenografts.

Methods: The binding affinity of SP7219 to recombinant human CD74 ECD (extracellular domain) and cynomolgus monkey (cyno) CD74 ECD was measured by SPR using a Biacore T200. Biotinylated SP7219 was used for immunohistochemistry (IHC) in NHL tumor specimens. Flow cytometry was used for measuring CD74 expression in NHL cell lines. STRO-001 was used to determine the EC₅₀ and percent span of killing in NHL cell lines. The anti-tumor activity of STRO-001 in NHL xenografts in severe combined immune deficient (SCID) mice was examined.

Results: Anti-CD74 antibody SP7219 binds to both human and cyno CD74 ECD with high affinity (K_D=0.85nM for human, K_D=2.4nM for cyno). Tissue samples from the Department of Pathology at Stanford University were used to evaluate CD74 expression by IHC after appropriate permissions were obtained. Medium to high CD74 expression in >70% of cells was observed in 118/118 (100%) diffuse large B-cell lymphoma (DLBCL), 148/151 (98%) follicular lymphoma and 19/21 (90%) mantle cell lymphoma (MCL) samples. In vitro cytotoxicity assays show potent activity of STRO-001 in a diverse panel of B-cell tumor lines with EC₅₀ values ranging from 0.17-13 nM (Table 1). CD74 cell surface expression is required for STRO-001 cytotoxicity but expression level does not correlate highly with in vitro potency (R² = 0.4154). STRO-001 exhibits dose-dependent tumor growth inhibition in rituximab-resistant germinal center B-cell-like (GCB)-DLBCL SU-DHL-6 xenografts starting at 2.5 mg/kg weekly x 3 doses. STRO-001 + bendamustine/rituximab (BR) further improves tumor suppression in SU-DHL-6 xenografts compared to vehicle (p = 0.002) or BR alone (p = 0.02). In the activated B-cell (ABC)-DLBCL U2932 model, single doses of STRO-001 produce dose-dependent growth inhibition at 1 and 3 mg/kg and complete tumor regression at 10 mg/kg in 7/7 animals with no tumor re-growth up to > 90 days post-treatment (Figure 1). Studies with an MCL model, Jeko-1, demonstrate robust STRO-001 anti-tumor activity compared to vehicle (p < 0.0001) starting at a single 3 mg/kg dose, with a single 10 mg/kg dose resulting in tumor regression for up to 64 days post treatment (Figure 2). STRO-001 treatment 14 days post tumor inoculation was used to evaluate disease progression in the disseminated Mino MCL model. Vehicle-treated animals developed advanced progressive disease, with median survival of 81 days. In contrast, all Mino xenografts treated with STRO-001 at 3 mg/kg or 10 mg/kg were alive and disease-free at the time of sacrifice 135 days post tumor inoculation (Figure 3).

Conclusions: CD74 is expressed with high frequency and intensity in DLBCL, follicular lymphoma and MCL, supporting the investigation of novel biologics targeting this antigen. STRO-001 demonstrates potent in vitro cytotoxicity in multiple NHL cell lines and anti-tumor activity in GCB-DLBCL, ABC-DLBCL and mantle cell lymphoma xenograft models, including prolongation of survival in the disseminated Mino MCL model and prevention of tumor regrowth in the U2932 ABC-DLBCL and Jeko-1 MCL subcutaneous models. Clinical studies of this novel ADC for treatment of B-cell malignancies are under development.

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