Neutropenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC) and we aimed to elucidate the potential mechanism of this toxicity. To investigate whether ADCs affect neutrophil production from bone marrow, an assay was developed in which hematopoietic stem cells (HSC) were differentiated to neutrophils. Several antibodies against targets absent in HSCs and neutrophils were conjugated to MMAE via a cleavable valine-citrulline linker (vcMMAE-ADC) or MMAF via a noncleavable maleimidocaproyl linker (mcMMAF-ADC), and their cytotoxicity was tested in the neutrophil differentiation assay. Results showed that HSCs had similar sensitivity to vcMMAE-ADCs and mcMMAF-ADCs; however, vcMMAE-ADCs were more cytotoxic to differentiating neutrophils than the same antibody conjugated to mcMMAF. This inhibitory effect was not mediated by internalization of ADC either by macropinocytosis or FcγRs. Our results suggested that extracellular proteolysis of the cleavable valine-citrulline linker is responsible for the cytotoxicity to differentiating neutrophils. Mass spectrometry analyses indicated that free MMAE was released from vcMMAE-ADCs in the extracellular compartment when they were incubated with differentiating neutrophils or neutrophil conditioned medium, but not with HSC-conditioned medium. Using different protease inhibitors, our data suggested that serine, but not cysteine proteases, were responsible for the cleavage. experiments demonstrated that the purified serine protease, elastase, was capable of releasing free MMAE from a vcMMAE-ADC. Here we propose that ADCs containing protease cleavable linkers can contribute to neutropenia via extracellular cleavage mediated by serine proteases secreted by differentiating neutrophils in bone marrow. .

OBJECTIVES:Interleukin-23 (IL-23) has emerged as a new therapeutic target for the treatment of inflammatory bowel disease (IBD). As biomarkers of disease state and treatment efficacy are becoming increasingly important in drug development, we sought to identify efficacy biomarkers for anti-IL-23 therapy in Crohn's disease (CD).METHODS:Candidate IL-23 biomarkers, downstream of IL-23 signaling, were identified using shotgun proteomic analysis of feces and colon lavages obtained from a short-term mouse IBD model (anti-CD40 Rag2−/−) treated preventively with monoclonal antibodies (mAbs) to the IL-23 receptor (IL-23R). The biomarkers were then measured in an IBD T-cell transfer model treated therapeutically with a mAb to IL-23 (p19), confirming their association with IBD. To assess the clinical relevance of these markers, we assessed their concentrations in clinical serum, colon tissue, and feces from CD patients.RESULTS:We identified 57 proteins up or downregulated in diseased animals that returned to control values when the mice were treated with mAbs to IL-23R. Among those, S100A8, S100A9, regenerating protein 3β (REG), REG3γ, lipocalin 2 (LCN2), deleted in malignant tumor 1 (DMBT1), and macrophage migration inhibitory factor (MIF) mRNA levels correlated with disease score and dose titration of mAbs to IL-23R or IL-23(p19). All biomarkers, except DMBT1, were also downregulated after therapeutic administration of mAbs to IL-23(p19) in a T-cell transfer IBD mouse model. In sera from CD patients, we confirmed a significant upregulation of S100A8/A9 (43%), MIF (138%), pancreatitis-associated protein (PAP, human homolog of REG3β/γ 49%), LCN2 (520%), and CCL20 (1280%), compared with control samples, as well as a significant upregulation of S100A8/A9 (887%), PAP (401%), and LCN2 (783%) in human feces from CD patients compared with normal controls.CONCLUSIONS:These studies identify multiple protein biomarkers downstream of IL-23 that could be valuable tools to assess the efficacy of this new therapeutic agent.

<p>Figure S1. Chemical structures of MMAF, MMAE and 13C5-MMAE. The asterisks indicate where Carbon-13 isotope has replaced Carbon-12; Figure S2. Expression of CD32 on mature neutrophils. Circulating neutrophils (AllCell) were incubated with anti-CD32 antibody conjugated with FITC (CD32-FITC) or control IgG1-FITC, and were subjected to FACS analysis. Typical results from one experiment were shown; Figure S3. Little expression of SLC44A4 and ENPP3 on neutrophils at different differentiation stages. Microarray analyses were done for SLC44A4 (AGS5 target) or ENPP3 (AGS16 target) in HSCs, differentiating neutrophils at Day 8 or Day 14, or circulating neutrophils (mature); Figure S4. AGS5-vcMMAF conjugation did not affect intracellular cleavage. PC3 cells overexpressing SLC44A4, PC3-SLC44A4 were incubated with indicated concentrations of AGS5-vcMMAE, AGS5-vcMMAF or AGS5-mcMMAF for 6 days; Table S1: Information about ADCs used in this report; Table S2: Chromatographic gradient; Table S3: MRM Transitions monitored; Table S4. CD66b expression during neutrophil differentiation; Table S5. Correlation between sensitivity and extracellular cleavage.</p>

<p>Figure S1. Chemical structures of MMAF, MMAE and 13C5-MMAE. The asterisks indicate where Carbon-13 isotope has replaced Carbon-12; Figure S2. Expression of CD32 on mature neutrophils. Circulating neutrophils (AllCell) were incubated with anti-CD32 antibody conjugated with FITC (CD32-FITC) or control IgG1-FITC, and were subjected to FACS analysis. Typical results from one experiment were shown; Figure S3. Little expression of SLC44A4 and ENPP3 on neutrophils at different differentiation stages. Microarray analyses were done for SLC44A4 (AGS5 target) or ENPP3 (AGS16 target) in HSCs, differentiating neutrophils at Day 8 or Day 14, or circulating neutrophils (mature); Figure S4. AGS5-vcMMAF conjugation did not affect intracellular cleavage. PC3 cells overexpressing SLC44A4, PC3-SLC44A4 were incubated with indicated concentrations of AGS5-vcMMAE, AGS5-vcMMAF or AGS5-mcMMAF for 6 days; Table S1: Information about ADCs used in this report; Table S2: Chromatographic gradient; Table S3: MRM Transitions monitored; Table S4. CD66b expression during neutrophil differentiation; Table S5. Correlation between sensitivity and extracellular cleavage.</p>