No abstract

Antibody-drug conjugates (ADC) utilizing noncleavable linker drugs have been approved for clinical use, and several are in development targeting solid and hematologic malignancies including multiple myeloma. Currently, there are no reliable biomarkers of activity for these ADCs other than presence of the targeted antigen. We observed that certain cell lines are innately resistant to such ADCs, and sought to uncover the underlying mechanism of resistance. The expression of 43 lysosomal membrane target genes was evaluated in cell lines resistant to ADCs bearing the noncleavable linker, pyrrolobenzodiazepine payload SG3376, The functional relevance of SLC46A3, a lysosomal transporter of noncleavable ADC catabolites whose expression uniquely correlated with SG3376 resistance, was assessed using EPHA2-, HER2-, and BCMA-targeted ADCs and isogenic cells overexpressing or genetically inactivated for expression was also examined in patient-derived xenograft and models of acquired T-DM1 resistance and multiple myeloma bone marrow samples by RT-PCR. Loss of expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376. Sensitivity was restored in refractory lines upon introduction of, suggesting that expression of may be more predictive of activity than target antigen levels alone. Interrogation of primary multiple myeloma samples indicated a range of expression, including samples with undetectable levels like multiple myeloma cell lines resistant to BCMA-targeting DM1 and SG3376 ADCs. Our findings support as a potential patient selection biomarker with immediate relevance to clinical trials involving these ADCs.

Immunogenic cell death (ICD) is the process by which certain cytotoxic drugs induce apoptosis of tumor cells in a manner that stimulates the immune system. In this study, we investigated whether antibody-drug conjugates (ADCS) conjugated with pyrrolobenzodiazepine dimer (PBD) or tubulysin payloads induce ICD, modulate the immune microenvironment, and could combine with immuno-oncology drugs to enhance antitumor activity. We show that these payloads on their own induced an immune response that prevented the growth of tumors following subsequent tumor cell challenge. ADCs had greater antitumor activity in immunocompetent versus immunodeficient mice, demonstrating a contribution of the immune system to the antitumor activity of these ADCs. ADCs also induced immunologic memory. In the CT26 model, depletion of CD8 T cells abrogated the activity of ADCs when used alone or in combination with a PD-L1 antibody, confirming a role for T cells in antitumor activity. Combinations of ADCs with immuno-oncology drugs, including PD-1 or PD-L1 antibodies, OX40 ligand, or GITR ligand fusion proteins, produced synergistic antitumor responses. Importantly, synergy was observed in some cases with suboptimal doses of ADCs, potentially providing an approach to achieve potent antitumor responses while minimizing ADC-induced toxicity. Immunophenotyping studies in different tumor models revealed broad immunomodulation of lymphoid and myeloid cells by ADC and ADC/immuno-oncology combinations. These results suggest that it may be possible to develop novel combinatorial therapies with PBD- and tubulysin-based ADC and immuno-oncology drugs that may increase clinical responses. .

Mutations in the STK11 (LKB1) gene regulate resistance to PD-1/PD-L1 blockade. This study evaluated this association in patients with nonsquamous non-small-cell lung cancer enrolled in three Phase 1/2 trials. STK11 mutations were associated with resistance to the anti-PD-L1 antibody durvalumab (alone/with the anti-CTLA-4 antibody tremelimumab) independently of KRAS mutational status, highlighting STK11 as a potential driver of resistance to checkpoint blockade. Retrospective assessments of tumor tissue, whole blood and serum revealed a unique immune phenotype in patients with STK11 mutations, with increased expression of markers associated with neutrophils (i.e. CXCL2, IL6), Th17 contexture (i.e. IL17A) and immune checkpoints. Associated changes were observed in the periphery. Reduction of STAT3 in the tumor microenvironment using an antisense oligonucleotide reversed immunotherapy resistance in preclinical STK11 knockout models. These results suggest that STK11 mutations may hinder response to checkpoint blockade through mechanisms including suppressive myeloid cell biology, which could be reversed by STAT3-targeted therapy. SIGNIFICANCEPatients with nonsquamous STK11mut NSCLC are less likely than STK11wt patients to respond to anti-PD-L1 ± anti-CTLA-4 immunotherapies, and their tumors show increased expression of genes and cytokines that activate STAT3 signaling. Preclinically, STAT3 modulation reverses this resistance, suggesting STAT3-targeted agents as potential combination partners for immunotherapies in STK11mut NSCLC.

Two cell lines, PICM-19H and PICM-19B, were derived from the bipotent PICM-19 pig liver stem cell line and assessed for their potential application in artificial liver devices (ALD). The study included assessments of growth rate and cell density in culture, morphological features, serum protein production, gamma-glutamyltranspeptidase (GGT) activity and hepatocyte detoxification functions, i.e., inducible P450 activity, ammonia clearance, and urea production. The PICM-19H cell line was derived by temperature selection at 33-34 degrees C. After each passage, PICM-19H cells grew to a nearly confluent monolayer of cells of hepatocyte morphology, i.e., cuboidal cells with centrally located nuclei joined by biliary canaliculi. No differentiation and self-organization into multi-cellular bile ductules, as observed in the parental PICM-19 cell line, occurred within the PICM-19H cell monolayers. The PICM-19H cells contained numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies and occasional lipid vacuoles. The cells had a doubling time of 48-72 h and reached a final density of 1.5 x 10(5) cells/cm(2) at approximately10 d post-passage from a 1:6 split ratio. PICM-19H cells displayed inducible P450 activity, cleared ammonia, and produced urea in a glutamine-free medium. The PICM-19B cells were colony-cloned after spontaneous generation from the PICM-19 parental cell line. PICM-19B cells grew as a tightly knit dome-forming monolayer with no visible biliary canaliculi. Their doubling time was 48-72 h with a final cell density of 2.6 x 10(5) cells/cm(2). Ultrastructural analysis of the PICM-19B monolayers showed the roughly cuboidal cells displayed basal-apical polarization and were joined by tight junction-like complexes. Other ultrastructure features were similar to those of PICM-19H cells except that they possessed numerous cell bodies resembling mucus vacuoles. The PICM-19B cells had relatively high levels of GGT activity, but did retain some inducible P450 activity, and some ammonia clearance and urea synthesis ability. PICM-19B cells produced markedly less serum proteins than PICM-19H cells. These data indicated that both cell lines, either together or alone, may be useful as the cellular substrate for an ALD.