Evaluation of PNU-159682 antibody drug conjugates (ADCs).

PNU-159682 is a highly potent secondary metabolite of nemorubicin belonging to the anthracycline class of natural products. Due to its extremely high potency and only partially understood mechanism of action, it was deemed an interesting starting point for the development of a new suite of linker drugs for antibody drug conjugates (ADCs). Structure activity relationships were explored on the small molecule which led to six linker drugs being developed for conjugation to antibodies. Herein we describe the synthesis of novel PNU-159682 derivatives and the subsequent linker drugs as well as the corresponding biological evaluations of the small molecules and ADCs.

A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo.

The high-grade pulmonary neuroendocrine tumors, small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), remain among the most deadly malignancies. Therapies that effectively target and kill tumor-initiating cells (TICs) in these cancers should translate to improved patient survival. Patient-derived xenograft (PDX) tumors serve as excellent models to study tumor biology and characterize TICs. Increased expression of delta-like 3 (DLL3) was discovered in SCLC and LCNEC PDX tumors and confirmed in primary SCLC and LCNEC tumors. DLL3 protein is expressed on the surface of tumor cells but not in normal adult tissues. A DLL3-targeted antibody-drug conjugate (ADC), SC16LD6.5, comprised of a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin, induced durable tumor regression in vivo across multiple PDX models. Serial transplantation experiments executed with limiting dilutions of cells provided functional evidence confirming that the lack of tumor recurrence after SC16LD6.5 exposure resulted from effective targeting of DLL3-expressing TICs. In vivo efficacy correlated with DLL3 expression, and responses were observed in PDX models initiated from patients with both limited and extensive-stage disease and were independent of their sensitivity to standard-of-care chemotherapy regimens. SC16LD6.5 effectively targets and eradicates DLL3-expressing TICs in SCLC and LCNEC PDX tumors and is a promising first-in-class ADC for the treatment of high-grade pulmonary neuroendocrine tumors.

PR-924, a selective inhibitor of the immunoproteasome subunit LMP-7, blocks multiple myeloma cell growth both in vitro and in vivo.

PR-924 is an LMP-7-selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N-terminal threonine active sites. In the present study, we show that PR-924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR-924-induced apoptosis in MM cells is associated with activation of caspase-3, caspase-8, caspase-9, BID, PARP and cytochrome-c release. In vivo administration of PR-924 inhibits tumour growth in human plasmacytoma xenografts. Results from SCID-hu model show a significant reduction in the shIL-6R levels in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumour-bearing mice with PR-924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP-7 subunit as a novel therapeutic target in MM.

A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis.

The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low-molecular mass polypeptide-7 (LMP7, encoded by Psmb8), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I-restricted antigens in vitro and in vivo. Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-gamma and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders.

Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (PR-047).

Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and non-Hodgkin's lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents. The lead compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (PR-047), selectively inhibited CT-L activity of both the constitutive proteasome (beta5) and immunoproteasome (LMP7) and demonstrated an absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral administration at doses resulting in >80% proteasome inhibition in most tissues and elicited an antitumor response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse syngeneic models. The favorable pharmacologic profile supports its further development for the treatment of malignant diseases.

Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome.

Clinical studies with bortezomib have validated the proteasome as a therapeutic target for the treatment of multiple myeloma and non-Hodgkin's lymphoma. However, significant toxicities have restricted the intensity of bortezomib dosing. Here we describe the antitumor activity of PR-171, a novel epoxyketone-based irreversible proteasome inhibitor that is currently in clinical development. In comparison to bortezomib, PR-171 exhibits equal potency but greater selectivity for the chymotrypsin-like activity of the proteasome. In cell culture, PR-171 is more cytotoxic than bortezomib following brief treatments that mimic the in vivo pharmacokinetics of both molecules. Hematologic tumor cells exhibit the greatest sensitivity to brief exposure, whereas solid tumor cells and nontransformed cell types are less sensitive to such treatments. Cellular consequences of PR-171 treatment include the accumulation of proteasome substrates and induction of cell cycle arrest and/or apoptosis. Administration of PR-171 to animals results in the dose-dependent inhibition of the chymotrypsin-like proteasome activity in all tissues examined with the exception of the brain. PR-171 is well tolerated when administered for either 2 or 5 consecutive days at doses resulting in >80% proteasome inhibition in blood and most tissues. In human tumor xenograft models, PR-171 mediates an antitumor response that is both dose and schedule dependent. The antitumor efficacy of PR-171 delivered on 2 consecutive days is stronger than that of bortezomib administered on its clinical dosing schedule. These studies show the tolerability, efficacy, and dosing flexibility of PR-171 and provide validation for the clinical testing of PR-171 in the treatment of hematologic malignancies using dose-intensive schedules.