PCA062, a P-cadherin Targeting Antibody-Drug Conjugate, Displays Potent Antitumor Activity Against P-cadherin-expressing Malignancies.

The cell surface glycoprotein P-cadherin is highly expressed in a number of malignancies, including those arising in the epithelium of the bladder, breast, esophagus, lung, and upper aerodigestive system. PCA062 is a P-cadherin specific antibody-drug conjugate that utilizes the clinically validated SMCC-DM1 linker payload to mediate potent cytotoxicity in cell lines expressing high levels of P-cadherin in vitro, while displaying no specific activity in P-cadherin-negative cell lines. High cell surface P-cadherin is necessary, but not sufficient, to mediate PCA062 cytotoxicity. In vivo, PCA062 demonstrated high serum stability and a potent ability to induce mitotic arrest. In addition, PCA062 was efficacious in clinically relevant models of P-cadherin-expressing cancers, including breast, esophageal, and head and neck. Preclinical non-human primate toxicology studies demonstrated a favorable safety profile that supports clinical development. Genome-wide CRISPR screens reveal that expression of the multidrug-resistant gene ABCC1 and the lysosomal transporter SLC46A3 differentially impact tumor cell sensitivity to PCA062. The preclinical data presented here suggest that PCA062 may have clinical value for treating patients with multiple cancer types including basal-like breast cancer.

GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells.

Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein-coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell-derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

Preclinical Antitumor Activity of a Novel Anti-c-KIT Antibody-Drug Conjugate against Mutant and Wild-type c-KIT-Positive Solid Tumors.

Purpose: c-KIT overexpression is well recognized in cancers such as gastrointestinal stromal tumors (GIST), small cell lung cancer (SCLC), melanoma, non-small cell lung cancer (NSCLC), and acute myelogenous leukemia (AML). Treatment with the small-molecule inhibitors imatinib, sunitinib, and regorafenib resulted in resistance (c-KIT mutant tumors) or limited activity (c-KIT wild-type tumors). We selected an anti-c-KIT ADC approach to evaluate the anticancer activity in multiple disease models.Experimental Design: A humanized anti-c-KIT antibody LMJ729 was conjugated to the microtubule destabilizing maytansinoid, DM1, via a noncleavable linker (SMCC). The activity of the resulting ADC, LOP628, was evaluated in vitro against GIST, SCLC, and AML models and in vivo against GIST and SCLC models.Results: LOP628 exhibited potent antiproliferative activity on c-KIT-positive cell lines, whereas LMJ729 displayed little to no effect. At exposures predicted to be clinically achievable, LOP628 demonstrated single administration regressions or stasis in GIST and SCLC xenograft models in mice. LOP628 also displayed superior efficacy in an imatinib-resistant GIST model. Further, LOP628 was well tolerated in monkeys with an adequate therapeutic index several fold above efficacious exposures. Safety findings were consistent with the pharmacodynamic effect of neutropenia due to c-KIT-directed targeting. Additional toxicities were considered off-target and were consistent with DM1, such as effects in the liver and hematopoietic/lymphatic system.Conclusions: The preclinical findings suggest that the c-KIT-directed ADC may be a promising therapeutic for the treatment of mutant and wild-type c-KIT-positive cancers and supported the clinical evaluation of LOP628 in GIST, AML, and SCLC patients. Clin Cancer Res; 24(17); 4297-308. ©2018 AACR.

A synthetic lethal screen reveals enhanced sensitivity to ATR inhibitor treatment in mantle cell lymphoma with ATM loss-of-function.

UNLABELLED: Mechanisms to maintain genomic integrity are essential for cells to remain viable. Not surprisingly, disruption of key DNA damage response pathway factors, such as ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia and RAD3-related (ATR) results in loss of genomic integrity. Here, a synthetic lethal siRNA-screening approach not only confirmed ATM but identified additional replication checkpoint proteins, when ablated, enhanced ATR inhibitor (ATRi) response in a high-content γ-H2AX assay. Cancers with inactivating ATM mutations exhibit impaired DNA double-stranded break (DSB) repair and rely on compensatory repair pathways for survival. Therefore, impairing ATR activity may selectively sensitize cancer cells to killing. ATR inhibition in an ATM-deficient context results in phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) and leads to induction of γ-H2AX. Using both in vitro and in vivo models, ATR inhibition enhanced efficacy in ATM loss-of-function mantle cell lymphoma (MCL) compared with ATM wild-type cancer cells. In summary, single-agent ATR inhibitors have therapeutic utility in the treatment of cancers, like MCL, in which ATM function has been lost. IMPLICATIONS: These data suggest that single-agent ATR inhibitors have therapeutic utility and that ATR uses a complex and coordinated set of proteins to maintain genomic stability that could be further exploited.

Vemurafenib cooperates with HPV to promote initiation of cutaneous tumors.

Treatment with RAF inhibitors such as vemurafenib causes the development of cutaneous squamous cell carcinomas (cSCC) or keratoacanthomas as a side effect in 18% to 30% of patients. It is known that RAF inhibitors activate the mitogen-activated protein kinase (MAPK) pathway and stimulate growth of RAS-mutated cells, possibly accounting for up to 60% of cSCC or keratoacanthoma lesions with RAS mutations, but other contributing events are obscure. To identify such events, we evaluated tumors from patients treated with vemurafenib for the presence of human papilloma virus (HPV) DNA and identified 13% to be positive. Using a transgenic murine model of HPV-driven cSCC (K14-HPV16 mice), we conducted a functional test to determine whether administration of RAF inhibitors could promote cSCC in HPV-infected tissues. Vemurafenib treatment elevated MAPK markers and increased cSCC incidence from 22% to 70% in this model. Furthermore, 55% of the cSCCs arising in vemurafenib-treated mice exhibited a wild-type Ras genotype, consistent with the frequency observed in human patients. Our results argue that HPV cooperates with vemurafenib to promote tumorigenesis, in either the presence or absence of RAS mutations.

Neutralization of prolactin receptor function by monoclonal antibody LFA102, a novel potential therapeutic for the treatment of breast cancer.

Numerous lines of evidence suggest that the polypeptide hormone prolactin (PRL) may contribute to breast and prostate tumorigenesis through its interactions with the prolactin receptor (PRLR). Here, we describe the biologic properties of LFA102, a humanized neutralizing monoclonal antibody directed against the extracellular domain of PRLR. This antibody was found to effectively antagonize PRL-induced signaling in breast cancer cells in vitro and in vivo and to block PRL-induced proliferation in numerous cell line models, including examples of autocrine/paracrine PRL activity. A single administration of LFA102 resulted in regression of PRL-dependent Nb2-11 tumor xenografts and significantly prolonged time to progression. Finally, LFA102 treatment significantly inhibited PRLR signaling as well as tumor growth in a carcinogen-induced, estrogen receptor-positive rat mammary cancer model as a monotherapy and enhanced the efficacy of the aromatase inhibitor letrozole when administered in combination. The biologic properties of LFA102, elucidated by the preclinical studies presented here, suggest that this antibody has the potential to be a first-in-class, effective therapeutic for the treatment of PRL-dependent cancers.

Cutaneous pythiosis in a nontravelled California horse.

An 18-year-old Arabian mare was examined with a large mass on the left hind pastern and fetlock. The mare was located in the Central Valley of northern California, and had never been out of the state. Routine histopathological processing and examination of biopsy samples from the mass showed several hyphal organisms that were delineated with a silver stain. Using immunohistochemistry the organism was diagnosed as Pythium insidiosum. The owner declined debulking surgery, and despite treatment with an immunotherapeutic vaccine, the horse's condition deteriorated leading to euthanasia.

Mechanisms underlying intranuclear rod formation.

Specific mutations within the alpha-skeletal actin gene (ACTA1) result in intranuclear rod myopathy (IRM), characterized by rod-like aggregates containing actin and alpha-actinin-2 inside the nucleus of muscle cells. The mechanism leading to formation of intranuclear aggregates containing sarcomeric proteins and their impact on cell function and contribution to disease pathogenesis is unknown. In this study, we transfected muscle and non-muscle cells with mutants of alpha-skeletal actin (Val163Leu, Val163Met) associated with intranuclear rod myopathy. By live-cell imaging we demonstrate that nuclear aggregates of actin form within the nuclear compartment, rather than entering the nucleus after formation in the cytoplasm, and are highly motile and dynamic structures. Thus, the nuclear environment supports the polymerization of actin and the movement and coalescence of the polymerized actin into larger structures. We show that the organization of actin within these aggregates is influenced by the binding of alpha-actinin, and that alpha-actinin is normally present in the nucleus of muscle and non-muscle cells. Furthermore, we demonstrate that, under conditions of cell stress (cytoskeletal disruption and ATP depletion), WT skeletal actin forms aggregates within the nucleus that are similar in morphology to those formed by the mutant actin, suggesting a common pathogenic mechanism for aggregate formation. Finally, we show that the presence of intranuclear actin aggregates significantly decreases the mitotic index and hence impacts on the function of the cell. Intranuclear aggregates thus likely contribute to the pathogenesis of muscle weakness in intranuclear rod myopathy.

Ultrastructure of in situ perfusion-fixed avian liver, with special reference to structure of the sinusoids.

Broiler chicken and laying hen livers were fixed using a simple technique of in situ puncture perfusion of cacodylate-buffered fixative, which allowed characterisation of the fine structure of hepatic parenchyma, hepatocytes, bile ductules, and, in particular, the sinusoidal cells including endothelial, Kupffer, and Ito cells. Sinusoidal endothelial cells with their bulging perinuclear cytoplasm, evident in both transmission and scanning electron micrographs, were easily distinguishable from Kupffer cells, which possessed numerous pseudopodia. Bile ductular epithelium and hepatocytes of the laying hens contained large amounts of lipid. The ultrastructural characteristics of intercalated cells (putative extra-sinusoidal macrophages of chicken liver) are described and their possible role as precursors of Kupffer cells is discussed.