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Supplemental Figures 1-5. Supplemental Figure 1. Kinase selectivity profile of ATRi. Supplemental Figure 2. Screening of inhibitors in cellular γ-H2AX assay in U2OS cells. Supplemental Figure 3. Flow cytometry profiles assessing γ-H2AX response to compare antimetabolite combinations with a fixed concentration of ATRi in U2OS cells. Supplemental Figure 4. γ-H2AX response and cell viability following ATRi treatment in U2OS cells. Supplemental Figure 5. In vivo pharmacokinetics and γ-H2AX pharmacodynamics of ATRi in GRANTA-519 (ATM mut) MCL tumor xenograft model.

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Figures S1, S2, s3, S4, Table S1

PDF file - 77KB, Pharmacodynamic effects of LFA102 in the rat mammary gland

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. Mol Cancer Ther; 12(3); 295–305. ©2012 AACR.

Supplemental Table 2. SiRNA sequences (Supplemental data)

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Supplemental Table 1. DiscoveRx ATR inhibitor profile

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.

Supplemental Figure legends. Supplemental Figure 1. Kinase selectivity profile of ATRi. Supplemental Figure 2. Screening of inhibitors in cellular γ-H2AX assay in U2OS cells. Supplemental Figure 3. Flow cytometry profiles assessing γ-H2AX response to compare antimetabolite combinations with a fixed concentration of ATRi in U2OS cells. Supplemental Figure 4. γ-H2AX response and cell viability following ATRi treatment in U2OS cells. Supplemental Figure 5. In vivo pharmacokinetics and γ-H2AX pharmacodynamics of ATRi in GRANTA-519 (ATM mut) MCL tumor xenograft model.

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. Mol Cancer Res; 13(1); 120–9. ©2014 AACR.

PDF file - 55KB, Pharmacokinetic properties of LFA102 in rodents

Cell line sensitivity table

Material and Methods

Figure S1. RNASeq data across multiple tumor types indicate c-KIT overexpression in a subset of cancer types such as AML, melanoma, testicular germ cell cancer and lung cancer. This was obtained from the TCGA data set. Figure S2. Western blot analysis of phosphorylated c-KIT and total c-KIT protein levels. Cells were starved overnight in 0.1% serum and then left untreated or treated with 10 ng/ml of SCF +/- 5 ug/ml of ADC together for 15 minutes before preparing cell lysates for immunoblotting. Figure S3. Correlation between c-KIT expression level and sensitivity to LOP628. GIST, SCLC and AML cell lines were analyzed for surface c-KIT expression by flow cytometry and assessed for sensitivity to LOP cell proliferation assays. Sensitivity to LOP628 was enriched if the cell line expressed >20,000 surface c-KIT receptors. Figure S4. Representative images of c-KIT immunostained GIST-T1, GIST430, NCI-H1048 and NCI-H2170 tumor xenografts in SCID-beige mice Figure S5. Lack of LOP628 efficacy in the c-KIT low/negative NCI-H2170 tumor xenograft model in SCID-beige mice after a single 10 mg/kg i.v. dose. Table S1. Activity of antagonist vs non-ligand blocking c-Kit ADCs in panel of GIST, AML and SCLC cell lines. Cell line sensitivity to the S-DM1-ME payload was also evaluated. There were no significant differences in activity between the two classes of ADCs. (A) refers to antagonist or ligand-blocking Abs. Table S2. Surface c-KIT expression on human and cynomolgus monkey mast cells and bone marrow cells as quantified by IHC or flow cytometry.

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.

PDF file - 507K, Koilocyte-like cells observed in an HPV negative verrucous papilloma (S1); H&E and pERK staining of gastric epithelium from mice treated for 60 days with 60 mg/kg Vemurafenib (S2); pERK staining of Vemurafenib and vehicle treated cSCC (S3); Reduced hyperkeratosis and improved grooming of K14-HPV16 mice treated with PD0325901 (S4); cSCC tumor response after treatment with 3 mg/kg PD0325901 (S5).

PDF file - 50K, RAS mutation and HPV status of cutaneous lesions by histological type.

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. Cancer Res; 74(8); 2238–45. ©2014 AACR.

BackgroundWhile CAR-T have demonstrated potent activity against hematologic tumors, less success has been seen with solid tumors. Here we report generation of TSCM-enriched allogeneic MUC1-C-specific CAR T cells, P-MUC1C-ALLO1, with potential for a broad range of solid tumors. The proliferative capacity and metabolic profile of TSCM CAR-T are well-suited to activity in the solid tumor setting. MUC1 is comprised of an N-terminal subunit (MUC1-N) tethered to a C-terminal subunit (MUC1-C), forming a stable complex on the cell surface. During tumorigenesis, MUC1 becomes both overexpressed and hypo-glycosylated on many carcinomas. Furthermore, MUC1 undergoes proteolytic cleavage in the tumor microenvironment, leaving behind a proteolytic ‘stump’ of MUC1-C that is over-represented in cancer, making it an attractive therapeutic target.MethodsP-MUC1C-ALLO1 is manufactured using the piggyBac® DNA Delivery System for CAR insertion and the Cas-CLOVER™ Gene Editing System to knockout both the TCR and MHC class I proteins. The addition of a selectable marker within the transposon allows for selection of a fully CAR-positive population while any residual TCR-positive cells are removed at the end of production to prevent TCR-mediated GvHD. Lastly, inclusion of a proprietary ‘booster molecule’ in our allogeneic process further improves cell expansion, along with phenotype and function, and enables the production of up to hundreds of patient doses from a single manufacturing run.ResultsSignificant doses of P-MUC1C-ALLO1 products made from multiple healthy donors were achieved and comprised of an exceptionally high-percentage of desirable TSCM cells. Preclinical evaluation of these products showed potent tumor killing and cytokine secretion against MUC1-C-positive breast and ovarian tumor cell lines. P-MUC1C-ALLO1 demonstrates potent cytotoxicity against tumor cells, and minimal killing of normal MUC1-C-positive human primary cells. In a triple negative breast cancer xenograft model, MUC1C CAR-T eliminated established MDA-MB-468 tumor cells, mounted robust T cell expansion in peripheral blood and maintained a favorable TSCM percentage over time. Likewise, in an orthotopic ovarian cancer xenograft model, intraperitoneally administered MUC1C CAR-T eliminated established OVCAR3 cells to levels below the limit of detection. All together, these data demonstrated the efficacy of the MUC1C CAR-T cells and the robustness of the allogeneic platform.ConclusionsP-MUC1C-ALLO1 is an allogeneic TSCM CAR-T therapy that has a potential to treat multiple MUC1-expressing indications. P-MUC1C-ALLO1 displayed specificity for tumor vs. normal cells as well as in vivo efficacy against xenograft models of breast and ovarian cancer. This allogeneic cell therapy is advancing rapidly towards the clinic.

Background MUC1 is a highly glycosylated protein that is expressed at the apical border of mucosal epithelium where it plays a protective role. MUC1 is comprised of an N-terminal subunit (MUC1N) tethered to a C-terminal subunit (MUC1C), forming a stable complex on the cell surface. A proteolytic ‘stump’ of MUC1C that may be aberrantly glycosylated is over-represented in cancer, making it an attractive therapeutic target. Here we report generation of allogeneic MUC1C-specific CAR T cells, P-MUC1C-ALLO1, that are designed to leverage the learnings of our P-BCMA-ALLO1 program. P-MUC1C-ALLO1 targets a MUC1C epitope and has the potential for efficacy against a wide range of solid tumors, without targeting normal epithelial cells. Methods mRNA-generated MUC1C CAR-T cells were evaluated for specificity and function by degranulation assay against various solid tumor and normal cells and cell lines. Autologous and allogeneic MUC1C CAR-T cells were produced using the piggyBac® DNA Modification System, a nonviral CAR-T manufacturing method that produces CAR-T products with an exceptionally high percentage of T stem cell memory (TSCM) cells. To produce allogeneic cells, multiplex editing of both TRBC and B2M was performed with the Cas-CLOVER™ Site-Specific Gene Editing System to reduce or eliminate GvHD and host versus graft alloreactivity, respectively. To determine in vivo antitumor efficacy of MUC1C CAR-T cells, we employed the MDA.MB.468 triple negative breast cancer model and the OVCAR3 disseminated ovarian cancer model. Results Specific degranulation of transiently-expressing CAR+ T cells was observed against multiple tumor cells, with no observable activity against normal human primary cells. In assay of stable P-MUC1C-101 CAR-T cells, more than 95% expressed CAR, and were comprised of an exceptionally high-percentage of TSCM cells (CD45RA+CD62L+CD45RO-). In vitro, P-MUC1C-ALLO1 cells specifically proliferated, lysed, and secreted IFN-γ against MUC1C+ breast and ovarian tumor cell lines. In breast cancer in vivo xenograft model, both unedited (MUC1C CAR-T) and edited (P-MUC1C-ALLO1) MUC1C CAR-T eliminated established, triple negative MDA.MB.468 tumor cells to undetectable levels, demonstrating the efficacy of the MUC1C CAR-T and the robustness of the allogeneic platform. In the OVCAR3 xenograft model, intraperitoneally administered MUC1C CAR-T eliminated established tumor cells to levels below the limit of detection. Conclusions P-MUC1C-ALLO1 is Poseida’s allogeneic CAR TSCM product that has a potential to treat multiple MUC1-expressing indications. P-MUC1C-ALLO1 displayed in vitro specificity for tumor vs normal cells, and in vivo efficacy against xenograft models of breast and ovarian cancer. We anticipate an IND filing and initiation of a Phase 1 clinical trial in 2021.

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.

P-BCMA-101 and P-BCMA-ALLO1, autologous and allogeneic BCMA targeting CAR-T cell therapies respectively, are manufactured using a novel transposon-based system called piggyBac (PB). They comprise a high percentage of desirable stem cell memory T-cells associated with efficacy and safety. Single agent P-BCMA-101 data was previously reported showing marked efficacy and minimal toxicity (PRIME study), and exploratory combination cohorts have also been evaluated. P-BCMA-ALLO1 is expected to be first assessed in patients by the time of this presentation. Here we report results for P-BCMA-101 exploratory cohorts and P-BCMA-ALLO1. PRIME is a Phase 1/2 clinical trial to assess the safety and efficacy of P-BCMA-101 in patients with RRMM (≥ 3 prior lines, including a proteasome inhibitor (PI) and an immunomodulatory agent (IMiD), or double refractory) (NCT03288493). Patients undergo apheresis to harvest T cells and P-BCMA-101 is then manufactured. Patients then receive a 3-day cyclophosphamide (300 mg/m 2/day) / fludarabine (30 mg/m 2/day) lymphodepletion (LDC) regimen. Following 2 days of rest, patients received a single administration of P-BCMA-101 in escalating doses starting at 0.75 x 10 6 CAR-T cells/kg. Similar P-BCMA-101 dose escalation was performed in combination cohorts with rituximab (Rit) or lenalidomide (Len). During the study, use of a nanoplasmid (NP) vector in CAR-T manufacturing was implemented to improve transposition efficiency and cell quality. As of June 30 th, 2021, 90 unique patients have been treated with P-BCMA-101 in 5 dose levels of single agent P-BCMA-101, 3 dose levels of P-BCMA-101 with Rit and 2 dose levels of P-BCMA-101 with Len. The median age is 61 years and 66/37% were M/F. Patients were heavily pre-treated with a median of 6 prior regimens (range 3-18), 100% had received prior PI and IMiD, 74% daratumumab and 63% ASCT. 13 patients were treated in combination with Rit and 9 in combination with Len. Like prior reports of P-BCMA-101, the safety profile compares favorably to other CAR-T. No dose limiting toxicities (DLT) were observed in the single agent or the combination cohorts. The addition of Rit or Len did not increase toxicity. Grade 1-2 CRS was seen in 25% (no G3/4) of patients and ICANS in 7% (2% G3). Tocilizumab was used in 11% of patients. There were no treatment related deaths or unexpected/off-target toxicities. The most common treatment emergent adverse events were cytopenias, infections and constitutional symptoms (≥ G3 neutropenia 74%, thrombocytopenia 30%, anemia 35%), as expected with CAR-T and LDC. The overall response rate (ORR) with the Rit and Len combinations was 73% and 71% respectively. The favorable safety profile allowed outpatient treatment in 23 (25%) patients. Founded on the P-BCMA-101 data, an allogeneic CAR-T (P-BCMA-ALLO1) using a similar PB construct was created and demonstrated excellent preclinical safety and efficacy leading to the initiation of a Phase 1 clinical trial (NCT04960579). P-BCMA-ALLO1 CAR-T cells are generated from healthy donor T-cells using Cas-CLOVER to knockout Beta-2 microglobulin to prevent host vs graft rejection, TCRβchain for graft vs host disease prevention. Proprietary booster molecules are utilized to increase manufacturing yield and cell quality. The CAR binding domain is a novel anti-BCMA VCAR that produced superior efficacy in tumor models. These constructs contain the same PB transposon transgene cassette, DHFR for gene selection and an iCasp9-based safety switch, successfully assessed in the PRIME study. The primary objective of the P-BCMA-ALLO1 phase 1 trial is to assess the safety and maximum tolerated dose (MTD) based on DLT in RRMM patients who have received a PI, IMid and CD38 mAb. Secondary objectives will assess the anti-myeloma effect of P-BCMA-ALLO1 and biomarkers. The protocol is designed to treat 40 RRMM patients in a standard 3+3 dose escalation with a P-BCMA-ALLO1 starting dose of 0.75 X 10 6 cells/kg. Patients will receive P-BCMA-ALLO1 following LDC with cyclophosphamide (300 mg/m 2/day) / fludarabine (30 mg/m 2/day X 3 days. In conclusion, transposon generated autologous CAR-T cells demonstrate excellent clinical activity with a favorable toxicity profile in RRMM, can be safely combined with Len and Rit, and administered in the outpatient setting. Allogeneic CAR-T cells generated from this platform represent a further advance. Current data from both clinical studies will be presented. Disclosures Derman: Sanofi: Membership on an entity's Board of Directors or advisory committees. Deol: Kite, a Gilead Company: Consultancy. Ali: BMS: Research Funding; Aduro: Research Funding; Poseida: Research Funding; Aduro: Consultancy; Amgen: Consultancy; Sanofi: Consultancy; Oncopeptides: Consultancy; BMS: Consultancy; Janssen: Consultancy. Dholaria: Pfizer: Research Funding; MEI: Research Funding; Poseida: Research Funding; Takeda: Research Funding; Janssen: Research Funding; Angiocrine: Research Funding; Jazz: Speakers Bureau; Celgene: Speakers Bureau. Berdeja: Bioclinica: Consultancy; BMS: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Lilly: Research Funding; Abbvie: Research Funding; EMD Sorono: Research Funding; Takeda: Consultancy, Research Funding; Prothena: Consultancy; Servier: Consultancy; Janssen: Consultancy, Research Funding; Karyopharm: Consultancy; Vivolux: Research Funding; Cellularity: Research Funding; Novartis: Research Funding; Poseida: Research Funding; CURIS: Research Funding; CRISPR Therapeutics: Consultancy, Research Funding; Acetylon: Research Funding; Amgen: Consultancy, Research Funding; Teva: Research Funding; Legend: Consultancy; Kite Pharma: Consultancy; Bluebird: Research Funding; Constellation: Research Funding; Glenmark: Research Funding; Genentech: Research Funding; Kesios: Research Funding. Cohen: GlaxoSmithKline: Consultancy, Research Funding; AstraZeneca: Consultancy; Genentech/Roche: Consultancy; BMS/Celgene: Consultancy; Novartis: Research Funding; Oncopeptides: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Patel: Oncopeptides: Consultancy; BMS Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Pfizer: Consultancy. Siegel: Karyopharm: Honoraria; Amgen Inc.: Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Honoraria, Speakers Bureau; GlaxoSmithKline: Honoraria, Speakers Bureau; Celularity: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau. Nath: Actinium: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. McArthur: Poseida: Current Employment, Current equity holder in publicly-traded company. McCaigue: Poseida: Current Employment, Current equity holder in publicly-traded company. Martin: Poseida: Current Employment, Current equity holder in publicly-traded company. Ghoddusi: Poseida: Current Employment, Current equity holder in publicly-traded company. Namini: Poseida: Current Employment, Current equity holder in publicly-traded company. Ostertag: Poseida: Current Employment, Current equity holder in publicly-traded company. Spear: Poseida: Current Employment, Current equity holder in publicly-traded company. Belani: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company; Amgen: Current equity holder in publicly-traded company. Shah: Indapta Therapeutics: Consultancy; CareDx: Consultancy; BMS/Celgene: Research Funding; Amgen: Consultancy; Janssen: Research Funding; Bluebird Bio: Research Funding; Precision Biosciences: Research Funding; Karyopharm: Consultancy; Nektar: Research Funding; Oncopeptides: Consultancy; Poseida: Research Funding; Sanofi: Consultancy; CSL Behring: Consultancy; Kite: Consultancy; GSK: Consultancy; Sutro Biopharma: Research Funding; Teneobio: Research Funding.

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.

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.

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. Mol Cancer Res; 13(1); 120–9. ©2014 AACR.

P-BCMA-101 is a novel chimeric antigen receptor (CAR)-T cell product targeting B Cell Maturation Antigen (BCMA). P-BCMA-101 is produced using the piggyBac® (PB) DNA Modification System instead of the viral vector that is used with most CAR-T cells, requiring only plasmid DNA and mRNA. This makes it less costly and produces cells with a high percentage of the favorable T stem cell memory phenotype (TSCM). The higher cargo capacity of PB permits the incorporation of multiple genes in addition to CAR(s), including a safety switch allowing for rapid CAR-T cell elimination with a small molecule drug infusion in patients if desired, and a selection gene allowing for enrichment of CAR+ cells. Rather than using a traditional antibody-based binder, P-BCMA-101 has a Centyrin™ fused to a CD3ζ/4-1BB signaling domain. Centyrins are fully human proteins with high specificity and a large range of binding affinities, but are smaller, more stable and potentially less immunogenic than traditional scFv. Cumulatively, these features are predicted to result in a greater therapeutic index. A Phase 1, 3+3 dose escalation from 0.75 to 15 x 106 P-BCMA-101 CAR-T cells/kg (RP2D 6-15 x 106 cells/kg) was conducted in patients with r/r MM (Blood 2018 132:1012) demonstrating excellent efficacy and safety of P-BCMA-101, including notably low rates and grades of CRS and neurotoxicity (maximum Grade 2 without necessitating ICU admission, safety switch activation or other aggressive measures). These results supported FDA RMAT designation and initiation of a pivotal Phase 2 study. A Phase 2 pivotal portion of this study has recently been designed and initiated (PRIME; NCT03288493) in r/r MM patients who have received at least 3 prior lines of therapy. Their therapy must have contained a proteasome inhibitor, an IMiD, and CD38 targeted therapy with at least 2 of the prior lines in the form of triplet combinations. They must also have undergone ≥2 cycles of each line unless PD was the best response, refractory to the most recent line of therapy, and undergone autologous stem cell transplant or not be a candidate. Patients are required to be >=18 years old, have measurable disease by International Myeloma Working Group criteria (IMWG; Kumar 2016), adequate vital organ function and lack significant autoimmune, CNS and infectious diseases. No pre-specified level of BCMA expression is required, as this has not been demonstrated to correlate with clinical outcomes for P-BCMA-101 and other BCMA-targeted CAR-T products. Interestingly, unlike most CAR-T products patients may receive P-BCMA-101 after prior CAR-T cells or BCMA targeted agents, and may be multiply infused with P-BCMA-101. Patients are apheresed to harvest T cells, P-BCMA-101 is then manufactured and administered to patients as a single intravenous (IV) dose (6-15 x 106 P-BCMA-101 CAR-T cells/kg) after a standard 3-day cyclophosphamide (300 mg/m2/day) / fludarabine (30 mg/m2/day) conditioning regimen. One hundred patients are planned to be treated with P-BCMA-101. Uniquely, given the safety profile demonstrated during Phase 1, no hospital admission is required and patients may be administered P-BCMA-101 in an outpatient setting. The primary endpoints are safety and response rate by IMWG criteria. With a 100-subject sample, the Phase 2 part of the trial will have 90% power to detect a 15-percentage point improvement over a 30% response rate (based on that of the recently approved anti-CD38 antibody daratumumab), using an exact test for a binomial proportion with a 1-sided 0.05 significance level. Multiple biomarkers are being assessed including BCMA and cytokine levels, CAR-T cell kinetics, immunogenicity, T cell receptor diversity, CAR-T cell and patient gene expression (e.g. Nanostring) and others. Overall, the PRIME study is the first pivotal study of the unique P-BCMA-101 CAR-T product, and utilizes a number of novel design features. Studies are being initiated in combination with approved therapeutics and earlier lines of therapy with the intent of conducting Phase 3 trials. Funding by Poseida Therapeutics and the California Institute for Regenerative Medicine (CIRM). Disclosures Costello: Takeda: Honoraria, Research Funding; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Gregory:Poseida: Research Funding; Celgene: Speakers Bureau; Takeda: Speakers Bureau; Amgen: Speakers Bureau. Ali:Celgene: Research Funding; Poseida: Research Funding. Berdeja:Amgen Inc, BioClinica, Celgene Corporation, CRISPR Therapeutics, Bristol-Myers Squibb Company, Janssen Biotech Inc, Karyopharm Therapeutics, Kite Pharma Inc, Prothena, Servier, Takeda Oncology: Consultancy; AbbVie Inc, Amgen Inc, Acetylon Pharmaceuticals Inc, Bluebird Bio, Bristol-Myers Squibb Company, Celgene Corporation, Constellation Pharma, Curis Inc, Genentech, Glenmark Pharmaceuticals, Janssen Biotech Inc, Kesios Therapeutics, Lilly, Novartis, Poseida: Research Funding; Poseida: Research Funding. Patel:Oncopeptides, Nektar, Precision Biosciences, BMS: Consultancy; Takeda, Celgene, Janssen: Consultancy, Research Funding; Poseida Therapeutics, Cellectis, Abbvie: Research Funding. Shah:University of California, San Francisco: Employment; Genentech, Seattle Genetics, Oncopeptides, Karoypharm, Surface Oncology, Precision biosciences GSK, Nektar, Amgen, Indapta Therapeutics, Sanofi: Membership on an entity's Board of Directors or advisory committees; Indapta Therapeutics: Equity Ownership; Celgene, Janssen, Bluebird Bio, Sutro Biopharma: Research Funding; Poseida: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Nkarta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees; Teneobio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ostertag:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Martin:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Ghoddusi:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Shedlock:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Spear:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Orlowski:Poseida Therapeutics, Inc.: Research Funding. Cohen:Poseida Therapeutics, Inc.: Research Funding.

Early clinical results using BCMA targeted CAR T cell therapies for advanced multiple myeloma (MM) have shown promise. However, BCMA expression can be variable, and BCMA downregulation has been correlated with relapse (Brudno J. JCO. 2018; Cohen A. ASH. 2017). Targeting multiple antigens may enhance response durability. We report that the orphan seven transmembrane G protein coupled receptor, GPRC5D, is an attractive additional target for CAR T cell therapy of MM. GPRC5D mRNA expression was previously identified in bone marrow cells from patients with MM; however its protein expression could not be detected with available FACS reagents (Frigyesi I. Blood. 2014). We evaluated 83 primary marrow samples by quantitative immunofluorescence (Q-IF) for CD138, BCMA, and GPRC5D. In 98% of the samples, CD138+ cells expressed surface GPRC5D. In most samples, the majority of CD138+ cells expressed both BCMA and GPRC5D, however, in several cases the dominant CD138+ population expressed only BCMA or GPRC5D, with GPRC5D expression independent of BCMA across samples (R2=0.156; Figure 1). The potential for "on target/off tumor" binding by targeting GPRC5D was evaluated by screening 30 essential normal tissue types by IHC (n=3 donors/type) followed by validation of any positive findings by RNA in situ hybridization and quantitative PCR. Results in non-plasma cell normal tissue were consistent with prior reports of GPRC5D off target expression restricted to cells from the hair follicle, a potentially immune privileged site. We developed GPRC5D-targeted CARs considering immunogenicity, spacer length, and tonic signaling. To minimize potential anti-CAR immunity, a human B cell-derived phage display library was screened. Seven diverse and highly specific human single chain variable fragments (scFvs) were identified. 42 CARs were derived from the 7 scFvs by modifying scFv orientation (VH/VL; VL/VH) and incorporating a short, medium, or long IgG4 based spacer. To monitor CAR-mediated signaling, we transduced each CAR into a Jurkat reporter line with RFP inserted in-frame, downstream of endogenous NR4A1 (Nur77). Nur77 expression is an immediate-early, specific marker of CD3z signaling (Ashouri J. J Immunol. 2017). Using this reporter, we observed that (1) a long spacer provided enhanced antigen-dependent activation across all anti-GPRC5D CARs; and (2) the use of different scFvs resulted in vastly different levels of tonic signaling. We selected potential lead and backup constructs and evaluated CAR activity on primary human T cells. When co-cultured specifically with MM cell lines, anti-GPRC5D CAR T cells secreted a polyfunctional cytokine profile, proliferated, and effectively lysed target cells. CD138+/CD38hi primary MM bone marrow aspirate cells were also specifically lysed. In vivo efficacy of GPRC5D-targeted CAR T cells was evaluated in NSG mice engrafted with a human MM cell line (ffLuc+) bone marrow xenograft. Donor T cells were gene-modified to express anti-GPRC5D CARs with either a 4-1BB or a CD28 co-stimulatory domain and membrane-anchored Gaussia luciferase (GLuc). Compared to control CAR T cells specific for an irrelevant target, anti-GPRC5D CAR T cells with either co-stimulatory domain proliferated and homed to the site of MM (Gluc imaging), eradicated MM xenograft (ffLuc imaging), and increased survival (Figure 2). One scFv that was highly functional in our GPRC5D CAR screen was evaluated for off-target binding against either >200 G protein-coupled receptors (cell based), or >4000 human transmembrane proteins (scFv-Fc based), and demonstrated binding only to GPRC5D. Studies with murine and cynomolgus cross-reactive GPRC5D targeting CARs did not show signs of alopecia or other unexpected toxicity in either species. In a murine model of post-BCMA CAR T cell treated antigen escape (CRISPR BCMA KO of a subpopulation of MM cells), anti-GPRC5D CAR T cells rescue BCMA- relapse. These results indicate that GPRC5D will be an important target for the immunotherapy of MM. We are translating this 4-1BB-containing, human-derived, GPRC5D-targeted CAR construct to the clinic. Disclosures Smith: Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Harrington:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Masakayan:Agentus Inc: Employment. Jones:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Long:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Ghoddusi:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Do:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Pham:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Wang:Eureka Therapeutics: Employment, Equity Ownership. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Xu:Eureka Therapeutics: Employment, Equity Ownership. Riviere:Juno Therapeutics, a Celgene Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics Inc.: Research Funding. Liu:Eureka Therapeutics, Inc.: Employment, Equity Ownership. Sather:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Brentjens:Juno Therapeutics, a Celgene Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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.

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.

This study examined the effects of a bout of low-intensity, prolonged downhill exercise on sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity, Ca(2+) uptake and release in rat red vastus muscle. Ionophore stimulation was determined to assess vesicle integrity by measuring the ratio of Ca(2+)-ATPase activities in the presence and absence of A23187. Observations of the muscle ultrastructure were made to evaluate muscle damage at the level of the myofibrils and SR. Adult male Sprague-Dawley rats (weight, 395 +/- 5.9 g) were either assigned as non-exercise controls or subjected to 90 min of downhill treadmill exercise (-16 deg; 15 m min(-1)), and then killed immediately, 4, 24, 48, 72 or 144 h after exercise (n = 7). Calcium uptake was significantly lower (P < 0.05) compared with control values (19.25 +/- 1.38 nmol min(-1) (mg protein)(-1)), by 29 and 36% immediately and 4 h postexercise, respectively, and remained depressed (P < 0.05) 24 h postexercise. Calcium release was also significantly lower (P < 0.05) compared with control values (31.06 +/- 2.36 nmol min(-1) (mg protein)(-1)), by 37 and 39% immediately and 4 h postexercise, respectively, and remained depressed (P < 0.05) 24 h postexercise. Ca(2+)-ATPase activity measured with ionophore was 31% lower (P < 0.05) 4 h postexercise, and remained lower (P < 0.05) 24 h postexercise. The ratio of Ca(2+)-ATPase activities in the presence and absence of A23187 was not significantly changed after exercise, indicating that membrane integrity was not altered by the exercise. Focal dilatations of the SR were observed immediately and 4 h following exercise, implying that SR may be susceptible to damage in the localized regions of overstretched sarcomeres. The results demonstrate that a bout of low-intensity, prolonged downhill exercise results in a long-lasting depression of SR function that is not fully restored after 2 days of recovery, which may underlie some functional impairments induced by eccentric exercise.

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is a variably severe neuromuscular disorder for which no effective treatment is available. Although a number of genes have been identified in which mutations can cause NM, the pathogenetic mechanisms leading to the phenotypes are poorly understood. To address this question, we examined gene expression patterns in an NM mouse model carrying the human Met9Arg mutation of alpha-tropomyosin slow (Tpm3). We assessed five different skeletal muscles from affected mice, which are representative of muscles with differing fiber-type compositions, different physiological specializations and variable degrees of pathology. Although these same muscles in non-affected mice showed marked variation in patterns of gene expression, with diaphragm being the most dissimilar, the presence of the mutant protein in nemaline muscles resulted in a more similar pattern of gene expression among the muscles. This result suggests a common process or mechanism operating in nemaline muscles independent of the variable degrees of pathology. Transcriptional and protein expression data indicate the presence of a repair process and possibly delayed maturation in nemaline muscles. Markers indicative of satellite cell number, activated satellite cells and immature fibers including M-Cadherin, MyoD, desmin, Pax7 and Myf6 were elevated by western-blot analysis or immunohistochemistry. Evidence suggesting elevated focal repair was observed in nemaline muscle in electron micrographs. This analysis reveals that NM is characterized by a novel repair feature operating in multiple different muscles.

Three canine osteosarcoma cell lines were established from spontaneous pelvic and radial osteosarcomas. The cell populations cultured exhibited characteristics of malignancy and consisted of adherent, pleomorphic, mostly large spindle-shaped or polyhedral cells, characterised by the presence of numerous cytoplasmic granules and vacuoles. The main ultrastructural features included the presence of abundant rough endoplasmic reticulum and numerous cytoplasmic vesicles, deposit vacuoles and small cytoplasmic protrusions. Zymography showed that the cell lines produce high levels of MMP-2 and MMP-9, enzymes directly involved in crucial aspects of the metastatic process. Consistent with their osteoblastic lineage and malignant phenotype, all cell lines were immunoreactive to vimentin, osteopontin, PCNA, p53, MMP-2 and MMP-9, while they were negative for cytokeratin, desmin, SMA, Factor VIII, NSE, GFAP, Rb and p21 protein. No retroviral particles or RNA were detected ultrastructurally or with RT-PCR, although the possibility of viral involvement in osteosarcoma cannot be excluded. The new cell lines provide excellent in vitro models that may allow further studies on the pathobiology of canine osteosarcoma to be undertaken.

We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development.

SUMMARYThe toadfishes Tetractenos hamiltoni and Torquigener pleurogramma (Tetraodontidae) possess occlusable yellow corneas. We examine the light transmission and location of the yellow/orange pigment throughout the cornea, the temporal properties of pigment migration and the ultrastructure of the pigmented processes during light- and dark-adaptation. Each species was dark-adapted during the day and light-adapted during the night and then exposed to either sun illumination or darkness for different lengths of time (0–70 min). Movement of corneal pigment could be induced in both species regardless of time of day or night. The pigment was able to migrate in a dorsal or ventral direction and changed from minimal to maximal pigmentation within 60 min. Three types of transmission curves were found with varying degrees of transmission in the 400–500 nm waveband, indicating that the pigment distribution is not uniform across the cornea; some areas of the cornea transmit near UV light, while others absorb blue light. The gradual change of the transmission characteristics in different areas of the cornea indicates the presence of different concentrations of a single type of pigment. Ultrastructural examination of the corneas showed that the layer containing the pigment is situated within the scleral cornea either surrounding (T. pleurogramma) or abutting (T. hamiltoni) an iridescent layer. Long sheet-like processes or chromatophores extending centrally from dorsal and ventral reservoirs are filled with pigment during the light-adapted state but empty in the dark-adapted state.

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. Cancer Res; 74(8); 2238–45. ©2014 AACR.

We have constructed a defined acapsular mutant in Pasteurella multocida X-73 (serogroup A:1) by disrupting the hexA gene through the insertion of a tetracycline resistance cassette. The genotype of the hexA :: tet (M) strain was confirmed by PCR and Southern hybridization, and the acapsular phenotype of this strain was confirmed by electron microscopy. The hexA :: tet (M) strain was attenuated in both mice and chickens. Complementation of the mutant with an intact hexAB fragment restored lethality in mice but not in chickens. In contrast to the results described previously for P. multocida serogroup B (J. D. Boyce and B. Adler, Infect. Immun. 68:3463–3468, 2000), the hexA :: tet (M) strain was sensitive to the bactericidal action of chicken serum, whereas the wild-type and complemented strains were both resistant. Following inoculation into chicken muscle, the bacterial count of the hexA :: tet (M) strain decreased significantly, while the wild-type and complemented strains both grew rapidly over 4 h. The capsule is thus an essential virulence determinant in the pathogenesis of fowl cholera.

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. Mol Cancer Ther; 12(3); 295–305. ©2012 AACR.

Oxazyme (OC4) is an orally administered formulation that has as an active component a recombinant mutant form of Bacillus subtilis oxalate decarboxylase (OxDC) enzyme C383S, designed to degrade dietary oxalate in the stomach. Fourteen-day repeat-dose studies were conducted in rats and dogs to evaluate toxicity and determine a no observed adverse effect level (NOAEL). Animals were administered OC4 by oral gavage twice daily for 14 consecutive days. Reversibility, progression, and delayed appearance of any observed changes were evaluated in a subset of animals that underwent a recovery of 7 days following 14 days of control or test-article. There were no test-article-related adverse effects or deaths in either species. Results indicate that the NOAEL under the conditions used in the studies was 720.8 mg/kg/d in rats and 187.2 mg/kg/d in dogs, the high dose tested in each species.

The cell surface glycoprotein P-cadherin is an attractive target for an antibody-drug conjugate (ADC) therapy as it is known to be highly expressed in a number of malignancies, including those arising in the epithelium of the breast, lung, bladder, esophagus, stomach, endometrium and colon, among others. In breast cancer, P-cadherin is frequently overexpressed in high grade invasive tumors and is a reliable marker of the basal-like breast cancer molecular subtype, a disease with no effective therapeutic treatment options. Based on the expression profile of P-cadherin in human cancer, a highly selective and potent ADC was developed to target cancer types overexpressing this glycoprotein. This ADC consists of a fully human anti-P-cadherin-specific antibody conjugated to the potent maytansine-derived microtubule-disruptor, DM1, via an SMCC non-cleavable thioether linkage (technology licensed from ImmunoGen, Inc.). In vitro, the ADC was demonstrated to selectively bind P-cadherin expressing cell lines, to rapidly internalize and traffic to lysosomes, and to release a sufficient amount of activated payload to potently induce a cytotoxic response in cell viability assays. Profiling of activity in a cell line panel indicated that this ADC can effectively target and kill P-cadherin-positive cancer cells representing breast, head and neck, and bladder carcinomas. In vivo, the ADC was highly efficacious in numerous relevant xenograft models of P-cadherin expressing cancers, including breast, head and neck, bladder and lung. From this promising cellular and in vivo activity, this ADC may be an effective treatment for patients with P-cadherin positive cancers of high unmet medical need. Citation Format: Daniel Menezes, Tinya J. Abrams, Christopher Karim, Yan Tang, Chi Ying, Kathy Miller, Christie Fanton, Majid Ghoddusi, Zhen Wang, Montesa Patawaran, Nancy Pryer, Emma Lees, Jason Damiano. Development and activity of a novel antibody-drug conjugate for the treatment of P-cadherin expressing cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1682. doi:10.1158/1538-7445.AM2015-1682

This study examined the effects of a bout of low-intensity, prolonged downhill exercise on sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity, Ca(2+) uptake and release in rat red vastus muscle. Ionophore stimulation was determined to assess vesicle integrity by measuring the ratio of Ca(2+)-ATPase activities in the presence and absence of A23187. Observations of the muscle ultrastructure were made to evaluate muscle damage at the level of the myofibrils and SR. Adult male Sprague-Dawley rats (weight, 395 +/- 5.9 g) were either assigned as non-exercise controls or subjected to 90 min of downhill treadmill exercise (-16 deg; 15 m min(-1)), and then killed immediately, 4, 24, 48, 72 or 144 h after exercise (n = 7). Calcium uptake was significantly lower (P0.05) compared with control values (19.25 +/- 1.38 nmol min(-1) (mg protein)(-1)), by 29 and 36% immediately and 4 h postexercise, respectively, and remained depressed (P0.05) 24 h postexercise. Calcium release was also significantly lower (P0.05) compared with control values (31.06 +/- 2.36 nmol min(-1) (mg protein)(-1)), by 37 and 39% immediately and 4 h postexercise, respectively, and remained depressed (P0.05) 24 h postexercise. Ca(2+)-ATPase activity measured with ionophore was 31% lower (P0.05) 4 h postexercise, and remained lower (P0.05) 24 h postexercise. The ratio of Ca(2+)-ATPase activities in the presence and absence of A23187 was not significantly changed after exercise, indicating that membrane integrity was not altered by the exercise. Focal dilatations of the SR were observed immediately and 4 h following exercise, implying that SR may be susceptible to damage in the localized regions of overstretched sarcomeres. The results demonstrate that a bout of low-intensity, prolonged downhill exercise results in a long-lasting depression of SR function that is not fully restored after 2 days of recovery, which may underlie some functional impairments induced by eccentric exercise.

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.

Patients with the inherited muscle disease nemaline myopathy experience prolonged muscle weakness following periods of immobility. We have examined endurance exercise as a means of improving recovery following muscle inactivity in our alpha-tropomyosin(slow)(Met9Arg)-transgenic mouse model of nemaline myopathy. Physical inactivity, mimicked using a hindlimb immobilization protocol, resulted in fiber atrophy and severe muscle weakness. Following immobilization, the nemaline mice (NM) were weaker than WT mice but regained whole-body strength with exercise training. The disuse-induced weakness and the regain of strength with exercise in NM were associated with the respective formation and resolution of nemaline rods, suggesting a role for rods in muscle weakness. Muscles from NM did not show the typical features of muscle repair during chronic stretch-immobilization of the soleus muscle (regeneration occurred with relative lack of centralized nuclei). This indicates that the normal process of regeneration may be altered in nemaline myopathy and may contribute to poor recovery. In conclusion, endurance exercise can alleviate disuse-induced weakness in NM. The altered myofiber repair process in the nemaline mice may be a response to primary myofibrillar damage that occurs in nemaline myopathy and is distinct from the classical repair in muscular dystrophy resulting from plasma membrane defects.

Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

A subset of melanoma patients (18-30%) treated with Vemurafenib (and other RAF inhibitors) develop spontaneous cutaneous squamous-cell carcinoma (cSCC) and/or keratoacanthoma (KA). Preclinical studies demonstrate that RAF inhibitors paradoxically activate the MAPK pathway and stimulate growth of RAS mutated cells. Based on these observations it has been proposed that drug induced MAPK activation in pre-cancerous tissues may account for the high incidence of cSCC and KA in treated patients. Because human cSCC is frequently positive for human papillomavirus (HPV) DNA, we hypothesized that RAF inhibitors promote tumorigenesis in HPV infected epidermis. To investigate this possibility, we used a transgenic murine model (K14-HPV16) of cSCC, driven by basal keratinocyte-specific expression (K14 promoter) of HPV type 16 early genes including E6 and E7, which inactivates p53 and pRB tumor suppressors. Exposure to Vemurafenib elevated MAPK markers in epidermis and increased cSCC incidence from 22% to 70%. Exome sequencing revealed that 100% of cSCCs from untreated mice harbored activating RAS mutations, yet 55% of cSCCs from Vemurafenib treated mice were RAS wild-type. Concomitant treatment with a MEK inhibitor reduced cSCC frequency, and MEK inhibitor alone was sufficient to completely regress established cSCCs. Together, these results suggest that cSCC is dependent upon MAPK activation induced either by a RAS oncogene or alternatively by exposure to RAF inhibitors in HPV infected keratinocytes. Citation Format: Matthew Holderfield, Edward Lorenzana, Ben Weisburd, Lisa Lomovasky, Majid Ghoddusi, Dylan Daniel, Nancy Pryer, Frank McCormick, Darrin Stuart. Vemurafenib promotes RAS wild-type tumor formation in a mouse model of HPV-driven cutaneous squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-61. doi:10.1158/1538-7445.AM2013-LB-61

Selective RAF inhibitors have significant activity in patients with metastatic melanoma whose tumors express BRAFV600E. However, not all patients respond equally well to treatment and the duration of response is often limited to less than 6 months. LGX818 was developed with the hypothesis that a more potent inhibitor with excellent pharmacological properties would maximize the degree and duration of patient response. LGX818 is a highly potent RAF inhibitor with selective anti-proliferative and apoptotic activity in cells expressing BRAFV600E. In the A375 (BRAFV600E) human melanoma cell line LGX818 suppresses phospho-ERK (EC50 = 3 nM) leading to potent inhibition of proliferation (EC50 = 4 nM). No significant activity was observed against a panel of 100 kinases (IC50 > 900 nM) and LGX818 did not inhibit proliferation of > 400 cell lines expressing wild-type BRAF. Contributing to the high potency of LGX818 is the extremely slow off-rate from BRAFV600E which is not observed with other RAF inhibitors. In biochemical assays the dissociation half-life was >24 hours which translated into sustained target inhibition in cells following drug wash-out. Single dose PK/PD studies in human melanoma xenograft models (BRAFV600E) indicated that LGX818 treatment at oral doses as low as 6 mg/kg resulted in strong (75%) and sustained (>24 hours) decrease in phospho-MEK, even following clearance of drug from circulation. Decreases in phospho-ERK were consistent with phospho-MEK but markers of downstream transcriptional output (DUSP6 and SPRY4) appeared to provide a more sensitive measure of pathway activation. LGX818 induced tumor regression in multiple BRAF mutant human tumor xenograft models grown in immune compromised mice and rats at doses as low as 1 mg/kg. Consistent with the in vitro data, LGX818 was inactive against BRAF wild-type tumors at doses up to 300 mg/kg bid, with good tolerability and linear increase in exposure. Efficacy was also achieved in a more disease-relevant spontaneous metastatic melanoma and a model of melanoma brain metastasis. LGX818 is a potent and selective RAF kinase inhibitor with unique biochemical properties that contribute to an excellent pharmacological profile. A Phase I clinical trial in patients with BRAF mutant tumors is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3790. doi:1538-7445.AM2012-3790