CRISPR screening identifies T cell-intrinsic regulators of CD3-bispecific antibody responses.

CD3-engaging bispecific antibodies (BsAbs) enable the formation of an immune synapse between T cells and tumor cells, resulting in robust target cell killing not dependent on a preexisting tumor specific T cell receptor. While recent studies have shed light on tumor cell-specific factors that modulate BsAb sensitivity, the T cell-intrinsic determinants of BsAb efficacy and response durability are poorly understood. To better clarify the genes that shape BsAb-induced T cell responses, we conducted targeted analyses and a large-scale unbiased in vitro CRISPR/Cas9-based screen to identify negative regulators of BsAb-induced T cell proliferation. These analyses revealed that CD8+ T cells are dependent on CD4+ T cell-derived signaling factors in order to achieve sustained killing in vitro. Moreover, the mammalian target of rapamycin (mTOR) pathway and several other candidate genes were identified as intrinsic regulators of BsAb-induced T cell proliferation and/or activation, highlighting promising approaches to enhancing the utility of these potent therapeutics.

Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with Genome Editing in Transgenic Mice.

Alpha-1 antitrypsin deficiency (AATD) is a hereditary liver disease caused by mutations in the SERPINA1 serine protease inhibitor gene. Most severe patients are homozygous for PiZ alleles (PiZZ; amino acid E324K), which lead to protein aggregates in hepatocytes and reduced circulating levels of AAT. The liver aggregates typically lead to fibrosis, cirrhosis, and hepatocellular carcinoma, and the reduced circulating AAT levels can lead to emphysema and chronic obstructive pulmonary diseases. In this study, two CRISPR/Cas9 gene editing approaches were used to decrease liver aggregates and increase systemic AAT-M levels in the PiZ transgenic mouse. In the first approach, AAT expression in hepatocytes was reduced more than 98% following the systemic delivery of AAV8-CRISPR targeting exon 2 of hSERPINA1, leading to reduced aggregates in hepatocytes. In the second approach, a second adeno-associated virus, which provided the donor template to correct the Z mutation, was also administered. These treated mice had reduced AAT expression (> 98%) and a low level (5%) of wildtype AAT-M mRNA. Taken together, this study shows that CRISPR gene editing can efficiently reduce liver expression of AAT-Z and restore modest levels of wildtype AAT-M in a mouse model of AATD, raising the possibility of CRISPR gene editing therapeutic for AATD.

H3B-6527 Is a Potent and Selective Inhibitor of FGFR4 in FGF19-Driven Hepatocellular Carcinoma.

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1-3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. Cancer Res; 77(24); 6999-7013. ©2017 AACR.

Pleiotropic stromal effects of vascular endothelial growth factor receptor 2 antibody therapy in renal cell carcinoma models.

The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non-endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)-specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma.

Tumor necrosis factor-alpha and interleukin-1 antagonists alleviate inflammatory skin changes associated with epidermal growth factor receptor antibody therapy in mice.

Cancer patients receiving epidermal growth factor receptor (EGFR) antibody therapy often experience an acneiform rash of uncertain etiology in skin regions rich in pilosebaceous units. Currently, this condition is treated symptomatically with very limited, often anecdotal success. Here, we show that a monoclonal antibody targeting murine EGFR, ME1, caused a neutrophil-rich hair follicle inflammation in mice, similar to that reported in patients. This effect was preceded by the appearance of lipid-filled hair follicle distensions adjacent to enlarged sebaceous glands. The cytokine tumor necrosis factor-alpha (TNFalpha), localized immunohistochemically to this affected region of the pilosebaceous unit, was specifically up-regulated by ME1 in skin but not in other tissues examined. Moreover, skin inflammation was reduced by cotreatment with the TNFalpha signaling inhibitor, etanercept, indicating the involvement of TNFalpha in this inflammatory process. Interleukin-1, a cytokine that frequently acts in concert with TNFalpha, is also involved in this process given the efficacy of the interleukin-1 antagonist Kineret. Our results provide a mechanistic framework to develop evidence-based trials for EGFR antibody-induced skin rash in patients with cancer.

Prioritization of EGFR/IGF-IR/VEGFR2 combination targeted therapies utilizing cancer models.

BACKGROUND: Rational strategies utilizing anticancer efficacy and biological principles are needed for the prioritization of specific combination targeted therapy approaches for clinical development, from among the many with experimental support. MATERIALS AND METHODS: Antibodies targeting epidermal growth factor receptor (EGFR) (cetuximab), insulin-like growth factor-1 receptor (IGF-IR) (IMC-A12) or vascular endothelial growth factor receptor 2 (VEGFR2) (DC101), were dosed alone or in combination, in 11 human tumor xenograft models established in mice. Efficacy readouts included the tumor burden and incidence of metastasis, as well as tumor active hypoxia inducible factor-1 (HIF-1), human VEGF and blood vessel density. RESULTS: Cetuximab and DC101 contributed potent and non-overlapping benefits to the combination approach. Moreover, DC101 prevented escape from IMC-A12 + cetuximab in a colorectal cancer model and cetuximab prevented escape from DC101 therapy in a pancreatic cancer model. CONCLUSION: Targeting VEGFR2 + EGFR was prioritized over other treatment strategies utilizing EGFR, IGF-IR and VEGFR2 antibodies. The criteria that proved to be valuable were a non-overlapping spectrum of anticancer activity and the prevention of resistance to another therapy in the combination.

In vivo method for establishing synergy between antibodies to epidermal growth factor receptor and vascular endothelial growth factor receptor-2.

Targeted therapy for cancer is shifting towards an approach of inhibiting multiple pathways, justified in part by the ability of cancer cells to overcome the inhibition of a single pathway. However the literature is replete with preclinical data supporting the anticancer potential of numerous combinations of targeted agents, making it difficult to select the combination strategies to invest in through clinical development. One characteristic of a combination strategy that can be utilized for prioritization is synergy. Synergy indicates that the effect of the combination is greater than that predicted from the monotherapy potencies. Here we describe a detailed method for establishing synergy between two treatments in vivo. We utilized this method to establish that antibodies targeting the epidermal growth factor receptor and vascular endothelial growth factor receptor-2 are synergistic with regard to antitumor effects, in a BxPC-3 subcutaneous xenograft model for pancreatic cancer.

Pharmacodynamics of RWJ-54428 against Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis in a neutropenic mouse thigh infection model.

RWJ-54428 (also known as MC-02,479) is a new cephalosporin with promising activity against gram-positive bacteria. The pharmacodynamics (PDs) of RWJ-54428 against Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis were studied in a neutropenic mouse thigh infection model. The RWJ-54428 MICs ranged from 0.25 to 1 mg/liter. Mice with ca. 10(6) CFU/thigh at the initiation of therapy were treated intraperitoneally with RWJ-54428 at doses that ranged from 3 to 1,200 mg/kg of body weight/day (in 2, 3, 4, 6, or 12 divided doses) for 24 h. The maximal reductions in bacterial counts in thigh tissues at 24 h for the methicillin-resistant S. aureus, penicillin-resistant S. pneumoniae, and E. faecalis strains were -2.8, -3.8, and -1.7 log10 CFU/thigh, respectively. The percentage of a 24-h dosing interval that the unbound serum RWJ-54428 concentrations exceeded the MIC (fT>MIC) was the pharmacokinetic (PK)-PD parameter that best described the efficacy of RWJ-54428. The fT>MICs for a bacteriostatic effect (no net change in the numbers of CFU/thigh over 24 h) ranged from 14 to 20% for staphylococci and streptococci; for maximal reductions in the numbers of CFU/thigh, the fT>MICs ranged from 22 to 36% for these strains. For E. faecalis, the ranges of fT>MICs for static and maximal effects were 30 to 46% and 55 to 60%, respectively. These data show that treatment with RWJ-54428 results in marked antibacterial effects in vivo, with the PK-PD parameters for efficacy being comparable to those for the efficacy of penicillins and carbapenems active against staphylococci and pneumococci.

Anti-vascular endothelial growth factor receptor-1 antagonist antibody as a therapeutic agent for cancer.

PURPOSE: Vascular endothelial growth factor receptor-1 (VEGFR-1) plays important roles in promotion of tumor growth by mediating cellular functions in tumor vascular endothelium and cancer cells. Blockade of VEGFR-1 activation has been shown to inhibit pathologic angiogenesis and tumor growth, implicating VEGFR-1 as a potential therapeutic target for the treatment of cancer. We have thus developed a VEGFR-1 antagonist human monoclonal antibody designated as IMC-18F1 and evaluated its antitumor activity in preclinical experimental models to show the therapeutic potential of the antibody for cancer treatment in clinic. EXPERIMENTAL DESIGN: Human IgG transgenic mice were used for generation of anti-VEGFR-1 antibodies. Anti-VEGFR-1-specific blocking antibodies were identified using solid-phase binding and blocking assays. Inhibitory antitumor cell activity of IMC-18F1 was assessed in cell-based kinase and growth assays. Pharmacokinetic/pharmacodynamic studies were done to determine the association of antibody blood level with antitumor efficacy of the antibody in vivo. Antitumor efficacy of the anti-VEGFR-1 antibodies as monotherapy and in combination with cytotoxic agents was evaluated in human breast cancer xenograft models. RESULTS: A fully human neutralizing antibody, IMC-18F1, was shown to be a high-affinity (KD=54 pmol) inhibitor of VEGFR-1 ligand binding (VEGF-A, VEGF-B, and placental growth factor). IMC-18F1 inhibited ligand-induced intracellular activation of VEGFR-1 and mitogen-activated protein kinase signaling and prevented ligand-stimulated in vitro growth of breast cancer cells. In vivo, IMC-18F1 suppressed the growth of human breast tumor xenografts in association with reduced mitogen-activated protein kinase and Akt activation, reduced tumor cell proliferation, and increased tumor cell apoptosis. Pharmacokinetic/pharmacodynamic studies established a plasma elimination half-life of 5 days for IMC-18F1 and a steady-state trough plasma therapeutic threshold of 88 microg/mL. Importantly, inhibition of mouse and human VEGFR-1 with MF1 and IMC-18F1, respectively, enhanced the antitumor efficacy of cytotoxic agents commonly used to treat breast cancer. CONCLUSIONS: Based on preclinical validation studies, IMC-18F1 anti-VEGFR-1 has potential to provide clinical benefit to cancer patients.

Synergistic antitumor effects of combined epidermal growth factor receptor and vascular endothelial growth factor receptor-2 targeted therapy.

PURPOSE: Combination therapies that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) pathways, are being actively tested for the treatment of cancer. In evaluating combination strategies, the ideal combination would be one in which the treatments interact in a way that is synergistic with regard to antitumor effects. Here, we have evaluated the interaction between anti-EGFR antibody Erbitux (cetuximab) and anti-VEGFR2 antibody, DC101, in preclinical models of pancreatic (BxPC-3) and colon (GEO) cancer. EXPERIMENTAL DESIGN: Analysis of the interaction between cetuximab and DC101 in vivo used a novel method for establishing the upper 95% confidence limits for the combination index (CI) of isobologram analyses, where CI < 1 indicates synergy. Assessment of tumor cell proliferation, apoptosis, VEGF production, and hypoxia, as well as tumor vascularization, was performed to gain insights into the mechanistic basis for synergy between agents targeting different tumor compartments. RESULTS: Monotherapy ED(50) values for tumor growth inhibition ranged from 1.8 to 2.3 mg/kg and 10.5 to 16.6 mg/kg for cetuximab and DC101, respectively. From the dose response of the combination treatment, it was determined that cetuximab and DC101 are synergistic in the BxPC-3 (CI = 0.1, P < 0.01) and GEO (CI = 0.1, P < 0.01) models. Overlapping effects on the tumor cell and vascular compartments form a basis for the interaction, with VEGF production and hypoxia-inducible factor 1alpha potentially acting as molecular links between EGFR and VEGFR2 inhibition. CONCLUSIONS: Results show antitumor synergy for combined EGFR and VEGFR2 targeted therapy, supporting the significant therapeutic potential of this combination strategy.

Pharmacodynamics of levofloxacin against Pseudomonas aeruginosa with reduced susceptibility due to different efflux pumps: do elevated MICs always predict reduced in vivo efficacy?

The Pseudomonas aeruginosa efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN play an important role in susceptibility to fluoroquinolones in vitro. To determine if levofloxacin MICs arising from different levels of expression of efflux pumps result in a proportional reduction in the response to levofloxacin in vivo, isogenic strains of P. aeruginosa were tested with levofloxacin in two mouse models of infection (sepsis and neutropenic mouse thigh models). The levofloxacin 50% effective doses (ED(50)s) increased proportionally with the MICs for most strains. Similarly, the 24-h area under the concentration-time curve (AUC)/MIC ratio that resulted in 90% of the maximum bactericidal activity (90% E(max)) exceeded 75 for all strains except those with elevated MICs due to MexEF-OprN overexpression. In these strains, levofloxacin ED(50)s were 2- to 10-fold lower than the ED(50)/MIC ratios in the other strains and 90% E(max) AUC/MIC ratios were 2- to 4-fold lower than those predicted from pharmacodynamic modeling of efficacy against other strains. These data show that while the MexEF-OprN efflux pump can provide P. aeruginosa resistance to levofloxacin in vitro, it appears to be less efficient in providing resistance to levofloxacin in animal models of infection.

1-[4-(1H-Benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea derivatives as small molecule heparanase inhibitors.

A novel class of 1-[4-(1H-benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-ureas are described as potent inhibitors of heparanase. Among them are 1,3-bis-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea (7a) and 1,3-bis-[4-(5,6-dimethyl-1H-benzoimidazol-2-yl)-phenyl]-urea (7d), which displayed good heparanase inhibitory activity (IC(50) 0.075-0.27 microM). Compound 7a showed good efficacy in a B16 metastasis model.

Targeting the platelet-derived growth factor receptor alpha with a neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: implications as a potential therapeutic target.

Platelet-derived growth factor receptor alpha (PDGFRalpha) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRalpha, which did not cross-react with the beta form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRalpha. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRalpha. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P=0.0004) and SKLMS-1 (P <0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRalpha.