Weiqun Li

R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors.

Retrovirally Delivered Random Cyclic Peptide Libraries Yield Inhibitors of Interleukin-4 Signaling in Human B Cells

Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a “split and inverted” configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line ε transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides.

Metabolism of Fostamatinib, the Oral Methylene Phosphate Prodrug of the Spleen Tyrosine Kinase Inhibitor R406 in Humans: Contribution of Hepatic and Gut Bacterial Processes to the Overall Biotransformation

The metabolism of the spleen tyrosine kinase inhibitor N4-(2,2-dimethyl-3-oxo-4-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine (R406) and its oral prodrug N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine disodium hexahydrate (R788, fostamatinib) was determined in vitro and in humans. R788 was rapidly converted to R406 by human intestinal microsomes, and only low levels of R788 were observed in plasma of human subjects after oral administration of 14C-R788. R406 was the major drug-related compound in plasma from human subjects, and only low levels of metabolites were observed in plasma. The plasma metabolites of R406 were identified as a sulfate conjugate and glucuronide conjugate of the para-O-demethylated metabolite of R406 (R529) and a direct N-glucuronide conjugate of R406. Elimination of drug-related material into the urine accounted for 19% of the administered dose, and the major metabolite in urine from all the human subjects was the lactam N-glucuronide of R406. On average, 80% of the total drug was recovered in feces. Two drug-related peaks were observed; one peak was identified as R406, and the other peak was identified as a unique 3,5-benzene diol metabolite of R406. The 3,5-benzene diol metabolite appeared to result from the subsequent O-demethylations and dehydroxylation of R529 by anaerobic gut bacteria because only R529 was converted to this metabolite after the in vitro incubation with human fecal samples. These data indicate that the major fecal metabolite of R406 observed in humans is a product of a hepatic cytochrome P450-mediated O-demethylation and subsequent O-demethylations and dehydroxylation by gut bacteria.

Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells

BACKGROUND Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes. RESULTS Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 microM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides. CONCLUSIONS Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.

Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment

BackgroundMyeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity.MethodsCB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively.ResultsCB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers.ConclusionsThese results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.

Abstract 552: Immuno-oncology agent CB-1158 is a potent and selective arginase inhibitor and causes an immune-mediated anti-tumor response

L-arginine is a critical metabolite for T-cell receptor signaling and subsequent T-cell proliferation, and depletion of arginine arrests T-cell growth. In the tumor microenvironment, infiltrating myeloid-derived suppressor cells (MDSCs), macrophages, and neutrophils produce arginase, which depletes local arginine concentrations and dampens T cell-mediated immune surveillance. Pharmacological inhibition of arginase is expected to restore arginine levels and allow T-cells to proliferate, thereby leading to an immune-mediated anti-tumor response. CB-1158 is a potent inhibitor of human arginase (IC50 = 98 nM). In culture, human granulocytes release arginase and deplete media arginine to levels that inhibit T-cell proliferation. In a co-culture system of human granulocytes and T-cells, CB-1158 potently blocks granulocyte-derived arginase activity, maintains extracellular arginine levels, and restores proliferation of T-cells. CB-1158 has high oral bioavailability in rodents and is very well tolerated. BID oral dosing of CB-1158 leads to dose-dependent pharmacodynamic increases in plasma and tumor arginine levels resulting in single agent anti-tumor efficacy in mouse syngeneic tumor models including Lewis Lung carcinoma (LLC) and Madison 109. The anti-tumor effects of CB-1158 are consistent with promoting a proinflammatory tumor microenvironment. Following CB-1158 treatment, multiple Th1 T-cell, NK-cell, and M1 macrophage-associated chemokines, cytokines, and activation markers are elevated in the LLC tumor microenvironment. The anti-tumor efficacy of CB-1158 requires an intact tumor microenvironment since CB-1158 has no effect on LLC cell growth in vitro. Furthermore, CB-1158 treatment of immunocompromised C57/SCID mice bearing LLC tumors has no anti-tumor effect, supporting an immune-mediated anti-tumor mechanism. Immunosuppression in the tumor microenvironment can occur via multiple mechanisms, including arginine depletion, and our data support the combination of checkpoint inhibitors and arginase inhibition by CB-1158. In mice bearing LLC tumors, CB-1158 in combination with checkpoint inhibitors reduced tumor growth, increased the number of tumor infiltrating CD8+ T-cells, and increased the level of Th1/NK/M1-associated chemokines, cytokines, and activation markers in the tumor microenvironment. In mice bearing 4T1 tumors, a tumor type that is highly refractory to checkpoint inhibition, the combination of CB-1158 with anti-PD-1 and anti-CTLA-4 reduces tumor growth and lung metastases. These results support the development of CB-1158, a first-in-class arginase inhibitor, as a novel immuno-oncology agent targeting the immunosuppressive effects of tumor-infiltrating myeloid cells. Citation Format: Melissa Works, Mark Bennett, Jason Chen, Ethan Emberley, Tony Huang, Julie Janes, Weiqun Li, Andy Mackinnon, Gisele Marguier, Silinda Neou, Alison Pan, Francesco Parlati, Mirna Rodriguez, Susanne Steggerda, Tracy Wang, Jing Zhang, Winter Zhang, Matthew Gross. Immuno-oncology agent CB-1158 is a potent and selective arginase inhibitor and causes an immune-mediated anti-tumor response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 552.

Discovery of dual Axl/VEGF-R2 inhibitors as potential anti-angiogenic and anti-metastatic drugs for cancer chemotherapy

Axl tyrosine kinase has been shown to be involved in multiple pathways contributing to tumor development, angiogenesis, and metastasis. High Axl expression has been observed in many human tumors where it appears to confer aggressive tumor behavior. Here we present several series of dual Axl-VEGF-R2 kinase inhibitors based on extensive optimization of an acyl diaminotriazole. It was hypothesized that dual inhibition of these two receptor tyrosine kinases may have a synergistic affect in inhibiting tumor angiogenesis and metastasis. One of these molecules, R916562 showed comparable activity to Sunitinib in two mouse tumor xenograft models and a mouse corneal micropocket model.

Abstract B045: Arginase inhibitor CB-1158 elicits immune-mediated antitumor responses as a single agent and in combination with other immunotherapies

Myeloid derived suppressor cells (MDSCs) and polymorphonuclear cells (PMNs) are mediators of tumor immune evasion, however there are no approved clinical agents that directly antagonize the immunosuppressive activity of these cells. One of the mechanisms by which MDSCs and PMNs suppress anti-tumor immunity is expression of the enzyme arginase, which reduces L-arginine levels rendering T-cells and natural killer (NK)-cells unable to proliferate and mount an effective anti-tumor response. As a rationale to pursue arginase inhibition as a potential therapeutic strategy, we investigated the prevalence of arginase in tumors and peripheral blood from patients with various types of cancer. Our results confirm reports that multiple tumor types have arginase-expressing PMN infiltrates and that cancer patients have higher levels of plasma arginase and lower levels of plasma arginine compared to healthy volunteers. We also confirmed that in vitro-activated primary human T-cells and NK-cells require arginine to proliferate, secrete pro-inflammatory cytokines, and express the intracellular signaling molecule CD3ζ and cell surface activation marker PD-1. We developed CB-1158 to be a potent, selective, and orally-bioavailable small molecule inhibitor of arginase. CB-1158 potently inhibits recombinant human arginase 1 (IC 50 = 98 nM), recombinant human arginase 2 (IC 50 = 249 nM), and endogenous arginase from human neutrophils (IC 50 = 160 nM). In a co-culture system, neutrophils strongly suppressed T-cell proliferation. The addition of CB-1158 blocked arginase activity, maintained arginine levels in the media, and allowed T-cells to proliferate in the presence of suppressive myeloid cells, suggesting that arginase is a major mechanism of myeloid cell suppression of lymphocyte proliferation and that CB-1158 can reverse the suppression. To extend these findings to MDSCs, we isolated peripheral monocytic MDSCs (M-MDSCs) or granulocytic MDSCs (G-MDSCs) from cancer patients and found that G-MDSCs expressed higher levels of arginase, depleted more arginine from cell-culture media, and were more suppressive to T-cell proliferation compared to M-MDSCs. Incubation of G-MDSCs with CB-1158 resulted in media arginine levels that could support T-cell proliferation. CB-1158 has high oral bioavailability in mice and rats. Twice-daily oral dosing of CB-1158 produced dose-dependent pharmacodynamic increases in plasma and tumor arginine levels and resulted in single-agent anti-tumor efficacy in the murine syngeneic tumor models Lewis Lung carcinoma (LLC1), Madison-109 lung carcinoma, and B16F10 melanoma. Antibody-mediated depletion of either CD8 + T-cells or NK-cells partially abrogated the anti-tumor effect of CB-1158 in the LLC1 and B16F10 models indicating that the mechanism of action of CB-1158 is immune cell-mediated through the actions of both T-cells and NK cells. Corroborating the immune-mediated anti-tumor efficacy in CB-1158-treated LLC1 tumors, following dosing with CB-1158 we observed increases in tumor infiltrating CD8 + T-cells; increased levels of tumor Th1 T-cell cytokines; increased expression of T-cell and NK-cell activation markers; and increased interferon-inducible genes. Based on the novel mechanism of action of CB-1158, there is potential for enhanced therapeutic benefit by combining CB-1158 with other immunotherapies. We observed improved anti-tumor activity by combining CB-1158 with either epacadostat or anti-PD-L1 in the B16F10 model, with low dose ionizing radiation in the Madison-109 model, and with gemcitabine in the LLC1 model. These results support the development of CB-1158, a first-in-class arginase inhibitor, as a novel immuno-oncology agent targeting the immunosuppressive effects of tumor-infiltrating myeloid cells. Citation Format: Susanne M. Steggerda, Mark Bennett, Jason Chen, Ethan Emberley, Matthew Gross, Tony Huang, Weiqun Li, Andy MacKinnon, Amani Makkouk, Gisele Marguier, Silinda Neou, Alison Pan, Tracy Wang, Melissa Works, Jing Zhang, Winter Zhang, Francesco Parlati. Arginase inhibitor CB-1158 elicits immune-mediated antitumor responses as a single agent and in combination with other immunotherapies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B045.

Abstract A195: CB-1158 inhibits the immuno-oncology target arginase and causes an immune mediated anti-tumor response

The role of myeloid derived suppressor cells (MDSCs) has become increasingly recognized as an important mechanism of tumor immune evasion. However, to date there are no effective means to antagonize the immunosuppressive activity of these cells in patients. MDSCs suppress cytotoxic T-cells and Natural Killer cells through the secretion of the enzyme arginase which depletes local arginine concentrations. The depletion of arginine in the tumor microenvironment renders cytotoxic T-cells unable to proliferate and therefore unable to effectively mount an anti-tumor attack. Similarly, M2 macrophages and polymorphonuclear cells (PMNs) express high levels of arginase and may contribute to the local suppression of immune responses. Restoration of arginine levels in the tumor microenvironment via arginase inhibition would be expected to allow T-cell activation and proliferation to occur and result in T-cell mediated anti-tumor responses. We have developed novel, potent, and specific inhibitors of arginase. Our clinical candidate CB-1158 has an IC 50 of less than 100 nM in a recombinant human arginase assay. As expected, inhibition of arginase in cell culture does not have a direct anti-proliferative effect on any cell type tested. However, in Peripheral Blood Mononuclear Cells (PBMCs) from a patient with renal cell carcinoma containing both suppressive MDSCs and cytotoxic T-cells, the addition of an arginase inhibitor to the MDSC/T-cell co-culture resulted in a dose-dependent increase in T-cell proliferation relative to vehicle controls. CB-1158 has high oral bioavailability in mice and rats. In mice bearing Lewis Lung Carcinoma (LLC) syngeneic tumors, treatment with CB-1158 results in a 3-4 fold increase in tumor arginine levels with a clear pharmacokinetic/pharmacodynamic relationship. The pharmacodynamic effect of arginase inhibition in tumors was sustained throughout a 24-hour period using a twice-daily oral dosing schedule and has been observed in multiple syngeneic models. Moreover, systemic plasma arginine levels are significantly increased in mice following dosing with CB-1158. Importantly, oral dosing with CB-1158 results in single agent anti-tumor efficacy in the LLC model in C57.Bl/6 mice. In contrast, treatment of immunocompromised C57/scid mice bearing LLC tumors with CB-1158 had no effect on tumor growth. This finding is consistent with the observed anti-tumor efficacy in immune competent mice being mediated through an immune mechanism. Evaluation of tumors treated with arginase inhibitors revealed an increase in CD3+ T-cell infiltrates further supporting an immune-based mechanism of action. CB-1158 has been very well tolerated in rodents with no impact on body weights or serum chemistry enzymes following multi-week dosing schedules. CB-1158 is a first-in-class arginase inhibitor that targets the immunosuppressive effects of myeloid cells in the tumor microenvironment and is currently in development as a novel immuno-oncology strategy. Based on this novel mechanism of action there is also a potential for enhanced therapeutic benefit by combining CB-1158 with other immune checkpoint inhibitors. Citation Format: Matthew Gross, Jason Chen, Ethan Emberley, Julie Janes, Weiqun Li, Andy Mackinnon, Alison Pan, Francesco Parlati, Mirna Rodriguez, Susanne Steggerda, Tracy Wang, Melissa Works, Jing Zhang, Winter Zhang, Mark Bennett. CB-1158 inhibits the immuno-oncology target arginase and causes an immune mediated anti-tumor response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A195.

Proteomics Approach for Identifying Interacting Partners of a C-Terminal Functional Peptide Derived from the Tumor Suppressor, p21 cip/waf1

With the recent technological innovations of proteomics, protein separation followed by mass spectrometry (MS) has become the technique of choice in identifying and validating potential drug targets. As a part of our drug discovery program, we tested the validity of this approach by conducting a simple affinity extraction followed by mass spectrometric (MS) analysis of the components isolated. For this purpose, we chose the C-terminal peptide of the tumor suppressor p21cip/waf1, which is known to bind proliferating cell nuclear antigen (PCNA) and cause cell cycle arrest [1]. Using Jurkat-E cell lysate, affinity extraction of PCNA and its binding partners was carried out by spiking streptavidin agarose beads pre-conjugated with biotinylated p21-derived peptide(s). Using tryptic digests of entire affinity extracts and differential micro-capillary LC/MS/MS, or difference 2D gels combined with in-gel tryptic digests and MALDI-TOF MS, we have identified binding partners of the p21 C-terminal peptide, or of its complex with PCNA. Results from the above experiments were confirmed either by reciprocal affinity extraction and/or Western blotting with respective antibodies. This study suggests that peptides obtained from intracellular functional screens could also serve as efficient baits to affinity extract target proteins and map mammalian cell protein interaction networks.