A trispecific antibody targeting HER2 and T cells inhibits breast cancer growth via CD4 cells.

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies1. Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies2-4. Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2+ breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.

Discovery of SARxxxx92, a pan-PIM kinase inhibitor, efficacious in a KG1 tumor model.

N-substituted azaindoles were discovered as potent pan-PIM inhibitors. Lead optimization, guided by structure and focused on physico-chemical properties allowed us to solve inherent hERG and permeability liabilities, and provided compound 27, which subsequently impacted KG-1 tumor growth in a mouse model.

Discovery of N-substituted 7-azaindoles as Pan-PIM kinases inhibitors - Lead optimization - Part III.

N-substituted azaindoles were discovered as promising pan-PIM inhibitors. Lead optimization is described en route toward the identification of a clinical candidate. Modulation of physico-chemical properties allowed to solve inherent hERG and permeability liabilities. Compound 17 showed tumor growth inhibition in a KG1 tumor-bearing mouse model.

Discovery of N-substituted 7-azaindoles as PIM1 kinase inhibitors - Part I.

Novel N-substituted azaindoles have been discovered as PIM1 inhibitors. X-ray structures have played a significant role in orienting the chemistry effort in the initial phase of hit confirmation. Disclosure of an unconventional binding mode for 1 and 2, as demonstrated by X-ray crystallography, is presented and was an important factor in selecting and advancing a lead series.

Discovery of N-substituted 7-azaindoles as Pan-PIM kinase inhibitors - Lead series identification - Part II.

N-Substituted azaindoles have been discovered as pan-PIM kinase inhibitors. Initial SAR, early ADME and PK/PD data of a series of compounds is described and led to the identification of promising pan-PIM inhibitors which validated our interest in the 7-azaindole scaffold and led us to pursue the identification of a clinical candidate.

PIM inhibitors target CD25-positive AML cells through concomitant suppression of STAT5 activation and degradation of MYC oncogene.

Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.

Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across multiple preclinical tumor models.

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.

SAR439859, a Novel Selective Estrogen Receptor Degrader (SERD), Demonstrates Effective and Broad Antitumor Activity in Wild-Type and Mutant ER-Positive Breast Cancer Models.

Primary treatment for estrogen receptor-positive (ER+) breast cancer is endocrine therapy. However, substantial evidence indicates a continued role for ER signaling in tumor progression. Selective estrogen receptor degraders (SERD), such as fulvestrant, induce effective ER signaling inhibition, although clinical studies with fulvestrant report insufficient blockade of ER signaling, possibly due to suboptimal pharmaceutical properties. Furthermore, activating mutations in the ER have emerged as a resistance mechanism to current endocrine therapies. New oral SERDs with improved drug properties are under clinical investigation, but the biological profile that could translate to improved therapeutic benefit remains unclear. Here, we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradation activities against both wild-type and mutant Y537S ER. Driven by its fluoropropyl pyrrolidinyl side chain, SAR439859 has demonstrated broader and superior ER antagonist and degrader activities across a large panel of ER+ cells, compared with other SERDs characterized by a cinnamic acid side chain, including improved inhibition of ER signaling and tumor cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ breast cancer models, including MCF7-ESR1 wild-type and mutant-Y537S mouse tumors, and HCI013, a patient-derived tamoxifen-resistant xenograft tumor. These findings indicate that SAR439859 may provide therapeutic benefit to patients with ER+ breast cancer, including those who have resistance to endocrine therapy with both wild-type and mutant ER.

Pan-TGFß inhibition by SAR439459 relieves immunosuppression and improves antitumor efficacy of PD-1 blockade.

TGFß is a pleiotropic cytokine that may have both tumor inhibiting and tumor promoting properties, depending on tissue and cellular context. Emerging data support a role for TGFß in suppression of antitumor immunity. Here we show that SAR439459, a pan-TGFß neutralizing antibody, inhibits all active isoforms of human and murine TGFß, blocks TGFß-mediated pSMAD signaling, and TGFß-mediated suppression of T cells and NK cells. In vitro, SAR439459 synergized with anti-PD1 to enhance T cell responsiveness. In syngeneic tumor models, SAR439459 treatment impaired tumor growth, while the combination of SAR439459 with anti-PD-1 resulted in complete tumor regression and a prolonged antitumor immunity. Mechanistically, we found that TGFß inhibition with PD-1 blockade augmented intratumoral CD8+ T cell proliferation, reduced exhaustion, evoked proinflammatory cytokines, and promoted tumor-specific CD8+ T cell responses. Together, these data support the hypothesis that TGFß neutralization using SAR439459 synergizes with PD-1 blockade to promote antitumor immunity and formed the basis for the ongoing clinical investigation of SAR439459 in patients with cancer (NCT03192345).

Discovery of 6-(2,4-Dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidin-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annulene-2-carboxylic acid (SAR439859), a Potent and Selective Estrogen Receptor Degrader (SERD) for the Treatment of Estrogen-Receptor-Positive Breast Cancer.

More than 75% of breast cancers are estrogen receptor alpha (ERα) positive (ER+), and resistance to current hormone therapies occurs in one-third of ER+ patients. Tumor resistance is still ERα-dependent, but mutations usually confer constitutive activation to the hormone receptor, rendering ERα modulator drugs such as tamoxifen and aromatase inhibitors ineffective. Fulvestrant is a potent selective estrogen receptor degrader (SERD), which degrades the ERα receptor in drug-resistant tumors and has been approved for the treatment of hormone-receptor-positive metastatic breast cancer following antiestrogen therapy. However, fulvestrant shows poor pharmacokinetic properties in human, low solubility, weak permeation, and high metabolism, limiting its administration to inconvenient intramuscular injections. This Drug Annotation describes the identification and optimization of a new series of potent orally available SERDs, which led to the discovery of 6-(2,4-dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidin-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annulene-2-carboxylic acid (43d), showing promising antitumor activity in breast cancer mice xenograft models and whose properties warranted clinical evaluation.

Oncogenic dependency on ß-catenin in liver cancer cell lines correlates with pathway activation.

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/ß-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (ß-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of ß-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on ß-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on ß-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) ß-catenin were significantly more sensitive to ß-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active ß-catenin. Finally, significant therapeutic benefit of ß-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. ß-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of ß-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant ß-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Concomitant Inhibition of PI3Kß and BRAF or MEK in PTEN-Deficient/BRAF-Mutant Melanoma Treatment: Preclinical Assessment of SAR260301 Oral PI3Kß-Selective Inhibitor.

Class IA PI3K pathway activation resulting from PTEN deficiency has been associated with lack of sensitivity of melanoma to BRAF kinase inhibitors. Although previous studies have shown synergistic activity when pan-PI3K inhibitors were combined with MAPK inhibitors in the treatment of melanoma exhibiting concurrent genetic abnormalities, overlapping adverse events in patients limit optimal dosing and clinical application. With the aim of specifically targeting PTEN-deficient cancers and minimizing the potential for on-target toxicity when inhibiting multiple PI3K isoforms, we developed a program to discover PI3Kß-selective kinase inhibitors and identified SAR260301 as a potent PI3Kß-selective, orally available compound, which is now in clinical development. Herein, we provide a detailed biological characterization of SAR260301, and show that this compound has outstanding biochemical and cellular selectivity for the PI3Kß isoform versus the α, δ, and γ isoforms and a large panel of protein and lipid kinases. We demonstrate that SAR260301 blocks PI3K pathway signaling preferentially in PTEN-deficient human tumor models, and has synergistic antitumor activity when combined with vemurafenib (BRAF inhibitor) or selumetinib (MEK inhibitor) in PTEN-deficient/BRAF-mutated human melanoma tumor models. Combination treatments were very well tolerated, suggesting the potential for a superior safety profile at optimal dosing using selective compounds to inhibit multiple signaling pathways. Together, these experiments provide a preclinical proof-of-concept for safely combining inhibitors of PI3Kß and BRAF or MEK kinase modulators to improve antitumor activity in PTEN-deficient/BRAF-mutant melanoma, and support the evaluation of SAR260301-based combinations in clinical studies. Mol Cancer Ther; 15(7); 1460-71. ©2016 AACR.

Information processing in bacterial chemotaxis.

Motile bacteria respond to attractants and repellents in their environment by changing their movement. Stock et al. describe the similarities of the bacterial chemotaxis signaling system to eukaryotic signaling cascades. Also included is a discussion of how the ordered signaling complex of the receptor, the kinase CheA, and the kinase regulator CheW can be thought of as a primitive "probrain" to allow the integration of signals to produce the optimal cellular response.

Use of carboxylated cellulose nanofibrils-filled magnetic chitosan hydrogel beads as adsorbents for Pb(II).

Novel magnetic hydrogel beads (m-CS/PVA/CCNFs), consisting of carboxylated cellulose nanofibrils (CCNFs), amine-functionalized magnetite nanoparticles and poly(vinyl alcohol) (PVA) blended chitosan (CS), were prepared by an instantaneous gelation method. SEM, XRD, and TGA techniques were applied to investigate the structure of the hydrogel materials. The magnetic hydrogels were employed as absorbents for removal of Pb(II) ions from aqueous solutions and the fundamental adsorption behavior was studied. Experimental results revealed that the m-CS/PVA/CCNFs hydrogels exhibit higher adsorption capacity with the value of 171.0mg/g, and the carboxylate groups on the CCNFs surface play an important role in Pb(II) adsorption. Moreover, adsorption isotherm data were reliably described by the Langmuir model and the adsorption kinetics closely followed pseudo-second order model. Additionally, the Pb(II)-loaded m-CS/PVA/CCNFs hydrogels could be easily regenerated in weak acid solution and the adsorption effectiveness of 90% can be maintained after the 4 cycles.

SAR103168: a tyrosine kinase inhibitor with therapeutic potential in myeloid leukemias.

SAR103168, a tyrosine kinase inhibitor of the pyrido [2,3-d] pyridimidine subclass, inhibited the kinase activities of the entire Src kinase family, Abl kinase, angiogenic receptor kinases (vascular endothelial growth factor receptor [VEGFR] 1 and 2), Tie2, platelet derived growth factor (PDGF), fibroblast growth factor receptor (FGFR) 1 and 3, and epidermal growth factor receptor (EGFR). SAR103168 was a potent Src inhibitor, with 50% inhibitory concentration (IC50) = 0.65 ± 0.02 nM (at 100 µM ATP), targeting the auto-phosphorylation of the kinase domain (Src(260-535)) and activity of the phosphorylated kinase. Phosphorylation of Src, Lyn and Src downstream signaling pathways (PYK2, P-130CAS, FAK, JNK and MAPK) were inhibited in a dose-dependent manner. SAR103168 inhibited the phosphorylation of STAT5 in KG1 cells and fresh cells from patients with acute myeloid leukemia (AML). SAR103168 inhibited proliferation and induced apoptosis in acute and chronic myeloid leukemic cells at nanomolar IC50. SAR103168 induced anti-proliferation of leukemic progenitors (CFU-L) from 29 patients with AML, and > 85% of AML patient samples were sensitive to SAR103168. These antagonist activities of SAR103168 were independent of FLT3 expression. SAR103168 treatment was effective in 50% of high-risk patient samples carrying chromosome 7 abnormalities or complex rearrangement. SAR103168 administration (intravenous or oral) impaired tumor growth and induced tumor regression in animals bearing human AML leukemic cells, correlating with potent inhibition of Src downstream signaling pathways in AML tumors. SAR103168 showed potent anti-tumor activity in SCID (severe combined immunodeficiency) mice bearing AML (KG1, EOL-1, Kasumi-1, CTV1) and chronic myeloid leukemia (CML) (K562) tumors. The combination of cytarabine and SAR103168 showed synergistic activity in AML and CML tumor models. These results highlight the therapeutic potential of SAR103168 in myeloid leukemias and support the rationale for clinical investigations.

Receptor methylation controls the magnitude of stimulus-response coupling in bacterial chemotaxis.

Motile prokaryotes employ a chemoreceptor-kinase array to sense changes in the media and properly adjust their swimming behavior. This array is composed of a family of Type I membrane receptors, a histidine protein kinase (CheA), and an Src homology 3-like protein (CheW). Binding of an attractant to the chemoreceptors inhibits CheA, which results in decreased phosphorylation of the chemotaxis response regulator (CheY). Sensitivity of the system to stimuli is modulated by a protein methyltransferase (CheR) and a protein methylesterase (CheB) that catalyze the methylation and demethylation of specific glutamyl residues in the cytoplasmic domain of the receptors. One of the most fundamental unanswered questions concerning the bacterial chemotaxis mechanism is the quantitative relationship between ligand binding to receptors and CheA inhibition. We show that the receptor glutamyl modifications cause adaptation by changing the gain (magnitude amplification) between attractant binding and kinase inhibition without substantially affecting ligand binding affinity. The mechanism adjusts receptor sensitivity to background stimulus intensity over several orders of magnitude of attractant concentrations. The cooperative effects of ligand binding appear to be minimal with Hill coefficients for kinase inhibition less than 2, independent of the state of glutamyl modification.

Organization of the receptor-kinase signaling array that regulates Escherichia coli chemotaxis.

Motor behavior in prokaryotes is regulated by a phosphorelay network involving a histidine protein kinase, CheA, whose activity is controlled by a family of Type I membrane receptors. In a typical Escherichia coli cell, several thousand receptors are organized together with CheA and an Src homology 3-like protein, CheW, into complexes that tend to be localized at the cell poles. We found that these complexes have at least 6 receptors per CheA. CheW is not required for CheA binding to receptors, but is essential for kinase activation. The kinase activity per mole of bound CheA is proportional to the total bound CheW. Similar results were obtained with the E. coli serine receptor, Tsr, and the Salmonella typhimurium aspartate receptor, Tar. In the case of Tsr, under conditions optimal for kinase activation, the ratio of subunits in complexes is approximately 6 Tsr:4 CheW:1 CheA. Our results indicate that information from numerous receptors is integrated to control the activity of a relatively small number of kinase molecules.

Interactions between Escherichia coli nucleoside-diphosphate kinase and DNA.

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (approximately 1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.