Activity In Tumor Xenograft Models Research Articles

A Marine-Derived Small Molecule Inhibits Prostate Cancer Growth by Promoting Endoplasmic Reticulum Stress Induced Apoptosis and Autophagy.

MHO7 (6-epi-ophiobolin G), a novel component extracted from a mangrove fungus, exhibits significant anticancer effects against breast cancer. However, the precise mechanism underlying the anticancer effects of MHO7 in prostate cancer (PCa) is yet to be fully elucidated. Therefore, this study was undertaken to assess the effect of MHO7 on PCa cells and elucidate its underlying mechanism. A series of in vitro experiments were conducted, including Cell Counting Kit-8, and plate clone formation assays, flow cytometry analysis, electron microscopy, immunofluorescence staining, western blotting, and molecular dynamics simulation. Additionally, in vivo tumor xenograft models were employed. Our findings revealed that MHO7 could induce cellular autophagy at low concentration (2 μM) and apoptosis at relatively high concentration (4 and 8 μM), leading to significant PCa cell growth inhibition. Furthermore, MHO7 triggered endoplasmic reticulum (ER) stress, which subsequently stimulated autophagy and apoptosis via IRE1α/XBP-1s signaling pathway activation. Notably, IRE1α knockdown markedly reduced MHO7-induced autophagy and apoptosis. Moreover, MHO7 targeted the IRE1α protein, thereby enhancing its stability. MHO7 also exhibited substantial anticancer activity in tumor xenograft models. Our study revealed that MHO7 holds considerable potential as an anticancer agent against PCa, attributable to its activation of ER stress-induced autophagy and apoptosis at different concentrations, facilitated by the upregulation of IRE1α expression.

Sequential Exposure to IL21 and IL15 During Human Natural Killer Cell Expansion Optimizes Yield and Function.

Natural killer (NK) cells are frequently expanded for the clinic using irradiated, engineered K562 feeder cells expressing a core transgene set of membrane-bound (mb) IL15 and/or mbIL21 together with 41BBL. Prior comparisons of mbIL15 to mbIL21 for NK expansion lack comparisons of key attributes of the resulting NK cells, including their high-dimensional phenotype, polyfunctionality, the breadth and potency of cytotoxicity, cellular metabolism, and activity in xenograft tumor models. Moreover, despite multiple rounds of K562 stimulation, studies of sequential use of mbIL15- and mbIL21-based feeder cells are absent. We addressed these gaps and found that using mbIL15- versus mbIL21-based feeder cells drove distinct phenotypic and functional profiles. Feeder cells expressing mbIL15 alone drove superior functionality by nearly all measures, whereas those expressing mbIL21 alone drove superior yield. In combination, most attributes resembled those imparted by mbIL21, whereas in sequence, NK yield approximated that imparted by the first cytokine, and the phenotype, transcriptome, and function resembled that driven by the second cytokine, highlighting the plasticity of NK cell differentiation. The sequence mbIL21 followed by mbIL15 was advantageous in achieving significant yields of highly functional NK cells that demonstrated equivalent in vivo activity to those expanded by mbIL15 alone in two of three xenograft models. Our findings define the impact of mbIL15 versus mbIL21 during NK expansion and reveal a previously underappreciated tradeoff between NK yield and function for which sequential use of mbIL21-based followed by mbIL15-based feeder cells may be the optimal approach in many settings.

The third-generation anti-CD30 CAR T-cells specifically homing to the tumor and mediating powerful antitumor activity

CAR T-cell therapy is well tolerated and effective in patients with Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). However, even second- generation anti-CD30 CAR T-cells with CD28 (28z) costimulatory domains failed to achieve the desired rate of complete responses. In the present study, we developed second-generation (CD28z) and third-generation (CD28BBz) CAR T-cells targeting CD30 and investigated their efficacy in vitro and in vivo. Both of CD28z and CD28BBz anti-CD30 CAR T cells were similar regarding amplification, T cell subsets distribution, T cell activity, effector/memory and exhaustion. However, we found that the 28BBz anti-CD30 CAR T-cells persist long-term, specifically homing to the tumor and mediating powerful antitumor activity in tumor xenograft models. Subsequently, we also demonstrated that the third generation anti-CD30 CAR T-cells have miner side effects or potential risks of tumorigenesis. Thus, anti-CD30 CAR T-cells represent a safe and effective treatment for Hodgkin lymphoma.

A phase 1 first-in-human study of XL092 administered alone or in combination with immune checkpoint inhibitors (ICIs) in patients (pts) with inoperable locally advanced or metastatic solid tumors: Description of genitourinary (GU) expansion cohorts.

TPS401 Background: XL092 is a novel oral multi-targeted inhibitor of receptor tyrosine kinases MET, VEGFR2, and TAM kinases (AXL, MER), which are important in tumor growth, neovascularization, and immune modulation of the tumor microenvironment. XL092 as a single-agent and in combination with an anti-PD-1 antibody showed antitumor activity in xenograft tumor models (Hsu et al. 2020). This first-in-human study is evaluating safety, pharmacokinetics (PK), and preliminary antitumor activity of XL092 with or without (w/wo) ICIs in pts with advanced solid tumors. Presented here are the trial design and details of GU cohorts. Methods: In this phase 1, multicenter, open-label, dose-escalation and expansion study (NCT03845166), pts will receive escalating doses of XL092 w/wo atezolizumab (ATEZO) 1200 mg Q3W or avelumab (AVE) 800 mg Q2W. The dose-escalation stage is enrolling pts with inoperable locally advanced or metastatic solid tumors using a 3+3 (single-agent) or rolling 6 (combination) design; the primary objective is to determine maximum tolerated dose and/or recommended dose of XL092 for further evaluation when administered w/wo ICIs; secondary objective is to evaluate plasma PK of XL092 and its metabolites alone and in combination with ICIs. Primary objectives for the expansion stage are to evaluate preliminary efficacy of XL092 w/wo ICIs by estimating objective response rate (ORR) by investigator (Inv) per RECIST 1.1 and for specific cohorts by estimating the % of pts with PFS at 6 months by Inv per RECIST 1.1; secondary objective is safety of XL092 w/wo ICIs via monitoring nonserious and serious adverse events (AEs) including immune related AEs and AEs of special interest. For GU cohorts in expansion stage, single-agent XL092 will be tested in: (i) previously treated advanced clear cell (cc) renal cell carcinoma (RCC; progression on 1-3 prior systemic regimens w/wo a sarcomatoid component); (ii) previously treated advanced non-cc RCC (progression on 1-3 prior systemic regimens); (iii) metastatic castration resistant prostate cancer (mCRPC; prior treatment with ≥1 novel hormonal therapy and taxane-based chemotherapy, ≤4 prior systemic regimens). XL092 + ATEZO will be tested in non-cc RCC and mCRPC cohorts. Pts with advanced urothelial carcinoma will be enrolled in 3 cohorts: (i) pts with ongoing CR/PR/SD after 4-6 cycles of first-line chemotherapy (gemcitabine + cisplatin and/or gemcitabine + carboplatin) will receive XL092 + AVE; (ii) pts with progression on/after prior ICI therapy (≤2 prior systemic therapies) will be randomized 1:1 to XL092 + AVE or single-agent XL092; (iii) pts with progression on/after first-line platinum-based chemotherapy will receive XL092 + AVE. The study is currently enrolling pts. Clinical trial information: NCT03845166.

Development of AO-176, a Next-Generation Humanized Anti-CD47 Antibody with Novel Anticancer Properties and Negligible Red Blood Cell Binding.

Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and overexpressed on many tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggers a "don't eat me" signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next-generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPα interaction to induce tumor cell phagocytosis, but also induces tumor cytotoxicity in hematologic and solid human tumor cell lines, but not normal noncancerous cells, by a cell autonomous mechanism (not ADCC). AO-176 also binds preferentially to tumor versus many normal cell types. In particular, AO-176 binds negligibly to RBCs in contrast to tumor cells, even at high concentrations up to 200 μg/mL and does not agglutinate RBCs up to 1 mg/mL in vitro These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. When tested in cynomolgus monkeys, AO-176 was well tolerated with no adverse effects. Finally, we show that AO-176 demonstrates dose-dependent antitumor activity in tumor xenograft models. Taken together, the unique properties and antitumor activity of our next-generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPα axis targeting agents in clinical development.

Abstract 4866: Development and characterization of novel CD40 antibody agonists for cancer immunotherapy

Abstract CD40 is a key molecule in the regulation of immune responses and its activity can be modulated using antibodies. In particular, agonist CD40 antibodies are highly effective in preclinical tumor models either through direct interaction with CD40-expressing lymphomas, or indirectly through the activation of an adaptive anti-tumor immune response. To date, limited clinical data have been reported with strong CD40 agonist antibodies; nonetheless it seems likely that targeting this pathway will require a balance between the benefits of immune stimulation to drive anti-tumor responses, and the damage that can result from non-specific immune cell activation. We set out to develop novel human anti-CD40 antibodies with different levels of agonist activity to identify a lead candidate for systemic application. Anti-CD40 monoclonal antibodies (mAbs) were generated by immunization of human Ig transgenic mice with recombinant and cell surface expressed human CD40. Hybridomas developed from these mice were screened using CD40 binding assays and activity on a reporter cell line engineered to express CD40 and NFêB-responsive luciferase. The variable regions of lead antibodies that displayed differential activity were cloned into vectors containing human IgG1 or IgG2 constant domains and expressed in CHO cells. These human CD40 mAbs were further characterized by analysis of binding affinity, CD40L blocking activity, B cell and dendritic cell activation, and anti-tumor activity in xenograft tumor models. We found a wide range of activities among the CD40 mAbs that is linked to epitope specificity as well as the isotype. In general, the IgG2 isotype mAbs had greater signaling activity than their IgG1 counterparts. The lead candidate mAbs are undergoing additional testing related to functional and toxicity parameters before a final candidate is nominated for clinical development. Citation Format: Laura A. Vitale, Thomas O’Neill, Jenifer Widger, Andrea Crocker, Li-Zhen He, Jeffrey Weidlick, Karuna Sundarapandiyan, James Storey, Lawrence Thomas, Joel Goldstein, Henry C. Marsh, Tibor Keler. Development and characterization of novel CD40 antibody agonists for cancer immunotherapy. [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 4866.

Effects of the novel BET inhibitor BI 894999 on upregulation of HEXIM1 in cancer cells and on antitumor activity in xenograft tumor models.

11574Background: BRD4 is a key epigenetic regulator facilitating transcriptional elongation of super-enhancer regulated oncogenes including MYC. BI 894999, a novel, potent and selective orally bioa...

A Novel Eg5 Inhibitor (LY2523355) Causes Mitotic Arrest and Apoptosis in Cancer Cells and Shows Potent Antitumor Activity in Xenograft Tumor Models.

Intervention of cancer cell mitosis by antitubulin drugs is among the most effective cancer chemotherapies. However, antitubulin drugs have dose-limiting side effects due to important functions of microtubules in resting normal cells and are often rendered ineffective by rapid emergence of resistance. Antimitotic agents with different mechanisms of action and improved safety profiles are needed as new treatment options. Mitosis-specific kinesin Eg5 represents an attractive anticancer target for discovering such new antimitotic agents, because Eg5 is essential only in mitotic progression and has no roles in resting, nondividing cells. Here, we show that a novel selective Eg5 inhibitor, LY2523355, has broad target-mediated anticancer activity in vitro and in vivo. LY2523355 arrests cancer cells at mitosis and causes rapid cell death that requires sustained spindle-assembly checkpoint (SAC) activation with a required threshold concentration. In vivo efficacy of LY2523355 is highly dose/schedule-dependent, achieving complete remission in a number of xenograft tumor models, including patient-derived xenograft (PDX) tumor models. We further establish that histone-H3 phosphorylation of tumor and proliferating skin cells is a promising pharmacodynamic biomarker for in vivo anticancer activity of LY2523355.

LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms.

CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1's role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe.

Therapy of B Cell Malignancies with CD19-Specific Chimeric Antigen Receptor-Modified T Cells of Defined Subset Composition

BACKGROUND:The adoptive transfer of CD19-specific chimeric antigen receptor-modified (CD19 CAR) T cells is a promising strategy for treating patients with CD19+ B cell acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL). Dramatic responses have been observed in a subset of patients receiving CD19 CAR T cell therapy, and prior studies suggest that persistence of transferred T cells may correlate with the extent of tumor regression. The use of unselected T cells to prepare CAR T cells results in variation in the phenotypic composition of the infused product in individual patients, making it difficult to determine whether particular T cell subsets contribute to efficacy and/or toxicity. Studies in our lab demonstrated that genetically modified effector T cells derived from purified T cell subsets differ in the capacity to persist in vivo after adoptive transfer, and that a combination of CAR-modified CD8+ central memory (TCM) and CD4+ T cells provides optimal antitumor activity in tumor xenograft models. Based on these data, we designed the first clinical trial in which patients with CD19+ B cell malignancies receive CD19 CAR T cells comprised of a defined composition of CD8+ TCM and CD4+T cells engineered to express a CD19 CAR.METHODS:Patients with relapsed or refractory CD19+ ALL, CLL or NHL are eligible for this phase I/II study. CD8+ TCM and CD4+ T cells were separately enriched by immunomagnetic selection from a leukapheresis product from each patient, and cryopreserved. The CD8+ TCM and CD4+ T cells were stimulated in independent cultures with anti-CD3/anti-CD28 paramagnetic beads, and transduced with a lentivirus encoding the murine FMC63 anti-CD19 scFv, 4-1BB and CD3 zeta signaling domains. After in vitro expansion, the cell product for infusion was formulated in a 1:1 ratio of CD4+:CD8+ CAR+ T cells. A truncated non-functional human epidermal growth factor receptor (EGFRt) encoded in the transgene cassette allowed identification of transgene-expressing T cells by flow cytometry. Lymphodepleting chemotherapy was administered followed by infusion of EGFRt+ CAR T cells at one of three dose levels (2 x 105 EGFRt+ cells/kg, 2 x 106 EGFRt+ cells/kg, 2 x 107 EGFRt+cells/kg).RESULTS:Twenty patients with relapsed or refractory ALL (n = 9), NHL (n = 10) or CLL (n = 1), including those who failed prior autologous (n = 4) or allogeneic (n = 4) stem cell transplant have been treated on the trial. Fifteen of 20 treated patients received a product that conformed to the prescribed CD8+ T­CM:CD4 composition. Five patients received a product manufactured using a modified strategy either due to low blood lymphocyte counts (n = 3) or due to failure to propagate T cells in culture (n = 2). CD8+ TCM and CD4+ T cells have been isolated from 12 additional patients and cryopreserved for therapy. Patients have been treated at all three dose levels without acute infusional toxicity. Severe cytokine release syndrome (sCRS) consisting of fever, hypotension, and reversible neurotoxicity associated with elevated serum IFN-γ and IL-6 was only observed in ALL patients with a high tumor burden. One ALL patient treated at the highest cell dose died of complications associated with sCRS. None of the NHL patients had sCRS. Of patients who are >6 weeks after CD19 CAR T cell therapy, best responses included complete (n=1) or partial (n=5) remission in 6/9 patients with NHL and complete remission in 5/7 patients with ALL. Both CD4+ and CD8+ CAR-T cells expanded in vivo and could be detected in blood, marrow and CSF. The peak level and duration of persistence of both CD4+ and CD8+ EGFRt+ T cells were associated with clinical response. TCRBV gene sequencing of flow sorted CD4+ and CD8+ EGFRt+CAR T cells from 2 patients showed that proliferating CAR T cells were polyclonal. A subset of NHL patients in whom CAR T cells became undetectable developed a T cell immune response to sequences in the murine CD19-specific scFv component of the CAR transgene.CONCLUSION:Adoptive immunotherapy with CD19 CAR T cells of defined subset composition is feasible and safe in a majority of heavily pretreated patients with refractory B cell malignancies and has potent anti-tumor activity. Persistence of CAR-T cells may be limited in some patients by transgene product immunogenicity. Data from this ongoing clinical trial will be updated at the meeting. DisclosuresTurtle:Juno Therapeutics: Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Hudecek:Juno Therapeutics: Patents & Royalties. Jensen:Juno: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Riddell:Juno Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Maloney:Juno Therapeutics: Research Funding.

Dendronized nanoconjugates of lysine and folate for treatment of cancer

Poly-l-lysine (PLL) dendrimers are currently being investigated as antiangiogenic agent for therapy of cancer. In this study, we report folate conjugated poly-l-lysine dendrimers (FPLL) as an efficient carrier for model anticancer drug, doxorubicin hydrochloride (Dox); for pH sensitive drug release, selective targeting to cancer cells, anticancer activity and antiangiogenic activity. This nanoconjugate of Dox showed initial rapid in vitro release followed by gradual slow release, and the drug release was found to be pH sensitive with greater release at acidic pH. In the CAM assay and tubule formation assay with HUVEC, Dox-FPLL formulation showed the significant antiangiogenic activity confirming that activity of PLL was not compromised by the presence of Dox and folic acid. The ex vivo investigations with human breast cancer cell lines MCF-7 showed enhanced cytotoxicity of Dox-FPLL with significantly enhanced intracellular uptake (p<0.001). The in vivo therapeutic potential of nanoconjugate was determined in MCF-7 breast cancer xenograft model in tumor-bearing mice. Dox-FPLL increased the concentration of Dox in tumor by 121.5-fold after 24h in comparison with free Dox formulation. The folate conjugated dendrimeric Dox showed superior anti-tumor activity in tumor xenograft model with significantly prolonged survival determined by Kaplan Meier survival analysis (p<0.001).

G-Quadruplexes as Potential Therapeutic Targets for Embryonal Tumors

Embryonal tumors include a heterogeneous group of highly malignant neoplasms that primarily affect infants and children and are characterized by a high rate of mortality and treatment-related morbidity, hence improved therapies are clearly needed. G-quadruplexes are special secondary structures adopted in guanine (G)-rich DNA sequences that are often present in biologically important regions, e.g. at the end of telomeres and in the regulatory regions of oncogenes such as MYC. Owing to the significant roles that both telomeres and MYC play in cancer cell biology, G-quadruplexes have been viewed as emerging therapeutic targets in oncology and as tools for novel anticancer drug design. Several compounds that target these structures have shown promising anticancer activity in tumor xenograft models and some of them have entered Phase II clinical trials. In this review we examine approaches to DNA targeted cancer therapy, summarize the recent developments of G-quadruplex ligands as anticancer drugs and speculate on the future direction of such structures as a potential novel therapeutic strategy for embryonal tumors of the nervous system.

Searching Drug-Like Anti-cancer Compound(s) Based on G-Quadruplex Ligands

G-quadruplex structure is a four-stranded form of DNA, which is associated with cancer cell proliferation. G-quadruplex-stabilized ligands have the potential to interfere with telomere replication by blocking the elongation procedure catalyzed by telomerase, and therefore have the potential to be anti-cancer drugs. A considerable number of novel compounds capable of targeting G-quadruplex at high affinity and specificity have been reported. Among them, several G-quadruplex ligands have shown promising anti-cancer activity in tumor xenograft models, and entered phase II clinical trials on cancer patients. This review summarized recent developments of G-quadruplex ligands as anti-cancer drugs and several powerful strategies to discover novel G-quadruplex ligands as anti-cancer drug candidates by screening natural product extracts and structural optimization of previously identified typical compounds.

Ugt1a is required for the protective effect of selenium against irinotecan-induced toxicity

Irinotecan (CPT-11) is widely used for the treatment of patients with colorectal cancer. However, the adverse effects associated with the treatment have hindered the efficacies of irinotecan. We have shown that organic selenium compounds could significantly attenuate irinotecan-associated toxicity and enhance antitumor activity in xenograft tumor models. The objective of this study is to determine the role of a specific group of uridine diphosphate glucuronosyltransferases, which is coded by UGT1A, in detoxification process of irinotecan as well as selenium-associated protective effect against irinotecan-induced toxicity. In this study, the toxicities of irinotecan, docetaxel and cisplatin in the Ugta1 mutant rats and their wild-type controls were compared. The plasma concentrations of irinotecan and SN-38 were measured. The modulatory effect of a selenium compound on irinotecan-induced toxicity was analyzed in these rats. We demonstrated that the maximum tolerated doses of irinotecan in the homozygous mutant rats were significantly lower than those in wild-type rats, 25 mg/kg × 1 versus 200 mg/kg × 1 and 3 mg/kg/day × 3 versus 100 mg/kg/day × 3, respectively. The enhanced sensitivity was specific to irinotecan and was not observed with other chemotherapeutic agents, such as docetaxel and cisplatin, where Ugt1a is not required for their metabolism. Our results also showed that selective protection against irinotecan-induced toxicity by 5-methylselenocysteine was achieved in the wild-type rats but not in the Ugt1a null rats. These data support the hypothesis that expression of UGT1A is critical for 5-methylselenocysteine to exert its protective effect against irinotecan-induced toxicity.

Abstract C38: CRLX101 shows significant tumor penetration and superior activity compared to first- and second-line agents in NSCLC xenograft tumor models.

Abstract Introduction: CRLX101 is a tumor-targeted nanopharmaceutical designed to localize and accumulate in tumors. A randomized Phase 2 study is ongoing evaluating safety and efficacy of CRLX101 as a second- and third-line single agent therapy for the treatment of advanced non-small cell lung cancer (NSCLC). CRLX101 has previously demonstrated broad spectrum anti-tumor activity in xenograft tumor models of lung, ovarian, colorectal, pancreatic, breast and lymphoma. Here we present efficacy data of CRLX101 in NSCLC tumor models representative of clinical subgroups with limited therapeutic options, including squamous cell, and KRAS and EGFR mutants. Additionally, we present microscopy data illustrating that CRLX101 penetrates tumor tissues and is taken up by cancer cells. Experimental procedures: CRLX101 is composed of camptothecin, a highly potent topoisomerase 1 inhibitor, conjugated to a cyclodextrin-poly(ethylene glycol) co-polymer and self-assembles into nanoparticles of 20–50 nm in diameter. For in vivo efficacy, tumor-bearing mice were administered either CRLX101 or a comparator at their respective maximum-tolerated dose (MTD) and schedule, and tumor growth inhibition and survival were measured. For tumor localization studies, xenograft tumors were excised and CRLX101 was visualized with standard staining methods. Summary of data: In H1299 NSCLC model, CRLX101 showed superior activity over approved 1st and 2nd line agents resulting in 100% (10/10) cures. Gemcitabine and topotecan achieved 50% (5/10) or 10% (1/10) cures, respectively; and no cure (0/10) was observed with docetaxel, erlotinib or pemetrexed. In H520 squamous-type NSCLC model, CRLX101 showed a greater median survival and tumor growth delay than carboplatin, docetaxel or gemcitabine. Finally, CRLX101 showed significant tumor growth delay activity and survival improvements in G12C and G12S KRAS mutation models (H2122 and A549 respectively) and in a T790M EGFR mutation model (H1975). BrdU staining of tumor sections from H1299 NSCLC xenografts treated with CRLX101 demonstrated that the nanoparticle penetrated deep and uniformly in the tumor tissues and inhibited cell proliferation. Conclusions: CRLX101 is a novel nanopharmaceutical that has shown encouraging activity in advanced NSCLC patients in a Phase 1/2b clinical trial. Our preclinical data suggests that CRLX101 may be efficacious in patient population harboring mutations and drug resistance that render them refractory to currently approved agents. Our imaging data further support that CRLX101 can potentially achieve superior anti-tumor activity by localizing and penetrating deep into tumor tissues and releasing camptothecin inside tumor cells. In sum, our pre-clinical and clinical data together support that CRLX101 is potentially a versatile therapeutic option for the management of advanced NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C38.

ENMD-2076 Is an Orally Active Kinase Inhibitor with Antiangiogenic and Antiproliferative Mechanisms of Action

ENMD-2076 is a novel orally active, small molecule kinase inhibitor with a mechanism of action involving several pathways key to tumor growth and survival: angiogenesis, proliferation, and the cell cycle. ENMD-2076 has selective activity against the mitotic kinase Aurora A, as well as kinases involved in angiogenesis (VEGFRs, FGFRs). ENMD-2076 inhibited the growth in vitro of a wide range of human solid tumor and hematopoietic cancer cell lines with IC(50) values ranging from 0.025 to 0.7 μmol/L. ENMD-2076 was also shown to induce regression or complete inhibition of tumor growth in vivo at well-tolerated doses in tumor xenograft models derived from breast, colon, melanoma, leukemia, and multiple myeloma cell lines. Pharmacodynamic experiments in vivo showed that in addition to inhibiting Aurora A, single doses of ENMD-2076 had sustained inhibitory effects on the activation of Flt3 as well as the angiogenic tyrosine kinases, VEGFR2/KDR and FGFR1 and 2. ENMD-2076 was shown to prevent the formation of new blood vessels and regress formed vessels in vivo at doses equivalent to those that gave substantial activity in tumor xenograft models. These results indicate that ENMD-2076 is a well-tolerated, orally active multitarget kinase inhibitor with a unique antiangiogenic/antiproliferative profile and provides strong preclinical support for use as a therapeutic for human cancers. Several phase 1 studies involving ENMD-2076 have been recently completed, and the compound is currently being evaluated in a phase 2 clinical trial in patients with platinum-resistant ovarian cancer.

Abstract 3624: A novel, selective FGFR inhibitor that causes blockade of FGFR autophosphorylation shows a broad-spectrum of anti-tumor activity in preclinical xenograft tumor models

Abstract Anti-angiogenic agents targeting VEGF/VEGFR including Bevacizumab, Sorafenib, and Sunitinib that target blood vessel formation in rapidly growing cancer cells are very effective cancer therapies in the clinic. However, these anti-angiogenic agents also have debilitating side effects, such as hypertension and bleeding, which thereby limit their use in many patients in which the anti-VEGF/VEGFR based therapies are contraindicated. Furthermore, the emergence of resistance to the anti-VEGF/VEGFR based therapies in the clinic due to over-expression of FGF2 or other growth factors could pose a serious challenge to their current use in the clinic. The FGFRs are receptor tyrosine kinases that are present in many types of tumor cells as well as endothelial cells, and play an important role in tumor cell growth, survival, and migration, and also in maintaining tumor angiogenesis. Over-expression of FGFRs or aberrant regulation of their activities has been implicated in many forms of human malignancies. Therefore, targeting FGFRs for cancer treatment represents an attractive strategy that has the potential to treat a variety of cancers by simultaneously inhibiting tumor cell growth, survival, and migration, and also tumor angiogenesis without the side effects associated with the VEGF/VEGFR based therapies. Here, we describe a selective small molecule inhibitor of FGFRs. The FGFR inhibitor is active against all four FGF receptors (IC50 = 2.6 to 7.7 nM) as revealed by biochemical autophosphorylation assays. It also exhibits a potent activity against FGFR or FGFR-mediated Erk phosphorylation in several cancer cell lines (IC50 = 0.6 to 1.5 nM). In addition, in vitro cell based assays have shown that this inhibitor is much more active at inhibiting the proliferation of FGFR-dependent than FGFR-independent cancer cell lines (IC50s = 3-2,600 nM). Furthermore, in vitro selectivity assays have established that this inhibitor exhibits a robust preferential activity in inhibiting FGF- over VEGF-mediated cord formation (IC50s = 0.6 nM for FGF and 3.6 nM for VEGF). Consistent with these in vitro selectivity data, in vivo target inhibition assays have also demonstrated that the FGFR inhibitor has a potent, yet much higher activity against FGF- than VEGF-mediated target phosphorylation in mice (TEC50s and TEC90s of 6 and 29 nM for FGFR, and 35 and 252 nM for VEGFR, respectively). Finally, the FGFR inhibitor shows an excellent broad-spectrum anti-tumor activity in xenograft tumor models representing several major cancer histologies including bladder cancer when dosed at TED50 (1.3 mg/kg) and especially at TED90 (3.2 mg/kg). Thus, the FGFR inhibitor exhibits a very well behaved PK/PD relationship. Currently, the FGFR inhibitor is being further evaluated for its potential utility in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3624.

Targeting Pim Kinases in Hematological Malignancies.

The three Pim kinases represent a small subfamily of serine/threonine kinases known to be involved in a number of signaling pathways as downstream effectors and potent inhibitors of apoptosis. Unlike most other kinases, Pim kinases lack a regulatory domain which means they are controlled largely at the transcriptional level and that the mRNA expression levels of Pim kinases in cells correlate with their activity. While normal expression of Pim-1 kinase is seen in cells of hematopoietic origin, examination of gene expression of Pim-1 in different malignancies using cDNA microarray analysis suggests that Pim-1 is overexpressed in a large percentage of ALL, AML, CML, DLBCL, Prostatic Adenocarcinoma, Bladder, and Oral cancers. Pim-2 is also largely expressed in ALL, AML, Squamous Cell Lung and Adenocarcinoma of the Lung while Pim-3 is restricted to Melanoma, Pancreatic Adenocarcinoma, Gastric cancers. This implicates Pim-1 and Pim-2 in the onset and progression in several of hematological malignancies and therefore they represent interesting potential targets for drug development. To evaluate the potential of Pim-1 and -2 as a drug targets we have identified and synthesized a series of Pim kinase inhibitors using our proprietary CLIMB™ drug discovery process. Through the use of CLIMB™, the published Pim-1 kinase crystal structure was used to build several models that were then used to predict potential small molecule inhibitors of Pim-1 and Pim-2 from a large virtual library. A subset of leads, based on calculated binding energies as well as additional physical chemical properties, were screened using a number of in silico physicochemical and ADMET prediction algorithms to determine which compounds were most likely to be successful in a biological context. Lead candidates were initially screened using biochemical enzyme-based and cell-based assays. Cell-based activity was determined in HEL (acute megakaryocytic leukemia), K562 (chronic myeloid leukemia), MO7e (myeloid leukemia), and other human leukemia and lymphoma cell lines. From several lead candidates a series of analogs were produced with improved inhibitory activity and pharmacokinetic characteristics. In the Pim-1 and Pim-2 in vitro kinase assay and in the cell-based assay a number of leads exhibited inhibitory activity with IC50 concentrations in the low nanomolar range. Here we present the details of the biochemical and cell-based assay results as well as the activity in tumor xenograft models of our Pim kinase inhibitors.

Efficient growth inhibition of ErbB2-overexpressing tumor cells by anti-ErbB2 ScFv-Fc-IL-2 fusion protein in vitro and in vivo

To investigate the antitumor activities of an anti-ErbB2 scFv-Fc-interleukin 2 (IL-2) fusion protein (HFI) in vitro and in vivo. Fusion protein HFI was constructed. The efficacy of HFI in mediating tumor cell lysis was determined by colorimetric lactate dehydrogenase release assays. The antitumor activity of HFI was evaluated in tumor xenograft models. The fusion protein was folded as a homodimer formed by covalently linking Fc portions and it retained ErbB2 specificity and IL-2 biological activity. HFI mediated antibody-dependent cell-mediated cytotoxicity (ADCC) at low effector-to-target ratios in vitro and improved the therapeutic efficacy of IL-2 in experiments in vivo. The genetically-engineered anti-ErbB2 scFv-Fc-IL-2 fusion protein exhibited high efficiency both in mediating ADCC in vitro and significant antitumor activity in tumor xenograft models.

Enhanced tumor killing by Apo2L/TRAIL and CPT-11 co-treatment is associated with p21 cleavage and differential regulation of Apo2L/TRAIL ligand and its receptors.

Apo2L/TRAIL exhibits enhanced apoptotic activity in tumor xenograft models when used in combination with the topoisomerase 1 inhibitor CPT-11. To investigate the cellular mechanisms involved in this increased tumor-killing activity, a series of in vitro experiments were conducted using the human colon carcinoma cell line (HCT116). Apo2L/TRAIL induced a transient upregulation of DR5 mRNA, while CPT-11 increased both death and decoy receptor expression. Upregulation of decoy receptors by CPT-11 was partially inhibited by co-administration of Apo2L/TRAIL. CPT-11 treatment resulted in accumulation of cells at G(2)M-phase and correlated with a substantial increase in the protein levels of the cyclin-dependent kinase inhibitor p21. However, cells co-treated with CPT-11 and Apo2L/TRAIL, or pretreated with CPT-11 for up to 24 h followed by 2 h Apo2L/TRAIL, resulted in a caspase-dependent degradation of p21, reversal of G(2)-M phase arrest with a concomitant increase in apoptosis. The sequential treatment produced the greatest induction of DR5 and DR4, caspase-3-like cleavage/activation and p21 degradation, as well as increased apoptosis. These data indicate that the up-regulation of Apo2L/TRAIL ligand and its death receptors as well as cleavage of p21 protein in the Apo2L/TRAIL plus CPT-11 treatment contributes to the positive cooperation between these agents in enhancing tumor cell apoptosis.