Growth Inhibition Studies Research Articles

Tislelizumab (BGB-A317) for Relapsed/Refractory Classical Hodgkin Lymphoma: Preliminary Efficacy and Safety Results from a Phase 2 Study

Background: Tislelizumab is a humanized IgG4 monoclonal antibody with high affinity/specificity for programmed cell death protein 1 (PD-1). Tislelizumab was specifically engineered to minimize binding to FcɤR on macrophages, thereby abrogating antibody-dependent phagocytosis, a potential mechanism of T-cell clearance and resistance to anti-PD-1 therapy (Dahan 2015). Results of tumor growth inhibition studies suggest that tislelizumab had superior antitumor activity compared with nivolumab in mice transplanted with human cancer cells and peripheral blood mononuclear cells. Favorable results with other PD-1 inhibitors in patients with relapsed or refractory (R/R) classical HL (cHL) provide a strong rationale to investigate tislelizumabin this disease.Methods: BGB-A317-203 (clinicaltrials.gov NCT03209973) is a single-arm, open-label, multicenter, phase 2 study of tislelizumab in Chinese patients with R/R cHL; all patients received tislelizumab 200 mg intravenously every 3 weeks until progression or unacceptable toxicity. Patients were eligible if they (a) failed to achieve a response or progressed after autologous stem cell transplant (ASCT) or (b) received ≥2 prior systemic chemotherapy regimens for cHL and were ineligible for ASCT. Diagnosis of cHL was confirmed in all patients by central pathologic review.The primary endpoint was overall response rate (ORR) determined using the Lugano criteria (Cheson, 2014) as assessed by an independent review committee (IRC). Key secondary endpoints included progression-free survival (PFS), duration of response, rate of complete response (CR), time to response, safety, and tolerability. Treatment emergent adverse events (TEAEs) were summarized according to NCI-CTCAE v4.03.Results: In total, 70 patients were enrolled from 11 Chinese centers; patient characteristics are shown in the Table. With a data cutoff date of 25 May 2018, the median follow-up was 7.9 months (range, 3.4 to 12.7). The IRC-assessed ORR was 85.7%, based on PET-CT scans. A total of 43 patients (61.4%) achieved CR, 38 of whom were in CR at the first on-study response assessment. At data cutoff, 53 patients remained on treatment and 17 had discontinued (11 for progressive disease [PD]; 4 for TEAEs; 1 withdrew consent; 1 due to pregnancy). The estimated 6-month PFS rate was 80%. The most frequently reported (≥15%) TEAEs due to any cause were pyrexia (52.9%), hypothyroidism (30.0%), increased weight (28.6%), upper respiratory tract infection (27.1%) and cough (17.1%). Grade ≥3 TEAEs reported in ≥2 patients were upper respiratory tract infection (2.9%) and pneumonitis (2.9%). Immune-related TEAEs were reported in 23 patients (32.9%); Grade ≥3 in 5 patients (7.1%): pneumonitis (n=2), organizing pneumonia, nephritis (focal segmental glomerulosclerosis) and increased creatine phosphokinase (each n=1). There were no Grade 5 TEAEs. TEAEs that led to treatment discontinuation in 4 patients (5.7%) included pneumonitis (n=2), organizing pneumonia (n=1), and focal segmental glomerulosclerosis (n=1). One patient died on study due to PD.Conclusions: In this study, tislelizumab therapy was shown to be highly active resulting in a high CR rate in patients with R/R cHL who had failed or were ineligible for ASCT. Tislelizumab was generally well-tolerated in Chinese patients with R/R cHL. The safety profile was generally consistent with that of other PD-1 inhibitors for the treatment of cHL. DisclosuresSong:Peking University Cancer Hospital (Beijing Cancer Hospital): Employment. Liu:West China Hospital of Sichuan University: Employment. Guo:BeiGene (Shanghai) Co., LTD: Employment. Yang:BeiGene (Beijing) Co., Ltd.: Employment. Elstrom:BeiGene (Beijing) Co., Ltd.: Employment, Equity Ownership. Huang:BeiGene (Beijing) Co., Ltd.: Employment, Equity Ownership. Novotny:BeiGene (Beijing) Co., Ltd.: Employment, Equity Ownership. Wei:BeiGene (Beijing) Co., Ltd.: Employment, Equity Ownership. Zhu:Beijing Cancer Hospital: Employment.

Abstract 2897: Therapeutic efficacy of antibodies targeting domain 1 of HSPG2 in breast cancer

Abstract Significant advancements in chemotherapy have improved the survival rates of patients presenting with local or regional breast cancer to as high as 99% and 85%, respectively. On the contrary, patients with metastatic breast cancer have a dismal 5-year survival rate of 26%. Thus, there is an urgent need for research strategies directed towards treatment of metastasis. Our lab used a phage display-based cell panning procedure to develop two fully humanized antibodies (Clone 6 and AM6) that are able to bind specifically to breast cancer metastatic cells. Target deconvolution revealed HSPG2/Perlecan Domain 1 as the cell surface antigen bound by the antibodies. Immunohistochemistry studies revealed high HSPG2 expression across various tumor subtypes including melanoma, bladder cancer, glioblastoma and ovarian cancer. There was a significant correlation between high HSPG2 and with poor survival in bladder and ovarian cancers. Interestingly, we observed significant tumor growth inhibition in the triple-negative MDA-MB-231 breast cancer xenograft model. This efficacy was significantly reduced when the tumor growth inhibition studies were repeated in NSG mice, suggesting NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) as the potential mechanism of action. The data presented here point to the relevance of HSPG2 as a novel target for not only breast cancer but also other malignancies. We also show the potential of Clone 6 and AM6 as therapeutic and targeting agents. Further studies are required to understand the significance of HSPG2 overexpression and its correlation with tumor progression as well as to confirm the mechanism of action with Clone 6 and AM6 antibodies. Citation Format: Vidhi Khanna, Stephen Kalscheuer, Jayanth Panyam, Da Yang, Sihan Li. Therapeutic efficacy of antibodies targeting domain 1 of HSPG2 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2897.

Addressing diversity in tuberculosis using multidimensional approaches.

Tuberculosis is a complex disease, which can affect many organs other than the lungs. Initial infection may be cleared without inducing immunological memory, or progress directly to primary disease. Alternatively, the infection may be controlled as latent TB infection, that may progress to active tuberculosis at a later stage. There is now a greater understanding that these infection states are part of a continuum, and studies using PET/CT imaging have shown that individual lung granulomas may respond to infection independently, in an un-synchronized manner. In addition, the Mycobacterium tuberculosis organisms themselves can exist in different states: as nonculturable forms, as 'persisters', as rapidly growing bacteria and a biofilm-forming cording phenotype. The 'omics' approaches of transcriptomics, metabolomics and proteomics can help reveal the mechanisms underlying these different infection states in the host, and identify biosignatures with diagnostic potential, that can predict the development of disease, in 'progressors' as early as 12-18months before it can be detected clinically, or that can monitor the success of anti-TB therapy. Further insights can be obtained from studies of BCG vaccination and new TB vaccines. For example, epigenetic changes associated with trained immunity and a stronger immune responses following BCG vaccination can be identified. These omics approaches may be particularly valuable when linked to studies of mycobacterial growth inhibition, as a direct read-out of the ability to control mycobacterial growth. The second generation of omics studies is identifying much smaller signatures based on as few as 3 or 4 genes. Thus, narrowing down omics-derived biosignatures to a manageable set of markers now opens the way to field-friendly point of care assays.

Nucleoside‐Based Self‐Assembling Drugs for Localized Drug Delivery

We have synthesized a range of gelators based on the nucleoside analogues gemcitabine and lamivudine, characterizing representative gels from the series using rheology and transmission electron microscopy. Growth inhibition studies of gemcitabine derivatives confirmed the feasibility of these compounds as novel treatments, indicating the potential of nucleoside-based gelators for localized drug delivery.

Co-delivery of docetaxel and gemcitabine using PEGylated self-assembled stealth nanoparticles for improved breast cancer therapy

The present report deals with conjugation of dual drug; docetaxel (DTX) and gemcitabine (GEM) with linker poly-ethylene-glycol (PEG) to develop amphiphilic molecule having self-assembled property. The synthesized conjugate (DTX-PEG-GEM) demonstrated critical micelle concentration (CMC) in the range of 5–10 μg/ml which self-assembled to form NPs with size 124.2 ± 5.7. Remarkably higher coumarin-6 (C-6) fluorescence signals observed in case of C-6 loaded NPs, suggested enhanced cellular uptake via clathrin mediated endocytosis. Developed NPs demonstrated 4.8-fold higher AUC(0-∞) value of GEM in comparison with Gemzar®. Tumor growth inhibition study demonstrated significant reduction in tumor volume and higher survival rate with NPs. Moreover, NPs demonstrated significantly lower hepato- and nephro-toxicity, evident from both histopathological sections and biochemical markers level estimation, and hemolytic toxicity. Data in hand suggest enhanced therapeutic efficacy and reduced toxicity of developed NPs over conventional drugs, resulting in efficient combinatorial chemotherapeutic-regimen and patient compliance, which is still an unmet task.

Tall oil fatty acid inclusion in the diet improves performance and increases ileal density of lactobacilli in broiler chickens

ABSTRACT1. Studies were conducted with tall oil fatty acids (TOFA) to determine their effect on broiler chicken performance and ileal microbiota. TOFA, a product originating from coniferous trees and recovered by fractional distillation of side-streams from pulp production, mainly comprises free long-chain fatty acids (~90%) and resin acids (~8%). Conjugated linolenic acids and pinolenic acid are characteristic fatty acid components of TOFA.2. TOFA products at 750 mg/kg feed were tested in two 35-day broiler chicken trials, each using a wheat soya-based diet and with 12 replicate pens per treatment. In both trials, TOFA improved body weight gain at all time points (P < 0.001) and feed conversion efficiency during the first 21 days (P < 0.01). Two different dry TOFA formulations (silica carrier and palm oil coating) were tested and showed performance effects similar to liquid TOFA.3. Ileal digesta of the broiler chickens was analysed for total eubacteria, Lactobacillus spp., Enterococcus spp., Escherichia coli and Clostridium perfringens on days 14 and 35. TOFA significantly increased total eubacteria and lactobacilli density on day 14 (P < 0.05). There was a significant positive correlation between these bacterial groups and broiler body weight on day 14 (P < 0.01).4. A numerical reduction in C. perfringens was observed. In vitro growth inhibition studies showed that C. perfringens was strongly inhibited by 10 mg/l TOFA (P < 0.001), while common lactobacilli were resistant to >250 mg/l. The in vitro results were thus in line with in vivo observations.5. The mechanisms behind the bacterial shifts and their role in performance improvement are unknown. Further purification of TOFA components is needed to identify the effective agents.

212Pb-Labeled Antibody 225.28 Targeted to Chondroitin Sulfate Proteoglycan 4 for Triple-Negative Breast Cancer Therapy in Mouse Models.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis. There is a clinical need for effective, targeted therapy strategies that destroy both differentiated TNBC cells and TNBC cancer initiating cells (CICs), as the latter are implicated in the metastasis and recurrence of TNBC. Chondroitin sulfate proteoglycan 4 (CSPG4) is overexpressed on differentiated tumor cells and CICs obtained from TNBC patient specimens, suggesting that CSPG4 may be a clinically relevant target for the imaging and therapy of TNBC. The purpose of this study was to determine whether α-particle radioimmunotherapy (RIT) targeting TNBC cells using the CSPG4-specific monoclonal antibody (mAb) 225.28 as a carrier was effective at eliminating TNBC tumors in preclinical models. To this end, mAb 225.28 labeled with 212Pb (212Pb-225.28) as a source of α-particles for RIT was used for in vitro Scatchard assays and clonogenic survival assays with human TNBC cells (SUM159 and 2LMP) grown as adherent cells or non-adherent CIC-enriched mammospheres. Immune-deficient mice bearing orthotopic SUM159 or 2LMP xenografts were injected i.v. with the targeted (225.28) or irrelevant isotype-matched control (F3-C25) mAbs, labeled with 99mTc, 125I, or 212Pb for in vivo imaging, biodistribution, or tumor growth inhibition studies. 212Pb-225.28 bound to adherent SUM159 and 2LMP cells and to CICs from SUM159 and 2LMP mammospheres with a mean affinity of 0.5 nM. Nearly ten times more binding sites per cell were present on SUM159 cells and CICs compared with 2LMP cells. 212Pb-225.28 was six to seven times more effective than 212Pb-F3-C25 at inhibiting SUM159 cell and CIC clonogenic survival (p < 0.05). Radiolabeled mAb 225.28 showed significantly higher uptake than radiolabeled mAb F3-C25 in SUM159 and 2LMP xenografts (p < 0.05), and the uptake of 212Pb-225.28 in TNBC xenografts was correlated with target epitope expression. 212Pb-225.28 caused dose-dependent growth inhibition of SUM159 xenografts; 0.30 MBq 212Pb-225.28 was significantly more effective than 0.33 MBq 212Pb-F3-C25 at inhibiting tumor growth (p < 0.01). These results suggest that CSPG4-specific 212Pb-225.28 is a useful reagent for RIT of CSPG4-expressing tumors, including metastatic TNBC.

Green Approach To Synthesize Crystalline Nanoscale ZnII-Coordination Polymers: Cell Growth Inhibition and Immunofluorescence Study.

Five new coordination polymers (CPs) namely, [{Zn(μ2-H2O)0.5(5N3-IPA)(2,2'-bpe)}]∞ (1), [{Zn(μ2-H2O)0.5(5N3-IPA)(1,10-phen)}]∞ (2), [{Zn(5N3-IPA)(1,2-bpe)}]∞ (3), [{Zn(5N3-IPA)(1,2-bpey)}]∞ (4), and [{Zn(H2O)(5N3-IPA)(4,4'-tme)}(H2O)0.5]∞ (5) (5N3-H2IPA = 5-azidoisophthalic acid, 2,2'-bpe= 2,2'-bipyridine, 1,10-phen = 1,10-phenanthroline, 1,2-bpe = 1,2-bis(4-pyridyl)ethane, 1,2-bpey = 1,2-bis(4-pyridyl)ethylene, 4,4'-tme = 4,4'-trimethylenedipyridine), have been synthesized based on a mixed ligand approach adopting a solvothermal technique. Depending upon the intrinsic structural flexibility of the bis-pyridyl coligands, interesting structural topologies have also been observed in the resulting CPs: Sra SrAl2 type topology for 3 and a 3-fold interpenetrated dmp topology for 4. A green hand grinding technique has been implemented to reduce the particle size of the CPs to generate nanoscale CPs (NCPs). SEM studies of NCPs reveal the formation of square and spherical particles for NCP 1 and 2, respectively, and nano rod for NCP 3, 4, and 5. Remarkably, when scaled down to nano range all the NCPs retain their crystalline nature. The cytotoxic activity of the NCPs (1-5) has been studied using human colorectal carcinoma cells (HCT 116). Significant cell death is observed for NCP 2, which is further corroborated by cell growth inhibition study. The observed cell death is likely to be due to mitochondrial-assisted apoptosis as is evident from immunofluorescence study.

Transferrin-Modified Vitamin-E/Lipid Based Polymeric Micelles for Improved Tumor Targeting and Anticancer Effect of Curcumin.

Transferrin receptor (TfR) is up-regulated in various malignant tumors not only to meet the iron requirement, but also to increase the cell survival via participation in various cellular signaling pathways. Here we explored transferrin as ligand for Poly(ethylene Glycol) (PEG)-ylated vitamin-E/lipid (PE) core micelles (VPM). Transferrin modified polymer was synthesized and drug loaded micelles were evaluated in 2D Hela and HepG2 cancer cells for cellular uptake and cytotoxicity and in 3D Hela spheroids for growth inhibition, uptake and penetration studies. Targeted (Tf-VPM) and non-targeted (VPM) micelles showed mean hydrodynamic diameter of 114.2 ± 0.64nm and 117.4 ± 0.72nm and zeta potential was -22.8 ± 0.62 and -14.8 ± 1.74mV, respectively. Cellular uptake study indicated that the Tf-CVPM were taken up by cancer cells (Hela and HepG2) with higher efficiency. Enhanced cytotoxicity was demonstrated for Tf-VPM compared to CVPM. Marked spheroid growth inhibition following treatment with Tf-CVPM was observed compared to the treatment with non-targeted CVPM. The developed transferrin-modified micelles have improved ability to solubilize the loaded drugs and could actively target solid tumors by its interaction with over-expressed transferrin receptors. Therefore, the nano-micelles could be further explored for its potential utilization in cancer therapy.

Transferrin-targeted, resveratrol-loaded liposomes for the treatment of glioblastoma

Glioblastomas (GBMs) are highly aggressive brain tumors with a very grim prognosis even after multi-modal therapeutic regimens. Conventional chemotherapeutic agents frequently lead to drug resistance and result in severe toxicities to non-cancerous tissues. Resveratrol (RES), a natural polyphenol with pleiotropic health benefits, has proven chemopreventive effects in all the stages of cancer including initiation, promotion and progression. However, the poor physico-chemical properties of RES severely limit its use as a free drug. In this study, RES was loaded into PEGylated liposomes (RES-L) to counter its drawbacks as a free drug. Since transferrin receptors (TfRs) are up-regulated in GBM, the liposome surface was modified with transferrin moieties (Tf-RES-L) to make them cancer cell-specific. The liposomal nanomedicines developed in this project were aimed at enhancing the physico-chemical properties of RES and exploiting the passive and active targeting capabilities of liposomes to effectively treat GBM.The RES-L were stable, had a good drug-loading capacity, prolonged drug-release in vitro and were easily scalable. Flow cytometry and confocal microscopy were used to study the association with, and internalization of, Tf-L into U-87 MG cells. The Tf-RES-Ls were significantly more cytotoxic and induced higher levels of apoptosis accompanied by activation of caspases 3/7 in GBM cells when compared to free RES or RES-L. The ability of RES to arrest cells in the S-phase of the cell cycle, and selectively induce production of reactive oxygen species in cancer cells were probably responsible for its cytotoxic effects. The therapeutic efficacy of RES formulations was evaluated in a subcutaneous xenograft mouse model of GBM. A tumor growth inhibition study and a modified survival study showed that Tf-RES-Ls were more effective than other treatments in their ability to inhibit tumor growth and improve survival in mice. Overall, the liposomal nanomedicines of RES developed in this project exhibited favorable in vitro and in vivo efficacies, which warrant their further investigation for the treatment of GBMs.

Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide

SummaryBackground The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of β5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional β5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the β5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.

Abstract P5-20-18: Comparison of paclitaxel plus T-DM1 and pertuzumab versus paclitaxel plus trastuzumab and pertuzumab with carboplatin for HER2 overexpressed breast cancer models

Abstract Background: Remarkable progressions have been made in the breast cancer treatments over the past years, however, patients getting relief from the toxicity of cancer treatments are still unmet needs. The neoadjuvant regimen of docetaxel, carboplatin, and trastuzumab plus pertuzumab is standard choice for patients with HER2 positive breast cancer (Phase III results of the KRISTINE trial, SA Hurvitz et al., 2016). Trastuzumab emtansine (T-DM1) plus pertuzumab (P) has shown superior anti-tumor effect comparing with trastuzumab/Herceptin (H) plus P in preclinical HER2 positive breast cancer models (Y Sun et al., 2013). In this study we aimed to evaluate the preclinical efficacy of paclitaxel (T) plus T-DM1 and P versus paclitaxel plus H and P with carboplatin (C) (TCHP) in HER2 positive breast cancer models. Methodology: 1) HER2 amplified/overexpressed with ER+ BT474 (H- sensitive) and BT474HerR, an H-resistant derivative from BT474, were used. 2) 6-7 week-old female NCr (nu/nu, 20-25g) athymic mice (Taconic) were used for in vivo tumor growth inhibition studies. 3) Mice were injected s.c. on the flank with 5 x 106 BT474 or BT474HerR cells. All mice were also received s.c. injection of 1 mg/kg estradiol valerate (JHP Pharmaceuticals) once weekly to promote tumor cell growth. 4) Mice bearing BT474 or BT474HerR tumors were treated with different combinations of T (20 mg/kg, i.v., every 4 days, total 7 doses), C (40 mg/kg, i.v., one dose), H (10 mg/kg, i.p., twice weekly for 4 weeks), T-DM1 (10 mg/kg, i.v., every 3 weeks, total 3 doses) and P (15 mg/kg, i.p., once weekly for 8 weeks) versus vehicle control. 5) In vivo data were analyzed by Student's t-test. A p-value of 0.05 was considered statistically significant. Results: We observed that 1) both regimens of TCHP and paclitaxel plus T-DM1 and P inhibited tumor growth in BT474 (TCHP p&amp;lt;0.0043 vs. T+T-DM1+P p&amp;lt;0.0025) and BT474HerR (TCHP p&amp;lt;0.00044 vs. T+T-DM1+P p&amp;lt;0.000037) xenograft models; 2) T+T-DM1+P caused almost complete regression in both models (comparing to 1 day before treatment started, the mean tumor volume at the end of study was decreased ˜ 91.13% in BT474 model and ˜ 100% in BT474HerR model) whereas TCHP caused significant tumor growth inhibition in both models (comparing to 1 day before treatment started, the mean tumor volume at the end of study was decreased ˜ 74.6% in BT474 model and ˜ 73.31% in BT474HerR model). Conclusion: Our in vivo data suggest that 1) T+T-DM1+P shows equivalent excellent antitumor activity to TCHP in HER2 overexpressed BT474 model; 2) T+T-DM1+P shows superior antitumor activity to TCHP (p&amp;lt;0.0045) in HER2 overexpressed BT474HerR model; 3) comparing to TCHP, the combination of T+T-DM1+P may have better safety profile in patients. Citation Format: Sun Y, Lin X, Jepperson T, Williams C, De P, Leyland-Jones B. Comparison of paclitaxel plus T-DM1 and pertuzumab versus paclitaxel plus trastuzumab and pertuzumab with carboplatin for HER2 overexpressed breast cancer models [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-20-18.

Establishing in vitro-in vivo correlation for antibody drug conjugate efficacy: a PK/PD modeling approach.

The objective of this manuscript was to establish in vitro-in vivo correlation (IVIVC) between the in vitro efficacy and in vivo efficacy of antibody drug conjugates (ADCs), using a PK/PD modeling approach. Nineteen different ADCs were used to develop IVIVC. In vitro efficacy of ADCs was evaluated using a kinetic cell cytotoxicity assay. The cytotoxicity data obtained from in vitro studies was characterized using a novel mathematical model, parameter estimates from which were used to derive an in vitro efficacy matrix for each ADC, termed as 'in vitro tumor static concentration' (TSCin vitro). TSCin vitro is a theoretical concentration at continuous exposure of which the number of cells will neither increase nor decrease, compared to the initial cell number in the experiment. The in vivo efficacy of ADCs was evaluated using tumor growth inhibition (TGI) studies performed on human tumor xenograft bearing mice. The TGI data obtained from in vivo studies was characterized using a PK/PD model, parameter estimates from which were used to derive an in vivo efficacy matrix for each ADC, termed as 'in vivo tumor static concentration' (TSCin vivo). TSCin vivo is a theoretical concentration if one were to maintain in the plasma of a tumor bearing mouse, the tumor volume will neither increase nor decrease compared to the initial tumor volume. Comparison of the TSCin vitro and TSCin vivo values from 19 ADCs provided a linear and positive IVIVC. The Spearman's rank correlation coefficient for TSCin vitro and TSCin vivo was found to be 0.82. On average TSCin vivo was found to be~27 times higher than TSCin vitro. The reasonable IVIVC for ADCs suggests that in vitro efficacy data was correctly able to differentiate ADCs for their in vivo efficacy. Thus, IVIVC can be used as a tool to triage ADC molecules in the discovery stage, thereby preventing unnecessary scaling-up of ADCs and waste of time and resources. An ability to predict the concentration of ADC that is efficacious in vivo using the in vitro data can also help in optimizing the experimental design of preclinical efficacy studies. As such, the novel PK/PD modeling method presented here to establish IVIVC for ADCs holds promise, and should be evaluated further using diverse set of cell lines and anticancer agents.

Abstract LB-B29: Combined inhibition of MAPK and PI3K signaling augments standard chemotherapy response in preclinical models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal cancer with mortality closely paralleling incidence despite substantial advances in biological understanding of the disease. Nab-paclitaxel (NPT) plus Gemcitabine (Gem) represents the current standard for systemic therapy of advanced PDAC. More than 90% of PDACs harbor an activating mutation in the KRAS oncogene. Presently, no therapeutics exist that effectively target this oncogene, but alternative strategies focus on inhibition of downstream effectors of KRAS signaling pathways. The RAF-MEK-ERK (MAPK) and the AKT-PI3K signaling pathways are well-described mediators of KRAS induced transformation and tumorigenesis in several cancers including PDAC and represent potential targets for combination therapy. We evaluated combination treatment benefits of NPT+Gem with the MEK inhibitor trametinib (Tra) and the AKT inhibitor MK-2206 (MK) in preclinical models to evaluate their therapeutic potential against PDAC. An animal survival study was performed in peritoneal dissemination model in NOD/SCID mice. Tumor growth inhibition studies were performed in subcutaneous PDAC cell-line-derived xenografts and PDAC patient-derived xenografts in mice. Intratumoral mechanism of action was determined by immunohistochemistry (IHC) and Immunoblot analysis. Median animal survival in peritoneal dissemination PDAC xenografts in mice revealed that the median survival was 21 days in controls, which was significantly improved by the NPT+Gem combination (35 days, a 67% increase over controls). Median survival was further increased by addition of trametinib or MK-2206 to the NPT+Gem chemotherapy regimen: NPT+Gem+Tra (43 days, a 105% increase over controls), NPT+Gem+MK (39 days, a 86% increase over controls) and NPT+Gem+MK+Tra (48 days, a 129% increase over controls). In human subcutaneous xenografts using AsPC-1 PDAC cells, trametinib and MK-2206 were also able to enhance NPT+Gem effects. Compared to controls (100±34.8), the percent net local tumor growth in different therapy groups was 21.8±5.9 for NPT+Gem, 7.1±11.2 for NPT+Gem+Tra, 17.2±4.8 for NPT+Gem+MK and 5.7±9.1 for NPT+Gem+MK+Tra. Effects of therapy on intratumoral proliferation and apoptosis corresponded with tumor growth inhibition data. Furthermore, in vivo effects of trametinib and MK-2206 correlated with reduced expression of phospho-ERK and phospho-AKT in tumor samples. More importantly, in a recently completed patient-derived xenograft study, we observed that the addition of trametinib or MK-2206 again augmented the NPT+Gem tumor growth inhibition response. In this experiment, compared to controls (350 mm3), the net local tumor growth in different therapy groups was 145.4 mm3 for NPT+Gem, 122 mm3 for NPT+Gem+Tra, 20.4 mm3 for NPT+Gem+MK and -43.7 mm3 (tumor regression) for NPT+Gem+MK+Tra. These findings from multiple preclinical model settings suggest that the effects of NPT+Gem can be enhanced through combined inhibition of MAPK and PI3K signaling, which supports the potential of these targets for clinical PDAC therapy. Citation Format: Niranjan Awasthi, Sheena Monahan, Alexis Stefaniak, Margaret A. Schwarz, Roderich E. Schwarz. Combined inhibition of MAPK and PI3K signaling augments standard chemotherapy response in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B29.

212Pb-labeled B7-H3-targeting antibody for pancreatic cancer therapy in mouse models

IntroductionWe recently validated monoclonal antibody (mAb) 376.96 as an effective carrier for targeted α-particle radioimmunotherapy (RIT) with 212Pb in ovarian cancer mouse models. In this study, we tested the binding of radiolabeled mAb 376.96 to human pancreatic ductal adenocarcinoma (PDAC) cells and localization in xenografts in immune-deficient mice and evaluated 212Pb-labeled 376.96 (212Pb-376.96) for PDAC therapy. MethodsIn vitro Scatchard assays assessed the specific binding of 212Pb-376.96 to human PDAC3 adherent differentiated cells and non-adherent cancer initiating cells (CICs) dissociated from tumorspheres. In vitro clonogenic assays were used to measure the proliferation of adherent PDAC3 cells and CIC-enriched tumorspheres treated with 212Pb-376.96 or the irrelevant isotype-matched 212Pb-F3-C25. Mice bearing patient derived pancreatic cancer Panc039 xenografts were i.v. injected with 0.17–0.70 MBq 212Pb-376.96 or isotype control 212Pb-F3-C25, and used for biodistribution and tumor growth inhibition studies. Mice bearing orthotopic PDAC3 xenografts were i.v. co-injected with 99mTc-376.96 and 125I–F3-C25 and used for biodistribution studies. Results212Pb-376.96 specifically bound to PDAC3 adherent and dissociated tumorsphere CICs; Kd values averaged 9.0 and 21.7 nM, respectively, with 104–105 binding sites/cell. 212Pb-376.96 inhibited the clonogenic survival of PDAC3 cells or CICs dissociated from tumorspheres 3–6 times more effectively than isotype-matched control 212Pb-F3-C25. Panc039 s.c. tumors showed significantly higher uptake of 212Pb-376.96 (14.0 ± 2.1% ID/g) compared to 212Pb-F3-C25 (6.5 ± 0.9% ID/g, p < .001) at 24 h after dosing. Orthotopic PDAC3 tumors showed significantly higher uptake of 99mTc-376.96 (6.4 ± 1.8% ID/g) compared to 125I–F3-C25 (3.9 ± 0.9% ID/g, p < .05) at 24 h after dosing. Panc039 tumor growth was significantly inhibited by 212Pb-376.96 compared to 212Pb-F3-C25 or non-treated control tumors (p < .05). ConclusionOur results provide evidence for the efficacy of B7-H3 targeted RIT against preclinical models of pancreatic ductal adenocarcinoma (PDAC) and support future studies with 212Pb-376.96 in combination with chemotherapy to potentiate efficacy against PDAC.

Is Cr(III) toxic to bacteria: toxicity studies using Bacillus subtilis and Escherichia coli as model organism.

The objective of this work is to detect the mode of damage caused by Cr(III), one of the widely used industrial pollutant on Bacillus subtilis-industrial strain 168 and Escherichia coli MTCC 40. Bioassays are very sensitive, precise, economical and rapid for detecting early stages of pollution. The detrimental effect of trivalent chromium becomes clear from the growth profile and growth inhibition studies. Mode of action of damage by trivalent chromium in bacterial model was found to be oxidative, as chromium is one of the redox active metals. The generation of reactive oxygen species (ROS) resulted in membrane damage which in turn had a detrimental effect on the membrane proteins as well as the DNA. The structural changes in the SEM and AFM images clearly reveals the damage caused by Cr(III) to the test bacterial models. Trivalent chromium causes greater DNA, protein and membrane damage in case of E. coli than B. subtilis. Membrane damage caused by ROS becomes evident from the production of Thiobarbituric acid reactive substances (TBARs) as the mechanism of killing followed by DNA damage and the production of elevated levels of stress proteins known as extracellular cellular proteins.

Anti-Tumour Effects of High Affinity L-type Amino Acid Transporter1 Inhibitors on Human Pancreatic Adencarcinoma Cells

Background: a system L transporter L-type amino acid transporter 1 (LAT1) is upregulated to support tumor cell growth in malignant tumor. Objective: The purpose of the present study was to investigate the growth inhibition and [14C] L-leucine transport in human pancreatic adenocarcinoma cells MAIPaCa-2 and BXPC3. Methodology: This animal study was carried out in Japan. The in vitro growth inhibition study was performed by using KYT0351 and KYT0353 which inhibited the tumor cell growth in dose dependent manner and uptake of [14C] L-leucine by MIAPaCa-2 and BXPC3 cells was Na+- independent and was strongly inhibited by KYT0351 and KYT0353. The in vivo tumor growth inhibition was also carried out by intra tumor injection of KYT0351 and KYT0353 at the concentration of 2.6mM of each on both the MIAPaCa-2 and BXPC3 nude mice tumor. Result: In a subsequent survival study with the intra peritoneal injection of ascites mice model, control mice had a mean life span of 20 ± 4.30 days and 21 ± 5 days in MIAPaCa-2 and BXPC3 cells respectively, whereas the intraperitoneal injection of 10mg/kg twice daily of KYT0351 and KYT0353 group had improved survival (mean life span 28.4 ± 8.5 and 34.4 ± 9.86 days, 26 ±4.52 and 31.8 ± 7.62 days respectively in MIAPaCa-2 and BXPC3 cells). Kaplan-Meier survival data of nude mice treated with KYT0351 and KYT0353 were significant. To study the mechanism of growth inhibition we investigated the MIB-1 proliferation assay and TUNEL assay. Significantly less MIB-1 staining and more apoptotic nuclei was detected in tumors treated with KYT0351 and KYT0353 in both MIAPaCa-2 and BXPC3 cells compared with saline treated group. Conclusion: In conclusion both the KYT0351 and KYT0353 is a potent LAT1-specific inhibitor and LAT1 could be one of the molecular target in pancreatic adenocarcinoma therapy. Journal of National Institute of Neurosciences Bangladesh, 2016;2(2): 55-68

Extended release delivery of erlotinib glutathione nanosponge for targeting lung cancer

Systemic and uncontrolled administration of erlotinib hydrochloride (ETB) is associated with severe toxicity. A novel targeted and extended release nanosponge (NS) was synthesized from glutathione (GHS) by a one-step reaction between β-cyclodextrin and pyromellitic dianhydride at room temperature for delivery of ETB in lung cancer. Characterization studies were performed using sophisticated instruments. In-vitro release study was performed in the presence of incremental concentrations of GHS which was analyzed using HPLC. Cell cytotoxicity study was evaluated on human lung cancer (A549) cell lines. In-vivo tumour inhibition and biodistribution of ETB-loaded GHS-NS (ETB-NS) were performed on BALB/c mice. NS obtained was spherical, size 212 ± 2.45 nm and high drug entrapment (92.34 ± 5.31%) (p < .001). In-vitro extended drug release (76.89 ± 0.1% release at 168 h), which was directly proportional to the concentration of GHS, demonstrated tumour targeting. There was enhanced in-vitro cytotoxicity and 97.5% inhibition in tumour growth on administering NS when compared to plain ETB (48% inhibition) indicating targeting of NS to the tumour site. Biodistribution study and in-vivo tumour growth inhibition study revealed drug release to the cancerous cell, thus preventing unnecessary drug exposure. ETB-NS exhibits extended drug release proportional to the external GSH concentration.

Abstract 2092: A potent and selective RET inhibitor with efficacy in RET-driven mouse models of medullary thyroid carcinoma and lung adenocarcinoma

Abstract Background: The aim of this CRUK-MI Drug Discovery project is to deliver a RET-selective inhibitor for the treatment of cancers with RET activating mutations, which include 1-2% of lung adenocarcinomas and medullary thyroid cancers (MTC). Recent data supports the hypothesis that the efficacy of vandetanib and cabozantinib, clinically approved multi-kinase inhibitors, is limited by toxicities associated with potent activity against KDR. Therefore, a RET-selective inhibitor would represent a best-in-class agent for the treatment of these cancers. Methods: We have established a robust screening cascade to develop a potent, selective RET inhibitor and developed several in vivo models to evaluate compound PKPD and antitumor efficacy. Tumor growth inhibition and PKPD studies were carried out in BaF3 mouse allograft models overexpressing KIF5B-RET or RETV804M and other disease relevant models, including an MTC xenograft (MZ-CRC-1), a KIF5B-RET lung cancer patient derived xenograft (PDX) model (CTG-0838, Champions Oncology) and a lung cancer control xenograft (Calu-6). Results: Two orally bioavailable compounds displaying nanomolar RET potency and &amp;gt;10 fold selectivity over KDR in cellular assays were selected from the lead series and further evaluated in our in vivo PD and efficacy models. Both compounds demonstrated efficacy in the BaF3 KIF5B-RET driven model (71% and 103% tumor growth inhibition (TGI), respectively), accompanied by reduced levels of pRET in the tumor tissue. Following further lead optimisation; a compound displaying an improved DMPK profile and additional nanomolar potency versus the gatekeeper mutation (RETV804M) was identified and accelerated through our DMPK/in vivo cascade. We consider this additional activity versus RETV804M beneficial since mutations at the gatekeeper residue in other tyrosine kinases (e.g. EGFR) have been shown to mediate acquired drug resistance in the clinic. This compound demonstrated significant TGI of 58% and 82% respectively in the BaF3 KIF5B-RET and BaF3 RETV804M allograft models. Moreover, tumor growth in the lung cancer PDX model was strongly inhibited (95% TGI) and tumor regression induced in the MTC xenograft model (109% TGI). As expected, this potent and selective RET inhibitor was not active in the Calu-6 model, which is sensitive to KDR inhibition, whereas vandetanib, a potent KDR inhibitor, significantly inhibited tumor growth (84% TGI). Additional in vitro and in vivo DMPK analyses further support the nomination of this compound as a preclinical candidate. Conclusions: The identification of selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of the currently available clinical compounds. We have made considerable progress towards this goal and show here the compelling data supporting our nomination of a preclinical development compound. Citation Format: Mandy Watson, Helen Small, Ben Acton, Habiba Begum, Samantha Hitchin, Allan Jordan, Paul Kelly, Rebecca Newton, Ian Waddell, Gina Paris, Donald Ogilvie. A potent and selective RET inhibitor with efficacy in RET-driven mouse models of medullary thyroid carcinoma and lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2092. doi:10.1158/1538-7445.AM2017-2092

Abstract 3092: U3-1402a, a novel HER3-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent antitumor efficacy

Abstract Background HER3 (human epidermal growth factor receptor 3) is a member of HER family, and overexpressed in breast cancer, NSCLC, melanoma, gastric cancer and pancreatic cancer patients` tissues. U3-1402a is an antibody-drug conjugate (ADC) comprised of a fully human anti-HER3 monoclonal immunoglobulin G1 (IgG1) antibody (U3-1287) covalently conjugated via a cleavable peptide linker to exatecan derivative (DXd). The DXd is released after internalization of U3-1402a and leads to apoptosis of the target tumor cells by the inhibition of topoisomerase I. This ADC achieves a high drug-to-antibody-ratio (DAR 7 to 8) with homogeneous conjugation with the topoisomerase I inhibitor. The aim of this study was to preclinically evaluate the efficacy of U3-1402a in breast cancer models. Materials and methods In order to evaluate the pharmacological potential of U3-1402a, in vitro and in vivo studies were performed. In vitro growth inhibition assay evaluated the sensitivity of U3-1402a in HER3-positive human breast cancer cell line (HCC1569) and HER3-negative human cervical carcinoma cell line (C33A). Cells were treated with U3-1402a or MAAA-1181 (payload of U3-1402a) depending on its concentration (U3-1402a: 0.153 to 10 000 ng/mL, MAAA-1181: 2.44 to 160,000 pg/mL). In vivo growth inhibition study evaluated the dose-dependent sensitivity of U3-1402a in HER3-positive breast cancer xenograft model, MDA-MB-453. In addition, several xenograft models with different HER3 expression were tested with its sensitivity to U3-1402a. These models were HCC1569 (human breast cancer cell line, HER3 IHC score 3+), MDA-MB-453 (human breast cancer cell line, HER3 IHC score 2+), NIBIO-G016 (human gastric cancer patient-derived xenograft, HER3 IHC score 1+) and MDA-MB-231 (human breast cancer cell line, HER3 IHC score 0). R esults In vitro study, U3-1402a exhibited anti-tumor killing activity in HER3-positive human breast cancer cell line, HCC1569. C-33A human cervical carcinoma cell line was not sensitive to U3-1402a even MAAA-1181 itself exhibited anti-tumor killing activity to this cell line. In vivo study, U3-1402a showed dose-dependent anti-tumor killing activity in a HER3-positive breast cancer MDA-MB-453 xenograft model. Finally, in vivo tumor regression was only observed in HER3 2+ and 3+ models. Conclusions U3-1402a preclinically exhibited its efficacy in breast cancer model in vitro and in vivo. In vivo efficacy was correlated with HER3 expression. These studies suggest that U3-1402a, a novel HER3-targeting ADC, would be efficacious in a broader patient population with HER3 expression like breast cancer, melanoma, NSCLC, gastric cancer and pancreatic cancer. Citation Format: Suguru Ueno, Kenji Hirotani, Reimar Abraham, Sabine Blum, Birgit Frankenberger, Mauricio Redondo-Muller, Johannes Bange, Yusuke Ogitani, Akiko Zembutsu, Koji Morita, Takashi Nakada, Shuji Majima, Yuki Abe, Toshinori Agatsuma. U3-1402a, a novel HER3-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2017-3092