Crown Bioscience Research Articles

Abstract 784: Nonclinical activity of the FGFR, VEGFR and PDGFR inhibitor lucitanib in FGFR3 translocated tumor models

Abstract Lucitanib (S 80881, E-3810, CO-3810) is an oral, potent inhibitor of the tyrosine kinase activity of fibroblast growth factor receptors 1 through 3 (FGFR1-3), vascular endothelial growth factor receptors 1 through 3 (VEGFR1-3) and platelet-derived growth factor receptors alpha and beta (PDGFRα-ß). A Phase 1/2 clinical study (Soria et al., 2014, Ann. Oncol. 25(11):2244-51) showed compelling activity of lucitanib in solid tumors and, in particular, a 50% (6 of 12 patients) RECIST partial response rate in breast cancer patients with FGF-aberrant (FGFR1 and/or FGF3/4/19 gene amplified) tumors. On-going clinical studies are further defining the activity of lucitanib in breast (NCT02053636; NCT02202746) and lung cancer (NCT02109016). FGFR gene translocations (fusions; e.g. FGFR3-TACC3) resulting in constitutively active FGFR signaling and tumor proliferation are observed in a wide variety of cancers (Stransky et al., 2014; Nat. Comm. (5):5006), and inhibition of FGFR signaling in FGFR translocated tumors with lucitanib may be a potential new therapeutic strategy. We evaluated the nonclinical activity of lucitanib in FGFR3 translocated models with the hypothesis that the combined blockade of FGFR and VEGFR signaling would be more effective than targeting each receptor tyrosine kinase independently. The in vitro activity of lucitanib was first evaluated in a panel of bladder carcinoma cell lines (n = 8) with and without FGFR3 translocation. Lucitanib preferentially inhibited the growth of cell lines with FGFR3 translocations in 2D and 3D cell culture, with GI50 values of 80-120 nM in a 72-hour cell viability assay, coincident with a reduction in phosphorylated FGFR3 with an IC50 of ∼60 nM. Minimal activity was observed in five wild-type FGFR3 cell lines (GI50 > 5 μM) with the exception of the JMSU1 bladder cell line that is reported to be dependent on FGFR1 signaling for proliferation (Tomlinson et al., 2009, Cancer Res. 69(11):4613-20). The anti-tumor activity of lucitanib dosed orally at 20 mg/kg/day was evaluated in three bladder carcinoma cell-line derived xenografts with FGFR3 translocation (RT112/84, RT4 and SW780) and a patient-derived xenograft (PDX) model of glioblastoma (BN2289; Crown Bioscience) harboring a FGFR3-TACC3 fusion. Administration of lucitanib resulted in significant (p<0.0001) tumor growth inhibition and, in some cases, tumor regression. The anti-tumor activity of lucitanib in these xenograft models was greater than that observed with either sunitinib (VEGFR/PDGFR inhibitor; 40 mg/kg/day) or BGJ398 (FGFR1-4 inhibitor; 20 mg/kg/day). Together, these results provide a strong nonclinical rationale for further exploration of lucitanib in tumors with FGFR translocations in clinical studies. Citation Format: Minh Nguyen, Kevin K. Lin, Mike F. Burbridge, Andrew D. Simmons, Thomas C. Harding. Nonclinical activity of the FGFR, VEGFR and PDGFR inhibitor lucitanib in FGFR3 translocated tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 784. doi:10.1158/1538-7445.AM2015-784

EYE ON CHINA

Response Biomedical Corp. expands Chinese distribution agreement for RAMP® cardiovascular products. Nanocarrier drugs for pancreatic cancer now being produced in China. Cellular Biomedicine Group acquires Chinese PLA General Hospital's CAR-T immuno-oncology technology and clinical data. VHS Group kicks off the integration of the EAP Industry in China by acquiring ZO's EAP business. Hua Medicine achieves positive Phase 1b results in diabetes with 4th-Generation glucokinase activator. 2nd World-China Biosimilars Forum 2015 coming back in March. World-China Generics Forum will take place in March, 2015. ACT Genomics raises $8 million in its first private funding round. TWi Biotechnology selects Medidata's cloud-based platform to advance cutting-edge clinical development. Crown Bioscience expands partnership with The National Resource Center for Mutant Mice (NRCMM) on developing novel immuno-oncology mouse models. Potential new breathalyzer for lung cancer screening. First decoded Neobatrachia genome provides new insight for amphibian evolutionary research.

KB004, a Novel Non-Fucosylated Humaneered® Antibody, Targeting EphA3, Is Active and Well Tolerated in a Phase I/II Study of Advanced Hematologic Malignancies

Background: EphA3 is a novel drug target involved in cell positioning in fetal development. In adults it is an oncofetal antigen, that is re-expressed in hematologic malignancies (blood and bone marrow, leukemic stem cells) and solid tumors. It is also upregulated in diseases characterized by abnormal proliferation and fibrosis, such as idiopathic pulmonary fibrosis and diabetic kidney disease. KB004 is a Humaneered® high affinity antibody (KD = 610 pM) targeting EphA3 with at least 3 possible mechanisms of action: direct apoptosis in tumor cells, activation of ADCC and disruption of tumor vasculature.Objectives: The primary objectives of the Phase I study component are to determine safety and MTD for KB004 in patients with hematologic malignancies, refractory to or unfit for chemotherapy. Secondary objectives are to characterize PK, immunogenicity, and preliminary clinical activity of KB004. Exploratory objectives include evaluation of EphA3 expression on tumor, stromal, and endothelial cells.Methods: Multicenter Phase I/II study. Key eligibility criteria: unsuitable for standard of care or relapsed or refractory hematologic malignancy, ECOG PS 0-1, adequate organ function, platelets ≥ 10,000/uL (untransfused for 7 days) and normal coagulation times. KB004 was administered as a 1-2 hr intravenous infusion on days 1, 8, and 15 of each 21-day cycle, at incremental doses of 20, 40, 70, 100, 140, 190, 250 and 330 mg. At 70 mg and above infusion reaction prophylaxis included H1 and H2 blockers, acetaminophen and IV steroids. Safety and activity by IWG response criteria were assessed. Peripheral blood and bone marrow biopsies for PK analysis and EphA3 expression were also collected.Results: A total of 50 patients (AML 39, MDS 7, DLBCL 1, MF 3) received KB004 in the phase I/dose finding component of the study, which has been completed.The most common toxicities were transient grade 1 and grade 2 transient infusion reactions (IRs) in 79% of patients. IRs were characterized by chills, elevated temperature, fever, rigors, back pain, nausea, vomiting, hypotension, hypertension and transient hypoxia (in 2 cases). No other significant KB004 related toxicity was observed. Two patients discontinued KB004 due to an IR. One of these (grade 3) defined a DLT at the 330mg dose level. A second patient at 330mg had grade 2 infusion reactions associated with multiple infusion delays. These observations prompted expansion of the next lowest dose cohort, 250mg. Six evaluable patients were treated at this dose level. No clinically significant IRs or DLTs were observed. This is therefore the recommended phase 2 dose (RP2D). At all dose levels observed Cmax for KB004 was approximately dose proportional. Sustained exposure above the predicted effective concentration (1ug/mL) to cover the 7-day interval between doses was achieved above 190mg.Responses according to IWG criteria were observed in patients with AML, MF and MDS at the 20 mg, 140g and 250mg dose levels, respectively. At 20mg, a 78 yr-old patient with relapsed AML achieved CRp. Remission was sustained for over 18 months and relapse was preceded by a rise in EphA3 expression. Serial bone marrow biopsies with KB004 treatment show decreased reticulin and collagen fibrosis. At 140mg, a 67 yr old patient with JAK2 V617F mutant previously untreated myelofibrosis whose predominant clinical problem at diagnosis was anemia achieved Clinical Improvement [CI]. Transfusion independency (both RBC and platelets) has been sustained for 8+ months with improvement in constitutional symptoms and improved splenomegaly. At 250 mg an 84 yr-old patient with MDS/MPN (intermediate risk) achieved a Hematologic Improvement [HI, erythroid]. A > 50% reduction in marrow blast percentage was seen in 8 patients.Bone marrow biopsies positive for EphA3 expression with a cut-off of 10% of nucleated cells were obtained in greater than 70% of AML patients. Of 20 patients for whom EphA3 expression data exists with time, 7 (35%) had at least a 50% reduction in expression with treatment.Conclusion: KB004 is a novel agent targeted against EphA3 that is well tolerated when given as a weekly 2 hour infusion. The promising clinical activity profile is postulated to be consistent with the antifibrotic mechanism. The Phase II component of the study is ongoing in which the activity of KB004 will be characterized in disease specific cohorts including AML, MDS and MF at the RP2D of 250mg. DisclosuresDurrant:KaloBios: Research Funding. Advani:KaloBios: Research Funding. Greenberg:Celgene: Research Funding; Novartis: Research Funding; GSK: Research Funding; Onconova: Research Funding; KaloBios: Research Funding. Cortes:KaloBios: Research Funding. Yarranton:KaloBios: Employment; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; Stemline Therapeutics: Equity Ownership. Walling:KaloBios, Corcept Therapeutics, Prothena, NewGen Therapeutics, Valent Technologies, LBC Pharmaceuticals: Consultancy, Equity Ownership; Amgen, BioMarin: Equity Ownership; Crown BioScience: Membership on an entity’s Board of Directors or advisory committees.

Abstract 2726: RET fusions identified in colorectal cancer PDX models are sensitive to the potent RET inhibitor ponatinib

Abstract Background: Chromosomal translocations resulting in formation of activating RET fusion genes have been identified in papillary thyroid cancer and 1-2% of NSCLC. We have previously shown that ponatinib, a pan-BCR-ABL and multi-targeted tyrosine kinase inhibitor (TKI), potently inhibits the most common NSCLC fusion variant, KIF5B-RET, at clinically-achievable concentrations. Here we used patient-derived (PDX) tumor models to explore the potential involvement of RET fusions, and the efficacy of ponatinib, in other cancers. Results: To identify potential RET fusion-positive PDX tumor models, we examined RET mRNA levels in 273 PDX tumors (Crown Bioscience) from various cancer types, looking for models with outlier RET expression. To search for RET fusions, we performed RNAseq on 4 tumors that had the highest RET levels. Interestingly, RET fusions were detected in tumors from 2 colorectal cancer (CRC) patients (pts) and not in tumors from 2 NSCLC pts that expressed similar levels of RET. The fusions detected in CRC pts, NCOA4-RET and CCDC6-RET, had previously been observed in NSCLC pts. Both CRC tumors were negative for other major hotspot mutations, including KRAS, BRAF and PI3K, suggesting that these RET-fusions might be primary oncogenic drivers in these tumors. To evaluate the cellular activity of ponatinib, we generated cell lines that were dependent on the activity of these fusions. Ponatinib potently inhibited the viability of Ba/F3 cells expressing NCOA4-RET and CCD6-RET with IC50s of 6 and 22 nM, respectively. Consistent with these effects being due to inhibition of RET, ponatinib inhibited RET phosphorylation with similar potency. Other TKIs with RET activity, vandetanib (IC50s: 564-1000 nM), cabozantinib (60-386 nM), sunitinib (277-584 nM), sorafenib (105-494 nM), and lenvatinib (68-257 nM), also inhibited viability of the Ba/F3 lines, but with potencies substantially reduced compared to ponatinib. Finally, we examined the efficacy of ponatinib in the RET fusion positive PDX colorectal models, compared to 2 RET fusion negative colorectal models. In the RET-negative models, daily oral dosing of ponatinib (20 mg/kg) inhibited tumor growth by 24-41%. In contrast, ponatinib exhibited much greater efficacy in the RET-fusion positive models, inhibiting tumor growth by 79% in the NCOA4-RET model and inducing near complete regression in the CCDC6-RET model. Conclusion: We identified, for the first time, oncogenic RET fusions in CRC patient samples, suggesting RET may be a driver in a subset of CRC patients. Ponatinib effectively inhibited these fusions with potency substantially exceeding that of other RET inhibitors and demonstrated significant anti-tumor activity in PDX models. These results provide strong support for the clinical evaluation of ponatinib in patients with RET-fusion positive cancers, including colorectal cancer. Citation Format: Joe M. Gozgit, Tzu-Hsiu Chen, Tim Clackson, Victor M. Rivera. RET fusions identified in colorectal cancer PDX models are sensitive to the potent RET inhibitor ponatinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2726. doi:10.1158/1538-7445.AM2014-2726

INSIDE INDUSTRY

Sanaria® PfSPZ malaria vaccine wins 2014 Vaccine Industry Excellence Award for “Best Prophylactic Vaccine”. Analytik Jena AG planning merger by absorption of CyBio AG. Sanguine to begin work with Mayo Clinic Bioservices to accelerate individualized medicine research and development. Thermo Fisher Scientific and the National University of Singapore Sign MOU to create strategic alliance to accelerate life science research in Singapore. Sun Pharma to acquire Ranbaxy in a US$ 4 billion landmark transaction. Meda and Valeant terminates joint ventures. Crown Bioscience enters Japanese market via strategic partnership with Shin Nippon Biomedical Laboratories. Quintiles and Biogen Idec enter comprehensive clinical development partnership. Registration opened for Oxford Global's Drug Discovery USA Congress. Speakers for Oxford Global's Microbiology & Infectious Diseases Congress released. Daiichi Sankyo and UC San Francisco announce collaboration in drug discovery research for neurodegenerative diseases. Funding crunch hits neglected diseases plan. Coca-Cola partners with A*STAR to expand technology presence in Singapore. DaVita expands kidney care services in Malaysia acquires Sinar Indentiti clinics in Seremban.

EYE ON CHINA

Epigenomics and BioChain enter into broad strategic collaboration in China. China Sunergy's Head of Research wins 2013 ACAA/IELTS Australia China Alumni Awards. WuXi PharmaTech enters agreement for patient-derived xenograft models with Mayo Clinic's Center for individualized medicine. Merck enters into further global co-development and commercialization agreement for PARP inhibitor with Chinese R&D company BeiGene. Creganna-Tactx Medical opens office in China. Honeywell to modernize petrochemical plants in China. WuXi PharmaTech to partner with Pharmacyclics. Novozymes and M&G Chemicals to collaborate on biomass-based plastics in China. Catalent's Clinical Trial Supply Facility in Shanghai, China opens for business. Shanghai Institute of Materia Medica and Crown Bioscience to develop mouse clinical center and PDX and Translational Oncology Platform.

Creation Of Patient Derived AML Xenografts Displaying Distinct Phenotypes and Geneotypes

We have recently successfully engrafted leukemic cells from bone marrow of several AML patients into immunocompromised NOD/SCID mice. One of them, AM7577, was reported in the last year ASH1. This model displayed typical aggressive AML disease of M5 subtype, which starts at bone marrow and gradually expand to peripheral (spleen, lymphonode and peripheral blood…). AM7577 also harbors interesting genotypes of mutations for IDH2-R140Q, FLT3-ITD, DNMT3A R882H and NPM1.Our second AML PDX model, AM8096, was established similarly using bone marrow from a recurrent patient with AML-M2 disease. AML8096 displayed similarly aggressive disease and the same 100% mortality, including typical symptoms (BW loss, hunched, inactivity, labored breathing, ruffled coat and eventual mortality) and with abundant leukemic cells in bone, etc. The leukemic cells can serially be passed in mice with 100% take-rate and cause consistent disease (even with < 1e4 cells). This also creates a renewable and potentially unlimited source of leukemia cells. The leukemic cells in mice are identical to those of the original patient leukemic cells (CD45+, CD33+, CD13+, CD123+, and CD19-. However, some aspects of disease presentations are vastly different from that of AM7577. First, the peripheral symptom is significant less characterized by lower leukemic counts in peripheral blood and only slightly enlarged spleen and smaller lymphonode. Second, the leukemic cell morphology is also rather different with AM8096 demonstrating less differentiated phenotype. Third, the genotype, in contrast to AM7577, is wild-types for all the above oncogenes. While we have not identified the likely leukemogenesis driver gene for this model, we are currently performing RNAseq of AM8096, along with AM7577, in order to explore the underlying molecular mechanisms that drive both diseases. In addition, we are also investigating the drug response to standard of care (SOC), as compared to those AM7577.In summary, the two AML models could serve as useful experimental models to investigate the diverse leukemogenesis, including molecular mechanisms by genetic profiling. Ultimately, they can be used to help to identify new treatment strategies for AML.1. Liu, e.a. A unique leukemia mouse model established from AML patient with IDH2 R140Q and FLT3-ITD mutations among other common AML mutations. ASH-2012 Annual Meeting (2012). Disclosures:Li:Crown Bioscience: Employment. Liu:Crown Bioscience: Employment. An:Crown Bioscience: Employment. Wu:Crown Bioscience: Employment. Cai:Crown Bioscience: Employment. Wery:Crown Bioscience: Employment.

An IHC Screen For EphA3 Positive FFPE Tumors

Eph receptors are the largest subgroup of the receptor tyrosine kinases (RTK). Unlike other RTK, these function principally during development. Quiescent in postembryonic tissues, Eph expression by adult tissues is abnormal and implicated in tumor initiation, invasion and metastasis.Aberrant EphA3 expression is seen in solid and hematologic tumors, particularly advanced stage lymphoproliferative and myeloproliferative diseases. In this regard, targeting EphA3 may constitute a novel treatment for hematological malignancy.With clinical utility in mind, i.e., patient selection for anti-EphA3 therapy, a panel of commercial and proprietary (KaloBios) antibodies was screened by Western Blot for reactivity to recombinant Eph receptors (EphA3, EphA4, EphA5, EphA7, EphB2, EphB3).Those with EphA3 binding selectivity were further screened (QualTek) by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded (FFPE) normal and diseased human bone marrow (NLBM, AML) as well the LK63 pre-B-ALL cell line from which EphA3 was originally isolated.Different tissue pretreatments were used for antigen/epitope retrieval of EphA3, including steam-heating in citrate-based or Tris & chelator-based buffers, subsequent/or protease digestion, or neither.Following each antigen/epitope retrieval procedure, antibody reactivity for EphA3 was assessed by light microscopy using enzymatic biotin, tyramide and polymer-based detection techniques. In each instance, the location of the EphA3/antibody complex was visualized with 3,3-diaminobenzidine that precipitates a discrete insoluble reaction product in presence of enzyme (HRP). Nuclei were counterstained with hematoxylin to assess cell/tissue morphology.From this screen, one antibody (with an epitope in the cytoplasmic CT-domain of EphA3) was chosen for further assay optimization based on its selective reactivity towards LK63 and AML and low selective reactivity towards NLBM.Assay optimization included, amongst other aspects, evaluation of EphA3 expression in a panel of NLBM and hematopoietic tumors (200+ specimens inclusive of acute & chronic leukemia, peripheral T-cell & B-cell neoplasm, Hodgkin & non-Hodgkin lymphoma, multiple myeloma, myelodysplasic syndrome, myeloproliferative neoplasm) by a board-certified hematopathologist.In NLBM, the frequency of EphA3+ immature-blast cells (CD34+ or CD117+) was insignificant. Less than 10% CD34+/CD117+ cells were EphA3+.Elevated EphA3 expression (percentage EphA3+ nucleated cells) was observed in most hematopoietic tumors.For example, in multiple myeloma, tumor cells were typically EphA3+/CD138+ plasma cells. For AML, leukemic CD34+ or CD117+ blasts/initiating cells were typically EphA3+. Some CD138- plasma cells or leukemic CD34-/CD117- cells were also EphA3+.Correlation was made between EphA3 expression and specific tumor maturation stages, differentiation status and/or tumor aggressiveness.Tumors in blast crisis presented elevated EphA3 expression, e.g., CML in accelerated or blastic crisis but not chronic phase.Elevated EphA3 expression was noted in pre-B-ALL & pro-B-ALL (early pre-B-ALL) rather than mature B-cell ALL.EphA3 expression for some peripheral B-cell neoplasms correlated well with tumor grade: high grade, poorly differentiated (typically aggressive) B-cell lymphomas or follicular lymphomas were EphA3+. A similar relationship was noted for non-Hodgkin lymphoma (Hodgkin lymphoma was EphA3-).Preclinical screening also provided evidence for EphA3 expression by stroma/fibroblast (mostly lymphoma) and vasculature/endothelium, further rationale for development of reliable tools for profiling EphA3 in hematologic tumors and other malignancies.Using well-characterized normal and diseased FFPE bone marrow biopsies, this IHC assay for EphA3 has subsequently been validated (QualTek) to provide data that is not directly available from routine histopathology review and that supports use of the assay for profiling EphA3 in specific hematologic tumors and for patient selection in early Phase clinical trial/s of an anti-EphA3 monoclonal antibody (KB004, KaloBios).Beyond this, EphA3 targeted therapy with KB004 is anticipated for treatment of solid tumors. Recent genome-wide surveys identify EphA3 amongst the most frequently overexpressed genes in pancreatic, colon and lung carcinoma, melanoma and glioblastoma. Disclosures:Locke:QualTek Molecular Labs: Employment. Bernstein:QualTek Molecular Laboratories: Employment, Equity Ownership. Lynch:QualTek Molecular Laboratories: Employment, Equity Ownership. Siami-Namini:QualTek Molecular Laboratories: Consultancy. Walling:KaloBios: Consultancy; Corcept Therapeutics: Consultancy; Prothena: Consultancy; New Gen Therapeutics: Consultancy; Valent Technologies: Consultancy; LBC Pharmaceuticals: Consultancy; Amgen: Equity Ownership; BioMarin: Equity Ownership; Crown BioScience: Membership on an entity's Board of Directors or advisory committees. Yonker:KaloBios: Employment, Equity Ownership. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; StemLine Therapeutics: Equity Ownership.

A Phase I Study Of KB004, a Novel Non-Fucosylated humaneered® Antibody, Targeted Against The Receptor Tyrosine Kinase EphA3, In Advanced Hematologic Malignancies

BackgroundEphA3 is involved in cell positioning in fetal development. In the adult it is an oncofetal antigen re-expressed in hematologic malignancies (blood and bone marrow, leukemic stem cells) and solid tumors (tumor stem cells, neovasculature and stroma) and may be prognostic. KB004 is a humaneered® high affinity antibody targeted against EphA3 with 3 putative mechanisms of action: direct induction of apoptosis in tumor cells, activation of ADCC and disruption of tumor vasculature by induction of endothelial cell rounding and subsequent blood vessel infarction. Herein, we describe results of an ongoing, phase 1 dose-escalation study of KB004 in adult patients with advanced hematologic cancers. Objectives1) primary: to determine safety and MTD for KB004 in patients with hematologic malignancies refractory to or unfit for chemotherapy; 2) secondary: to examine PK profile, immunogenicity, and clinical activity of KB004. Exploratory objectives include evaluation of EphA3 expression on tumor, stromal, and endothelial cells. MethodsThis is a multicenter phase 1 study. Key eligibility criteria include: 1) relapsed or refractory hematologic malignancy 2) ECOG PS 0-1; 3) adequate end-organ function. Additional eligibility criteria amended later to protect against hemorrhagic events, included platelets ≥ 10,000/uL (untransfused) and normal coagulation times. A standard 3 + 3 escalation study design (amended to allow up to 6 pts. per cohort in the absence of a DLT) was utilized. KB004 was administered as a 1 or 2 hr infusion on days 1, 8, and 15 of each 21-day cycle, at incremental doses of 20, 40, 70, 100, 140, 190, 250 mg and thereafter 33% increments up to a planned maximum of 700 mg. At 70 mg and above infusion reaction (IR) prophylaxis included an H1 blocker, H2 blocker, acetaminophen and IV steroids. Peripheral blood and bone marrow biopsy specimens for PK analysis and EphA3 expression, respectively, were collected during cycle 1. ResultsTo date, 37 patients (AML 32, MDS 2, lymphoma 1, myelofibrosis 2) received KB004; 20 mg: 9 pts, 40 mg: 3pts, 70 mg: 8 pts, 100 mg: 7pts, 140 mg: 5pts, 190 mg 5pts. At 70 mg, two patients had intracranial hemorrhages 5 and 18 days after last KB004 dose in the context of thrombocytopenia and hyperleukocytosis. A causal relationship to KB004 could not be excluded. One occurred in course 1 and was considered a DLT. KB004 blood levels were near the limit of quantitation at 48 and 96 hours. Following the change to entry criteria no further episodes of serious bleeding or other DLTs or significant changes in soluble clotting factors have been observed.Overall KB004 is well tolerated. The most common toxicity was mild to moderate transient IRs in 28 (76%) patients characterized by chills, elevated temperature, fever, rigors, back pain, nausea, vomiting, hypotension, hypertension, transient hypoxia (in 2 cases). Fourteen % of infusions were slowed due to an adverse event, two (1.2%) were prematurely discontinued but no patient discontinued KB004 secondary to an IR. No other significant KB004-related toxicity has been observed. KB004 Cmax at all dose levels was above the predicted effective concentration (1 ug/ml) and was approximately dose proportional. However at dose levels below 190 mg sustained exposure to cover the entire interval between doses was not achieved.One CRp was observed at the 20mg dose level in a 78 yr-old patient with relapsed AML, who remains on study and in sustained remission for over a year. Serial bone marrow biopsies with KB004 treatment show decreased reticulin and collagen fibrosis. A > 50% reduction in marrow blast percentage was seen in 14% of AML patients, and 59% had overall stable disease beyond 1 cycle.Bone marrow biopsies positive for EphA3 expression with a cut-off of 10% of nucleated cells were obtained in 75% of AML patients. EphA3 was expressed in at least 30% of nucleated cells in the baseline sample of the patient with an ongoing CRp. ConclusionKB004 is a novel agent targeted against EphA3 that is well tolerated with evidence of clinical activity. It is anticipated that 190 mg given over 2 hours will provide sufficient plasma exposure to achieve sustained efficacy over the interval between doses. The study is ongoing. Additional PK, PD, and clinical data will be presented. Disclosures:Durrant:KaloBios: Research Funding. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; StemLine Therapeutics: Equity Ownership. Walling:KaloBios: Consultancy; Corcept Therapeutics: Consultancy; Prothena: Consultancy; New Gen Therapeutics: Consultancy; Valent Technologies: Consultancy; LBC Pharmaceuticals: Consultancy; Amgen: Equity Ownership; BioMarin: Equity Ownership; Crown BioScience: Membership on an entity's Board of Directors or advisory committees.

Analysis Of EphA3 Expression In Hematologic Malignancies By Quantitative PCR

EphA3 is a receptor tyrosine kinase that plays an important role in cell positioning and tissue organization during fetal development, but is not thought to play a significant role in healthy adults. However, aberrant EphA3 expression has been reported in a variety of hematologic and solid tumors. KaloBios is conducting a clinical study (KB004-01) of the anti-EphA3 monoclonal antibody KB004 in patients with hematologic malignancies. To gain further insight into the expression of EphA3 in human cancer, we are analyzing EphA3 mRNA expression in a broad range of hematologic diseases using quantitative PCR analysis. The goal of this research is to determine EphA3 expression in hematologic diseases, to identify cytogenetic and/or molecular mutations associated with aberrant EphA3 expression, and to identify EphA3 positive patient populations for inclusion in the ongoing KB004-01 clinical trial. To date, a total of 45 bone marrow aspirate samples have been analyzed from a cohort of patients with AML, MDS, multiple myeloma (PCM), PMF, CML, ALL, or CLL and the study is ongoing. Relative quantification of EphA3 mRNA was determined by normalization to GAPDH and B2M control genes, and expression calculated relative to the level of EphA3 found in normal bone marrow. Patients were designated EphA3+ if the relative EphA3 expression was >2-fold above normal bone marrow. In AML, 8 out of 14 (57.1%) were EphA3+ with expression levels ranging from 2 - 203 fold. The two AML samples with the highest EphA3 expression (43 and 203 fold) were from refractory AMLs. In MDS, 7 out of 15 (46.7%) were EphA3+ with expression ranging from 2 - 33 fold. In PCM, 2 out of 3 (66.6%) were EphA3+ with expression ranging from 10 - 55 fold. The sample with high EphA3 expression of 55 fold had involvement of both PCM and PMF disease. In PMF, 2 out of 3 (66.6%) were EphA3+ with expression levels ranging from 8 - 977 fold. The PMF sample with 977 fold EphA3 expression is the highest level of EphA3 measured in the study to date. The remaining samples analyzed consisted of 1 unspecified MPN, 3 CML, 2 ALL, and 4 CLL all of which had EphA3 mRNA levels equal to or lower than normal bone marrow. In summary, elevated levels of EphA3 mRNA were detected in a number of bone marrow aspirates from AML, MDS, PCM, and PMF patients suggesting these diseases as possible candidates for anti-EphA3 targeted therapy. Disclosures:Woronicz:KaloBios: Employment. Cortes:KaloBios: Research Funding. Jorgensen:KaloBios: Research Funding. Challagundla:KaloBios: Research Funding. Walling:Amgen: Equity Ownership; KaloBios: Consultancy; Corcept Therapeutics: Consultancy; Prothena: Consultancy; New Gen Therapeutics: Consultancy; Valent Technologies: Consultancy; LBC Pharmaceuticals: Consultancy; BioMarin: Equity Ownership; Crown BioScience: Membership on an entity's Board of Directors or advisory committees. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; StemLine Therapeutics: Equity Ownership.

A Unique Leukemia Mouse Model Established From AML Patient with IDH2 R140Q and FLT3-ITD Mutations Among Other Common AML Mutations

Abstract 3579Somatic mutations in isocitrate dehydrogenase 1 (IDH1, cytosolic) and IDH2 (mitochondrial homolog) have been associated with acute myeloblast leukemia (AML, 7% and 15% respectively), as well as other malignancies, e.g. gliomas, etc1,2. These mutations lead to 2-hydroxyglutarate (2HG) production in leukemic cells, which can be detected both in leukemic cells and plasma of AML patients. This association has led to speculation that IDH mutations are important in AML leukemogenesis, or oncogenic drivers, and could potentially be used as drug targets for AML treatment. However, these assumptions have never been verified for lack of experimental models. We recently successfully engrafted leukemic cells from bone marrow of an AML patient into immunocompromised NOD/SCID mice. This patient has aggressive disease of poor prognosis, with initial response to standard of care of chemotherapy and subsequent fast recurrence, followed by death. The patient was diagnosed as M5 subtype AML with heterozygous IDH2 R140Q mutation, and also mutations of FLT3-ITD, DNMT3A R882H and NPM1. The transplanted mice (AM7577) developed AML leukemia with typical symptoms (BW loss, hunched, inactivity, labored breathing, ruffled coat and eventual mortality) and with leukemic cells in bone and peripheral organs (e.g. spleen, blood, etc) (gradual detection of leukemic cells in peripheral blood after 30 days). The leukemic cells can serially be passed in mice with 100% take-rate and cause 100% leukemia induced mortality (even with < 1e5 cells), thus creating a renewable and potentially unlimited source of leukemia cells. The leukemic cells in mice are identical to those of the original patient leukemic cells (CD45+, CD33+, CD13+, CD123+, and CD19−, heterozygous IDH2 R140Q mutation, along with FLT3-ITD; DNMT3A R882H and NPM1. This mutation pattern is somewhat in contrast to the previous belief that IDH mutations are mutually exclusive to other common AML mutation3. The engrafted animals produce 2HG in leukemic cells and in mouse plasma, both of which can be readily detected and quantified by high-performance liquid chromatography and mass spectrometry (HPLC/MS). 2HG and cytokines could be useful biomarkers to monitor the disease progression as well as treatment response. The treatment leukemic models in vivo with standard of care (SOC: Ara-C, 5day-on/2day-off dosing scheme) resulted in significant response, manifested by reduction of leukemic load to undetectable in peripheral blood and serum biomarkers for both tested doses (2 and 10mg/kg), as well as extended survivals. Leukemia, however, rapidly relapse after the treatment withdrawal, suggesting that AraC has effect only on the leukemic blasts, but not on leukemic stem cells. The appearance of leukemic blasts in peripheral blood after araC withdrawal can be effectively re-suppressed by araC. Further tests on Flt3 inhibitor for anti-leukemic activities are currently ongoing. In summary, the engrafted mice could serve as a useful experimental model to investigate the role of IDH mutation in leukemogenesis and help to identify new treatment strategies for AML, including compounds targeting IDH mutations and FLT3, etc. This is, to our knowledge, the first reported AML experimental model of featuring IDH mutation. Disclosures:Liu:Crown Bioscience, Inc.: Employment. Wu:Crown Bioscience, Inc.: Employment. Cai:Crown Bioscience: Employment. Wang:Crown Bioscience: Employment. Chen:Crown Bioscience: Employment. Wery:Crown Bioscience: Employment. Chen:Crown Bioscience: Employment. Li:Crown Bioscience, Inc.: Employment.

Abstract 3603: Development of novel small molecule PIM kinase inhibitors

Abstract The PIM family of serine/threonine kinases, regulated by cytokines and growth factors, is over-expressed in multiple cancer types, including prostate tumors and hematologic malignancies. Recent reports of PIM kinase inhibitors have been implicated with anti-tumor growth activity in cancer cells and xenograft models, thus providing evidence that PIM kinases are attractive targets for anticancer drug development. Jasco Pharmaceuticals has developed a proprietary focused screening library of small molecules designed to inhibit PIM kinase activity. Kinase panel screening of the focused set identified nanomolar inhibitors from a novel chemical series. To further characterize the compounds, Jasco Pharma has established a research partnership with Crown BioScience for access to its cancer cell line and human primary tumor (HuPrime) xenograft models. The initial efforts of this partnership involved the identification of cancer cell lines which express high levels of PIM kinases for anti-proliferation studies and MOA exploration. Medicinal chemistry optimization provided a cluster of compounds with low single-digit micromolar anti-proliferation activity. These improved compounds have been demonstrated to act through a PIM driven mechanism of action, decreasing the levels of the proto-oncogene c-myc and the phosphorylation of the proteins BAD and 4E-BP1. In order to select the compounds with better in vivo drug activity, we performed in vivo PK followed by plasma protein binding assay (PPB) and drug-drug interaction (DDI) analysis instead of the whole series of in vitro ADME property assays. Four compounds were picked up for in vivo efficacy study according to their potency, in vivo pk characteristics, results of PPB and DDI. Preliminary results indicated that one compound had significant anti-tumor activity in MV-4-11 tumor models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3603. doi:10.1158/1538-7445.AM2011-3603

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