Jean Gudas

Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma

Interleukin-8 (IL-8) has recently been shown to contribute to human melanoma progression by functioning as a mitogenic and angiogenic factor. In the present study, we investigated whether targeting IL-8 by a fully human anti-IL-8 antibody (ABX-IL8) could be a potential therapeutic strategy to control angiogenesis, growth, and metastasis of melanoma. The human melanoma cells A375SM (high IL-8 producer) and TXM-13 (intermediate IL-8 producer) were injected subcutaneously into nude mice, which were then treated with ABX-IL8 (1 mg/3 times weekly, i.p., for 3 weeks). Tumor growth of both melanomas in ABX-IL8-treated mice was significantly inhibited when compared with control IgG-treated animals. ABX-IL8 treatment also suppressed experimental metastasis when the melanoma cells were injected intravenously. IL-8 blockade by ABX-IL8 significantly inhibited the promoter activity and the collagenase activity of matrix metalloproteinase-2 in human melanoma cells, resulting in decreased invasion through reconstituted basement membrane in vitro. In vivo, ABX-IL8 treatment resulted in decreased expression of matrix metalloproteinase-2, and decreased vascularization (angiogenesis) of tumors concomitant with increased apoptosis of tumor cells. Moreover, in an in vitro vessel formation assay, ABX-IL8 directly interfered with the tubule formation by human umbilical vein endothelial cells. Taken together, these results point to the potential utility of ABX-IL8 as a modality to treat melanoma and other solid tumors either alone or in combination with conventional chemotherapy or other anti-tumor agents.

Monoclonal Antibodies to Six-Transmembrane Epithelial Antigen of the Prostate-1 Inhibit Intercellular Communication In vitro and Growth of Human Tumor Xenografts In vivo

Six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) is a novel cell surface protein highly expressed in primary prostate cancer, with restricted expression in normal tissues. In this report, we show STEAP-1 expression in prostate metastases to lymph node and bone and in the majority of human lung and bladder carcinomas. We identify STEAP-1 function in mediating the transfer of small molecules between adjacent cells in culture, indicating its potential role in tumor cell intercellular communication. The successful generation of two monoclonal antibodies (mAb) that bind to cell surface STEAP-1 epitopes provided the tools to study STEAP-1 susceptibility to naked antibody therapy. Both mAbs inhibited STEAP-1-induced intercellular communication in a dose-dependent manner. Furthermore, both mAbs significantly inhibited tumor growth in mouse models using patient-derived LAPC-9 prostate cancer xenografts and established UM-UC-3 bladder tumors. These studies validate STEAP-1 as an attractive target for antibody therapy in multiple solid tumors and provide a putative mechanism for mAb-induced tumor growth inhibition.

Pet imaging of cytotoxic human T cells using an 89Zr-labeled anti-CD8 minibody.

Meeting abstracts Major challenges to advancing the new wave of cancer immunotherapies are to select patients who will respond to single immunotherapies, identify those who need more tailored combination regimens, and then, determine early during treatment whether the therapy is working. “

Abstract 104: Preclinical microPET/CT imaging of 89Zr-Df-SGN-35 in mice bearing xenografted CD30 expressing and non-expressing tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Positron emission tomography (PET) imaging is a non-invasive tool to quantitatively describe the drug distribution in vivo. To answer the question whether radiolabeled antibody-drug conjugates (ADCs) can be utilized as PET imaging probes, and to explore PET imaging sensitivity for detecting antigen expression, we conducted preclinical microPET/CT imaging studies with radiolabeled SGN-35 in SCID mice bearing tumors with varying levels of CD30 expression. Methods: SGN-35 was either directly labeled with 124Iodine or conjugated with the chelator desferrioxamine (DFO) and subsequently labeled with 89Zirconium. Karpas 299, TF1-α, and Daudi were selected as the representative high (CD30high), low (CD30low) and negative (CD30neg) CD30 expressing tumor cell lines, respectively. Expression of CD30 and receptor copy number on each cell line was confirmed by quantitative FACS assay. One mouse from each CD30 expression group was administered a single IV bolus dose of either 124I-SGN-35 (0.5 mg/kg) or 89Zr-Df-SGN-35 (1 mg/kg), and serially imaged 0 - 2 hours (dynamic scan), and at 4, 24, 48, 72, 96 and 144 hours (10 min static scans). An additional 3 mice with CD30high and CD30low tumors, and 2 mice with CD30neg tumors, were dosed and imaged at 72, 96 and 144 hours (10 min static scans). Blood samples (10 μL) were taken from mice at 0, 4, 24, 48, 72, 96 hours and by cardiac puncture at 144 hours for pharmacokinetic (PK) analysis. Additional mice (n=4 per group) were sacrificed at 72 and 144 hours to determine tissue biodistribution via gamma counting. Results: The elimination of 124I-SGN-35 and 89Zr-Df-SGN-35 followed 2-compartment PK, and the calculated elimination T1/2 was 3.6-3.9 days. In mice bearing CD30high tumors with ∼291,000 CD30 copies per cell, the tumor:blood ratio of 124I-radioactivity after a dose of 124I-SGN-35 fell from 1.24 after 72 hours to 0.78 after 144 hours, whereas after a dose of 89Zr-Df-SGN-35, mean tumor:blood ratio of 89Zr-radioactivity increased from 7.38 at 72 hours to 15.05 (157.5 %ID/g) at 144 hours. The 124I-radioactivity and 89Zr-radioactivity tumor:blood ratio in the CD30neg tumors was 0.2 and 0.5 after 144 hours, respectively. The tumor:blood ratio of 89Zr-radioactivity continued to increase from 0.57 after 72 hours to 0.90 (13.4 %ID/g) at 144 hours after a dose of 89Zr-Df-SGN-35 in TF1-α (CD30low) tumor bearing mice. Conclusions: These results indicate that 89Zr-Df-SGN-35 retains the specific anti-CD30 immune-reactivity and PK of the parent SGN-35 molecule. 89Zr labeling generated superior imaging results compared with 124I labeling. With respect to specificity, a favorable tumor:blood ratio was observed in the CD30high tumor bearing mice in contrast to the lack of accumulation of 89Zr-radioactivity in the CD30 negative tumors. The accumulation of 89Zr-radioactivity in CD30low TF1-α tumors suggests that 89Zr-Df-SGN-35 is a very sensitive CD30 targeted PET/CT imaging agent. Citation Format: Aaron Moss, Jean Gudas, Tina Albertson, Nancy Whiting, Che-Leung Law. Preclinical microPET/CT imaging of 89Zr-Df-SGN-35 in mice bearing xenografted CD30 expressing and non-expressing tumors. [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 104. doi:10.1158/1538-7445.AM2014-104

Abstract 4393: ASG-5ME is a novel antibody drug conjugate (ADC) for treating prostate cancers

AGS-5 is a novel transmembrane antigen discovered by Agensys using transcriptional profiling that is highly expressed in a variety of epithelial tumors. A fully human IgG2k monoclonal antibody that binds to AGS-5 with high affinity was conjugated with the anti-microtubulin drug, monomethyl auristatin E (MMAE), via a conditionally labile valine-citrulline (vc) maleimidocaproyl linker to yield an average drug: antibody ratio of 3.7. Following cell surface binding, ASG-5ME is internalized and trafficked through the endocytic pathway, where proteolytic cleavage releases free active drug that subsequently binds and disrupts microtubules. We evaluated the expression of AGS-5 protein in a large number of prostate patient tumors using IHC. ASG-5ME was evaluated for its cell cytoxicity in vitro, and its anti-tumor activity was investigated on different established xenograft tumors, using both patient-derived and cell line models of prostate cancer. The pharmacokinetics of ASG-5ME in mice was also evaluated. Our studies indicated AGS-5 protein was expressed in more than 95% of primary prostate cancer specimens and distant metastases, and on more than 90% and 80% of pancreatic and gastric cancer specimens, respectively. ASG-5ME, bound with high affinity (Kd = 0.5 nM) to both human and cynomolgus monkey AGS-5 and mediated potent dose dependent cell cytotoxicity in vitro. ASG-5ME showed significant long term tumor regression and eradication of multiple established prostate cancer xenografts, including those grown orthotopically. Phamacokinetic (PK) analysis of ASG-5ME in mice indicated that the ADC was stabile with long T1/2 of ∼12 days. When considered together these data indicate that ASG-5ME is a promising therapeutic ADC for the treatment of prostate and other AGS-5 expressing cancers. 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 4393.

Abstract LB-188: Sensitivity of 89Zr-labeled anti-CD8 minibody for PET imaging of infiltrating CD8+ T cells

Background- The ability to monitor CD8 positive tumor infiltrating lymphocytes (TILs) in vivo is important for evaluating response to immunotherapies and assisting in the development of more effective immune cell targeted single and combination therapies. “ImmunoPET” imaging of tumor infiltrating T cells can provide a specific and sensitive modality to aid selection of patients for specific immunotherapy regimens and determine whether the therapy is working. Here, we report initial results to define the number of CD8+ T cells that can be detected with 89 Zr-Df-IAB22M2C, an anti-CD8 immunoPET probe, using different animal models Methods- IAB22M2C, a humanized anti-CD8 minibody, was conjugated with desferrioxamine (Df) and radiolabeled with 89 Zr. NOD scid mice were implanted with varying ratios of CD8+ T and tumor cell admixtures either intramuscularly (IM) without Matrigel or subcutaneously (SC) with Matrigel. One or six days later, CD8+ T-cells were visualized with 89 Zr-Df-IAB22M2C. The same probe was used to detect CD8+ T cells in NSG TM mice engrafted with human PBMCs for 1 and 4 weeks to monitor the temporal progression of Graft versus Host Disease (GvHD). Results - CD8+ T cells implanted in the muscles of mice were imaged one day later and SC implanted Matrigel plugs imaged 6 days later. Both approaches yielded similar results and indicated that the lower limit of detection was between 1.6 and 4 million CD8+ T cells in a volume of ∼480 mm 3 in the presence of normal tissue background activity. The sensitivity of detection increased 10-fold when ex vivo radiolabeled CD8+ T cells were implanted SC with Matrigel. NSG TM mice engrafted with human PBMCs provide a reliable model for xenogeneic T cell driven Graft versus Host Disease (GvHD). Human CD8 + T cells were readily detectable in the spleens of mice 1 week post PBMC engraftment using 89 Zr-Df-IAB22M2C. As GvHD progressed 4 weeks later, expansion and trafficking of the engrafted T cells to extra-lymphoid tissues including lungs could be followed. Terminal biodistribution showed a 2-3 fold increase in radioactivity uptake in lungs by week 4 post-engraftment; a result that was confirmed by IHC analysis. T cell enumeration and IHC analyses are in progress to further define the sensitivity range using an optimal dose and specific activity of 89 Zr-Df-IAB22M2C. Conclusion- These studies show that the lower limit of CD8+ T cell detection by 89 Zr-Df-IAB22M2C is between 1.6-4.0 million cells in the presence of normal tissue background activity and that the probe can be used to monitor CD8+ T cell trafficking in a GvHD model in vivo. 89 Zr-Df-IAB22M2C has sensitivity properties that may enable the detection of CD8+ T cells in human tumors. Clinical trials with 89 Zr-Df-IAB22M2C in melanoma patients will commence later this year. Citation Format: Tove Olafsen, Ziyue Karen Jiang, Jason Romero, Charles Zamilpa, Filippo Marchioni, Green Zhang, Michael Torgov, Daulet Satpayev, Jean M. Gudas. Sensitivity of 89 Zr-labeled anti-CD8 minibody for PET imaging of infiltrating CD8+ T cells. [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 LB-188.

Abstract LB-163: Development of an immunoPET tracer for imaging human CD8+ T cells

Background: Infiltration of CD8+ T cells into human tumors is associated with an increased disease-specific survival in many cancers. A better understanding of T cell trafficking and characterization of the subset of CD8+ T cells with the highest localized activity would improve the ability to schedule and select patients for chemo- and immuno-therapy regimens. To facilitate this objective, we have engineered a humanized anti-CD8 antibody fragment and used it to image human CD8+ T cells in a tumor xenograft and humanized mouse model. Methods: The VH and VL sequences of a murine anti-CD8 antibody were cloned by RT-PCR, engineered into minibody (Mb) fragments (scFv-CH3 dimer) of 80 kDa in size and humanized by CDR grafting onto a human germline framework. Multiple humanized variants were evaluated and the lead Mb candidate was selected based on ELISA, flow cytometry and SPR binding properties. The lead Mb, IAb22G3M1 was transiently expressed in CHO cells and purified by Protein L chromatography. PET imaging was performed with desferrioxamine (Df) conjugated IAb22G3M1 radiolabeled with Zr-89 (T1/2 3.3 d). SCID mice bearing subcutaneous HPB-ALL (CD8+ve) or Daudi (CD8-ve) xenografts were serially imaged at 4, 24 and 41 h after i.v. administration of 89Zr-IAb22G3M2 and tissues harvested and counted to determine the biodistribution at the time of sacrifice. The Mb was also evaluated in NOD-SCID-Gamma (NSG) mice that were engrafted with 20 million human PBMCs. In this latter study IAb22G3M1 was radiolabeled with Cu-64 (T1/2 12.7 hrs) following NODAGA conjugation. Mice were imaged at 4 and 7 hrs followed by tissue collection for biodistribution. NSG mice that were not grafted with PBMCs served as experimental controls. Results: Purification of IAb22G3M1 yielded a product that migrated at the expected MW of 80 kDa with very low ( Conclusion: We have successfully generated a functional anti-CD8 imaging agent that can be used to detect and monitor T cell trafficking and expansion in vivo. Pre-clinical PET imaging studies suggest that IAb22G3M1 is a promising tracer for detecting CD8 positive immune cells in vivo in suitable models paving the way for clinical translation. Citation Format: Tove Olafsen, David Ho, Eric Epin, Green Zhang, Michael Torgov, Charlie Beigarten, Giti Agahi, Edward Cabral, Anna M. Wu, Jean M. Gudas. Development of an immunoPET tracer for imaging human CD8+ T cells. [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 LB-163. doi:10.1158/1538-7445.AM2014-LB-163

Abstract LB-351: Evaluation of two engineered antibody fragments derived from a human CD20 antibody as tracers for immunoPET .

Background: CD20 is a cell surface antigen that is expressed on the majority of B-cell lymphomas and B-cell leukemias. Several monoclonal antibodies (mAbs), including the fully human IgG1, Ofatumumab, have been used successfully to target human CD20. Ofatumumab is FDA-approved for the treatment of patients with chronic lymphocytic leukemia (CLL) and under ongoing clinical development for other hematological malignancies and autoimmune diseases. In this study Ofatumumab was engineered into smaller fragments including a minibody (scFv-C H 3; 80kDa) and a cys-diabody (scFv dimer; 50kDa) to enhance pharmacokinetic parameters and optimize possible application for immunoPET imaging. Methods: The variable (V) genes of Ofatumumab were reformatted into two minibodies (Mb) and four cys-diabodies (Cys-Db), differing in orientation of the V genes and linker lengths. Following in vitro characterization of each variant, a lead candidate from each format was produced at larger scale for in vivo evaluation. Ofatumumab, Mb and Cys-Db were radioiodinated with I-131 (t 1/2 8d) for biodistribution and I-124 (t 1/2 4.2d) for PET imaging studies using the Iodogen method. Female SCID mice bearing Ramos (CD20 + ) and BC-1 (CD20 − ) tumors were used. Blocking studies were performed by pre-injecting 0.2-1mg of unlabeled Ofatumumab 24h prior to imaging. The percent injected dose per gram (%ID/g) was determined in harvested tumor, blood and organs. Results: The lead Cys-Db and Mb were shown to have similar binding affinity (0.8 nM) to that of the parental Ofatumumab. Receptor specific binding was confirmed by competitive cell binding assays using both fluorophore-conjugated and radiolabeled antibody fragments. Blood clearances resulted in MRTs of 3.8h, 8.3h and 24.9h for Cys-Db, Mb and Ofatumumab, respectively. Due to its slower initial distribution, the Mb had a higher blood AUC (306%ID/g*h) than that of Ofatumumab (234% ID/g*h) and Cys-Db (102% ID/g*h). Biodistribution studies of Ofatumumab revealed significant uptake in spleen, bone and liver, which was attributed to Fc-mediated clearance as this was not observed with the antibody fragments. Imaging studies with Mb and Cys-Db showed clear delineation of Ramos xenografts; 1.3% ID/g Cys-Db at 8h and 2.7% ID/g Mb at 24h. This resulted in a tumor to blood ratio of 1.7 for the Cys-Db at 8h and 1.1 for the Mb at 24h. Prior administration of excess unlabeled Ofatumumab reduced tumor uptake of the Cys-Db by 50% at 8h and decreased uptake of the Mb by 17% at 24h. Conclusion: Our preliminary results show that antibody fragments derived from Ofatumumab can be used to target CD20 positive cells in vitro and in vivo . The favorable pharmacokinetics of the Cys-Db resulted in higher tumor to blood ratios at 8h and 24h compared to the Mb or Ofatumumab and support the further development of this fragment as a CD20-targeted immunoPET agent. Citation Format: Jean Gudas, Tove Olafsen, David Ho, Derek Bartlett, Arye Lipman, Jean Gudas, Katy Sorenson, Jennifer Keppler, Christian Behrenbruch, Anna Wu, Sharon Ashworth, Jan Passchier, Roger Gunn, Paul Matthews. Evaluation of two engineered antibody fragments derived from a human CD20 antibody as tracers for immunoPET . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-351. doi:10.1158/1538-7445.AM2013-LB-351

Abstract 2590: Preclinical evaluation of ASG-5ME, a novel antibody-drug conjugate for pancreatic cancer

Antibody-drug conjugates (ADCs) have demonstrated promising activity and tolerability profiles in oncology clinical trials, including for Hodgkin lymphoma (brentuzumab vedotin) and breast cancer (trastuzumab-DM1), motivating the development of new ADCs targeting antigens in other prevalent cancers. An immunohistochemical survey of expression of the novel tumor antigen AGS-5 revealed strong staining in 90% of pancreatic cancer specimens tested. Importantly, this staining was largely uniform, with essentially all transformed cells demonstrating membranous staining. This observation along with the observation of equally abundant expression of AGS-5 in prostate and other cancers motivated the development of ASG-5ME, an ADC targeting AGS-5. The ADC is comprised of a fully human IgG2 monoclonal antibody conjugated to the potent tubulin-binding drug monomethylauristatin E, conjugated with an average of 3.7 drug molecules per antibody. Conjugation and analysis of this IgG2 ADC will be discussed. The resulting conjugate demonstrated antigen-dependent internalization and in vitro cytotoxicity at sub-saturating ADC concentrations, as well as potent in vivo antitumor activity in patient-derived xenograft models of pancreatic cancer. ASG-5ME has a long (12 day) T1/2 in mice. Taken together, these results support the clinical evaluation of ASG-5ME for treatment of pancreatic cancer 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 2590.

Abstract 2436: AGS-16M8F is a novel antibody drug conjugate (ADC) for treating renal and liver cancers

AGS-16 is a novel transmembrane antigen discovered by Agensys using transcription profiling and is expressed in kidney, liver and other cancers. A fully human IgG2k monoclonal antibody that binds to AGS-16 with high affinity was conjugated to the anti-microtubulin drug monomethyl auristatin F (MMAF) via a noncleavable maleimidocaproyl (mc) linker to yield the antibody drug conjugate, AGS-16M8F. AGS-16M8F was evaluated for binding affinity, species cross reactivity and cell cytotoxicity in vitro. Its’ anti-tumor activity was investigated using multiple established patient-derived and cell line models of renal clear cell carcinomas. The pharmacokinetics (PK) of AGS-16M8F was evaluated in mice. In addition, the expression of AGS-16 protein in patient kidney and liver cancer specimens was confirmed using IHC. AGS-16M8F bound with high affinity (Kd = 0.3 nM) to both human and cynomolgus monkey AGS-16 orthologs. Following cell surface binding, AGS-16M8F was internalized and trafficked to lysosomes leading to catabolism and release of active drug metabolite. AGS-16M8F mediated potent cell cytotoxicity in vitro and led to significant and prolonged growth inhibition or complete eradication of multiple established renal cancer xenograft models, including those grown orthotopically. Pharmacokinetic analysis of AGS-16M8F in mice demonstrated ADC stability. IHC analysis confirmed that AGS-16 was highly expressed in over 80% of renal clear cell cancers and 33% of hepatocellular carcinomas. When considered together these data indicate that AGS-16M8F is a promising therapeutic ADC for the treatment of renal and other AGS-16 expressing cancers. 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 2436.