Glenn Pacheco

Quantifying Antivascular Effects of Monoclonal Antibodies to Vascular Endothelial Growth Factor: Insights from Imaging

Purpose: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti–vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials. Experimental Design: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab. Results: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (P = 0.048). Consistent with the preclinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient colorectal cancer metastases within 48 h after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (P = 0.0023) and tumor shrinkage in 9 of 26 tumors at day 12. Conclusion: These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant antitumor activity within one cycle of therapy. This finding has important implications for the design of early-phase clinical trials that incorporate physiologic imaging. The study shows how animal data help interpret clinical imaging data, an important step toward the validation of image biomarkers of tumor structure and function. (Clin Cancer Res 2009;15(21):6674–82)Purpose Little is known concerning the onset, duration and magnitude of direct therapeutic effects of anti-VEGF therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early phase clinical trials.

Vessel imaging with viable tumor analysis for quantification of tumor angiogenesis

Imaging of tumor microvasculature has become an important tool for studying angiogenesis and monitoring antiangiogenic therapies. Ultrasmall paramagnetic iron oxide contrast agents for indirect imaging of vasculature offer a method for quantitative measurements of vascular biomarkers such as vessel size index, blood volume, and vessel density. Here, this technique is validated with direct comparisons to ex vivo micro‐CT angiography and histologic vessel measurements, showing significant correlations between in vivo vascular MRI measurements and ex vivo structural vessel measurements. The sensitivity of the MRI vascular parameters is also demonstrated, in combination with a multispectral analysis technique for segmenting tumor tissue to restrict the analysis to viable tumor tissue and exclude regions of necrosis. It is shown that this viable tumor segmentation increases sensitivity for detection of significant effects on blood volume and vessel density by two antiangiogenic therapeutics (anti‐VEGF and anti‐neuropilin‐1) on an HM7 colorectal tumor model. Anti‐VEGF reduced blood volume by 36 ± 3% (P < 0.0001) and vessel density by 52 ± 3% (P < 0.0001) at 48 h posttreatment; the effects of anti‐neuropilin‐1 were roughly half as strong with a reduction in blood volume of 18 ± 6% (P < 0.05) and a reduction in vessel density of 33 ± 5% (P < 0.05) at 48 h posttreatment. Magn Reson Med 63:1637–1647, 2010.

An Antimesothelin-Monomethyl Auristatin E Conjugate with Potent Antitumor Activity in Ovarian, Pancreatic, and Mesothelioma Models

Mesothelin (MSLN) is an attractive target for antibody–drug conjugate therapy because it is highly expressed in various epithelial cancers, with normal expression limited to nondividing mesothelia. We generated novel antimesothelin antibodies and conjugated an internalizing one (7D9) to the microtubule-disrupting drugs monomethyl auristatin E (MMAE) and MMAF, finding the most effective to be MMAE with a lysosomal protease-cleavable valine–citrulline linker. The humanized (h7D9.v3) version, αMSLN-MMAE, specifically targeted mesothelin-expressing cells and inhibited their proliferation with an IC50 of 0.3 nmol/L. Because the antitumor activity of an antimesothelin immunotoxin (SS1P) in transfected mesothelin models did not translate to the clinic, we carefully selected in vivo efficacy models endogenously expressing clinically relevant levels of mesothelin, after scoring mesothelin levels in ovarian, pancreatic, and mesothelioma tumors by immunohistochemistry. We found that endogenous mesothelin in cancer cells is upregulated in vivo and identified two suitable xenograft models for each of these three indications. A single dose of αMSLN-MMAE profoundly inhibited or regressed tumor growth in a dose-dependent manner in all six models, including two patient-derived tumor xenografts. The robust and durable efficacy of αMSLN-MMAE in preclinical models of ovarian, mesothelioma, and pancreatic cancers justifies the ongoing phase I clinical trial. Mol Cancer Ther; 13(11); 2630–40. ©2014 AACR.

Dose dependent pharmacokinetics, tissue distribution, and anti-tumor efficacy of a humanized monoclonal antibody against DLL4 in mice

Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of 125Iodide and 111Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.

Preclinical Efficacy of an Antibody–Drug Conjugate Targeting Mesothelin Correlates with Quantitative 89Zr-ImmunoPET

Antibody–drug conjugates (ADC) use monoclonal antibodies (mAb) as vehicles to deliver potent cytotoxic drugs selectively to tumor cells expressing the target. Molecular imaging with zirconium-89 (89Zr)-labeled mAbs recapitulates similar targeting biology and might help predict the efficacy of these ADCs. An anti-mesothelin antibody (AMA, MMOT0530A) was used to make comparisons between its efficacy as an ADC and its tumor uptake as measured by 89Zr immunoPET imaging. Mesothelin-targeted tumor growth inhibition by monomethyl auristatin E (MMAE), ADC AMA-MMAE (DMOT4039A), was measured in mice bearing xenografts of ovarian cancer OVCAR-3×2.1, pancreatic cancers Capan-2, HPAC, AsPC-1, and HPAF-II, or mesothelioma MSTO-211H. Ex vivo analysis of mesothelin expression was performed using immunohistochemistry. AMA-MMAE showed the greatest growth inhibition in OVCAR-3×2.1, Capan-2, and HPAC tumors, which showed target-specific tumor uptake of 89Zr-AMA. The less responsive xenografts (AsPC-1, HPAF-II, and MSTO-211H) did not show 89Zr-AMA uptake despite confirmed mesothelin expression. ImmunoPET can demonstrate the necessary delivery, binding, and internalization of an ADC antibody in vivo and this correlates with the efficacy of mesothelin-targeted ADC in tumors vulnerable to the cytotoxic drug delivered. Mol Cancer Ther; 16(1); 134–42. ©2016 AACR.

Quantifying Antivascular Effects of Monoclonal Antibodies to Vascular Endothelial Growth Factor

Purpose: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti–vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials. Experimental Design: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab. Results: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (P = 0.048). Consistent with the preclinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient colorectal cancer metastases within 48 h after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (P = 0.0023) and tumor shrinkage in 9 of 26 tumors at day 12. Conclusion: These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant antitumor activity within one cycle of therapy. This finding has important implications for the design of early-phase clinical trials that incorporate physiologic imaging. The study shows how animal data help interpret clinical imaging data, an important step toward the validation of image biomarkers of tumor structure and function. (Clin Cancer Res 2009;15(21):6674–82)Purpose Little is known concerning the onset, duration and magnitude of direct therapeutic effects of anti-VEGF therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early phase clinical trials.

Abstract 4502: Nonclinical characterization and tolerability of a surrogate anti-mesothelin-MMAE antibody-drug conjugate

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mesothelin (MSLN) is a cell surface glycoprotein widely expressed in several cancers with normal expression limited to the serosal mesothelia, features of an ideal target for antibody-based therapy. SS1P, a (dsFv)-PE38 immunotoxin to MSLN that kills cells by inhibition of protein synthesis, exhibits minor clinical responses as monotherapy, but was dose-limited by pleuritis, likely an antigen-dependent toxicity (Hassan et al., (2007) Clin Cancer Res 13 p3144). As mesothelial cells divide infrequently, we evaluated whether an anti-mitotic antibody-drug conjugate (ADC) directed to MSLN would be better tolerated. Our humanized lead anti-MSLN-MMAE (mc-vc-PAB-monomethylauristatin E) conjugate shows excellent preclinical activity (see accompanying abstract by Scales et al), but is specific to human MSLN, so a surrogate ADC that cross-reacts with cynomolgus monkey and rat MSLN was generated for non-clinical toxicity studies. While both the lead and the surrogate antibodies recognize human MSLN, they bound to different epitopes and the surrogate binding was sensitive to glycosylation, only recognizing a subset of cell lines expressing human MSLN with high affinity. The affinity of the surrogate antibody for cynomolgus monkey MSLN is 5 to 22-fold lower than that of the lead antibody for human MSLN, depending on its glycosylation pattern in the human parental cell line to which it was compared. Nonetheless, the surrogate antibody detects endogenous MSLN in monkey pleura and the surrogate ADC exhibits robust cytotoxic activity against monkey MSLN-expressing cells in vitro. Importantly, the surrogate ADC demonstrated comparable in vivo efficacy to the lead ADC against BJAB xenografts expressing monkey or human MSLN respectively, thus validating its use in safety studies. We thus conducted a repeat-dose monkey toxicity study with a clinically relevant (q3w x5) dosing schedule, which yielded similar results for both surrogate and lead ADCs (the primary finding being reversible myelotoxicity, an antigen-independent toxicity similar to those of other IgG1-MMAE ADCs (Li et al., (2013), Mol Can Thera 12 p1255)). Unlike SS1P, there was no evidence of target-dependent pleuritis, nor any other serositis. Our data suggest that anti-MSLN-MMAE ADCs may be safer than SS1P and helped define the Phase I starting dose. Citation Format: Nidhi Gupta, Willy A. Solis, Reina N. Fuji, Amy Oldendorp, Glenn Pacheco, Elizabeth Luis, Josefa Chuh, Dorothy M. French, Elizabeth Drake, Mark S. Dennis, Katherine R. Kozak, Sarajane Ross, Jay Tibbitts, Susan D. Spencer, Suzie J. Scales. Nonclinical characterization and tolerability of a surrogate anti-mesothelin-MMAE antibody-drug conjugate. [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 4502. doi:10.1158/1538-7445.AM2014-4502

Abstract 4494: A clinical candidate anti-mesothelin-MMAE antibody-drug conjugate (ADC) for therapy of mesothelin-expressing cancers

Mesothelin (MSLN) is a cell surface glycoprotein widely expressed in a variety of cancers, with prevalence levels by IHC of 85% in ovarian, 75% in pancreatic and 45% in mesothelioma, and normal expression limited to the mesothelia, suggesting it could be an ideal target for antibody-drug conjugate therapy of these cancers. We have generated a high affinity (subnanomolar), humanized antibody to MSLN and conjugated it to auristatin anti-mitotic drugs (monomethylauristatin E and F, Seattle Genetics) via an uncleavable linker (anti-MSLN-mc-MMAF) or a cathepsin-cleavable valine-citrulline linker (anti-MSLN-mc-vc-PAB-MMAE/F) for comparison. The in vivo efficacy obtained in an ovarian transplant model was superior with the anti-MSLN-MMAE ADC. Anti-MSLN-MMAE was specifically internalized by MSLN-expressing cells in vitro, resulting in cell death compared to control ADCs. Pancreatic, ovarian and mesothelioma tumor cell lines endogenously expressing physiological levels of MSLN were identified and established as xenografts in mice. A single dose of anti-MSLN ADC was sufficient to inhibit or shrink tumor growth in models of each of the three indications in vivo, as well as inducing complete regressions in primary human pancreatic models, even those expressing low levels of MSLN typical of most human pancreatic tumors. Additionally, anti-MSLN-MMAE (at suboptimal doses) appeared to synergize with gemcitabine at clinically relevant doses in an HPAC xenograft model. Furthermore, anti-MSLN-MMAE was well tolerated in non-clinical toxicity studies (see accompanying abstract by Gupta et al.). Our data suggest that anti-MSLN-vc-MMAE is a promising clinical candidate for the treatment of several types of mesothelin-positive cancers. Citation Format: Suzie J. Scales, Nidhi Gupta, Glenn Pacheco, Ron Firestein, Dorothy M. French, Josefa Chuh, Yin Zhang, Leanne Berry, Jenny Bostrom, Elizabeth Luis, Aimee Fourie O9Donohue, Katherine R. Kozak, Sarajane Ross, Mark S. Dennis, Jay Tibbitts, Susan D. Spencer. A clinical candidate anti-mesothelin-MMAE antibody-drug conjugate (ADC) for therapy of mesothelin-expressing cancers. [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 4494. doi:10.1158/1538-7445.AM2014-4494

Abstract 1362: Mechanistic evaluation of combination anti-angiogenic effects of anti-neuropilin-1 and anti-VEGF by in vivo imaging

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction. Anti-neuropilin 1 (anti-NRP1) has been shown preclinically to have an additive effect when used in combination with anti-VEGF. This has been hypothesized to be due in part to the inhibition of vascular maturation that results from anti-NRP1 treatment, which leaves vasculature in a highly anti-VEGF sensitive state. In this study, we examined the effects of sequencing these agents to evaluate this hypothesis. We investigated the effect of pre-treating tumors with anti-NRP1 before treating with anti-VEGF, and vice versa. To monitor tumor vasculature during ongoing treatment, in vivo magnetic resonance imaging was used to reveal information about therapeutic effects on tumor vasculature beyond simple tumor volume measurements. The vessel imaging MRI technique determines the fractional blood volume, mean vessel size, and Q (a dimensionless parameter related to vessel density) on a voxel-by-voxel basis. Methods. MRI scans were performed on a 4.7 T Varian MRI system (Varian Inc, Palo Alto, CA), for 25 mice with Calu6 human lung cancer tumors grown subcutaneously on the leg. Mice were randomized into three groups based on tumor volume caliper measurements: Group 1 (N=8) was treated with appropriate control antibodies twice a week for 3 weeks, Group 2 (N=8) was treated first with anti-VEGF twice a week for 1.5 weeks and then with anti-NRP1 twice a week for 1.5 weeks, and Group 3 (N=9) was treated first with anti-NRP1 twice a week for 1.5 weeks and then with anti-VEGF twice a week for 1.5 weeks. All mice were imaged after the first 10 days of treatment and again after the second 10 days of treatment. Vessel size index maps, blood volume maps, and Q maps were calculated voxel-by-voxel, and mean values of each parameter were calculated across the viable tumor tissue as determined using tissue segmentation analysis on the images. Results & Discussion. After 3 weeks, both treated groups had significantly reduced viable tumor volume relative to the control group. There was no difference in mean vessel size between the three groups. However, Group 3 had significantly reduced blood volume and Q (vessel density) relative to both Groups 1 & 2. This may indicate that initial treatment with anti-NRP1 makes tumor vasculature more susceptible to later treatment with anti-VEGF, while tumors that had been treated first with anti-VEGF did not benefit from later treatment with anti-NRP1. A repeat study with an additional group that was treated with both antibodies for the entire study duration confirmed these results and showed that the group pre-treated with anti-NRP1 showed equivalent reductions in blood volume and Q to the group treated with both antibodies for the full three weeks. 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 1362.

Quantifying anti-vascular effects of monoclonal antibodies to VEGF: insights from multi-modality cross species imaging in colorectal cancer

Purpose: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti–vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials. Experimental Design: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab. Results: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (P = 0.048). Consistent with the preclinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient colorectal cancer metastases within 48 h after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (P = 0.0023) and tumor shrinkage in 9 of 26 tumors at day 12. Conclusion: These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant antitumor activity within one cycle of therapy. This finding has important implications for the design of early-phase clinical trials that incorporate physiologic imaging. The study shows how animal data help interpret clinical imaging data, an important step toward the validation of image biomarkers of tumor structure and function. (Clin Cancer Res 2009;15(21):6674–82)Purpose Little is known concerning the onset, duration and magnitude of direct therapeutic effects of anti-VEGF therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early phase clinical trials.