Michael Torgov

Development of potent monoclonal antibody auristatin conjugates for cancer therapy

We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.

Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97

Identifying factors that determine the sensitivity or resistance of cancer cells to cytotoxicity by antibody-drug conjugates is essential in the development of such conjugates for therapy. Here the monoclonal antibody L49 is used to target melanotransferrin, a glycosylphosphatidylinositol-anchored glycoprotein first identified as p97, a cell-surface marker in melanomas. L49 was conjugated via a proteolytically cleavable valine-citrulline linker to the antimitotic drug, monomethylauristatin F (vcMMAF). Effective drug release from L49-vcMMAF likely requires cellular proteases most commonly located in endosomes and lysosomes. Melanoma cell lines with the highest surface p97 expression (80,000–280,000 sites per cell) were sensitive to L49-vcMMAF whereas most other cancer cell lines with lower p97 expression were resistant, as were normal cells with low copy numbers (≤20,000 sites per cell). Cell line sensitivity to L49-vcMMAF was found by immunofluorescence microscopy to correlate with intracellular fate of the conjugate. Specifically, L49-vcMMAF colocalized with the lysosomal marker CD107a within sensitive cell lines such as SK-MEL-5 and A2058. In contrast, in resistant cells expressing lower p97 levels (H3677; 72,000 sites per cell), L49-vcMMAF colocalized with caveolin-1, a protein prominent in caveolae, but not with CD107a. Thus, for antibody-drug conjugates targeting p97, antigen level and trafficking to the lysosomes are important factors for achieving robust in vitro cytotoxicity against cancer cells. Immunohistochemical analysis with L49 revealed that 62% of metastatic melanoma tumors had strong staining for p97. Overexpression of p97 in melanoma as compared with normal tissue, in conjunction with the greater sensitivity of tumor cells to L49-vcMMAF, supports further evaluation of antibody-drug conjugates for targeting p97-overexpressing tumors. [Mol Cancer Ther 2006;5(6):1474–82]

Pharmacologically enhanced expression of GPNMB increases the sensitivity of melanoma cells to the CR011-vcMMAE antibody-drug conjugate

GPNMB is a melanoma‐associated glycoprotein that is targeted by the CR011‐vcMMAE antibody‐drug conjugate (ADC). Previous studies have shown that CR011‐vcMMAE induces the apoptosis of GPNMB‐expressing tumor cells in vitro and tumor regression in xenograft models. This ADC is currently in clinical trials for melanoma. In the present investigation, a variety of compounds were examined for their ability to increase the expression of GPNMB by cancer cells. These experiments lead to the identification of three distinct groups of compounds that increased GPNMB, some of which were shown to enhance the sensitivity of melanoma cells to CR011‐vcMMAE. These data indicate that it may be possible to increase the anticancer activity of CR011‐vcMMAE through pharmacological enhancement of GPNMB expression for potential therapeutic benefit.

Cerenkov Luminescence Imaging as a Modality to Evaluate Antibody-based Positron Emission Tomography Radiotracers.

Antibodies, and engineered antibody fragments, labeled with radioisotopes are being developed as radiotracers for the detection and phenotyping of diseases such as cancer. The development of antibody-based radiotracers requires extensive characterization of their in vitro and in vivo properties, including their ability to target tumors in an antigen-selective manner. In this study, we investigated the use of Cerenkov luminescence imaging (CLI) as compared with PET as a modality for evaluating the in vivo behavior of antibody-based radiotracers. Methods: The anti–prostate-specific membrane antigen (PSMA) huJ591 antibody (IgG; 150 kDa) and its minibody (Mb; 80 kDa) format were functionalized with the chelator 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) and radiolabeled with the positron-emitting radionuclide 64Cu (half-life, 12.7 h). Immunoreactive preparations of the radiolabeled antibodies were injected into NCr nu/nu mice harboring PSMA-positive CWR22Rv1 and PSMA-negative PC-3 tumor xenografts. Tumor targeting was evaluated by both PET and CLI. Results: 64Cu-NODAGA-PSMA-IgG and 64Cu-NODAGA-PSMA-Mb retained the ability to bind cell surface PSMA, and both radiotracers exhibited selective uptake into PSMA-positive tumors. Under the experimental conditions used, PSMA-selective uptake of 64Cu-NODAGA-PSMA-IgG and 64Cu-NODAGA-PSMA-Mb was observed by CLI as early as 3 h after injection, with tumor-to-background ratios peaking at 24 (IgG) and 16 (Mb) h after injection. Targeting data generated by CLI correlated with that generated by PET and necropsy. Conclusion: CLI provided a rapid and simple assessment of the targeting specificity and pharmacokinetics of the antibody-based PET radiotracers that correlated well with the behavior observed by standard PET imaging. Moreover, CLI provided clear discrimination between uptake kinetics of an intact IgG and its small-molecular-weight derivative Mb. These data support the use of CLI for the evaluation of radiotracer performance.

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 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 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.