C Andrew Boswell,
Eduardo E Mundo,
Crystal Zhang,
Daniela Bumbaca,
Nicole R Valle,
Katherine R Kozak,
Aimee Fourie,
Josefa Chuh,
Neelima Koppada,
Ola Saad,
Herman Gill,
Ben-Quan Shen,
Bonnee Rubinfeld,
Jay Tibbitts,
Surinder Kaur,
Frank-Peter Theil,
Paul J Fielder,
Leslie A Khawli,
Kedan Lin
Department of Pharmacokinetic & Pharmacodynamic Sciences, Genentech Research & Early Development, South San Francisco, California 94080, United States.
Antibody-drug conjugates (ADCs) are designed to combine the exquisite specificity of antibodies to target tumor antigens with the cytotoxic potency of chemotherapeutic drugs. In addition to the general chemical stability of the linker, a thorough understanding of the relationship between ADC composition and biological disposition is necessary to ensure that the therapeutic window is not compromised by altered pharmacokinetics (PK), tissue distribution, and/or potential organ toxicity. The six-transmembrane epithelial antigen of prostate 1 (STEAP1) is being pursued as a tumor antigen target. To assess the role of ADC composition in PK, we evaluated plasma and tissue PK profiles in rats, following a single dose, of a humanized anti-STEAP1 IgG1 antibody, a thio-anti-STEAP1 (ThioMab) variant, and two corresponding thioether-linked monomethylauristatin E (MMAE) drug conjugates modified through interchain disulfide cysteine residues (ADC) and engineered cysteines (TDC), respectively. Plasma PK of total antibody measured by enzyme-linked immunosorbent assay (ELISA) revealed ∼45% faster clearance for the ADC relative to the parent antibody, but no apparent difference in clearance between the TDC and unconjugated parent ThioMab. Total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal effects on PK from cysteine mutation. An ELISA specific for MMAE-conjugated antibody indicated that the ADC cleared more rapidly than the TDC, but total antibody ELISA showed comparable clearance for the two drug conjugates. Furthermore, consistent with relative drug load, the ADC had a greater magnitude of drug deconjugation than the TDC in terms of free plasma MMAE levels. Antibody conjugation had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascularized organs. Liver uptakes of ADC and TDC at 5 days postinjection were 2-fold and 1.3-fold higher, respectively, relative to the unmodified antibodies. Taken together, these results indicate that the degree of overall structural modification in anti-STEAP1-MMAE conjugates has a corresponding level of impact on both PK and tissue distribution.
Other papers by authors:
Br J Pharmacol. 2012 Aug 13;:
22889168
C Andrew Boswell,
Eduardo E Mundo,
Ron Firestein,
Crystal Zhang,
Weiguang Mao,
Herman Gill,
Cynthia Young,
Nina Ljumanovic,
Shannon Stainton,
Sheila Ulufatu,
Aimee Fourie,
Katherine R Kozak,
Reina Fuji,
Paul Polakis,
Leslie A Khawli,
Kedan Lin
Genentech Research and Early Development, South San Francisco, CA 94080.
Background and purpose: The success of antibody drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualise target expression in normal tissues could reveal causes for target mediated clearance observed in pharmacokinetic characterisation. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. Experimental approach: A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by noninvasive SPECT-CT imaging and immunohistochemistry. Key results: The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As proof-of-concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. Conclusions and implications: Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues. © 2012 Genentech, Inc. British Journal of Pharmacology © 2012 The British Pharmacological Society.
Cinthia V Pastuskovas,
Eduardo E Mundo,
Simon P Williams,
Tapan K Nayak,
Jason Ho,
Sheila Ulufatu,
Suzanna Clark,
Sarajane Ross,
Eric Cheng,
Kathryn Parsons-Reponte,
Gary Cain,
Marjie Van Hoy,
Nicholas Majidy,
Sheila Bheddah,
Josefa dela Cruz Chuh,
Katherine R Kozak,
Nicholas Lewin-Koh,
Peter Nauka,
Daniela Bumbaca,
Mark Sliwkowski,
Jay Tibbitts,
Frank-Peter Theil,
Paul J Fielder,
Leslie A Khawli,
C Andrew Boswell
Genentech Research and Early Development, South San Francisco, California, USA.
Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumab was administered alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography-X-ray computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastuzumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-VEGF treatment, with no significant differences in the other physiologic parameters measured despite immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to achieve improved clinical outcomes.
J Nucl Med. 2012 Aug 7;:
22872740
C Andrew Boswell,
Eduardo E Mundo,
Crystal Zhang,
Shannon L Stainton,
Shang-Fan Yu,
Jennifer A Lacap,
Weiguang Mao,
Katherine R Kozak,
Aimee Fourie,
Paul Polakis,
Leslie A Khawli,
Kedan Lin
Genentech Research and Early Development, South San Francisco, California.
TENB2, also known as tomoregulin or transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is a transmembrane proteoglycan overexpressed in human prostate tumors. This protein is a promising target for antimitotic monomethyl auristatin E (MMAE)-based antibody-drug conjugate (ADC) therapy. Nonlinear pharmacokinetics in normal mice suggested that antigen expression in normal tissues may contribute to targeted mediated disposition. We evaluated a predosing strategy with unconjugated antibody to block ADC uptake in target-expressing tissues in a mouse model while striving to preserve tumor uptake and efficacy. METHODS: Unconjugated, unlabeled antibody was preadministered to mice bearing the TENB2-expressing human prostate explant model, LuCaP 77, followed by a single administration of (111)In-labeled anti-TENB2-MMAE for biodistribution and SPECT/CT studies. A tumor-growth-inhibition study was conducted to determine the pharmacodynamic consequences of predosing. RESULTS: Preadministration of anti-TENB2 at 1 mg/kg significantly increased blood exposure of the radiolabeled ADC and reduced intestinal, hepatic, and splenic uptake while not affecting tumor accretion. Similar tumor-to-heart ratios were measured by SPECT/CT at 24 h with and without the predose. Consistent with this, the preadministration of 0.75 mg/kg did not interfere with efficacy in a tumor-growth study dosed at 0.75 mg or 2.5 mg of ADC per kilogram. CONCLUSION: Overall, the potential to mask peripheral, nontumor antigen uptake while preserving tumor uptake and efficacy could ameliorate toxicity and may significantly affect future dosing strategies for ADCs.
Daniela Bumbaca,
Hong Xiang,
C Andrew Boswell,
Ruediger E Port,
Shannon L Stainton,
Eduardo E Mundo,
Sheila Ulufatu,
Anil Bagri,
Frank-Peter Theil,
Paul J Fielder,
Leslie A Khawli,
Ben-Quan Shen
Department of Pharmacokinetic and Pharmacodynamic Sciences, Genentech Research and Early Development, South San Francisco, CA, USA Department of Investigational Safety Assessment, Genentech Research and Early Development, South San Francisco, CA, USA Department of Molecular Biology, Genentech Research and Early Development, South San Francisco, CA, USA.
BACKGROUND AND PURPOSE Neuropilin-1 (NRP1) is a VEGF receptor that is widely expressed in normal tissues and is involved in tumour angiogenesis. MNRP1685A is a rodent and primate cross-binding human monoclonal antibody against NRP1 that exhibits inhibition of tumour growth in NPR1-expressing preclinical models. However, widespread NRP1 expression in normal tissues may affect MNRP1685A tumour uptake. The objective of this study was to assess MNRP1685A biodistribution in tumour-bearing mice to understand the relationships between dose, non-tumour tissue uptake and tumour uptake. EXPERIMENTAL APPROACH Non-tumour-bearing mice were given unlabelled MNRP1685A at 10 mg·kg(-1). Tumour-bearing mice were given (111) In-labelled MNRP1685A along with increasing amounts of unlabelled antibody. Blood and tissues were collected from all animals to determine drug concentration (unlabelled) or radioactivity level (radiolabelled). Some animals were imaged using single photon emission computed tomography - X-ray computed tomography. KEY RESULTS MNRP1685A displayed faster serum clearance than pertuzumab, indicating that target binding affected MNRP1685A clearance. I.v. administration of (111) In-labelled MNRP1685A to tumour-bearing mice yielded minimal radioactivity in the plasma and tumour, but high levels in the lungs and liver. Co-administration of unlabelled MNRP1685A with the radiolabelled antibody was able to competitively block lungs and liver radioactivity uptake in a dose-dependent manner while augmenting plasma and tumour radioactivity levels. CONCLUSIONS AND IMPLICATIONS These results indicate that saturation of non-tumour tissue uptake is required in order to achieve tumour uptake and acceptable exposure to antibody. Utilization of a rodent and primate cross-binding antibody allows for translation of these results to clinical settings.
PLoS One. 2011 ;6 (3):e17874
21436893
C Andrew Boswell,
Gregory Z Ferl,
Eduardo E Mundo,
Daniela Bumbaca,
Michelle G Schweiger,
Frank-Peter Theil,
Paul J Fielder,
Leslie A Khawli
Department of Pharmacokinetic and Pharmacodynamic Sciences, Genentech Inc., South San Francisco, California, United States of America. boswell.andy@gene.com
The identification of clinically meaningful and predictive models of disposition kinetics for cancer therapeutics is an ongoing pursuit in drug development. In particular, the growing interest in preclinical evaluation of anti-angiogenic agents alone or in combination with other drugs requires a complete understanding of the associated physiological consequences. Technescan™ PYP™, a clinically utilized radiopharmaceutical, was used to measure tissue vascular volumes in beige nude mice that were naïve or administered a single intravenous bolus dose of a murine anti-vascular endothelial growth factor (anti-VEGF) antibody (10 mg/kg) 24 h prior to assay. Anti-VEGF had no significant effect (p>0.05) on the fractional vascular volumes of any tissues studied; these findings were further supported by single photon emission computed tomographic imaging. In addition, apart from a borderline significant increase (p = 0.048) in mean hepatic blood flow, no significant anti-VEGF-induced differences were observed (p>0.05) in two additional physiological parameters, interstitial fluid volume and the organ blood flow rate, measured using indium-111-pentetate and rubidium-86 chloride, respectively. Areas under the concentration-time curves generated by a physiologically-based pharmacokinetic model changed substantially (>25%) in several tissues when model parameters describing compartmental volumes and blood flow rates were switched from literature to our experimentally derived values. However, negligible changes in predicted tissue exposure were observed when comparing simulations based on parameters measured in naïve versus anti-VEGF-administered mice. These observations may foster an enhanced understanding of anti-VEGF effects in murine tissues and, in particular, may be useful in modeling antibody uptake alone or in combination with anti-VEGF.
Curr Drug Metab. 2012 Mar 28;:
22475269
Cit:1
Ben-Quan Shen,
Daniela Bumbaca,
Ola Saad,
Qin Yue,
Cinthia V Pastuskovas,
S Cyrus Khojasteh,
Jay Tibbitts,
Surinder Kaur,
Bei Wang,
Yu-Waye Chu,
Patricia M Lorusso,
Sandhya Girish
Genentech, Department of Pharmacokinetics and Pharmacodynamics, South San Francisco, CA 94080; shen.ben@gene.com.
Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in clinical development for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. Herein, we describe a series of studies to assess T-DM1 absorption, distribution, metabolism, and excretion (ADME) in rats as well as to assess human exposure to T-DM1 catabolites. Following administration of unlabeled and radiolabeled T-DM1 in female Sprague Dawley rats as a single dose, plasma, urine, bile and feces were assessed for mass balance, profiling and identification of catabolites. In rats, the major circulating species in plasma was T-DM1, while DM1 concentrations were low (1.08 to 15.6 ng/mL). The major catabolites found circulating in rat plasma were DM1,[N-maleimidomethyl] cyclohexane-1- carboxylate-DM1 (MCC-DM1), and Lysine-MCC-DM1. These catabolites identified in rats were also detected in plasma samples from patients with HER2-positive metastatic breast cancer who received single-agent T-DM1 (3.6 mg/kg every 3 weeks) in a phase 2 clinical study. There was no evidence of tissue accumulation in rats or catabolite accumulation in human plasma following multiple dosing. In rats, T-DM1 was distributed nonspecifically to the organs without accumulation. The major pathway of DM1-containing catabolite elimination in rats was the fecal/biliary route, with up to 80% of radioactivity recovered in the feces and 50% in the bile. The rat T-DM1 ADME profile is likely similar to the human profile, although there may be differences since trastuzumab does not bind the rat HER2- like receptor. Further research is necessary to more fully understand the T-DM1 ADME profile in humans.
MAbs. 2012 Mar 1;4 (2):
22453096
Yanan Zheng,
Devin B Tesar,
Lisa Benincosa,
Herbert Birnböck,
C Andrew Boswell,
Daniela Bumbaca,
Kyra J Cowan,
Dimitry M Danilenko,
Ann L Daugherty,
Paul J Fielder,
Hans Peter Grimm,
Amita Joshi,
Nicole Justies,
Gerry Kolaitis,
Nicholas Lewin-Koh,
Jing Li,
Sami McVay,
Jennifer O'Mahony,
Michael Otteneder,
Michael Pantze,
Wendy S Putnam,
Zhihua J Qiu,
Jane Ruppel,
Thomas Singer,
Oliver Stauch,
Frank-Peter Theil,
Jennifer Visich,
Jihong Yang,
Yong Ying,
Leslie A Khawli,
Wolfgang F Richter
Research and Early Development; Genentech; South San Francisco, CA USA; These authors contributed equally to this work.
Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated:(1) minipig is predictive of human linear clearance;(2) the SC bioavailabilities in minipigs are weakly correlated with those in human;(3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.
Mol Pharm. 2010 Aug 12;:
20704296
C Andrew Boswell,
Gregory Z Ferl,
Eduardo E Mundo,
Michelle G Schweiger,
Jan Marik,
Michael P Reich,
Frank-Peter Theil,
Paul J Fielder,
Leslie A Khawli
Department of Pharmacokinetic and Pharmacodynamic Sciences, Department of Investigative Safety Assessment, and Department of Biomedical Imaging, Genentech Research and Early Development, South San Francisco, California 94080.
Identification of clinically predictive models of disposition kinetics for antibody therapeutics is an ongoing pursuit in drug development. To encourage translation of drug candidates from early research to clinical trials, clinical diagnostic agents may be used to characterize antibody disposition in physiologically relevant preclinical models. TechneScan PYP was employed to measure tissue vascular volumes (V(v)) in healthy mice. Two methods of red blood cell (RBC) labeling were compared: a direct in vivo method that is analogous to a clinical blood pool imaging protocol, and an indirect method in which radiolabeled blood was transfused from donor mice into recipient mice. The indirect method gave higher precision in RBC labeling yields, lower V(v) values in most tissues, and lower (99m)Tc uptake in kidneys and bladder by single photon emission computed tomographic (SPECT) imaging relative to the direct method. Furthermore, the relative influence of each method on the calculated area under the first 7 days of the concentration-time curve (AUC(0-7)) of an IgG in nude mice was assessed using a physiologically based pharmacokinetic model. The model was sensitive to the source of V(v) values, whether obtained from the literature or measured by either method, when used to predict experimental AUC(0-7) values for radiolabeled trastuzumab in healthy murine tissues. In summary, a novel indirect method for preclinical determination of V(v) offered higher precision in RBC labeling efficiency and lower renal uptake of (99m)Tc than the direct method. In addition, these observations emphasize the importance of obtaining accurate physiological parameter values for modeling antibody uptake.
AAPS J. 2012 Sep ;14 (3):612-8
22648903
Genentech Research and Early Development, South San Francisco, California 94080, USA. boswell.charles@gene.com
Monoclonal antibodies have provided many validated and potential new therapeutic candidates for various diseases encompassing the realms of neurology, ophthalmology, immunology, and especially oncology. The mechanism of action for these biological molecules typically involves specific binding to a soluble ligand or cell surface protein in order to block or alter a molecular pathway, induce a desired cellular response, or deplete a target cell. Many antigens reside within the interstitial space, the fluid-filled compartment that lies between the outer endothelial vessel wall and the plasma membranes of cells. This mini-review examines the concepts relevant to the kinetics and behavior of antibodies within the interstitium with a special emphasis on radiometric measurement of quantitative pharmacology. Molecular probes are discussed to outline chemical techniques, selection criteria, data interpretation, and relevance to the study of antibody pharmacokinetics. The importance of studying the tissue uptake of antibodies at a compartmental level is highlighted, including a brief overview of receptor occupancy and its interpretation in radiotracer studies. Experimental methods for measuring the spatial composition of tissues are examined in terms of relative vascular, interstitial, and cellular volumes using solid tumors as a representative example. Experimental methods and physiologically based pharmacokinetic modeling are introduced as distinct approaches to distinguish between free and bound fractions of interstitial antibody. Overall, the review outlines the available methods for pharmacokinetic measurements of antibodies and physiological measurements of the compartments that they occupy, while emphasizing that such approaches may not fully capture the complexities of dynamic, heterogeneous tumors and other tissues.
AAPS J. 2012 Sep ;14 (3):554-8
22610647
Department of Pharmacokinetic and Pharmacodynamic Sciences, Genentech, Inc., South San Francisco, California 94080, USA. bumbaca.daniela@gene.com
Monoclonal antibodies are increasingly being developed to treat multiple disease areas, including those related to oncology, immunology, neurology, and ophthalmology. There are multiple factors, such as charge, size, neonatal Fc receptor (FcRn) binding affinity, target affinity and biology, immunoglobulin G (IgG) subclass, degree and type of glycosylation, injection route, and injection site, that could affect the pharmacokinetics (PK) of these large macromolecular therapeutics, which in turn could have ramifications on their efficacy and safety. This minireview examines how characteristics of the antibodies could be altered to change their PK profiles. For example, it was observed that a net charge modification of at least a 1-unit shift in isoelectric point altered antibody clearance. Antibodies with enhanced affinity for FcRn at pH 6.0 display longer serum half-lives and slower clearances than wild type. Antibody fragments have different clearance rates and tissue distribution profiles than full length antibodies. Fc glycosylation is perceived to have a minimal effect on PK while that of terminal high mannose remains unclear. More investigation is warranted to determine if injection route and/or site impacts PK. Nonetheless, a better understanding of the effects of all these variations may allow for the better design of antibody therapeutics.
Latest similar papers:
Biotechnol J. 2012 Nov 1;:
23125076
Novartis Pharma AG, Novartis Institute of Biomedical Research, Basel, Switzerland. karen.vincent@novartis.com.
Antibody engineering technologies are constantly advancing to improve the clinical effectiveness of monoclonal antibodies (mAbs). Effector functions may be modified by engineering the Fc region, for example to improve or reduce binding to Fc gamma receptors (FcγRs) or complement factors. Other examples for Fc engineering include modification of the half-life of immunoglobulin G (IgG); various studies have shown that half-life can be prolonged by increasing the affinity of Fc for the Fc neonatal receptor (FcRn). Furthermore, engineered pH-dependent antigen binding can be applied to enhance the recycling of IgG via FcRn, enabling binding to additional target molecules. Since bispecific approaches may elicit desired effects on disease targets, a variety of bispecific formats have been developed, including variants that structurally mimic IgG. Finally, antibody-drug conjugates (ADCs) create new opportunities for treatment of certain diseases. Advances in antibody generation, selection of highly cytotoxic molecules and production of stable linkers have paved the way to the development of many ADCs that can be tested in clinical trials. This review covers current antibody engineering strategies for the modification of therapeutic antibodies in the areas of Fc engineering and pH-dependent antigen binding, bispecific antibodies and ADCs.
Jun Y Axup,
Krishna M Bajjuri,
Melissa Ritland,
Benjamin M Hutchins,
Chan Hyuk Kim,
Stephanie A Kazane,
Rajkumar Halder,
Jane S Forsyth,
Antonio F Santidrian,
Karin Stafin,
Yingchun Lu,
Hon Tran,
Aaron J Seller,
Sandra L Biroc,
Aga Szydlik,
Jason K Pinkstaff,
Feng Tian,
Subhash C Sinha,
Brunhilde Felding-Habermann,
Vaughn V Smider,
Peter G Schultz
Department of Chemistry, Department of Molecular Biology, Department of Molecular and Experimental Medicine, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037.
Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2(+) cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.
Anal Chem. 2012 Aug 22;:
22913809
Early Stage Pharmaceutical Development and ‡Protein Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States.
Antibody drug conjugates enable the targeted delivery of potent chemotherapeutic agents directly to cancerous cells. They are made by the chemical conjugation of cytotoxins to monoclonal antibodies, which can be achieved by first reducing interchain disulfide bonds followed by conjugation of the resulting free thiols with drugs. This process yields a controlled, but heterogeneous, population of conjugated products that contains species with various numbers of drugs linked to different former interchain disulfide cysteine residues on the antibodies. We have developed a mathematical approach using inputs from capillary electrophoresis and hydrophobic interaction chromatography to determine the positional isomer distribution within a population of antibody drug conjugates. The results are confirmed by analyzing isolated samples of specific drug-to-antibody ratio species. The procedure is amenable to rapid determination of positional isomer distributions and features low material requirements. A survey of several antibody drug conjugates based on the same IgG framework and small molecule drug combination has shown a very similar distribution of isomers among all of the molecules using this technique, suggesting a robust conjugation process.
MAbs. 2012 Nov 1;4 (6):
22909934
Centre d'Immunologie Pierre Fabre; Saint-Julien-en-Genevois, France.
The 4th World Antibody Drug Conjugate (WADC) Summit, organized by Hanson Wade was held on February 29‑March 1, 2012 in Frankfurt, Germany, which was also the location for the 3rd WADC held in February 2011. During the one year since that meeting, antibody drug conjugates (ADCs) have confirmed their technological maturity and their clinical efficacy in oncology. Brentuximab vedotin (ADCETRIS (TM)) gained approval by the US Food and Drug Administration in August 2011 and trastuzumab emtansine (T-DM1) confirmed impressive clinical efficacy responses in a large cohort of breast cancer patients. During the 4th WADC meeting, antibody-maytansinoid conjugates were showcased by representatives of ImmunoGen (T-DM1, SAR3419, lorvotuzumab mertansine/IMGN801, IMGN529 and IMG853) and Biotest (BT-062). Data on antibody-auristatin conjugates were presented by scientists and clinicians from Seattle Genetics and Takeda (brentuximab vedotin), Pfizer (5T4-MMAF), Agensys/Astella (AGS-16M8F), Progenics (PSMA-ADC) and Genmab (anti-TF ADCs). Alternative payloads such as calicheamicins and duocarmycin used for preparation of ADCs were discussed by Pfizer and Synthon representatives, respectively. In addition, emerging technologies, including site-directed conjugation (Ambrx), a protein toxin as payload (Viventia), hapten-binding bispecific antibodies (Roche), and use of light activated drugs (Photobiotics), were also presented. Last but not least, progresses in solving Chemistry Manufacturing and Control, and pharmacokinetic issues were addressed by scientists from Genentech, Pfizer, Novartis and Pierre Fabre.
Br J Pharmacol. 2012 Aug 13;:
22889168
C Andrew Boswell,
Eduardo E Mundo,
Ron Firestein,
Crystal Zhang,
Weiguang Mao,
Herman Gill,
Cynthia Young,
Nina Ljumanovic,
Shannon Stainton,
Sheila Ulufatu,
Aimee Fourie,
Katherine R Kozak,
Reina Fuji,
Paul Polakis,
Leslie A Khawli,
Kedan Lin
Genentech Research and Early Development, South San Francisco, CA 94080.
Background and purpose: The success of antibody drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualise target expression in normal tissues could reveal causes for target mediated clearance observed in pharmacokinetic characterisation. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. Experimental approach: A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by noninvasive SPECT-CT imaging and immunohistochemistry. Key results: The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As proof-of-concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. Conclusions and implications: Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues. © 2012 Genentech, Inc. British Journal of Pharmacology © 2012 The British Pharmacological Society.
AAPS J. 2012 Aug 9;:
22875610
ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts, 02451, USA, Hans.Erickson@immunogen.com.
The concept of treating cancer with antibody-drug conjugates (ADCs) has gained momentum with the favorable activity and safety of trastuzumab emtansine (T-DM1), SAR3419, and lorvotuzumab mertansine (IMGN901). All three ADCs utilize maytansinoid cell-killing agents which target tubulin and suppress microtubule dynamics. Each ADC utilizes a different optimized chemical linker to attach the maytansinoid to the antibody. Characterizing the absorption, distribution, metabolism, and excretion (ADME) of these ADCs in preclinical animal models is important to understanding their efficacy and safety profiles. The ADME properties of these ADCs in rodents were inferred from studies with radio-labeled ADCs prepared with nonbinding antibodies since T-DM1, SAR3419, IMGN901 all lack cross-reactivity with rodent antigens. For studies exploring tumor localization and activation in tumor-bearing mice, tritium-labeled T-DM1, SAR3419, and IMGN901 were utilized. The chemical nature of the linker was found to have a significant impact on the ADME properties of these ADCs-particularly on the plasma pharmacokinetics and observed catabolites in tumor and liver tissues. Despite these differences, T-DM1, SAR3419, and IMGN901 were all found to facilitate efficient deliveries of active maytansinoid catabolites to the tumor tissue in mouse xenograft models. In addition, all three ADCs were effectively detoxified during hepatobiliary elimination in rodents.
J Nucl Med. 2012 Aug 7;:
22872740
C Andrew Boswell,
Eduardo E Mundo,
Crystal Zhang,
Shannon L Stainton,
Shang-Fan Yu,
Jennifer A Lacap,
Weiguang Mao,
Katherine R Kozak,
Aimee Fourie,
Paul Polakis,
Leslie A Khawli,
Kedan Lin
Genentech Research and Early Development, South San Francisco, California.
TENB2, also known as tomoregulin or transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is a transmembrane proteoglycan overexpressed in human prostate tumors. This protein is a promising target for antimitotic monomethyl auristatin E (MMAE)-based antibody-drug conjugate (ADC) therapy. Nonlinear pharmacokinetics in normal mice suggested that antigen expression in normal tissues may contribute to targeted mediated disposition. We evaluated a predosing strategy with unconjugated antibody to block ADC uptake in target-expressing tissues in a mouse model while striving to preserve tumor uptake and efficacy. METHODS: Unconjugated, unlabeled antibody was preadministered to mice bearing the TENB2-expressing human prostate explant model, LuCaP 77, followed by a single administration of (111)In-labeled anti-TENB2-MMAE for biodistribution and SPECT/CT studies. A tumor-growth-inhibition study was conducted to determine the pharmacodynamic consequences of predosing. RESULTS: Preadministration of anti-TENB2 at 1 mg/kg significantly increased blood exposure of the radiolabeled ADC and reduced intestinal, hepatic, and splenic uptake while not affecting tumor accretion. Similar tumor-to-heart ratios were measured by SPECT/CT at 24 h with and without the predose. Consistent with this, the preadministration of 0.75 mg/kg did not interfere with efficacy in a tumor-growth study dosed at 0.75 mg or 2.5 mg of ADC per kilogram. CONCLUSION: Overall, the potential to mask peripheral, nontumor antigen uptake while preserving tumor uptake and efficacy could ameliorate toxicity and may significantly affect future dosing strategies for ADCs.
MAbs. 2012 May 1;4 (3):
22531451
Department of Medicinal Chemistry; University of Washington; Seattle, Washington USA.
Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHISNLC and aHISTPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.
Anal Chem. 2012 Mar 20;84 (6):2843-9
22384990
Seattle Genetics, Bothell, Washington 98021, United States. jdouglass@seagen.com
We present here a method for the rapid determination of the intact mass of noncovalently associated antibody heavy chains (HC) and light chains (LC) which result from the attachment of drug conjugates to interchain cysteine residues. By analyzing the antibody-drug conjugate (ADC) using native desalting conditions, we maintain the intact bivalent structure of the ADC, which ordinarily would decompose as a consequence of denaturing chromatographic conditions typically used for liquid chromatographic-mass spectrometric (LC-MS) analysis. The mass of the desalted ADC is subsequently determined using standard desolvation and ionization conditions. Methods presented previously in the literature for analyzing interchain cysteinyl-linked ADCs are either not amenable to online mass spectrometry or result in the denaturing dissociation of conjugated HC and LC during chromatographic separation and subsequent mass measurement. We have avoided this outcome with our method and have successfully and routinely obtained intact mass measurement of IgG1 mAbs conjugated with maleimidocaproyl-monomethyl Auristatin F (mcMMAF) and valine-citrulline-monomethyl Auristatin E (vcMMAE) at interchain cysteine residues. Our results thus represent the first reported direct measurement of the intact mass of an ADC conjugated at interchain cysteine residues.
Ben-Quan Shen,
Keyang Xu,
Luna Liu,
Helga Raab,
Sunil Bhakta,
Margaret Kenrick,
Kathryn L Parsons-Reponte,
Janet Tien,
Shang-Fan Yu,
Elaine Mai,
Dongwei Li,
Jay Tibbitts,
Jakub Baudys,
Ola M Saad,
Suzie J Scales,
Paul J McDonald,
Philip E Hass,
Charles Eigenbrot,
Trung Nguyen,
Willy A Solis,
Reina N Fuji,
Kelly M Flagella,
Darshana Patel,
Susan D Spencer,
Leslie A Khawli,
Allen Ebens,
Wai Lee Wong,
Richard Vandlen,
Surinder Kaur,
Mark X Sliwkowski,
Richard H Scheller,
Paul Polakis,
Jagath R Junutula
Genentech Inc., 1 DNA Way, S. San Francisco, California, USA.
The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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