Publications by authors named "Douglas Leipold"

25 Publications

  • Page 1 of 1

A Novel Calicheamicin Antibody-Drug Conjugate with Improved Properties.

Mol Cancer Ther 2021 Mar 15. Epub 2021 Mar 15.

Research and Early Development, Genentech, Inc.

Calicheamicin antibody-drug conjugates (ADCs) are effective therapeutics for leukemias with two recently approved in the United States, Mylotarg (gemtuzumab ozogamicin) targeting CD33 for AML and Besponsa (inotuzumab ozogamicin) targeting CD22 for ALL. Both of these calicheamicin ADCs are heterogeneous, aggregation-prone, and have a shortened half-life due to the instability of the acid sensitive hydrazone linker in circulation. We hypothesized that we could improve upon the heterogeneity, aggregation and circulation stability of calicheamicin ADCs by directly attaching the thiol of a reduced calicheamicin to an engineered cysteine on the antibody via a disulfide bond to generate a linkerless and traceless conjugate. We report herein that the resulting homogeneous conjugates possess minimal aggregation and display high in vivo stability with 50% of the drug remaining conjugated to the antibody after 21 days. Furthermore, these calicheamicin ADCs are highly efficacious in mouse models of both solid tumor (HER2+ breast cancer) and hematologic malignancies (CD22+ non-Hodgkin lymphoma). Safety studies in rats with this novel calicheamicin ADC revealed an increased tolerability compared to that reported for Mylotarg. Overall, we demonstrate that applying novel linker chemistry with site-specific conjugation affords an improved, next generation calicheamicin ADC.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0035DOI Listing
March 2021

An Anti-CD22--CBI-Dimer Antibody-Drug Conjugate (ADC) for the Treatment of Non-Hodgkin Lymphoma That Provides a Longer Duration of Response than Auristatin-Based ADCs in Preclinical Models.

Mol Cancer Ther 2021 02 3;20(2):340-346. Epub 2020 Dec 3.

Research and Early Development, Genentech Inc., South San Francisco, California.

We are interested in developing a second generation of antibody-drug conjugates (ADCs) for the treatment of non-Hodgkin lymphoma (NHL) that could provide a longer duration of response and be more effective in indolent NHL than the microtubule-inhibiting ADCs pinatuzumab vedotin [anti-CD22-vc-monomethyl auristatin E (MMAE)] and polatuzumab vedotin (anti-CD79b-vc-MMAE). Pinatuzumab vedotin (anti-CD22-vc-MMAE) and polatuzumab vedotin (anti-CD79b-vc-MMAE) are ADCs that contain the microtubule inhibitor MMAE. Clinical trial data suggest that these ADCs have promising efficacy for the treatment of NHL; however, some patients do not respond or become resistant to the ADCs. We tested an anti-CD22 ADC with a -CBI-dimer payload, thio-Hu anti-CD22-(LC:K149C)-SN36248, and compared it with pinatuzumab vedotin for its efficacy and duration of response in xenograft models and its ability to deplete normal B cells in cynomolgus monkeys. We found that anti-CD22-(LC:K149C)-SN36248 was effective in xenograft models resistant to pinatuzumab vedotin, gave a longer duration of response, had a different mechanism of resistance, and was able to deplete normal B cells better than pinatuzumab vedotin. These studies provide evidence that anti-CD22-(LC:K149C)-SN36248 has the potential for longer duration of response and more efficacy in indolent NHL than MMAE ADCs and may provide the opportunity to improve outcomes for patients with NHL.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0046DOI Listing
February 2021

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Bioorg Med Chem Lett 2020 02 18;30(4):126907. Epub 2019 Dec 18.

WuXi AppTec, Waigaoqiao Free Trade Zone, 288 Fute Zhong Road, Shanghai 200131, China.

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.
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http://dx.doi.org/10.1016/j.bmcl.2019.126907DOI Listing
February 2020

Antibody Conjugation of a Chimeric BET Degrader Enables in vivo Activity.

ChemMedChem 2020 01 14;15(1):17-25. Epub 2019 Nov 14.

Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.
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http://dx.doi.org/10.1002/cmdc.201900497DOI Listing
January 2020

Prediction of Human Pharmacokinetics of Antibody-Drug Conjugates From Nonclinical Data.

Clin Transl Sci 2019 09 11;12(5):534-544. Epub 2019 Jun 11.

Genentech Inc., South San Francisco, California, USA.

Prediction of human pharmacokinetics (PK) based on preclinical information for antibody-drug conjugates (ADCs) provide important insight into first-in-human (FIH) study design. This retrospective analysis was conducted to identify an appropriate scaling method to predict human PK for ADCs from animal PK data in the linear range. Different methods for projecting human clearance (CL) from animal PK data for 11 ADCs exhibiting linear PK over the tested dose ranges were examined: multiple species allometric scaling (CL vs. body weight), allometric scaling with correction factors, allometric scaling based on rule of exponent, and scaling from only cynomolgus monkey PK data. Two analytes of interest for ADCs, namely total antibody and conjugate (measured as conjugated drug or conjugated antibody), were assessed. Percentage prediction errors (PEs) and residual sum of squares (RSS) were compared across methods. Human CL was best estimated using cynomolgus monkey PK data alone and an allometric scaling exponent of 1.0 for CL. This was consistently observed for both conjugate and total antibody analytes. Other scaling methods either underestimated or overestimated human CL, or produced larger average absolute PEs and RSS. Human concentration-time profiles were also reasonably predicted from the cynomolgus monkey data using species-invariant time method with a fixed exponent of 1.0 for CL and 1.0 for volume of distribution. In conclusion, results from this retrospective analysis of 11 ADCs indicate that allometric scaling of CL with an exponent of 1.0 using cynomolgus monkey PK data alone can successfully project human PK profiles of an ADC within linear range.
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http://dx.doi.org/10.1111/cts.12649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742937PMC
September 2019

Antibody-Drug Conjugates Derived from Cytotoxic seco-CBI-Dimer Payloads Are Highly Efficacious in Xenograft Models and Form Protein Adducts In Vivo.

Bioconjug Chem 2019 05 22;30(5):1356-1370. Epub 2019 Apr 22.

Genentech Inc. , 1 DNA Way , South San Francisco , California 94080 , United States.

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00133DOI Listing
May 2019

Phase I study of the anti-FcRH5 antibody-drug conjugate DFRF4539A in relapsed or refractory multiple myeloma.

Blood Cancer J 2019 02 4;9(2):17. Epub 2019 Feb 4.

Sarah Cannon Research Institute, Nashville, TN, USA.

FcRH5 is a cell surface marker enriched on malignant plasma cells when compared to other hematologic malignancies and normal tissues. DFRF4539A is an anti-FcRH5 antibody-drug conjugated to monomethyl auristatin E (MMAE), a potent anti-mitotic agent. This phase I study assessed safety, tolerability, maximum tolerated dose (MTD), anti-tumor activity, and pharmacokinetics of DFRF4539A in patients with relapsed/refractory multiple myeloma. DFRF4539A was administered at 0.3-2.4 mg/kg every 3 weeks or 0.8-1.1 mg/kg weekly as a single-agent by intravenous infusion to 39 patients. Exposure of total antibody and antibody-conjugate-MMAE analytes was linear across the doses tested. There were 37 (95%) adverse events (AEs), 8 (21%) serious AEs, and 15 (39%) AEs ≥ grade 3. Anemia (n = 10, 26%) was the most common AE considered related to DFRF4539A. Two cases of grade 3 acute renal failure were attributed to DFRF4539A. There were no deaths; the MTD was not reached. DFRF4539A demonstrated limited activity in patients at the doses tested with 2 (5%) partial response, 1 (3%) minimal response, 18 (46%) stable disease, and 16 (41%) progressive disease. FcRH5 was confirmed to be expressed and occupied by antibody post-treatment and thus remains a valid myeloma target. Nevertheless, this MMAE-based antibody-drug-conjugate targeting FcRH5 was unsuccessful for myeloma.
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http://dx.doi.org/10.1038/s41408-019-0178-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362066PMC
February 2019

Pharmacokinetic and Pharmacodynamic Considerations in the Design of Therapeutic Antibodies.

Clin Transl Sci 2019 03 27;12(2):130-139. Epub 2018 Dec 27.

Preclinical and Translational Pharmacokinetics/Pharmacodynamics, Genentech, South San Francisco, California, USA.

The design and development of therapeutic monoclonal antibodies (mAbs) through optimizing their pharmacokinetic (PK) and pharmacodynamic (PD) properties is crucial to improve efficacy while minimizing adverse events. Many of these properties are interdependent, which highlights the inherent challenges in therapeutic antibody design, where improving one antibody property can sometimes lead to changes in others. Here, we discuss optimization approaches for PK/PD properties of therapeutic mAbs.
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http://dx.doi.org/10.1111/cts.12597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440574PMC
March 2019

Preclinical pharmacokinetics and pharmacodynamics of DCLL9718A: An antibody-drug conjugate for the treatment of acute myeloid leukemia.

MAbs 2018 Nov-Dec;10(8):1312-1321. Epub 2018 Oct 2.

a Preclinical Translational Pharmacokinetics Department , Genentech Inc. , South San Francisco , CA , USA.

Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker. Here, we characterize the in vitro and in vivo stability, the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A and MCLL0517A in rodents and cynomolgus monkeys. Three key PK analytes were measured in these studies: total antibody, antibody-conjugated PBD dimer and unconjugated PBD dimer. In vitro, DCLL9718A, was stable with most (> 80%) of the PBD dimer payload remaining conjugated to the antibody over 96 hours. This was recapitulated in vivo with antibody-conjugated PBD dimer clearance estimates similar to DCLL9718A total antibody clearance. Both DCLL9718A and MCLL0517A showed linear PK in the non-binding rodent species, and non-linear PK in cynomolgus monkeys, a binding species. The PK data indicated minimal impact of conjugation on the disposition of DCLL9718A total antibody. Finally, in cynomolgus monkey, MCLL0517A showed target engagement at all doses tested (0.5 and 20 mg/kg) as measured by receptor occupancy, and DCLL9718A (at doses of 0.05, 0.1 and 0.2 mg/kg) showed strong PD activity as evidenced by notable reduction in monocytes and neutrophils.
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http://dx.doi.org/10.1080/19420862.2018.1517565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284592PMC
June 2019

Nonclinical Safety Assessment of Anti-Factor D: Key Strategies and Challenges for the Nonclinical Development of Intravitreal Biologics.

J Ocul Pharmacol Ther 2018 Jan/Feb;34(1-2):204-213. Epub 2017 Nov 17.

1 Department of Safety Assessment, Genentech, Inc. , South San Francisco, California.

Purpose: The nonclinical toxicology program described here was designed to characterize the safety profile of anti-factor D (AFD; FCFD4514S, lampalizumab) to support intravitreal (ITV) administration in patients with geographic atrophy (GA).

Methods: The toxicity of AFD was assessed in a single-dose and 6-month repeat-dose study in monkeys at doses up to 10 mg/eye. Toxicity was assessed by clinical ophthalmic examinations, intraocular pressure measurements, ocular photography, electroretinography, fluorescein angiography, optical coherence tomography, and anatomic pathology.

Results: Systemic exposure to AFD generally increased with the increase in dose level. The increases in mean maximal concentration and area under the curve values were roughly dose proportional. No accumulation of AFD was observed following 10 doses, and drug exposures were not affected by anti-drug antibodies. AFD was locally and systemically well tolerated in monkeys following ITV doses of up to 10 mg/eye. Ocular effects associated with AFD were limited to transient, reversible, dose-related, aqueous cell responses and injection-related, mild, vitreal cell responses. In the 6-month repeat-dose study, 2 monkeys had a nonspecific immune response to AFD that resulted in severe ocular inflammation, attributed to administration of a heterologous (humanized) protein.

Conclusions: The comprehensive toxicology program in monkeys described here was designed to evaluate the safety profile of AFD and to support multiple ITV injections in the clinic. Administration of a heterologous (humanized) protein presents a challenge, and immunogenicity in nonclinical species is not predictive of immunogenicity in humans. Taken together, the results of the nonclinical program described here support the use of AFD in patients with GA.
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http://dx.doi.org/10.1089/jop.2017.0063DOI Listing
October 2019

Attachment Site Cysteine Thiol pK Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates.

Bioconjug Chem 2017 10 22;28(10):2538-2548. Epub 2017 Sep 22.

Genentech Incorporated , 1 DNA Way, South San Francisco, California 94080, United States.

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pK. We measured the cysteine thiol pK using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pK (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pK and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pK of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pK. The influence of cysteine thiol pK on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00365DOI Listing
October 2017

Development of Efficient Chemistry to Generate Site-Specific Disulfide-Linked Protein- and Peptide-Payload Conjugates: Application to THIOMAB Antibody-Drug Conjugates.

Bioconjug Chem 2017 08 13;28(8):2086-2098. Epub 2017 Jul 13.

WuXi AppTec Co., Ltd , 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, PR China.

Conjugation of small molecule payloads to cysteine residues on proteins via a disulfide bond represents an attractive strategy to generate redox-sensitive bioconjugates, which have value as potential diagnostic reagents or therapeutics. Advancement of such "direct-disulfide" bioconjugates to the clinic necessitates chemical methods to form disulfide connections efficiently, without byproducts. The disulfide connection must also be resistant to premature cleavage by thiols prior to arrival at the targeted tissue. We show here that commonly employed methods to generate direct disulfide-linked bioconjugates are inadequate for addressing these challenges. We describe our efforts to optimize direct-disulfide conjugation chemistry, focusing on the generation of conjugates between cytotoxic payloads and cysteine-engineered antibodies (i.e., THIOMAB antibody-drug conjugates, or TDCs). This work culminates in the development of novel, high-yielding conjugation chemistry for creating direct payload disulfide connections to any of several Cys mutation sites in THIOMAB antibodies or to Cys sites in other biomolecules (e.g., human serum albumin and cell-penetrating peptides). We conclude by demonstrating that hindered direct disulfide TDCs with two methyl groups adjacent to the disulfide, which have heretofore not been described for any bioconjugate, are more stable and more efficacious in mouse tumor xenograft studies than less hindered analogs.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00258DOI Listing
August 2017

Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody-Drug Conjugates with Self-Immolative Disulfide Linkers.

Mol Cancer Ther 2017 05 21;16(5):871-878. Epub 2017 Feb 21.

Genentech, Inc., South San Francisco, California.

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody-drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0641DOI Listing
May 2017

Development and Translational Application of an Integrated, Mechanistic Model of Antibody-Drug Conjugate Pharmacokinetics.

AAPS J 2017 01 27;19(1):130-140. Epub 2016 Sep 27.

Genentech Research and Early Development, 1 DNA Way, South San Francisco, California, 94080, USA.

Antibody drug conjugates (ADC), in which small molecule cytotoxic agents are non-specifically linked to antibodies, can enable targeted delivery of chemotherapeutics to tumor cells. ADCs are often produced and administered as a mixture of conjugated antibodies with different drug to antibody ratios (DAR) resulting in complex and heterogeneous disposition kinetics. We developed a mechanism-based platform model that can describe and predict the complex pharmacokinetic (PK) behavior of ADCs with protease-cleavable valine-citrulline (VC) linker linked to Monomethylmonomethyl auristatin F/E by incorporating known mechanisms of ADC disposition. The model includes explicit representation of all DAR species; DAR-dependent sequential deconjugation of the drug, resulting in the conversion of higher DAR to lower DAR species; and DAR-dependent antibody/ADC clearance. PK profiles of multiple analytes (total antibody, drug-conjugated antibody, and/or antibody-conjugated drug) for different ADC molecules and targets in rodents and cynomolgus monkeys were used for model development. The integrated cross-species model was successful in capturing the multi-analyte PK profiles after administration of purified ADCs with defined DAR species and ADCs with mixtures of DAR. Human PK predictions for DSTP3086S (anti-STEAP1-vc-MMAE) with the platform model agreed well with PK (total antibody and antibody-conjugated drug concentrations) measurements in the dose-ranging phase I clinical study. The integrated model is applicable to various other ADCs with different formats, conjugated drugs, and linkers, and provides a valuable tool for the exploration of mechanisms governing disposition of ADCs and enables translational predictions.
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http://dx.doi.org/10.1208/s12248-016-9993-zDOI Listing
January 2017

Site-specific trastuzumab maytansinoid antibody-drug conjugates with improved therapeutic activity through linker and antibody engineering.

J Med Chem 2014 Oct 18;57(19):7890-9. Epub 2014 Sep 18.

Genentech, Inc. , 1 DNA Way, South San Francisco, California 94080, United States.

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.
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http://dx.doi.org/10.1021/jm500552cDOI Listing
October 2014

Mechanistic pharmacokinetic/pharmacodynamic modeling of in vivo tumor uptake, catabolism, and tumor response of trastuzumab maytansinoid conjugates.

Cancer Chemother Pharmacol 2014 Nov 4;74(5):969-80. Epub 2014 Sep 4.

Quantitative Solutions, Menlo Park, CA, USA.

Purpose: Trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) comprised of trastuzumab linked to the antimitotic agent DM1, has shown promising results in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. Investigations of the mechanisms of the action of ADCs, including T-DM1, have been primarily descriptive or semiquantitative. However, quantitative pharmacokinetic/pharmacodynamic (PK/PD) analysis may provide insights into their complex behavior. The analyses described herein applied PK/PD modeling to nonclinical studies of maytansinoid conjugates.

Methods: The maytansinoid conjugates T-DM1 and T-SPP-DM1, with thioether and disulfide linkers, respectively, were tested in mouse efficacy, PK, and tumor uptake studies. (3)[H]DM1-bearing ADCs were used to facilitate the quantitation of the ADCs in plasma, as well as ADC and ADC catabolites in tumors. Three mechanistic PK/PD models were used to characterize plasma ADC, tumor ADC, and tumor catabolite concentrations. Tumor catabolite concentrations were used to fit tumor response. Model parameters were estimated using R software and nonlinear least squares regression.

Results: Plasma ADC-associated DM1 concentrations of T-DM1 decreased more slowly than those of T-SPP-DM1, likely due to slower DM1 release. A comparison of the mechanistic models found that the best model allowed catabolism and catabolite exit rates to differ between ADCs, that T-DM1 exhibited both faster tumor catabolism and catabolite exit rate from tumors than T-SPP-DM1; findings inconsistent with expected behavior based on the physicochemical nature of the respective catabolites. Tumor catabolite concentrations adequately described tumor response with both ADCs showing similar potency.

Conclusion: Mechanistic PK/PD studies described herein provided results that confirmed and challenged current hypotheses, and suggested new areas of investigation.
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http://dx.doi.org/10.1007/s00280-014-2561-2DOI Listing
November 2014

A mechanistic pharmacokinetic model elucidating the disposition of trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) for treatment of metastatic breast cancer.

AAPS J 2014 Sep 11;16(5):994-1008. Epub 2014 Jun 11.

Department of Clinical Pharmacology, Genentech Inc., South San Francisco, California, USA,

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) therapeutic for treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. The T-DM1 dose product contains a mixture of drug-to-antibody ratio (DAR) moieties whereby the small molecule DM1 is chemically conjugated to trastuzumab antibody. The pharmacokinetics (PK) underlying this system and other ADCs are complex and have not been elucidated. Accordingly, we have developed two PK modeling approaches from preclinical data to conceptualize and understand T-DM1 PK, to quantify rates of DM1 deconjugation, and to elucidate the link between trastuzumab, T-DM1, and DAR measurements. Preclinical data included PK studies in rats (n = 34) and cynomolgus monkeys (n = 18) at doses ranging from 0.3 to 30 mg/kg and in vitro plasma stability. T-DM1 and total trastuzumab (TT) plasma concentrations were measured by enzyme-linked immunosorbent assay. Individual DAR moieties were measured by affinity capture liquid chromatography-mass spectrophotometry. Two PK modeling approaches were developed for T-DM1 using NONMEM 7.2 software: a mechanistic model fit simultaneously to TT and DAR concentrations and a reduced model fit simultaneously to TT and T-DM1 concentrations. DAR moieties were well described with a three-compartmental model and DM1 deconjugation in the central compartment. DM1 deconjugated fastest from the more highly loaded trastuzumab molecules (i.e., DAR moieties that are ≥3 DM1 per trastuzumab). T-DM1 clearance (CL) was 2-fold faster than TT CL due to deconjugation. The two modeling approaches provide flexibility based on available analytical measurements for T-DM1 and a framework for designing ADC studies and PK-pharmacodynamic modeling of ADC efficacy- and toxicity-related endpoints.
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http://dx.doi.org/10.1208/s12248-014-9618-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147041PMC
September 2014

Immunogenicity assays for antibody-drug conjugates: case study with ado-trastuzumab emtansine.

Bioanalysis 2013 May;5(9):1007-23

Department of Bioanalytical Sciences, Genentech, South San Francisco, CA 94080, USA.

Background: Antibody-drug conjugates (ADCs) such as Kadcyla™ (ado-trastuzumab emtansine [T-DM1]) present covalently bound cytotoxic drugs, which may influence their immunogenicity potential compared with antibody therapies. Therefore, ADCs require assay strategies that allow measurement of responses to all the molecular components.

Results: The immunogenicity strategy for T-DM1 used a risk-based, tiered approach that included screening and titration to detect antitherapeutic antibodies; confirmation of positive responses; and characterization to assess whether the immune response is primarily to the antibody or to the linker-drug and/or new epitopes in trastuzumab resulting from conjugation.

Conclusion: The tiered immunogenicity assay strategy for T-DM1 allowed detection of antitherapeutic antibodies to all components of the ADC in multiple nonclinical and clinical studies. Characterization strategies implemented in clinical studies provided additional insights into the specificity of the immune response.
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http://dx.doi.org/10.4155/bio.13.64DOI Listing
May 2013

American Association of Pharmaceutical Scientists National Biotechnology Conference Short Course: Translational Challenges in Developing Antibody-Drug Conjugates: May 24, 2012, San Diego, CA.

MAbs 2013 Jan-Feb;5(1):5-12. Epub 2012 Dec 19.

Novartis Pharmaceuticals, Clinical Pharmacology, Oncology Business Unit, Florham Park, NJ, USA.

The American Association of Pharmaceutical Scientists (AAPS) National Biotechnology Conference Short Course "Translational Challenges in Developing Antibody-Drug Conjugates (ADCs)," held May 24, 2012 in San Diego, CA, was organized by members of the Pharmacokinetics, Pharmacodynamics and Drug Metabolism section of AAPS. Representatives from the pharmaceutical industry, regulatory authorities, and academia in the US and Europe attended this short course to discuss the translational challenges in ADC development and the importance of characterizing these molecules early in development to achieve therapeutic utility in patients. Other areas of discussion included selection of target antigens; characterization of absorption, distribution, metabolism, and excretion; assay development and hot topics like regulatory perspectives and the role of pharmacometrics in ADC development. MUC16-targeted ADCs were discussed to illustrate challenges in preclinical development; experiences with trastuzumab emtansine (T-DM1; Genentech) and the recently approved brentuximab vedotin (Adcetris; Seattle Genetics) were presented in depth to demonstrate considerations in clinical development. The views expressed in this report are those of the participants and do not necessarily represent those of their affiliations.
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http://dx.doi.org/10.4161/mabs.22909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564886PMC
July 2013

Antibody delivery of drugs and radionuclides: factors influencing clinical pharmacology.

Ther Deliv 2011 Jun;2(6):769-91

Early Development Pharmacokinetic & Pharmacodynamic Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

The therapeutic rationale of antibody conjugates is the selective delivery of a cytotoxin to tumor cells via binding and internalization of the monoclonal antibodies to a specific cell-surface antigen, thereby enhancing the therapeutic index of the cytotoxin. The key structural and functional components of an antibody conjugate are the antibody, the linker and the cytotoxin (chemical or radionuclide) with each component being critical for the successful development of the conjugate. Considerable efforts have been made in understanding the pharmacokinetics, pharmacodynamics, tissue distribution, metabolism and pharmacologic effects of these complex macromolecular entities. The purpose of this article is to discuss the properties and various structural components of antibody conjugates that influence their clinical pharmacology.
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http://dx.doi.org/10.4155/tde.11.41DOI Listing
June 2011

The effect of different linkers on target cell catabolism and pharmacokinetics/pharmacodynamics of trastuzumab maytansinoid conjugates.

Mol Cancer Ther 2012 May 9;11(5):1133-42. Epub 2012 Mar 9.

ImmunoGen, Inc., Waltham, MA, USA.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the anti-HER2 antibody trastuzumab linked via a nonreducible thioether linker to the maytansinoid antitubulin agent DM1. T-DM1 has shown favorable safety and efficacy in patients with HER2-positive metastatic breast cancer. In previous animal studies, T-DM1 exhibited better pharmacokinetics (PK) and slightly more efficacy than several disulfide-linked versions. The efficacy findings are unique, as other disulfide-linked antibody-drug conjugates (ADC) have shown greater efficacy than thioether-linked designs. To explore this further, the in vitro and in vivo activity, PK, and target cell activation of T-DM1 and the disulfide-linked T-SPP-DM1 were examined. Both ADCs showed high in vitro potency, with T-DM1 displaying greater potency in two of four breast cancer cell lines. In vitro target cell processing of T-DM1 and T-SPP-DM1 produced lysine-N(ε)-MCC-DM1, and lysine-N(ε)-SPP-DM1 and DM1, respectively; in vivo studies confirmed these results. The in vitro processing rates for the two conjugate to their respective catabolites were similar. In vivo, the potencies of the conjugates were similar, and T-SPP-DM1 had a faster plasma clearance than T-DM1. Slower T-DM1 clearance translated to higher overall tumor concentrations (conjugate plus catabolites), but unexpectedly, similar levels of tumor catabolite. These results indicate that, although the ADC linker can have clear impact on the PK and the chemical nature of the catabolites formed, both linkers seem to offer the same payload delivery to the tumor.
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http://dx.doi.org/10.1158/1535-7163.MCT-11-0727DOI Listing
May 2012

Characterization of intact antibody-drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography-mass spectrometry.

Anal Biochem 2011 May 7;412(1):56-66. Epub 2011 Jan 7.

Genentech, South San Francisco, CA 94080, USA.

Antibody-drug conjugates (ADCs) are designed to facilitate the targeted delivery of cytotoxic drugs to improve their tumor fighting effects and minimize systemic toxicity. However, efficacy and safety can potentially be compromised due to the release of conjugated drugs from the ADC with time while in circulation, resulting in changes in the drug-to-antibody ratio (DAR). Current understanding of this process is limited because existing methods such as immunoassays fail to distinguish ADCs with different DARs. Here we demonstrate a novel method with bead-based affinity capture and capillary liquid chromatography-mass spectrometry to allow direct measurement of drug release by quantifying DAR distributions of the ADC in plasma/serum. This method successfully identified individual intact conjugated antibody species produced due to drug loss from ADCs (e.g., an engineered site-specific anti-MUC16 THIOMAB-drug conjugate) and measured the corresponding DAR distributions in vitro and in vivo. Information obtained can provide insights into the mechanisms involved in drug loss and help to optimize ADC therapeutics. Other potential applications of the method may include characterization of posttranslational modifications, protein adducts, and immunogenicity.
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http://dx.doi.org/10.1016/j.ab.2011.01.004DOI Listing
May 2011

Modeling the efficacy of trastuzumab-DM1, an antibody drug conjugate, in mice.

J Pharmacokinet Pharmacodyn 2010 Jun 28;37(3):221-42. Epub 2010 Apr 28.

Department of Pharmacokinetics, Pharmacodynamics, and Bioanalytical Sciences, Genentech, Inc., 1 DNA Way, MS463, South San Francisco, CA 94080, USA.

Trastuzumab-DM1 (T-DM1) is a novel antibody-drug conjugate under investigation for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. One challenge in oncologic drug development is determining the optimal dose and treatment schedule. A novel dose regimen-finding strategy was developed for T-DM1 using experimental data and pharmacokinetic/pharmacodynamic modeling. To characterize the disposition of T-DM1, pharmacokinetic studies were conducted in athymic nude and beige nude mice. The pharmacokinetics of T-DM1 were described well by a two-compartment model. Tumor response data were obtained from single-dose, multiple-dose and time-dose-fractionation studies of T-DM1 in animal models of HER2-positive breast cancer, specifically engineered to be insensitive to trastuzumab. A sequential population-based pharmacokinetic/pharmacodynamic modeling approach was developed to describe the anti-tumor activity of T-DM1. A cell-cycle-phase nonspecific tumor cell kill model incorporating transit compartments captured well the features of tumor growth and the activity of T-DM1. Key findings of the model were that tumor cell growth rate played a significant role in the sensitivity of tumors to T-DM1; anti-tumor activity was schedule independent; and tumor response was linked to the ratio of exposure to a concentration required for tumor stasis.
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http://dx.doi.org/10.1007/s10928-010-9156-2DOI Listing
June 2010

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.

Nat Biotechnol 2008 Aug 20;26(8):925-32. Epub 2008 Jul 20.

Genentech Inc, South San Francisco, California 94080, USA.

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.
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http://dx.doi.org/10.1038/nbt.1480DOI Listing
August 2008

Bioinversion of R-flurbiprofen to S-flurbiprofen at various dose levels in rat, mouse, and monkey.

Chirality 2004 Jul;16(6):379-87

Encore Pharmaceuticals, Inc, Riverside, California 92507, USA.

Information about the potential and extent of bioinversion of chiral drugs in laboratory animal species and humans is critical to the interpretation of preclinical pharm-tox studies with these drugs. Unlike in the dog, guinea pig, and rabbit, in humans the 2-arylpropionic acid (APA) R-flurbiprofen (R-FB) undergoes very little bioinversion to S-flurbiprofen. The primary objective of this research was to identify laboratory animal species with an R- to S-bioinversion profile similar to humans. Detailed evaluations of the pharmacokinetics parameters of R-flurbiprofen in male and female rats and mice, and male nude rats and monkeys demonstrated R- to S-bioinversion of 30% (average) in monkeys, 15-24% in mice, and approximately 4% in rats. To date, no laboratory animal species has been identified with an R-flurbiprofen bioinversion profile identical to humans. However, the rat has a bioinversion profile sufficiently similar to humans to be useful for preclinical.
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http://dx.doi.org/10.1002/chir.20053DOI Listing
July 2004