Article pubs.acs.org/molecularpharmaceutics
Phenotype of TPBG Gene Replacement in the Mouse and Impact on the Pharmacokinetics of an Antibody−Drug Conjugate George Hu,†,‡ Mauricio Leal,‡,§ Qingcong Lin,∥,□ Timothy Affolter,⊥ Puja Sapra,# Brian Bates,∥,¶ and Marc Damelin*,# †
Drug Safety Research & Development, Pfizer Inc., Pearl River, New York 10965, United States Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Pearl River, New York 10965, United States ∥ Global Biotherapeutic Technologies, Pfizer Inc., Cambridge, Massachusetts 02139, United States ⊥ Drug Safety Research & Development, Pfizer Inc., La Jolla, California 92121, United States # Oncology Research Unit, Pfizer Inc., Pearl River, New York 10965, United States §
S Supporting Information *
ABSTRACT: The use of predictive preclinical models in drug discovery is critical for compound selection, optimization, preclinical to clinical translation, and strategic decision-making. Trophoblast glycoprotein (TPBG), also known as 5T4, is the therapeutic target of several anticancer agents currently in clinical development, largely due to its high expression in tumors and low expression in normal adult tissues. In this study, mice were engineered to express human TPBG under endogenous regulatory sequences by replacement of the murine Tpbg coding sequence. The gene replacement was considered functional since the hTPBG knockin (hTPBG-KI) mice did not exhibit clinical observations or histopathological phenotypes that are associated with Tpbg gene deletion, except in rare instances. The expression of hTPBG in certain epithelial cell types and in different microregions of the brain and spinal cord was consistent with previously reported phenotypes and expression patterns. In pharmacokinetic studies, the exposure of a clinical-stage anti-TPBG antibody−drug conjugate (ADC), A1mcMMAF, was lower in hTPBG-KI versus wild-type animals, which was evidence of target-related increased clearance in hTPBG-KI mice. Thus, the hTPBG-KI mice constitute an improved system for pharmacology studies with current and future TPBG-targeted therapies and can generate more precise pharmacokinetic and pharmacodynamic data. In general the strategy of employing gene replacement to improve pharmacokinetic assessments should be broadly applicable to the discovery and development of ADCs and other biotherapeutics. KEYWORDS: antibody−drug conjugate, ADC, TPBG, 5T4, A1mcMMAF, pharmacokinetics, humanized mouse, gene replacement
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INTRODUCTION Trophoblast glycoprotein (TPBG), also known as 5T4, is an oncofetal protein with higher expression in embryonic development and cancer and lower expression in normal adult tissues.1,2 During mouse development, the expression of TPBG is generally limited to extraembryonic tissues and cycling undifferentiated epithelial progenitor cells in the embryo.3 TPBG is an integral plasma membrane protein that consists of a ∼42 kDa peptide core and ∼30 kDa of glycosylation. The extracellular domain contains seven leucine-rich repeats (LRRs) clustered in two LRR domains. TPBG functions in cell adhesion and migration and is associated with the epithelial−mesenchymal transition (EMT), a process that occurs during development and tumor metastasis.4−6 The promigratory functions of TPBG along with its interaction with the cytoskeleton manifest in the promotion of renal podocyte motility,7 the modulation of chemotaxis and cell signaling mediated by the CXCL12-CXCR4 axis,8,9 and the regulation of activity-dependent dendritic development in the olfactory bulb.10 In zebrafish embryos, TPBG regulates Wnt pathway selection in signal receiving cells by acting as a feedback © XXXX American Chemical Society
inhibitor of canonical Wnt signaling while enhancing noncanonical Wnt signaling.11 TPBG expression and function in cancer parallels that in normal tissues. In non-small cell lung cancer, TPBG is associated with EMT and tumor progenitor cells, also known as tumorinitiating cells and cancer stem cells.12 The expression of TPBG is associated with worse clinical outcome and/or more aggressive disease in lung, colorectal, ovarian, and gastric cancers12−15 and has been observed in many solid tumor types16,17 as well as childhood adult lymphoblastic leukemia.18 Due to its overexpression and function in tumors, TPBG is the therapeutic target of several anticancer agents currently in clinical development. MVA-5T4 (TroVax) is a vaccine based on a Special Issue: Antibody-Drug Conjugates Received: September 19, 2014 Revised: November 13, 2014 Accepted: November 25, 2014
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Figure 1. Generation and validation of hTPBG-KI mice. (A) Gene targeting strategy. The entire mouse Tpbg CDS was replaced with the human TPBG CDS without affecting the endogenous regulatory sequences. Homologous recombination was detected by Southern blot with probes to both the 5′ and 3′ sides. (B) Expression of human TPBG but not murine Tpbg in the knockin mice. TaqMan RT-PCR assays for hTPBG mRNA (left) and mTpbg mRNA (right) were applied to brain, lung, and ovary tissues from wild-type and hTPBG knockin animals. Measurements were normalized to GAPDH mRNA. Note that KI-1 was male so there is no ovary sample.
modified vaccinia virus engineered to express TPBG/5T4.19 ABR-217620 (Anyara) consists of superantigen fused to a moiety of anti-5T4 antibody.20 A1mcMMAF, also known as PF06263507, is an antibody−drug conjugate (ADC) that consists of a microtubule disrupting agent, auristatin, conjugated to an anti-5T4 antibody.21 Notably, the preclinical models for some of these agentsand subsequently the pharmacokinetics/pharmacodynamics (PK/PD) modelingare compromised by the lack of cross-reactivity of the TPBG-binding moiety to the murine antigen. We sought to develop a mouse model to enable improved preclinical pharmacology of TPBG-targeted agents. Here we describe the generation and characterization of the hTPBG knockin (hTPBG-KI) mouse, in which the human TPBG coding sequence (CDS) replaces the murine Tpbg CDS while all of the endogenous regulatory sequences are retained. We demonstrate that human TPBG is functional in mice, which could be used to improve the preclinical pharmacological characterization of compounds such as A1mcMMAF.
homologous recombination in 12 ES clones (Figure S1 in the Supporting Information). Mouse lines were derived from targeted ES clones by blastocyst microinjection, and then bred to a Cre-recombinase-deleter strain in order to remove the Neo gene. All mice were maintained on a pure C57Bl/6 background. For quantitative reverse-transcription polymerase chain reaction (RT-PCR), RNA was extracted from brain, lung, and ovary tissues and treated with DNase I to remove contamination of genomic DNA. 20 ng of cDNA was synthesized from each sample, and TaqMan assays were used for PCR: #Hs00272649_s1 (human TPBG), #Mm00495741_s1 (murine Tpbg), and #4352339E (mouse GAPDH). The results for hTPBG and mTpbg were normalized to GAPDH. Phenotyping. A panel of tissues was collected and prepared for standard histological analysis. The panel was composed of brain, pituitary gland, spinal column (cervical and thoracic/ lumbar), eyes (bilateral), tongue, pancreas, salivary gland, thymus, lungs, heart, liver, liver with gallbladder, spleen, kidney (bilateral), mesenteric lymph node, adrenals (bilateral), urinary bladder, thyroid, parathyroid, esophagus, diaphragm, sciatic nerve (cross and longitudinal), quadriceps muscle (cross and longitudinal), stomach (glandular and nonglandular), duodenum, jejunum, ileum, cecum, colon, hind limb (bone, joint, marrow), testis (bilateral)male animals, epididymis (bilateral)male animals, ovary (bilateral)female animals, vaginafemale animals, and uterusfemale animals. Brain sections only from an additional 13 animals of an older age (21−32 weeks) were also included for histopathology evaluation, due to the report of hydrocephalus and structural disorganization in the brain of TPBG knockout mouse.9 At necropsy, the tissues listed above were collected and fixed in 10% neutral buffered formalin (NBF). After the tissues were fixed
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MATERIALS AND METHODS Generation of hTPBG-KI Mice. All procedures using mice were approved by the Pfizer Institutional Animal Care and Use Committee (IACUC) according to established guidelines. The targeting vector was constructed using recombineering technology to replace the entire mouse coding sequence (CDS), which resides entirely in Exon 2, with the entire human TPBG CDS followed by a LoxP-Neo-LoxP cassette (Figure 1A). The DNA was electroporated into C57BL/6 embryonic stem (ES) cells, and clones with homologous recombination were identified by Southern blot analysis. Southern blots with targeting vector external probes to both the 5′ and 3′ ends confirmed the correct B
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reagent and then washed with wash buffer by further centrifugation. Fluorescence of analyte was measured by a laser embedded in the workstation. The quantitation range of the assay is 0.050 to 50.0 μg/mL plasma, as defined by quality control samples. The lower limit of quantitation (LLOQ) was 0.050 μg/ mL plasma. All data was processed by the Watson v 7.4 LIMS system. Noncompartmental PK parameters were calculated using a Watson v7.4 LIMS system. Statistical evaluation (unpaired t test) was done using the Analysis option within GraphPad Prism v6.03.
for 24 h in 10% NBF, they were cassetted individually to maintain identity and processed whole on a Sakura VIP 5 series by dehydrating through a series of graded ethanol solutions, cleared with xylene, and impregnated with paraffin. The tissues were embedded in block and sectioned in 4 μm thickness. Sectioned tissues were heated in a 60 °C oven for a minimum of 1 h, stained via automated linear stainer with hematoxylin−eosin (H&E) and coverslipped. Slides were evaluated by a board certified pathologist. Immunohistochemistry. Formalin fixed, paraffin embedded (FFPE) tissue blocks were sectioned by microtome, deparaffinized in xylene substitute, and rehydrated with graded alcohols to distilled water. To expose antigenic sites, tissue sections were heated in EDTA buffer pH 8.0 (Invitrogen) in a pressure cooker (Retriever; Electron Microscopy Sciences) and cooled to room temperature. Endogenous peroxidase activity was inactivated with 3% hydrogen peroxide for 15 min. Nonspecific protein interactions were blocked by a 10 min incubation with UV Block (Labvision). Tissue sections were incubated with rabbit anti-TPBG (5278-1, Epitomics; 0.36 μg/ mL) for 1 h. Primary antibodies were detected with Signalstain Boost Reagent (8114, Cell Signaling Technologies) for 30 min. TPBG immunostaining was developed with DAB+ (DAKO) for 5 min. Finally, sections were counterstained with Hematoxylin QS (Vector Laboratories), washed in tap water, dehydrated in graded alcohols, cleared in xylene substitute, and mounted with Permount Mounting Medium (Fisher Chemicals, Fair Lawn, NJ). Rabbit IgG (Jackson Immunoresearch) served as a negative control. All processed glass slides were evaluated under an Olympus light microscope, and the images were captured by Spot Insight Firewire Camera and analyzed by Spotsoftware Advanced (Diagnostic Instruments, Inc.). Criteria for assessing the expression levels of TPBG were as follows: grade 1, approximately up to 25% of the tissue section positive with minimal minimum staining intensity; grade 2, approximately up to 50% of the tissue section positive with minimum to mild staining intensity; grade 3, approximately up to 75% of the tissue section positive with minimum to moderate staining intensity. Pharmacokinetics. For studies in hTPBG-KI and wild-type (WT) mice, A1mcMMAF was administered at a single dose of 0.3 or 3 mg/kg as a slow intravenous bolus infusion, with n = 3 or 4 per group. Whole blood samples (10 μL diluted with 190 μL of HEPES-buffered saline [HBS]) were drawn at time points up to 504 h after the dose. Quantitation of the concentrations of total Ab (conjugated and unconjugated anti-TPBG antibody) and ADC (conjugated Ab) in mouse plasma was achieved using the Gyrolab workstation with fluorescence detection. Briefly, isolation and detection of total Ab and ADC in biological matrix was achieved on streptavidin coupled microcolumns located on Bioaffy compact discs (CDs) within the workstation. The capture protein, biotinylated recombinant human TPBG/5T4 (Pfizer Inc.) at 100 μg/mL, was automatically loaded onto the columns, and the CDs were centrifuged to remove excess reagent and then washed with wash buffer by centrifugation. The ADC plasma calibration standards, quality control samples, and mouse plasma samples diluted with SuperBlock buffer at the minimum required dilution (MRD) of 1:40 were loaded onto the CDs. The microcolumns were again washed with wash buffer, and the detection antibodies were loaded onto the microcolumns at 5 μg/mL (goat anti-human IgG AlexaFluor647 [Bethyl Laboratories] for total Ab or AlexaFluor647 anti-MMAF mAb [Pfizer Inc.] for ADC). The CDs were centrifuged to remove excess
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RESULTS Generation of hTPBG-KI Mouse. Standard gene targeting techniques were used to generate mice in which the Tpbg coding sequence (CDS) was replaced with human TPBG CDS while endogenous regulatory sequences remained intact. In the murine Tpbg and human TPBG genes, the entire CDS is contained within one exon, which enabled a targeting strategy to replace the entire CDS without affecting the endogenous mouse promoter, intron-exon structure, 3′ UTR, or polyadenylation sequences (Figure 1A). After removal of the Neo selection gene by breeding to a protamine Cre-recombinase-deleter mouse, a single 34basepair LoxP sequence following the TPBG stop codon was the only remnant of the targeting vector besides the hTPBG CDS (Figure 1A). Thus, the expression of hTPBG in the hTPBG-KI mouse would be regulated by endogenous sequences and would reflect endogenous mTpbg expression patterns and levels as closely as possible. Notably, in many other cases of gene replacement, more complex genomic structure does not enable this approach and requires elimination of introns and introduction of exogenous regulatory sequences, which can impact transgene expression. The expression of hTPBG and not mTpbg in the transgenic animals was confirmed by RT-PCR with a TaqMan assay. The expression levels of mTpbg and hTPBG were determined in selected tissues harvested from wild-type C57BL/6 (WT) and hTPBG-KI animals; ovary, lung, and brain were chosen based on published data.3,22 In WT animals, mTpbg mRNA was detected but hTPBG was not, and conversely in hTPBG-KI animals, hTPBG was detected but mTpbg was not (Figure 1B). One hTPBG-KI animal was male, and thus there is no data for ovary. These results confirmed that hTPBG had replaced mTpbg and was expressed in the hTPBG-KI animals. Clinical Observations of hTPBG KI Mice. No behavioral anomalies, breeding problems, or any other deficiencies were observed for hTPBG-KI mice. To further evaluate the functionality of the hTPBG gene replacement, the hTPBG-KI animals were closely monitored for hydrocephalus since that was the primary phenotype finding reported for Tpbg knockout animals. In a previous report, approximately 13% of Tpbg knockout animals exhibited hydrocephalus and required euthanasia between the ages of 38−83 days.9 The hTPBG-KI animals in this study were generated in the same strain background (C57BL/6) as the knockout animals, which enabled a controlled comparison of the phenotype. Over the course of the project, 61 homozygous hTPBG-KI animals were monitored for over 100 days, which was older than the age range in which hydrocephalus was observed in the knockout animals. There were no signs indicative of hydrocephalus in 60 out of 61 animals; one animal showed clinical signs at 46 days. No other clinical signs were observed for the hTPBG-KI animals. Given the isolated case of hydrocephalus, it is unclear whether it was incidental or knockin-related. Taken C
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Table 1. Summary of Histological Phenotypes in hTPBG Knockin Mice
a
The incidences of unilateral hydronephrosis and of bilateral hydrometra were observed in the same animal. Box shading highlights the incidences of phenotypes.
Figure 2. Histopathological phenotypes observed in hTPBG-KI mice. (A) Brain phenotype (1 out of 33 hTPBG-KI animals): various degrees of the expansion of lateral ventricles and third ventricle. (B) Kidney phenotypes (3 out of 20 hTPBG-KI animals). (i) Transverse section of normal kidney from a wild-type mouse. (ii, iii) Transverse sections show large central dilated space (ii) and a cystic space (iii) possibly contiguous with the hilar region. There was a minimal amount of medullary tissues (ii, iii) and distorted cortical architecture (ii).
Expression of hTPBG Protein. The expression pattern of TPBG in the hTPBG-KI animals was assessed by immunohistochemistry (IHC) with a commercial monoclonal anti-TPBG antibody. The crisp staining in the hTPBG-KI animals together with the absence of staining in the littermate WT animals indicated the specificity of the antibody for human TPBG. Specificity of the antibody for human TPBG was further demonstrated by IHC and immunoblotting of human and murine samples (data not shown). Moreover, the extent of antibody staining of various human cell lines correlated with the level of TPBG expression as determined by orthogonal methods such as immunoblotting. Immunohistochemistry in tissues from hTPGB-KI mice revealed plasma membrane staining of TPBG in transitional epithelial cells of the urinary bladder (Figure 3A), mucosal epithelial cells of the uterus, granulosa cells of the ovaries, and rarely medullary cells of the adrenals. Mild staining was also observed in the kidney (Figure S2 in the Supporting Information). TPBG expression is best characterized in trophoblasts,17 but placental tissue was not evaluated in the current study.
together these results indicate that the hTPBG gene replacement was functional and able to substitute for the developmental functions of the endogenous gene. Histopathological Phenotyping of hTPBG KI Mice. A panel of 35 tissues from each of 20 age-matched WT and 20 hTPBG-KI mice were evaluated by gross examination of whole organs and by histopathology. Ten animals (5 male/5 female) from each group were sacrificed at 7−9 weeks of age, and another ten animals (5 male/5 female) from each group were sacrificed at 17−19 weeks of age. Overall, phenotypic alterations were minimal between hTPBG-KI and WT mice in both age groups. While phenotypes were observed in the brain, kidney, and uterus of hTPBG-KI mice, each finding was confined to one or two animals in the entire survey (Table 1). Moderate bilateral hydrocephalus was observed in the brain of one female (Figure 2A). Unilateral hydronephrosis was observed in one female (Figure 2B), and bilateral hydrometra in the uterus was observed in the same animal. Minimal cortical irregularities in the kidney were observed in two animals. A detailed pathology report is provided in the Supporting Information. D
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Figure 3. hTPBG expression in hTPBG-KI mice. (A) Urinary bladder. Grade 3+ TPBG immunohistochemical stain was identified in the cell membrane of the transitional epithelial cells. (B) Spinal cord. Strong TPBG expression was observed in the neuropil and occasional neurons of gray matter in the ventral horns, while no expression was observed in the white matter. (C) Hypothalamus area. TPBG expression was observed in the neuropil and occasionally the neurons in the bilateral hypothalamic nucleus. (D) Cochlear nucleus. Strong TPBG expression on the membrane and in the cytoplasm of the neurons and neuropil of the superficial glial zone and dorsal cochlear nucleus. (E) Cerebrum. Minimal to mild TPBG expression was observed in the neuropil and neurons in the cerebral cortex, caudate putamen, and lateral thalamic nucleus as well as cell membrane of the epithelial cells in the coroid plexus of the brain ventricles. (F) Retina. TPBG expression was observed in the inner/outer plexiform layers and the ganglion cell layer, including multipolar neurons and Muller cells.
Figure 4. Altered pharmacokinetics of A1mcMMAF ADC in hTPBG-KI mice. (A) Concentrations of total antibody (conjugated and unconjugated) following a single dose administration of A1mcMMAF anti-TBPG ADC in WT and hTPBG-KI mice. Data points indicate mean ± standard deviation (n = 3−4). (B) Exposure (AUC0−504) of total antibody following a single dose administration of A1mcMMAF anti-TBPG ADC in WT and hTPBG-KI mice (corresponds to the curves in panel A). * denotes significantly lower exposure in KI mouse than corresponding gender WT mouse group (p = 0.005 to p = 0.0001). (C) Traces for individual animals that were averaged for panels A and B.
Consistent with historical reports on TPBG expression in the brain and the brain-related phenotypes of TPBG-knockout mice,3,9,10,22 immunohistochemistry revealed hTPBG expression in the brain of the hTPBG-KI mice. There was minimal to mild (grade 1+ to 2+) TPBG expression in the neuropil, axon, and/or
neurocytoplasm in multiple microanatomic regions of the central nervous system (Figure 3B−E). The histological regions include inner cortex, bilateral superficial and dorsal cochlear nucleus, bilateral caudate/putamen (striatum), bilateral medial geniculate nucleus, bilateral thalamic nucleus, bilateral hypothalamic E
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level and limited animal numbers; since the difference was observed in both WT and hTPBG-KI animals and since A1mcMMAF does not bind to murine TPBG, the effect appears independent of the TPBG target. Taken together, the results demonstrate that the expression of TPBG in normal tissues has an effect on the PK of A1mcMMAF in plasma.
nucleus, substantia nigra, facial nucleus, lateral septal nucleus, raphe nucleus, commissure of the inferior colliculus, vesticulocerebellar nucleus, posterolateral amygdalohippocampal areas, and geniculate nucleus as well as cell membrane of the choroid plexus epithelial cells in the ventricles of the brain. Interestingly, many of these microanatomic regions in the central nerve system are associated with hearing. In the spinal cord, mild (grade 2+) expression of TPBG was detected in the neuropil and neurons in the ventral horn of the gray matter, but no expression was observed in the white matter. In the pituitary glands, TPBG expression was detected in the cytoplasm of the neurons in the par neurosa and negative in par distalis and intermedia. In the eye, mild to moderate TPBG expression was observed in inner/outer plexiform layers, ganglion cell layer (including multipolar neurons and muller cells), and optic fiber (Figure 3F). The positivity was mostly located in the cytoplasm, cell membrane, and neurofibers. In addition, some corneal stromal cells were cytoplasmic positive. In summary, the expression profile of hTPBG in the hTPBGKI mice was morphologically and functionally consistent with historical findings and the phenotypes of KI and knockout mice. The results further characterized and validated the hTPBG-KI mouse strain. Pharmacokinetics of A1mcMMAF in hTPBG-KI and WT Mice. Since the phenotyping and immunohistochemical analyses of the hTPBG-KI mice indicated that the gene replacement was functional, PK studies were conducted with the anti-TPBG ADC A1mcMMAF. An ADC consists of an antibody such as one directed to a tumor antigen, a potent cytotoxic drug, and a chemical linker; thus the antibody delivers the drug preferentially to the tumor and minimizes its exposure in normal tissues.23,24 Due to the complexity of the ADC structure, PK assessment typically involves multiple analytes.25 A1mcMMAF does not cross-react to murine antigen,21 and thus a comparison of its PK in WT vs hTPBG-KI mice would reveal the potential effects of target expression in normal tissues on drug exposure. A1mcMMAF was administered at a single dose of 0.3 or 3 mg/kg as a slow intravenous bolus infusion, and the quantities of total Ab (conjugated and unconjugated antibody) and ADC (conjugated antibody) were determined at several time points up to 504 h after the dose administration. No clinical observations, body weight loss, or adverse events were observed in any of the animals during the 3-week in life study. In both the WT and hTPBG-KI mice, the concentration profiles over time of both total Ab and ADC were biphasic in nature, with an initial decline within the first 6 h followed by a prolonged elimination phase. In addition, the concentration profile of the ADC indicated lower overall systemic exposure (AUC0−504) than that of the total Ab. Both of these observations are consistent with the previously characterized PK profile of A1mcMMAF.21,26 The exposures (AUC0−504) of both total Ab and ADC were notably reduced in the hTPBG-KI animals relative to WT animals, at both dose levels. Total Ab exposures in hTPBG-KI mice were 31% and 48% of those in WT mice at the 0.3 mg/kg and 3 mg/kg dose levels respectively (Figure 4). Similarly, ADC exposure in the hTPBG-KI mice was 38% and 58% of that in WT mice at the 0.3 mg/kg and 3 mg/kg dose levels respectively (Figure S3 in the Supporting Information). Total exposures for both analytes were proportional to dose level in both WT and hTPBG-KI animals, especially in males. Interestingly, the data suggested a trend toward lower exposures in females, though this difference was observed only at the lower (more sensitive) dose
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DISCUSSION The use of predictive preclinical models in drug discovery is critical for increasing the clinical success rate of therapeutics. This study has described the generation and characterization of hTPBG-KI mice that could be used in pharmacology studies to yield improved PK/PD-based predictions for current and future TPBG-targeted therapies. The genetic strategy was designed to generate the most physiologically relevant gene replacement possible by replacing only the coding sequence and retaining the endogenous 5′ and 3′ regulatory sequences. In other cases of gene replacement, exogenous promoters and/or polyadenylation sequences are used either for convenience or due to complex genomic structure; such exogenous sequences can significantly impact expression of the gene product. The phenotypic and immunohistochemical analyses of the hTPBG-KI mice indicated that the gene replacement was functional. However, we cannot rule out that the single incidence of hydrocephalus and the other rare histopathologic phenotypes were related to a nonequivalence of the novel hTPBG allele. Notably, the incidence of hydrocephalus was substantially lower than in the knockout mice in the same strain background.9 Expression of TPBG in the hTPBG-KI mice was observed primarily in the central nervous system and in epithelial cells in various tissues. These results are consistent with historical findings and phenotypes of the KI and knockout mice. The observation of TPBG expression in many microanatomic regions involved in hearing may suggest previously unknown functions of TPBG. Interestingly, TPBG expression and function in the olfactory bulb were observed in neonatal mice at 3 weeks of age;10 we did not observe hTPBG expression in the hTPBG-KI mice in the younger cohort (7−9 weeks) or the older cohort (17−19 weeks), which suggests that the function of TPBG in the olfactory bulb is most prominent in, or even restricted to, neonatal development. The expression of TPBG in normal human tissues is not well characterized in the literature, but some similarities were observed between the published report17 and the hTPBG-KI mice in this study. In particular, expression was reported in the cervix and endometrium, which is consistent with our observation of hTPBG in the uterus. Expression was noted in the esophagus,17 whereas we did not observe it; faint or equivocal staining was also noted in other tissues. No expression was detected in the brain (notably, only one sample evaluated);17 interestingly, the expression in brain that we observed in hTPBGKI mice was consistent with several reported studies in mice.3,9,10,22 Due to the different preservation of samples (frozen versus formalin fixation), different antibodies (epitopes) and staining methods, and the limited number of samples evaluated for some tissues,17 additional studies are required to determine whether dissimilarities in staining are attributable to actual differences in expression between humans and hTPBG-KI mice. The observation of altered PK of a TPBG-targeted agent in hTPBG-KI mice demonstrates the utility of preclinical pharmacology studies in the hTPBG-KI mice. The data suggest a component of target-mediated drug disposition that affects the F
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exposure of A1-mcMMAF and potentially other TPBG-targeted compounds. Additional PK studies in hTPBG-KI mice would enable more precise estimations of dosing and pharmacodynamics of TPBG-targeted therapies. Toxicology studies with compounds such as A1mcMMAF could be conducted and the results compared to those obtained in other nonclinical species; a special consideration in the case of A1mcMMAF is that mice exhibit a greater degree of tolerance of some microtubule inhibitors including auristatins than do rats and monkeys.27,28 Future studies could also include breeding the TPBG-KI animals into an immune-compromised background to enable efficacy studies with human tumor xenografts. Genetically humanized mice offer advantages in many aspects of drug development as well as basic research.29 Preclinical studies inform critical decisions of whether to advance a compound into the clinic and, if so, how to develop the compound. The generation of target-specific knockin mice early in the drug discovery process would enable more precise pharmacological evaluation of compounds especially when one or more of the lead candidates does not engage the mouse target. The gene replacement strategy employed in this study enabled the expression of the human gene under the mouse endogenous regulatory sequences and thus provided the most precise model possible for such studies. Overall this strategy could improve the PK/PD-based predictions of human efficacious dose and be used to inform preclinical decisions as well as clinical development strategies.
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REFERENCES
(1) Hole, N.; Stern, P. L. A 72 kD trophoblast glycoprotein defined by a monoclonal antibody. Br. J. Cancer 1988, 57, 239−46. (2) Hole, N.; Stern, P. L. Isolation and characterization of 5T4, a tumour-associated antigen. Int. J. Cancer 1990, 45, 179−84. (3) Barrow, K. M.; Ward, C. M.; Rutter, J.; Ali, S.; Stern, P. L. Embryonic expression of murine 5T4 oncofoetal antigen is associated with morphogenetic events at implantation and in developing epithelia. Dev. Dyn. 2005, 233, 1535−1545. (4) Carsberg, C. J.; Myers, K. A.; Stern, P. L. Metastasis-associated 5T4 antigen disrupts cell-cell contacts and induces cellular motility in epithelial cells. Int. J. Cancer 1996, 68, 84−92. (5) Eastham, A. M.; Spencer, H.; Soncin, F.; Ritson, S.; Merry, C. L.; Stern, P. L.; Ward, C. M. Epithelial-mesenchymal transition events during human embryonic stem cell differentiation. Cancer Res. 2007, 67, 11254−11262. (6) Spencer, H. L.; Eastham, A. M.; Merry, C. L.; Southgate, T. D.; Perez-Campo, F.; Soncin, F.; Ritson, S.; Kemler, R.; Stern, P. L.; Ward, C. M. E-cadherin inhibits cell surface localization of the pro-migratory 5T4 oncofetal antigen in mouse embryonic stem cells. Mol. Biol. Cell 2007, 18 (8), 2838−51. (7) Murakami, T.; Abe, H.; Nagai, K.; Tominaga, T.; Takamatsu, N.; Araoka, T.; Kishi, S.; Takahashi, T.; Mima, A.; Takai, Y.; Kopp, J. B.; Doi, T. Trophoblast glycoprotein: possible candidate mediating podocyte injuries in glomerulonephritis. Am. J. Nephrol. 2010, 32 (6), 505−21 DOI: 10.1159/000321366. (8) McGinn, O. J.; Marinov, G.; Sawan, S.; Stern, P. L. CXCL12 receptor preference, signal transduction, biological response and the expression of 5T4 oncofoetal glycoprotein. J. Cell Sci. 2012, 125 (Part 22), 5467−78 DOI: 10.1242/jcs.109488. (9) Southgate, T. D.; McGinn, O. J.; Castro, F. V.; Rutkowski, A. J.; AlMuftah, M.; Marinov, G.; Smethurst, G. J.; Shaw, D.; Ward, C. M.; Miller, C. J.; Stern, P. L. CXCR4 mediated chemotaxis is regulated by 5T4 oncofetal glycoprotein in mouse embryonic stem cells. PLoS One 2010, 5, e9982. (10) Yoshihara, S.; Takahashi, H.; Nishimura, N.; Naritsuka, H.; Shirao, T.; Hirai, H.; Yoshihara, Y.; Mori, K.; Stern, P. L.; Tsuboi, A. 5T4 Glycoprotein Regulates the Sensory Input-Dependent Development of a Specific Subtype of Newborn Interneurons in the Mouse Olfactory Bulb. J. Neurosci. 2012, 32 (6), 2217−26. (11) Kagermeier-Schenk, B.; Wehner, D.; Ozhan-Kizil, G.; Yamamoto, H.; Li, J.; Kirchner, K.; Hoffmann, C.; Stern, P.; Kikuchi, A.; Schambony, A.; Weidinger, G. Waif1/5T4 Inhibits Wnt/beta-Catenin Signaling and Activates Noncanonical Wnt Pathways by Modifying LRP6 Subcellular Localization. Dev. Cell 2011, 21, 1129−43. (12) Damelin, M.; Geles, K. G.; Follettie, M. T.; Yuan, P.; Baxter, M.; Golas, J.; DiJoseph, J. F.; Karnoub, M.; Huang, S.; Diesl, V.; Behrens, C.; Choe, S. E.; Rios, C.; Gruzas, J.; Sridharan, L.; Dougher, M.; Kunz, A.; Hamann, P. R.; Evans, D.; Armellino, D.; Khandke, K.; Marquette, K.; Tchistiakova, L.; Boghaert, E. R.; Abraham, R. T.; Wistuba, I. I.; Zhou, B. B. Delineation of a cellular hierarchy in lung cancer reveals an oncofetal antigen expressed on tumor-initiating cells. Cancer Res. 2011, 71, 4236− 46. (13) Naganuma, H.; Kono, K.; Mori, Y.; Takayosh, S.; Stern, P. L.; Tasaka, K.; Matsumoto, Y. Oncofetal antigen 5T4 expression as a prognostic factor in patients with gastric cancer. Anticancer Res. 2002, 22, 1033−8. (14) Starzynska, T.; Marsh, P. J.; Schofield, P. F.; Roberts, S. A.; Myers, K. A.; Stern, P. L. Prognostic significance of 5T4 oncofetal antigen expression in colorectal carcinoma. Br. J. Cancer 1994, 69, 899−902. (15) Wrigley, E.; McGown, A. T.; Rennison, J.; Swindell, R.; Crowther, D.; Starzynska, T.; Stern, P. L. 5T4 oncofetal antigen expression in ovarian carcinoma. Int. J. Gynecol. Cancer 1995, 5, 269−74. (16) Al-Taei, S.; Salimu, J.; Lester, J. F.; Linnane, S.; Goonewardena, M.; Harrop, R.; Mason, M. D.; Tabi, Z. Overexpression and potential targeting of the oncofoetal antigen 5T4 in malignant pleural mesothelioma. Lung Cancer 2012, 77 (2), 312−8 DOI: 10.1016/ j.lungcan.2012.03.008.
ASSOCIATED CONTENT
* Supporting Information S
Description of the pathology of the hTPBG-KI animals and accompanying figures. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Pfizer Worldwide Research & Development, 401 N. Middletown Road, Building 200-4611, Pearl River, NY 10965. Tel: 845602-7985. E-mail: marc.damelin@pfizer.com. Present Addresses ¶
(B.B.) Pfizer Inc., Centers for Therapeutic Innovation, 3 Blackfan Circle, Boston, MA, 02115. □ (Q.L.) Shenogen Pharma Group Ltd., Changping, Beijing PRC 102206. Author Contributions ‡
G.H. and M.L. made equal contributions.
Notes
The authors declare the following competing financial interest(s): Drs. Hu, Leal, Affolter, Sapra, Bates, and Damelin are current employees and shareholders of Pfizer Inc.
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ACKNOWLEDGMENTS The authors gratefully acknowledge contributions from Jonathon Golas, Theresa Paradis, Musen Liang, Joann Wentland, Timothy Coskran, the Investigational Pathology Laboratory, Johnny Yao, the ORU In Vivo Pharmacology Group, Jen Sadlier, Mary Bauchmann, and Pfizer Worldwide Comparative Medicine, as well as Adam Cole at Charles River Laboratories. The authors acknowledge Seattle Genetics Inc. and Oxford BioMedica for access to technology regarding A1mcMMAF. G
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(17) Southall, P. J.; Boxer, G. M.; Bagshawe, K. D.; Hole, N.; Bromley, M.; Stern, P. L. Immunohistological distribution of 5T4 antigen in normal and malignant tissues. Br. J. Cancer 1990, 61, 89−95. (18) Castro, F. V.; McGinn, O. J.; Krishnan, S.; Marinov, G.; Li, J.; Rutkowski, A. J.; Elkord, E.; Burt, D. J.; Holland, M.; Vaghjiani, R.; Gallego, A.; Saha, V.; Stern, P. L. 5T4 oncofetal antigen is expressed in high risk of relapse childhood pre-B acute lymphoblastic leukemia and is associated with a more invasive and chemotactic phenotype. Leukemia 2012, 26 (7), 1487−98 DOI: 10.1038/leu.2012.18. (19) Amato, R. J.; Stepankiw, M. Evaluation of MVA-5T4 as a novel immunotherapeutic vaccine in colorectal, renal and prostate cancer. Future Oncol. 2012, 8 (3), 231−7 DOI: 10.2217/fon.12.7. (20) Forsberg, G.; Skartved, N. J.; Wallén-Ohman, M.; Nyhlén, H. C.; Behm, K.; Hedlund, G.; Nederman, T. Naptumomab estafenatox, an engineered antibody-superantigen fusion protein with low toxicity and reduced antigenicity. J. Immunother. 2010, 33 (5), 492−9. (21) Sapra, P.; Damelin, M.; Dijoseph, J.; Marquette, K.; Geles, K. G.; Golas, J.; Dougher, M.; Narayanan, B.; Giannakou, A.; Khandke, K.; Dushin, R.; Ernstoff, E.; Lucas, J.; Leal, M.; Hu, G.; O’Donnell, C. J.; Tchistiakova, L.; Abraham, R. T.; Gerber, H. P. Long-term tumor regression induced by an antibody-drug conjugate that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells. Mol. Cancer Ther. 2013, 12 (1), 38−47 DOI: 10.1158/1535-7163.MCT-12-0603. (22) Woods, A. M.; Wang, W. W.; Shaw, D. M.; Ward, C. M.; Carroll, M. W.; Rees, B. R.; Stern, P. L. Characterization of the murine 5T4 oncofoetal antigen: a target for immunotherapy in cancer. Biochem. J. 2002, 366 (Part 1), 353−65. (23) Sapra, P.; Hooper, A. T.; O’Donnell, C. J.; Gerber, H. P. Investigational antibody drug conjugates for solid tumors. Expert Opin. Invest. Drugs 2011, 20, 1131−49. (24) Lambert, J. M. Drug-conjugated antibodies for the treatment of cancer. Br. J. Clin. Pharmacol. 2013, 76 (3), 248−62. (25) Kaur, S.; Xu, K.; Saad, O. M.; Dere, R. C.; Carrasco-Triguero, M. Bioanalytical assay strategies for the development of antibody-drug conjugate biotherapeutics. Bioanalysis 2013, 5 (2), 201−26. (26) Shah, D. K.; King, L. E.; Han, S.; Wentland, J.; Zhang, J.; Lucas, J.; Haddish-Berhane, N.; Betts, A.; Leal, M. A Priori Prediction of Tumor Payload Concentration: Preclinical Case Study with an Auristatin Based Anti-5T4 Antibody Drug Conjugate. AAPS J. 2014, 16 (3), 452−63. (27) Mirsalis, J. C.; Schindler-Horvat, J.; Hill, J. R.; Tomaszewski, J. E.; Donohue, S. J.; Tyson, C. A. Toxicity of dolastatin 10 in mice, rats and dogs and its clinical relevance. Cancer Chemother. Pharmacol. 1999, 44, 395−402. (28) Polson, A. G.; Calemine-Fenaux, J.; Chan, P.; Chang, W.; Christensen, E.; Clark, S.; de Sauvage, F. J.; Eaton, D.; Elkins, K.; Elliott, J. M.; Frantz, G.; Fuji, R. N.; Gray, A.; Harden, K.; Ingle, G. S.; Kljavin, N. M.; Koeppen, H.; Nelson, C.; Prabhu, S.; Raab, H.; Ross, S.; Slaga, D. S.; Stephan, J. P.; Scales, S. J.; Spencer, S. D.; Vandlen, R.; Wranik, B.; Yu, S. F.; Zheng, B.; Ebens, A. Antibody-drug conjugates for the treatment of non-Hodgkin’s lymphoma: target and linker-drug selection. Cancer Res. 2009, 69 (6), 2358−64. (29) Scheer, N.; Snaith, M.; Wolf, C. R.; Seibler, J. Generation and utility of genetically humanized mouse models. Drug Discovery Today 2013, 18 (23−24), 1200−11.
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Phenotype of TPBG Gene Replacement in the Mouse and Impact on the Pharmacokinetics of an Antibody−Drug Conjugate George Hu,†,‡ Mauricio Leal,‡,§ Qingcong Lin,∥,□ Timothy Affolter,⊥ Puja Sapra,# Brian Bates,∥,¶ and Marc Damelin*,# †
Drug Safety Research & Development, Pfizer Inc., Pearl River, New York 10965, United States Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Pearl River, New York 10965, United States ∥ Global Biotherapeutic Technologies, Pfizer Inc., Cambridge, Massachusetts 02139, United States ⊥ Drug Safety Research & Development, Pfizer Inc., La Jolla, California 92121, United States # Oncology Research Unit, Pfizer Inc., Pearl River, New York 10965, United States §
S Supporting Information *
ABSTRACT: The use of predictive preclinical models in drug discovery is critical for compound selection, optimization, preclinical to clinical translation, and strategic decision-making. Trophoblast glycoprotein (TPBG), also known as 5T4, is the therapeutic target of several anticancer agents currently in clinical development, largely due to its high expression in tumors and low expression in normal adult tissues. In this study, mice were engineered to express human TPBG under endogenous regulatory sequences by replacement of the murine Tpbg coding sequence. The gene replacement was considered functional since the hTPBG knockin (hTPBG-KI) mice did not exhibit clinical observations or histopathological phenotypes that are associated with Tpbg gene deletion, except in rare instances. The expression of hTPBG in certain epithelial cell types and in different microregions of the brain and spinal cord was consistent with previously reported phenotypes and expression patterns. In pharmacokinetic studies, the exposure of a clinical-stage anti-TPBG antibody−drug conjugate (ADC), A1mcMMAF, was lower in hTPBG-KI versus wild-type animals, which was evidence of target-related increased clearance in hTPBG-KI mice. Thus, the hTPBG-KI mice constitute an improved system for pharmacology studies with current and future TPBG-targeted therapies and can generate more precise pharmacokinetic and pharmacodynamic data. In general the strategy of employing gene replacement to improve pharmacokinetic assessments should be broadly applicable to the discovery and development of ADCs and other biotherapeutics. KEYWORDS: antibody−drug conjugate, ADC, TPBG, 5T4, A1mcMMAF, pharmacokinetics, humanized mouse, gene replacement
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INTRODUCTION Trophoblast glycoprotein (TPBG), also known as 5T4, is an oncofetal protein with higher expression in embryonic development and cancer and lower expression in normal adult tissues.1,2 During mouse development, the expression of TPBG is generally limited to extraembryonic tissues and cycling undifferentiated epithelial progenitor cells in the embryo.3 TPBG is an integral plasma membrane protein that consists of a ∼42 kDa peptide core and ∼30 kDa of glycosylation. The extracellular domain contains seven leucine-rich repeats (LRRs) clustered in two LRR domains. TPBG functions in cell adhesion and migration and is associated with the epithelial−mesenchymal transition (EMT), a process that occurs during development and tumor metastasis.4−6 The promigratory functions of TPBG along with its interaction with the cytoskeleton manifest in the promotion of renal podocyte motility,7 the modulation of chemotaxis and cell signaling mediated by the CXCL12-CXCR4 axis,8,9 and the regulation of activity-dependent dendritic development in the olfactory bulb.10 In zebrafish embryos, TPBG regulates Wnt pathway selection in signal receiving cells by acting as a feedback © XXXX American Chemical Society
inhibitor of canonical Wnt signaling while enhancing noncanonical Wnt signaling.11 TPBG expression and function in cancer parallels that in normal tissues. In non-small cell lung cancer, TPBG is associated with EMT and tumor progenitor cells, also known as tumorinitiating cells and cancer stem cells.12 The expression of TPBG is associated with worse clinical outcome and/or more aggressive disease in lung, colorectal, ovarian, and gastric cancers12−15 and has been observed in many solid tumor types16,17 as well as childhood adult lymphoblastic leukemia.18 Due to its overexpression and function in tumors, TPBG is the therapeutic target of several anticancer agents currently in clinical development. MVA-5T4 (TroVax) is a vaccine based on a Special Issue: Antibody-Drug Conjugates Received: September 19, 2014 Revised: November 13, 2014 Accepted: November 25, 2014
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Figure 1. Generation and validation of hTPBG-KI mice. (A) Gene targeting strategy. The entire mouse Tpbg CDS was replaced with the human TPBG CDS without affecting the endogenous regulatory sequences. Homologous recombination was detected by Southern blot with probes to both the 5′ and 3′ sides. (B) Expression of human TPBG but not murine Tpbg in the knockin mice. TaqMan RT-PCR assays for hTPBG mRNA (left) and mTpbg mRNA (right) were applied to brain, lung, and ovary tissues from wild-type and hTPBG knockin animals. Measurements were normalized to GAPDH mRNA. Note that KI-1 was male so there is no ovary sample.
modified vaccinia virus engineered to express TPBG/5T4.19 ABR-217620 (Anyara) consists of superantigen fused to a moiety of anti-5T4 antibody.20 A1mcMMAF, also known as PF06263507, is an antibody−drug conjugate (ADC) that consists of a microtubule disrupting agent, auristatin, conjugated to an anti-5T4 antibody.21 Notably, the preclinical models for some of these agentsand subsequently the pharmacokinetics/pharmacodynamics (PK/PD) modelingare compromised by the lack of cross-reactivity of the TPBG-binding moiety to the murine antigen. We sought to develop a mouse model to enable improved preclinical pharmacology of TPBG-targeted agents. Here we describe the generation and characterization of the hTPBG knockin (hTPBG-KI) mouse, in which the human TPBG coding sequence (CDS) replaces the murine Tpbg CDS while all of the endogenous regulatory sequences are retained. We demonstrate that human TPBG is functional in mice, which could be used to improve the preclinical pharmacological characterization of compounds such as A1mcMMAF.
homologous recombination in 12 ES clones (Figure S1 in the Supporting Information). Mouse lines were derived from targeted ES clones by blastocyst microinjection, and then bred to a Cre-recombinase-deleter strain in order to remove the Neo gene. All mice were maintained on a pure C57Bl/6 background. For quantitative reverse-transcription polymerase chain reaction (RT-PCR), RNA was extracted from brain, lung, and ovary tissues and treated with DNase I to remove contamination of genomic DNA. 20 ng of cDNA was synthesized from each sample, and TaqMan assays were used for PCR: #Hs00272649_s1 (human TPBG), #Mm00495741_s1 (murine Tpbg), and #4352339E (mouse GAPDH). The results for hTPBG and mTpbg were normalized to GAPDH. Phenotyping. A panel of tissues was collected and prepared for standard histological analysis. The panel was composed of brain, pituitary gland, spinal column (cervical and thoracic/ lumbar), eyes (bilateral), tongue, pancreas, salivary gland, thymus, lungs, heart, liver, liver with gallbladder, spleen, kidney (bilateral), mesenteric lymph node, adrenals (bilateral), urinary bladder, thyroid, parathyroid, esophagus, diaphragm, sciatic nerve (cross and longitudinal), quadriceps muscle (cross and longitudinal), stomach (glandular and nonglandular), duodenum, jejunum, ileum, cecum, colon, hind limb (bone, joint, marrow), testis (bilateral)male animals, epididymis (bilateral)male animals, ovary (bilateral)female animals, vaginafemale animals, and uterusfemale animals. Brain sections only from an additional 13 animals of an older age (21−32 weeks) were also included for histopathology evaluation, due to the report of hydrocephalus and structural disorganization in the brain of TPBG knockout mouse.9 At necropsy, the tissues listed above were collected and fixed in 10% neutral buffered formalin (NBF). After the tissues were fixed
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MATERIALS AND METHODS Generation of hTPBG-KI Mice. All procedures using mice were approved by the Pfizer Institutional Animal Care and Use Committee (IACUC) according to established guidelines. The targeting vector was constructed using recombineering technology to replace the entire mouse coding sequence (CDS), which resides entirely in Exon 2, with the entire human TPBG CDS followed by a LoxP-Neo-LoxP cassette (Figure 1A). The DNA was electroporated into C57BL/6 embryonic stem (ES) cells, and clones with homologous recombination were identified by Southern blot analysis. Southern blots with targeting vector external probes to both the 5′ and 3′ ends confirmed the correct B
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reagent and then washed with wash buffer by further centrifugation. Fluorescence of analyte was measured by a laser embedded in the workstation. The quantitation range of the assay is 0.050 to 50.0 μg/mL plasma, as defined by quality control samples. The lower limit of quantitation (LLOQ) was 0.050 μg/ mL plasma. All data was processed by the Watson v 7.4 LIMS system. Noncompartmental PK parameters were calculated using a Watson v7.4 LIMS system. Statistical evaluation (unpaired t test) was done using the Analysis option within GraphPad Prism v6.03.
for 24 h in 10% NBF, they were cassetted individually to maintain identity and processed whole on a Sakura VIP 5 series by dehydrating through a series of graded ethanol solutions, cleared with xylene, and impregnated with paraffin. The tissues were embedded in block and sectioned in 4 μm thickness. Sectioned tissues were heated in a 60 °C oven for a minimum of 1 h, stained via automated linear stainer with hematoxylin−eosin (H&E) and coverslipped. Slides were evaluated by a board certified pathologist. Immunohistochemistry. Formalin fixed, paraffin embedded (FFPE) tissue blocks were sectioned by microtome, deparaffinized in xylene substitute, and rehydrated with graded alcohols to distilled water. To expose antigenic sites, tissue sections were heated in EDTA buffer pH 8.0 (Invitrogen) in a pressure cooker (Retriever; Electron Microscopy Sciences) and cooled to room temperature. Endogenous peroxidase activity was inactivated with 3% hydrogen peroxide for 15 min. Nonspecific protein interactions were blocked by a 10 min incubation with UV Block (Labvision). Tissue sections were incubated with rabbit anti-TPBG (5278-1, Epitomics; 0.36 μg/ mL) for 1 h. Primary antibodies were detected with Signalstain Boost Reagent (8114, Cell Signaling Technologies) for 30 min. TPBG immunostaining was developed with DAB+ (DAKO) for 5 min. Finally, sections were counterstained with Hematoxylin QS (Vector Laboratories), washed in tap water, dehydrated in graded alcohols, cleared in xylene substitute, and mounted with Permount Mounting Medium (Fisher Chemicals, Fair Lawn, NJ). Rabbit IgG (Jackson Immunoresearch) served as a negative control. All processed glass slides were evaluated under an Olympus light microscope, and the images were captured by Spot Insight Firewire Camera and analyzed by Spotsoftware Advanced (Diagnostic Instruments, Inc.). Criteria for assessing the expression levels of TPBG were as follows: grade 1, approximately up to 25% of the tissue section positive with minimal minimum staining intensity; grade 2, approximately up to 50% of the tissue section positive with minimum to mild staining intensity; grade 3, approximately up to 75% of the tissue section positive with minimum to moderate staining intensity. Pharmacokinetics. For studies in hTPBG-KI and wild-type (WT) mice, A1mcMMAF was administered at a single dose of 0.3 or 3 mg/kg as a slow intravenous bolus infusion, with n = 3 or 4 per group. Whole blood samples (10 μL diluted with 190 μL of HEPES-buffered saline [HBS]) were drawn at time points up to 504 h after the dose. Quantitation of the concentrations of total Ab (conjugated and unconjugated anti-TPBG antibody) and ADC (conjugated Ab) in mouse plasma was achieved using the Gyrolab workstation with fluorescence detection. Briefly, isolation and detection of total Ab and ADC in biological matrix was achieved on streptavidin coupled microcolumns located on Bioaffy compact discs (CDs) within the workstation. The capture protein, biotinylated recombinant human TPBG/5T4 (Pfizer Inc.) at 100 μg/mL, was automatically loaded onto the columns, and the CDs were centrifuged to remove excess reagent and then washed with wash buffer by centrifugation. The ADC plasma calibration standards, quality control samples, and mouse plasma samples diluted with SuperBlock buffer at the minimum required dilution (MRD) of 1:40 were loaded onto the CDs. The microcolumns were again washed with wash buffer, and the detection antibodies were loaded onto the microcolumns at 5 μg/mL (goat anti-human IgG AlexaFluor647 [Bethyl Laboratories] for total Ab or AlexaFluor647 anti-MMAF mAb [Pfizer Inc.] for ADC). The CDs were centrifuged to remove excess
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RESULTS Generation of hTPBG-KI Mouse. Standard gene targeting techniques were used to generate mice in which the Tpbg coding sequence (CDS) was replaced with human TPBG CDS while endogenous regulatory sequences remained intact. In the murine Tpbg and human TPBG genes, the entire CDS is contained within one exon, which enabled a targeting strategy to replace the entire CDS without affecting the endogenous mouse promoter, intron-exon structure, 3′ UTR, or polyadenylation sequences (Figure 1A). After removal of the Neo selection gene by breeding to a protamine Cre-recombinase-deleter mouse, a single 34basepair LoxP sequence following the TPBG stop codon was the only remnant of the targeting vector besides the hTPBG CDS (Figure 1A). Thus, the expression of hTPBG in the hTPBG-KI mouse would be regulated by endogenous sequences and would reflect endogenous mTpbg expression patterns and levels as closely as possible. Notably, in many other cases of gene replacement, more complex genomic structure does not enable this approach and requires elimination of introns and introduction of exogenous regulatory sequences, which can impact transgene expression. The expression of hTPBG and not mTpbg in the transgenic animals was confirmed by RT-PCR with a TaqMan assay. The expression levels of mTpbg and hTPBG were determined in selected tissues harvested from wild-type C57BL/6 (WT) and hTPBG-KI animals; ovary, lung, and brain were chosen based on published data.3,22 In WT animals, mTpbg mRNA was detected but hTPBG was not, and conversely in hTPBG-KI animals, hTPBG was detected but mTpbg was not (Figure 1B). One hTPBG-KI animal was male, and thus there is no data for ovary. These results confirmed that hTPBG had replaced mTpbg and was expressed in the hTPBG-KI animals. Clinical Observations of hTPBG KI Mice. No behavioral anomalies, breeding problems, or any other deficiencies were observed for hTPBG-KI mice. To further evaluate the functionality of the hTPBG gene replacement, the hTPBG-KI animals were closely monitored for hydrocephalus since that was the primary phenotype finding reported for Tpbg knockout animals. In a previous report, approximately 13% of Tpbg knockout animals exhibited hydrocephalus and required euthanasia between the ages of 38−83 days.9 The hTPBG-KI animals in this study were generated in the same strain background (C57BL/6) as the knockout animals, which enabled a controlled comparison of the phenotype. Over the course of the project, 61 homozygous hTPBG-KI animals were monitored for over 100 days, which was older than the age range in which hydrocephalus was observed in the knockout animals. There were no signs indicative of hydrocephalus in 60 out of 61 animals; one animal showed clinical signs at 46 days. No other clinical signs were observed for the hTPBG-KI animals. Given the isolated case of hydrocephalus, it is unclear whether it was incidental or knockin-related. Taken C
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Table 1. Summary of Histological Phenotypes in hTPBG Knockin Mice
a
The incidences of unilateral hydronephrosis and of bilateral hydrometra were observed in the same animal. Box shading highlights the incidences of phenotypes.
Figure 2. Histopathological phenotypes observed in hTPBG-KI mice. (A) Brain phenotype (1 out of 33 hTPBG-KI animals): various degrees of the expansion of lateral ventricles and third ventricle. (B) Kidney phenotypes (3 out of 20 hTPBG-KI animals). (i) Transverse section of normal kidney from a wild-type mouse. (ii, iii) Transverse sections show large central dilated space (ii) and a cystic space (iii) possibly contiguous with the hilar region. There was a minimal amount of medullary tissues (ii, iii) and distorted cortical architecture (ii).
Expression of hTPBG Protein. The expression pattern of TPBG in the hTPBG-KI animals was assessed by immunohistochemistry (IHC) with a commercial monoclonal anti-TPBG antibody. The crisp staining in the hTPBG-KI animals together with the absence of staining in the littermate WT animals indicated the specificity of the antibody for human TPBG. Specificity of the antibody for human TPBG was further demonstrated by IHC and immunoblotting of human and murine samples (data not shown). Moreover, the extent of antibody staining of various human cell lines correlated with the level of TPBG expression as determined by orthogonal methods such as immunoblotting. Immunohistochemistry in tissues from hTPGB-KI mice revealed plasma membrane staining of TPBG in transitional epithelial cells of the urinary bladder (Figure 3A), mucosal epithelial cells of the uterus, granulosa cells of the ovaries, and rarely medullary cells of the adrenals. Mild staining was also observed in the kidney (Figure S2 in the Supporting Information). TPBG expression is best characterized in trophoblasts,17 but placental tissue was not evaluated in the current study.
together these results indicate that the hTPBG gene replacement was functional and able to substitute for the developmental functions of the endogenous gene. Histopathological Phenotyping of hTPBG KI Mice. A panel of 35 tissues from each of 20 age-matched WT and 20 hTPBG-KI mice were evaluated by gross examination of whole organs and by histopathology. Ten animals (5 male/5 female) from each group were sacrificed at 7−9 weeks of age, and another ten animals (5 male/5 female) from each group were sacrificed at 17−19 weeks of age. Overall, phenotypic alterations were minimal between hTPBG-KI and WT mice in both age groups. While phenotypes were observed in the brain, kidney, and uterus of hTPBG-KI mice, each finding was confined to one or two animals in the entire survey (Table 1). Moderate bilateral hydrocephalus was observed in the brain of one female (Figure 2A). Unilateral hydronephrosis was observed in one female (Figure 2B), and bilateral hydrometra in the uterus was observed in the same animal. Minimal cortical irregularities in the kidney were observed in two animals. A detailed pathology report is provided in the Supporting Information. D
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Figure 3. hTPBG expression in hTPBG-KI mice. (A) Urinary bladder. Grade 3+ TPBG immunohistochemical stain was identified in the cell membrane of the transitional epithelial cells. (B) Spinal cord. Strong TPBG expression was observed in the neuropil and occasional neurons of gray matter in the ventral horns, while no expression was observed in the white matter. (C) Hypothalamus area. TPBG expression was observed in the neuropil and occasionally the neurons in the bilateral hypothalamic nucleus. (D) Cochlear nucleus. Strong TPBG expression on the membrane and in the cytoplasm of the neurons and neuropil of the superficial glial zone and dorsal cochlear nucleus. (E) Cerebrum. Minimal to mild TPBG expression was observed in the neuropil and neurons in the cerebral cortex, caudate putamen, and lateral thalamic nucleus as well as cell membrane of the epithelial cells in the coroid plexus of the brain ventricles. (F) Retina. TPBG expression was observed in the inner/outer plexiform layers and the ganglion cell layer, including multipolar neurons and Muller cells.
Figure 4. Altered pharmacokinetics of A1mcMMAF ADC in hTPBG-KI mice. (A) Concentrations of total antibody (conjugated and unconjugated) following a single dose administration of A1mcMMAF anti-TBPG ADC in WT and hTPBG-KI mice. Data points indicate mean ± standard deviation (n = 3−4). (B) Exposure (AUC0−504) of total antibody following a single dose administration of A1mcMMAF anti-TBPG ADC in WT and hTPBG-KI mice (corresponds to the curves in panel A). * denotes significantly lower exposure in KI mouse than corresponding gender WT mouse group (p = 0.005 to p = 0.0001). (C) Traces for individual animals that were averaged for panels A and B.
Consistent with historical reports on TPBG expression in the brain and the brain-related phenotypes of TPBG-knockout mice,3,9,10,22 immunohistochemistry revealed hTPBG expression in the brain of the hTPBG-KI mice. There was minimal to mild (grade 1+ to 2+) TPBG expression in the neuropil, axon, and/or
neurocytoplasm in multiple microanatomic regions of the central nervous system (Figure 3B−E). The histological regions include inner cortex, bilateral superficial and dorsal cochlear nucleus, bilateral caudate/putamen (striatum), bilateral medial geniculate nucleus, bilateral thalamic nucleus, bilateral hypothalamic E
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level and limited animal numbers; since the difference was observed in both WT and hTPBG-KI animals and since A1mcMMAF does not bind to murine TPBG, the effect appears independent of the TPBG target. Taken together, the results demonstrate that the expression of TPBG in normal tissues has an effect on the PK of A1mcMMAF in plasma.
nucleus, substantia nigra, facial nucleus, lateral septal nucleus, raphe nucleus, commissure of the inferior colliculus, vesticulocerebellar nucleus, posterolateral amygdalohippocampal areas, and geniculate nucleus as well as cell membrane of the choroid plexus epithelial cells in the ventricles of the brain. Interestingly, many of these microanatomic regions in the central nerve system are associated with hearing. In the spinal cord, mild (grade 2+) expression of TPBG was detected in the neuropil and neurons in the ventral horn of the gray matter, but no expression was observed in the white matter. In the pituitary glands, TPBG expression was detected in the cytoplasm of the neurons in the par neurosa and negative in par distalis and intermedia. In the eye, mild to moderate TPBG expression was observed in inner/outer plexiform layers, ganglion cell layer (including multipolar neurons and muller cells), and optic fiber (Figure 3F). The positivity was mostly located in the cytoplasm, cell membrane, and neurofibers. In addition, some corneal stromal cells were cytoplasmic positive. In summary, the expression profile of hTPBG in the hTPBGKI mice was morphologically and functionally consistent with historical findings and the phenotypes of KI and knockout mice. The results further characterized and validated the hTPBG-KI mouse strain. Pharmacokinetics of A1mcMMAF in hTPBG-KI and WT Mice. Since the phenotyping and immunohistochemical analyses of the hTPBG-KI mice indicated that the gene replacement was functional, PK studies were conducted with the anti-TPBG ADC A1mcMMAF. An ADC consists of an antibody such as one directed to a tumor antigen, a potent cytotoxic drug, and a chemical linker; thus the antibody delivers the drug preferentially to the tumor and minimizes its exposure in normal tissues.23,24 Due to the complexity of the ADC structure, PK assessment typically involves multiple analytes.25 A1mcMMAF does not cross-react to murine antigen,21 and thus a comparison of its PK in WT vs hTPBG-KI mice would reveal the potential effects of target expression in normal tissues on drug exposure. A1mcMMAF was administered at a single dose of 0.3 or 3 mg/kg as a slow intravenous bolus infusion, and the quantities of total Ab (conjugated and unconjugated antibody) and ADC (conjugated antibody) were determined at several time points up to 504 h after the dose administration. No clinical observations, body weight loss, or adverse events were observed in any of the animals during the 3-week in life study. In both the WT and hTPBG-KI mice, the concentration profiles over time of both total Ab and ADC were biphasic in nature, with an initial decline within the first 6 h followed by a prolonged elimination phase. In addition, the concentration profile of the ADC indicated lower overall systemic exposure (AUC0−504) than that of the total Ab. Both of these observations are consistent with the previously characterized PK profile of A1mcMMAF.21,26 The exposures (AUC0−504) of both total Ab and ADC were notably reduced in the hTPBG-KI animals relative to WT animals, at both dose levels. Total Ab exposures in hTPBG-KI mice were 31% and 48% of those in WT mice at the 0.3 mg/kg and 3 mg/kg dose levels respectively (Figure 4). Similarly, ADC exposure in the hTPBG-KI mice was 38% and 58% of that in WT mice at the 0.3 mg/kg and 3 mg/kg dose levels respectively (Figure S3 in the Supporting Information). Total exposures for both analytes were proportional to dose level in both WT and hTPBG-KI animals, especially in males. Interestingly, the data suggested a trend toward lower exposures in females, though this difference was observed only at the lower (more sensitive) dose
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DISCUSSION The use of predictive preclinical models in drug discovery is critical for increasing the clinical success rate of therapeutics. This study has described the generation and characterization of hTPBG-KI mice that could be used in pharmacology studies to yield improved PK/PD-based predictions for current and future TPBG-targeted therapies. The genetic strategy was designed to generate the most physiologically relevant gene replacement possible by replacing only the coding sequence and retaining the endogenous 5′ and 3′ regulatory sequences. In other cases of gene replacement, exogenous promoters and/or polyadenylation sequences are used either for convenience or due to complex genomic structure; such exogenous sequences can significantly impact expression of the gene product. The phenotypic and immunohistochemical analyses of the hTPBG-KI mice indicated that the gene replacement was functional. However, we cannot rule out that the single incidence of hydrocephalus and the other rare histopathologic phenotypes were related to a nonequivalence of the novel hTPBG allele. Notably, the incidence of hydrocephalus was substantially lower than in the knockout mice in the same strain background.9 Expression of TPBG in the hTPBG-KI mice was observed primarily in the central nervous system and in epithelial cells in various tissues. These results are consistent with historical findings and phenotypes of the KI and knockout mice. The observation of TPBG expression in many microanatomic regions involved in hearing may suggest previously unknown functions of TPBG. Interestingly, TPBG expression and function in the olfactory bulb were observed in neonatal mice at 3 weeks of age;10 we did not observe hTPBG expression in the hTPBG-KI mice in the younger cohort (7−9 weeks) or the older cohort (17−19 weeks), which suggests that the function of TPBG in the olfactory bulb is most prominent in, or even restricted to, neonatal development. The expression of TPBG in normal human tissues is not well characterized in the literature, but some similarities were observed between the published report17 and the hTPBG-KI mice in this study. In particular, expression was reported in the cervix and endometrium, which is consistent with our observation of hTPBG in the uterus. Expression was noted in the esophagus,17 whereas we did not observe it; faint or equivocal staining was also noted in other tissues. No expression was detected in the brain (notably, only one sample evaluated);17 interestingly, the expression in brain that we observed in hTPBGKI mice was consistent with several reported studies in mice.3,9,10,22 Due to the different preservation of samples (frozen versus formalin fixation), different antibodies (epitopes) and staining methods, and the limited number of samples evaluated for some tissues,17 additional studies are required to determine whether dissimilarities in staining are attributable to actual differences in expression between humans and hTPBG-KI mice. The observation of altered PK of a TPBG-targeted agent in hTPBG-KI mice demonstrates the utility of preclinical pharmacology studies in the hTPBG-KI mice. The data suggest a component of target-mediated drug disposition that affects the F
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exposure of A1-mcMMAF and potentially other TPBG-targeted compounds. Additional PK studies in hTPBG-KI mice would enable more precise estimations of dosing and pharmacodynamics of TPBG-targeted therapies. Toxicology studies with compounds such as A1mcMMAF could be conducted and the results compared to those obtained in other nonclinical species; a special consideration in the case of A1mcMMAF is that mice exhibit a greater degree of tolerance of some microtubule inhibitors including auristatins than do rats and monkeys.27,28 Future studies could also include breeding the TPBG-KI animals into an immune-compromised background to enable efficacy studies with human tumor xenografts. Genetically humanized mice offer advantages in many aspects of drug development as well as basic research.29 Preclinical studies inform critical decisions of whether to advance a compound into the clinic and, if so, how to develop the compound. The generation of target-specific knockin mice early in the drug discovery process would enable more precise pharmacological evaluation of compounds especially when one or more of the lead candidates does not engage the mouse target. The gene replacement strategy employed in this study enabled the expression of the human gene under the mouse endogenous regulatory sequences and thus provided the most precise model possible for such studies. Overall this strategy could improve the PK/PD-based predictions of human efficacious dose and be used to inform preclinical decisions as well as clinical development strategies.
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ASSOCIATED CONTENT
* Supporting Information S
Description of the pathology of the hTPBG-KI animals and accompanying figures. This material is available free of charge via the Internet at http://pubs.acs.org.
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Article
AUTHOR INFORMATION
Corresponding Author
*Pfizer Worldwide Research & Development, 401 N. Middletown Road, Building 200-4611, Pearl River, NY 10965. Tel: 845602-7985. E-mail: marc.damelin@pfizer.com. Present Addresses ¶
(B.B.) Pfizer Inc., Centers for Therapeutic Innovation, 3 Blackfan Circle, Boston, MA, 02115. □ (Q.L.) Shenogen Pharma Group Ltd., Changping, Beijing PRC 102206. Author Contributions ‡
G.H. and M.L. made equal contributions.
Notes
The authors declare the following competing financial interest(s): Drs. Hu, Leal, Affolter, Sapra, Bates, and Damelin are current employees and shareholders of Pfizer Inc.
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ACKNOWLEDGMENTS The authors gratefully acknowledge contributions from Jonathon Golas, Theresa Paradis, Musen Liang, Joann Wentland, Timothy Coskran, the Investigational Pathology Laboratory, Johnny Yao, the ORU In Vivo Pharmacology Group, Jen Sadlier, Mary Bauchmann, and Pfizer Worldwide Comparative Medicine, as well as Adam Cole at Charles River Laboratories. The authors acknowledge Seattle Genetics Inc. and Oxford BioMedica for access to technology regarding A1mcMMAF. G
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