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Effects of Systemic Administration of Free Payload Compared with Conventional HER2-vc0101 on Indicators of Bone Marrow Toxicity and Bone Marrow Payload Levels in Rats

ADC retention times by hydrophobic interaction chromatography (HIC).

Conjugation of vc0101 does not alter anti-HER2 mAb binding.

Effects of a Conventional and a Site-Specific Conjugate Targeting HER2 on Indicators of Bone Marrow Toxicity and Serum ADC/Payload Exposures in Rats

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody–drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug–antibody ratio (DAR) ADC with this linker–payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site–specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo–acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.

Immunohistochemical evaluation of HER2 expression in vivo cell line and patient-derived xenograft models.

In vivo efficacy of site-specific anti-HER2 vc0101 4 DAR ADCs in N87 xenograft tumor model.

In vitro cytotoxicity of PF-06804103 across a panel of cancer cell lines.

Includes Supplementary Materials and Methods, Data and Figure Legends

Supplementary Figures 1-4. Supplementary Figure 1. Analysis of EFNA4 copy number in breast and hepatocellular carcinoma. Supplementary Figure 2. Characterization of PF-06647263 conjugates. Supplementary Figure 3. Biomarkers of PF-06647263 activity in breast PDX tumors. Supplementary Figure 4. Characterization of EFNA4 affinity for EphA and EphB receptors.

Purpose: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival.Experimental Design: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody–drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification.Results: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification.Conclusions: These findings demonstrate the potential of PF-06647263 (anti–EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development. Clin Cancer Res; 21(18); 4165–73. ©2015 AACR.

Table S1. Mean ({plus minus}SD) Toxicokinetic Parameters for Ipilimumab and Novolumab in Cynomolgus Monkeys Table S2 - Differentially expressed genes in the heart of ipilimumab and nivolumab treated cynos (adjusted P-value {less than or equal to} 0.05) Table S3 - Top 50 enriched pathways identified by IPA when using the data set of 882 analysis ready differentially expressed genes (adjusted P-value {less than or equal to} 0.05). Ratio: number of DE genes in a pathway divided by the number of genes comprised in the same pathway. Table S4: Top 25 networks identified based upon differentially expressed genes (adjusted P-value {less than or equal to} 0.05) in ipilimumab and nivolumab treated cynos. Table S5: Regulator effects networks generated using genes that are differentially expressed (adjusted P-value {less than or equal to} 0.05) in ipilimumab and nivolumab treated cynos. Upstream regulator and diseae& function cutoffs were each log P-value {less than or equal to} 10 and a z-score {greater than or equal to} |3| Table S6 - Differential expression of myocarditis- associated genes in the heart of ipilimumab and nivolumab treated cynos

Supplemental Figure S1. Concentration versus time profiles of ipilimumab and nivolumab following four weekly intravenous dose administrations of 15 mg/kg or 20 mg/kg, respectively. Supplementary Figure S2. Hematoxylin and eosin stained sections of tissues from control and monkeys dosed with ipilimumab and nivolumab. Supplementary Figure S3. Quantification of mononuclear cell infiltrates from IHC images of heart sections.

Purpose:Immune checkpoint inhibitors (ICI) targeting PD1, PDL1, or CTLA4 are associated with immune-related adverse events (irAE) in multiple organ systems including myocarditis. The pathogenesis and early diagnostic markers for ICI-induced myocarditis are poorly understood, and there is currently a lack of laboratory animal model to enhance our understanding. We aimed to develop such a model using cynomolgus monkeys.Experimental Design:Chinese-origin cynomolgus monkeys were dosed intravenously with vehicle or nivolumab 20 mg/kg plus ipilimumab 15 mg/kg once weekly and euthanized on day 29.Results:Multiple organ toxicities were observed in cynomolgus monkeys, and were characterized by loose feces, lymphadenopathy, and mononuclear cell infiltrations of varying severity in heart, colon, kidneys, liver, salivary glands, and endocrine organs. Increased proliferation of CD4+ and CD8+ T lymphocytes as well as an increase in activated T cells and central memory T cells in the blood, spleen, and lymph nodes, were observed. Transcriptomic analysis suggested increased migration and activation of T cells and increased phagocytosis and antigen presentation in the heart. Mononuclear cell infiltration in myocardium was comprised primarily of T cells, with lower numbers of macrophages and occasional B cells, and was associated with minimal cardiomyocyte degeneration as well as increases in cardiac troponin-I and NT-pro-BNP. Morphologically, cardiac lesions in our monkey model are similar to the reported ICI myocarditis in humans.Conclusions:We have developed a monkey model characterized by multiple organ toxicities including myocarditis. This model may provide insight into the immune mechanisms and facilitate biomarker identification for ICI-associated irAEs.

Purpose: Immune checkpoint inhibitors (ICI) targeting PD1, PDL1, or CTLA4 are associated with immune-related adverse events (irAE) in multiple organ systems including myocarditis. The pathogenesis and early diagnostic markers for ICI-induced myocarditis are poorly understood, and there is currently a lack of laboratory animal model to enhance our understanding. We aimed to develop such a model using cynomolgus monkeys. Experimental Design: Chinese-origin cynomolgus monkeys were dosed intravenously with vehicle or nivolumab 20 mg/kg plus ipilimumab 15 mg/kg once weekly and euthanized on day 29. Results: Multiple organ toxicities were observed in cynomolgus monkeys, and were characterized by loose feces, lymphadenopathy, and mononuclear cell infiltrations of varying severity in heart, colon, kidneys, liver, salivary glands, and endocrine organs. Increased proliferation of CD4+ and CD8+ T lymphocytes as well as an increase in activated T cells and central memory T cells in the blood, spleen, and lymph nodes, were observed. Transcriptomic analysis suggested increased migration and activation of T cells and increased phagocytosis and antigen presentation in the heart. Mononuclear cell infiltration in myocardium was comprised primarily of T cells, with lower numbers of macrophages and occasional B cells, and was associated with minimal cardiomyocyte degeneration as well as increases in cardiac troponin-I and NT-pro-BNP. Morphologically, cardiac lesions in our monkey model are similar to the reported ICI myocarditis in humans. Conclusions: We have developed a monkey model characterized by multiple organ toxicities including myocarditis. This model may provide insight into the immune mechanisms and facilitate biomarker identification for ICI-associated irAEs.

Antibody-drug conjugates (ADCs) represent a rapidly evolving area of drug development and hold significant promise. To date, nine ADCs have been approved by the US Food and Drug Administration (FDA). These conjugates combine the target specificity of monoclonal antibodies with the anticancer activity of small-molecule therapeutics (also referred to as payload). Due to the complex structure, three analytes, namely ADC conjugate, total antibody, and unconjugated payload, are typically quantified during drug development; however, the benefits of measuring all three analytes at later stages of clinical development are not clear. The cytotoxic payloads, upon release from the ADC, are considered to behave like small molecules. Given the relatively high potency and low systemic exposure of cytotoxic payloads, drug-drug interaction (DDI) considerations for ADCs might be different from traditional small molecule therapeutics. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) convened an ADC working group to create an IQ ADC database that includes 26 ADCs with six unique payloads. The analysis of the ADC data in the IQ database, as well as nine approved ADCs, supports the strategy of pharmacokinetic characterization of all three analytes in early-phase development and progressively minimizing the number of analytes to be measured in the late-phase studies. The systemic concentrations of unconjugated payload are usually too low to serve as a DDI perpetrator; however, the potential for unconjugated payloads as a victim still exists. A data-driven and risk-based decision tree was developed to guide the assessment of a circulating payload as a victim of DDI.

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody–drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug–antibody ratio (DAR) ADC with this linker–payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site–specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo–acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.

A modeling and simulation approach was used for quantitative comparison of a new generation HER2 antibody drug conjugate (ADC, PF-06804103) with trastuzumab-DM1 (T-DM1). To compare preclinical efficacy, the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of PF-06804103 and T-DM1 was determined across a range of mouse tumor xenograft models, using a tumor growth inhibition model. The tumor static concentration was assigned as the minimal efficacious concentration. PF-06804103 was concluded to be more potent than T-DM1 across cell lines studied. TSCs ranged from 1.0 to 9.8 µg/mL (n = 7) for PF-06804103 and from 4.7 to 29 µg/mL (n = 5) for T-DM1. Two experimental models which were resistant to T-DM1, responded to PF-06804103 treatment. A mechanism-based target mediated drug disposition (TMDD) model was used to predict the human PK of PF-06804103. This model was constructed and validated based on T-DM1 which has non-linear PK at doses administered in the clinic, driven by binding to shed HER2. Non-linear PK is predicted for PF-06804103 in the clinic and is dependent upon circulating HER2 extracellular domain (ECD) concentrations. The models were translated to human and suggested greater efficacy for PF-06804103 compared to T-DM1. In conclusion, a fit-for-purpose translational PK/PD strategy for ADCs is presented and used to compare a new generation HER2 ADC with T-DM1. Electronic supplementary material The online version of this article (10.1007/s10928-020-09702-3) contains supplementary material, which is available to authorized users.

CD3 bispecific antibody constructs recruit cytolytic T cells to kill tumor cells, offering a potent approach to treat cancer. T cell activation is driven by the formation of a trimolecular complex (trimer) between drugs, T cells, and tumor cells, mimicking an immune synapse. A translational quantitative systems pharmacology (QSP) model is proposed for CD3 bispecific molecules capable of predicting trimer concentration and linking it to tumor cell killing. The model was used to quantify the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a CD3 bispecific targeting P-cadherin (PF-06671008). It describes the disposition of PF-06671008 in the central compartment and tumor in mouse xenograft models, including binding to target and T cells in the tumor to form the trimer. The model incorporates T cell distribution to the tumor, proliferation, and contraction. PK/PD parameters were estimated for PF-06671008 and a tumor stasis concentration (TSC) was calculated as an estimate of minimum efficacious trimer concentration. TSC values ranged from 0.0092 to 0.064 pM across mouse tumor models. The model was translated to the clinic and used to predict the disposition of PF-06671008 in patients, including the impact of binding to soluble P-cadherin. The predicted terminal half-life of PF-06671008 in the clinic was approximately 1 day, and P-cadherin expression and number of T cells in the tumor were shown to be sensitive parameters impacting clinical efficacy. A translational QSP model is presented for CD3 bispecific molecules, which integrates in silico, in vitro and in vivo data in a mechanistic framework, to quantify and predict efficacy across species. Electronic supplementary material The online version of this article (10.1208/s12248-019-0332-z) contains supplementary material, which is available to authorized users.

There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a "linker-less" conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs.

Purpose: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. Experimental Design: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody–drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. Results: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. Conclusions: These findings demonstrate the potential of PF-06647263 (anti–EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development. Clin Cancer Res; 21(18); 4165–73. ©2015 AACR.

As the antibody drug conjugate (ADC) community continues to shift towards site-specific conjugation technology, there is a growing need to understand how the site of conjugation impacts the biophysical and biological properties of an ADC. In order to address this need, we prepared a carefully selected series of engineered cysteine ADCs and proceeded to systematically evaluate their potency, stability, and PK exposure. The site of conjugation did not have a significant influence on the thermal stability and in vitro cytotoxicity of the ADCs. However, we demonstrate that the rate of cathepsin-mediated linker cleavage is heavily dependent upon site and is closely correlated with ADC hydrophobicity, thus confirming other recent reports of this phenomenon. Interestingly, conjugates with high rates of cathepsin-mediated linker cleavage did not exhibit decreased plasma stability. In fact, the major source of plasma instability was shown to be retro-Michael mediated deconjugation. This process is known to be impeded by succinimide hydrolysis, and thus, we undertook a series of mutational experiments demonstrating that basic residues located nearby the site of conjugation can be a significant driver of succinimide ring opening. Finally, we show that total antibody PK exposure in rat was loosely correlated with ADC hydrophobicity. It is our hope that these observations will help the ADC community to build “design rules” that will enable more efficient prosecution of next-generation ADC discovery programs.

The objective of this manuscript was to establish in vitro–in vivo correlation (IVIVC) between the in vitro efficacy and in vivo efficacy of antibody drug conjugates (ADCs), using a PK/PD modeling approach. Nineteen different ADCs were used to develop IVIVC. In vitro efficacy of ADCs was evaluated using a kinetic cell cytotoxicity assay. The cytotoxicity data obtained from in vitro studies was characterized using a novel mathematical model, parameter estimates from which were used to derive an in vitro efficacy matrix for each ADC, termed as ‘in vitro tumor static concentration’ (TSCin vitro). TSCin vitro is a theoretical concentration at continuous exposure of which the number of cells will neither increase nor decrease, compared to the initial cell number in the experiment. The in vivo efficacy of ADCs was evaluated using tumor growth inhibition (TGI) studies performed on human tumor xenograft bearing mice. The TGI data obtained from in vivo studies was characterized using a PK/PD model, parameter estimates from which were used to derive an in vivo efficacy matrix for each ADC, termed as ‘in vivo tumor static concentration’ (TSCin vivo). TSCin vivo is a theoretical concentration if one were to maintain in the plasma of a tumor bearing mouse, the tumor volume will neither increase nor decrease compared to the initial tumor volume. Comparison of the TSCin vitro and TSCin vivo values from 19 ADCs provided a linear and positive IVIVC. The Spearman’s rank correlation coefficient for TSCin vitro and TSCin vivo was found to be 0.82. On average TSCin vivo was found to be ~ 27 times higher than TSCin vitro. The reasonable IVIVC for ADCs suggests that in vitro efficacy data was correctly able to differentiate ADCs for their in vivo efficacy. Thus, IVIVC can be used as a tool to triage ADC molecules in the discovery stage, thereby preventing unnecessary scaling-up of ADCs and waste of time and resources. An ability to predict the concentration of ADC that is efficacious in vivo using the in vitro data can also help in optimizing the experimental design of preclinical efficacy studies. As such, the novel PK/PD modeling method presented here to establish IVIVC for ADCs holds promise, and should be evaluated further using diverse set of cell lines and anticancer agents.

As antibody-drug conjugate (ADC) design is evolving with novel payload, linker, and conjugation chemistry, the need for sensitive and precise quantitative measurement of conjugated payload to support pharmacokinetics (PK) is in high demand. Compared to ADCs containing noncleavable linkers, a strategy specific to linkers which are liable to pH, chemical reduction, or enzymatic cleavage has gained popularity in recent years. One bioanalytical approach to take advantage of this type of linker design is the development of a PK assay measuring released conjugated payload. For the ADC utilizing a dipeptide ValCit linker studied in this report, the release of payload PF-06380101 was achieved with high efficiency using a purified cathepsin B enzyme. The subsequent liquid chromatography mass spectrometry (LC/MS) quantitation leads to the PK profile of the conjugated payload. For this particular linker using a maleimide-based conjugation chemistry, one potential route of payload loss would result in an albumin adduct of the linker-payload. While this adduct's formation has been previously reported, here, for the first time, we have shown that payload from a source other than ADC contributes only up to 4% of total conjugated payload while it accounts for approximately 35% of payload lost from the ADC at 48 h after dosing to rats.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

Aim: Complex nature of bioconjugates require multiple bioanalytical approaches to support PK and absorption, distribution, metabolism and excretion characterization. For antibody-drug conjugate (ADC) bioanalysis both LC–MS and ligand-binding assays (LBAs) are employed. Results: A method consisting of immunocapture extraction of ADC from biomatrices followed by LC–MS analysis of light and heavy chain is described. Drug antibody ratio (DAR) profiles of ADC Tras-mcVC-PF06380101 dosed at 0.3, 1 and 3 mg/kg in Sprague Dawley rats were obtained. Combined with total antibody (monoclonal antibody) measurement by LBA, conjugated payload concentration was calculated. Conclusion: PK profiles from LBA, ADC and calculated conjugated payload (DAR × monoclonal antibody) were in good agreement. We present a new tool for PK assessment of ADCs while also exploring ADC metabolism and DAR sensitivity of LBA ADC assay.

Aim: The application of high-resolution MS to antibody–drug conjugate (ADC) drug development may provide insight into their safety and efficacy. Quantification of unconjugated cytotoxic drug (payload) and characterization of drug-to-antibody ratio distribution were determined in plasma using orthogonal acceleration quadrupole-time-of-flight MS. Results: Unconjugated payload quantification determined by quadrupole-time-of-flight-based MRMhighresolution and triple quadrupole-based multiple reaction monitoring were comparable and achieved detection limits of 0.030 and 0.015 ng/ml, respectively. As determined by immunocapture and TOF-MS, drug-to-antibody ratio remained unchanged up to 3-weeks postdose for an ADC containing engineered glutamine linkers, but declined from four to three over 2 weeks in an ADC containing engineered cysteine linkers. Conclusion: The use of high-resolution MS in ADC drug discovery confirms its utility within the bioanalytical discipline.

The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.

A bispecific immunomodulatory biotherapeutic molecule (P-cadherin LP-DART) based on the Dual Affinity Re-Targeting (DART) scaffold has been developed as a potential antitumor treatment showing efficacy in preclinical testing. A minimal anticipated biological effect level (MABEL) approach was applied to project the first-in-human (FIH) dose, because of its immune agonistic properties following target engagement. The pharmacological activity of P-cadherin LP-DART is driven by binding to both P-cadherin on the tumor cells and CD3 on T cells. Therefore, the concentration of the tri-molecular synapse formed between drug, T cell, and tumor cell, rather than drug concentration, is responsible for efficacy. A mechanistic pharmacokinetic/pharmacodynamic (PK/PD)-driven approach was explored to understand the exposure-response relationship based on the synapse concentration to project the MABEL dose. Orthogonal approaches including PK-driven and receptor occupancy calculations were also investigated. This study showcases the application of PK/PD modeling in immune-oncology, and could potentially be implemented for other bispecific biotherapeutics.

The current study sought to define the molecular mechanisms involved in the cross talk between 1,25-dihydroxyvitamin D3[1,25(OH)2D3] and activators of PKC in the regulation of 25(OH)D324-hydroxlyase [24(OH)ase]. Transfection of the h24(OH)ase promoter construct [-5,500/-22 luciferase; vitamin D response elements at -294/-274 and -174/-151; AP-1 site at -1,167/-1,160] in vitamin D receptor (VDR)-transfected COS-7 cells resulted in strong activation by 1,25(OH)2D3. In these cells, cotreatment with the PKC activator TPA and 1,25(OH)2D3yielded a 27-fold increase in luciferase activity, which was 2- to 3-fold greater than activation obtained with 1,25(OH)2D3alone ( P < 0.05). Similar results were observed using LLCPK-1 kidney cells, suggesting that the previously observed enhancement of 1,25(OH)2D3-induced renal 24(OH)ase mRNA and activity by PKC activation occurs at the level of transcription. The functional cooperation between PKC activation and VDR was not found to be mediated by the AP-1 site in the h24(OH)ase promoter or by enhanced binding of GRIP or DRIP205 to VDR and was also not due to PKC-mediated phosphorylation of VDR on Ser51. Our study demonstrates that, in LLCPK-1 kidney cells, the PKC enhancement of 1,25(OH)2D3-stimulated transcription may be due, in part, to an increase in VDR concentration. In addition, inhibitors of the MAPK pathway were found to decrease the TPA enhancement ( P < 0.05). Because activation of MAPK has been reported to result in the phosphorylation of SRC-1 and in functional cooperation between SRC-1 and CREB binding protein, we propose that the potentiation of VDR-mediated transcription may also be mediated through changes in the phosphorylation of specific VDR coregulators.

Recent findings have indicated that calbindin-D(28k), the first known target of vitamin D action, is present in osteoblasts and protects against TNF and glucocorticoid induced apoptosis of osteoblastic cells. Cytokine mediated destruction of pancreatic beta cells, a cause of insulin dependent diabetes, is also inhibited by calbindin-D(28k). In calbindin-D(28k) transfected pancreatic beta cells free radical formation by cytokines is inhibited by calbindin. Thus, besides its role as a facilitator of calcium diffusion, calbindin has a major role in protecting against cellular degeneration in different cell types. Besides calbindin, the other known pronounced effect of 1,25(OH)(2)D(3) in intestine and kidney is increased synthesis of 25(OH)D(3) 24-hydroxylase (24(OH)ase) which is involved in the catabolism of 1,25(OH)(2)D(3). We have noted that CCAAT enhancer binding protein beta (C/EBPbeta) is induced by 1,25(OH)(2)D(3) in kidney and osteoblastic cells and can enhance the transcriptional response of 24(OH)ase to 1,25(OH)(2)D(3). These studies establish C/EBPbeta as a novel 1,25(OH)(2)D(3) target gene and indicate a role for C/EBPbeta in 24(OH)ase transcription. These studies extend our previous studies related to factors that affect vitamin D receptor (VDR) mediated 24(OH)ase transcription (YY1, TFIIB, CBP) and the effect of signaling pathways on 24(OH)ase transcription and cofactor recruitment.

When UMR-106 osteoblastic cells, LLCPK1 kidney cells, and VDR transfected COS-7 cells were transfected with the rat 24-hydroxylase [24(OH)ase] promoter (1,367/74) or the mouse osteopontin (OPN) promoter (777/79), we found that the response to 1,25dihydroxyvitamin D3 [1,25-(OH)2D3] could be significantly enhanced 2- to 5-fold by the protein phosphatase inhibitor, okadaic acid (OA). Enhancement of 1,25-(OH)2D3-induced transcription by OA was also observed using a synthetic reporter gene containing either the proximal 24(OH)ase vitamin D response element (VDRE) or the OPN VDRE, suggesting that the VDRE is sufficient to mediate this effect. OA also enhanced the 1,25-(OH)2D3-induced levels of 24(OH)ase and OPN mRNA in UMR osteoblastic cells. The effect of OA was not due to an up-regulation of VDR or to an increase in VDR-RXR interaction with the VDRE. To determine whether phosphorylation regulates VDR-mediated transcription by modulating interactions with protein partners, we examined the effect of phosphorylation on the protein-protein interaction between VDR and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex, using glutathione-S-transferase pull-down assays. Similar to the functional studies, OA treatment was consistently found to enhance the interaction of VDR with DRIP205 3- to 4-fold above the interaction observed in the presence of 1,25-(OH)2D3 alone. In addition, studies were done with the activation function-2 defective VDR mutant, L417S, which is unable to stimulate transcription in response to 1,25-(OH)2D3 or to interact with DRIP205. However, in the presence of OA, the mutant VDR was able to activate 24(OH)ase and OPN transcription and to recruit DRIP205, suggesting that OA treatment may result in a conformational change in the activation function-2 defective mutant that creates an active interaction surface with DRIP205. Taken together, these findings suggest that increased interaction between VDR and coactivators such as DRIP205 may be a major mechanism that couples extracellular signals to vitamin D action. (Molecular Endocrinology 16: 301–314, 2002)

The stability of the connection between the antibody and the toxin can have a profound impact on ADC safety and efficacy. There has been increasing evidence in recent years that maleimide-based ADCs are prone to payload loss via a retro-Michael type reaction. Herein, we report a mild method for the hydrolysis of the succinimide-thioether ring which results in a “ring-opened” linker. ADCs containing this hydrolyzed succinimide linker show equivalent cytotoxicity, improved in vitro stability, improved PK exposure, and improved efficacy as compared to their nonhydrolyzed counterparts. This method offers a simple way to improve the stability, exposure, and efficacy of maleimide-based ADCs.

Author for correspondence: Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, Eastern Point Rd, Groton, CT 06340, USA Tel.: +1 860 715 0641 Fax: +1 860 715 9501 E-mail: tracey.clark@pfizer.com Xiaogang Han Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, CT, USA Lindsay King Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, CT, USA Frank Barletta Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer, NY, USA Antibody–drug conjugate (ADC) therapeutics utilize the specificity of monoclonal antibodies (mAbs) and potency of highly toxic small molecules. ADCs are typically composed of a mAb with a cytotoxin conjugated to it, resulting in a heterogeneous mixture of mAb with various numbers of toxins. Due to this heterogeneity, characterized by the therapeutic drug-to-antibody ratio (DAR), the selection of bioanalytical tools, biomeasure assays and analytes used to understand and develop ADCs can be challenging [1]. Since the therapeutic has both largeand small-molecule components, one can use bioanalytical tools in both spaces. Questions for ADC programs are typically: what should be measured, when, and by which method? Since all assays have limitations [1], a combination of bioanalytical tools is typically used to understand the ADC in vitro/in vivo, understand payload delivery to the site of action and to establish an exposure–response relationship.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor regrowth and metastasis. Here we identify Protein Tyrosine Kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase, as an antigen that is enriched on TICs in low-passage patient-derived xenografts (PDX) of TNBC, NSCLC and other tumor types. An anti-PTK7 antibody-drug conjugate (ADC) was generated from a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker and the Aur0101 auristatin microtubule inhibitor. The anti-PTK7 ADC induced sustained regressions of TNBC, NSCLC and ovarian cancer PDX, with improved activity over standard-of-care chemotherapy, and reduced the frequency of TICs as determined by serial transplantation experiments. Moreover, the ADC may have additional mechanisms of action, including an anti-angiogenic effect, that promote anti-tumor immune responses. Together these preclinical results indicate the potential of the anti-PTK7 ADC to improve the long-term survival of cancer patients. The ADC is currently being tested in a Phase 1 clinical trial, from which interim results will be presented. Citation Format: Marc Isaac Damelin, Alex Bankovich, Jeff Bernstein, Justin Lucas, Liang Chen, Sam Williams, Albert Park, Jorge Aguilar, Elana Ernstoff, Manoj Charati, Russell Dushin, Amy Jackson-Fisher, Monette Aujay, Christina Lee, Hanna Ramoth, Milly Milton, Johannes Hampl, Sasha Lazetic, Virginia Pulito, Douglas Armellino, Edward Rosfjord, Magali Guffroy, Hadi Falahatpisheh, Lindsay King, Frank Barletta, Robert Stull, Marybeth Pysz, Paul Escarpe, David Liu, Orit Foord, Brenda Gibson, Eric Powell, Christopher O’Donnell, Xiaohua Xin, Hans Peter Gerber, Puja Sapra, Scott Dylla. A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1220.

Antibody-drug conjugates (ADCs) have emerged as an important class of cancer therapeutics. The FDA approval of Kadcyla (T-DM1), a single agent for treatment of HER2-positive advanced metastatic breast cancer, was a significant milestone in the field of targeted therapy, as the first and only ADC for treatment of solid tumors. Despite the 3-month improvement over standard of care in the median survival, almost all the patients eventually became refractory to T-DM1. We have identified several possible areas for improvement: 1) The potency of T-DM1 as confirmed by the Phase III clinical data is restricted to high HER2 tumors which leaves moderate or low HER2 expressing patients without access to T-DM1 treatment; 2) The 48% overall response rate is indicative of intrinsic resistance to T-DM1 and all T-DM1 treated patients eventually relapse. 3) The randomized lysine conjugation in T-DM1 generates heterogeneity of the product. We have developed a novel, site-specific anti-HER2 ADC (NG-HER2 ADC) and evaluated it in comparative preclinical studies with T-DM1. The results show that the NG-HER2 ADC is ∼ 10 fold more potent than T-DM1 in HER2 3+ xenograft models of breast and gastric cancers. Our proprietary cleavable and permeable linker-payload can mediate bystander effect and this enables potent anti-tumor activity in non-HER2 amplified breast cancer and heterogeneous low HER2 NSCLC PDX models, where T-DM1 is ineffective. Our ADC can overcome T-DM1 resistance in in vitro and in vivo models.. Our site-specific ADC at HNSTD of 9 mg/kg in cynomolgus monkeys showed high AUC, long half-life and had normal clinical observations with no marked neutropenia. On the contrary, conventional conjugates with cleavable linker payloads typically have severe bone marrow toxicity as DLT above 5 mg/kg. The therapeutic index for NG-HER2 ADC is significantly greater than T-DM1 in all models tested. NG-HER2 ADC has a projected clinical efficacious dose of ∼1 mg/kg, compared to 3-5 mg/kg for T-DM1, based on PK/PD modeling. In addition, the activity of the NG-HER2 ADC shows increased infiltration of CD8 positive effector cells, an essential component for immuno-oncology (IO) efficacy, in a syngeneic HER2 overexpressing model. This property potentially allows the combination of the ADC with IO drugs to improve the long-term, overall survival. Our data provides preclinical proof of concept for NG-HER2 ADC with best-in-class potential and is currently being tested in preparation for clinical trials for treatment of HER2 solid tumors. Citation Format: Dangshe Ma, Bitha Narayanan, Kim Marquette, Edmund Graziani, Frank Loganzo, Manoj Charati, Nadira Prashad, Nathan Tumey, Jon Golas, Christine Hosselet, George Hu, Frank Barletta, Alison Betts, Judy Lucas, Chris O’Donnell, Lioudmila Tchistiakova, Hans-Peter Gerber, Puja Sapra. Creating a superior, site-specific anti-HER2 antibody-drug conjugate (NG-HER2 ADC) for treatment of solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 868.

Site-specific conjugation of toxic payload to antibody yields homogeneous ADCs resulting in improved therapeutic index over conventional conjugation. To understand the role of conjugation site on pharmacokinetics, off-target toxicity, cysteine(cys) was engineered into a humanized anti-IL13Rα2 antibody, hAB08, at various sites within the IgG1/k constant domains. Single cys mutants L443C and Q347C with DAR 2, and double cys mutants K392C+L443C, L443C/kK183C and Q347C/kK183C with DAR 4 were conjugated using auristatin 0101 via maleimidocapronic - valine-citruline-p-aminobenzyloxycarbonyl (vc, cleavable linker). All cys mutants and their conjugates maintained their binding properties compared to parental hAb08. Cysteine substitution sites were selected based on conjugation efficiency and in vitro stability. In vivo efficacy was evaluated in mouse xenograph tumor models. Pharmacokinetics (PK) and off target toxicity (TOX) were assessed in both mouse and rat in vivo studies. Our data demonstrate that that all site specific conjugates show improved in vitro stability, comparable or better in vivo efficacy in mice xenograph models and in vivo stability (ratio of ADC-AUC/Ab-AUC) compared to conventional conjugates. However PK and off-target toxicity of antibody drug conjugates had been significantly impacted by conjugation site and animal species. L443 mutants show higher ADC-AUC, lower clearance and longer half life than Q347 mutants in mice while a reverse TK behavior was observed in rat. All site specific conjugates show higher dose tolerance and less off-target toxicity relative to conventional conjugates in rat. This off-target toxicity improvement depends on sites of conjugation. The L443 mutants exhibited a better off-target TOX profile relative to Q347 mutants. Taken together, site-specific conjugation improved in vivo efficacy and off-target toxicity relative to conventionally conjugated ADC. PK differences and off-target toxicity improvements depend on conjugation sites and animal species. Note: This abstract was not presented at the meeting. Citation Format: Dangshe Ma, Fang Jin, Frank Barletta, George Hu, Nathan Tumey, Haige Zhang, Tao He, Eric Sousa, Manoj Charti, Kiran Khadke, Judy Lucas, Darren Ferguson, Christoper Brown, Weijun Ma, Scott Gatto, William Brady, Edmund Graziani, Hans-Peter Gerber, Puja Sapra, Lioudmila Tchistikova. Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 644. doi:10.1158/1538-7445.AM2015-644

Triple-negative breast cancer (TNBC) and ovarian cancer comprise heterogeneous tumors, and neither targeted therapies nor traditional chemotherapies have provided consistent clinical benefit. Novel therapies that target and actively eradicate the subpopulation of tumor cells that mediate drug resistance and tumor relapse could significantly improve patient survival. Tumor-initiating cells (TIC) are functionally defined as the subpopulation of cells that drive long-term tumor growth, resistance to therapy and disease relapse. We herein identified CD324 as a surface antigen able to reproducibly enrich for TIC in well annotated, low passage TNBC and ovarian cancer patient-derived xenografts (PDXs). Gene expression analysis of TIC led to the identification of Ephrin-A4 as a prospective therapeutic TIC target. Humanized Ephrin-A4-specific monoclonal antibodies (mAbs) were generated and demonstrated to internalize to mediate the delivery of potent cytotoxins. An antibody-drug conjugate (ADC) comprising a humanized anti-Ephrin-A4 mAb conjugated to the DNA damaging agent calicheamicin achieved sustained tumor regressions in vivo in both TNBC and ovarian cancer PDX. Anti-Ephrin-A4-ADC (PF-06647263) actively reduced TIC frequency as evidenced by limiting dilution analysis in serial transplantation assays. Unexpectedly, TNBC tumors of the non-Claudin low molecular subtype exhibited higher Ephrin-A4 expression and more robust responses to the ADC than other breast cancer subtypes, which suggests a specific translational application for breast tumor subtype classification. Together these findings demonstrate the potential of the Ephrin-A4-targeted calicheamicin conjugate as a first-in-class compound designed to eradicate TIC and improve long-term survival of cancer patients. PF-06647263 is currently being evaluated in a Phase I clinical trial. Citation Format: Marc Damelin, Alex Bankovich, Albert Park, Jorge Aguilar, Wade Anderson, Marianne Santaguida, Sarah Fong, Monette Aujay, Kiran Khandke, Virginia Pulito, Elana Ernstoff, Paul Escarpe, Jeff Bernstein, Marybeth A. Pysz, Wenyan Zhong, Erik Upeslacis, Judy Lucas, Justin Lucas, Timothy Nichols, Kathryn Loving, Orit Foord, Johannes Hampl, Robert Stull, Frank Barletta, Hadi Falahatpisheh, Puja Sapra, Hans Peter Gerber, Scott J. Dylla. An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5425. doi:10.1158/1538-7445.AM2015-5425

In this study, we have evaluated the molecular mechanism of Src activation after its interaction with estrogen receptor α (ERα) and a newly identified scaffold protein, called MNAR (modulator of nongenomic activity of ER). Under basal condition, Src enzymatic activity is inhibited by intramolecular interactions. The enzyme can be activated by interaction between the SH2 domain of Src and phosphotyrosine-containing sequences and/or by interaction between the SH3 domain of Src and proteins containing PXXP motifs. Mutational analysis and functional evaluation of MNAR and the use of ERα and cSrc mutants revealed that MNAR interacts with Src’s SH3 domain via its N-terminal PXXP motif. Mutation of this motif abolished both the MNAR-induced activation of Src and the stimulation of ER transcriptional activity. ER interacts with Src’s SH2 domain using phosphotyrosine 537, and this complex was further stabilized by MNAR-ER interaction. Mapping studies reveal that both the A/B domain and Y537 of ERα are required for MNAR-induced activation of ER transcriptional activity. The region responsible for MNAR interaction with ER maps to two N-terminal LXXLL motifs of MNAR. Mutation of these motifs prevented ER-MNAR complex formation and eliminated activation of the Src/MAPK pathway. These data explicate how the coordinate interactions between MNAR, ER, and Src lead to Src activation. Our findings also demonstrate that MNAR is a scaffold protein that mediates ER-Src interaction and plays an important role in the integration of ER action in Src-mediated signaling.

Ying Yang transcription factor (YY1) can repress or activate transcription. 25-Hydroxyvitamin D(3)-24-hydroxylase [24(OH)ase], an enzyme involved in the catabolism of 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], is up-regulated at the transcriptional level by 1,25-(OH)(2)D(3) to self-induce its deactivation. Here we report that YY1 can repress 1,25-(OH)(2)D(3)-induced 24(OH)ase transcription in CV1 cells transfected with vitamin D receptor (VDR) expression vector or in LLCPK(1) cells that contain VDR endogenously. With increasing amounts of YY1 DNA transfected (500 ng to 2 microg), ligand-dependent VDR activation of 24(OH)ase transcription was steadily repressed (maximum repression was 10-fold). Thus, YY1 may be a key modulator preventing activation at times that do not require the enzyme to be expressed. Relief of YY1 repression was observed in the presence of TFIIB or CBP (CREB binding protein) suggesting that YY1 may exert repression, in part, by sequestering TFIIB/CBP. Glutathione-S-transferase (GST) pull-down assays identified regions in the N and C termini of CBP that can bind YY1. In addition, the N-terminal region of CBP that interacts with YY1 can inhibit YY1 from binding to TFIIB. Thus, CBP may alleviate YY1-mediated repression, in part, by preventing YY1 from binding to TFIIB, which is required for VDR-mediated transcription. In summary, our results suggest that YY1 represses 24(OH)ase transcription, at least in part, by sequestering activator proteins involved in VDR-mediated transcription. In addition, our findings demonstrate a role for CBP in relief of repression of VDR-mediated transcription.

The 25-hydroxyvitamin D-24-hydroxylase enzyme (24-OHase) is responsible for the catabolic breakdown of 1,25-dihydroxyvitamin D[ 1,25(OH)2D], the active form of vitamin D. The 24-OHase enzyme can also act on the 25-hydroxyvitamin D substrate to generate 24,25-dihydroxyvitamin D, a metabolite whose physiological importance remains unclear. We report that mice with a targeted inactivating mutation of the 24-OHase gene had impaired 1,25(OH)2D catabolism. Surprisingly, complete absence of 24-OHase activity during development leads to impaired intramembranous bone mineralization. This phenotype was rescued by crossing the 24-OHase mutant mice to mice harboring a targeted mutation in the vitamin D receptor gene, confirming that the elevated 1,25(OH)2D levels, acting through the vitamin D receptor, were responsible for the observed accumulation of osteoid. Our results confirm the physiological importance of the 24-OHase enzyme for maintaining vitamin D homeostasis, and they reveal that 24,25-dihydroxyvitamin D is a dispensable metabolite during bone development.

Calcium has been known to be a potent second messenger for a wide range of cellular processes from fertilization to cell death. It has been implicated in the regulation of protein kinases, phosphatases, protease activity, chromatin structure and transcription as well as in the regulation of muscle contraction, nerve transmission, cytoskeletal organization, cell cycle progression and differentiation (Berridge, 1997; Berridge et al., 1998). Calcium homeostasis is tightly regulated such that any exogenous or internally generated calcium load is rapidly controlled to maintain calcium balance. Calcium ions signal from outside to inside by raising the intracellular cytosolic calcium concentration. An increase in cytosolic calcium can also occur inside the cell by release from stores. The mitochondria and the endoplasmic reticulum cross talk with each other to modulate the cytosolic calcium concentration. Thus an interplay among membrane components, intracellular organelles, calcium pumps and ion channels exists. Although calcium signaling is complex and incorporates multiple factors, it has been suggested that the ability of the calcium ion to interact with a family of calcium binding proteins (Kd = 10−8–10−10 M), known as EF-hand proteins, can play an important role in the transduction of the calcium signal into a biological response (Christakos et al., 1989, 1997; Heizmann and Braun, 1992; Heizmann and Hunziker, 1991; Schafer and Heizmann, 1996; Zimmer et al., 1995). This family of calcium binding proteins consists of over 200 members and is characterized by the EF-hand structural motif.