Use of Payload Binding Selectivity Enhancers to Improve Therapeutic Index of Maytansinoid-Antibody-Drug Conjugates.

Systemic exposure to released cytotoxic payload contributes to the dose-limiting off-target toxicities of anticancer antibody-drug conjugates (ADC). In this work, we present an "inverse targeting" strategy to optimize the therapeutic selectivity of maytansinoid-conjugated ADCs. Several anti-maytansinoid sdAbs were generated via phage-display technology with binding IC50 values between 10 and 60 nmol/L. Co-incubation of DM4 with the anti-maytansinoid sdAbs shifted the IC50 value of DM4 up to 250-fold. Tolerability and efficacy of 7E7-DM4 ADC, an anti-CD123 DM4-conjugated ADC, were assessed in healthy and in tumor-bearing mice, with and without co-administration of an anti-DM4 sdAb. Co-administration with anti-DM4 sdAb reduced 7E7-DM4-induced weight loss, where the mean values of percentage weight loss at nadir for mice receiving ADC+saline and ADC+sdAb were 7.9% ± 3% and 3.8% ± 1.3% (P < 0.05). In tumor-bearing mice, co-administration of the anti-maytansinoid sdAb did not negatively affect the efficacy of 7E7-DM4 on tumor growth or survival following dosing of the ADC at 1 mg/kg (P = 0.49) or at 10 mg/kg (P = 0.9). Administration of 7E7-DM4 at 100 mg/kg led to dramatic weight loss, with 80% of treated mice succumbing to toxicity before the appearance of mortality relating to tumor growth in control mice. However, all mice receiving co-dosing of 100 mg/kg 7E7-DM4 with anti-DM4 sdAb were able to tolerate the treatment, which enabled reduction in tumor volume to undetectable levels and to dramatic improvements in survival. In summary, we have demonstrated the utility and feasibility of the application of anti-payload antibody fragments for inverse targeting to improve the selectivity and efficacy of anticancer ADC therapy.

Mechanisms of ADC Toxicity and Strategies to Increase ADC Tolerability.

Anti-cancer antibody-drug conjugates (ADCs) aim to expand the therapeutic index of traditional chemotherapy by employing the targeting specificity of monoclonal antibodies (mAbs) to increase the efficiency of the delivery of potent cytotoxic agents to malignant cells. In the past three years, the number of ADCs approved by the Food and Drug Administration (FDA) has tripled. Although several ADCs have demonstrated sufficient efficacy and safety to warrant FDA approval, the clinical use of all ADCs leads to substantial toxicity in treated patients, and many ADCs have failed during clinical development due to their unacceptable toxicity profiles. Analysis of the clinical data has demonstrated that dose-limiting toxicities (DLTs) are often shared by different ADCs that deliver the same cytotoxic payload, independent of the antigen that is targeted and/or the type of cancer that is treated. DLTs are commonly associated with cells and tissues that do not express the targeted antigen (i.e., off-target toxicity), and often limit ADC dosage to levels below those required for optimal anti-cancer effects. In this manuscript, we review the fundamental mechanisms contributing to ADC toxicity, we summarize common ADC treatment-related adverse events, and we discuss several approaches to mitigating ADC toxicity.

Payload-Binding Fab Fragments Increase the Therapeutic Index of MMAE Antibody-Drug Conjugates.

Monomethyl auristatin E (MMAE) is a potent tubulin inhibitor that is used as the payload for four FDA-approved antibody-drug conjugates (ADC). Deconjugated MMAE readily diffuses into untargeted cells, resulting in off-target toxicity. Here, we report the development and evaluation of a humanized Fab fragment (ABC3315) that enhances the therapeutic selectivity of MMAE ADCs. ABC3315 increased the IC50 of MMAE against human cancer cell lines by > 500-fold with no impact on the cytotoxicity of MMAE ADCs, including polatuzumab vedotin (PV) and trastuzumab-vc-MMAE (TvcMMAE). Coadministration of ABC3315 did not reduce the efficacy of PV or TvcMMAE in xenograft tumor models. Coadministration of ABC3315 with 80 mg/kg TvcMMAE significantly (P < 0.0001) increased the cumulative amount of MMAE that was excreted in urine 0 to 4 days after administration from 789.4±19.0 nanograms (TvcMMAE alone) to 2625±206.8 nanograms (for mice receiving TvcMMAE with coadministration of ABC3315). Mice receiving 80 mg/kg TvcMMAE and PBS exhibited a significant drop in white blood cell counts (P = 0.025) and red blood cell counts (P = 0.0083) in comparison with control mice. No significant differences, relative to control mice, were found for white blood cell counts (P = 0.15) or for red blood cell counts (P = 0.23) for mice treated with 80 mg/kg TvcMMAE and ABC3315. Coadministration of ABC3315 with 120 mg/kg PV significantly (P = 0.045) decreased the percentage body weight loss at nadir for treated mice from 11.9%±7.0% to 4.1%±2.1%. Our results demonstrate that ABC3315, an anti-MMAE Fab fragment, decreases off-target toxicity while not decreasing antitumor efficacy, increasing the therapeutic window of MMAE ADCs.

Transient Inhibition of Trastuzumab-Tumor Binding to Overcome the "Binding-Site Barrier" and Improve the Efficacy of a Trastuzumab-Gelonin Immunotoxin.

We have recently shown that coadministration of mAbs with anti-idiotypic distribution enhancers (AIDE) that inhibit mAb binding to tumor antigens enabled increased intratumoral mAb distribution and increased efficacy of an antibody-drug conjugate (trastuzumab emtansine, T-DM1). In this article, a pharmacokinetic/pharmacodynamic (PK/PD) model was applied to predict the impact of this optimization strategy on the within-tumor distribution and antitumor efficacy of trastuzumab-gelonin, where the released payload (gelonin) is expected to exhibit negligible bystander activity. Immunofluorescence histology was used to investigate trastuzumab-gelonin distribution in solid tumors following dosing with or without coadministration of anti-trastuzumab AIDEs. Antitumor efficacy of trastuzumab-gelonin, with or without coadministration of AIDEs, was also evaluated in tumor-bearing mice. Trastuzumab-gelonin efficiently induced cytotoxicity when applied to NCI-N87 cells in culture (IC50: 0.224 ± 0.079 nmol/L). PK/PD simulations predicted that anti-idiotypic single-domain antibodies AIDEs with dissociation rate constants between 0.03 and 0.2 per hour would provide optimal enhancement of trastuzumab-gelonin efficacy. LE8 and 1HE, anti-trastuzumab AIDEs, were selected for evaluation in vivo. Coadministration of trastuzumab-gelonin with the inhibitors increased the portion of tumor area that stained positive for trastuzumab-gelonin by 58% (P = 0.0059). In addition, LE8 or 1HE coadministration improved trastuzumab-gelonin efficacy in NCI-N87 xenograft-bearing mice by increasing the percent increase in life span (%ILS) from 27.8% (for trastuzumab-gelonin administered alone) to 62.5% when administered with LE8 (P = 0.0007) or 83.3% (P = 0.0007) when administered with 1HE. These findings support the hypothesis that transient, competitive inhibition of mAb-tumor binding can improve the intratumoral distribution and efficacy of immunotoxins when applied for treatment of solid tumors.

Development and Evaluation of Competitive Inhibitors of Trastuzumab-HER2 Binding to Bypass the Binding-Site Barrier.

Our group has developed and experimentally validated a strategy to increase antibody penetration in solid tumors through transient inhibition of antibody-antigen binding. In prior work, we demonstrated that 1HE, an anti-trastuzumab single domain antibody that transiently inhibits trastuzumab binding to HER2, increased the penetration of trastuzumab and increased the efficacy of ado-trastuzumab emtansine (T-DM1) in HER2+ xenograft bearing mice. In the present work, 1HE variants were developed using random mutagenesis and phage display to enable optimization of tumor penetration and efficacy of trastuzumab-based therapeutics. To guide the rational selection of a particular 1HE mutant for a specific trastuzumab-therapy, we developed a mechanistic pharmacokinetic (PK) model to predict within-tumor exposure of trastuzumab/T-DM1. A pharmacodynamic (PD) component was added to the model to predict the relationship between intratumor exposure to T-DM1 and the corresponding therapeutic effect in HER2+ xenografts. To demonstrate the utility of the competitive inhibition approach for immunotoxins, PK parameters specific for a recombinant immunotoxin were incorporated into the model structure. Dissociation half-lives for variants ranged from 1.1 h (for variant LG11) to 107.9 h (for variant HE10). Simulations predicted that 1HE co-administration can increase the tumor penetration of T-DM1, with inhibitors with longer trastuzumab binding half-lives relative to 1HE (15.5 h) further increasing T-DM1 penetration at the expense of total tumor uptake of T-DM1. The PK/PD model accurately predicted the response of NCI-N87 xenografts to treatment with T-DM1 or T-DM1 co-administered with 1HE. Model predictions indicate that the 1HE mutant HF9, with a trastuzumab binding half-life of 51.1 h, would be the optimal inhibitor for increasing T-DM1 efficacy with a modest extension in the median survival time relative to T-DM1 with 1HE. Model simulations predict that LG11 co-administration will dramatically increase immunotoxin penetration within all tumor regions. We expect that the mechanistic model structure and the wide range of inhibitors developed in this work will enable optimization of trastuzumab-cytotoxin penetration and efficacy in solid tumors.

Targeted Delivery of Endosomal Escape Peptides to Enhance Immunotoxin Potency and Anti-cancer Efficacy.

This work describes use of anti-carcinoembryonic antigen antibodies (10H6, T84.66) for targeted delivery of an endosomal escape peptide (H6CM18) and gelonin, a type I ribosome inactivating protein. The viability of colorectal cancer cells (LS174T, LoVo) was assessed following treatment with gelonin or gelonin immunotoxins, with or without co-treatment with T84.66-H6CM18. Fluorescent microscopy was used to visualize the escape of immunoconjugates from endosomes of treated cells, and efficacy and toxicity were assessed in vivo in xenograft tumor-bearing mice following single- and multiple-dose regimens. Application of 25 pM T84.66-H6CM18 combined with T84.66-gelonin increased gelonin potency by ~ 1,000-fold and by ~ 6,000-fold in LS174T and LoVo cells. Intravenous 10H6-gelonin at 1.0 mg/kg was well tolerated by LS174T tumor-bearing mice, while 10 and 25 mg/kg doses led to signs of toxicity. Single-dose administration of PBS, gelonin conjugated to T84.66 or 10H6, T84.66-H6CM18, or gelonin immunotoxins co-administered with T84.66-H6CM18 were evaluated. The combinations of T84.66-gelonin + 1.0 mg/kg T84.66-H6CM18 and 10H6-gelonin + 0.1 mg/kg T84.66-H6CM18 led to significant delays in LS174T growth. Use of a multiple-dose regimen allowed further anti-tumor effects, significantly extending median survival time by 33% and by 69%, for mice receiving 1 mg/kg 10H6-gelonin + 0.1 mg/kg T84.66-H6CM18 (p = 0.0072) and 1 mg/kg 10H6-gelonin + 1 mg/kg T84.66-H6CM18 (p = 0.0017). Combined administration of gelonin immunoconjugates with antibody-targeted endosomal escape peptides increased the delivery of gelonin to the cytoplasm of targeted cells, increased gelonin cell killing in vitro by 1,000-6,000 fold, and significantly increased in vivo efficacy.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging for the Prediction of Monoclonal Antibody Tumor Disposition.

The prediction of monoclonal antibody (mAb) disposition within solid tumors for individual patients is difficult due to inter-patient variability in tumor physiology. Improved a priori prediction of mAb pharmacokinetics in tumors may facilitate the development of patient-specific dosing protocols and facilitate improved selection of patients for treatment with anti-cancer mAb. Here, we report the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), with tumor penetration of the contrast agent gadobutrol used as a surrogate, to improve physiologically based pharmacokinetic model (PBPK) predictions of cetuximab pharmacokinetics in epidermal growth factor receptor (EGFR) positive xenografts. In the initial investigations, mice bearing Panc-1, NCI-N87, and LS174T xenografts underwent DCE-MRI imaging with the contrast agent gadobutrol, followed by intravenous dosing of an 125Iodine-labeled, non-binding mAb (8C2). Tumor concentrations of 8C2 were determined following the euthanasia of mice (3 h-6 days after 8C2 dosing). Potential predictor relationships between DCE-MRI kinetic parameters and 8C2 PBPK parameters were evaluated through covariate modeling. The addition of the DCE-MRI parameter Ktrans alone or Ktrans in combination with the DCE-MRI parameter Vp on the PBPK parameters for tumor blood flow (QTU) and tumor vasculature permeability (σTUV) led to the most significant improvement in the characterization of 8C2 pharmacokinetics in individual tumors. To test the utility of the DCE-MRI covariates on a priori prediction of the disposition of mAb with high-affinity tumor binding, a second group of tumor-bearing mice underwent DCE-MRI imaging with gadobutrol, followed by the administration of 125Iodine-labeled cetuximab (a high-affinity anti-EGFR mAb). The MRI-PBPK covariate relationships, which were established with the untargeted antibody 8C2, were implemented into the PBPK model with considerations for EGFR expression and cetuximab-EGFR interaction to predict the disposition of cetuximab in individual tumors (a priori). The incorporation of the Ktrans MRI parameter as a covariate on the PBPK parameters QTU and σTUV decreased the PBPK model prediction error for cetuximab tumor pharmacokinetics from 223.71 to 65.02%. DCE-MRI may be a useful clinical tool in improving the prediction of antibody pharmacokinetics in solid tumors. Further studies are warranted to evaluate the utility of the DCE-MRI approach to additional mAbs and additional drug modalities.

Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies.

Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated KD of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated KD of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation (KD of 66.9 nM and 30.3 nM, respectively). Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo.

Transient Competitive Inhibition Bypasses the Binding Site Barrier to Improve Tumor Penetration of Trastuzumab and Enhance T-DM1 Efficacy.

Poor penetration of mAbs in solid tumors is explained, in part, by the binding site barrier hypothesis. Following extravasation, mAbs rapidly bind cellular antigens, leading to the observation that, at subsaturating doses, therapeutic antibody in solid tumors localizes around tumor vasculature. Here we report a unique strategy to overcome the binding site barrier through transient competitive inhibition of antibody-antigen binding. The anti-trastuzumab single domain antibody 1HE was identified through in vitro binding assays as a model inhibitor. Coadministration of 1HE did not alter the plasma pharmacokinetics of trastuzumab or ado-trastuzumab emtansine (T-DM1) in vivo. Administration of 1HE alone was rapidly eliminated with a terminal plasma half-life of 1.2 hours, while coadministrations of 1HE with trastuzumab had a terminal half-life of 56 hours. In mice harboring SKOV3 xenografts, coadministration of 1HE with trastuzumab led to significant increases in both penetration of trastuzumab from vasculature and the percentage of tumor area that stained positive for trastuzumab. 1HE coadministered with a single dose of T-DM1 to NCI-N87 xenograft-bearing mice significantly enhanced T-DM1 efficacy, increasing median survival. These results support the hypothesis that transient competitive inhibition can improve therapeutic antibody distribution in solid tumors and enhance antibody efficacy. SIGNIFICANCE: This study describes the development of a transient competitive inhibition strategy that enhances the tumor penetration and efficacy of anticancer antibodies.See related commentary by van Dongen, p. 3956.

Strategies to enhance monoclonal antibody uptake and distribution in solid tumors.

Despite the significant resources dedicated to the development of monoclonal antibody (mAb) therapies for solid tumors, the clinical success, thus far, has been modest. Limited efficacy of mAb in solid tumors likely relates to unique aspects of tumor physiology. Solid tumors have an aberrant vasculature and a dense extracellular matrix that slow both the convective and diffusive transport of mAbs into and within tumors. For mAbs that are directed against cellular antigens, high antigen expression and rapid antigen turnover can result in perivascular cells binding to and eliminating a significant amount of extravasated mAb, limiting mAb distribution to portions of the tumor that are distant from functional vessels. Many preclinical investigations have reported strategies to improve mAb uptake and distribution; however, to our knowledge, none have translated into the clinic. Here, we provide an overview of several barriers in solid tumors that limit mAb uptake and distribution and discuss approaches that have been utilized to overcome these barriers in preclinical studies.

Hedgehog Proteins Consume Steroidal CYP17A1 Antagonists: Potential Therapeutic Significance in Advanced Prostate Cancer.

Abiraterone, a potent inhibitor of the human enzyme CYP17A1 (cytochrome P450c17), provides a last line of defense against ectopic androgenesis in advanced prostate cancer. Herein we report an unprecedented off-target interaction between abiraterone and oncogenic hedgehog proteins. Our experiments indicate that abiraterone and its structural congener, galeterone, can replace cholesterol as a substrate in a specialized biosynthetic event of hedgehog proteins, known as cholesterolysis. The off-target reaction generates covalent hedgehog-drug conjugates. Cell-based reporter assays indicate that these conjugates activate hedgehog signaling when present in the low nanomolar range. Because hedgehog signaling is implicated in prostate cancer progression, and abiraterone is administered to treat advanced stages of the disease, this off-target interaction may have therapeutic significance.

Förster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor.

Hedgehog (Hh) proteins function in cell/cell signaling processes linked to human embryo development and the progression of several types of cancer. Here, we describe an optical assay of hedgehog cholesterolysis, a unique autoprocessing event critical for Hh function. The assay uses a recombinant Förster resonance energy transfer (FRET)-active Hh precursor whose cholesterolysis can be monitored continuously in multi-well plates (dynamic range=4, Z'=0.7), offering advantages in throughput over conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assays. Application of the optical assay in a pilot small molecule screen produced a novel cholesterolysis inhibitor (apparent IC50=5×10(-6)M) that appears to inactivate hedgehog covalently by a substitution nucleophilic aromatic (SNAr) mechanism.