Abstract
Pet dogs with naturally occurring cancers play an important role in studies of cancer biology and drug development. We assessed tolerability, efficacy, and pharmacokinetic/pharmacodynamic relationships with a first-in-class small molecule inhibitor of valosin-containing protein (VCP/p97), CB-5339, administered to 24 tumor-bearing pet dogs. Tumor types assessed included solid malignancies, lymphomas, and multiple myeloma. Through a stepwise dose and schedule escalation schema, we determined the maximum tolerated dose to be 7.5 mg/kg when administered orally on a 4 days on, 3 days off schedule per week for 3 consecutive weeks. Adverse events were minimal and mainly related to the gastrointestinal system. Pharmacokinetic/pharmacodynamic data suggest a relationship between exposure and modulation of targets related to induction of the unfolded protein response, but not to tolerability of the agent. An efficacy signal was detected in 33% (2/6) of dogs with multiple myeloma, consistent with a mechanism of action relating to induction of proteotoxic stress in a tumor type with abundant protein production. Clinical trials of CB-5339 in humans with acute myelogenous leukemia and multiple myeloma are ongoing.
Introduction
Cancer cells carry numerous mutations and supernumerary chromosomes, making them likely to produce abnormal or excess proteins. It has therefore been suggested that cancer cells are more dependent on components of the protein degradation machinery to maintain cellular homeostasis critical for survival (1). Valosin containing protein (VCP), also known as p97, is a member of the ATPase Associated with diverse cellular Activities (AAA) family of proteins and is an important cellular enzyme that uses energy derived from ATP hydrolysis to reshape the proteome (2, 3).
VCP has a well-described role in the ubiquitin proteasome system (UPS), where it chaperones subsets of proteins to the proteasome for degradation, providing energy for the transportation of degraded substrates during endoplasmic-reticulum-associated degradation (ERAD) by hydrolyzing ATP (4). VCP also functions in the cell's alternate protein disposal pathway, the autophagy lysosome system (ALS; refs. 5, 6). Evidence suggests that cancer cells can become dependent on these protein disposal pathways; and therefore, inhibition of VCP is expected to have meaningful anticancer activity through the induction of irreversible proteotoxic stress (7).
Targeting protein homeostasis has become a clinically demonstrated anticancer strategy since the introduction of proteasome inhibitors as treatment for multiple myeloma (8). Although their development provided the rationale for targeting of the UPS, the clinical investigation of proteasome inhibitors was associated with high rates of drug resistance. As a class, these agents were found to be ineffective in solid tumors, illustrating a need for drugs targeting novel regulatory components of the UPS pathway (9–11). Additional concerns for issues relating to adverse events with proteosome inhibitors in patients with multiple myeloma, such as peripheral neuropathy and dose-limiting thrombocytopenia, support investigation into alternate approaches (12). To that end, inhibitors of VCP have been developed and assessed for advancement into human patients with cancer.
An early generation small-molecule inhibitor, CB-5083, was screened for activity across a wide variety of human and canine cell lines and preclinical models, with a particular emphasis on tumors that are known to generate disproportionately large quantities of proteins (e.g., multiple myeloma and B-cell lymphoma; ref. 13). In preclinical comparative studies, CB-5083 treatment also resulted in preferential cytotoxicity in canine lymphoma cell lines over peripheral blood mononuclear cells (PBMC; ref. 14). In these experiments, CB-5083 rapidly disrupted the UPS, causing accumulation of cellular polyubiquitinated proteins, and consequent endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). This mechanism of action (MOA) was supported by an increase in eIF2α phosphorylation and increased transcription of ATF4, leading to apoptosis through activation of the caspase cascade (14).
However, unfavorable off-target effects, hypothesized to involve inhibition of phosphodiesterase 6 (PDE6) resulted in the discontinuation of CB-5083 from further clinical evaluation. Therefore, a next generation VCP inhibitor, CB-5339, was developed to improve the therapeutic index and pharmacokinetic profile of the first-generation molecule (15). CB-5339 possesses several favorable characteristics that support its advancement to first-in-human use over its predecessor. These include a comparable in vitro and in vivo potency profile, but with 40X reduced effects on PDE6 and improved physiochemical and pharmacokinetic properties.
The comparative oncology clinical trial reported herein was intended to define the safety, pharmacokinetics, and pharmacodynamic modulation of CB-5339 in tumor-bearing pet dogs. This trial was designed as a dose escalation study wherein tolerability, clinical efficacy, and pharmacokinetic/pharmacodynamic data are collected in pet dogs with naturally-occurring cancers. The primary objectives were to correlate tolerable exposures of orally-administered CB-5339 in pet dogs sufficient for activation of the UPR and generation of clinically evident anticancer activities, and leverage these high-value preclinical findings to support the rationale design of follow-on human trials. Additional objectives were the exploration of pharmacodynamic biomarkers assessed serially in normal PBMC and tumor tissues, and definition of the timing of collection of biologic samples wherein relationships between pharmacokinetic and pharmacodynamic could be confirmed. Increasingly, such data is recognized as a valuable, complementary component to an Investigational New Drug (IND) filing for new human anticancer agents (16, 17).
Materials/Methods/Patient Enrollment
Comparative Oncology Trials Consortium
The Comparative Oncology Trials Consortium (COTC) infrastructure, data reporting, and goals have been previously described. The canine clinical trial described herein was conducted through a multi-institutional consortium (17, 18). Three COTC sites (University of Illinois at Urbana-Champaign, University of Missouri, and the University of Wisconsin-Madison) participated in the study, and all dogs were recruited and evaluated following a defined protocol and standard operating procedures. The study protocol was reviewed and approved by each participating site's Institutional Animal Care and Use Committee and, where applicable, Clinical Trials Review Board. All study data were managed by the National Cancer Institute Comparative Oncology Program utilizing the Cancer Central Clinical Database (C3D).
Trial eligibility and enrollment
Client-owned pet dogs weighing ≥15 kg with cytologically or histologically confirmed macroscopic peripheral lymphoma or solid malignancies were included in the study. Dogs with multiple myeloma were also recruited and enrolled, with a diagnosis confirmed through documentation of at least two major criteria (presence of Bence–Jones proteinuria, lytic bone lesions, serum monoclonal gammopathy, evidence of plasma cell infiltration in bone marrow or tissue; ref. 19). Eligibility criteria required dogs with lymphoma to have a nodal presentation (stage 2 or greater) with at least three nodes measuring a minimum of 3 cm in longest diameter that were amenable to repeated biopsies. Both newly diagnosed and previously treated dogs were eligible with a 2-week washout for chemotherapy and radiation therapy. Dogs previously treated with corticosteroids or L-asparaginase required a 7-day washout prior to study initiation. All dogs received a physical examination, laboratory evaluations (complete blood count, serum biochemical profile, and urinalysis), and thoracic radiographs as part of the eligibility screening within 10 days prior to study enrollment. Dogs determined to have any significant comorbid illness or with specific tumor types not amenable to serial biopsy (mast cell tumors, hemangiosarcoma) were excluded. In addition, dogs with any of the following were considered ineligible for enrollment: creatinine >3.0 mg/dL, total bilirubin >2.0 mg/dL or elevated bile acids, HCT <25%, platelets <50,000/μL, or any other > grade 2 hematologic or biochemical abnormality based on the Veterinary Cooperative Oncology Group Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 (20). Eligibility criteria included a performance status of 0 or 1 [modified Eastern Cooperative Oncology Group (ECOG); ref. 21] and written informed owner consent.
Study schema
A schedule of patient evaluations, diagnostics, sample collection, and treatments performed at each study time point are summarized in Fig. 1. CB-5339 was initially assessed within a dose-escalation schema that included 2 weeks of consecutive dosing on Monday to Thursday with Friday/Saturday/Sunday off, followed by a week of rest, with assessment at the end of the cycle on Day 22 (Fig. 1A). After review of the first 17 dogs enrolled to the protocol, an additional week of treatment on Monday through Thursday was added, followed by a week of rest, then assessment at the end of the 4-week cycle on Day 29 (Fig. 1B).
Clinical trial schema for the comparative study of CB-5339 in tumor-bearing pet dogs. Two-week (A) and 3-week (B) dosing schedules were utilized in this clinical trial. BMA, bone marrow aspiration cytology; CBC, complete blood count; chem, serum biochemical panel; PE, physical examination; PK, pharmacokinetic assessment; PO, per os/oral dosing; UA, urinalysis.
Clinical trial schema for the comparative study of CB-5339 in tumor-bearing pet dogs. Two-week (A) and 3-week (B) dosing schedules were utilized in this clinical trial. BMA, bone marrow aspiration cytology; CBC, complete blood count; chem, serum biochemical panel; PE, physical examination; PK, pharmacokinetic assessment; PO, per os/oral dosing; UA, urinalysis.
Dosing strategy
Dosing of CB-5339 was escalated in a standard 3+3 dose escalation design. The starting dose of 5 mg/kg was determined through review of data generated within multiweek IND-enabling GLP safety studies of CB-5339 conducted in healthy Beagle dogs, wherein the no-observed-adverse-effect level (NOAEL) was 10 mg/kg/day. Dose cohorts for CB-5339 were outlined as follows: dose level −1 (if de-escalation was necessary): 2.5 mg/kg, dose level 1: 5 mg/kg, dose level 2: 7.5 mg/kg, dose level 3: 10 mg/kg. Three dogs were enrolled in the first dosing cohort and observed for dose-limiting toxicities (DLT). Severity of toxicity of CB-5339 was assessed using the VCOG-CTCAE v1.1 at each study time point, and toxicity attributions were designated as due to drug, disease, research, or other cause (20). The certainty of attributions was further characterized as unrelated, unlikely, possible, probable, or definite. DLT was defined as any grade 3 nonhematologic or grade 4 hematologic toxicity. If no DLTs were observed in the first cohort of 3 dogs within 2 weeks of CB-5339 administration, a second cohort of dogs was treated at an increased dose. If a DLT was observed in one dog, the cohort was expanded up to a total of six dogs. If no additional DLTs were noted in the expanded cohort of six dogs, dose escalation was continued with a higher dosage of CB-5339. If ≥2 DLTs were observed in the initial or expanded cohort, case accrual was stopped and the MTD was determined to be the highest dosage used in a cohort where <2 DLTs were noted.
Two different formulations of CB-5339 were assessed in this clinical trial, provided by Cleave Therapeutics Inc. The first 11 dogs received a powder in capsule [PIC, consisting of free-base active pharmaceutical ingredient (API) only]. During execution of the canine trial, the human clinical formulation (HCF) became available in preparation for human phase I testing (containing the API as tosylate salt, plus inactive ingredients: microcrystalline cellulose, mannitol, croscarmellose sodium, polaxamer, silicone dioxide, and magnesium stearate). A switch to using the HCF was made due to improved pharmacokinetic in Beagle dogs. One pet dog in this study received both PIC and HCF, whereas 12 dogs received HCF exclusively. On the basis of analysis of data obtained from the 2-week dosing cohort, as well as interest in exploring tolerability of an extended drug exposure period to mirror eventual human clinical studies, the study schedule was modified. Within the 3-week dosing cohort, an additional pharmacokinetic curve was added on Day 15 with a biopsy collected at either 1, 3, or 6 hours (one time point per dog enrolled, see Fig. 1A and B).
Plasma pharmacokinetics
A 6-point pharmacokinetic timecourse of plasma CB-5339 levels was performed with samples obtained pretreatment (0 minutes), and at 30 minutes, 1, 2, 3, and 6 hours post-drug administration on Day 1. All plasma pharmacokinetic collections were listed as time after oral administration of drug. A second pharmacokinetic 6-hour curve was added to Day 15 for dogs enrolled to the 3-week dosing cohort.
An HPLC/MS-MS method was developed at Alturas Analytics, Inc. to determine concentrations of CB-5339 in dog K2EDTA plasma in accordance with internal Standardized Operating Procedures. Following protein precipitation with acetonitrile, an aliquot of the extract was injected onto an HPLC/MS-MS triple quadrupole mass spectrometer (Sciex API4000). A C18 HPLC column and gradient elution with acetonitrile/water in 1% formic acid was used to separate CB-5339 and the internal standard (IS), CB-5339-d5, from any interfering compounds present in the sample extract. The peak area of the product ion of the compound (CB-5339) was measured against the peak area of the product ion of the internal standard. A calibration curve ranging from 1.00 to 1,500 ng/mL (eight concentrations in duplicate) was used to quantify CB-5339 in the samples. Interbatch accuracy was ±6% and interbatch precision was 2.8% to 10.8%. Analyte recovery was ∼73% and stability assessments were within acceptable range.
Tumor biopsies
Lymph node or tumor biopsies were collected from dogs with lymphoma or solid tumors, respectively, to measure K48 (K48 = ubiquitin chains branched at lysine 48) and CHOP (DNA damage-inducible transcript 3, also known as C/EBP Homologous Protein) levels pre- and posttreatment. Incisional lymph node biopsies were performed in triplicate using a 14-gauge Tru-cut needle prior to treatment and on Day 1 posttreatment, initially at 1, 6, and 24 hours following the first dose of drug administration on Day 1. Biopsies were also collected on Day 8 prior to dosing. A tumor biopsy was also collected on Day 22, but only if tumor response was categorized as stable or progressive disease. Biopsy cores were placed individually in prechilled cryogenic vials, frozen within 2 minutes of collection, transported on dry ice, and stored at ≤−80°C until extractions were performed for pharmacodynamic evaluation. Two biopsy samples were flash frozen and stored at −80°C per time point. Formalin-fixed samples were only collected prior to treatment. The frozen samples were held at -80°C until study participation was completed. For dogs with multiple myeloma, if an accessible lesion confirmed to be a site of malignant plasma cell proliferation was available, such target lesions were also subjected to serial biopsy as described above.
PBMC collection and processing
PBMCs were collected at every time point at which a tumor biopsy was collected to determine if circulating leukocytes could serve as an accurate surrogate for tumor tissue pharmacodynamic modulation. PBMC collections were discontinued when the schedule was modified to 3-week dosing. Peripheral blood was collected into two to three 7 mL green top tubes (Na-heparin), kept at room temperature and enrichment of mononuclear cells performed within 2 hours of venipuncture. Blood was pooled into one 50 mL tube and diluted 1:1 (one-part blood, one-part PBS) with 1× PBS and mixed slowly by gentle tube inversion. Diluted blood (15 mL) was overlayed on top of a Ficoll layer (GE Healthcare, Catalog No. 17144002) without mixing the blood and Ficoll. The 50 mL tube was centrifuged at 800 × g for 30 minutes at room temperature without rotor brakes. The resultant buffy layer was transferred to a new 50 mL tube, diluted with 1× PBS to a total volume of 50 mL and centrifuged at 300 × g for 7 minutes at room temperature. Supernatant was carefully removed and discarded. The resultant cell pellet was resuspended in 2.5 mL of 1× PBS, with a small aliquot being used for cell counting and the remainder of cells were pelleted in a 1.5 mL microcentrifuge tube at 10,000 × g for 5 minutes at 4°C. PBS was completely removed, and the final PBMC cell pellets were flash frozen in dry ice and transported for biomarker analysis.
Preparation of PBMC and tumor biopsy cell lysates
Fractionated cell lysates were prepared from PBMC and tumor biopsies by procedures described previously (22) with minor modifications to the extraction buffers (both MIM & Buffer A) by adding iodoacetic acid (10 μmol/L) and vinyl sulfone (0.5 μmol/L, supplied as 250 μmol/L solution in 50 mmol/L MES buffer pH 6.0) to stabilize ubiquitinated substrates in cell lysates. Two major crude fractions representing cytosol and mitochondria were generated from PBMCs and tumor tissue biopsies. Total protein concentrations of fractionated lysates were determined by BCA Protein Assay Kit (Thermo Fisher Scientific, Catalog No. 23227) using BSA (Thermo Fisher Scientific, Catalog No. 23209) as calibrator. Lysates were stored at −80°C until analyzed in a batched manner.
Pharmacodynamic assays
The biomarkers were developed as sandwich immunoassays on a Luminex multiplex platform as described below. Cytosol and nuclear fractions from cell and tumor tissues were prepared as described previously (22). CB-5083 was used to assess responsiveness of the biomarkers in vitro and in vivo. Although pharmacodynamic biomarkers were also developed to support the first-in-human clinical trial (23), the fitness of biomarkers to monitor pharmacodynamic response in canine cell lines and canine xenograft tumors was demonstrated for the purposes of this canine clinical trial. Prior to use in canine clinical trial samples, pharmacodynamic assays were developed as follows. The canine cell line DH82 (macrophage like, malignant histiocytosis) was obtained from ATCC (CRL-10389). A canine B cell lymphoma cell line 17–71 was kindly provided by Dr. Douglas Thamm of the Colorado State University Flint Animal Cancer Center and MC.KOS (canine osteosarcoma) was obtained from the intramural National Cancer Institute Comparative Oncology Program (24). Cell lines were confirmed as canine origin based upon short tandem repeat profiling (17–71) or karyotyping (MC-KOS). The DH82 and 17–71 lines were tested for Mycoplasma sp. using MycoAlert Mycoplasma Detection Kit (Lonza, Catalog No. LT07-118); 17–71 was positive and subsequently treated with the MycoZap Mycoplasma elimination reagent (Lonza, Catalog No. LTo7-918). MC-KOS was confirmed as Mycoplasma negative by PCR. The experimental therapeutic agent CB-5083 (NSC786100) was obtained from the Division of Cancer Treatment and Diagnosis (DCTD) Investigational Drug Repository, whereas the experimental therapeutic agent CB-5339 was provided by Cleave Therapeutics. Materials used in sample preparation and assays are specified in the subsequent Method sections. Purity of antibodies and recombinant proteins were assessed by SDS-PAGE and protein concentrations measured by the Bradford method (Bio-Rad) with BSA as a calibrator (Thermo Fisher Scientific). Conjugation of antibodies to Luminex biotin beads using Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) has been reported previously (22).
Cellular viability assay to determine the potency of CB-5083 and CB-5339
Canine lymphoma (17–71) and malignant histiocytosis (DH82) cells were plated in 384-well plates (Greiner) at a density of 750 cells per 30 μL per well in DMEM medium containing 5% FBS and 1% penicillin/streptomycin. Twenty-four hours after seeding, 8 μL medium containing DMSO (solvent control) or serially diluted CB-5083 or CB-5339 were added, and plates were incubated for another 48 hours. Cellular viabilities were measured using Cell Titer Glo Luminescent Cell Viability Assay (Promega), and IC50 values were calculated using the percentage of growth of compound-treated wells versus DMSO control.
Canine xenograft treatment with p97 inhibitor
All animal studies were conducted in accordance with an approved animal care and use committee protocol accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and followed the USPHS Policy for the Care and Use of Laboratory Animals. The canine osteosarcoma cell line MC.KOS was subcutaneously (1 × 107 cells/mouse) implanted in female nod-SCIDγ (NSG) mice (obtained from the NCI Division of Cancer Therapy and Diagnosis Biologic Testing Branch) and allowed to grow until tumor weights reached 150 to 200 mg. Mice were then randomized and treated with a single intraperitoneal dose of vehicle (citrate buffer, pH 5.0, isotonic) or CB-5083 at 100 mg/kg. Tumors were resected at 1-, 6-, and 24-hours posttreatment, flash frozen within 2 minutes of collection as quarters and stored at −80°C until processed into cell lysates.
K48 sandwich immunoassay on Luminex platform
The K48 immunoassay was developed on a Luminex xMAP multiplex technology platform using MagPlex polystyrene-coated magnetic bead (Luminex) and performed in a flat-bottomed black 96-well plate (Bio-Rad) as described previously (22) with some modification. Briefly, anti-Ubiquitin K48 linkage specific antibody (Catalog No. M02848-1, BosterBio) was conjugated to magnetic beads and an anti-ubiquitin antibody (BML-PW0930-1000, Enzo) was used as detection of K48-ubiquitinated substrates. A recombinant K48-linked tetra-ubiquitin (Boston Biochem, Catalog No. UC-250) was used as recombinant K48 calibrator. The biopsy lysate processing procedure was modified to include a denaturing step with urea and heat. Calibrator, controls, and tumor biopsy lysates were adjusted to a final concentration of 3M urea. Tumor biopsy lysates were diluted between 500 and 125 μg/mL of total protein concentrations (exact dilution depended on type of ubiquitin pathway modulator drug in terms of whether it decreases or increases K48-substrates post treatment) in sample buffer and a uniform 3M urea concentration. Calibrator, controls, and cell lysate samples were then heated for 5 minutes at 60°C (60 ± 1°C) in a water bath, placed on ice for 2 minutes to stop the heating process, and lysates were kept at room temperature for 10 minutes before added to 96-well plates to initiate Luminex assay. First, 10 μL of a blocker solution was added to plates. Second, calibrator, controls, and samples were loaded on plates at 30 μL/well, and finally MagPlex beads were loaded at 10 μL/well (2,500 beads/well). Plates were incubated for 2 hours at 25 ± 5°C with orbital shaking at 850 rpm. Plates were washed three times using a BioTek 405 TS magnetic plate washer with 300 μL/well wash buffer (1× PBS, 0.05% Tween-20, and 0.05% ProClin 300). After washing, 40 μL/well of 4 μg/mL antibody–biotin conjugate in detection buffer was added and the plates were incubated with shaking for 1 hour. Without washing step, 20 μL/well of 100 μg/mL R-phycoerythrin–labeled streptavidin (Invitrogen, in wash buffer) was added and the plates were incubated with shaking for 30 minutes. Plates were washed as described above, and then the beads were resuspended in 100 μL/well of the wash buffer and incubated with shaking for 1 to 2 minutes. Plates were read on Luminex 200 readers within 2 hours of completion of last step. Data were analyzed on Bio-Plex Manager Software v6.2 (Bio-Rad).
Analytical validation of K48 immunoassay
To determine reproducibility, lysate matrix interference (dilution recovery) and accuracy (spike recovery was used as surrogate for accuracy), control specimens were prepared using canine cancer cell lines DH82, 17–71, and MC.KOS, with low and high concentrations of K48 at baseline. The K48 signal was 2- to 3-fold lower in absence of urea and heat denaturing step, therefore, denaturation step was critical to assay implementation. Typical calibrator curve for K48 assay is shown in Supplementary Fig. S1A. The within assay variability was CV <10% determined by analyzing 10 replicates of two tumor lysates at high and low concentrations of K48. Mean spike recovery was 131 ± 37% determined by spiking human K48 calibrator at three representative concentrations in six canine tumor tissue lysate samples in four different experiments. Dilution of tumor lysates by 1:2 and 1:4 using assay buffer was assessed using multiple samples, a r2 value between 0.96 and 99 was observed for correlation between observed and expected concentrations in five different experiments. The sensitivity or lower limit of quantitation (LLOQ) for K48 assay was established at the lowest concentration of calibrator, 0.05 ng/mL. Hemolysis in tumor or PBMC lysates interfered in the K48 assays (produced low or undetectable levels of K48) and hemolyzed samples were excluded from analysis. Overall, the analytical performance of the canine K48 assay was well suited for batched analysis of cytosolic K48 in response to p97 inhibitors.
Fit-for-purpose validation of K48 immunoassay for pharmacodynamic evaluation of p97 inhibitors in canine cell lines and tumor tissue
The K48 assay fitness to assess the pharmacodynamics of p97 inhibitors was first demonstrated in vitro by treating two cell lines of canine origin, DH82 and 17 to 71 with 0.3 μmol/L of CB-5083 (representative data from the canine 17 to 71 cell line are shown in Supplementary Fig. S1B). Cellular viability studies for both 17 to 71 and DH82 treated with both CB-5083 and CB-5339 are shown in Supplementary Fig. S2. A time course was established and maximum accumulation of K48-ubiquitinated substrates was observed at 1 hour posttreatment. This timing is in good accordance with studies in human and murine cell lines, in which the elevation of levels of K48-ubiquitinated substrates are an early sign of proteasome misfunction. The pharmacodynamic effect lasted 4 to 6 hours and declined slightly over next 24 hours.
In xenograft studies, 1 hour after the intraperitoneal dose, there was a 4- to 5-fold increase in the K48-substrates in cytosol fraction (Supplementary Fig. S1C). Over the next 24 hours, the K48-substrates partially recovered but remained significantly higher than the vehicle-treated group. The changes in K48-substrate over time was consistent with pharmacokinetics of the p97 inhibitor CB-5083. Overall, the pharmacodynamic response measured by K48 immunoassays was well above the within-group biological variability.
qPCR assay
Canine clinical trial samples (tumor or PBMCs) were resuspended in DPBS/TRizol-LS mixture (Ambion, Catalog No. 10296010; v/v 1:3) by homogenizing with a microgrinder pestle mixer. Total RNAs were extracted with Direct-zol RNA MiniPrep Kit (Zymo Research, Catalog No. R2072) by following the manufacturer's instructions. RNA concentrations were determined with NanoDrop (Thermo Fisher Scientific). One microgram of total RNAs were aliquoted for cDNA synthesis using SensiFAST cDNA Synthesis Kit (Bioline, Catalog No. BIO-65054). qRT-PCR reactions were carried out using cDNA samples, qPCR probes (CHOP, Catalog No. Cf02654858_m1; ZC3H14, Catalog No. Cf02638760_g1) and SensiFAST Probe HI-ROX Mix (Bioline, Catalog No. BIO-82020) on the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). 2ˆ(−∆CT) values were calculated by comparing CHOP levels (target gene) to ZC3H14 levels (reference gene), and the data were further normalized to pretreatment samples. The reactions of each sample were carried out in four replicates.
Clinical response assessment
Caliper measurements of the longest dimension of one to five target and up to five nontarget lymph node or solid tumor measurements were performed independently by two clinicians and recorded in millimeters at each weekly visit. Although not a primary study endpoint, tumor response was assessed at each weekly study visit, and responses determined using the Response Evaluation Criteria for Peripheral Nodal Lymphoma in Dogs v1.0. or the veterinary RECIST solid tumor guidelines, depending on the patient's diagnosis (25, 26). Progressive disease (PD) was defined as at least a 20% increase in the sum of the mean lymph node measurements or the development of new lesions. Partial response (PR) was defined as a minimal decrease of 30% in the sum of mean lymph node measurements; dogs were considered to have stable disease (SD) if they did not meet the criteria for either PD or PR. Dogs were determined to have a complete response (CR) if all peripheral lesions were determined to be nonpathologic in size by the supervising clinicians. Dogs achieving an objective response (CR or PR) at the end of a treatment cycle were eligible for subsequent cycles of treatment. Dogs that developed PD at any point of a treatment cycle were removed from study and allowed to pursue alternative treatments. Dogs with SD were allowed to continue study if no adverse events were noted and/or could be alleviated with institution of either dose reduction or drug holiday.
Dogs with multiple myeloma were evaluated using applied International Myeloma Working Group (IMWG) criteria, initially developed for humans. Changes in serum paraprotein (M‐protein) and serum globulin concentration were calculated, and response category assigned using serum‐specific IMWG response criteria (27). M‐protein was documented by serum protein electrophoresis (SPE) ± immunofixation (IF) in an initial sample and subsequent electrophoretic evaluation of serial samples according to the study schema.
Data availability
The data generated in this study are available within the article and its Supplementary Data files.
Results
A total of 24 dogs were enrolled from March 2019 to January 2021, representing a range of solid tumors, lymphoma, and multiple myeloma diagnoses (Table 1).
Dogs enrolled in the dose-escalation cohorts.
. | Patient ID . | Cohort . | Formulation . | Tumor type . | Breed . | Age (years) . | Sex . | Weight (kg) . | Prior therapy . | Doses received/planned . | Schedule . | Response . | DLT . |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 0301 | 5 mg/kg | PIC | STS | Labrador Retriever | 13 | MC | 32.1 | Surgery, corticosteroid therapy | 8/8 | 2 weeks | SD (18% increase) | No |
2 | 0302 | 5 mg/kg | PIC | LSA B cell | Great Dane | 4 | MC | 76.5 | Corticosteroid therapy | 8/8 | 2 weeks | PD (21% increase) | No |
3 | 0501 | 5 mg/kg | PIC | Histiocytic sarcoma | Mixed breed | 10.5 | FS | 21.6 | Surgery, chemotherapy (lomustine, vinorelbine) | 8/8 | 2 weeks | SD (19% increase) | No |
4 | 0701 | 7.5 mg/kg | PIC | STS | Mixed breed | 9 | FS | 24.6 | None | 16/16 | 2 weeks | C1: SD (9% decrease) C2: PD (20% increase) | No |
5 | 0702 | 7.5 mg/kg | PIC | LSA B cell | Mixed breed | 10 | FS | 20.4 | None | 8/8 | 2 weeks | SD (5% decrease) | No |
6 | 0303 | 7.5 mg/kg | PIC | STS | Mixed breed | 8 | FS | 41.8 | Surgery | 8/8 | 2 weeks | SD (15% increase) | No |
7 | 0502 | 10 mg/kg | PIC | Plasma cell tumor | Brittany | 10 | MC | 20.3 | Radiotherapy, chemotherapy, corticosteroid therapy | 8/8 | 2 weeks | PD (8% increase), new lesion | No |
8 | 0503 | 10 mg/kg | PIC | STS | Labrador Retriever | 9.5 | MC | 29 | Corticosteroid therapy | 8/8 | 2 weeks | PD (19% increase), new lesion | No |
9 | 0704 | 10 mg/kg | PIC | STS | Mixed breed | 9 | MC | 49.5 | None | 8/8 | 2 weeks | PD (20% increase) | No |
10 | 0304 | 7.5 mg/kg | HCF | LSA w/hypercalcemia | Red Heeler | 9 | FS | 26.9 | None | 8/8 | 2 weeks | SD (11% increase) | No |
11 | 0505 | 7.5 mg/kg | HCF | STS | Whippet | 11 | FS | 16.3 | Surgery | 8/8 | 2 weeks | SD (15% decrease) | No |
12 | 0305 | 7.5 mg/kg | HCF | LSA B cell | Golden Retriever | 5 | FS | 24.8 | None | 8/8 | 2 weeks | SD (12% decrease) | No |
13 | 0710 | 7.5 mg/kg | HCF | STS | Puggle | 7 | MC | 17.2 | None | 12/12 | 3 weeks | SD (5% increase) | No |
14 | 0708 | 7.5 mg/kg | HCF | LSA | Golden Retriever | 5 | MC | 37.6 | Multiagent chemotherapy (CHOP) | 12/12 | 3 weeks | PD (42% increase) | No |
15 | 0506 | 7.5 mg/kg | HCF | Melanoma | Golden Doodle | 9 | FS | 27.7 | Surgery | 6/12 | 3 weeks | N/A | No |
16 | 0713 | 10 mg/kg | HCF | STS | Mixed breed | 12 | FS | 21.3 | Surgery, immunotherapy | 24/24 | 3 weeks | SD both cycles (6% increase, 14% increase) | Yes |
17 | 0714 | 10 mg/kg | HCF | STS | Labrador Retriever | 11 | FS | 31.5 | Corticosteroid therapy | 2/12 | 3 weeks | N/A | No |
18 | 0507 | 10 mg/kg | HCF | Metastatic SCC | Saluki | 11 | MC | 30.1 | Surgery, chemotherapy, corticosteroid therapy | 3/12 | 3 weeks | N/A | Yes |
. | Patient ID . | Cohort . | Formulation . | Tumor type . | Breed . | Age (years) . | Sex . | Weight (kg) . | Prior therapy . | Doses received/planned . | Schedule . | Response . | DLT . |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 0301 | 5 mg/kg | PIC | STS | Labrador Retriever | 13 | MC | 32.1 | Surgery, corticosteroid therapy | 8/8 | 2 weeks | SD (18% increase) | No |
2 | 0302 | 5 mg/kg | PIC | LSA B cell | Great Dane | 4 | MC | 76.5 | Corticosteroid therapy | 8/8 | 2 weeks | PD (21% increase) | No |
3 | 0501 | 5 mg/kg | PIC | Histiocytic sarcoma | Mixed breed | 10.5 | FS | 21.6 | Surgery, chemotherapy (lomustine, vinorelbine) | 8/8 | 2 weeks | SD (19% increase) | No |
4 | 0701 | 7.5 mg/kg | PIC | STS | Mixed breed | 9 | FS | 24.6 | None | 16/16 | 2 weeks | C1: SD (9% decrease) C2: PD (20% increase) | No |
5 | 0702 | 7.5 mg/kg | PIC | LSA B cell | Mixed breed | 10 | FS | 20.4 | None | 8/8 | 2 weeks | SD (5% decrease) | No |
6 | 0303 | 7.5 mg/kg | PIC | STS | Mixed breed | 8 | FS | 41.8 | Surgery | 8/8 | 2 weeks | SD (15% increase) | No |
7 | 0502 | 10 mg/kg | PIC | Plasma cell tumor | Brittany | 10 | MC | 20.3 | Radiotherapy, chemotherapy, corticosteroid therapy | 8/8 | 2 weeks | PD (8% increase), new lesion | No |
8 | 0503 | 10 mg/kg | PIC | STS | Labrador Retriever | 9.5 | MC | 29 | Corticosteroid therapy | 8/8 | 2 weeks | PD (19% increase), new lesion | No |
9 | 0704 | 10 mg/kg | PIC | STS | Mixed breed | 9 | MC | 49.5 | None | 8/8 | 2 weeks | PD (20% increase) | No |
10 | 0304 | 7.5 mg/kg | HCF | LSA w/hypercalcemia | Red Heeler | 9 | FS | 26.9 | None | 8/8 | 2 weeks | SD (11% increase) | No |
11 | 0505 | 7.5 mg/kg | HCF | STS | Whippet | 11 | FS | 16.3 | Surgery | 8/8 | 2 weeks | SD (15% decrease) | No |
12 | 0305 | 7.5 mg/kg | HCF | LSA B cell | Golden Retriever | 5 | FS | 24.8 | None | 8/8 | 2 weeks | SD (12% decrease) | No |
13 | 0710 | 7.5 mg/kg | HCF | STS | Puggle | 7 | MC | 17.2 | None | 12/12 | 3 weeks | SD (5% increase) | No |
14 | 0708 | 7.5 mg/kg | HCF | LSA | Golden Retriever | 5 | MC | 37.6 | Multiagent chemotherapy (CHOP) | 12/12 | 3 weeks | PD (42% increase) | No |
15 | 0506 | 7.5 mg/kg | HCF | Melanoma | Golden Doodle | 9 | FS | 27.7 | Surgery | 6/12 | 3 weeks | N/A | No |
16 | 0713 | 10 mg/kg | HCF | STS | Mixed breed | 12 | FS | 21.3 | Surgery, immunotherapy | 24/24 | 3 weeks | SD both cycles (6% increase, 14% increase) | Yes |
17 | 0714 | 10 mg/kg | HCF | STS | Labrador Retriever | 11 | FS | 31.5 | Corticosteroid therapy | 2/12 | 3 weeks | N/A | No |
18 | 0507 | 10 mg/kg | HCF | Metastatic SCC | Saluki | 11 | MC | 30.1 | Surgery, chemotherapy, corticosteroid therapy | 3/12 | 3 weeks | N/A | Yes |
Note: Two- and 3-week dosing using both the PIC and HCF.
Abbreviations: CHOP, cyclophosphamide/doxorubicin/vincristine/prednisone; DLT, dose-limiting toxicity; FS, female/spayed; LSA, lymphoma; MC, male/castrated; N/A, not applicable; PD, progressive disease; SCC, squamous cell carcinoma; SD, stable disease; STS, soft-tissue sarcoma.
Dose escalation and determination of MTD
Using the PIC formulation, 12 dogs were enrolled across all three initial dosing cohorts (5.0, 7.5, and 10 mg/kg), and dose de-escalation was not necessary. These cohorts included 9 dogs with either a solid tumor or lymphoma, and 3 dogs with multiple myeloma. Two of the 3 patients with multiple myeloma in this initial phase of dose escalation received 10 mg/kg of the PIC.
One dog in the first dosing cohort of 5 mg/kg (0301), a 13-year-old castrated male Labrador Retriever, with a distal extremity soft tissue sarcoma experienced significant tumoral necrosis after receiving eight doses of drug, necessitating partial amputation of the affected limb. The extent of necrosis was so severe that a partial limb amputation had to be performed 12 days later to gain control of the open wounds within the tumor site, prevent further infection and regain function/cosmesis. Caliper measurements of the tumor indicated stable disease but were considered somewhat under-representative of the volume of viable tumor.
At this point in the trial, the HCF became available, and we chose to convert to using this formulation due to improved pharmacokinetic compared with PIC in nonclinical studies. Subsequently, dogs were then enrolled into a 7.5 mg/kg bridging cohort, which included 3 dogs with lymphoma/solid tumors and 3 dogs with myeloma. Because no AEs were noted in the bridging cohort, we added an additional week of therapy, for a total of 3 weeks of treatment at 7.5 mg/kg, followed by a week of rest (Fig. 1B) in three additional dogs. No AEs were noted within this cohort; thus, we escalated to 10 mg/kg with the HCF with 3 weeks of treatment. Only 1/3 dogs in this cohort completed the cycle, due to AEs. Two of three dogs in the 10 mg/kg, 3-week treatment cohort experienced DLTs. One dog experienced dose-limiting gastrointestinal side effects, whereas another developed dose-limiting Grade IV liver enzymopathy during Cycle 2 of drug treatment. Further dose and schedule changes were halted at that point, and the MTD of CB-5339 was determined to be 7.5 mg/kg when administered Monday to Thursday for 3 consecutive weeks, using the HCF.
Clinical response to CB-5339 therapy
Clinical response data are recorded in Table 1. Of the 24 dogs enrolled, there were no complete responses, one PR (myeloma, patient #0706), one minimal response (MR; myeloma patient #0504), 13 dogs with SD, and 6 dogs with PD. Three patients were not evaluable for treatment response because they exited the study prior to Day 22 or Day 29, depending on whether they were enrolled in the 2- or 3-week dosing cohorts.
Patient #0706, a castrated male Standard Poodle with biclonal IgA myeloma and hepatic involvement, achieved a partial response to the drug administered at 10 mg/kg (PIC) and 7.5 mg/kg (HCF), receiving five total cycles of treatment (Fig. 2). The peak response occurred on Day 8 of Cycle 3 with PIC dosing, based on a 70% reduction in M protein. A slow reemergence of rising M protein level was noted during Cycle 4 with appearance of new liver lesions detected by abdominal ultrasonography but not confirmed cytologically as malignant plasma cell tumors. This canine patient was given treatment with HCF for Cycle 5 at a dosage of 7.5 mg/kg but exhibited ongoing rising M protein and subsequently exited the study.
Serial SPE measurements of M protein and other serum proteins in patient 0706, with a biclonal IgA multiple myeloma. The * denotes date of best overall response, a PR, which occurred on Cycle 3 Day 8 and represents a 70% reduction in M protein relative to baseline.
Serial SPE measurements of M protein and other serum proteins in patient 0706, with a biclonal IgA multiple myeloma. The * denotes date of best overall response, a PR, which occurred on Cycle 3 Day 8 and represents a 70% reduction in M protein relative to baseline.
Patient #0504, an intact female Boxer with a monoclonal gammopathy of unknown Ig subclass and lytic bone lesions, had a MR based on 46% reduction in β-globulin from 5.59 to 2.98 g/dL from baseline to end of Cycle 1. The dog then developed an oral mucosal lesion diagnosed cytologically as a soft tissue plasmacytoma. On the basis of this appearance of a new lesion, the dog exited the study on Day 8 of Cycle 2 to pursue other therapies.
Pharmacokinetic assessment of CB-5339 in tumor-bearing dogs
In the dose escalation cohorts using the PIC formulation, plasma concentrations of CB-5339 were highly variable between animals. When the HCF became available, subsequent animals were dosed with this material based on the more consistent pharmacokinetic observed in preclinical studies with Beagle dogs. The plasma pharmacokinetic curves for all dogs receiving PIC and HCF are shown in Figs. 3 and 4, examples also given in Supplementary Fig. S1E. Repeat pharmacokinetic curves for selected dogs enrolled in the 3-week exposure cohort are presented in Supplementary Fig. S3. Even with the use of HCF, both the extent and time course of CB-5339 absorption was highly variable in these animals. The broad range of breed, size, and disease status in these dogs may have contributed to the observed variability. On the basis of prior studies conducted in Beagle dogs that did not indicate any significant impact of feeding on pharmacokinetic, this was not thought to be a major factor in affecting absorption of CB-5339 in pet dogs.
Comparisons of pharmacokinetic exposures obtained from n = 12 patients with canine cancer receiving the PIC formulation of CB-5339. Each canine patient is represented by an identifier number and a corresponding pharmacokinetic sampling curve, obtained for 6 consecutive hours after oral dosing of CB-5339 on Day 1 of the clinical trial.
Comparisons of pharmacokinetic exposures obtained from n = 12 patients with canine cancer receiving the PIC formulation of CB-5339. Each canine patient is represented by an identifier number and a corresponding pharmacokinetic sampling curve, obtained for 6 consecutive hours after oral dosing of CB-5339 on Day 1 of the clinical trial.
Comparisons of pharmacokinetic exposures obtained from n = 12 patients with canine cancer receiving the HCF of CB-5339. Each canine patient is represented by an identifier number and a corresponding pharmacokinetic sampling curve, obtained for 6 consecutive hours after oral dosing of CB-5339 on Day 1 of the clinical trial.
Comparisons of pharmacokinetic exposures obtained from n = 12 patients with canine cancer receiving the HCF of CB-5339. Each canine patient is represented by an identifier number and a corresponding pharmacokinetic sampling curve, obtained for 6 consecutive hours after oral dosing of CB-5339 on Day 1 of the clinical trial.
Potency and PDs of CB-5083 and CB-5339 in canine cancer cell lines, PBMCs, and tumor biopsies
Cellular viability assays were performed to demonstrate comparative potency of CB-5083 and CB-5339 and to determine their suitability for further pharmacodynamic assay development. The canine lymphoma cell line 17–71 appeared more sensitive to drug treatment compared with the malignant histiocytosis line DH82, but both cell lines exhibited similar sensitivity to CB-5083 and CB-5339 as evidenced by comparable IC50 values (Supplementary Fig. S2).
Clinical fitness of K48 assays was demonstrated in tumor biopsies collected during dose escalation and bridging cohort phases (Table 2, example also given in Supplementary Fig. S1D). PBMCs were also evaluated from healthy canine donors during dose escalation phase to determine if they can serve as surrogate for tumor tissue (Supplementary Fig. S1B). However, the K48 modulation in PBMCs from canine patients were largely found to be independent of tissue response. Therefore, evaluation of K48 levels in PBMCs were not pursued at later stages of the trial. Another important observation made during the dose escalation phase biopsies was that core biopsies collected from same lesion produced highly hemolyzed lysates that were unusable for K48 assays, therefore, biopsy collection procedure was modified to completely avoid hemolysis. K48 modulation was observed to correlate with increasing plasma exposure in this limited dataset gathered from dogs receiving both drug formulations (Table 2; Supplementary Figs. S1D and S1E). These data demonstrated that modulation of K48 levels may provide an indication of p97 blockage and link the drug response to its mechanism-of-action. Serial multi-time point biopsies from 6 canine patients that received highest doses of CB-5339 were evaluated with assays for induction of K48 and CHOP (Fig. 5). Importantly, the reversal of K48 levels even after multiple doses indicated that stress induced by p97 blockage was not sustained for prolonged periods and could potentially explain a lack of antitumor response of CB-5339 in this very limited dataset. Pharmacodynamic assessment of target modulation in tumor tissue was not performed in dogs with multiple myeloma, as no accessible lesions were available for repeated sampling in these patients. Clinical responses were monitored through serum M protein measurements.
Dogs enrolled in the multiple myeloma cohort.
. | Patient ID . | Cohort . | Formulation . | Tumor type . | Breed . | Age (years) . | Sex . | Weight (kg) . | Prior therapy . | Doses received/planned . | Schedule . | Response . |
---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 0703 | 7.5 mg/kg | PIC | Multiple myeloma | Labrador Retriever | 11 | MC | 27.4 | Yes (steroids) | 16/16 | 2 weeks | C1: SD (5% increase), C2: SD (17% increase) |
2 | 0705 | 10 mg/kg | PIC | Multiple myeloma | American Staffordshire Terrier | 10 | FS | 44 | Yes (steroids) | 24/24 | 2 weeks | C1: SD (11% decrease), C2: SD (6% decrease), C3: SD (20% decrease on Cycle 3 Day 8; 10% decrease at end of Cycle 3) |
3 | 0706 | 10 mg/kg | PIC/HCF | Multiple myeloma | Standard Poodle | 9 | MC | 23.7 | None | 40/40 | 2 weeks | C1: PR (51% decrease), C2: PR (66% decrease), C3: PR (70% decrease), C4: MR (42% decrease), C5: SD (8% decrease) |
4 | 0707 | 7.5 mg/kg | HCF | Multiple myeloma | Labrador Mix | 8 | FS | 25.7 | Melphalan | 8/8 | 2 weeks | C1: PD (41% increase) |
5 | 0504 | 7.5 mg/kg | HCF | Multiple myeloma | Boxer | 4 | F | 29.9 | Yes (steroids) | 16/16 | 2 weeks | C1: MR (46% decrease in total globulins), C2: PD (new oral lesion) |
6 | 0709 | 7.5 mg/kg | HCF | Multiple myeloma | Labrador Retriever | 13 | MC | 29.5 | Yes (steroids) | 12/12 | 3 week | C1: SD (17% increase) |
. | Patient ID . | Cohort . | Formulation . | Tumor type . | Breed . | Age (years) . | Sex . | Weight (kg) . | Prior therapy . | Doses received/planned . | Schedule . | Response . |
---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 0703 | 7.5 mg/kg | PIC | Multiple myeloma | Labrador Retriever | 11 | MC | 27.4 | Yes (steroids) | 16/16 | 2 weeks | C1: SD (5% increase), C2: SD (17% increase) |
2 | 0705 | 10 mg/kg | PIC | Multiple myeloma | American Staffordshire Terrier | 10 | FS | 44 | Yes (steroids) | 24/24 | 2 weeks | C1: SD (11% decrease), C2: SD (6% decrease), C3: SD (20% decrease on Cycle 3 Day 8; 10% decrease at end of Cycle 3) |
3 | 0706 | 10 mg/kg | PIC/HCF | Multiple myeloma | Standard Poodle | 9 | MC | 23.7 | None | 40/40 | 2 weeks | C1: PR (51% decrease), C2: PR (66% decrease), C3: PR (70% decrease), C4: MR (42% decrease), C5: SD (8% decrease) |
4 | 0707 | 7.5 mg/kg | HCF | Multiple myeloma | Labrador Mix | 8 | FS | 25.7 | Melphalan | 8/8 | 2 weeks | C1: PD (41% increase) |
5 | 0504 | 7.5 mg/kg | HCF | Multiple myeloma | Boxer | 4 | F | 29.9 | Yes (steroids) | 16/16 | 2 weeks | C1: MR (46% decrease in total globulins), C2: PD (new oral lesion) |
6 | 0709 | 7.5 mg/kg | HCF | Multiple myeloma | Labrador Retriever | 13 | MC | 29.5 | Yes (steroids) | 12/12 | 3 week | C1: SD (17% increase) |
Note: Two- and 3-week dosing using both the PIC and human formulation. For assessment of response within multiple myeloma cases, all percentage increases/decreases are relative to baseline measurements of M protein unless otherwise specified.
Abbreviations: F, female/intact; MR, minimal response; PR, partial response.
Summary of pharmacodynamic modulation data from dogs receiving PIC or HCF of CB-5339.
Patient ID . | 0301 . | 0302 . | 0501 . | 0701 . | 0702 . | 0303 . | 0703 . | 0502 . | 0503 . | 0704 . | 0705 . | 0706 . |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Dose cohort | 5 mg/kg | 5 mg/kg | 5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 10 mg/kg | 10 mg/kg | 10 mg/kg | 10 mg/kg | 10 mg/kg |
Formulation | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC |
Tumor type | STS | LSA | Histiocytic sarcoma | STS | LSA | STS | Multiple myeloma | Plasma cell tumor | STS | STS | Multiple myeloma | Multiple myeloma |
AUC (h*ng/mL) | 2,311 | 1,580 | 1,925 | 4,673 | 174 | 19 | 164 | 1,179 | 572 | 292 | 1,420 | 2,278 |
K48 upregulation | 1 hour: − | 1 hour: +++ | 1 hour: + | 1 hour: +++ | 1 hour: + | 1 hour: + | nd | nd | nd | nd | nd | nd |
6 hours: + | 6 hours: − | 6 hours: + | 6 hours: +++ | 6 hours: + | 6 hours: − | |||||||
CHOP mRNA induction | 1 hour: − | 1 hour: − | 1 hour: +++ | 1 hour: − | 1 hour: ++ | 1 hour: + | a1 hour: − | 1 hour: − | 1 hour: − | 1 hour: +++ | *1 hr: − | nd |
6 hours: + | 6 hours: + | 6 hours: +++ | 6 hours: + | 6 hours: + | 6 hours: + | a6 hours: + | 6 hours: +++ | 6 hours: − | 6 hours: ++ | *6 hr: − | ||
Patient ID | 0304 | 0505 | 0305 | 0710 | 0708 | 0506 | 0707 | 0504 | 0709 | 0713 | 0714 | 0507 |
Dose cohort | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 10 mg/kg | 10 mg/kg | 10 mg/kg |
Formulation | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF |
Tumor type | LSA | STS | LSA | STS | LSA | Melanoma | Multiple myeloma | Multiple myeloma | Multiple myeloma | STS | STS | SCC |
AUC (h*ng/mL) | 3,847 | 4,183 | 232 | 76 | 11,065 | 2,701 | 412 | 345 | 2,246 | 60 | 146 | 512 |
K48 upregulation | nd | nd | nd | + | +++ | +++ | nd | nd | nd | + | + | ++ |
CHOP mRNA induction | 1 hour: +++ | 1 hour: − | 1 hour: − | + | +++ | +++ | a1 hour: ++ | a1 hour: +++ | nd | + | ++ | ++ |
6 hours: +++ | 6 hours: − | 6 hours: − |
Patient ID . | 0301 . | 0302 . | 0501 . | 0701 . | 0702 . | 0303 . | 0703 . | 0502 . | 0503 . | 0704 . | 0705 . | 0706 . |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Dose cohort | 5 mg/kg | 5 mg/kg | 5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 10 mg/kg | 10 mg/kg | 10 mg/kg | 10 mg/kg | 10 mg/kg |
Formulation | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC | PIC |
Tumor type | STS | LSA | Histiocytic sarcoma | STS | LSA | STS | Multiple myeloma | Plasma cell tumor | STS | STS | Multiple myeloma | Multiple myeloma |
AUC (h*ng/mL) | 2,311 | 1,580 | 1,925 | 4,673 | 174 | 19 | 164 | 1,179 | 572 | 292 | 1,420 | 2,278 |
K48 upregulation | 1 hour: − | 1 hour: +++ | 1 hour: + | 1 hour: +++ | 1 hour: + | 1 hour: + | nd | nd | nd | nd | nd | nd |
6 hours: + | 6 hours: − | 6 hours: + | 6 hours: +++ | 6 hours: + | 6 hours: − | |||||||
CHOP mRNA induction | 1 hour: − | 1 hour: − | 1 hour: +++ | 1 hour: − | 1 hour: ++ | 1 hour: + | a1 hour: − | 1 hour: − | 1 hour: − | 1 hour: +++ | *1 hr: − | nd |
6 hours: + | 6 hours: + | 6 hours: +++ | 6 hours: + | 6 hours: + | 6 hours: + | a6 hours: + | 6 hours: +++ | 6 hours: − | 6 hours: ++ | *6 hr: − | ||
Patient ID | 0304 | 0505 | 0305 | 0710 | 0708 | 0506 | 0707 | 0504 | 0709 | 0713 | 0714 | 0507 |
Dose cohort | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 7.5 mg/kg | 10 mg/kg | 10 mg/kg | 10 mg/kg |
Formulation | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF | HCF |
Tumor type | LSA | STS | LSA | STS | LSA | Melanoma | Multiple myeloma | Multiple myeloma | Multiple myeloma | STS | STS | SCC |
AUC (h*ng/mL) | 3,847 | 4,183 | 232 | 76 | 11,065 | 2,701 | 412 | 345 | 2,246 | 60 | 146 | 512 |
K48 upregulation | nd | nd | nd | + | +++ | +++ | nd | nd | nd | + | + | ++ |
CHOP mRNA induction | 1 hour: +++ | 1 hour: − | 1 hour: − | + | +++ | +++ | a1 hour: ++ | a1 hour: +++ | nd | + | ++ | ++ |
6 hours: +++ | 6 hours: − | 6 hours: − |
Abbreviations: CHOP, DNA damage-inducible transcript 3, also known as C/EBP homologous protein; LSA, lymphoma; nd, no data available; SCC, squamous cell carcinoma; STS, soft-tissue sarcoma; −, unchanged or decrease in signal relative to baseline; +, <30% signal relative to baseline; ++, <50% signal relative to baseline; +++, >50% signal relative to baseline; K48, ubiquitin chains branched at lysine 48.
aDenotes pharmacodynamic data obtained from circulating PBMC.
Comparison of timecourse samples representative of induction of pharmacodynamic markers K48 linkage-specific ubiquitin (left) and CHOP (right, by mRNA) in serial tumor biopsy samples taken from six selected dogs receiving the HCF of CB-5339.
Comparison of timecourse samples representative of induction of pharmacodynamic markers K48 linkage-specific ubiquitin (left) and CHOP (right, by mRNA) in serial tumor biopsy samples taken from six selected dogs receiving the HCF of CB-5339.
Discussion
Development and validation of noninvasive biomarkers of drug effect and response are increasingly important as cancer therapy enters the era of precision medicine. Pharmacodynamic assays that confirm a drug's MOA at the tumor tissue level provide proof of mechanism (POM) in vivo. The use of pharmacodynamic tools to confirm the preclinical MOA enhances the drug development process and are a foundational step toward full pharmacologic characterization. Biomarkers in cancer drug development can provide an indicator of primary pharmacodynamic effect (e.g., the first action of the drug in question on a biologic system, such as emergence of the phosphoprotein product of a tyrosine kinase), the secondary pharmacodynamic effect (the biochemical changes occurring immediately downstream of the intended molecular target, such as a reduction in phospho-ERK levels after drug inhibition of Raf kinase activity), or the tertiary pharmacodynamic effect (drug effects on cell-cycle progression, apoptosis, effector T-cell-mediated tumor cell cytolysis, and tumor cell migration/invasiveness; ref. 28).
The primary objectives of this comparative oncology clinical trial were to establish the tolerability, pharmacokinetic/pharmacodynamic relationships, and antitumor activity in dogs with naturally occurring cancers receiving the novel VCP inhibitor CB-5339. Several comparative oncology studies have sought to establish these relationships, spanning small molecules, biologics, and cell-based therapies, with the goal of optimizing the human drug development path (16).
We have determined that the MTD of CB-5339 in the tumor bearing dog to be 7.5 mg/kg/day for three 4-day cycles, which was similar to observations made in laboratory Beagle dogs. Although the sample size is somewhat limited, it appears that the 10 mg/kg dose when using the HCF has limited tolerability in tumor-bearing pet dogs. Adverse events noted in these canine patients were related mainly to gastrointestinal effects (inappetence, nausea, and vomiting) which were characterized as dose-limiting in one patient enrolled in the 10 mg/kg HCF cohort. Limited tolerability of 10 mg/kg HCF also limited the ability to discern pharmacokinetic/pharmacodynamic relationships in this cohort.
Pharmacokinetic parameters of CB-5339 in both formulations appeared highly variable in tumor-bearing dogs compared with similar measurements in laboratory Beagle dogs. This observed difference in pharmacokinetic profiles is likely due to presence of subclinical comorbidities that could exist within an aged pet dog population comprising various breeds and physical sizes (weights ranging from 16.3 to 76.5 kg) receiving a variety of diets, but not likely to be present in young, healthy Beagle dogs. These include but are not limited to insufficient drug absorption due to subclinical gastrointestinal disease, impaired renal clearance, or suboptimal hepatic function/metabolism (29). In general, a strong association between pharmacokinetic and pharmacodynamic modulation in solid tumor tissue could not be established with either formulation. This is likely due to small number of dogs in each dosing cohort, highly variable pharmacokinetic, and the lack of sufficient or available tumor tissue at each time point. In addition, the role of unmeasured active metabolites and differences in inter-dog drug metabolizing activity remain possible causes for the inconsistent relationship between drug exposure, antitumor activity, and adverse events.
CHOP and K48 were chosen as pharmacodynamic markers for this canine trial, which required creation and validation of assays for application to this species. This is the first application of such assays to clinical samples, in this case collected from spontaneous canine malignancies within the context of a veterinary clinical trial. The experience with these samples and assays in this setting paves the way for application of similar techniques to human clinical studies. These pharmacodynamic markers were chosen for their proximity to the actions of VCP. When VCP is inhibited, accumulation of proteosome-specific (K48) ubquitinated proteins occurs in the cytosol, with induction of ER stress and UPR leading to induction of expression of the transcription factor CHOP in the nuclear fraction of treated cells (30).
Several important lessons were learned within this canine trial that have bearing on the design and conduct of follow-on human studies. For example, PBMCs were evaluated from canine patients during the dose escalation phase to determine if they could serve as a surrogate for tumor tissue during monitoring of the pharmacodynamic response in vivo. However, the K48 modulation in PBMCs from canine patients were largely found to be independent of tissue response. This is likely because normal cells, functioning without overabundant protein production, are less reliant on the functions for VCP for homeostasis and thus are not appropriate surrogates for evidence of VCP inhibition in vivo.
In cases where matched pharmacokinetic and pharmacodynamic canine samples were available, there is evidence of target modulation, particularly in patients 0701, 0304, 0506, and 0708 (Table 2), which provides rationale for the use of these assays in human trials of CB-5339. Further, optimal timing of tissue collection for assessment of pharmacodynamic response was explored in this study as we moved through the dose-escalation and extended schedules of the trial design. In dogs for which multiple time point (1- and 6-hour post-dose) pharmacodynamic biopsy samples were available, comparable findings were noted between these data and prior in vitro and in vivo (mouse) effects of CB-5339 and its predecessor CB-5083, with the effect lasting only several hours (Fig. 5; refs. 14, 15). One would expect to see the immediate response of K48 ubiquitination at 1-hour post-dose, followed by transcriptional induction of CHOP at 6 hours or later, however insufficient paired samples were available to observe this as a consistent finding. In addition, multiple time point samples taken from a single malignant lesion in canine patients introduced iatrogenic procedure-associated hemorrhage and K48 assay interference, leading to incomplete datasets. Future sample collection techniques should attempt to avoid this and perhaps suggest an alternate method would be valuable in cases where biopsy samples are inherently vascular.
Although sample limitations made correlations between pharmacokinetic/pharmacodynamic and clinical response difficult, it is possible that differential pharmacokinetic exposures are linked to differential drug mechanisms of action and thus differential pharmacodynamic effects with CB-5339. This was observed with in vitro treatment of AML cell lines using CB-5339, where lower drug concentrations (0.2 μmol/L) impaired DNA repair whereas higher concentrations (0.4–>1.6 μmol/L) resulted in UPR activation and ER stress, as evidenced by polyubiquitin protein, spliced XBP-1, and ATF-4 accumulation (15). These data suggest that a concentration-dependent increase in proteotoxic stress may be occurring during CB-5339 treatment, and that antitumor activity associated with impaired DNA repair could occur at lower drug exposures. Canine tumor tissue drug levels would be needed to correlate exposures to specific mechanistic changes in this context.
The efficacy signal observed in 2/6 dogs with myeloma is encouraging for application of this agent to humans, given the role of p97/VCP in management of protein degradation through the ubiquitination–proteosome pathway and the overabundance of protein production in MM cells. It is also noteworthy that disease stabilization occurred in 14/24 (58%) of all patients enrolled in the trial, yet with the caveat that the clinical and biological significance of disease stabilization is highly dependent on the natural course of disease. It is possible that additional clinical responses may have been observed with a longer duration of CB-5339 treatment, but this was outside the bounds of this canine study. It is likely that dogs with low-to-intermediate grade STS that were not noted to be rapidly progressive at the time of enrollment experienced limited clinical benefit from CB-5339, but for dogs with histiocytic sarcoma (0501) or lymphoma (0702, 0304, 0305) maintaining stable disease is clinically significant, as these cancer types are known to be rapidly progressive without treatment or while receiving ineffective therapy (31, 32). In patient #0304, a dog with high-grade T-cell lymphoma with hypercalcemia, strong CHOP induction was evident alongside disease stabilization, which persisted for the entire 22-day study period. This is clinically significant given the aggressive nature of this lymphoma subtype in dogs (33). Indeed, a recent study of steroid-only palliation of intermediate or large-cell canine lymphoma found a median survival time of 50 days (34).
In the multiple myeloma cohort described herein, 4 dogs had IgA biclonal gammopathy, 1 dog had IgG gammopathy, and for 1 dog a specific Ig subtype immunofixation data was not available. One dog with biclonal IgA gammopathy and 1 dog with an undetermined Ig subclass exhibited a clinical response (PR and MR, respectively), 3 dogs (2 IgA, 1 IgG) had stable disease, and 1 dog with IgA gammopathy had pharmacodynamic. Interestingly, the dog with 46% reduction in serum globulin (Patient #0504) classified as an MR occurred at the end of Cycle 1 (Day 22) after only receiving 8 doses of drug. This patient did not have Ig-specific typing performed.
The observed clinical efficacy in canine multiple myeloma is likely linked to CB-5339’s MOA, which relates to induction of proteotoxic stress. However, dogs with multiple myeloma enrolled in this trial did not have easily accessible lesions from which serial biopsies could be taken for pharmacodynamic measurements. Thus, we are only able to extrapolate data from solid tumor/lymphoma pharmacodynamic samples for explanation of CB-5339’s effects on canine tumor tissue. Pet dogs spontaneously develop myeloma-related disorders (MRD), which encompass a spectrum of clinical syndromes caused by clonal expansions of neoplastic plasma cells. These include multiple myeloma, extramedullary (bone and soft tissue) plasmacytomas, IgM (Waldenstrom's) macroglobulinemia, and Ig-secreting lymphomas and leukemias (19). Dogs with multiple myeloma may present with varied clinical and pathologic abnormalities, including secreted M protein, infiltration of bone/bone marrow, tissue or organs with malignant plasma cells. The overabundant secretion of immunoglobulin (Ig) of a specific type, the M protein, can be represented by any of the Ig subclasses, either by the entire molecule or a portion thereof (i.e., light chain or Bence–Jones protein). Canine IgG and IgA myelomas occur in nearly equal frequency or with a predisposition for the IgA subclass (27, 35, 36).
A recent retrospective study evaluated how the IMWG consensus response criteria for serum paraprotein monitoring in humans could be applied to dogs with secretory multiple myeloma has been reported (27). This approach evaluated densitometric M‐protein as well as previously used markers, radial immunodiffusion (RID) or direct measurement of serum globulins, as quantifiable surrogates of disease burden, and current recommendations based on this report indicate that both SPE and IF be evaluated to quantify M protein(s) and monitor response to therapy in dogs.
Reduction in M protein reflects both the elimination of the Ig-producing malignant cells, and the serum half-lives of the specific Ig subclasses in the dog. For canine IgA the circulating half-life is 5 to 6 days, significantly shorter than IgG (approximately 21 days). Given the lifespan of circulating immunoglobulins, it would then be expected that patients receiving CB-5339 would need at least 1 cycle of treatment to be evaluable for response. IgA has previously been shown to have a short circulating half-life (<1 day to ∼4 days) in multiple species. This is not surprising given that, unlike IgG, IgA does not bind the neonatal receptor, FcRn, and therefore, cannot undergo endosomal recycling and escape from lysosomal degradation (37, 38).
Conclusions
The novel p97 inhibitor CB-5339 appears well tolerated in tumor-bearing pet dogs, with an efficacy signal identified in naturally-occurring canine multiple myeloma. Although drug pharmacokinetics were variable in the studied canine patients, evidence of pharmacodynamic target modulation was present in serial tumor biopsies procured during the clinical trial. The canine cancer patient represents a novel complementary species in which biologically-rich investigations can be conducted to define pharmacokinetic–pharmacodynamic relationships in the context of relevant clinical trial schema for humans. Such data hold significant value for translational cancer drug development efforts.
Authors' Disclosures
D.M. Vail reports grants from NIH during the conduct of the study. B.K. Flesner reports grants from Leidos during the conduct of the study. J.N. Bryan reports grants from Leidos during the conduct of the study and personal fees from ELIAS Animal Health outside the submitted work. S. Harris reports other support from Cleave Therapeutics, Inc. during the conduct of the study. J. Vargas reports other support from Cleave Therapeutics outside the submitted work and also has a patent for US 9828363 and US 10174005 issued. T. Chou reports a patent for p97/VCP inhibition impacts cell-cycle regulators and coronaviral replication in human cells pending and p97/VCP ATPase inhibitors that can prevent P97 mutation-linked motor neuron degeneration pending. No disclosures were reported by the other authors.
Authors' Contributions
A.K. LeBlanc: Conceptualization, resources, data curation, formal analysis, supervision, investigation, writing–original draft, writing–review and editing. C.N. Mazcko: Resources, data curation, formal analysis, supervision, visualization, project administration, writing–review and editing. T.M. Fan: Resources, data curation, investigation, writing–review and editing. D.M. Vail: Resources, data curation, investigation, writing–review and editing. B.K. Flesner: Resources, data curation, investigation, writing–review and editing. J.N. Bryan: Resources, data curation, investigation, writing–review and editing. S. Li: Resources, data curation, software, formal analysis, investigation, visualization, methodology, writing–review and editing. F. Wang: Resources, data curation, software, formal analysis, investigation, visualization, methodology, writing–review and editing. S. Harris: Conceptualization, resources, supervision, writing–review and editing. J.D. Vargas: Conceptualization, resources, writing–review and editing. J.P. Govindharajulu: Resources, data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–review and editing. S. Jaganathan: Resources, data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–review and editing. F. Tomaino: Resources, data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–review and editing. A.K. Srivastava: Resources, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. T. Chou: Resources, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. G.M. Stott: Resources, data curation, software, formal analysis, visualization, methodology, writing–review and editing. J.M. Covey: Data curation, software, formal analysis, visualization, methodology, writing–review and editing. B. Mroczkowski: Resources, supervision, funding acquisition, project administration, writing–review and editing. J.H. Doroshow: Conceptualization, resources, supervision, funding acquisition, writing–review and editing.
Acknowledgments
Canine SPE and IF assays were conducted at the Colorado State University Veterinary Clinical Pathology Laboratory, with special thanks to Dr. Russell Moore. NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals (National Research Council; 1996; National Academy Press; Washington, DC).” This project has been funded in whole or in part with federal funds from the NCI, NIH, under Contract Nos. HHSN261201500003 and 75N91019D00024 and Task Order No. 75N091019F00129. This work was supported by the Intramural Program of the NCI, NIH (Z01-BC006161). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).