62^ND SOT ANNUAL MEETING— ALTASCIENCES’ SCIENTIFIC PRESENTATIONS

On Wednesday, March 22, at 9:00 a.m., Room 101A, Dr. Norbert Makori, Vice President, Toxicology, and Dr. Kriscelle Mendoza, Director, Veterinary Services, will present on CNS and ocular delivery in nonhuman primates for new drug modalities.

To learn about our early phase drug development solutions, pre-book time with one of our experts or meet with us at booth #633.

ABSTRACTS:

Minipigs (MPs) are recognized as offering advantages over other established non-rodent models, such as beagle dogs, based on substantial evidence of similarities to humans with regard to anatomy, physiology, and biochemistry. Currently, MPs are used increasingly in nonclinical CROs and by the biopharmaceutical industry to support IND-enabling toxicology studies. However, similarities and differences in toxicological reference data between the commonly used Göttingen^TM and Sinclair^TM breeds have not been reported. To provide scientific justification for selection of the most appropriate strain of MP for a drug development program, and as part of the Altasciences Historical Control Database initiative, this study was performed to compare reference baseline/background data for a battery of standard toxicological parameters obtained from Sinclair^TM and Göttingen^TM MP studies conducted at Altasciences.

Data for Göttingen^TM MPs was extracted from the electronic data capture system (Pristima^®) and compared with the reference data of downsized Sinclair^TM MPs (Book of Normals 2021; SBR), including body weight, clinical pathology (hematology, serum chemistry, coagulation, urinalysis), organ weights, and histopathology background lesions of a panel of tissues from nine physiological organ systems. Multiple statistical analyses, including mean and Standard Deviation (SD), range (min, max), fold difference of average, quartile, interquartile range (IQR) and Tukey fence (upper and lower limit), were used for data comparison.    

Body weights were similar between these two MP strains up to approximately three months of age. There was considerable overlap of mean and statistical clinical pathology values between Sinclair^TM vs. Göttingen^TM MPs, except that globulin, lymphocyte, and monocyte values were significantly higher (two to threefold) in Sinclair^TM MPs compared to Göttingen^TM MPs. In addition, brain and thymus weights for Sinclair^TM MPs were higher than Göttingen^TM MPs in both males (1.4-fold and 2.5-fold, respectively) and females (1.3-fold and 1.4-fold, respectively). The sex differences of organ weight to brain or body weight ratio were also observed, as the heart and adrenal weights to brain ratio were lower, while thymus weight to body weight ratio was higher in male (only) Sinclair^TM MPs as compared to Göttingen^TM MPs. The most common microscopic finding noted in Sinclair^TM MPs was multifocal lymphohistiocytic infiltration in various tissues. The detailed descriptions of differences, as well as similarities of spontaneous histopathological findings and incidence rate, will be reported and discussed.    

Based on data comparison in this study, all apparent differences among clinical pathology, organ weight, and background microscopic findings between Sinclair^TM vs. Göttingen^TM MPs were considered minor in magnitude and biological significance. This study, for the first time, provides assessment criteria for minipig strain selection, data quality control, and interpretation of results in preclinical toxicity studies using downsized Sinclair^TM MPs, which have similar toxicological reference data to Göttingen^TM, and are more cost effective and readily available at different ages for customized study design for a drug development strategy.

The objective of this study was to examine the in vivo retinal microanatomy of findings observed during pretest ophthalmic examination in laboratory rabbits. Five to 14-month-old Dutch-Belted rabbits were evaluated via complete ophthalmic examinations that included slit lamp and indirect ophthalmoscopy, fundus photography and confocal scanning laser ophthalmoscopy, and spectral domain-optical coherence tomography (cSLO/sd-OCT). Normal and affected eyes were collected and evaluated microscopically after in vivo imaging. Five male and two female rabbits presented unilateral or bilateral, oval, pink hued lesions superior to the optic nerve head. Lesions were autofluorescent (AF) under AF mode. OCT showed focal retinal detachment, AF material, likely lipofuscin, accumulated between photoreceptor and RPE layers. Hematoxylin and Eosin-stained histology sections showed retinal detachment, outer retinal atrophy, and subretinal accumulation of amorphous amphophilic material. Serial two-photon plus system (STP²) showed the location of the lesions and detached retina with material accumulation in the subretinal space in 2D and 3D images. In conclusion, fundus abnormalities often observed during pretest ophthalmic examination and broadly categorized as chorioretinal scars have been further characterized to identify specific microarchitecture changes. This is the first report correlating in vivo microanatomical and histological description of these retinal findings in Dutch-Belted rabbits.

Preclinical studies provide a wealth of information for organizations looking to submit their Investigational New Drug for clinical trials. Questions often asked relate to which assay to employ (i.e., multiplex or single-plex), which platform to use, and the reliability of the results generated. The most challenging assays (single-plex or multiplex) to validate are ones that look for endogenous markers. We retrospectively examined and validated an assay for the determination of complement protein SC5b-9, using a Quidel^® MicroVue™ SC5b-9 Plus EIA kit and with cynomolgus monkey K2EDTA plasma. The instrumentation that was used for conducting the analysis was a Synergy H1 System. Our analysis allowed us to fully validate and implement a new analytical method for quantifying SC5b-9 that can be used on both Good Laboratory Practice (GLP) and non-GLP studies for nonhuman primate (NHP) K2EDTA plasma samples. 

SC5b-9, known as the terminal complement complex (TCC), is an assembly of proteins created through the joining of protein C5 and C9. SC5b-9 can be formed from either the classical, lectin, or alternative pathways. The advantage of validating this kit is that it provides a full picture of what is occurring in the NHPs complement system. 

The kit contains a pre-coated plate that uses a monoclonal antibody that attaches to the TCC’s C9 ring, which then captures the complex. After an incubation with the samples, horseradish peroxidase (HRP)-conjugated antibodies are added and bind to the SC5b-9 complex antigens. 3,3’,5,5’-tetramethylbenzidene (TMB) is added after the incubation followed by the stop solution, and then the plate is analyzed on the Synergy H1 at 450 nm. 

For our SC5b-9 method validation, the following parameters for testing included intra/inter-accuracy and precision, dilutional linearity, specificity, selectivity, sensitivity, parallelism, 4 ⁰C stability (benchtop), freeze/thaw stability, and long-term stability. The intra/inter accuracy and precision were within the +/- 25% bias for ULQC and LLQC, and HQC and LQC (both provided reconstituted by the kit) were in range provided by the Certificate of Analysis, and 4.5 hours of benchtop, three freeze/thaw cycles and 66 days of stability were established. Parallelism will be assessed if a native sample can be found with high enough SC5b-9 concentrations. 

As expected, the most difficult parameter by far was selectivity, as this kit was very sensitive to picking up endogenous SC5b-9. All samples were removed from the freezer (-20 ⁰C for individual plasma/5% hemolyzed plasma for selectivity and specificity, -80 ⁰C for stability samples) and then placed on dry ice. Samples were then thawed rapidly in a 37 ⁰C water bath, and then placed on wet ice (equivalent to 4 ⁰C) until added to the analysis plate. Samples were processed in a timely fashion to prevent any endogenous SC5b-9 complexes from clumping together. In the original set of 10 non-hemolyzed NHP plasma samples, 5 males and 5 females, along with a 5% hemolyzed male plasma sample, 3 males and 1 female, did not meet the acceptance criteria as stated in the Bioanalytical Method Validation Guideline (May 2018) for the Lower Limit of Quantitation (LLOQ), nor did the 5% hemolyzed plasma. For these original 11 selectivity samples, nine out of 11 met acceptance criteria for the HQC level. Additional individual samples were tested at the LLOQ level, along with a different 5% hemolyzed plasma (female for this plasma). Of the new samples that were tested, 80% of the individual plasma samples met acceptance criteria for LLOQ. The original 11 Selectivity samples were also tested for Specificity, i.e., blank plasma is tested to see if it will fall into the range of the assay. Ten out of 11 samples met acceptance criteria. Only one male (one that failed both LLOQ and HQC for selectivity) failed to meet acceptance criteria for Specificity. 

Quantitation of phospho-STAT5 (pSTAT5) is a simple and effective way to assess T cell proliferation and is a useful tool for toxicological studies. Flow cytometry has recently become a common platform for pSTAT5 detection which, unlike more labor-intensive methods, such as western blotting, is able to report endogenous pSTAT5 levels in multiple cell populations in a precise and high-throughput manner. This is achieved by measuring levels of a detector fluorophore conjugated to anti-pSTAT5 antibody coupled with a panel of other immunophenotyping markers. Flow cytometry pSTAT5 assays have previously been described in literature in primary and secondary cell lines for discovery or diagnostic purposes. However, to date, have not been developed in Macaca fascicularis for the purposes of a toxicological study. Here, we detail our method development of a flow-cytometry based pSTAT5 assay in nonhuman primates (NHPs) where we isolated peripheral blood from six naïve Macaca fascicularis and stimulated ex vivo with human recombinant IL-2, followed by staining with a pSTAT5 immunophenotyping panel. pSTAT5 levels were quantitated in CD4+ T cells, PD1+CD4+ T cells, CD8+ T cells, PD1+CD8+ T cells, and CD25+CD4+ T cells as median fluorescent intensity (MFI) and relative percentage of parent. In this method development, we 1) measured assay feasibility on the LSR Fortessa flow cytometer, 2) measured reproducibility between analysts and individual replicates, 3) compared pSTAT5 activation between peripheral blood and PBMCs, and 4) tested a fluorescence minus one control (FMO) for each fluorophore. Our results indicated successful upregulation of pSTAT5 following stimulation with IL-2 as well as a high degree of reproducibility among analysts and replicates. Addition of IL-2 to whole blood resulted in a 16- to 118-fold increase of pSTAT5, varying based on T cell subset. Reproducibility was determined by comparing percent coefficient of variance (CV) between replicates for all cell populations: high prevalence populations were < 20% CV; low prevalence populations were < 25% CV; and very low populations were < 30% CV for all replicates, except Total CD3+ cells in one female. PBMCs displayed a one to 13-fold increase in pSTAT5 upregulation compared to whole blood after incubation with IL-2. FMOs displayed < 2% of positive cells for each control, indicating minimum background staining for this panel. This method development provided a novel technique of measuring pSTAT5 by flow cytometry in Macaca fascicularis blood and has practical applications for future NHP-based preclinical toxicology studies.

With the continued increase in development of biological and immune-modulating therapeutics, there is a need to assess potential toxicity of the immune system, especially in relation to other toxicity parameters. Assessment of the immune system is routinely achieved through addition of endpoints, such as immunophenotyping of circulating cells. Due to the nature of these therapeutics, the cynomolgus macaque is often the most appropriate test system to use. A new development is that body weights of cynomolgus monkeys available for preclinical studies have decreased significantly due to high demand and use of animals in research. Literature suggests that nutritional inadequacy can negatively impact the immune system resulting in an increased incidence of infection in humans. 

To improve the assessment of drug-related impact on the immune system, the typical phenotype of circulating immune cells was established in both young (two to three years) and mature (≥ 3 years) test article-naïve cynomolgus macaque. Blood was collected while animals were conscious and restrained. T cells, helper T cells, cytotoxic T cells, total B cells, natural killer (NK) cells, and monocytes were quantified using the BD FACSCanto™ II flow cytometer. Linear regression was performed to determine if there was a correlation between animal body weight and the relative circulating immune cell populations. The initial analysis revealed that there was no correlation between body weight and the relative cell populations in the young and mature animals. Furthermore, there was no difference in the relative cell population counts when comparing the young and mature animals.

Recently, there have been significant advances in gene therapy utilizing viral and non-viral vectors at both the preclinical and clinical stages of drug development. In the preclinical space, safety assessment of adeno-associated viral (AAV) vector-based therapeutics requires use of nonhuman primates (NHPs) as the main species; the presence of naturally occurring neutralizing antibodies (nAbs) in the primate population necessitates screening of a large number of animals to obtain an adequate negative titer cohort for the study. Additionally, pre-treatment with corticosteroids prior to AAV vector administration is often required to counter any adverse reactions. Given the unique challenges of working with AAV vector-based test articles, we reviewed data collected in a large number of toxicology studies conducted in the past few years with the aims of (1) establishing ranges for the number of animals to screen for nAbs; (2) establishing the dosage range for commonly used corticosteroid given prior to AAV vector administration; and (3) describing the most common in-life findings. Approximately 37% (182/491) of animals screened for nAbs against AAV8 were suitable for study assignment based on established criteria for negative or low viral titers by AAV neutralizing antibody assay (≤ 5 nAb50 in HEK293 cells). Pretreatment with 2 mg/kg of dexamethasone at approximately one to two hours prior to AAV administration was adequate to mediate immune related responses. There was no discernable effect of AAV administration on body weight, and most abnormal post-dose clinical signs were minor and not directly attributable to the AAV vector. In conclusion, this historical dataset serves as a guide for more informed study designs for AAV vector-based therapeutics, and allows for potential reduction and refinement of animal use in their safety testing. 

It is essential that the scientific community continually re-evaluates study designs and technology that will enable reduction in the number of subjects used for toxicology studies, an approach that aligns with the 3Rs of experimental animal welfare (replacement, reduction, and refinement).

In rodents, we adopted blood microsampling as a refinement to the more common technique of needle and syringe by leveraging the Mitra^TM volumetric absorptive microsampling (VAMS^®) device for accurate and precise collection of low sample volume. For example, by microsampling only 10 μL of whole blood, an entire cohort of study animals in the traditional needle and syringe collection was eliminated from the project since serial samples could also be collected from the main study animals. This represented a reduction in the number of study animals by 55% and 100% for mouse and rat studies, respectively. Collection of samples from the same cohort of animals allows correlations between PD findings and the actual drug exposure profile. 

In nonhuman primates (NHPs) and dogs, we carefully reviewed the number of animals in control and recovery cohorts in chronic studies that had data from prior subacute studies. This enabled reduction by > 25% in number of control group, with no recovery cohort included in the low-dose group. The NHP study design was reviewed and accepted for conduct by a regulatory agency. These approaches will be discussed to highlight the pros and cons of each for a more informed decision when designing toxicity studies.

Physical or chemical restraint has been historically used in the laboratory setting to allow for humane and efficient handling of nonhuman primates (NHPs), while minimizing animal stress or distress. When stress occurs, there are alterations to physiological homeostasis or psychological wellbeing of the animal. Among the forms of restraint, chairs have been shown to be the preferred method for research studies with NHPs. For this procedure, animals are fitted with a neck collar to which a pole can be attached and used to guide the animals from their home cage to the restraint chair termed pole and collar/restraint chair for this study (PC/restraint chair). This method allows for the application of positive restraint techniques with limited use of negative reinforcement (NRT), which increases animal cooperation, reduces distress and use of anesthesia, and encourages increased cognitive stimulation. The other method of interest for this study, the procedure cage, relies primarily on physical (hand) contact and aspects of negative reinforcement training (NRT), where the animal moves into position for a procedure at the front of the cage, and releasing the squeeze-back as soon as the desired movement toward the cage front is achieved. The goal of this study was to investigate the differences in outcomes of use of these methods on toxicological assessment in NHP studies and provide evidence for best practice in restraint in a laboratory setting. A retrospective analysis on acclimation data from six studies was performed: three studies with 83 males and 87 females that employed the procedure cage, and three studies with 45 males and 75 females that used the pole and collar and restraint chair. The animals were acclimated over at least six sessions to ensure that they were adequately adapted to the restraint method with provision of visual access to restraint devices where applicable (i.e., chair and pole). Surrogate measures of stress (neutrophil, eosinophil, monocyte, and lymphocyte count, i.e., stress leucogram), inflammation (albumin, fibrinogen, and prothrombin time), and muscle damage (creatine kinase) levels, which are typically evaluated in toxicological assessments, were collected during the acclimation period, and compared between the two restraint methods. A two-way ANOVA followed by the Šídák's multiple comparisons test was used to evaluate differences in these clinical pathology parameters for each restraint method. Neutrophilia (p= 0.4684) and lymphopenia (p=0.0026) indicative of the stress leucogram were present when the procedure cage was used compared to the use of the PC/restraint chair method. Levels of creatine kinase and fibrinogen were significantly higher in procedure cage vs. PC/restraint chair animals (p=0.0027) and (p=0.0041) respectively, suggestive of acute muscle damage and inflammation. This study is a direct clinical evaluation of the procedure cage vs. PC/restraint chair, and provides evidence that the PC/restraint chair leads to less destress and inflammation, resulting in limited alterations to physiological homeostasis translating to psychological wellbeing of animals.

Session Description    
New drug modalities require specialized delivery methods to the central nervous and ocular systems during early discovery. This, in turn, requires development of unique techniques and training. This presentation provides detailed examples of some of these methods and applications in gene and cell therapy testing in nonhuman primates. 

Educational Learning Objective    
The unique nature of enhanced delivery and availability of age-appropriate nonhuman primates have potential to impact timeline to market for a drug. This presentation aims to inform the audience on best practices based on case studies. The data will confirm successful development of delivery methods that have a wide application.

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• The Altascientist: Navigating the IND Submission Process
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