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Targeting immune checkpoint receptors expressed in the T cell synapse induces active and long-lasting antitumor immunity in preclinical tumor models and oncology patients. However, traditional nonhuman primate (NHP) studies in healthy animals have thus far demonstrated little to no pharmacological activity or toxicity for checkpoint inhibitors (CPIs), likely due to a quiescent immune system. We developed a NHP vaccine challenge model in Mauritius cynomolgus monkey (MCMs) that elicits a strong CD8+ T cell response to assess both pharmacology and safety within the same animal. MHC I-genotyped MCMs were immunized with three replication incompetent adenovirus serotype 5 (Adv5) encoding Gag, Nef and Pol simian immunodeficiency virus (SIV) proteins administered 4 weeks apart. Immunized animals received the anti-PD-L1 atezolizumab or an immune checkpoint-targeting bispecific antibody (mAbX) in early development. After a single immunization, Adv5-SIVs induced T-cell activation as assessed by the expression of several co-stimulatory and co-inhibitory molecules, proliferation, and antigen-specific T-cell response as measured by a Nef-dependent interferon-γ ELIspot and tetramer analysis. Administration of atezolizumab increased the number of Ki67+ CD8+ T cells, CD8+ T cells co-expressing TIM3 and LAG3 and the number of CD4+ T cells co-expressing 4–1BB, BTLA, and TIM3 two weeks after vaccination. Both atezolizumab and mAbX extended the cytolytic activity of the SIV antigen-specific CD8+ T cell up to 8 weeks. Taken together, this vaccine challenge model allowed the combined study of pharmacology and safety parameters for a new immunomodulatory protein-based therapeutic targeting CD8+ T cells in an NHP model.

The demonstration that antibodies can be used to treat cancers has revolutionized the field of cancer immunotherapy and gave hope to many patients. Originally targeting tumor-specific antigen, new therapeutic antibodies target now immune checkpoint markers expressed on T cells. The advantage is to generate an active and long-lasting antitumor immunity with the achievement of a broad and polyclonal antitumor immunity, addressing better the heterogeneity of cancers and reducing the chances of immune escape. The development of such highly specific molecules is particularly challenging however when it comes to preclinical safety assessment and efficacy studies. An ideal animal model needs to possess the target so that the drug product is pharmacologically active. Moreover, such model should ideally provide further insight in determining the pharmacological action of the product and the determination of safety to maximize the usefulness in animal studies. Intriguingly, no equivalent of the traditional T-cell-dependent antibody response (TDAR) assay, which evaluates mainly the CD4+ T cells response, is currently available for the CD8+ T cell response. The goal of this study is to fill this gap with a Non-Human Primate (NHP) challenge model that will elicit a potent CD8+ T cell response. MHC I-genotyped Mauritian cynomolgus macaques (MCMs) were immunized with 3 replication incompetent recombinant adenovirus serotype 5 (Ad5) vectors encoding Gag, Nef or Pol SIV proteins. MCMs were distributed into 3 groups and received two intramuscular injections of adenoviruses 4 weeks apart. One group received the anti-PD-L1 Atezolizumab and a second received a molecule in early development targeting another immune checkpoint molecule. T cell activation was monitored with blood samples taken on a weekly basis for 8 weeks. Blood samples were used to characterize the T cell immunophenotypes and to correlate them with some functional assays. Vaccination was well tolerated and could be conducted on standard safety toxicology studies without compromising other endpoints. Adv5-SIVs modulated total T cells 2 weeks after a single vaccination, induced T-cell activation, T-cell proliferation and the expression of several checkpoint molecules. Moreover, functional assays confirmed that T-cell activation profile correlated with an antigen-specific CD8 T-cell response. Compounds administered differently enhanced the profile of T-cell activation, the upregulation of immune checkpoint molecules and antigen specific responses of their T cells. Moreover, both compounds extended the cytolytic activity of the antigen-specific CD8+ T cell up to 8 weeks. Taken together, the vaccination model we developed seems appropriate to study the pharmacology of new protein-based therapeutics targeting CD8+ T cells. Citation Format: Richard Graveline, Morad Haida, Carolyne Dumont, Dominic Poulin, Florence Poitout-Belissent, Rana Samadfan, Sven Kronenberg, Franziska Regenass-Lechner, Rodney Prell, Marie-Soleil Piche. A simian challenge model to evaluate the CD8-positive T cell response with cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB097.

Method validation is a cornerstone on which biomarker development and utilization rest. However, given the abundance of biomarker candidates that are being identified and characterized, validation of these entities for the use in nonclinical studies can be complex. The objective of this continuing education course was to review current practices and challenges encountered during the validation of methods for the analysis of novel biomarkers. Additionally, the importance of biological validation and correlation with pathology end points for biomarker candidates was discussed. This article is a summary of the materials presented at the 36th Annual Symposium of the Society of Toxicologic Pathology for a continuing education course titled “Current Practices and Challenges in Method Validation.” The speakers were subject-matter experts in the validation of quantitative mass spectrometry, multiplex binding assays, biological biomarkers, and immunophenotyping and anatomic and clinical pathology considerations in biomarker qualification.

In October 2013, the International Life Sciences Institute - Health and Environmental Sciences Institute Immunotoxicology Technical Committee (ILSI-HESI ITC) held a one-day workshop entitled, “Workshop on Cytokine Release: State-of-the-Science, Current Challenges and Future Directions”. The workshop brought together scientists from pharmaceutical, academic, health authority, and contract research organizations to discuss novel approaches and current challenges for the use of in vitro cytokine release assays (CRAs) for the identification of cytokine release syndrome (CRS) potential of novel monoclonal antibody (mAb) therapeutics. Topics presented encompassed a regulatory perspective on cytokine release and assessment, case studies regarding the translatability of preclinical cytokine data to the clinic, and the latest state of the science of CRAs, including comparisons between mAb therapeutics within one platform and across several assay platforms, a novel physiological assay platform, and assay optimization approaches such as determination of FcR expression profiles and use of statistical tests. The data and approaches presented confirmed that multiple CRA platforms are in use for identification of CRS potential and that the choice of a particular CRA platform is highly dependent on the availability of resources for individual laboratories (e.g. positive and negative controls, number of human blood donors), the assay through-put required, and the mechanism-of-action of the therapeutic candidate to be tested. Workshop participants agreed that more data on the predictive performance of CRA platforms is needed, and current efforts to compare in vitro assay results with clinical cytokine assessments were discussed. In summary, many laboratories continue to focus research efforts on the improvement of the translatability of current CRA platforms as well explore novel approaches which may lead to more accurate, and potentially patient-specific, CRS prediction in the future.

Introduction Manipulating the immune system to achieve therapeutic efficacy in cancer treatment has led to a new class of drugs that brought lasting remissions to many patients who had run out of options. The development of new immunotherapy aimed at targeting cancer cells requires the ability to monitor specifically the immune response. Although CD8 T cells play a crucial role in this process, no equivalent of the traditional T-cell-dependent antibody response (TDAR) assay, which evaluates the T-helper immune response, is currently available. Since cancer immunotherapy agents are often tested in non-tumor bearing NHP for safety assessment, the animal models currently available do not allow monitoring of potential exaggerated pharmacology and efficacy of these new drug candidates. Objectives The goal of this study was to develop an NHP vaccination model that specifically elicits a CTL response, in order to evaluate the efficacy in exacerbating the Cytotoxic T Lymphocyte (CTL) response. Experimental Procedures To reach this goal, MHC-genotyped Mauritian cynomolgus macaques (MCMs) were immunized with 3 replication incompetent recombinant adenovirus serotype 5 (Ad5) vectors, each containing the coding sequence for Gag, Nef or Pol SIV proteins. Such model allowed monitoring of the CD8 T cell activation in lieu of a tumor or live virus model. MCMs were distributed into 3 groups: one control group and 2 groups which received two intramuscular injection of the adenovirusrs spaced by 4 or 8 weeks. The immune response was monitored with blood samples taken on a weekly basis for up to 12 weeks. Blood samples were used to 1) characterize the different CD8-positive sub-populations by Tetramer staining and Immunophenotyping; 2) to correlate the immunophenotyping profile obtained with functional assays such as ex vivo recall response and IFNγ ELIspot. Results Of the three groups tested, MCM monkeys receiving 2 injections 8 weeks apart showed the more robust CD8 T cell response. This response was mainly characterized by a proliferation profile (Ki67-positive cells), the expression of activation markers at the surface of CD8 T cells and antigen specific responses of the CD8 T cells. Of the three Gag, Pol and Nef proteins, immunization with Nef gave the most robust response, as observed by the IFNγ ELIspot results and by the presence of a Nef-specific CD8-specific subpopulation observed by tetramer staining. No changes in the distribution of T, B, NK, Treg or Memory T cells was observed between the 3 groups. Conclusion The results obtained during the course of this study suggests that the described CD8-specific vaccination model using Mauritius cynomolgus macaques is a promising model to evaluate the efficacy and potential exaggerated pharmacology of new drug candidates targeting CD8 T cells. Citation Format: Richard Graveline, Morad Haida, Carolyne Dumont, Rana Samadfam, Dominic Poulin, Marie-Soleil Piche. Development of a vaccine model to track the CD8-specific response in Mauritius cynomolgus monkey [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-269.

Many therapeutic monoclonal antibodies (mAbs) are in development for a wide array of indications. Because of the nature of the product and/or the intended pharmacology, mAbs are assessed for their potential immunomodulation, whether intended or not. Immunomodulation and immunotoxicology (i.e., immunosuppression, immunogenicity, immunostimulation) can be related. The intended immunomodulatory effect of mAbs is the intended pharmacology for clinical efficacy. Exaggerated or prolonged pharmacological activity may, however, result in adverse immunomodulation, leading to unpredictable and unintended immunotoxicology outcomes. Nonhuman primates (NHPs) are often considered as the most relevant species to perform preclinical studies for the testing of mAb-induced immunomodulation and immunotoxicity. This chapter focuses on regulatory considerations associated with the testing of mAbs in NHPs, the mAb product and biomanufacturing process characteristics to take into consideration for immunogenicity testing, and the preclinical study designs in adult and juvenile NHPs to monitor immunomodulation.