Development of T cell engagers (TCEs) for oncology indications is regulated mainly by ICH S6 and S9. Investigational new drug applications are usually supported by a 1-month toxicology study in a relevant animal species. Before the start of registrational clinical trials, a longer-duration toxicology study, typically 3 months, is performed in the same species to support marketing. As longer-term studies with human-specific biologics in animal species can be hampered by development of anti-drug antibodies and TCEs are quick-acting molecules, we analyzed the value of 3-month toxicology studies for some of our TCEs. We present data from 1- and, where applicable, 3-month toxicology studies for 4 TCE programs and describe our interactions with regulatory agencies. In none of the cases did the 3-month studies reveal new information to influence further the respective clinical development plans. Considering 3Rs (Replacement, Reduction and Refinement) in pharmaceutical development, we highlight that 3-month studies with TCEs don't always offer additional safety insights. Therefore, alternative strategies should be evaluated and conduct of 3-month studies carefully considered in the light of 3Rs and discussed case-by-case with health authorities.

Supplementary Figure 2 Representative photomicrographs of DLL3 immunostaining (brown color) in normal human tissues using immunohistochemistry.

Supplementary Figure 5 A, DLL3 RNA expression levels in a panel of SCLC patient-derived xenograft (PDX) models compared with positive (NCI-H82) and negative (NCI-H460) control SCLC cell lines. Data are reported as relative quantification normalized to NCI-H460 cell line expression. B, DLL3 protein expression levels in a panel of SCLC PDX models compared with positive (NCI-H82) and negative (NCI-H460) control SCLC cell lines. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Supplementary Figure 6 A, Expression of human CD25, CD69, PD-1, and 4-1BB on human CD4+ T cells isolated from mouse lungs harboring SHP-77 tumors 168 hours after treatment with AMG 757 or a control HLE BiTE® molecule. B, Expression of human CD25, CD69, PD-1, and 4-1BB on human CD8+ T cells isolated from mouse lungs harboring SHP-77 tumors 168 hours after treatment with either AMG 757 or a control HLE BiTE® molecule. *P < 0.05; **P < 0.01; *** P< 0.001;

Background Checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) have demonstrated clinical efficacy in advanced melanoma, but only a subset of patients with inflamed tumors are responsive. Talimogene laherparepvec (T-VEC), a modified herpes simplex virus type 1 (HSV-1) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), is a first-in-class oncolytic immunotherapy approved for the treatment of melanoma and has been shown to inflame the tumor microenvironment. To evaluate the potential and mechanisms of T-VEC to elicit systemic antitumor immunity and overcome resistance to checkpoint inhibitors in murine tumor models, OncoVEX mGM-CSF was developed similarly to T-VEC, except the human GM-CSF transgene was replaced with murine GM-CSF. Previous work had demonstrated that OncoVEX mGM-CSF generated systemic antitumor immunity dependent on CD8+ T cells in an immune checkpoint-sensitive tumor cell model. Methods A novel B16F10 syngeneic tumor model with both HSV-1−permissive subcutaneous tumors and HSV-1−refractory experimental lung metastasis was used to study the local and systemic effects of OncoVEX mGM-CSF treatment alone or in combination with checkpoint inhibitors. Results Intratumoral injection of OncoVEX mGM-CSF in combination with an anti-CTLA-4 or anti-PD-1 blocking antibody led to increased tumor growth inhibition, a reduction in the number of lung metastases, and prolonged animal survival. OncoVEX mGM-CSF induced both neoantigen-specific and tumor antigen-specific T-cell responses. Furthermore, cured mice from the combination treatment of OncoVEX mGM-CSF and anti-CTLA-4 antibody rejected tumor rechallenges. Conclusions These data support the concept that T-VEC and checkpoint inhibition may be an effective combination to treat patients with advanced melanoma.

The advancement of an investigational new drug in humans is a significant developmental milestone. In first-in-human (FIH)-enabling toxicology studies, the highest dose without a test article–related adverse effect (no-observed-adverse-effect-level [NOAEL]) serves as the basis for deriving a safe FIH starting dose. For anticancer pharmaceuticals, the FIH dose may be calculated using the highest non-severely toxic dose (HNSTD) in nonrodent models or the dose severely toxic to 10% (STD10) in rodents. Given the practice of reporting the NOAEL, but the lack of regulatory requirements to do so for anticancer pharmaceuticals, we conducted an informal survey of 20 companies to answer the question “How is our industry reporting toxic/adverse dose levels in FIH-enabling toxicology studies for anticancer indications?” The data indicated 4 reporting approaches, each providing a path to regulatory acceptance. Within the integrated toxicology study report, 45% of respondents report the HNSTD/STD10, 25% report the NOAEL, 20% report both the HNSTD/STD10 and NOAEL, and 10% do not define either, reserving definitions for regulatory submissions. One reporting approach may be preferred over another for reasons including consistency across indications, repurposing pharmaceuticals, regulatory feedback, or simplicity. The reporting approach should be defined in advance of study initiation, and the pathologist should provide context to support the chosen approach.

Supplementary FIgure 4 AMG 757 demonstrated antitumor activity in the WM266-4 melanoma xenograft model.

Supplementary Figure 1 DLL3 is differentially expressed in SCLC tumors and cell lines compared with normal tissues.

AMG 337 KINOME scan binding data

Supplementary methods of tumor xenograft studies and light microscopic evaluation of tumor xenografts

Supplementary Figure 3 Evaluation of DLL3 expression in tumor types other than SCLC.

High-level focal amplification of MET is associated with sensitivity to AMG 337

Figure S1. Kinase interaction map for AMG 337; Figure S2. In a large unbiased cancer cell line viability screen only MET-amplified cell lines were sensitive to treatment with an analogue of AMG 337 (Compound 5); Figure S3: AMG 337 inhibits the phosphorylation of MET and but not its downstream effectors in MET-amplified, KRAS mutant NSCLC cell line NCI-H1573; Figure S4. Cell lines harboring MET FISH scores >3 exhibited sensitivity to AMG 337. MET FISH analysis was performed on a subset of cancer cell lines exhibiting elevated MET gene copy number; Figure S5. Selective inhibition of MET exhibits partial effects on the viability of U-87 MG glioblastoma cells harboring an HGF/MET autocrine loop; Figure S6. Increases in MET gene number correlate with high levels of total MET protein; Figure S7. AMG 337 inhibits Gab-1 phosphorylation in a concentration dependent manner in the TPR-MET mouse tumor model.

Enzymatic and cellular potency of Amgen Compound 5

This file contains Supplementary Tables 1-5

Sexually mature nonhuman primates are often used in nonclinical safety testing when evaluating biopharmaceuticals; however, there is limited information in historical control databases or in the published literature on the spontaneous findings in the male reproductive system. This review evaluated digital slides from the male reproductive tract (testes, epididymides, prostate, and seminal vesicles) in sexually mature cynomolgus macaques ( Macaca fascicularis; n = 255) from vehicle control groups in nonclinical toxicology studies and compared the observations with body weight, organ weight, and geographical origin. The most common microscopic findings were hypospermatogenesis and tubular dilatation in the testes; inflammatory cell infiltrate, cellular debris, and decreased sperm in the epididymides; inflammatory cell infiltrate and acinar dilatation in the prostate; and corpora amylacea and atrophy in the seminal vesicles. There were a few correlative observations in animals when grouped by weight or geographical origin: animals with lower terminal body weights (<5 kg) often displayed features of late puberty despite having sperm in the epididymis, while animals originating from Mauritius had a lower incidence of inflammatory cell infiltrates than those from Southeast Asia/China. This review provides incidence, descriptions, and photomicrographs of the common spontaneous microscopic findings in the reproductive system of mature male cynomolgus macaques.

The potential for effector functions of therapeutic antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), is a biological activity of interest for characterization, regardless of if ADCC is an intended primary pharmacological effect. The composition of the conserved antibody Fc glycan can vary as a function of post-translational processing which may affect the binding affinity to Fc receptors, leading to a change of effector activity. Ordesekimab (AMG 714 or PRV-015), a fully human immunoglobulin G1-kappa anti-interleukin (IL)-15 monoclonal antibody, is in clinical development for celiac disease. The binding of ordesekimab to IL-15 inhibits the interaction of IL-15 with the IL-2Rβ and common γ chain of the IL-15 receptor complex, but not with the IL-15Rα chain. Therefore, the simultaneous binding of ordesekimab to the Fcγ receptor (R) IIIα expressed on natural killer (NK) cells and to the IL-15/IL-15Rα complex on cells such as monocytes may theoretically enable ADCC toward the IL-15Rα-expressing cells. The high mannose (HM) levels on the Fc glycan were found to vary in different lots of ordesekimab resulting from refinements to the manufacturing process, and the impact on ordesekimab-mediated ADCC activity was evaluated in in vivo and in vitro studies. A review of nonclinical and clinical data found no evidence of ordesekimab-induced depletion of monocytes, or cytotoxicity in organs with wide IL-15Rα expression, suggesting a lack of in vivo ADCC activity. In addition, in vitro peripheral blood mononuclear cells-based ADCC assay did not reveal any cytolytic effect of ordesekimab with various levels of HM content when cocultured with recombinant human IL-15. Taken together, these data demonstrate that ADCC is not a potential liability for ordesekimab and does not contribute to the reduction of IL-15-mediated inflammation, the intended pharmacological effect.

The sexual maturity status of animals in nonclinical safety studies can have a significant impact on the microscopic assessment of the reproductive system, the interpretation of potential test article–related findings, and ultimately the assessment of potential risk to humans. However, the assessment and documentation of sexual maturity for animals in nonclinical safety studies is not conducted in a consistent manner across the pharmaceutical and chemical industries. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology convened an international working group of pathologists and nonclinical safety scientists with expertise in the reproductive system, pathology nomenclature, and Standard for Exchange of Nonclinical Data requirements. This article describes the best practices for documentation of the light microscopic assessment of sexual maturity in males and females for both rodent and nonrodent nonclinical safety studies. In addition, a review of the microscopic features of the immature, peripubertal, and mature male and female reproductive system and general considerations for study types and reporting are provided to aid the study pathologist tasked with documentation of sexual maturity.