With multiple papers published in prestigious, peer-reviewed journals over the last decade, the scientists at Adaptin are experts in the biology of bispecific antibodies (bscAB) and regulator T cells (Tregs)
Human Regulatory T Cells Kill Tumor Cells through Granzyme-Dependent Cytotoxicity upon Retargeting with a Bispecific Antibody
Bryan D. Choi, Patrick C. Gedeon, James E. Herndon II, Gary E. Archer, Elizabeth A. Reap, Luis Sanchez-Perez, Duane A. Mitchell, Darell D. Bigner, and John H. Sampson
A major mechanism by which human regulatory T cells (Treg) have been shown to suppress and kill autologous immune cells is through the granzyme-perforin pathway. However, it is unknown whether Tregs also possess the capacity to kill tumor cells using similar mechanisms. Bispecific antibodies (bscAb) have emerged as a promising class of therapeutics that activate T cells against tumor antigens without the need for classical MHC-restricted Tcell receptor (TCR) recognition. Here, we show that a bscAb targeting the tumor-specific mutation of the EGF receptor, EGFRvIII, redirects human CD4þCD25þFoxP3þ Tregs to kill glioblastoma cells. This activity was significantly abrogated by inhibitors of the granzyme-perforin pathway. Notably, analyses of human primary
glioblastoma also displayed diffused infiltration of granzyme-expressing FoxP3þ T cells. Together, these data suggest that despite their known suppressive functions, tumor-infiltrating Tregs possess potent cytotoxic
mechanisms that can be co-opted for efficient tumor cell lysis.
Cancer Immunol Res; 1(3); 163–7. 2013 AACR.
A Rationally Designed Fully Human EGFRvIII:CD3-Targeted Bispecific Antibody Redirects Human T Cells to Treat Patient-derived Intracerebral Malignant Glioma
Patrick C. Gedeon, Teilo H. Schaller, Satish K. Chitneni, Bryan D. Choi, Chien-Tsun Kuan, Carter M. Suryadevara, David J. Snyder, Robert J. Schmittling, Scott E. Szafranski, Xiuyu Cui, Patrick N. Healy, James E. Herndon II, Roger E. McLendon, Stephen T. Keir, Gary E. Archer, Elizabeth A. Reap, Luis Sanchez-Perez, Darell D. Bigner, and John H. Sampson
Purpose: Conventional therapy for malignant glioma fails
to specifically target tumor cells. In contrast, substantial evidence indicates that if appropriately redirected, T cells can
precisely eradicate tumors. Here we report the rational development of a fully human bispecific antibody (hEGFRvIII-CD3 bi-scFv) that redirects human T cells to lyse malignant glioma
expressing a tumor-specific mutation of the EGFR (EGFRvIII).
Experimental Design: We generated a panel of bispecific single-chain variable fragments and optimized design through
successive rounds of screening and refinement. We tested the
ability of our lead construct to redirect na€ ve T cells and induce target cell–specific lysis. To test for efficacy, we evaluated tumor growth and survival in xenogeneic and syngeneic models of glioma. Tumor penetrance following intravenous drug administration was assessed in highly invasive, orthotopic
glioma models.
Results: Ahighly expressed bispecific antibody with specificity
to CD3 and EGFRvIII was generated (hEGFRvIII-CD3 bi-scFv). Antibody-induced T-cell activation, secretion of proinflammatory cytokines, and proliferation was robust and occurred exclusively in the presence of target antigen. hEGFRvIII-CD3 bi-scFv
was potent and target specific, mediating significant lysis of
multiple malignant glioma cell lines and patient-derived malignant glioma samples that heterogeneously express EGFRvIII. In both subcutaneous and orthotopic models, well-engrafted,
patient-derived malignant glioma was effectively treated despite heterogeneity of EGFRvIII expression; intravenous hEGFRvIIICD3
bi-scFv administration caused significant regression of
tumor burden (P < 0.0001) and significantly extended survival (P < 0.0001). Similar efficacy was obtained in highly infiltrative, syngeneic glioma models, and intravenously administered hEGFRvIII-CD3 bi-scFv localized to these orthotopic tumors.
Conclusions: We have developed a clinically translatable bispecific antibody that redirects human T cells to safely and
effectively treat malignant glioma.Onthe basis of these results, we have developed a clinical study of hEGFRvIII-CD3 bi-scFv for patients with EGFRvIII-positive malignant glioma.
Clin Cancer Res; 24(15); 3611–31. 2018 AACR.
Pharmacokinetic analysis of a novel human EGFRvIII:CD3 bispecific antibody in plasma and whole blood using a high-resolution targeted mass spectrometry approach
Teilo H Schaller1, Matthew W Foster, J Will Thompson, Ivan Spasojevic, Deimante Normantaite, M. Arthur Moseley, Luis Sanchez-Perez, John H Sampson
Bi-specific single chain antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressing T cells to recognize and destroy malignant, EGFRvIII-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we developed a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ~8 minutes and a terminal half-life of ~2.5 hours. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and Phase I studies of EGFRvIII:CD3 bi-scFv.
J Proteome Res. 2019 August 02; 18(8): 3032–3041
First in human dose calculation of a single-chain bispecific antibody targeting glioma using the MABEL approach
Teilo H Schaller, David J Snyder, Ivan Spasojevic, Patrick C Gedeon, Luis Sanchez-Perez, John H Sampson
Background
First-in-human (FIH) clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal model. However, with the advent of target-specific and highly active immunotherapeutics, both the Food and Drug Administration and the European Medicines Agency have provided guidance that recommend determining a safe starting dose based on a minimum anticipated biological effect level (MABEL) approach.
Methods
We recently developed a T cell activating bispecific antibody that effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma in mice (hEGFRvIII:CD3 bi-scFv). hEGFRvIII:CD3 bi-scFv is comprized of two single chain antibody fragments (bi-scFvs) that bind mutant epidermal growth factor receptor variant III (EGFRvIII), a mutation frequently seen in malignant glioma, and human CD3 on T cells, respectively. In order to establish a FIH dose, we used a MABEL approach to select a safe starting dose for hEGFRvIII:CD3 bi-scFv, based on a combination of in vitro data, in vivo animal studies, and theoretical human receptor occupancy modeling.
Results
Using the most conservative approach to the MABEL assessment, a dose of 57.4 ng hEGFRvIII:CD3 bi- scFv/kg body weight was selected as a safe starting dose for a FIH clinical study.
Conclusions
The comparison of our MABEL-based starting dose to our in vivo efficacious dose and the theoretical human receptor occupancy strongly supports that our human starting dose of 57.4 ng hEGFRvIII:CD3 bi-scFv/patient kg will be safe.
GLP toxicology study of a fully-human T cell redirecting CD3:EGFRvIII binding immunotherapeutic bispecific antibody
Patrick C. Gedeon, Michael A. Streicker, Teilo H. Schaller, Gary E. Archer, Micheal P. Jokinen, John H. Sampson
We recently reported the development of a fully-human, CD3-binding bispecific antibody for immunotherapy of malignant glioma. To translate this therapeutic (hEGFRvIII-CD3- bi-scFv) to clinical trials and to help further the translation of other similar CD3-binding therapeutics, some of which are associated with neurologic toxicities, we performed a good laboratory practice (GLP) toxicity study to assess for potential behavioral, chemical, hematologic, and pathologic toxicities including evaluation for experimental autoimmune encephalomyelitis (EAE). To perform this study, male and female C57/BL6 mice heterozygous for the human CD3 transgene (20/sex) were allocated to one of four designated groups. All animals were administered one dose level of hEGFRvIII-CD3 bi-scFv or vehicle control. Test groups were monitored for feed consumption, changes in body weight, and behavioral disturbances including signs of EAE. Urinalysis, hematologic, and clinical chemistry analysis were also performed. Vehicle and test chemical-treated groups were humanely euthanized 48 hours or 14 days following dose administration. Complete gross necropsy of all tissues was performed, and selected tissues plus all observed gross lesions were collected and evaluated for microscopic changes. This included hematoxylin-eosin histopathological evaluation and Fe-ECR staining for myelin sheath enumeration. There were no abnormal clinical observations or signs of EAE noted during the study. There were no statistical changes in food consumption, body weight gain, or final body weight among groups exposed to hEGFRvIII-CD3 bi-scFv compared to the control groups for the 2- and 14-day timepoints. There were statistical differences in some clinical chemistry, hematologic and urinalysis endpoints, primarily in the females at the 14-day timepoint (hematocrit, calcium, phosphorous, and total protein). No pathological findings related to hEGFRvIII-CD3 bi-scFv administration were observed. A number of gross and microscopic observations were noted but all were considered to be incidental background findings. The results of this study allow for further translation of this and other important CD3 modulating bispecific antibodies.
