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Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors.

ABSTRACT: To enhance the therapeutic index of T-cell engagers (TCEs), we engineered masked, precision-activated TCEs (XPAT proteins), targeting a tumor antigen (human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR)) and CD3. Unstructured XTEN polypeptide masks flank the N and C termini of the TCE and are designed to be released by proteases in the tumor microenvironment. In vitro, unmasked HER2-XPAT (uTCE) demonstrates potent cytotoxicity, with XTEN polypeptide masking providing up to 4-log-fold protection. In vivo, HER2-XPAT protein induces protease-dependent antitumor activity and is proteolytically stable in healthy tissues. In non-human primates, HER2-XPAT protein demonstrates a strong safety margin (>400-fold increase in tolerated maximum concentration versus uTCE). HER2-XPAT protein cleavage is low and similar in plasma samples from healthy and diseased humans and non-human primates, supporting translatability of stability to patients. EGFR-XPAT protein confirmed the utility of XPAT technology for tumor targets more widely expressed in healthy tissues.

SUBMITTER: Cattaruzza F

PROVIDER: S-EPMC10132983 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors.

Cattaruzza Fiore F Nazeer Ayesha A To Milton M Hammond Mikhail M Koski Caitlin C Liu Lucas Y LY Pete Yeung V V Rennerfeldt Deena A DA Henkensiefken Angela A Fox Michael M Lam Sharon S Morrissey Kari M KM Lange Zachary Z Podust Vladimir N VN Derynck Mika K MK Irving Bryan A BA Schellenberger Volker V

Nature cancer 20230330 4

To enhance the therapeutic index of T-cell engagers (TCEs), we engineered masked, precision-activated TCEs (XPAT proteins), targeting a tumor antigen (human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR)) and CD3. Unstructured XTEN polypeptide masks flank the N and C termini of the TCE and are designed to be released by proteases in the tumor microenvironment. In vitro, unmasked HER2-XPAT (uTCE) demonstrates potent cytotoxicity, with XTEN polypeptide masking ...[more]

PMID: 36997747

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Project description:IntroductionWe have recently developed a novel T cell engager concept by utilizing γ9δ2TCR as tumor targeting domain, named gamma delta TCR anti-CD3 bispecific molecule (GAB), targeting the phosphoantigen-dependent orchestration of BTN2A1 and BTN3A1 at the surface of cancer cells. GABs are made by the fusion of the ectodomains of a γδTCR to an anti-CD3 single chain variable fragment (scFv) (γδECTO-αCD3), here we explore alternative designs with the aim to enhance GAB effectivity.MethodsThe first alternative design was made by linking the variable domains of the γ and δ chain to an anti-CD3 scFv (γδVAR-αCD3). The second alternative design was multimerizing γδVAR-αCD3 proteins to increase the tumor binding valency. Both designs were expressed and purified and the potency to target tumor cells by T cells of the alternative designs was compared to γδECTO-αCD3, in T cell activation and cytotoxicity assays.Results and discussionThe γδVAR-αCD3 proteins were poorly expressed, and while the addition of stabilizing mutations based on finding for αβ single chain formats increased expression, generation of meaningful amounts of γδVAR-αCD3 protein was not possible. As an alternative strategy, we explored the natural properties of the original GAB design (γδECTO-αCD3), and observed the spontaneous formation of γδECTO-αCD3-monomers and -dimers during expression. We successfully enhanced the fraction of γδECTO-αCD3-dimers by shortening the linker length between the heavy and light chain in the anti-CD3 scFv, though this also decreased protein yield by 50%. Finally, we formally demonstrated with purified γδECTO-αCD3-dimers and -monomers, that γδECTO-αCD3-dimers are superior in function when compared to similar concentrations of monomers, and do not induce T cell activation without simultaneous tumor engagement. In conclusion, a γδECTO-αCD3-dimer based GAB design has great potential, though protein production needs to be further optimized before preclinical and clinical testing.

Project description:BackgroundLiver cancer is a major global health concern due to the steady increases in its incidence and mortality. Transcription factors, yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) have emerged as critical regulators in human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), the two major types of primary liver cancer. However, our study as well as other previous reports have shown that activation of YAP and TAZ (YAP/TAZ) in adult murine livers is insufficient for the development of liver cancer, suggesting a requirement for an additional oncogenic collaborator for liver carcinogenesis in adulthood. Therefore, we sought to identify the oncogenic partners of YAP/TAZ that promote hepatocarcinogenesis in adults.MethodsData analysis of the transcriptome of patients with liver cancer was performed using the national center for biotechnology information (NCBI) gene expression omnibus (GEO) database and the cancer genome atlas (TCGA). The cancer therapeutics response portal (CTRP) was used to investigate the correlation between sensitivity to chemicals and the copy number of TAZ in human cancer cell lines. Transposons encoding constitutively activated forms of TAZ (TAZS89A), BRAF (BRAFV600E), and PIK3CA (PI3KE545K) were used for hydrodynamic tail vein injection. Mice were monitored at least twice per week and sacrificed when moribund. Tumor-bearing livers were formalin fixed for hematoxylin-eosin staining and immunohistochemistry.ResultsThrough database analyses, we identified EGFR/HER2 signaling to be essential in human cancers with high TAZ activity. Furthermore, immunohistochemical analyses showed that human HCC and CC tissues with high YAP/TAZ activities exhibited concomitant activation of EGFR/HER2 signaling pathways. To demonstrate that EGFR/HER2 signaling promotes YAP/TAZ-mediated hepatocarcinogenesis, TAZS89A was simultaneously expressed in murine adult livers with BRAFV600E or PI3KE545K, activated forms of effector molecules downstream of EGFR/HER2 signaling pathways. Expression of TAZS89A plus BRAFV600E induced HCC, whereas TAZS89A and PI3KE545K led to the development of CC-like cancer.ConclusionsOur study demonstrates that TAZ collaborates with EGFR/HER2 signaling pathways to induce both HCC and CC.

Project description:Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.

Dataset Information

Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors.

Publications

Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors.

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