Overcoming tumor heterogeneity by ex vivo arming of T cells using multiple bispecific antibodies.

Tumorous heterogeneity is a hallmark of tumor evolution and cancer progression, being a longstanding challenge to targeted immunotherapy. Ex vivo armed T cells (EATs) using IgG-(L)-scFv bispecific antibodies (BsAbs) are potent tumor-specific cytotoxic effectors. To improve the anti-tumor efficacy of EATs against heterogeneous solid tumors, we explored multi-antigen targeting approaches. Ex vivo expanded T cells were armed with BsAbs built on the IgG-(L)-scFv platform, where an anti-CD3 (huOKT3) scFv was attached to the carboxyl end of both light chains of a tumor specific IgG. Multispecificity was created by combining monospecific EATs, combining BsAbs on the same T cell, or combining specificities on the same antibody. Three multi-antigens targeting EAT strategies were tested: (1) pooled-EATs (EATs each with unique specificity administered simultaneously) or alternate-EATs (EATs each with unique specificity administered in an alternating schedule), (2) dual-EATs or multi-EATs (T cells simultaneously armed with ≥2 BsAbs), and (3) TriAb-EATs (T cells armed with BsAb specific for two targets besides CD3 (TriAb)). The properties and efficiencies of these three strategies were evaluated by flow cytometry, in vitro cytotoxicity, cytokine release assays, and in vivo studies performed in BALB-Rag2-/-IL-2R-γc-KO (BRG) mice xenografted with cancer cell line (CDX) or patient-derived tumor (PDX). Multi-EATs retained target antigen specificity and anti-tumor potency. Cytokine release with multi-EATs in the presence of tumor cells was substantially less than when multiple BsAbs were mixed with unarmed T cells. When tested against CDXs or PDXs, dual-EATs or multi-EATs effectively suppressed tumor growth without clinical toxicities. Most importantly, dual-EATs or multi-EATs were highly efficient in preventing clonal escape while mono-EATs or TriAb- EATs were not as effective. Multi-EATs have the potential to increase potency, reduce toxicity, and overcome tumor heterogeneity without excessive cytokine release. Arming T cells with multiple BsAbs deserves further exploration to prevent or to treat cancer resistance.

Park JA ，Cheung NV 《Journal for ImmunoTherapy of Cancer》

Potent ex vivo armed T cells using recombinant bispecific antibodies for adoptive immunotherapy with reduced cytokine release.

T cell-based immunotherapies using chimeric antigen receptors (CAR) or bispecific antibodies (BsAb) have produced impressive responses in hematological malignancies. However, major hurdles remained, including cytokine release syndrome, neurotoxicity, on-target off-tumor effects, reliance on autologous T cells, and failure in most solid tumors. BsAb armed T cells offer a safe alternative. We generated ex vivo armed T cells (EATs) using IgG-[L]-scFv-platformed BsAb, where the anti-CD3 (huOKT3) scFv was attached to the light chain of a tumor-binding IgG. BsAb density on EAT, in vitro cytotoxicity, cytokine release, in vivo trafficking into tumors, and their antitumor activities were evaluated in multiple cancer cell lines and patient-derived xenograft mouse models. The efficacy of EATs after cryopreservation was studied, and gamma delta (γδ) T cells were investigated as unrelated alternative effector T cells. The antitumor potency of BsAb armed T cells was substantially improved using the IgG-[L]-scFv BsAb platform. When compared with separate BsAb and T cell injection, EATs released less TNF-α, and infiltrated tumors faster, while achieving robust antitumor responses. The in vivo potency of EAT therapy depended on BsAb dose for arming, EAT cell number per injection, total number of EAT doses, and treatment schedule intensity. The antitumor efficacy of EATs was preserved following cryopreservation, and EATs using γδ T cells were safe and as effective as αβ T cell-EATs. EATs exerted potent antitumor activities against a broad spectrum of human cancer targets with remarkable safety. The antitumor potency of EATs depended on BsAb dose, cell number and total dose, and schedule. EATs were equally effective after cryopreservation, and the feasibility of third-party γδ-EATs offered an alternative for autologous T cell sources.

Park JA ，Santich BH ，Xu H ，Lum LG ，Cheung NV ... -  《Journal for ImmunoTherapy of Cancer》

GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma.

Park JA ，Cheung NV 《Journal of Hematology & Oncology》

Targeting tumor vasculature to improve antitumor activity of T cells armed ex vivo with T cell engaging bispecific antibody.

Success of T cell immunotherapy hinges on the tumor microenvironment (TME), and abnormal tumor vasculature is a hallmark of most solid tumors and associated with immune evasion. The efficacy of T cell engaging bispecific antibody (BsAb) treatment relies on the successful trafficking and cytolytic activity of T cells in solid tumors. Normalization of tumor vasculature using vascular endothelial growth factor (VEGF) blockades could improve efficacy of BsAb-based T cell immunotherapy. Anti-human VEGF (bevacizumab, BVZ) or anti-mouse VEGFR2 antibody (DC101) was used as VEGF blockade, and ex vivo armed T cells (EATs) carrying anti-GD2, anti-HER2, or anti-glypican3 (GPC3) IgG-(L)-scFv platformed BsAb were used. BsAb-driven intratumoral T cell infiltration and in vivo antitumor response were evaluated using cancer cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) carried out in BALB-Rag2 -/-IL-2R-γc-KO (BRG) mice. VEGF expression on human cancer cell lines was analyzed by flow cytometry, and VEGF levels in mouse serum were measured using VEGF Quantikine ELISA Kit. Tumor infiltrating lymphocytes (TILs) were evaluated using flow cytometry and by bioluminescence; both TILs and tumor vasculature were studied using immunohistochemistry. VEGF expression on cancer cell lines increased with seeding density in vitro. BVZ significantly reduced serum VEGF levels in mice. BVZ or DC101 increased high endothelial venules (HEVs) in the TME and substantially enhanced (2.1-8.1 fold) BsAb-driven T cell infiltration into neuroblastoma and osteosarcoma xenografts, which was preferential for CD8(+) TILs versus CD4(+) TILs, leading to superior antitumor effects in multiple CDX and PDX tumor models without added toxicities. VEGF blockade using specific antibodies against VEGF or VEGFR2 increased HEVs in the TME and cytotoxic CD8(+) TILs, significantly improving the therapeutic efficacy of EAT strategies in preclinical models, supporting the clinical investigation of VEGF blockades to further enhance BsAb-based T cell immunotherapies.

Park JA ，Espinosa-Cotton M ，Guo HF ，Monette S ，Cheung NV ... -  《Journal for ImmunoTherapy of Cancer》

Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma.

EGFR and/or HER2 expression in pancreatic cancers is correlated with poor prognoses. We generated homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (T-BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors. EGFR and HER2 T-BsAbs were constructed using the 2 + 2 IgG-[L]-scFv T-BsAbs format bearing two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. A Fab arm exchange strategy was used to generate EGFRxHER2 heterodimeric T-BsAb carrying one Fab specific for EGFR and one for HER2. EGFR and HER2 T-BsAbs were also heterodimerized with a CD33 control T-BsAb to generate 'tumor-monovalent' EGFRxCD33 and HER2xCD33 T-BsAbs. T-BsAb avidity for tumor cells was studied by flow cytometry, cytotoxicity by T-cell mediated 51Chromium release, and in vivo efficacy against cell line-derived xenografts (CDX) or patient-derived xenografts (PDX). Tumor infiltration by T cells transduced with luciferase reporter was quantified by bioluminescence. The EGFRxEGFR, HER2xHER2, and EGFRxHER2 T-BsAbs demonstrated high avidity and T cell-mediated cytotoxicity against human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They were highly efficient in driving human polyclonal T cells into subcutaneous PDAC xenografts and mediated potent T cell-mediated anti-tumor effects. Both EGFRxCD33 and HER2xCD33 tumor-monovalent T-BsAbs displayed substantially reduced avidity by SPR when compared to homodimeric EGFRxEGFR or HER2xHER2 T-BsAbs (∼150-fold and ∼6000-fold respectively), tumor binding by FACS (8.0-fold and 63.6-fold), and T-cell mediated cytotoxicity (7.7-fold and 47.2-fold), while showing no efficacy against CDX or PDX. However, if either EGFR or HER2 was removed from SW1990 by CRISPR-mediated knockout, the in vivo efficacy of heterodimeric EGFRxHER2 T-BsAb was lost. EGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. Two arm Fab binding to either one or both targets was critical for robust anti-tumor effect in vivo. By engaging both targets, EGFRxHER2 heterodimeric T-BsAb exhibited potent anti-tumor effects if CDX or PDX were EGFR+HER2+ double-positive with the potential to spare single-positive normal tissue.

Long AW ，Xu H ，Santich BH ，Guo H ，Hoseini SS ，de Stanchina E ，Cheung NV ... -  《Journal of Hematology & Oncology》