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》

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

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

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》

A potent tetravalent T-cell-engaging bispecific antibody against CD33 in acute myeloid leukemia.

Acute myeloid leukemia (AML), the most common acute leukemia in adults and the second most common cancer in children, is still a lethal disease in the majority of patients, but immunologic approaches have improved outcome. Bispecific antibodies (BsAbs) are novel immunotherapeutics that can redirect immune cells against AML. We now report a tetravalent (2+2) humanized BsAb in the immunoglobulin G light chain single chain fragment variable [IgG(L)-scFv] format to engage polyclonal T cells to kill CD33+ AML targets. In vitro, this BsAb demonstrated strong antigen-specific T-cell-dependent cell-mediated cytotoxicity (TDCC) with an 50% effective concentration (EC50) in the femtomolar range that translated into treatment of established human AML IV xenografts in vivo. Importantly, it could redirect intraperitoneally injected T cells to ablate established and rapidly growing extramedullary subcutaneous AML xenografts in vivo. Furthermore, internalization of CD33 upon BsAb binding was identical to that of a bivalent (1+1) heterodimer, both being substantially less than anti-CD33 IgG. In contrast to the heterodimer, the tetravalent IgG-scFv BsAb was >10-fold more efficient in TDCC of AML cells in vitro and in vivo. This BsAb did not react with and did not kill CD38-CD34+ hematopoietic stem cells from cord blood. We conclude that the novel anti-CD33 IgG(L)-scFv BsAb construct reported here is a potential candidate for clinical development.

Hoseini SS ，Guo H ，Wu Z ，Hatano MN ，Cheung NV ... -  《-》

A novel glycoengineered bispecific antibody format for targeted inhibition of epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor type I (IGF-1R) demonstrating unique molecular properties.

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.

Schanzer JM ，Wartha K ，Croasdale R ，Moser S ，Künkele KP ，Ries C ，Scheuer W ，Duerr H ，Pompiati S ，Pollman J ，Stracke J ，Lau W ，Ries S ，Brinkmann U ，Klein C ，Umana P ... -  《-》