PF207 TARGETING WILMS TUMOR 1 WITH A T CELL BISPECIFIC ANTIBODY (WT1‐TCB): EX VIVO AND IN VIVO POTENCY BY BIVALENT RECOGNITION OF PEPTIDE‐MHC COMPLEXES FROM AN INTRACELLULAR TUMOR ANTIGEN
Authors: Augsberger C. , Klein C. , Heitmüller C. , Xu W. , Hanisch L.J. , Sam J. , Pulko V. , Schönle A. , Challier J. , Carpy A. , Konstandin N. , Rothe M. , Bücklein V. , Tunger A. , Schmitz M. , von Bergwelt M. , Spiekermann K. , Umaña P. , Subklewe M. 
Published: June 2019

Background:

Antibody‐based immunotherapy represents a promising strategy to target chemo‐resistant leukemic cells. However, classical antibody‐based approaches are restricted to cell surface antigens, even though targeting of intracellular antigens would allow further access to a large number of tumor‐specific target structures.

Aims:

In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody based on T cell receptor (TCR)‐like antibodies for immunotherapy of acute myeloid leukemia (AML). The TCB targets the intracellular tumor antigen Wilms Tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A∗02:01 (HLA‐A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR‐specificity.

Methods:

WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real‐time PCR, western blot and immunohistochemical staining. WT1‐TCB‐mediated cytotoxicity was analyzed by co‐cultivation of WT1‐expressing HLA‐A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCB‐mediated cytotoxicity against primary AML cells was evaluated in our pre‐established feeder layer‐based ex vivo long‐term culture system. For in vivo testing, NSG mice (NOD.Cg‐Prkdcscid‐Il2rgtm1Wjl/SzJ) were humanized with human HLA‐A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1‐expressing HLA‐A2+ SKM‐1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1‐TCB.

Results:

In accordance with previous reports, we observed WT1 expression in 79% (n = 38) of cancer cell lines and in 92% (n = 65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n = 26), whereas WT1 was expressed again at time of relapse (n = 21). WT1‐TCBs elicited antibody‐mediated T cell cytotoxicity against peptide‐pulsed T2 cells and AML cell lines in a WT1 and HLA‐restricted manner. Equally, TCR downstream signaling was observed in a WT1‐restrictive manner by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67 ± 6% after 13–14 days;  ± SEM; n = 18). Correspondingly, up‐regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3 ± 1.5, PD‐1: 5.1 ± 0.7, TIM‐3: 4.7 ± 0.6;  ± SEM; n = 22). WT1‐TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38 ± 13% after 13–14 days;  ± SEM; n = 5). In comparison with WT1RMF‐specific T cells, only bivalent binding by WT1‐TCB induced efficient lysis of primary AML cells. Furthermore, WT1‐TCB‐treated humanized mice bearing SKM‐1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control.

Summary/Conclusion:

TCR‐like TCBs targeting of intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide‐MHC complex on the cell surface validating WT1‐TCB as a promising therapeutic agent for the treatment of AML.