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Supplementary figure legend

Supplementary Figure 1: Murine SAR expression and immunophenotyping of transduced T cells

Supplementary Figure 5: Effect of pre-activated SAR T cells on hepatocellular carcinoma and glioblastoma cells


Purpose:Genetically engineered T cells are powerful anticancer treatments but are limited by safety and specificity issues. We herein describe an MHC-unrestricted modular platform combining autologous T cells, transduced with a targetable synthetic agonistic receptor (SAR), with bispecific antibodies (BiAb) that specifically recruit and activate T cells for tumor killing.Experimental Design:BiAbs of different formats were generated by recombinant expression. T cells were retrovirally transduced with SARs. T-cell activation, proliferation, differentiation, and T-cell–induced lysis were characterized in three murine and human tumor models in vitro and in vivo.Results:Murine T cells transduced with SAR composed of an extracellular domain EGFRvIII fused to CD28 and CD3ζ signaling domains could be specifically recruited toward murine tumor cells expressing EpCAM by anti-EGFRvIII × anti-EpCAM BiAb. BiAb induced selective antigen-dependent activation, proliferation of SAR T cells, and redirected tumor cell lysis. Selectivity was dependent on the monovalency of the antibody for EGFRvIII. We identified FAS ligand as a major mediator of killing utilized by the T cells. Similarly, human SAR T cells could be specifically redirected toward mesothelin-expressing human pancreatic cancer cells. In vivo, treatment with SAR T cells and BiAb mediated antitumoral activity in three human pancreatic cancer cell xenograft models. Importantly, SAR activity, unlike CAR activity, was reversible in vitro and in vivo.Conclusions:We describe a novel ACT platform with antitumor activity in murine and human tumor models with a distinct mode of action that combines adoptive T-cell therapy with bispecific antibodies.

Supplementary Figure 4: Human SAR T cells are specifically stimulated by anti-human mesothelin x anti-EGFRv3 2 + 1 BiAb and redirect lysis to mesothelin+ target cells in vitro and in vivo

Supplementary Figure 3: In contrast to SAR T cells, CAR T cells rely on the release of perforin for efficient killing.

Supplementary Figure 2: Soluble and bound EGFR ligands do not trigger unwanted SAR T cell activation, while antibodies monovalent and bivalent for SAR can trigger T cell activation to the same extent.

Introduction Matched donor hematopoietic stem cell transplantation (HSCT) cures most patients with inborn hematopoietic disorders, but haploidentical HSCT was historically associated with graft rejection, GVHD and infections. In leukemia, in vivo depletion of alloreactive T-cells with post-transplant cyclophosphamide (pTCy) results in outcomes comparable to those with matched related or unrelated donors. Objectives To assess safety and efficacy of haploidentical HSCT with pTCy in patients with inborn errors of hematopoiesis who lack a suitable matched donor or have failed a previous matched donor HSCT. Methods Nineteen patients (10 primary immunodeficiencies, 7 hemoglobinopathies, 1 congenital erythropoietic porphyria, 1 bone marrow failure) with a median age of 9 years (0-22) were treated prospectively on an institutional protocol with a conditioning regimen of alemtuzumab 0.4mg/kg, rituximab 375mg/m², fludarabine 150mg/m², treosulfan 42g/m², thiotepa 10mg/kg and cyclophosphamide 29mg/kg followed by haploidentical bone marrow transplantation, pTCy and tacrolimus/MMF. In four patients who were in bone marrow aplasia after non-engraftment of a first HSCT, thiotepa was omitted and treosulfan reduced to 24g/m². Results After a median follow-up of 42 months (3-87), overall survival is 95% (18/19). One patient died on day +7 from suspected fludarabine neurotoxicity. Two patients experienced autologous reconstitution, one of whom had a successful second HSCT. Two patients have stable mixed chimerism predominantly of donor origin and remain disease free. All others exhibit complete donor chimerism. This results in event free survival of 84% (16/19) and disease free survival of 89% (17/19). Engraftment occurred after 17days (6-61) for neutrophils and 28 days (14-191) for platelets. The incidences of acute GVHD II-IV° and of chronic GVHD were 0%, respectively. Two patients (11%) developed GVHD I° of the skin.Viral reactivations were observed in 53% (10/19) of patients (8 CMV, 1ADV, 1 VZV), but only one patient developed overt disease (ADV). Immune reconstitution was swift with mean CD3+ and CD19+ counts of 793/µl and 271/µl on day +100. Other complications were bacterial bloodstream infections in 5 patients (26%) and moderate severity VOD in one (5%). No renal or hepatic toxicities greater than CTCAE grade 2 were observed. No patient developed hemorrhagic cystitis. Four patients had anti-donor HLA antibodies pre-HSCT, three of whom were treated with plasmapheresis; all engrafted. Conclusion Chemotherapy based conditioning and haploidentical HSCT with pTCy was safe and resulted in good disease free survival in this cohort of patients with inborn errors in the absence of relevant GHVD. These results compare well with outcomes achieved with matched donors in similar cohorts of patients, but larger multi-center studies are warranted to further evaluate this approach.