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[Abstract]: Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells. [Methods]: We evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing. [Results]: The presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells. [Conclusions]: Taken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts., The experimental work was supported by grants awarded by Ricerca Finalizzata GR-2013 02359212 (CQ), GR-2016-02364546 (BDA), RF-2016- 02364388 (FL), Accelerator Award-Cancer Research UK/AIRC/AECC-INCAR project (FL and AO), Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC)-Special Project 5×1000 no. 9962 (FL), AIRC IG 2018 id. 21724 (FL), Ricerca Corrente (FL, CQ, BDA), Ministero dell’Università e della Ricerca (Grant PRIN 2017 to FL); Italian Healthy Ministry project on CAR T RCR-2019-23669115 (coordinator FL), Independent Research grant AIFA (FL PI: 2016 call).

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

Based on the clinical success observed in acute lymphoblastic leukemia (ALL) with chimeric antigen receptor engineered T (CAR T), we hypothesized that combining the specificity of a CAR with the innate allo-reactivity of KIR-mismatched NK cells might provide a powerful tool for adoptive cell therapy. The use of a third-party bank of CAR-NK cells offers the advantage of an immediate availability to be exploited in the allogenic setting and could be associated with a lower toxicity profile than CAR-T cells. In order to overcome regulatory and manufacturing hurdles associated with generation of CAR-NK cells, we developed a feeder-free culture resulting in a 3.2-log expansion after 20 days of culture. Specifically, natural cytotoxicity receptors (NCR) expressed on NK cells are stimulated in the presence of pleiotropic cytokines and expanded in GMP grade bioreactors. Expanded NK cells from healthy donors preserve a high percentage of CD56+ CD57- cells (85±13%), associated with high proliferative capability, and maintain the surface expression and the responsiveness of NCR and CD16. We proved that NK cells generated from 10 different healthy donors have high ability to recognize and eliminate different tumor types, including acute myeloid leukemia (AML) and ALL. After genetic modification with a retroviral vector encoding a CAR specific for CD19 antigen, transduction of activated NK cells averaged 38%±15% and the CAR.CD19 expression was stable over extended in vitro culture (60 days). Detailed phenotypic characterization of CAR-NK cells showed that CAR expression was not limited to the more mature NKG2A-/KIR+ cells, but rather was distributed across different NK subsets. We also demonstrated that NK and CAR-NK cells display significant anti-leukemia activity towards CD19+ leukemia and lymphoma cell lines (LCL 721.221, DAUDI and BV173) and primary blasts obtained from patients with B-cell precursor ALL (Bcp-ALL). Co-culture experiments using a 1:5 E/T ratio, showed that, while the anti-tumor activity was already remarkable with non-modified effector NK cells (60±30%, 71±33% and 54±23% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p An in vivo model of leukemia xenograft immunodeficient mice was used to evaluate whether CAR-NK cells are associated with a lower toxicity profile compared to CAR-T cells. While the in vivo antileukemia activity was superimposable between CAR-T and CAR-NK cells (mouse bioluminenscence at 20 days, 4.9x105 vs 6.6x105 photons/second, respectively; p=n.s. Figure A), mice treated with two i.v. infusions (day 0 and day 15) of 10x106 CAR.CD19 NK cells had a 100% overall survival (OS of 5 out of 5 mice) at 50 days compared to 20% of mice (1 out of 5) receiving 10x106 CAR.CD19 T cells (Figure B; p=0.01). Cytokine plasma level monitoring, performed on day +7 and +30 after effector cell infusion in the absence of leukemia persistence (as evidenced by a lack of bioluminescence signal), showed that mice engrafted with CD19+ leukemia and treated with CAR.CD19-NK cells have lower levels of circulating hIFN-g cytokine compared to mice treated with CAR.CD19-T cells at both day 7 (42±82 vs 330±346 ng/ml; p=0.05) and day 30 (0.9±0.7 vs 4148±667 ng/ml; p=0.05). These in vitro and in vivo data demonstrate the feasibility of clinical scale feeder-free expansion of non-modified NK cells and stably transduced CAR-NK cells. Both non-modified and gene-modified cells were capable of significant tumor killing, suggesting a multi-modal adoptive cell approach to treatment of leukemia. Since NK cells have been shown to be safely used in third-party setting (St. Jude Children's Research Hospital, USA; NCT00640796), we suggest that ex-vivo expanded, feeder-free NK cells can be universally applied for 'off-the-shelf' immuno-gene-therapy, and that their innate allo-reactivity can be safely harnessed to potentiate allogeneic cell therapy. Figure. Figure. Disclosures Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees.