T lymphocytes expressing Chimeric Antigen Receptor (CAR) has proven to be an auspicious strategy in treatment of hematological malignancies, but with low efficacy in solid tumors due to their complex microenvironment. From this perspective, macrophages become promising cells for CAR insertion (CARMac) due to their ability to infiltrate the dense tumor matrix, promote phagocytose and antigen presentation and to secrete pro-inflammatory cytokines. However, sources for obtaining macrophages are limited due to their low proliferative capacity. Knowing that umbilical cord blood CD34+ cells are potential progenitors for macrophages, our main aim here is to generate off-the-shelf CARMac fromumbilical cord blood CD34+ cells to treat solid tumors.

We use cryopreserved umbilical cord blood CD34+ cells to first expand in vitro and to insert a reporter protein (GFP) using the piggyBAC transposon system (pB) prior the differentiation into CD34+ derived human macrophages (MacCD34). Subsequently, MacCD34 were generated using distinct protocols combining the use of growth factors and cytokines: Protocol 1 = SCF, GM-CSF, TNF-α + M-CSF; Protocol 2 = GM-CSF, M-CSF, IL-3 + M-CSF in vitro. Them, MacCD34 were tested formorphology,phenotype characterization and phagocytosis capabilities.

We obtained around 12.9% of GFP-expressing cells within 24 hours after electroporation with a viability of 63.4%. Subsequently for MacCD34 phenotyping using flow cytometry, in protocol 1 about 85% and 53.9% of CD14+CD64+ MacCD34 were obtained using two distinct culture media (RPMI 1640 (RP) and StemSpan SFEN II (SS)), respectively. Those cells presented CD34neg CD1cneg HLA-DRhigh CD163low CD16+CD86low and showed a typical morphology of macrophage when compared to monocytes derived macrophages (MacMono). In protocol 2 about 75% and 36.4% of CD14+CD64+ MacCD34 were obtained using RP and SS, respectively. Those cells presented CD34neg CD1clow HLA-DRhigh CD163neg CD16high CD86+. During differentiation, cells showed a considerable expansion fold, evidenced by the dilution of Cell Trace Violet dye. In addition, the polarization of CD14+CD34neg cells with M-CSF and IL-4 induced high expression of CD64, HLA-DR, CD86 and CD1c, showing a mixed morphologic MacMono/DC phenotype. For functional assays, MacCD34 from protocol 2 showed a reduced percentage of phagocytosis of SKBR3 tumor cells and a higher percentage for Nalm6 tumor cell phagocytosis, when compared to blood monocytes and MacMono. Beside that, MacCD34 can efficiently phagocyte pHrodo™ Zymosan microbeads and produce TNF-α (RP = 33.2%; SS = 24,0%) and IL-1β α (RP = 21.9%; SS = 10.4%) in response to LPS activation.

Our data indicate that pB is not an effective protocol for insertion of CAR molecules in CD34+ cells. In addition, differentiated MacCD34 showed phenotypic and functional similarities with classical macrophages differentiated from blood monocytes. Our ongoing steps are A) the generation of CAR-expressing CD34+ cells using lentiviral vectors and B) the differentiation into CARMac for their functional characterization.