Engineering human HSPCs to produce b12, a broadly neutralizing antibody against HIV-1 gp120, after in vitro maturation to B lymphocytes. (A) A 3-stage, in vitro human B lymphopoiesis culture system was established. Lentiviral constructs were introduced to the HSPCs to achieve B-cell programming. During the 5 days of stage 1, IL-3 (10 ng/mL), Flt3 ligand (Flt3-L; 10 ng/mL), thrombopoietin (TPO; 10 ng/mL), SCF (5 ng/mL), and G-CSF (5 ng/mL) primed the HSPCs for B-lineage commitment. The 6-week stage 2 used stromal support (MS5) and generated a mixture of pre-B and immature B cells from CD34⁺ HSPCs. The 3-week stage 3 promoted B-cell activation, proliferation, and differentiation into antibody-secreting plasmablasts and plasma cells by the presence of IL-2 (10 ng/mL), IL-10 (100 ng/mL), CpG (2 μM), and MS5 cells stably expressing a low level of CD40L (MS40L-low cells). The surface markers at different stages of B-cell development are shown. (B) Three lentiviral constructs were generated that carried the secretory form of b12-IgG₁ driven by either the ubiquitin-C (U-b12), Ig heavy chain (MH-b12), or Ig light chain promoter (EEK-b12). The MH promoter contained the human μ chain promoter (VHp) preceded by the iEμ enhancer flanked by matrix association regions (MAR). The EEK promoter contained the κ light chain promoter (VKp) preceded by an intronic enhancer (iEκ), an MAR, and a 3′ enhancer (3′Eκ). The secretory γ (γ_s) heavy chain and κ light chain genes with b12 variable regions were linked through F2A (2A sequence from foot-and-mouth disease virus) sequences. LTR indicates long terminal repeats; F, HIV-1 flap element; WRE, woodchuck hepatitis virus posttranscriptional regulatory element.