Coexpression of antibody and fusogenic protein on the surface of the virus packaging cell line. (A) The class I fusion protein HAmu derived from influenza A (FPV) HA. HA contains two glycoproteins after maturation: HA1 for binding to cell surface receptor, sialic acid; HA2 for triggering membrane fusion. Three point mutations within the receptor binding sites (a1, Y106F; a2, E199Q; a3, G237K) (21) were introduced to generate the binding-defective but fusion-competent HAmu. Single letter amino acid abbreviations are as follows: A, alanine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; K, lysine; M, methionine; P, proline; Q, glutamine; V, valine; Y, tyrosine. (B) FACS analysis of virus-producing cells. 293T cells were transiently transfected with plasmids encoding the following: the lentiviral vector FUGW; the membrane-bound antibody αCD20; the accessory proteins for antibodies, Igα and Igβ; the fusion protein HAmu; and viral gag, pol, and rev genes. Expression of αCD20 and HAmu was detected by using anti-human IgG antibody and anti-FPV HA antibody. (C) The class II fusion protein SINmu derived from SIN. SIN contains two membrane glycoproteins (E1 and E2) and a signal peptide (E3): E1 for mediating fusion, E2 for receptor binding, and E3 as a signal sequence for processing of E2 glycoprotein. A 10-residue tag sequence (MYPYDVPDYA) was inserted between amino acids 71 and 74 of the E2 glycoprotein. A series of alterations (a4: deletion of amino acids 61–64 of E3; a5: mutations of 68SLKQ71 into 68AAAA71; mutations of 157KE158 into 157AA158) (16) was introduced to yield the binding-defective and fusion-competent SINmu. (D) Directly analogous to B, except that SINmu was used for the fusion protein and was detected by an anti-tag antibody.

Targeting of lentivectors bearing both antibody and fusion protein to 293T/CD20 cells in vitro. (A) 293T/CD20 cells (2 × 10⁵) were transduced with 500 μl of fresh unconcentrated FUGW/αCD20 (no HAmu), FUGW/HAmu (no αCD20), or FUGW/αCD20+HAmu. 293T cells (no expression of CD20) were included as controls. The resulting GFP expression was analyzed by FACS. The specific transduction titer for FUGW/αCD20+HAmu was estimated to be ≈1 × 10⁵ TU/ml. (B) A similar transduction experiment was performed by using unconcentrated FUGW/SINmu (no αCD20) or FUGW/αCD20+SINmu. For comparison of targeting specificity, cells were also transduced with FUGW/HAmu. The specific transduction titer for FUGW/αCD20+SINmu was estimated to be ≈1 × 10⁶ TU/ml. (C) Evidence of pH-dependent fusion of HAmu and SINmu by a cell–cell fusion assay. 293T cells (0.1 × 10⁶) transiently transfected to express GFP and surface αCD20 and fusion protein (either HAmu or SINmu), and 293T/CD20 cells were mixed together, washed once with normal PBS (pH 7.4), and incubated in low pH PBS (pH 5.0) or normal pH PBS (as a control) for half an hour at 37°C. The cells were then washed and cultured in the regular medium for 1 day. Cells were visualized by epifluorescence microscope equipped with a GFP filter set. (D and E) Effect of addition of soluble αCD20 (D) or NH₄Cl (E). αCD20 or NH₄Cl was added into viral supernatants during transduction for 8 h. Then, the supernatants were replaced with fresh medium. The cells were analyzed for GFP expression after 2 days. Isotype-matched antibody was used as a control for D.

Virus–cell binding assay to study the codisplay of antibody and fusogenic protein on the lentiviral surface. (A) FACS analysis of target cell line 293T/CD20. CD20 expression was detected by using anti-CD20 antibody. Solid line, expression of CD20 in 293T/CD20; shaded area, CD20 expression in 293T cells (as a control). (B) Schematic representation of three-staining scheme used for analyzing virus–cell binding assay. Three stainings were used to detect the presence of CD20, αCD20, and the fusogenic molecule (HAmu or SINmu), respectively. (C Left) FACS plots of 293T/CD20 cells incubated with FUGW/αCD20+HAmu. The binding of virus to 293T/CD20 cells was probed with antibody against αCD20 (anti-IgG) and HAmu. Solid line, analysis on 293T/CD20; shaded area, analysis on 293T (as a control). (C Right) FACS plots of 293T/CD20 cells incubated with FUGW/aCD20+SINmu. The binding of virus to 293T/CD20 cells was detected by antibody against αCD20 and SINmu. Solid line, analysis on 293T/CD20; shaded area, analysis on 293T (as a control). (D) Codisplay of antibody and fusogenic protein was analyzed by a density plot correlating the presence of the two proteins.

Targeting CD20⁺ human primary B cells in vitro and in vivo using engineered lentivectors. (A) Fresh, unfractionated human PBMCs (2 × 10⁶) were transduced by coculturing with concentrated FUGW/αCD20+SINmu, CCMV/αCD20+SINmu, or CPGK/αCD20+SINmu (10 × 10⁶ TU). LPS (50 μg/ml) was added into the culture media for B cells to survive and grow. After 2 days, the B cell population was identified by costaining of CD19 and CD20. Solid line, analysis on transduced cells; shaded area, analysis on cells without transduction (as a control). (B) Fresh human PBMCs were transferred into irradiated RAG2^−/−γ_c^−/− mice (100 × 10⁶ per mouse) via tail vein injection. Six hours later, concentrated virus (100 × 10⁶ TU per mouse) was injected through the tail vein. Two days later, whole blood was collected from these mice via heart puncture, and the cells were stained for human CD3 and CD20 and then analyzed by FACS for GFP expression. Shaded area, no virus treatment; dashed line, treated with FUGW/b12+SINmu; solid line, treated with FUGW/αCD20+SINmu.