Engineered Antigen-Specific T Cells Secreting Broadly Neutralizing Antibodies: Combining Innate and Adaptive Immune Response against HIV

Abstract

While antiretroviral therapy (ART) can completely suppress viremia, it is not a cure for HIV. HIV persists as a latent reservoir of infected cells, able to evade host immunity and re-seed infection following cessation of ART. Two promising immunotherapeutic strategies to eliminate both productively infected cells and reactivated cells of the reservoir are the adoptive transfer of potent HIV-specific T cells and the passive administration of HIV-specific broadly neutralizing antibodies also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). The simultaneous use of both as the basis of a single therapeutic has never been explored. We therefore sought to modify HIV-specific T cells from HIV-naive donors (to allow their use in the context of allotransplant, a promising platform for sterilizing cures) so they are able to secrete a broadly neutralizing antibody (bNAb) directed against the HIV envelope to elicit ADCC. We designed an antibody construct comprising bNAb 10-1074 heavy and light chains, fused to IgG3 Fc to elicit ADCC, with truncated cluster of differentiation 19 (CD19) as a selectable marker. HIV-specific T cells were expanded from HIV-naive donors by priming with antigen-presenting cells expressing overlapping HIV antigens in the presence of cytokines. T cells retained specificity against Gag, Nef, and Pol peptides (218.55 ± 300.14 interferon γ [IFNγ] spot-forming cells [SFC]/1 × 10⁵) following transduction (38.92 ± 25.30) with the 10-1074 antibody constructs. These cells secreted 10-1074 antibodies (139.04 ± 114.42 ng/mL). The HIV-specific T cells maintained T cell function following transduction, and the secreted 10-1074 antibody bound HIV envelope (28.13% ± 19.42%) and displayed ADCC activity (10.47% ± 4.11%). Most critically, the 10-1074 antibody-secreting HIV-specific T cells displayed superior in vitro suppression of HIV replication. In summary, HIV-specific T cells can be engineered to produce antibodies mediating ADCC against HIV envelope-expressing cells. This combined innate/adaptive approach allows for synergy between the two immune arms, broadens the target range of the immune therapy, and provides further insight into what defines an effective anti-HIV response.

Keywords: HIV; antigen-specific T cells; broadly neutralizing antibodies.

Graphical abstract

Figure 1

Antibody Construct and Gene Modification of T Cells (A) Schematic of the transgene introduced to T cells via an Moloney murine leukemia virus (M-MLV) retroviral vector. The entire product is under the control of the constitutively active cytomegalovirus (CMV) promoter. The entire light chain variable region (LC) and heavy chain variable region (HC) of 10-1074 antibody were expressed as a single polypeptide separated by a 2A cleavage sequence. HC was attached to the Fc region. Each chain was directed to be secreted by a signal peptide. Transduction efficiency was measured by truncated CD19 (ΔCD19), separated from the LC and HC regions by a 2A cleavage peptide. (B) Mean transduction of mitogenically stimulated T cells (gray bar) was measured by co-expression of CD3 and CD19 (a product of the Ab construct) on the surface. Error bars depict standard deviation. Each peripheral blood donor is shown by a black circle; n = 4. (C) Phenotype of mitogenically stimulated T cells is shown as percent expression of lymphocytes in both nontransduced (black circles) and transduced (gray circles) cells. The long horizontal bar is the mean expression, and error bars depict standard deviation; n = 4. (D) Mean secretion of 10-1074 antibody in T cell supernatant of nontransduced (black rectangle) and transduced (gray rectangle) cells was measured by ELISA. Error bars depict standard deviation, black circles represent individual peripheral blood donors; n = 4.

Figure 2

T Cell-Secreted Antibodies Bind to HIV Envelope Expressed on Cells Summary of detectable bound IgG from supernatant of nontransduced (black) and transduced (gray) T cells on the surface of envelope-expressing HeLa cells; n = 3. Error bars depict standard deviation. Nontransduced and transduced cell supernatants were analyzed by paired t test, where ∗∗p = 0.0063.

Figure 3

HIV-Specific T Cells Can Be Modified to Secrete 10-1074 Antibodies (A) Mean transduction of HIV-specific T cells (gray bar) was measured by co-expression of CD3 and CD19 (expressed from construct, see schematic Figure 1A) on the surface. Error bars depict standard deviation, and each HIV-specific T cell line is shown as black circles; n = 10. (B) Mean concentration of 10-1074 antibody in supernatants from nontransduced (black rectangle) and transduced (gray rectangle) cells was quantified by ELISA. Error bars depict standard deviation, and each HIV-specific T cell line is shown as black circles; n = 9. (C) Phenotypes of HIV-specific T cells, shown here as percent expression of lymphocytes in both nontransduced (black circles) and transduced (gray circles) cells; n = 10. Long horizontal bars represent mean expression, and error bars depict standard deviation. ns denotes p > 0.05 by paired t test. (D) Mean surface expression of activation markers CD25 and CD69 of transduced HIV-specific T cells (black bar) and CD19+ (dark gray) and CD19− (light gray) fractions within the transduced T cell population were measured by flow cytometry; n = 4. Error bars depict standard deviation. Paired t tests were used for statistical analysis, where ns denotes p > 0.05, ∗p = 0.0135, ∗∗p = 0.0061. (E) Mean surface expression of exhaustion markers LAG3 and PD1 of transduced HIV-specific T cells (black bar) and CD19+ (dark gray) and CD19− (light gray) fractions within the transduced T cell population were measured by flow cytometry; n = 4. Error bars depict standard deviation. Paired t tests were used for statistical analysis, where ns denotes p > 0.05.

Figure 4

Transduced HIV-Specific T Cells Maintain T Cell Function (A) Fold expansion of transduced and non-transduced HIV-specific T cells is shown 7 days following stimulation 3 (day 23). The black line shows mean fold-expansion of non-transduced cells (black circles), while the gray line shows mean fold-expansion of non-transduced cells (gray squares); n = 8. Error bars depict standard deviation. Wilcoxon signed rank tests were used for statistical analysis, where ns denotes p > 0.05. (B) IFNγ ELISPOT (shown as the number of SFCs per 100,000 cells) measures HIV-specific T cell specificity. Rectangles depict mean responses to media (cytotoxic T lymphocyte [CTL] only), irrelevant antigen (actin), and HIV antigens gag, nef, and pol (GNP) of non-transduced cells (black) and transduced cells (gray); n = 10. Error bars depict standard deviation. Wilcoxon signed rank tests were used for statistical analysis, where ns denotes p > 0.05, ∗∗p = 0.0078, ∗∗∗p = 0.0010. (C) Multiplex ELISA of T cell responses following stimulation with antigen were used to measure secretion of cytokines by nontransduced (black) and transduced (gray) HIV-specific T cells; n = 6. Circles show individual T cell donors, and error bars depict the standard deviation. Paired t tests were used for statistical analysis, where ns denotes p > 0.05.

Figure 5

T Cell-Secreted Antibodies from HIV-Specific T Cells Elicit ADCC (A) Sample flow cytometry figure shown. HIV gp120-coated CEM.NKr.CCR5 cells were used as target cells. Binding to HIV envelope of secreted 10-1074 antibody was measured by expression of secondary anti-human IgG (H+L) on the cell surface by flow cytometry; n = 3. Error bars denote standard deviation. (B) Cytotoxicity assays from four donors show higher killing (over allo-based killing) of HIV envelope-expressing cells that were co-cultured with NK cells in the presence of nontransduced (black lines) or transduced supernatant (gray lines) from the donors indicated. Error bars denote standard deviation. (C) ADCC as measured by the change in NK cell killing of HIV envelope expressing target cells (HeLa) upon addition of antibody containing transduced HIV-specific T cell supernatant. Each graph represents one donor. Circles represent mean of 3 technical replicates. Error bars depict standard deviation. Student’s t tests were used for statistical analysis, where ns denotes p > 0.05, ∗p < 0.005. (C) Change in NK cell cytotoxicity upon addition of antibody containing transduced HIV-specific T cell supernatant (gray bar) at 2:1 E:T ratio was compared with change in cytotoxicity upon addition of WT 10-1074 antibody (black bar, 250 ng/mL). Circles depict individual donors; n = 4. Error bars depict the standard deviation. Student’s t tests were used for statistical analysis, where ns denotes p > 0.05, ∗p = 0.015.

Figure 6

Genetic Modification of HIV-Specific T Cells to Secrete 10-1074 Antibody Increases Anti-Viral Efficacy against HIV-Infected Targets (A) HIV p24 concentration in cell supernatant was measured by p24 ELISA and shown in pg/mL. Mean p24 levels are shown from SF162 HIV-infected autologous CD4+ T cells (black bar, no effectors), nontransduced HIV-specific T cells (dark gray bar), and transduced HIV-specific T cells (light gray bar) plated at 10:1 E:T ratios; n = 4. Error bars denote standard deviation. (B) Mean p24 levels are shown from SF162 HIV-infected autologous CD4+ T cells (black bar, no effectors), WT 10-1074 antibody (dark gray bar, 250 ng/mL), and HIV-specific T cell-secreted antibody (light gray bar); n = 4. Error bars denote standard deviation. (C) Mean p24 levels are shown from SF162 HIV-infected autologous CD4+ T cells (black bar, no effectors), nontransduced HIV-specific T cell (dark gray bar), transduced HIV-specific T cell (light gray bar), nontransduced HIV-specific T cell + autologous NK cells (dark gray bar, stripes), transduced HIV-specific T cell + autologous NK cells (light gray bar, stripes); n = 4. Error bars denote standard deviation. (A–C) One-way ANOVA was used to test differences in control (CD4+ HIV infect) versus treatment groups, followed by a Dunnett’s multiple comparison test, where ∗∗∗∗p < 0.0001. Paired t tests were used for statistical analysis between treatment groups, where ns denotes p > 0.05.