HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies

Abstract

Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8(+) T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1.

Importance: While chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1.

FIG 1

CAR structure and construction strategy. The parental vector contained a CAR based on a single-chain antibody. This vector was modified with a silent mutation to create an ApaI site (GGCCCT→GGGCCC; mutated nucleotides are underlined) in the hinge region of the CAR gene (within a sequence-confirmed XbaI-SmaI intermediate plasmid vector). New CAR genes were generated by the synthesis of single-chain antibody genes that were substituted into this vector via XbaI-ApaI restriction fragments.

FIG 2

Confirmation of novel CAR expression via Western blotting of transduced Jurkat cells. Western blotting for CD3 ζ was performed on Jurkat cells after transduction with CAR expression lentiviral vectors. The open arrow indicates the expected size of the native CD3 ζ chain, and the closed arrow indicates the approximate expected size of the CAR (including the single-chain antibody, hinge, 4-1BB signaling, and CD3 ζ signaling domains). Lanes: M, marker; 1 to 8, CAR-10E8, CAR-3BN117, CAR-PG9, CAR-PGT126, CAR-PGT128, CAR-VRC01, CAR-X5, and a nontransduced Jurkat control, respectively.

FIG 3

Confirmation of novel CAR expression via flow cytometry for cell surface immunoglobulin of transduced Jurkat cells. Transduced Jurkat cells were stained with goat antibody against human Fab and assessed by flow cytometry. Histogram negative gating was set on nontransduced control cells (not shown).

FIG 4

Selective functional expansion by binding of the single-chain antibody domain of the novel CARs. After transduction of primary CD8⁺ T lymphocytes with each of the seven CAR vectors, the cells were serially passaged (10 days each passage) by using stimulation with a goat anti-human Fab antibody with irradiated allogeneic feeder PBMCs and interleukin-2. The percentage of cells determined to express CAR was determined by flow cytometry as described in the legend of Fig. 3.

FIG 5

Proliferation mediated by novel CAR interactions with HIV-1-infected target cells. Primary CD8⁺ T lymphocytes transduced with the panel of CARs were enriched to >90% purity and labeled with CellTrace Violet and then cocultured with irradiated HIV-1 NL4-3-infected T2 cells. CellTrace Violet fluorescence was assessed by flow cytometry after 7 days. The open histograms indicate transduced cells exposed to control uninfected cells, while the shaded histograms indicate those exposed to infected cells.

FIG 6

Specific killing of HIV-1-infected target cells mediated by novel CARs. CAR-transduced primary CD8⁺ T lymphocytes were cocultured with HIV-1-infected T2 cells in standard 4-h chromium release assays to assess killing mediated by the CARs. PGT128- and PG9-based CARs were tested for killing in an experiment separate from those for the other CARs. The relative efficiencies of the CARs varied between experiments, and no single CAR was consistently superior.

FIG 7

Sample calculation of percent log efficiency of suppression by CAR-transduced cells. T1-CCR5 cells were infected with the indicated viruses at a multiplicity of infection of 10⁻¹ tissue culture infective doses/cell and cultured with or without CAR 10E8-transduced CD8⁺ T cells (>90% enriched) at an effector-to-target cell ratio of 1:4. (Left) The HIV-1 p24 antigen level was measured by an ELISA on day 7. Log units of p24 antigen (log₁₀ picograms per milliliter) are indicated above each bar. Virus suppression by CAR-transduced cells ranged from 5.44 − 4.73 = 0.71 log₁₀ units to 5.77 − 3.52 = 2.25 log₁₀ units (80.5% to 99.4%) for HIV-1_33931N and HIV-1_NL4-3, respectively. In general, replication without effector cells reached 3 to 6 log₁₀ pg/ml (10³ to 10⁶ pg/ml). (Right) Virus suppression is normalized to total replication without effector cells as the percent reduction in log₁₀ units of p24 antigen comparing cultures with and those without added effector cells: for HIV-1_NL4-3, (5.77 − 3.52)/5.77 = 0.390 = 39.0%; for HIV-1₈₇₃, (5.06 −3.77)/5.06 = 0.255 = 25.5%; and for HIV-1_33931N, (5.44 − 4.73)/5.44 = 0.130 = 13.0%.

FIG 8

Efficiencies of novel CAR-transduced primary CD8⁺ T cells against a panel of HIV-1 isolates. CAR-transduced primary CD8⁺ T cells were tested against a panel of 4 subtype B viruses and one subtype C virus (TZA246) to determine the percent efficiency of log suppression, as shown in Fig. 7. For each virus, bars represent the medians for all replicates across 1 to 6 (mean, 2.9) independent experiments, each with triplicates, with standard-error bars. Note that a 30% log efficiency of suppression for a typical experiment with control viral replication of 5 log₁₀ pg/ml would correspond to a reduction of 1.5 log₁₀ units, or 96.8% suppression of viral replication.