Allogeneic, off-the-shelf, SARS-CoV-2-specific T cells (ALVR109) for the treatment of COVID-19 in high-risk patients

Abstract

Defects in T-cell immunity to SARS-CoV-2 have been linked to an increased risk of severe COVID-19 (even after vaccination), persistent viral shedding and the emergence of more virulent viral variants. To address this T-cell deficit, we sought to prepare and cryopreserve banks of virus-specific T cells, which would be available as a partially HLA-matched, off-the-shelf product for immediate therapeutic use. By interrogating the peripheral blood of healthy convalescent donors, we identified immunodominant and protective T-cell target antigens, and generated and characterized polyclonal virus-specific T-cell lines with activity against multiple clinically important SARS-CoV-2 variants (including 'delta' and 'omicron'). The feasibility of making and safely utilizing such virus-specific T cells clinically was assessed by administering partially HLA-matched, third-party, cryopreserved SARS-CoV-2-specific T cells (ALVR109) in combination with other antiviral agents to four individuals who were hospitalized with COVID-19. This study establishes the feasibility of preparing and delivering off-the-shelf, SARS-CoV-2-directed, virus-specific T cells to patients with COVID-19 and supports the clinical use of these products outside of the profoundly immune compromised setting (ClinicalTrials.gov number, NCT04401410).

Figure 1.

Immunogenicity of SARS-CoV-2-derived antigens, target antigen selection, specificity and polyclonality of ex vivo-expanded SARS-CoV-2-specific T cells. (A, B) Reactivity against 18 SARS-CoV-2-derived antigens tested in peripheral blood mononuclear cells (A) and ex vivo-expanded SARS-CoV-2-VST (B) of 16 convalescent donors as measured by IFNγ ELIspot using all 18 antigens as a stimulus. Data are shown in box plots as spot-forming cells (SFC); mean and median values are indicated. (C) Schematic of the ALVR109 manufacturing process using the five selected immunodominant antigens. (D-G) Characterization of ex vivo-expanded SARS-CoV-2-VST. (D) Fold expansion. (E) Specificity as measured by IFNγ ELIspot for 16 lines generated using all five antigens as a stimulus. Data are shown as SFC ± standard error of mean (SEM) and each color represents an individual antigenic specificity. (F) Phenotype and memory/activation profile. Data are shown in box plots; mean and median values are indicated. (G) T-cell receptor vβ repertoire of ex vivo-expanded SARS-CoV-2-VST; representative donor (left) and summary data are shown as mean ± SEM (right). SFC: spot-forming cell; IFNγ: interferon gamma; S: spike; E: envelope; M: membrane; N: nucleocapsid; PBMC: peripheral blood mononuclear cells; IL: interleukin; AP: accessory protein; TEM: effector memory T cells; TCM: central memory T cells; TEMRA: terminally differentiated effector memory T cells; TCR: T-cell receptor; VST: virus-specific T cells; SARS-CoV-2; severe acute respiratory syndrome coronavirus-2.

Figure 2.

Ex vivo-expanded SARS-CoV-2-VST are Th1-polarized, polyfunctional and specifically kill virus-loaded targets. (A) SARS-CoV-2-directed IFNγ production detected within the CD4⁺ and CD8⁺ compartments by ICS; representative donor (left) and summary data shown as mean ± standard error of mean (SEM) (right). (B, C) Th1-polarized effector molecule production by SARS-CoV-2-VST as measured by Luminex (B) and Granzyme B ELIspot (C). ELIspot data have been normalized to background levels and are shown as SFC ± SEM. (D, E) Simultaneous production of multiple effector molecules by SARS-CoV-2-VST as measured by intracellular cytokine staining (D): representative donor [left] and summary data [right]) and multi-parametric FluoroSpot analysis (E). (F, G) Specific lysis of virus-expressing targets by SARS-CoV-2-VST (F) and lack of cytolytic activity against autologous or allogeneic targets (G). *Statistically significant differences (P<0.05). SARS-CoV-2; severe acute respiratory syndrome coronavirus-2; IFNγ: interferon gamma; GM-CSF; granulocyte-macrophage colony-stimulating factor; TNFα: tumor necrosis factor alpha; MIP: macrophage inflammatory protein; IL: interleukin; SFC: spot-forming cells; S: spike; M: membrane; N: nucleocapsid; GrB: granzyme B; PBMC: peripheral blood mononuclear cells; PHA: phytohemagglutinin; E:T: effector to target ratio; VST: virus-specific T cell.

Figure 3.

Ex vivo-expanded SARS-COV-2-VST provide coverage against clinically important viral variants. (A) SARS-CoV-2-VST generated against the parental strain maintain Spike-directed reactivity against all variant strains as measured by IFNγ ELIspot. Data are shown as spot-forming cells (SFC) ± standard error of mean (SEM). (B) Selected mutations of the Spike protein and their prevalence among the different viral variants. (C) Example of a representative donor with a strong response to Spike antigen and the two unique immunogenic epitopes and varying levels of reactivity against variant peptides, as shown by IFNγ ELIspot. Data are reported as SFC ± SEM. (D) Summary results of all 16 donors tested. Each donor retains activity against unique immunogenic epitopes and to multiple mutated Spike peptides. SFC: spike-forming cells; IFNγ: interferon gamma; SARS-CoV-2; severe acute respiratory syndrome coronavirus-2; UIE: unique immunogenic epitope; WT: wildtype; Var: variant; ID: identity; VST: virus-specific T cell.

Figure 4.

Detection of SARS-CoV-2-reactive T cells before and afer infusion in the four infused patients. Specific cells are measured by ELIspot using the five targeted antigens as a stimulus. Results are reported as spot-forming cells ± standard error of mean and each color represents an individual antigenic specificity. In addition, the red arrows indicate each time point at which T-cell receptor deep sequencing confirmed the presence of infused virus-specific T cells in the patients. SFC: spot-forming cells; D1: day 1; Wk: week; Mo: month; N: nucleocapsid; M: membrane; S: spike; TCR: T-cell receptor.