Antigen-specific B cell depletion for precision therapy of mucosal pemphigus vulgaris

Abstract

Desmoglein 3 chimeric autoantibody receptor T cells (DSG3-CAART) expressing the pemphigus vulgaris (PV) autoantigen DSG3 fused to CD137-CD3ζ signaling domains, represent a precision cellular immunotherapy approach for antigen-specific B cell depletion. Here, we present definitive preclinical studies enabling a first-in-human trial of DSG3-CAART for mucosal PV. DSG3-CAART specifically lysed human anti-DSG3 B cells from PV patients and demonstrated activity consistent with a threshold dose in vivo, resulting in decreased target cell burden, decreased serum and tissue-bound autoantibodies, and increased DSG3-CAART engraftment. In a PV active immune model with physiologic anti-DSG3 IgG levels, DSG3-CAART inhibited antibody responses against pathogenic DSG3 epitopes and autoantibody binding to epithelial tissues, leading to clinical and histologic resolution of blisters. DSG3 autoantibodies stimulated DSG3-CAART IFN-γ secretion and homotypic clustering, consistent with an activated phenotype. Toxicology screens using primary human cells and high-throughput membrane proteome arrays did not identify off-target cytotoxic interactions. These preclinical data guided the trial design for DSG3-CAART and may help inform CAART preclinical development for other antibody-mediated diseases.

Keywords: Autoimmune diseases; Autoimmunity; B cells; Immunotherapy; Therapeutics.

Figure 1. DSG3-CAART specifically depletes primary human anti-DSG3 IgG B cells from PV patients.

B cells purified from PV or healthy donor (HD) PBMCs were cocultured with DSG3-CAART, CART19, or NTD cells, and then stimulated with IL-2 and R848 before incubation on plates coated with rhDSG3 and anti–human IgG (anti-hIgG) to quantify anti-DSG3 and total IgG B cells, respectively. BSA served as a negative control antigen. ELISpot analysis shows that anti-DSG3 IgG B cells, but not total IgG B cells, are depleted by DSG3-CAART in PV patients 1 and 2 (columns 1 and 4). In PV patient 3, anti-DSG3 B cells were not detectable (column 7). CART19 eliminates all IgG B cells from PV patients and the HD (rows 3 and 6). Number of spots detected in each well is shown on the top right corner of each well. Quantification discussed in Methods.

Figure 2. DSG3-CAART demonstrates variable activity related to dose.

Luciferase-expressing anti-DSG3 hybridomas AK18, AK19, and AK23 (2 × 10⁵ total) were injected intravenously into NSG mice, followed 4 days later by injection with 3 × 10⁷, 1 × 10⁷, 3 × 10⁶ DSG3-CAART or 3 × 10⁷ NTD T cells. (A and B) Serial bioluminescence imaging indicates 24-fold, 302-fold, and 9-fold relative decreases in target cell burden in mice treated with 3 × 10⁷, 1 × 10⁷, and 3 × 10⁶ DSG3-CAART cells, respectively (black line, median). (C) Direct immunofluorescence staining of buccal mucosa with anti–human IgG indicates epithelial IgG binding (arrows) in 5 of 5 mice treated with NTD T cells and 4 of 5 mice treated with 3 × 10⁶ DSG3-CAART cells, compared with no mice treated with higher DSG3-CAART doses. Original magnification, ×20. (D) Anti-DSG3 ELISA performed on day 10 serum samples indicates that the 3 × 10⁷ and 1 × 10⁷ DSG3-CAART doses effectively abrogated serum anti-DSG3 IgG production relative to NTD or the 3 × 10⁶ DSG3-CAART dose. NS, nonsignificant. (E) Flow cytometric quantification of peripheral blood CD3⁺ T cells (left panel) and qPCR analysis of spleen (day 17, right panel) demonstrate dose-related DSG3-CAART engraftment. Average marking per human cell (mean ± standard deviation) reflects CAAR transgene copy number relative to p21 reference. NA, not applicable (not evaluated). Statistical analysis performed using Kruskal-Wallis (panels B and D) with post hoc Dunn’s test; ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 3. DSG3-CAART reverses rising serum antibody titers and improves blistering in a PV active immune model.

DSG3^–/–DSG1^tg/tg (DSG3-KO) mice were immunized with rhDSG3, followed by splenocyte transfer into syngeneic RAG2^–/– mice. Twenty days after adoptive transfer of splenocytes, 10 and 5 mice were treated with DSG3-CAART or NTD T cells, followed by euthanasia on day 39 (5 mice in each treatment group) or day 69 (5 DSG3-CAART–treated mice), or earlier based on humane endpoints. (A and B) DSG3-CAART (images 6–15) improves hair loss, erosions, and histologic acantholysis (arrows), which persists in NTD-treated mice (images 1–5). Scale bars: 200 μm. (C) Mouse reagents were normalized for use in the human clinical DSG3 ELISA to calculate an anti-DSG3 antibody index value (RU/mL) for all mice with remaining serum samples. Serum anti-DSG3 antibody levels in the active immune model match or exceed those observed in human PV patients. (D) ELISA (mean OD with standard deviation) normalized to week 3 values indicates that DSG3-CAART treatment reduces serum anti-DSG3 antibody titers during the period of DSG3-CAART persistence. (E) T cell persistence by flow cytometry of peripheral blood, weeks 6–8 after splenocyte transfer.

Figure 4. High-throughput membrane proteome array (MPA) plus targeted cell screening did not identify off-target DSG3-CAART cytotoxic interactions.

(A) More than 5300 membrane proteins were expressed in HEK293T cells, and then screened with soluble Fc-tagged proteins to assess binding. Soluble Fc-tagged DSG3-CAAR ectodomain bound weakly to CLEC4M. Px44, anti-DSG3 positive control; FCGR1A, anti-Fc internal control. (B) MPA validation, indicating DSG3-CAAR-Fc binding to CLEC4M-overexpressing HEK293T relative to Fc-isotype control. (C) K562 cells were transduced with lentivectors encoding CLEC4M isoform v1 or v8, sorted to select for high CLEC4M expression, and then incubated with DSG3-CAART or CART19 cells. IFN-γ levels were undetectable in culture supernatants after 16 hours of coincubation of DSG3-CAART and CLEC4M-K562 cells, whereas IFN-γ was detected in positive control cocultures of DSG3-CAART with Nalm6 B cells expressing anti-DSG3 BCR PVB28, or CART19 cells cocultured with CD19⁺ Nalm6 cells. (D) qPCR verifies CLEC4M mRNA expression in pulmonary microvascular but not hepatic sinusoidal endothelial cells. HaCat keratinocytes, negative control. (E) Luminex cytokine analysis performed on DSG3-CAART and primary human cell coculture supernatants indicates no cytotoxic cytokine (IFN-γ, TNF-α) production. Red/blue × = below/above the range of detection. (F) xCELLigence cellular impedance assay of DSG3-CAART and primary human cell cocultures indicates no detectable cytolysis of CLEC4M-expressing pulmonary microvascular endothelial cells. DSG3-CAART induced changes in hepatic endothelial cell impedance only at a 50:1 effector-to-target ratio, a ratio not likely to be achieved in vivo.