Fig 2: The fully human anti-CD229 antibody 2D3 binds to MM cells and B and T cells.a Binding of the fully human IgG1 antibody 2D3 to 293 cells individually expressing 5300 full-length, human membrane proteins as determined by flow cytometry. Fc gamma receptor 1A (FCGR1A) serves as a positive control. b K562 cells were stably transduced with a human CD229 expression construct and stained with 2D3. Binding was determined by flow cytometry. c Left: The extracellular domain of CD229 consists of two variable and two constant Ig-like domains. In a human CD229 expression construct, each of these domains was replaced with the corresponding mouse homolog as 2D3 did not show any reactivity to mouse CD229 (Supplementary Fig. 2A). Right: Binding by 2D3 and the commercially available CD229-specific antibody HLy9.1.2533 to the four chimeric proteins expressed on 293T cells was determined by flow cytometry. d MM cells were isolated from primary bone marrow samples from three patients with MM using a commercially available MM cell purification kit (Stemcell Technologies). Purified MM cells were stained with 2D3 and binding was determined by flow cytometry. e Bone marrow samples from seven patients with newly diagnosed (ND) and eight patients with relapsed/refractory (RR) MM were stained with 2D3 and analyzed by flow cytometry. Data represent mean ± standard deviation and statistical significance was determined by two-sided Student’s t-test. f CD34+ hematopoietic progenitors, neutrophils, monocytes, g natural killer (NK) cells, B cells, and T cells were isolated from peripheral blood from a healthy donor using commercially available purification kits (Stemcell Technologies). Cells were stained with 2D3 and analyzed by flow cytometry. Source data are provided as a Source Data file.

Fig 3: CD229 CAR T cells target MM plasma cells and MM-propagating cells.a Killing of MM cell lines by CD229 CAR T cells or ∆scFv CAR T cells as determined by luciferase-based cytotoxicity assay after 16 h co-culture. Data represent mean ± SD from three independent experiments. b Cytotoxic activity of CD229 CAR T cells or ∆scFv CAR T cells against malignant cells from three patients with plasma cell leukemia as determined by flow cytometry after 2–4 h co-culture. Data indicate mean ± SD from three patient samples. c Secretion of cytokines by CAR T cells during co-cultures with primary plasma cell leukemia cells as determined by cytometric bead assay. Data indicate mean ± SD from three co-cultures. Significance was calculated using two-sided Student’s t-test. NSG mice were injected with d 3 × 106 U-266 or e 1 × 106 RPMI-8226 cells expressing luciferase. After 1 week, mice were injected with 3 × 106 CAR T cells and bioluminescence was determined weekly. Differences in survival were determined by log-rank test. Data are representative of two independent experiments. f Killing of purified healthy B cells by CD229 CAR T cells or GFP T cells as determined by flow cytometry-based cytotoxicity assay. Data represent mean ± SD of three independent experiments. NSG mice were intravenously injected with 107 healthy PBMCs. After two days, mice were injected with 1 × 106 CD19 or CD229 CAR T cells or PBS and sacrificed after 4 days. B-cell numbers were determined in the animals’ g peripheral blood and h bone marrow. Data represent mean ± SD from three animals per group. Significance was determined by two-sided Student’s t-test. i In vitro cytotoxicity of CD229, ∆scFv, or BCMA CAR T cells against purified memory or naive B cells as determined by flow cytometry. Data represent mean ± SD from three independent experiments. j Bone marrow mononuclear cells from seven MM patients were co-cultured with CD229, ∆scFv, or BCMA CAR T cells for 4–6 h, depleted of CD3+ CAR T cells, and plated in methylcellulose. Colonies were counted after 14–21 days of culture. Data represent mean ± SD from seven patient samples. Statistical significance was determined by two-sided Student’s t-test. Source data are provided as a Source Data file.

Fig 4: Production of CD229 CAR T cells and T-cell targeting.a CAR T-cell expansion as determined by automated cell counting. Data represent mean ± SD from four healthy donors. Significance of differences between cell numbers on day 11 was determined by two-sided Student’s t-test. b Killing of K562 cells transduced with a CD229 expression construct or parental CD229- K562 cells by CD229 CAR T cells as determined by luciferase-based cytotoxicity assay. Data represent mean ± SD from three independent experiments. c Cytotoxic activity of CD229 CAR T cells against T cells that had undergone CAR T-cell manufacturing for 7 days except for addition of lentiviral particles (Tact) as determined by flow cytometry. Data represent mean from two independent experiments. d CD229 expression in whole cell lysates from T cells, NK cells, and Tact cells, as determined by western blot. Blots show representative results from two independent experiments. e CD229 expression in T cells, NK cells, and Tact cells as determined by quantitative RT-PCR. Data represent mean ± SD from three replicates. Statistical significance was determined by two-sided Student’s t-test. f Binding of 2D3 to Tact cells. Histogram shows representative result for one healthy donor from at least 3 independent experiments. g In vitro cytotoxicity of CD229 CAR T cells or GFP T cells against purified healthy T cells as determined by flow cytometry. Data represent mean ± SD of three independent experiments. h NSG mice were intravenously injected with 107 healthy PBMCs. After 2 days, mice were injected with 1 × 106 CD19 or CD229 CAR T cells or PBS and sacrificed after 4 days. Data represent mean ± SD of healthy non-CAR T cell numbers in the peripheral blood from three animals per group. Significance was determined by two-sided Student’s t-test. i CD229 expression on T cells from the same animals as determined by flow cytometry. j T cells from healthy donors were sorted for CD229 expression (Supplementary Fig. 3E) and stimulated with CD3/CD28 beads and 40 IU/ml IL-2 for 4 days. IFNG secretion by CD229low and CD229high T cells was determined by cytometric bead array (BD). Data represent mean ± SD from three healthy donors. Significance was determined by two-sided Student’s t-test. k Expansion of CD229low and CD229high T cells as determined by automated cell counting. Data represent mean ± SD from six healthy donors. Significance was determined by two-sided Student’s t-test. Source data are provided as a Source Data file.