Chimeric antigen receptor T cells to target CD79b in B-cell lymphomas

Abstract

Background: Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent and durable effects in B-cell malignancies. However, antigen loss or downregulation is a frequent cause of resistance. Here, we report development of a novel CAR T-cell therapy product to target CD79b, a pan B-cell antigen, widely expressed in most B-cell lymphomas.

Methods: We generated a novel anti-CD79b monoclonal antibody by hybridoma method. The specificity of the antibody was determined by testing against isogenic cell lines with human CD79b knock-in or knock-out. A single-chain variable fragment derived from the monoclonal antibody was used to make a panel of CD79b-targeting CAR molecules containing various hinge, transmembrane, and co-stimulatory domains. These were lentivirally transduced into primary T cells and tested for antitumor activity in in vitro and in vivo B-cell lymphoma models.

Results: We found that the novel anti-CD79b monoclonal antibody was highly specific and bound only to human CD79b and no other cell surface protein. In testing the various CD79b-targeting CAR molecules, superior antitumor efficacy in vitro and in vivo was found for a CAR consisting CD8α hinge and transmembrane domains, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. This CD79b CAR specifically recognized human CD79b-expressing lymphoma cell lines but not CD79b knock-out cell lines. CD79b CAR T cells, generated from T cells from either healthy donors or patients with lymphoma, proliferated, produced cytokines, degranulated, and exhibited robust cytotoxic activity in vitro against CD19⁺ and CD19^- lymphoma cell lines and patient-derived lymphoma tumors relapsing after prior CD19 CAR T-cell therapy. Furthermore, CD79b CAR T cells were highly efficient at eradicating pre-established lymphoma tumors in vivo in three aggressive lymphoma xenograft models, including two cell line-derived xenografts and one patient-derived xenograft. Notably, these CAR T cells did not demonstrate any significant tonic signaling activity or markers of exhaustion.

Conclusion: Our results indicated that this novel CD79b CAR T-cell therapy product has robust antitumor activity against B-cell lymphomas. These results supported initiation of a phase 1 clinical trial to evaluate this product in patients with relapsed or refractory B-cell lymphomas.

Keywords: B-Lymphocytes; Immunotherapy; Immunotherapy, Adoptive; Receptors, Chimeric Antigen; T-Lymphocytes.

Figure 1

Schematic of different CD79b CAR constructs and transduction efficiency in primary T cells. (A) Schematic design of CAR constructs to target CD19 or CD79b. (B and C) CAR constructs shown in panel A were transduced lentivirally into primary human T cells and transduction efficiency was determined by flow cytometry by assessing eGFP expression and CAR expression is shown by demonstrating binding to fluorescently labeled recombinant human CD79b-Fc protein. Data is representative of CAR T cells generated from two different donors. CAR, chimeric antigen receptor; eGFP, enhanced green fluorescent protein; LTR, long terminal repeat; LTRtr, LTR truncated; scFv, single chain variable fragment; sp, signal peptide; VL, variable light chain; VH, variable heavy chain; TM, transmembrane domain.

Figure 2

CD79b CAR T cells proliferated and produced cytokines in response to lymphoma cells in vitro. CAR T cells targeting CD19 or CD79b generated from healthy donor T cells were co-cultured with Daudi Burkitt lymphoma tumor cells at an effector:target ratio of 1:1. Untransduced T cells were used as controls. (A) Representative flow cytometric plots illustrating proliferation of CD4⁺ and CD8⁺ CAR T cells after 96 hours of co-culture. (B) Summary results showing proliferation from replicate wells (N=2). (C) Cytokine production by CAR T cells in response to Daudi lymphoma cells in replicate wells (N=2) after 24 hours of co-culture. Data represents mean±SD and is representative of at least three separate experiments from three different donors. ***p<0.001; **p<0.01; *p<0.05; (ns) p>0.05. CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor.

Figure 3

CD79b CAR T cells degranulated and exhibited cytotoxic activity against lymphoma cells in vitro. CAR T cells targeting CD19 or CD79b were generated from healthy donor T cells and co-cultured with Daudi Burkitt lymphoma tumor cells at an effector:target ratio of 1:1. Untransduced T cells were used as controls. (A) Representative flow cytometric plots illustrating CD107a/b degranulation on CD4⁺ and CD8⁺ CAR T cells after 6 hours of co-culture are shown. (B) Summary results showing degranulation from replicate wells (N=2). (C–E) CAR T cells labeled with CellTrace Far Red were co-cultured with Daudi lymphoma tumor cells labeled with CellTrace Violet at an effector:target ratio of 1:1. Cytotoxicity assay was assessed after 96 hours by flow cytometry by counting live tumor cells using TruCOUNT beads. Untransduced T cells were used as controls. (C) Representative flow cytometric plots showing percentage of live tumor and T cells after end of co-culture. Summary results of cytotoxicity assay from replicate wells (N=2) showing absolute live tumor cell count (D) or specific lysis (E) of Daudi tumor cells. Data represents mean±SD and is representative of at least three separate experiments from three different donors. ***p<0.001; **p<0.01; *p<0.05; (ns)p>0.05. CAR, chimeric antigen receptor.

Figure 4

CD79b CAR T cells exert antitumor effects in vivo. (A) Schematic overview of xenograft mouse model. Luciferase-labeled Daudi Burkitt lymphoma cells were injected intravenously into NSG mice at 2×10⁴ tumor cells/mouse. After 11 days, mice were treated with untransduced T cells, CD19 CAR T cells, or CD79b CAR T cells via tail vein injection at 5×10⁶ CAR⁺ T cells/mouse. (B) Tumor burden assessed by bioluminescence imaging at the indicated time points is shown. (C) Average radiance (photons/sec/cm²/steradian) of groups of mice at different time points is shown. Significance among treatment groups was determined by comparing area under the curve for each CAR-T group to untransduced control T-cell group in an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (n=5 mice per group, mean±SD is shown). (D) Kaplan-Meier survival curves of groups of mice treated with different T cells. (****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; (ns) p>0.05, ANOVA). ANOVA, analysis of variance; CAR, chimeric antigen receptor; I.V., intravenous.

Figure 5

CD79b CAR T cells generated from patients with lymphoma relapsing after autologous CD19 CAR T-cell therapy are functional. CAR T cells targeting CD79b were generated from T cells isolated from two patients with large B-cell lymphoma relapsing after autologous CD19 CAR T-cell therapy and co-cultured with Daudi Burkitt lymphoma tumor cells at an effector:target ratio of 1:1. Untransduced T cells were used as controls. Representative data from one patient is shown. (A) Representative flow cytometric plots showing transduction efficiency of 79b CAR and proportion of CD4⁺ and CD8⁺ T cells. (B) Representative flow cytometric plots illustrating CD107a/b degranulation by CD4⁺ and CD8⁺ CAR T cells after 6 hours of co-culture. (C) Summary results showing degranulation from replicate wells (N=2). (D) Representative flow cytometric plots illustrating proliferation of CD4⁺ and CD8⁺ CAR T cells after 96 hours of co-culture. (E) Summary results showing proliferation from replicate wells (N=2). (F) Summary results of cytotoxicity assay from replicate wells (N=2) showing absolute live tumor cell count (left panel) or specific lysis (right panel). (G) Cytokines production by CAR T cells co-cultured with Daudi lymphoma cells in replicate wells (N=2) after 24 hours of co-culture. Data is representative of at least three separate experiments with T cells from two different patients. ***p<0.001; **p<0.01; *p<0.05; (ns) p>0.05. CAR, chimeric antigen receptor; IFN, interferon; IL, interleukin; GFP, green fluorescent protein; GM-CSF, granulocyte-macrophage colony-stimulating factor.

Figure 6

Efficacy of CD79b CAR T cells against patient-derived lymphoma tumor cells. CAR T cells targeting CD79b were generated from primary healthy donor T cells, labeled with CellTrace Far Red and co-cultured with Daudi Burkitt lymphoma tumor cells or lymphoma tumor cells derived from two PDXs (PDX203 and PDX300) for 96 hours. Untransduced T cells were used as controls. (A) Histograms showing expression of CD19 and CD79b in the two tumor samples. (B) Representative flow cytometric plots illustrating proliferation of CD4⁺ and CD8⁺ CARs T cells in response to tumor cells at an effector:target (E:T) ratio of 0.6:1. (C) Summary results showing proliferation from replicate wells (N=2). (D) Summary results of cytotoxicity assay from replicate wells (N=2) showing specific lysis at E:T ratio of 0.6:1. (E and F) Pooled data from 17 experiments testing CD79b CAR T cells generated from five donors is shown for per cent change in absolute number of live tumor cells on day 4 in an in vitro cytotoxicity assay compared with day 0 (E) and fold change in interferon-γ production compared with untransduced T cells after 24 hours of co-culture (F). Target tumor cells in these experiments included Daudi, Jeko-1, SUDHL6, or PDX203. Each dot represents data from one experiment and mean values of data from replicate wells from each experiment are shown. (G–J) Luciferase-labeled PDX203 5D4 tumor cells (0.2$\times$10⁶ /mouse) were injected via tail vein into NSG mice on day −4 and CD79b CAR T cells or untransduced T cells (5$\times$10⁶ /mouse) derived from two different donors were injected via tail vein on day 0. Tumor burden was monitored by bioluminescence imaging (G and I). Average radiance (photons/sec/cm²/steradian) of groups of mice on days 35 and 29 are shown (H and J). (n=5 mice per group, mean±SD is shown). ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; (ns) p>0.05. CAR, chimeric antigen receptor; PDX, patient-derived xenografts.

Figure 7

CD79b CARs specially recognized human CD79b protein. (A) CAR T cells targeting CD79b were generated from primary healthy donor T cells, stained with fluorochrome labeled recombinant human CD19-Fc, CD79a-Fc, or CD79b-Fc proteins, and assessed by flow cytometry. Transduction efficiency determined by eGFP expression and binding of recombinant proteins is shown. (B–D) CAR constructs targeting CD19 or CD79b were transduced lentivirally into Jurkat-Lucia NFAT reporter cell line and cultured alone or with the indicated lymphoma cell lines at an effector:target (E:T) ratio of 1:1 or with anti-CD3 and anti-CD28 monoclonal antibodies. NFAT activation was determined after 24 hours by measuring luciferase activity. Percentage of Jurkat-Lucia NFAT reporter cells expressing CD19 or CD79b CAR was determined by assessing eGFP transduction marker by flow cytometry (B). Luciferase activity shown in Jurkat-Lucia NFAT reporter cells transduced with 79b CAR construct and cultured alone or with anti-CD3/CD28 antibodies or Daudi Burkitt lymphoma cells (C). Luciferase activity shown in Jurkat-Lucia NFAT reporter cells transduced with the indicated CD19 or CD79b CAR constructs and cultured alone or with isogenic SUDHL6 lymphoma cell lines (parent, CD19^KO, CD79b^KO, or CD19^KOCD79b^KO). (E) Cytotoxic activity of CD19 or CD79b CAR T cells generated from healthy donor T cells against isogenic cell lines of SUDHL6 (parent, CD19^KO, CD79b^KO, or CD19^KOCD79b^KO). (F) Cytotoxic activity of CD79b CAR T cells against wild type Jeko-1 and PDX203 5D4 (CD19⁺CD79b⁺), Jeko-1 CD19^KO, Jeko-1 CD79b^KO, or NALM6 acute lymphoblastic leukemia (CD19⁺CD79b^–) cell lines at an E:T ratio of 1:1. The absolute number of live tumor cells at the end of culture period from this experiment are shown in online supplemental figure 10. (G and H) Luciferase-labeled Jeko-1 CD19^KO or Jeko-1 CD79b^KO tumor cells (0.2$\times$10⁶ /mouse) were injected via tail vein into NSG mice on day −4 and CD79b CAR T cells or untransduced T cells (5$\times$10⁶ /mouse) were injected via tail vein on day 0. Tumor burden was monitored by bioluminescence imaging at the indicated time points (G) and average radiance (photons/sec/cm²/steradian) of groups of mice on day 39 is shown (H). Data represent the mean±SD of replicate wells (N=2) where applicable and is representative of at least three separate experiments from two different donors (C–F). ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; (ns) p>0.05. CAR, chimeric antigen receptor; eGFR, enhanced green fluorescent protein; HD, healthy donor; KO, knock-out; RLU, relative luminescence units.