A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity

Abstract

To generate chimeric Ag receptors (CARs) for the adoptive immunotherapy of cancer patients with ErbB2-expressing tumors, a single-chain Ab derived from the humanized mAb 4D5 Herceptin (trastuzumab) was initially linked to T cell signaling domains derived from CD28 and the CD3zeta to generate a CAR against ErbB2. Human PBLs expressing the 4D5 CAR demonstrated Ag-specific activities against ErbB2(+) tumors. However, a gradual loss of transgene expression was noted for PBLs transduced with this 4D5 CAR. When the CD3zeta signaling domain of the CAR was truncated or mutated, loss of CAR expression was not observed, suggesting that the CD3zeta signaling caused the transgene decrease, which was supported by the finding that T cells expressing 4D5 CARs with CD3zeta ITAM mutations were less prone to apoptosis. By adding 4-1BB cytoplasmic domains to the CD28-CD3zeta signaling moieties, we found increased transgene persistence in 4D5 CAR-transduced PBLs. Furthermore, constructs with 4-1BB sequences demonstrated increased cytokine secretion and lytic activity in 4D5 CAR-transduced T cells. More importantly, PBLs expressing this new version of the 4D5 CAR could not only efficiently lyse the autologous fresh tumor digests, but they could strongly suppress tumor growth in a xenogenic mouse model.

FIGURE 1.

Recognition of ErbB2-expressing tumor cell lines by Herceptin 4D5 CAR-transduced PBLs. A, ErbB2 expression of tumor cell lines. An Affibody reagent specific for ErbB2 was used to determine expression on tumor cell lines (solid line), and an isotype Ab was used as negative control (dotted line). B, A schematic representation of the retroviral vector MSGV-4D5–28Z (4D5–28Z) encoding the Herceptin (4D5)-based CAR against ErbB2. SP, signal peptide; VL, variable L chain, VH, variable H chain. Supplemental Fig. 1 shows the complete sequence of the CAR and primers used for synthesizing the scFv. C, Cytokine production of 4D5 CAR-transduced T cells. IFN-γ secretion of 4D5–28Z-transduced T cells following coculture with the indicated tumor lines. Nontransduced T cells (NV) were used as a negative control. D, Specific tumor cell lysis by 4D5 CAR-transduced T cells. 4D5 CAR-transduced PBLs (4D5–28Z) were tested in a ⁵¹Cr release assay, where nontransduced PBLs (NV) served as a control. PBLs were cocultured with ⁵¹Cr-labeled control tumor line MDA468 (ErbB2^–), and ErbB2⁺ tumor lines SK-OV3, SK-BR3, and 624.38mel at the indicated E:T ratio for 4 h, after which the percentage lysis of target cells was calculated. E, Proliferation of 4D5 CAR-transduced PBLs. 4D5–28Z or NY-ESO-1 TCR (1G4-AIB)-transduced PBLs were cocultured with tumor lines MDA468 (ErbB2^–/NY-ESO-1^–), MDA231 (ErbB2⁺/NY-ESO-1^–), and 624.38mel (ErbB2⁺/NY-ESO-1⁺) for 3 days. [³H]thymidine ([³H]TdR) was added for the final 17 h of culture and incorporation was measured. Data are representative of three experiments.

FIGURE 2.

Recognition of anti-ErbB2 CAR-transduced T cells is Ag specific and correlates with Ag expression levels. Ab-blocking experiments of 4D5 CAR (A) and control NY-ESO-1 TCR (B) are shown. The transduced PBLs were cocultured with tumor cell lines in the presence of two anti-ErbB2 Abs: mouse mAb N29(15), or a commercially available anti-ErbB2 IgG fraction (ErbB2) as described in Materials and Methods. Controls included an anti-MAGE1 mAb (MAGE1) and a culture without Ab (No AB). Twenty hours after coculture, IFN-γ secretion was measured by ELISA. C, ErbB2 expression of the clones selected from ErbB2 cDNA plasmid-transfected NIH3T3 cells. D, IFN-γ production of 4D5 CAR (4D5–28Z)-transduced PBLs or control NY-ESO-1 TCR (1G4-AIB) transduced PBLs were cocultured with clones from ErbB2 cDNA-transfected NIH3T3 cells as shown in C for 20 h, and IFN-γ production was measured by ELISA. E, Correlation of ErbB2 expression (mean fluorescence intensity (MFI)) on ErbB2-transfected NIH3T3 clones was plotted vs the production of IFN-γ by 4D5 CAR-transduced PBLs cocultured with these NIH3T3 clones. Data are representative of two experiments.

FIGURE 3.

Transgene decrease was correlated with loss of transduced cells from the culture and was associated with CD3ζ signaling in the CAR. A, A schematic representation of the 4D5-based CAR constructs, including scFv, hinge region (Hinge), transmembrane region (TM), CD28 intracellular domain (CD28 IC), CD3ζ intracellular domain (CD3 ζ IC), and the hinge and transmembrane region of CD8α (CD8). All of the 4D5 mutant scFvs and CD3ζ mutations were generated by PCR-based site-directed mutagenesis using the parent 4D5–28Z as template according to the published sequences (25). The published affinity of the different 4D5 Ab variants is: parent 4D5–28Z (3 × 10^–10 M), 4D5–1 (2.5 × 10^–8 M), 4D5–3 (4.4 × 10^–9 M), 4D5–5 (1.1 × 10^–9 M), and 4D5–7 (6.2 × 10^–10 M). The vertical line indicates the approximate location of the mutation. B, Transgene expression over time after transduction. PBLs were transduced with 4D5–28Z-based CARs with scFv at different affinities (4D5–28Z, 4D5–1, 4D5–3, 4D5–5, 4D5–7), a 4D5 CAR with hinge and transmembrane regions from human CD8α without CD28 signaling domain (4D5-CD8HTZ), and a 4D5 CAR with both CD28 and CD3ζ signaling domains being truncated (4D5–28D). Construct organization was as depicted in A. Controls included a CAR against CD19 (FMC63–28Z) and an anti-NY-ESO-1 TCR (1G4-AIB). Transgene expression was monitored at days 8, 21, and 35 after transduction by flow cytometry staining of the transduced PBLs using gene-specific reagents. Data shown are percentages of day 8 transgene expression of each transduced PBL population. C, PBLs were transduced with 4D5-based CARs with various signaling domain alterations: 4D5 CAR with CD28 and CD3ζ signaling domains being truncated (4D5–28D), 4D5 CAR with all three ITAMs within CD3ζ being mutated (4D5–28ZM), CD28 signaling domain being truncated from 4D5–28Z (4D5–28HTZ), 4D5 CAR without CD28 signaling domain and hinge/transmembrane region from CD8α (4D5-CD8HTZ), and the parent 4D5–28Z (4D5–28Z). As controls, PBLs were transduced with anti-NY-ESO-1 TCR (1G4-AIB), LNGFR CAR (LNGFR-28Z), SP6 CAR (SP6–28Z), and a CD19 CAR (FMC63–28Z). All mutant constructs were as depicted in A. Transgene expression was detected at indicated time by flow cytometry for protein using gene-specific reagents (left panel), for RNA using real-time quantitative RT-PCR (middle panel), and for DNA copy number determined by real-time quantitative PCR (right panel). Data shown are percentages of day 8 transgene expression of each transduced PBL population. Data are representative of three experiments.

FIGURE 4.

Effect of specific ITAM mutations on expression, function, and AICD. A, Schematic representation of 4D5–28Z-based CARs with different ITAMs in CD3ζ signaling moiety being mutated (tyrosine to phenylalanine). B, Transgene expression of PBLs transduced with CD3ζ ITAM-mutated 4D5 CAR constructs at days 7 and 55 after transduction. C, Annexin V and PI staining of PBLs transduced with 4D5 CARs with CD3ζ ITAM mutants following coculture with ErbB2⁺ tumor lines SK-OV3 or SK-BR3, with an ErbB2^– tumor line MDA468, and without a tumor line (Medium). Percentages of positive cells were as shown in each quadrate.

FIGURE 5.

Adding 4–1BB signaling moiety enhanced function and maintained transgene expression of 4D5 CAR-transduced PBLs. A, A schematic representation of variant 4D5 CAR constructs with hinge and transmembrane region from either CD8α or CD28 preceding the intracellular signaling domains of CD28, 4–1BB, or CD3ζ. The signaling domains were combined in the different orders as shown. B, Transgene expression. PBLs were transduced with 4D5 CAR-based constructs with different protein domains as diagrammed in A. Transgene expression was monitored at days 10 and 30 posttransduction by flow cytometry, and the percentage of transduction was as shown. C, PBLs transduced with a 4–1BB containing CARs were less prone to AICD. PBLs were transduced with 4D5 CARs with transmembrane region and hinge region from CD8α followed by CD3ζ (CD8HTZ), both CD28 and CD3ζ (CD8–28Z), or by a construct with 4–1BB inserted between CD28 and CD3ζ (CD8–28BBZ). Controls were an unrelated CAR, CD19 CAR (FMC63–28Z), and untransduced cells (NV). Three days posttransduction, PBLs were subjected to staining with PI to determine the percentage of dead cells. The flow cytometry results shown were the cells without any gating; the number shows the percentage of PI⁺ cells. D, Effector cytokine production. PBLs transduced with retroviral vectors expressing the constructs shown in A were tested by coculture with ErbB2⁺ (SK-BR3) and ErbB2^– (MDA468 and CEM) cell lines. Effector cytokine production (IFN-γ) was determined by ELISA following overnight coculture. E, Specific lysis by 4D5 CAR-transduced T cells. 4D5-CD8–28BBZ (CD8–28BBZ)- and 4D5-CD8–28Z (CD8–28Z)-transduced PBLs, with control nontransduced PBLs (NV), were cocultured with ⁵¹Cr-labeled tumor lines MDA361 (ErbB2⁺), SK-BR3 (ErbB2⁺), MDA468 (ErbB2^–), and CEM (ErbB2^–) at the indicated E:T ratio. Data were the percentage lysis of specific target cells as calculated (see Materials and Methods). Significant differences (where p < 0.01 by two-way ANOVA test) between the mean values for 4D5-CD8–28BBZ and 4D5-CD8–28Z are indicated by asterisks. Data are representative of three experiments.

FIGURE 6.

Adding 4–1BB signaling moiety in the CAR increased both Bcl-x_L and NKG2D expression upon specific Ag stimulation. A, Increased Bcl-x_L expression in cells transduced with 4–1BB containing CAR. PBLs were transduced with 4D5-CD8–28BBZ (CD8–28BBZ), 4D5–28Z (28Z), or nontransduced (NV) and then cocultured with ErbB2⁺ tumor SK-OV3 or ErbB2^– tumor MDA468 for 20 h. PBLs were separated from tumor cells by enriching for T cells with CD3 magnetic beads and subjected to Western blot for the detection of Bcl-x_L. Data were quantitated and expression relative to β-actin was plotted. B, Increased NKG2D expression in cells transduced with 4–1BB containing CAR. Transduced PBLs as indicated were stimulated with ErbB2-Fc-coated plates for 20 h. The cells were harvested and stained with NKG2D and ErbB2-Fc (for 4D5 CAR) and subjected to flow cytometry. Dead cells were excluded by PI gating and CAR-transduced cells were analyzed for NKG2D expression by gating on ErbB2-Fc⁺ population. Percentages of positive cells were as indicated. Data are representative of two experiments.

FIGURE 7.

Reactivity against fresh tumor and in vivo tumor treatment efficiency of Herceptin-based 4D5-CAR-transduced T cells. A, ErbB2 expression on fresh tumor samples. ErbB2 expression was determined using flow cytometry using an anti-ErbB2 Affibody moleculer (filled histogram) and an isotype control (open histogram) on fresh tumor digests from three melanoma patients (donors 1–3 FrTu), two ErbB2⁺ tumor lines (SK-OV3 and SK-BR3), and one ErbB2^– line (CEM) as control. B, Tumor cell lysis. Lytic activity of 4D5 CAR-transduced PBLs against tumor cell lines and autologous fresh tumor digests (FrTu Digest). 4D5-CD8–28BBZ-transduced PBLs from different donors (D1, D2, and D3) were cocultured with ⁵¹Cr-labeled tumor lines at the indicated E:T ratios using SK-OV3 (ErbB2⁺), SK-BR3 (ErbB2⁺), CEM (ErbB2^–), and autologous fresh tumor digests (FrTu) as targets. Data were the percentages of lysis of specific target cells as calculated (see Materials and Methods). Nontransduced PBLs from the same donors served as negative controls (NV). C, Treatment of a breast cancer xenograft by ErbB2-CAR-transduced PBLs. SCID mice bearing human breast tumor line BT-474, implanted in the mammary gland 10 days before treatment, were established as described in Materials and Methods. Adoptively transferred (i.v.) 4D5–28Z or 4D5-CD8–28BBZ- or SP6–28Z-transduced PBLs were administered to animals with palpable tumors. No treatment control mice received HBSS (n = 7–10/group). Tumor volume was monitored at time points as indicated. Significant difference (where p < 0.05 by two-way ANOVA test) between the mean values at day 72 for 4D5–28Z and 4D5-CD8–28BBZ is indicated by asterisks.