A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform

Abstract

Recent treatments of leukemias with chimeric antigen receptor (CAR) expressing T cells underline their impressive therapeutic potential. However, once adoptively transferred into patients, there is little scope left to shut them down after elimination of tumor cells or in case adverse side effects occur. This becomes of special relevance if they are directed against commonly expressed tumor associated antigens (TAAs) such as receptors of the ErbB family. To overcome this limitation, we recently established a modular CAR platform technology termed UniCAR. UniCARs are not directed against TAAs but instead against a unique peptide epitope on engineered recombinant targeting modules (TMs), which guide them to the target. In the absence of a TM UniCAR T cells are inactive. Thus an interruption of any UniCAR activity requires an elimination of unbound TM and the TM complexed with UniCAR T cells. Elimination of the latter one requires a disassembly of the UniCAR-TM complexes. Here, we describe a first nanobody (nb)-based TM directed against EGFR. The novel TM efficiently retargets UniCAR T cells to EGFR positive tumors and mediates highly efficient target-specific and target-dependent tumor cell lysis both in vitro and in vivo. After radiolabeling of the novel TM with ⁶⁴Cu and ⁶⁸Ga, we analyzed its biodistribution and clearance as well as the stability of the UniCAR-TM complexes. As expected unbound TM is rapidly eliminated while the elimination of the TM complexed with UniCAR T cells is delayed. Nonetheless, we show that UniCAR-TM complexes dissociate in vitro and in vivo in a concentration-dependent manner in line with the concept of a repeated stop and go retargeting of tumor cells via the UniCAR technology.

Keywords: CAR; EGFR; T cell; T cell therapy; retargeting.

Figure 1.

Redirection of T cells to EGFR-positive tumor cells via the UniCAR platform. The UniCAR system consists of two components: (i) the UniCAR effector T cells and (ii) TMs directed to a cell surface structure. In the absence of a TM UniCAR T cells are inert. In the presence of a TM UniCAR T cells can form an immune complex via a peptide epitope (E5B9) that is fused to the TM and recognized by the antibody domain of the UniCAR. So far, TMs were constructed from IgG type Abs resulting in scFv-based molecules (left panel). In the current manuscript we aimed to develop a TM based on a camelide Ab-derived nb (right panel) directed against EGFR.

Figure 2.

Development of the novel nb-based α-EGFR TM. (A) Two α-EGFR TM constructs (A I, α-EGFR TM (eu); A II, α-EGFR TM (pro)) were cloned for expression either in CHO cells (α-EGFR TM (eu)) or in E. coli (α-EGFR TM (pro)). As schematically shown, both nb-based α-EGFR TM constructs consist of the open reading frame encoding the EGFR-specific nb. For binding to the UniCAR the E5B9-tag is fused to the C-terminus. Furthermore, both TMs are tagged with 6xhis residues at the C-terminus for protein purification and detection. To enable eukaryotic expression, the α-EGFR TM (eu) construct additionally contains an N-terminal signal peptide (SP). To facilitate the interaction of UniCAR T cells with the TM the E5B9 tag was N- and C-terminally flanked with a glycine (4x)-serine (1x) linker (G₄S). (B) The elution fraction of the purified α-EGFR TM (eu) (lane 1) and α-EGFR TM (pro) (lane 2) was separated via SDS-PAGE and subsequently stained with Coomassie brilliant blue G-250 (BI) or transferred onto a nitrocellulose membrane for detection of the purified α-EGFR TM (eu) (lane 1) and α-EGFR TM (pro) (lane 2) via its C-terminal his-tag (BII). M, molecular weight marker. (C) Both TMs were further analyzed by size exclusion HPLC using either 15 µL of the α-EGFR TM (eu) (10 µg) or 7.5 µL of the α-EGFR TM (pro) (15 µg).

Figure 3.

Binding of the novel TMs to EGFR-expressing tumor cells. To analyze binding properties of the α-EGFR TMs, A431 and FaDu cells were stained with the respective TM (20 ng/μL). The specific binding was detected via the E5B9-tag using an α-E5B9 mAb and a PE-conjugated α-mouse-IgG mAb. As positive control, cells were labeled with mAb α-EGFR/PE-Cy7. The histograms show cells stained with either the control Ab (dark gray graphs, upper panel) or the α-EGFR TM (eu) (light gray graphs, lower panel) or α-EGFR TM (pro) (dark gray graphs, lower panel) and their respective controls (transparent graphs). The numbers represent the percentage of antigen-positive cells and mean fluorescence intensity (MFI) of stained cells.

Figure 4.

Estimation of the K_d values of the novel α-EGFR TMs. Increasing amounts of the α-EGFR TM (eu) (left panel) or α-EGFR TM (pro) (right panel) were used for staining of A431 cells. Binding was detected via the E5B9-tag using an α-E5B9 mAb and a PE-conjugated α-mouse-IgG mAb. The respective K_d value was calculated from the resulting binding curve (see also Materials and methods).

Figure 5.

Target-specific lysis of EGFR-positive tumor cells via the novel UniCAR system. In standard chromium release assays (A) A431 or (B) FaDu cells were incubated with T cells engrafted with either the vector control encoding the EGFP marker protein (vector control), the UniCAR Stop construct lacking intracellular signaling domains (UniCAR Stop) or the α-E5B9 signaling construct (UniCAR 28/ζ). The A431 and FaDu cells were cultivated with the respective genetically engineered T cells in the presence or absence of 50 nM α-EGFR TM (eu) (A I, B I) or α-EGFR TM (pro) (A II, B II) for 48 h. Mean specific lysis and SD for seven (A431 and FaDu, e:t ratio of 5:1) or three (A431, e:t ratio of 1:1) independent T cell donors are shown (**p < 0.01 and ***p < 0.001; with respect to controls: vector control ± α-EGFR TM, UniCAR Stop ± α-EGFR TM, UniCAR 28/ζ w/o Ab; one-way ANOVA with Bonferroni multiple-comparison test).

Figure 6.

Estimation of range of working concentrations and EC₅₀ values for the novel α-EGFR TMs. For a direct comparison of the capability to mediate a UniCAR-dependent lysis of EGFR-positive tumor cells A431 cells and UniCAR 28/ζ T cells were incubated with increasing amounts of (A) the α-EGFR TM (eu) or (B) the α-EGFR TM (pro). Chromium release was measured after 48 h of incubation. Data shown represent mean specific lysis and SD for 6 independent donors (***p < 0.001; with respect to control: UniCAR 28/ζ w/o TM; one-way ANOVA with Bonferroni multiple-comparison test). (C) For estimation of the respective EC₅₀ value (half maximal effective concentration) UniCAR 28/ζ engrafted T cells were incubated with A431 cells in the presence of increasing concentrations of the respective α-EGFR TM for 48 h. Mean specific lysis and SEM for six independent T cell donors are shown.

Figure 7.

Cytokine release from EGFR-redirected UniCAR T cells as estimated by either (A) a multiplex assay or (B) ELISA. (A and B) A431 cells were incubated in the presence of genetically engineered UniCAR 28/ζ T cells either in the absence (UniCAR CD28/ζ + A431, white bars) or presence of 50 nM of the α-EGFR TM (eu) (gray bars) or α-EGFR TM (pro) (black bars) at an e:t ratio of 5:1 for 48 h. Cytokines secreted into cell culture supernatants were estimated for three individual donors. As controls UniCAR T cells were co-cultivated with A431 cells in the absence of 50 nM TM as indicated in (A). As additional controls T cells were engrafted with the vector control or the UniCAR Stop construct and incubated in the presence of 50 nM TM as indicated in (B). (A) Using the MACSPlex Cytokine 12 Kit, variable amounts of the cytokines GM-CSF, IFNγ, IL-2, IL-4, IL-9, and TNF-α were detected (x, not detectable, n.d. not defined) but not the other cytokines IFN-α , IL-5, IL-6, IL-9, IL-10, IL-17A (Data not shown). (B) The concentration of selected cytokines including IFNγ (left), IL-2 (middle) and TNF (right) in co-culture supernatants was also estimated by ELISA. Mean cytokine concentrations and SD of triplicates for three independent donors are shown (x, not detectable).

Figure 8.

Retargeting of EGFR-positive tumor cells in experimental mice. A431 cells were transduced to express firefly luciferase resulting in A431-Luc cells. Per mouse, 1.5×10⁶ A431-Luc cells were mixed with 1.5×10⁶ UniCAR 28/ζ T cells and 100 µg of the α-EGFR TM (pro). As “untreated” control served 1.5×10⁶ A431-Luc cells mixed with 1.5×10⁶ UniCAR 28/ζ T cells without any TM. The respective mixture (100 µL) was injected subcutaneously into SCID/beige mice resulting in two groups of animals representing untreated (group A) or treated (group B) mice. Luminescence imaging of anesthetized mice was performed 10 min after i.p. injection of 200 µL of D-luciferin potassium salt (15 mg/mL) starting at day one (D1), and followed at day 2 (D2), and day 8 (D8).

Figure 9.

MALDI-TOF MS analysis (A) and SDS-PAGE followed by autoradiography (B) of ⁶⁴Cu-radiolabeled α-EGFR TM (pro). (A) A mixture of non-modified and NODAGA modified TMs were analyzed with MALDI-TOF MS. The molecular mass of the unmodified α-EGFR TM (pro) was determined with 16676.611 (m/z). The mean of the molecular mass of the NODAGA conjugated TMs was estimated with 17486.367 (m/z), which yields an average of 1.2 NODAGA chelator groups per molecule of the α-EGFR TM (pro) (NODAGA)_1.2. (B) After SDS-PAGE, the autoradiogram of the [⁶⁴Cu]Cu-α-EGFR TM (pro) (NODAGA)_1.2 shows a main fraction with a molecular mass of approximately 18 kDa.

Figure 10.

Biodistribution of ⁶⁴Cu- and ⁶⁸Ga-radiolabeled α-EGFR TM (pro). After conjugation of the α-EGFR TM (pro) with NODAGA the resulting α-EGFR TM (pro) (NODAGA)_1.2 was radiolabeled with either ⁶⁴Cu or ⁶⁸Ga. The biodistribution of the [⁶⁴Cu]Cu-α-EGFR TM (pro) (NODAGA)_1.2 complex is shown in (A and C). The biodistribution of the [⁶⁸Ga]Ga-α-EGFR TM (pro) (NODAGA)_1.2 complex is shown in (B and D). The biodistribution is given as percentage of the total activity of the injected dose (%ID) and the activity concentration (SUV) based on four A431-Luc tumor-bearing mice. (E) Target to background ratios including tumor to muscle-, and tumor to blood ratios.

Figure 11.

Small animal PET/CT. Orthogonal sections (left) scaled to either maximum (upper) or visualize the tumor (lower) or maximum intensity projections (right) of a selected A431-Luc tumor-bearing mouse at 2 h after single intravenous injection (ki, kidneys, tu, tumor, li, liver, bl, bladder).

Figure 12.

Time-activity curves (TAC) of regions of interest (ROI). The TAC curves are derived from PET studies of four A431-Luc tumor-bearing mice. The data points were collected over 20 h to 35 h after injection of the [⁶⁴Cu]Cu-α-EGFR TM (pro) (NODAGA)_1.2. (A) TAC of the ROI over the heart representing primarily the blood activity concentration (SUV_mean) supporting the fast elimination of the TM with half-lifes of 4.2 min and 23.2 min of the fast and slow distribution and elimination phase, respectively. (B) TAC of the tumor activity concentration with a maximum at 22 min p.i. and the clearance with half-life of 3.7 h. (C) TAC of the total activity (%ID) in the tumor with 3.2% ID at the maximum. (D), (E) TAC of the tumor ratios to muscle and blood, respectively, showing the increasing image contrast up to 2 h. (F) accumulation of the ⁶⁴Cu-activity in the kidneys with a maximum of 46.4% ID after 2 h.

Figure 13.

Evidence for disassembly of UniCAR-TM complexes in vitro. To analyze the stability of UniCAR-TM complexes T cells engrafted with the UniCAR CD28/ζ construct were pre-incubated with 50 nM α-EGFR TM (eu) (A) or α-EGFR TM (pro) (B) and washed several times as indicated before co-cultivation with A431 target cells. After washing standard chromium release assays were performed at an e:t ratio of 5:1. As controls non pre-incubated T cells were cultivated with A431 cells in the presence or absence of the respective TM. After 48 h chromium release assays were performed. As negative controls served T cells transduced with the vector control or the UniCAR Stop construct. Mean specific lysis and SD for three independent T cell donors are shown (*p <0.05 and ***p <0.001; with respect to controls: vector control ± α-EGFR TM, UniCAR Stop ± α-EGFR TM, UniCAR 28/ζ w/o Ab; ^##p <0.01 and ^###p < 0.001; with respect to UniCAR 28/ζ + Ab; one-way ANOVA with Bonferroni multiple-comparison test).

Figure 14.

Evidence for disassembly of UniCAR-TM complexes in vivo. (A) Small animal PET/CT scans of mice taken 5 or 90 min after s.c. injection of [⁶⁴Cu]Cu-α-EGFR (pro) TM (NODAGA)_1.2 (left panel) or the TM pre-incubated with A431 cells (right panel) (ki, kidneys, li, liver, s.c. injected material). (B) Dynamic PET analysis of either free TM (TM) or TM pre-incubated with A431 cells (TM+A431) or TM pre-incubated with UniCAR T cells (TM+UniCAR) or TM pre-incubated with A431 cells and UniCAR T cells (TM+A431+UniCAR). (C) Based on the elimination curves half-lifes for free and complexed TMs were calculated.