Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T lymphocytes

Abstract

Adoptive transfer of chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR-T cells) has had less striking therapeutic effects in solid tumors than in lymphoid malignancies. Although active tumor-mediated immunosuppression may have a role in limiting the efficacy of CAR-T cells, functional changes in T lymphocytes after their ex vivo manipulation may also account for the reduced ability of cultured CAR-T cells to penetrate stroma-rich solid tumors compared with lymphoid tissues. We therefore studied the capacity of human in vitro-cultured CAR-T cells to degrade components of the extracellular matrix (ECM). In contrast to freshly isolated T lymphocytes, we found that in vitro-cultured T lymphocytes lack expression of the enzyme heparanase (HPSE), which degrades heparan sulfate proteoglycans, the main components of ECM. We found that HPSE mRNA is downregulated in in vitro-expanded T cells, which may be a consequence of p53 (officially known as TP53, encoding tumor protein 53) binding to the HPSE gene promoter. We therefore engineered CAR-T cells to express HPSE and showed their improved capacity to degrade the ECM, which promoted tumor T cell infiltration and antitumor activity. The use of this strategy may enhance the activity of CAR-T cells in individuals with stroma-rich solid tumors.

Figure 1. LTE-T show reduced invasion of ECM and loss of the enzyme HPSE

(a) ECM invasion assay of CD14⁺ monocytes (M), freshly isolated T lymphocytes (FI-T), briefly activated T cells (BA-T) and long-term ex vivo expanded T cells (LTE-T). Monocytes freshly isolated from peripheral blood showed the highest capacity to degrade ECM (63% ± 23%). BA-T showed superior invasion of ECM compared to FI-T (*p=0.05). Conversely, LTE-T had significantly reduced ability to degrade ECM compared to both BA-T (**p=0.01) and FI-T (***p=0.022). Data summarize means ± SD of 5 donors. We compared all four cell subsets for each donor. (b) Western blot showing the expression of HPSE in M, CD4⁺ and CD8⁺ T cells at different time points of culture. Data are representative of 4 donors. Positive controls are HPSE transfected 293T cells. (c) Immunofluorescence staining for HPSE in MCF-7 cells, M, FI-T and LTE-T. Nuclei are stained with DAPI and shown in blue, while HPSE is stained with red-fluorescent dye (Alexa Fluor 555). Magnification is 20× (50 micron scale bar). (d) qRT-PCR of HPSE in CD4⁺ and CD8⁺ T cells at different time points of culture. Data summarize means ± SD of 4 donors. (e) HPSE enzymatic activity assessed in supernatants collected from CD4⁺ and CD8⁺ T cells at different time points of culture. Monocytes and tumor cell lines CHLA-255, A549 and DU-145 are positive controls. Data summarize means ± SD of 4 donors. In (b), (d), (e) the condition “day 15” indicates HPSE expression in LTE-T cultured for 14 days, and re-stimulated with immobilized OKT3 and anti-CD28 Abs for 24 hrs to assess whether TCR re-stimulation can re-induce HPSE expression. (f) Western blot showing the expression of HPSE and full-length p53 protein in T cells before (T=0) and after activation with immobilized OKT3 and anti-CD28 Abs for 18 and 72 hrs. Shown are results from 1 representative of 3 donors. (g) p53 ChIP in LTE-T cultured for 14 days, and (h) in CD45RA⁺ cells before (T=0) and after stimulation with OKT3 and anti-CD28 mAbs (T=72h). Input is DNA sonicated but not immunoprecipitated; IgG and p53 are DNA immunoprecipitated by the isotype and p53-specific Ab that recognizes the full-length protein. Relative quantification was performed comparing the intensities of PCR bands of IgG and p53 to input PCR band. For this representative sample relative quantifications are: IgG 20% and p53 90% for LTE-T (g); IgG 2% and p53 4% at T=0 and IgG 53% and p53 100% at T=72h for CD45RA⁺ cells (h). Shown is 1 representative of 3 donors.

Figure 2. LTE-T modified to express HPSE show enhanced degradation of ECM

We transduced LTE-T with a retroviral vector encoding HPSE and GFP [HPSE(I)GFP]. (a) GFP expression by both CD4⁺ and CD8⁺ LTE-T at day 12 – 14 of culture. (b) Western blot showing the expression of HPSE in control and transduced LTE-T at day 12 – 14 of culture. (c) qRT-PCR for HPSE in control and HPSE(I)GFP⁺ LTE-T starved in culture from IL-2 for 3, 7 and 10 days. Data are representative of 2 donors. (d) ECM invasion assay of control and HPSE(I)GFP⁺ LTE-T, with or without selection based on GFP expression. Data summarize mean ± SD of 9 donors, *p=0.025; **p<0.001. (e) ECM invasion assay of HPSE-transduced LTE-T in the presence or in the absence of the inhibitor, heparin H1. Data summarize mean ± SD of 4 donors, ***p<0.01.

Figure 3. LTE-T co-expressing HPSE and GD2-specific CAR retain GD2 specificity and have enhanced capacity to degrade ECM

We transduced LTE-T with retroviral vectors encoding either the GD2-specific CAR alone (CAR) or both the GD2-specific CAR and HPSE [CAR(I)HPSE]. (a) Flow cytometry analysis to detect CAR expression by control and transduced LTE-T. (b) Western blot to detect HPSE in control and transduced LTE-T. Data are representative of 5 donors. (c) Cytotoxic activity of control, CAR⁺ and CAR⁺HPSE⁺ LTE-T assessed by ⁵¹Cr-release assay at a 20:1 effector:target ratio. We used LAN-1 and CHLA-255 (GD2⁺), and Raji (GD2⁻) as target cells. (d) Transduced LTE-T release both IL-2 and IFN-γ in response to GD2⁺ tumor cells. (e) Invasion of ECM by control, CAR⁺ and CAR⁺HPSE⁺ LTE-T. Data in (c-e) summarize mean ± SD from 5 donors, *p=0.004. (f,g) We plated control and transduced LTE-T in the upper part of either ECM assay or insert assay, and LAN-1-GFP⁺ cells in the lower chamber. After day 3 of culture, we collected cells in the lower chamber to quantify CD3⁺ T cells and GFP⁺ tumor cells by flow cytometry. (f) illustrates representative dot plots, while (g) summarizes mean ± SD of 5 donors, **p=0.009. In all cases we analyzed LTE-T by day 12 –14 of culture.

Figure 4. CAR-GD2⁺ LTE-T co-expressing HPSE show enhanced tumor infiltration and improve overall survival in xenograft tumor models

(a) Kaplan-Meier analysis of mice engrafted i.p. with the tumor cell line CHLA-255 and treated i.p. with control, CAR⁺ and CAR(I)HPSE⁺ LTE-T. Shown are data from 3 independent experiments using LTE-T generated from 3 donors; control n=16, CAR n=22, CAR(I)HPSE n=26 mice; *p<0.007, **p<0.0001. (b) Kaplan-Meier analysis of mice engrafted i.p. with the tumor cell line LAN-1 and treated i.p. with control, CAR⁺ and CAR(I)HPSE⁺ LTE-T. For these experiments, we generated LTE-T from 2 donors; control n=12, CAR n=18, CAR(I)HPSE n=14 mice; *p=0.039, **p<0.0001. (c) Flow cytometry analysis of CD3⁺ T cells detected within the tumor samples. Dot plots are representative of 3 mice per group from mice infused with LTE-T generated from the same donor. (d) Weight of the tumors collected from euthanized mice engrafted with LAN-1 tumor. (e,f) Immunohistochemistry showing CD3⁺ T-cell infiltration in tumors implanted in the kidney of mice infused with either CAR⁺ or CAR-GD2⁺HPSE⁺ LTE-T. 100× magnification (e) and 200× magnification (f) (100 micron scale bar). (g) The graph shows the numbers of infiltrating CD3⁺ T cells per 10 high power fields in tumors collected from mice treated with either CAR⁺ or CAR(I)HPSE⁺ LTE-T, *p=0.028. (h) Kaplan-Meier analysis of tumor-bearing mice in the kidney infused i.v. with either CAR⁺ or CAR(I)HPSE⁺ LTE-T. For these experiments, we generated LTE-T from 2 donors; CAR n=21, CAR(I)HPSE n=21 mice; *p=0.0006.