Dual Antigen T Cell Engagers Targeting CA9 as an Effective Immunotherapeutic Modality for Targeting CA9 in Solid Tumors

Abstract

Glioblastomas (GBM), the most common malignant primary adult brain tumors, are uniformly lethal and are in need of improved therapeutic modalities. GBM contain extensive regions of hypoxia and are enriched in therapy resistant brain tumor-initiating cells (BTICs). Carbonic anhydrase 9 (CA9) is a hypoxia-induced cell surface enzyme that plays an important role in maintenance of stem cell survival and therapeutic resistance. Here we demonstrate that CA9 is highly expressed in patient-derived BTICs. CA9⁺ GBM BTICs showed increased self-renewal and proliferative capacity. To target CA9, we developed dual antigen T cell engagers (DATEs) that were exquisitely specific for CA9-positive patient-derived clear cell Renal Cell Carcinoma (ccRCC) and GBM cells. Combined treatment of either ccRCC or GBM cells with the CA9 DATE and T cells resulted in T cell activation, increased release of pro-inflammatory cytokines and enhanced cytotoxicity in a CA9-dependent manner. Treatment of ccRCC and GBM patient-derived xenografts markedly reduced tumor burden and extended survival. These data suggest that the CA9 DATE could provide a novel therapeutic strategy for patients with solid tumors expressing CA9 to overcome treatment resistance. .

Keywords: CA9; clear cell renal cell carcinoma; dual antigen T cell engagers; glioblastoma; hypoxic niche; immunotherapy.

Figure 1

CA9 as a therapeutic target in GBM. (A) Transcriptomic dataset shows significant upregulation of CA9 in GBM samples (n=163) when compared to non-tumor (n=207) (GEPIA2) (P value: * < 0.05). (B) CA9 has higher expression in mesenchymal (Mes) subtype (n=51) compared to proneural (PN) (n=46) and classical (Cla) (n=59) of GBM (GlioVisTCGA) (P value:***< 0.001). (C) Survival data from the TCGA dataset for CA9 high (n=77) transcript expression of GBM samples illustrating a significant increase in survival when compared to CA9 low (n=78) samples (Median CA9 mRNA expression (log2) cut off: 7.66; HR: 0.72 (0.5-1.02); Wilcoxon p value: 0.0288). (D) Characterisation of surface CA9 expression of GBM samples along with normal stem cells (NSCs) and normal human astrocytes (NHAs) in normoxic condition by flow cytometry reveals varying expression of CA9 in GBM lines, but low levels in normal cells. (E) GBM samples (n=11) from Fig 1.D. were grouped into either CA9low (red, n=5) or CA9high (blue, n=4) expression based on a flow cytometric median of 20%. Log-rank (Mantel-Cox Test) analysis demonstrated a significant survival benefit for patients bearing CA9Iow tumors with a median survival of 33 and 13.5 months for patients bearing CA9Iow and patients bearing CA9high tumors, respectively (P value: * =0.0283).

Figure 2

CA9 expression elevates in hypoxic condition and is associated with higher level of self-renewal and proliferation. (A) CA9 surface expression was evaluated after cells were cultured in normoxia or hypoxia (1% O₂) for 5 days; and the results indicated a dramatic increase in CA9 expression on both GBM BTIC lines upon exposure to hypoxia. (B) Significant increase of self-renewal capacity as measured by secondary sphere formation assay and (C) proliferative potential as measured by PrestoBlue proliferation assay is seen in CA9+ when compared to CA9- cells. (P value: **** <~0.0001, * 0.01, ns: non-significant) (mean±SEM, two-tailed t-test).

Figure 3

Generation and assessment of human anti-CA9 Dual specific T cell engagers (DATEs) on Renal Cell Carcinoma (ccRCC). (A) CA9 expression level on RCC243, RCC243 CA9-KO, Renca and Renca hCA9 cells. (B) Increasing concentrations of CA9 DATE binding to RCC243 vs RCC243 CA9-KO, (C) Renca hCA9 vs Renca WT cells and (E) CD3 expressing Jurkat cells were measured using flow cytometry. Error bars: mean ± SEM. (D) okt3 expression level on Jurkat cells. (F) Addition of CA9 DATEs (1 nM) to the co-culture of human CD3+ T cells with CA9 expressing target cells (Luciferase-expressing RCC 243 cell lines and the CA9 knockout counterpart [CA9-KO]) for 48 hours at the E:T ratio, 1:5 resulted in a significant elevation of CD25 expression on both CD4+ and eng+ T cells population confirmed by flow cytometry analysis. (n=4) (P value: **** < 0.0001) (2 way ANOVA) (G) Enzyme-linked immunosorbent assay (ELISA) indicated increased secretion of Interferon-gamma (IFN-y) by T cells only in the presence ofCA9 DATE and CA9 expression on target cells. (P value:**** < 0.0001) (2 way ANOVA).

Figure 4

Assessing the efficacy of CA9 DATE in therapeutic targeting of CA9 expressing RCC lines and xenografted immunocompromised mice: (A) CA9 DATE (1 nM) induced potent cytolysis in antigen expressing target cells when cocultured with human CD3+ T cells at an E:T ratio of 5:1 for 48 hours quantified by luminescence assay. Phase contrast images of the in vitro cytotoxicity assay confirmed potent cytolytic effect of CA9 DATE on CA9 antigen expression. (n=4) (Scale bar: 1000 µ) (P value: ****< 0.0001, ns, non-significant) (mean SEM, two-tailed t-test). (B) CA9 DATE effectively induced target lysis across a panel of kidney cancer cell lines when co-cultured with CD3+ T cells at E:T ratio of 10:1 (In vitro cytotoxicity assay setup as described earlier). lactate dehydrogenase (LDH) release assay on the supernatant indicated a drastic increase in target cell cytotoxicity in the presence of CA9 DATE and CA9 antigen expression. The phase contrast images of light microscope confirmed the cytolytic effect of CA9 DATE on CA9 expressing RCC lines (n=2). (P value:****< 0.0001) (2 way ANOVA). (C) NSG mice were subcutaneously implanted with human CA9+ RCC 243 VHL mut cells. Upon successful engraftment and having a palpable tumor, mice were intratumorally treated with 2x10⁶ T cells isolated from human PBMCs either with CA9 DATE or CA9 DATE control (50µg) for a total of 12 doses over 6 weeks. Mouse xenografts generated after CA9 DATE treatment had less tumor burden (n=6) (P value: **** < 0.0001) (mean±SEM, two-tailed t-test) and (D) maintained a significant survival advantage over control mice (n=7) (P value: *** < 0.0004) (Log-rank Mantel-Cox Test).

Figure 5

Assessment of anti-CA9 Dual specific T cell engagers (DATEs) dual specificity and its effect on GBM model. (A) Dual specificity of CA9 DATEs on CA9hi GBM BTIC (BT241), CA9low GBM BTIC (BT667) and (B) human PBMC derived T cells by flow cytometry. (P value: **<0.01, **** <0.0001) (2-way RM AVOVA). (C) Addition of CA9 DATE (1µg/mL) (13nM) to the co-culture of CA9hi GBM BTICs (BT241, BT935, BT428) and T cells (E:T ratio, 1:1) (overnight incubation) caused T cells activation as confirmed by increased expression of CD25 and CD69 by flow cytometry analysis (n=2). (P value: *= 0.04, **= 0.006) (2 way ANOVA) (D) CD8+ T cells were the main subset of activated T cells. (P value: ***< 0.001, **** < 0.0001) (2 way ANOVA) (E) Enzyme-linked immunosorbent assay (ELISA) shows elevated secretion of IFN-γ and TNF-α cytokines in supernatant collected from co-cultures ofT cells and GBM BTICs treated with CA9 DATEs. (n=2) (P value: **** < 0.0001).

Figure 6

Therapeutic targeting of CA9hi GBM BTICs using CA9 DATEs at in vitro level and in patient derived xenograft model of GBM. (A) Dose response study performed on BT 241, CA9hi GBM BTIC, identified 1 nM as the optimal dose for GBM cytotoxicity assay. (B) DATEs significantly induced cytotoxicity of CA9hi GBM BTICs (BT241, BT935, BT428) but not CA9- cells (HEK) when co-cultured with T cells and DATEs (1n M) for 16 hours at different E:T ratios. (n=3) (P value:*< 0.05, **** < 0.0001) (2 way ANOVA). (C) Micrographs of GBM BTICs and T-cell co-culture with and without DATEs. CA9hi GBM BTIC lines (BT 935 and BT 428) were incubated with either T cells (E:T ratio, 1 :2) or CA9 DATE alone or with both. GBM BTIC lysis were observed only in the presence of both T cells and CA9 DATEs (Scale bar: 400 m). (D) NSG mice were intracranially implanted with human CA9hi GBM BTICs (BT935 and BT241). Upon successful engraftment, mice were intracranially treated with 1x10⁶ T cells isolated from human PBMCs either with CA9 DATE or CA9 DATE control (50µg = 667nM) for a total of four doses over two weeks. Mouse xenografts generated after CA9 DATE treatment had less tumor burden (n=6) (P value: * < 0.03, **** < 0.0001) (mean±SEM, two-tailed t-test) and maintained a significant survival advantage over control mice in BT935 engrafted mice (P value: * < 0.03) (Log-rank Mantel-Cox Test); however, DATE treatment on BT241 engrafted mice only showed increased pattern of survival (n=6).