Constitutive GP130 activity improves CAR-T cell expansion, cytotoxicity, and anti-tumor potential

Therapy with chimeric antigen receptor (CAR) T cells is one of the most promising emerging treatments for B-cell malignancies. Despite the good efficacy outcomes that have been reported with CAR T cells, they still require further optimization to reduce not only CAR T-related toxicities like cytokine release syndrome (CRS) but also to improve responses to therapy. It is widely accepted that cytokine expression patterns and levels can improve or impair CAR T-cell expansion, migration, and persistence, thus directly affecting the efficacy of this treatment.¹ Interleukin (IL)-6 has been identified as one of the key cytokines which correlates with CAR T-cell activity and CRS. It binds either directly to a membrane-bound IL-6 receptor and glycoprotein 130 (GP130; classical signaling) or forms a complex with soluble IL-6 receptor, which is then recruited to ubiquitously expressed GP130 on cells (trans-signaling).

Based on this, Zhiwu Jiang and colleagues sought to evaluate the effect of IL-6 trans-signaling on CAR-T expansion, anti-tumor potential, and safety profile. The results of this study were published in Leukemia¹ and are summarized below.

Study design

• T cells isolated from peripheral blood mononuclear cells (PBMCs) were cultured and transduced in vitro with one or more of the following lentiviral-based CAR constructs that had CD28 and Toll-like receptor 2 (TLR2)-based costimulatory domains:
  • Third generation anti-CD19 (CAR19)
  • Anti-GPC3 (CARGPC3)
  • Anti-MUC1 (CARMUC1)
• To mimic IL-6 trans-signaling, a human recombinant fusion protein of IL-6 protein and its receptor (HIL-6) was used
• To understand the relevance of GP130 signaling, a constitutively active form of GP130 was co-expressed with CAR constructs in T cells
• Tumor models: Xenograft model using immunodeficient NSI mice were intravenously injected with:

• • NALM6-GL cells, a leukemia cell line
  • A549 cells, a MUC1-expressing human lung cancer cell line
  • Huh-7 cells, a GPC3-expressing hepatocellular cancer cell line

Results

In vitro HIL-6 results

• Addition of HIL-6 did not affect T-cell proliferation or survival following CD3/CD28 stimulation in vitro
• However, HIL-6 addition to CAR19-expressing T cells led to a significant increase in their proliferation and survival rate, following activation with a CD19-expressing B-cell leukemia cell line (NALM6)
• Concomitant overexpression of HIL-6 and CAR19 or CARMUC1 in T cells led to increased secretion of pro-inflammatory cytokines and enhanced cytotoxic potential against two independent CD19-expressing and MUC1-expressing leukemia cell lines

In vivo HIL-6 xenograft models

In the leukemia xenograft model:

• Infusion of CAR19-/HIL-6-expressing T-cells in the xenograft mouse models was more successful at inhibiting leukemic cell progression and promoting CAR T-cell survival and mouse overall survival than infusion with CAR19 T cells
• Increasing doses of CAR19/HIL-6 T cells did not significantly improve efficacy, however, mice treated at the highest dose showed significant weight loss indicative of graft-versus-host disease (GvHD)

In the solid tumor xenograft model:

• CARMUC1/HIL-6 T cells were more efficient at reducing tumor growth and at persisting in the blood than CARMUC1 T cells

In the hepatocellular carcinoma xenograft model:

• Similarly, CARGPC3/HIL-6 T cells expanded more and led to lower tumor burden in the xenograft model of hepatocellular carcinoma (GPC3-expressing Huh cell line) than CARCPC3 T cells

Mechanism of action of HIL-6 stimulation

• RNA sequencing and subsequent clustering analyses of CAR19 T cells with or without HIL-6 and with or without stimulation (NALM6 cells) showed that HIL-6 led to the upregulation of genes involved in:
  • T-cell migration (CCL5, CCR5, CXCR6)
  • T-cell memory (CD62L, IL-7R, CCR7, TCF7)
  • IL-6/GP130/STAT3 signaling pathway (SOCS3, JAK3, IL-6ST)
• Pharmacological block of IL-6 signaling with the GP130 inhibitor SC144 led to a significant increase in apoptotic T cells, irrespective of their activation status
• Similarly, when blocking STAT3 signaling with the BP-1-102 inhibitor, the cytotoxic capacity of CAR19/HIL-6 T cells was significantly reduced

Effects of co-expressing constitutively active GP130

• The cytotoxicity and peripheral persistence of CAR T cells were improved when co-expressing a GP130 constitutively active construct (CAR19/L-GP130) both in vitro and in the in vivo NAML6-GL leukemia xenograft model.
  • Mice injected with CAR19/L-GP130 CAR T cells had less tumor load and longer survival compared to mice injected with CAR19 or LGP130 CAR T cells only
  • Both CAR19/L-GP130 and CAR19/HIL-6 T cells led to similar cytokine and genome expression profiles

• A549 tumor bearing mice infused with CARMUC1/L-GP130 cells and Huh-7 tumor bearing mice infused with CARGPC3/L-GP130 cells showed superior expansion of CAR T cells and significantly reduced tumor load, compared with mice injected with the CAR T cells or L-GP130 cells only
• Although both CAR19/HIL-6 and CAR19/L-GP130 T cells led to similar outcomes in vitro and in vivo, one main difference was the fact that CAR/L-GP130 T cells did not lead to signs of severe GvHD, unlike the CAR19/HIL-6 cells

Conclusion

The results of this study indicate that IL-6 trans-signaling promotes CAR T-cell survival, expansion, and cytotoxicity both in vitro and in leukemic and solid-tumor xenograft mouse models in vivo. When exploring the mechanism of action further, the authors concluded that these positive effects are mediated by the IL-6/GP130/STAT3 signaling pathway. Indeed, CAR T cells with constitutively active GP130 (CAR19/L-GP130) behaved very similar to those with IL-6 trans-signaling stimulation (CAR19/HIL-6) in terms of promoting CAR T-cell expansion, survival, cytotoxicity, and tumor suppressing potential. Nevertheless, the authors showed that, although similar in terms of anti-tumor potential and cytotoxicity, CAR T cells co-expressing GP130 led to a lower risk of CRS and severe GvHD, indicating their superiority over CAR T cells with direct GP130 signaling stimulation.