High-affinity PD-1-CD28 switch receptor enhances sustained antitumor activity of CAR-T cells in both lymphoma and ewing sarcoma.

Abstract Background: Although CAR-T cell therapy has revolutionized the treatment of hematologic malignancies, durable responses remain limited by T cell exhaustion and poor tumor infiltration. These challenges are even more pronounced in solid tumors such as Ewing sarcoma, where physical and immunosuppressive barriers impede therapeutic efficacy. Engagement of PD-1 by PD-L1 on tumor cells plays a key role in CAR-T cell dysfunction. To overcome this, we engineered a high-affinity PD-1-CD28 switch receptor (HA PD-1-CD28) that converts inhibitory PD-1 signals into CD28-mediated costimulatory activation.Methods: Utilizing AlphaFold3, we predicted PD-1/PD-L1 interaction interfaces to engineer high-affinity binding.The intracellular inhibitory domain of PD-1 was replaced with the activating domain of CD28, constructing a high-affinity PD-1-CD28 switch receptor. This therapeutic strategy was functionally validated through in vitro and in vivo studies in both solid tumors (Ewing sarcoma) and hematological malignancies (relapsed/refractory B-cell lymphoma).Results:Upon co-culture with tumor cells, HA PD-1-CD28–expressing CAR-T cells exhibited enhanced activation of the NF-κB signaling pathway, cytoskeletal plasticity and strengthened T cell–tumor cell interactions, leading to increased T cell proliferation and potentiated CAR-T antitumor activity. In an allogeneic Ewing sarcoma NCG subcutaneous model, 57% (4/7) of mice treated with HA PD-1-CD28 CAR-T (anti-LINGO1) achieved complete tumor regression at endpoint. Tumor volume (54.96 ± 40.74 mm³ vs. 260.10 ± 65.08 mm³, P = 0.02) and overall tumor weight (0.02 ± 0.01 g vs. 0.11 ±0.03 g, P = 0.03) were significantly lower compared to standard PD-1-CD28 CAR-T (anti-LINGO1). In an orthotopic B-cell lymphoma model, the HA PD-1-CD28 CAR-T (anti-CD22) reduced 50% mortality risk at Day 88 and demonstrated doubled median overall survival (mOS) versus standard PD-1-CD28 CAR-T (anti-CD22) (81 days vs. 42 days, P < 0.05). Furthermore, human CD3+immunohistochemistry (IHC) revealed a 3.38-fold increase in tumor-infiltrating lymphocyte (TIL) density in HA PD-1-CD28 CAR-T (anti-LINGO1) groups and a 1.43-fold increase in HA PD-1-CD28 CAR-T (anti-CD22) groups (P < 0.05), accompanied by a 1.15-fold upregulation of tumor PD-L1 expression (P = 0.01), suggesting a self-amplifying therapeutic loop. Flow cytometry confirmed enhanced T cell fitness, with significantly elevated activation markers (CD25, CD69, 4-1BB; P < 0.001), increased persistence markers including IL-7Rα cells (P < 0.0001). HA PD-1-CD28 CAR-T showed equivalent organ somatic indices (P > 0.05) and normal histoarchitecture in all examined organs (liver, spleen, kidney, lung, heart, brain, intestine) versus controls, supporting its favorable safety profile in vivo.Conclusions: The HA PD-1-CD28 switch receptor enhances tumor infiltration of CAR-T cells, even in solid tumors with per se low PD-L1 expression, significantly improving anti-tumor efficacy and long-term persistence. This strategy demonstrates substantial translational potential for improving CAR-T and TCR-T cell therapy in cancer treatment.