Generating Nbs that differentially bind tumor cells and empower CAR T cells to kill the tumor cells. (A) Flowchart of AML-specific CAR-compatible Nbs in vivo screening. A llama was immunized with the AML cell line THP-1. An Nb library was generated from the llama PBMCs by molecular cloning. Two rounds of conventional cell-based phage display were applied, which took the T-acute lymphoblastic leukemia cell line Jurkat and the chronic myelogenous leukemia cell line K562 as negative absorption. Thereafter, 1 round of counter-selection was applied to obtain the nanobodies with high affinity. The resultant THP-1–specific Nbs were inserted into a CAR-expressing lenti-vector to generate the Nb–sub-lib CAR (Nb-CAR) library. Human primary T cells were transduced by the Nb-CAR library and injected into NSG mice with THP-1 or K562 tumors to perform the in vivo selection. Nbs that can redirect T cells to enrich in the tumor were amplified using polymerase chain reaction and sequenced. (B) Ten million THP-1 cells or 5 million K562 cells were transplanted into NSG mice subcutaneously, followed by treatment with UTD T cells or Nb–sub-lib CAR T cells. Two weeks later, Nbs from tumor-infiltrated T cells were isolated and identified using polymerase chain reaction amplification (n = 3). (C) The 5 most frequent Nbs in the THP-1 tumor are shown. The Nb-expressing phage was directly used to test the binding to THP-1 cells, Jurkat cells, or K562 cells using a flow cytometry assay, in which the red line was flow with Nb-expressing phage, and the blue line was isotype control. bp, base pair.