Supplementary Figure S7: Analysis of differentially accessible genes in NK cell resistant tumour cells.

Supplementary Table 1: Primers. Columns show primer name, sequence, usage, annealing temperature (in °C), and used polymerase.

Supplementary Figure S8: Secondary resistant B-ALL cell clones.

Supplementary Figure S1: Flow cytometric analysis and gating strategies.

Supplementary Figure S2: Flow cytometric analysis and gating strategies shown in Figure 5.

Supplementary Figure S4: B-ALL cell line characterisation and details about the barcode library.

Supplementary Figure S11: The expression of LY6E on K562 does not impact on IFN-γ or TNF-α production in NK cells.

Supplementary Figure S5: Extended quantification of NK cell-mediated cancer immunoediting in vitro.

Supplementary Figure S6: Analysis of differentially expressed genes in NK cell resistant tumour cells.

Supplementary Table 2: Antibodies and viability dyes used for flow cytometric analyses and immunoblotting. Indicated are target antigens, conjugates, clones, suppliers, catalogue numbers, and used working dilutions.

Supplementary Figure S10: Generation of Plaat3 KO B-ALL clones and their characterisation.

Supplementary Figure S3: Flow cytometric analyses of human data sets and the NK-cell receptor ligand analyses on mouse B-ALL cell lines.

Supplementary Figure S9: Profiles of tumour cells co-cultured with WT, Prf1-/- or Ifng-/- NK cells.

Abstract The term cancer immunoediting describes the dual role by which the immune system can suppress and promote tumor growth and is divided into three phases: elimination, equilibrium, and escape. The role of NK cells has mainly been attributed to the elimination phase. Here, we show that NK cells play a role in all three phases of cancer immunoediting. Extended co-culturing of DNA-barcoded mouse BCR/ABLp185+ B-cell acute lymphoblastic leukemia (B-ALL) cells with NK cells allowed for a quantitative measure of NK cell–mediated immunoediting. Although most tumor cell clones were efficiently eliminated by NK cells, a certain fraction of tumor cells harbored an intrinsic primary resistance. Furthermore, DNA barcoding revealed tumor cell clones with secondary resistance, which stochastically acquired resistance to NK cells. NK cell–mediated cytotoxicity put a selective pressure on B-ALL cells, which led to an outgrowth of primary and secondary resistant tumor cell clones, which were characterized by an IFNγ signature. Besides well-known regulators of immune evasion, our analysis of NK cell–resistant tumor cells revealed the upregulation of genes, including lymphocyte antigen 6 complex, locus A (Ly6a), which we found to promote leukemic cell resistance to NK cells. Translation of our findings to the human system showed that high expression of LY6E on tumor cells impaired their physical interaction with NK cells and led to worse prognosis in patients with leukemia. Our results demonstrate that tumor cells are actively edited by NK cells during the equilibrium phase and use different avenues to escape NK cell–mediated eradication.

While CAR T cells have revolutionized the treatment of certain hematologic malignancies, they can cause severe toxicities, which are expected to be exacerbated with next-generation CAR Ts engineered for improved proliferation, persistence, and efficacy. Therefore, regulatory systems are urgently needed to be able to control these living drugs directly in patients. Here, we engineered a molecular switch, in which the interaction of two human proteins is efficiently induced with the orally available and non-toxic drug A1120. We demonstrate the versatility of this switch by regulating CAR signaling and transcriptional activity in human T cells in vitro and in vivo . Both systems were tightly controlled in the absence of the drug but strongly activated upon administration of the small molecule. Since this switch enables the regulation of diverse systems including CARs and transcription factors, we anticipate that it represents an important step towards next-generation cellular therapies with improved safety and efficacy. ### Competing Interest Statement M.L. and M.W.T. receive funding from Miltenyi Biotec. E.S., C.U.Z., M.W.T. and M.L. are inventors on patents related to hRBP4- and nanobody-based molecular switches. J.Z. is a founder, shareholder, and scientific adviser of Quantro Therapeutics. The Zuber Lab receives research support and funding from Boehringer Ingelheim. A.M-L., F.E. and B.E. are full-time employees of Miltenyi Biotec. J.M. was an employee of Miltenyi Biotec at the time of this study. The remaining authors declare no competing interests. Austrian Federal Ministry of Economy and Tourism, the National Foundation for Research, Technology and Development, the Christian Doppler Research Association (Christian Doppler Laboratory for Next Generation CAR T Cells), Christian Doppler Laboratory for Next Generation CAR T Cells Rete ACC - Sviluppo successivo per il progetto CAR-T rete Oncologica: ponte alla traslazione clinica, RCR-2023-23684268 FWF Austrian Science Fund, https://ror.org/013tf3c58, W1224–Doctoral Program on Biomolecular Technology of Proteins–BioToP, EFP 45, Devising Advanced TCR-T cells to eradicate OsteoSarcoma (DART2OS), 10.55776/PAT8789924 Austrian Academy of Sciences, DOC Fellowships 26323 Boehringer Ingelheim (Austria), https://ror.org/026vtvm28 Austrian Research Promotion Agency, headquarter grant FFG-852936 Boehringer Ingelheim, Discovery Research global post-doc program

Traditionally, chimeric antigen receptor (CAR) T cells employ single-chain variable fragments (scFvs) as binding entities. While scFvs represent a convenient option due to their broad availability, they also come with drawbacks, in particular their tendency to cluster and their relatively large size. Moreover, most scFvs used in the CAR field are of non-human origin, potentially causing immunogenicity. Therefore, we established an engineering platform for minimalistic CAR binding domains (miniCARbids), which combine several critical advantages: (i) human origin, (ii) small size, (iii) efficient expression in T cells and (iv) single-domain architecture, among others. We demonstrate that miniCARbids can be engineered to recognize various antigens with antibody-like affinities, while being stable and aggregation-resistant. When miniCARbids are incorporated into CARs, they induce high anti-tumor potency in both adapter and conventional CAR formats. Remarkably, CD22-directed miniCARbid-based CARs showed similar or even more efficient tumor clearance in leukemia-bearing mice when compared with a CAR comprising the clinically tested m971-1xG4S scFv. Together, we introduce the miniCARbid engineering platform, enabling the generation of small, human antigen-binding domains with high potency in CAR T cells against virtually any target antigen. ### Competing Interest Statement M.L. and M.W.T. receive funding from Miltenyi Biotec. M.T., M.L. and M.W.T. have filed two patent applications related to the technologies described in this study. E.M., U.B. and A.M.-L. are full time employees of Miltenyi Biotec. J.M. was employee of Miltenyi Biotec at the time of this study. The remaining authors declare no competing interests. FWF Austrian Science Fund, https://ror.org/013tf3c58, W1224, 10.55776/P34832, ESP 465-B, EFP 45 Austrian Academy of Sciences, 26323, 25905 Christian Doppler Research Association, https://ror.org/00mv8h305, Christian Doppler Laboratory for Next Generation CAR T Cells

The limited controllability of CAR T cells in patients represents a key challenge of this highly potent immunotherapy. A molecular ON-switch, which can be regulated with a non-toxic and readily available small molecule drug, would represent a major advance towards controllable CAR T therapeutics. For that purpose, we engineered caffeine-responsive heterodimeric ON-switches (CaffSwitches) and demonstrate their high caffeine-dependency and virtually absent leakiness. When incorporating these CaffSwitches into CARs, the resulting CaffCARs were efficiently activated by caffeine concentrations achieved in human plasma after drinking one cup of coffee. Moreover, CaffCAR T cells also showed efficient tumor clearance in an in vivo mouse model, which was completely abolished in the absence of caffeine. This tight control was even observed with c-Jun overexpressing CaffCAR T cells, despite their massive expansion. Together, we anticipate that these novel CaffSwitches will be valuable tools for the development of safe and efficient next generation CAR T cells. ### Competing Interest Statement M.L. and M.W.T. receive funding from Miltenyi Biotec. E.S, B.S., C.U.Z., M.L. and M.W.T. have filed a patent application related to this work. J.Z. is a founder, shareholder, and scientific adviser of Quantro Therapeutics. The Zuber Lab receives research support and funding from Boehringer Ingelheim. A.M.-L., F.E. and B.E. are full-time employees of Miltenyi Biotec. J.M. was employee of Miltenyi Biotec at the time of this study. The remaining authors declare no competing interests. Federal Ministry for Digital and Economic Affairs of Austria National Foundation for Research, Technology and Development of Austria to the Christian Doppler Research Association, Christian Doppler Laboratory for Next Generation CAR T Cells Austrian Science Fund, 10.55776/P34832, W1224–Doctoral Program on Biomolecular Technology of Proteins–BioToP, EFP 45, Devising Advanced TCR-T cells to eradicate OsteoSarcoma, DART2OS, FWF, 10.55776/PAT4163824 Rete ACC, RCR-2023-23684268 DOC Fellowships of the Austrian Academy of Sciences, 26323, 25905 Boehringer Ingelheim (Austria), https://ror.org/026vtvm28, Boehringer Ingelheim Discovery Research global post-doc program, Boehringer Ingelheim Fonds (BIF) Austrian Research Promotion Agency, headquarter grant FFG-852936 European Research Council, ERC-2023-STG 101116251