Table 1

Overview of published methods in high throughput screening of novel CAR domains

Ref	Library variable	Library generation	Cell type	Modification method	Stimulation method	Enrichment method
36	scFv (CDR)	Degenerate HDR template synthesis	B3Z cell line (IL2-EGFP reporter)	CRISPR-Cas9	Soluble rhHER2 antigen, or SKBR3 cell line	3× rhHER2 binding sort, or 3× GFP expression sort
38	scFv (CDR)	Degenerate primer PCR and restriction-ligation cloning	Jurkat cell line (NFkB-ECFP & NFAT-EGFP reporter)	Transposon	JeKo cell line	rhROR1 binding sort and ECFP⁺EGFP⁺ expression sort
39	scFv (VL/VH)	Not specified	HEK293F cell line (Gal4-VP64-BFP reporter)	Lentiviral	Healthy PBMC and CD38⁺ K562 cell line	BFP expression sort
40	scFv (VL/VH)	Human B cell cDNA nested PCR and overlap extension PCR	Primary T cells	Retroviral	NY-ESO-1₁₅₇/HLA-A2-modified K562 or T2 cell line Raji cell line (CD19)	Repeated antigen stimulation followed by tetramer binding sort (NY-ESO-1₁₅₇/HLA-A2), or Repeated antigen stimulation followed by rhCD19 binding sort
41	scFv (full)	Degenerate primer PCR and restriction-ligation cloning	Jurkat cell line	Lentiviral	CEA-modified HeLa cell line	3× EGFP⁺CD69⁺ expression sort
42	scFv (full)	Human B cell cDNA PCR and restriction-ligation cloning	Jurkat cell line (NFAT-mCherry reporter)	Lentiviral	HEK293-6E cell line, or H-226 cell line, or AsPC-1 cell line	mCherry^- expression sort followed by 2× GFP⁺mCherry⁺ expression sort
43	scFv (full)	Human PBMC cDNA, murine spleen or BM cDNA nested PCR and restriction-ligation cloning	Primary T cells; NK-92 cell line	Lentiviral	Subcutaneous mouse models with an EGFR⁺-modified MCF-7, HER2⁺-modified MCF-7, SW480, A549, SK-OV-3, SK-BR-3, N3 or BN16 cell line	In vivo enrichment for tumor-reactive CARs of unknown specificity
44	scFv (full)	Phage display library PCR and restriction-ligation cloning	Jurkat cell line	Retroviral	Plate-bound CEA	3× CD69⁺ expression sort with subsequent gDNA PCR and recloning
45	ISD	Gene synthesis, PCR and restriction-ligation cloning	Primary T cells	CRISPR-Cas9	SKBR3 cell line	CAR⁺ expression sort
46	ISD	Gene synthesis, PCR and Golden Gate assembly	Primary T cells	Lentiviral	CD19⁺-modified K562 cell line	IFN-γ⁺, IL-2⁺ or CD69⁺ expression or proliferation sort
47	ISD	Gene synthesis, PCR, overlap extension PCR and restriction-ligation cloning	Jurkat cell line	Lentiviral	rhCD19 antigen	3× CD69⁺ and/or CD69⁺PD-1⁻ expression sort
48	ISD	cDNA PCR and restriction-ligation cloning	Jurkat cell line	Retroviral	Plate-bound c-Myc antibody	CD69⁺ expression sort
49	ISD	Gene synthesis and In-Fusion cloning	Primary T cells	Lentiviral	Nalm-6 cell line	NA
51	ABD, HD, ISD	Gene synthesis and restriction-ligation cloning	Jurkat cell line; Primary T cells	Retroviral	NA	CD69⁺ expression sort proliferation sort
53	ISD	Site-directed mutagenesis	Jurkat cell line (NFAT-GFP and NFkB-CFP reporter)	Retroviral	Soluble rhB7H6	GFP⁺CFP⁺ expression sort

AA, amino acid; ABD, antigen-binding domain; BFP, blue fluorescent protein; BM, bone marrow; CD, cluster of differentiation; cDNA, complementary DNA; CDR, complementary determining region; CEA, carcinoembryonic antigen; ECFP, enhanced cyan fluorescent protein; EGFP, enhanced green fluorescent protein; HD, hinge domain; HDR, homology directed repair; HER2, human epidermal growth factor receptor 2; IFN, interferon; IL, interleukin; ISD, intracellular signaling domain; ML, machine learning; NA, not applicable; NFAT, nuclear factor for the activation of T cells; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; NGFR, nerve growth factor receptor; NK, natural killer; PBMC, peripheral blood mononuclear cell; PD-1, programmed cell death protein-1; rh, recombinant human; scFv, single-chain variable fragment.