Table 1.

Overview of tACPA molecules used in this study

Name	Format	Isotype	Derived from	Features	Target	NET inhibition	Pharmacological activity (mouse models)
h-tACPA	Human	hIgG1/κ	Human scFv RA library	NA	citH2A and citH4	Yes	CAIA
m-tACPA	Mouse	mIgG1/κ	Hybridoma screen	NA	citH2A and citH4	Yes	CAIA, PF, colitis
ch-tACPA	Chimerized	h-mIgG1/κ	m-tACPA	Mouse variable and human constant domains	citH2A and citH4	Yes	CAIA
hz-tACPA	Humanized	hIgG1/κ	ch-tACPA	CDR grafted and germlined	citH2A and citH4	Yes	CAIA, sepsis, CIA
dc-tACPA	Development candidate	hIgG1/κ	hz-tACPA	Isomerization removed in light chain CDR1	citH2A and citH4	Yes	CAIA, peritonitis

NA not applicable

We engineered different tACPA molecules that have distinct features. Each time an improved molecule became available, we used it in our experiments. The development of these antibodies occurred step-by-step as follows: (1) h-tACPA was obtained from a human scFv RA library screen. The tACPA target was discovered using h-tACPAs; (2) m-tACPA was derived from a hybridoma screen; (3) ch-tACPA was generated from m-tACPA; (4) hz-tACPA was generated through ch-tACPA optimization; and (5) hz-tACPA optimization finally resulted in dc-tACPA. During the course of lead optimization efforts of tACPA, we tested the individual molecules, which all demonstrated NET-inhibiting capacities in vitro as well as in vivo pharmacological activity in the CAIA mouse model of IA