Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Mar 10;8(81):eadf1426.
doi: 10.1126/sciimmunol.adf1426. Epub 2023 Mar 3.

CD19 CAR antigen engagement mechanisms and affinity tuning

Changhao He  1 , Jorge Mansilla-Soto  2   3 , Nandish Khanra  1 , Mohamad Hamieh  2   3 , Victor Bustos  4 , Alice J Paquette  5 , Andreina Garcia Angus  2   3 , Derek M Shore  1   6 , William J Rice  5   7 , George Khelashvili  1   6 , Michel Sadelain  2   3 , Joel R Meyerson  1
Affiliations

CD19 CAR antigen engagement mechanisms and affinity tuning

Changhao He et al. Sci Immunol. .

Abstract

Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), the affinity of which is central to determining CAR T cell function and therapeutic success. CAR T cells targeting CD19 were the first to achieve marked clinical responses in patients with relapsed/refractory B cell malignancies and to be approved by the U.S. Food and Drug Administration (FDA). We report cryo-EM structures of CD19 antigen with the binder FMC63, which is used in four FDA-approved CAR T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi), and the binder SJ25C1, which has also been used extensively in multiple clinical trials. We used these structures for molecular dynamics simulations, which guided creation of lower- or higher-affinity binders, and ultimately produced CAR T cells endowed with distinct tumor recognition sensitivities. The CAR T cells exhibited different antigen density requirements to trigger cytolysis and differed in their propensity to prompt trogocytosis upon contacting tumor cells. Our work shows how structural information can be applied to tune CAR T cell performance to specific target antigen densities.

PubMed Disclaimer

Conflict of interest statement

Competing interests:

The authors declare no competing interests.

Supplementary Materials

Materials and Methods

Figures

Fig. 1.
Fig. 1.. Structures of CAR binders in complex with CD19.
(A) Illustration of a CAR on a CAR-T cell in complex with CD19 antigen displayed on a tumor cell. (B) Cryo-EM density map (left) and molecular model (right) of FMC63 scFv in complex with CD19. (C) Surface view of the FMC63-CD19 complex as shown perpendicular to the membrane (top). CD19 is also shown in surface view with the footprint of FMC63 colored by contact with the VL or VH region (bottom). (D) Cryo-EM density map (left) and molecular model (right) of SJ25C1 Fab in complex with CD19. (E) Surface view of the SJ25C1-CD19 complex as shown perpendicular to the membrane (top) but with the Fab constant regions (CH and CL) omitted for clarity. CD19 is also shown in surface view with the footprint of SJ25C1 colored by contact with the VL or VH region (bottom). In (C) and (E), the footprints of the binders on CD19 correspond to CD19 residues within 4 Å of each respective binder. TM, transmembrane; CL, light chain constant domain; CH, heavy chain constant domain.
Fig. 2.
Fig. 2.. Binding interface between CAR binders and CD19.
(A to D) Molecular model of FMC63 in complex with CD19 (A). The VL domain of FMC63 contacts CD19 with residues of CDRL1 and CDRL2 (B). The VH domain forms most of the contacts for FMC63, and these are mediated by residues in CDRH2 and CDRH3 (C and D). (E to I) Molecular model of SJ25C1 in complex with CD19 (E). The VH domain forms extensive contacts with CD19 through CDRH1, CDRH2, and CDRH3 (F to I), with more limited contact from CDRL1 of the VL domain (I).
Fig. 3.
Fig. 3.. Affinity measurements of CAR binder mutants.
(A and B) Binding energies for residues from FMC63-CD19 (A) and SJ25C1-CD19 (B) complexes obtained from MM/GBSA analysis of the MD trajectories. Residues were ordered by decreasing magnitude of per-residue binding energy at the interface with CD19 (left to right). The binding energies are quantified by averaging six independent MM/GBSA simulation decompositions for each residue. Error bars represent means ± SEM. Residues from the VL and VH domain are labeled with “(L)” and “(H),” respectively. Data for the VL domain residues are shown using lighter colored bars. (C) Illustrated SPR sensorgram depicting the experimental protocol used to measure CD19 binding to Fabs. Single-cycle kinetics was performed by injecting CD19 protein at five concentrations (0.5, 4, 20, 100, and 500 nM) followed by one long dissociation step and reported with response units (RUs). (D and E) SPR sensorgrams of binding kinetics between CD19 and FMC63 (D) and SJ25C1 (E) binders. Experimental data are shown in red. Fitted curves calculated using 1:1 kinetics Langmuir binding model are shown with black dashed lines. Dissociation constant (KD) for each binder is displayed as an average of triplicates with SEM.
Fig. 4.
Fig. 4.. CD19-interacting residues differentially shape CAR T cell function.
(A and F) CAR expression analyses for FMC63 (A) and SJ25C1 (F) CAR T cells. Data are from two independent experiments using different T cell donors. (B and G) Binding activity for FMC63 (B) and SJ25C1 (G) CAR T cells. Data for FMC63 CAR T cells are from two independent experiments using different T cell donors (one replicate in each experiment). Data for SJ25C1 CAR T cells are from two independent experiments using different T cell donors (two replicates in each experiment). (C to E and H to J) CAR T cell cytotoxic activity against NALM6/WT (~27,000 CD19 molecules; C), NALM6/12–39 (~2000 CD19 molecules; D), or NALM6/12–4 (~200 CD19 molecules; E) target cells. In (C) to (E), data are means ± SEM (n = 3). In (H) to (J), data are means ± SEM (n = 5, with three replicates from T cell donor 1 and two replicates from T cell donor 2). (K) Illustration of the trogocytosis assay. (L and M) Percentage of FMC63 (L) and SJ25C1 (M) CAR T cells (both CD4 and CD8 cells) with CD19-mCherry signal after coculture with CD19-mCherry–expressing NALM6 cells. Unpaired t test P value for FMC63 CAR T cells (L) or SJ25C1 CAR T cells (M). Data are representative of two independent experiments using two different T cell donors, with each experiment performed with three replicates (n = 3). Error bars are SEM.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sadelain M, Rivière I, Riddell S, Therapeutic T cell engineering. Nature. 545, 423–431 (2017). - PMC - PubMed
    1. Guedan S, Calderon H, Posey AD, Maus MV, Engineering and design of chimeric antigen receptors. Mol. Ther. Methods Clin. Dev 12, 145–156 (2019). - PMC - PubMed
    1. Srivastava S, Riddell SR, Engineering CAR-T cells: Design concepts. Trends Immunol. 36, 494–502 (2015). - PMC - PubMed
    1. Wang X, Rivière I, Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol. Ther. Oncolytics 3, 16015 (2016). - PMC - PubMed
    1. Brentjens RJ, Latouche J-B, Santos E, Marti F, Gong MC, Lyddane C, King PD, Larson S, Weiss M, Rivière I, Sadelain M, Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. Nat. Med 9, 279–286 (2003). - PubMed

Publication types