A CD1c lipid agnostic T cell receptor bispecific engager redirects T cells against CD1c+ cells

Abstract

Introduction: Immunotherapy is emerging as an efficacious treatment for some cancers, complementing traditional chemo-radiation therapies. Specific markers at the cell surface of cancer cells can be used as immunotherapy targets. However, many of these markers are defined by a patient's genetic background, limiting their use across the human population.

Methods: Here, we investigated the non-polymorphic antigen presenting molecule, CD1c, that is only expressed on subsets of mature hematopoietic cells, as a potential immunotherapy target with reduced risk of off-tumor on-target toxicity in healthy tissues.

Results and discussion: We identified a T cell receptor (TCR) which recognises CD1c in a lipid independent manner and determined the crystal structure of the TCR-CD1c complex which revealed flexibility around the lipid binding region, and a new binding mechanism of auto-antigen recognition. We generated affinity enhanced variants of the TCR and fused them to an anti-CD3 antibody for T cell redirection. Lipidomic analysis revealed promiscuous lipid recognition of CD1c by the affinity enhanced TCR variants, with preference for larger lipid head group, a finding which is supported by the crystal structure. The bispecific molecule induced potent re-directed T cell killing of CD1c positive cell lines. These proof-of-concept findings demonstrate that CD1c targeting TCR bispecific engagers might be good candidates for the development of non-MHC restricted, universal therapeutics for the treatment of CD1c+ leukemias.

Keywords: CD1c; T cell engager; T cell receptor; bispecifics; immunotherapy; leukemia; lipids.

Figure 1

Crystal structures of CD1c-S2c TCR complexes. (A). Cartoon overlay of phage-isolated S2c^WT and high affinity S2c^a5b6 TCRs in complex with CD1c-endo. TCRα, TCRβ, CD1c and β2m chains are coloured as orange, cyan, grey and brown respectively. The S2c^a5b6 TCR chains are coloured in darker shades. The arrows indicate the changes in CDR1α and CDR3β loop conformations between S2c^WT and S2c^a5b6. (B). Cartoon overlay of CD1c-S2c^WT and CD1c-3C8 TCR (PDB) complexes. The 3C8 TCR chains are coloured in magenta. (C). Top view showing the CD1c as grey surface and S2c^WT CDRs as ribbons. The position of disulphide bonds in the TCR variable domains are indicated by spheres and the line connecting them represents the vector used for calculating the docking angle. (D). CDR sequences of S2c^WT and S2c^a5b6 TCRs. Mutations introduced in S2c^a5b6 variant are highlighted in red. TCR chains: S2c (TRAV9-2*02/TRBV7-9*01); 3C8 (TRAV29/TRBV 7-2).

Figure 2

Comparative interaction analysis of S2c^WT and S2c^a5b6 TCRs with CD1c-endo. Side-by-side comparison of residues from the S2c^WT and S2c^a5b6 TCRs interacting with the helices of CD1c-endo. (A, C) The S2c^WT TCR α-chain (orange), dominated by CDR3α, forms substantial contacts along the length of CD1c helices 1 and 2 (grey), respectively. (B, D) The S2^ca5b6 TCR α-chain (darker orange) engaging CD1c helices 1 and 2, respectively. Y28α and L30α form additional van der Waals contact with CD1c. (E, G) The S2c^WT TCR β-chain (cyan) interacting with CD1c helices 1 and 2, respectively, contributed by CDR2β and CDR3β residues. (F, H) The S2c^a5b6 TCR β-chain (darker cyan) interacting with CD1c helices 1 and 2, respectively. M98β makes additional van der Waal interactions with CD1c. Interface residues within 4 Å are shown as sticks. Dotted lines denote polar contacts.

Figure 3

Comparison of Fo-Fc electron density in the lipid binding pockets of CD1c-S2c TCR structures. (A, B) Lipid binding region of the S2c^WT-CD1c complex showing variations in lipid density within the two asymmetric unit molecules in the crystal structure. CD1c F67 side chain conformation flip and associated TCR Y48β displacement are indicated by arrows (B). TCR residues that are near the F-pocket and conformational differ between the two asymmetric unit molecules are displayed as sticks. The Fo-Fc omit map (green mesh) is contoured at 3σ. The spacer lipids (decanes) are shown as black sticks. (C) Lipid binding region of the S2c^a5b6-CD1c complex with highly similar electron density and conformation of residues within the four asymmetric unit molecules in the crystal structure.

Figure 4

Lipidomic analysis of CD1c-lipid complexes trapped by CD1c–specific ImmTACs. (A) CD1c-endo molecules expressed in Expi293F cells were complexed with CD1c-specific ImmTACs, the CD1c-ImmTAC complex and the unbound CD1c fractions were separated by size exclusion chromatography. (B) CD1c-ImmTAC mixes were loaded onto size exclusion chromatography column and the separation was monitored by ultraviolet light and gel electrophoresis to determine the CD1c to ImmTAC ratio, and allowing normalization of lipid eluents based on protein abundance. (C) Shotgun lipidomics and data analysis were carried out comparing CD1c-endo, WT and High affinity ImmTAC+CD1c complexes and unbound fractions. HLA-A2 and ImmTACs alone were used as background. (D) Lipid composition of S2c^WT-CD1c and S2c^a5b6-CD1c complexes. (E) Lipid chain length profile of the CD1c-associated lipid ligands recognised by the phage-isolated low affinity TCR and affinity enhanced ImmTACs.

Figure 5

CD1c restricted ImmTAC molecules activate pan T-cells against cancer cell lines expressing variable levels of CD1c. CD2 enriched T-cells were co-cultured with the indicated cancer cell lines to assess potency of the S2c-^a5b6 ImmTAC molecule. (A, B) Dose response curve of CD4 (A) or CD8 (B) T cell activation to C1R, C1R CD1c or C1R CD1d targets. (C, D) Dose response curve of CD4 (C) or CD8 (D) T cell activation to SKW3, OCIM1, HPB-ALL, NALM6, CCRFSB, monocytes and B cells. CD2 cells in the absence of targets are also depicted. Panels A and B depict the percentage of CD25 expressing T cells after overnight activation in pre presence (dotted lines) or absence (solid lines) of anti-CD1d blocking antibody L161. The blocking data relative to panels (C, D) are shown in Supplementary Figure S5 . One experiment of two, performed in triplicates.