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A single-domain bispecific antibody targeting CD1d and the NKT T-cell receptor induces a potent antitumor response

Abstract

Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.

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Fig. 1: CD1d-specific VHH1D12 activates type I NKT cells.
Fig. 2: VHH1D12 selectively activates type I NKT cells and enhances their avidity toward (weakly)agonistic antigens presented by CD1d.
Fig. 3: Differential docking of VHH1D5, VHH1D22 and VHH1D12 over CD1d.
Fig. 4: VHH1D5 and VHH1D22 dock over the A′-pocket and flank of the F′-pocket of CD1d, respectively.
Fig. 5: The bispecific properties of VHH1D12 stabilize the CD1d-type I NKT TCR interface.
Fig. 6: VHH1D12 triggers type I NKT cell proliferation and cytokine production and lysis of CD1d-expressing tumor cells in vitro.
Fig. 7: VHH1D12 induces type I NKT cell activation and cytotoxicity toward patient tumor cells that correlates with CD1d expression.
Fig. 8: VHH1D12 induces type I NKT cell antitumor activity in vivo and increases survival in an MM model.

Data availability

The crystal structures have been deposited in the PDB: VHH1D5-hCD1d(α-GalCer) under PDB accession code 6V7Y; VHH1D22-hCD1d(α-GalCer) under PDB accession code 6V7Z; and VHH1D12-hCD1d(α-GalCer)-NKT12-TCR under PDB accession code 6V80. The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported by CCA-VICI grant no. 2000483 from the VU University Medical Center, grant no. 140343 from Worldwide Cancer Research, funding from LAVA Therapeutics (R.L., J.V. and H.J.V.), the National Health and Medical Research Council of Australia (NHMRC; grant nos. 1113293 and 1140126), the Australian Research Council (ARC; grant no. CE140100011) and the Cancer Council of Victoria (J.R., D.I.G.). S.G. is supported by an NHMRC Senior Research Fellowship (no. GNT#1159272). A.R.H. is supported by National Institutes of Health (grant no. R01 GM111849). D.I.G. is supported by an NHMRC Senior Principal Research Fellowship (no. 1117766). A.P.U. is supported by an ARC Future Fellowship (no. FT140100278). J.L.N is supported by an ARC Future Fellowship (no. FT160100074). J.R. is supported by an ARC Laureate Fellowship (no. FL160100049). We thank the Monash Macromolecular Crystallization Facility staff for assistance with crystallization, the Australian Synchrotron for assistance with data collection, the University of Melbourne, Department of Microbiology and Immunology Flow Cytometry facility for flow cytometry support, L. Smit and K. K. Sivaramanand for assistance and P.W.H.I. Parren for comments.

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R.L., A.S., J.V., S.J.J.R. and S.M.Q-P. performed the research. R.L. and A.S. analyzed the data. R.L., A.S., S.Z., D.I.G., T.D.G., J.R. and H.J.V. designed the research and interpreted the data. S.G., D.G.P., A.P.U., J.L.N., S.K.R., A.R.H. and R.W.J.G. provided unique reagents. R.L., A.S., D.I.G., T.D.G., J.R. and H.J.V. wrote the manuscript.

Corresponding authors

Correspondence to Jamie Rossjohn or Hans J. van der Vliet.

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Competing interests

H.J.V. is Chief Scientific Officer of LAVA Therapeutics, a company working on bispecific antibodies. H.J.V. and T.D.G. are shareholders of LAVA Therapeutics. D.I.G. is a member of the scientific advisory board and a shareholder of Avalia Immunotherapies, a company working on NKT cell-based vaccines.

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Extended data

Extended Data Fig. 1 CD1d-specific VHH1D12 activates type I NKT cells.

a-f, Expression (%) of CD25 or CD107a on type I NKT cells after 24 h or 4 h co-culture with either HeLa wild type (WT), HeLa.CD1d (a,c,f), MM.1 s.WT, MM.1 s.CD1d cells (b), A431 (d) or CCRF-CEM (e), plus NC, α-GalCer (100 ng/ml), VHH1D control (100 nM or concentration range (e)) and/or VHH1D12 (100 nM or concentration range (e)) (n = 3 (a-d, f) or n = 4 (e) independent experiments with type I NKT cells obtained from individual donors), analysed by flow cytometry. Transwell; type I NKT cells were placed in the upper chamber. g, Representative histogram depicting expression of CD1d (by means of binding of biotinylated VHH1D22) to MM.1S.CD1d after 24 h incubation with negative control (NC) or VHH1D12 (100 nM). h, Exemplifying gating strategy used for type I NKT cell CD107a expression analysis (here after co-culture with CCRF-CEM cells). a-d,f, The bars indicate the mean +s.d.; e, nonlinear regresssion with 95% confidence bands (dotted lines). a,d, One-way analysis of variance (ANOVA) with Tukey multiple comparisons test; b,c, two-way ANOVA with Šídák; f, Tukey multiple comparisons test.

Source data

Extended Data Fig. 2 VHH1D12 selectively activates type I NKT cells.

a, Fold change in CD69 expression on gated Jurkat-76 (J76) or SKW3 cell lines transduced with the CD1d(α-GalCer)-restricted NKT15-, 9B3- or 9C1-TCRs after overnight co-culture with either C1R.WT (CD1d negative) or C1R.GFP.CD1d plus NC, α-GalCer (100 ng/ml), VHH1D5 (1 µM), VHH1D12 (1 µM), VHH1D22 (1 µM) or a combination thereof (n = 4 (NKT15) or n = 3 (9B3-, and 9C1 TCR) independent experiments), analysed by flow cytometry. MF, median fluorescence. b, Fold change in CD69 expression on gated Jurkat J.RT3-T3.5 (JRT3) cell line transduced with the CD1d(sulfatide)-restricted DP10.7 TCR after overnight co-culture with either C1R.WT (CD1d negative) or C1R.CD1d plus NC, sulfatide (25 µg/ml), VHH1D22 (1 µM) or a combination thereof (n = 3 independent experiments), analysed by flow cytometry. c, Histograms depicting binding of lipid loaded CD1d-tetramers, pre-incubated with (red histograms) or without (dark grey histograms) VHH1D12 (~5 times molar excess), to JRT3.DP10.7 cell line. Number in each histogram indicates MF. Data are representative of 3 separate experiments. d, Type I NKT cell binding of lipid loaded CD1d tetramers, simultaneously (+) or after 60 minutes (>) incubated with or without VHH1D12 and/or 1D22 (~5 times molar excess) as indicated (n = 3 independent experiments with type I NKT cells obtained from individual donors). a,b,d, The bars indicate the mean +s.d. a,b,d, One-way analysis of variance (ANOVA) with Tukey multiple comparisons test.

Source data

Extended Data Fig. 3 VHH1D5 and VHH1D22 dock over the A’-pocket and flank of the F′-pocket of CD1d respectively.

a, Binding of VHH1D5 to CD1d wild-type (WT) and alanine-substitution mutants expressed on C1R cells (normalized for GFP expression and made relative to CD1d WT) (n = 5 independent experiments). b, Footprint of NKT12 TCR (yellow), VHH1D22 (dark-red) and overlapping area (orange). c, Binding of VHH1D22 to CD1d WT and alanine-substitution mutants expressed on C1R cells (normalized for GFP expression and made relative to CD1d WT) (n = 5 independent experiments). a,c, The bars indicate mean +s.d.

Source data

Extended Data Fig. 4 Bispecific properties of VHH1D12 stabilize the CD1d-type I NKT-TCR interface.

a, Enlarged view of VHH1D12 CDR1 (green), CDR3 (blue), and CDR1 and CDR3 loop interactions with CD1d(α-GalCer). Residues involved in contacts are represented as sticks, with hydrogen bonds represented as red dashed lines. Nitrogen, oxygen, and sulphate are coloured in blue, red, and yellow respectively. VHH1D12 residues are labelled in purple, CD1d and β2m residues in black. b, Sensorgrams and saturation plot of concentrations series for VHH1D12 passed over CD1d(endogenous). Sensorgram data are representative of 2 independent experiments performed in duplicate, saturation plot indicates data points from both independent experiments.. RU, response unit, KD, equilibrium dissociation constant, Ka, association rate constant, Kd, dissociation rate constant. c, Footprint of NKT12 TCR CDR regions on CD1d(α-GalCer) molecular surface and enlarged view of CDR2β (orange), CDR3β (yellow), CDR1α (teal), CDR2α (green) and CDR3α (purple) loop interactions with CD1d(α-GalCer) (grey). Residues and bonds are represented as above. d, Expression (%) of CD107a on type I NKT cells after 4 h co-culture with MM.1S.mCherry/luc.CD1d cells plus 10 nM VHH1D12 wild type (WT) or indicated alanine substitution mutants (n = 4 independent experiments), and e, histogram depicting binding of biotinylated VHH1D12 and alanine substitution mutants to C1R.GFP.CD1d, analysed by flow cytometry. f, Histogram depicting binding of lipid loaded CD1d-tetramer, whether or not pre-incubated with VHH1D12 (red histogram) or VHH1D12 R96A (blue histogram) (~5 times molar excess), to type I NKT cells. Data are representative of 3 separate experiments with type I NKT cells obtained from individual donors. g,h, Sensorgrams and saturation plots of concentrations series for NKT12 TCR passed over VHH1D12 R45A-CD1d(α-GalCer) (g) or VHH1D12 R96A (i). Sensorgram data are representative of 2 independent experiments performed in duplicate, saturation plot indicates data points from both independent experiments. d, The bars indicate mean +s.d.

Source data

Extended Data Fig. 5 VHH1D12 triggers type I NKT cell cytokine production, anti-tumor cytotoxicity and expansion.

a, Cytokine secretion by type I NKT cells after 24 h co-culture with HeLa.CD1d cells plus negative control (NC), α-GalCer (100 ng/ml), OCH (100 ng/ml), VHH1D12 (100 nM) or a combination thereof, analysed by CBA (n = 4 independent experiments with type I NKT cells obtained from individual donors). b, Living (annexin V and 7-AAD) CCRF-CEM cells after 16 h co-culture with type I NKT cells (effector:target ratio 1:2) plus NC or VHH1D12 (100 nM), quantified by flow cytometric counting beads and made relative to CCRF-CEM cells only (n = 5 independent experiments with type I NKT cells obtained from individual donors). c, Cytotoxicity of type I NKT cells plus NC, α-GalCer (100 ng/ml), VHH1D12 (100 nM) or a combination thereof towards CCRF-CEM cells as determined by annexin V+ and 7-AAD+ after 18 h co-culture (effector:target as indicated) analysed by flow cytometry (n = 4 independent experiments with type I NKT cells obtained from individual donors). d, Exemplifying gating strategy used to analyse type I NKT cell cytotoxicity towards tumor cells (here CCRF-CEM cells). e, Mean percentage CD4+, CD8+, and CD4CD8 type I NKT cell (fresh isolated) after 7-day co-culture with MM.1S.mCherry/luc.CD1d cells plus NC, α-GalCer (100 ng/ml) or VHH1D12 (100 nM) (effector:target ratio of 1:2) (n = 3 independent experiments with type I NKT cells obtained from individual donors). a, The box and whisker plots indicate the median and 25th to 75th percentiles, and minimun to maximum; b,c, the bars indicate the mean +s.d. a, One-way analysis of variance (ANOVA) with Tukey multiple comparisons test; b, two-tailed paired t-test; c, two-way ANOVA with Šídák multiple comparisons test.

Source data

Extended Data Fig. 6 Type I NKT cell cytokine secretion induced by VHH1D12 and CD1d expression and type I NKT cell frequency in MM and AML patient tumor samples.

a, Cytokine secretion by type I NKT cells after 16 h co-culture with fresh bone marrow (BM) derived MM patient samples plus NC, α-GalCer (100 ng/ml) or VHH1D12 (100 nM) (n = 7 individual patients), analysed by CBA. BM, indicates bone marrow cells without added effector cells. b, Exemplary histograms depicting CD1d expression on MM and AML cells. c,d, Exemplifying gating strategy used to analyse BM derived MM and AML patient samples (here after co-culture with type I NKT cells). e, type I NKT cell frequency (% of CD3pos cells) in BM samples of MM and AML patients. a,e, The box and whisker plots indicate the median and 25th to 75th percentiles, and minimum to maximum. a, One-way analysis of variance (ANOVA) with Tukey multiple comparisons test.

Source data

Extended Data Fig. 7 CD1d expression on tumor cells in mice treated with VHH1D12 and type I NKT cells.

a, Histogram depicting CD1d expression on pre-infusion MM.1S.mCherry/luc.CD1d and intraperitoneal plasmacytomas found in 2 mice treated with type I NKT cells plus VHH1D12 (from left to right mouse 2 and 4 in Fig. 5b). b, Exemplifying gating strategy used to analyse tumor-infiltrating type I NKT cells and tumor CD1d expression after tumor dissociation of mouse 2.

Extended Data Fig. 8 Surface expression of CD1a, CD1b, CD1c or CD1d and lipid antigen structures.

a,b, Histograms depicting expression of CD1a, CD1b, CD1c or CD1d on C1R transfectants, K562 transfectant, HeLa wild type (WT), HeLa transfectant, A431, MM.1S.WT, MM.1S transfectant, CCRF-CEM, MOLM-13 and NOMO-1. FMO, fluorescence minus one control. MF, median fluorescence. c, Structures of lipid antigens used in CD1d tetramers. PC, phosphatidylcholine.

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Lameris, R., Shahine, A., Pellicci, D.G. et al. A single-domain bispecific antibody targeting CD1d and the NKT T-cell receptor induces a potent antitumor response. Nat Cancer 1, 1054–1065 (2020). https://doi.org/10.1038/s43018-020-00111-6

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