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Bispecific antibodies increase the therapeutic window of CD40 agonists through selective dendritic cell targeting

Nature Cancer volume 3, pages 287–302 (2022)Cite this article

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Abstract

Therapeutic use of agonistic anti-CD40 antibodies is a potentially powerful approach for activation of the immune response to eradicate tumors. However, the translation of this approach to clinical practice has been substantially restricted due to the severe dose-limiting toxicities observed in multiple clinical trials. Here, we demonstrate that conventional type 1 dendritic cells are essential for triggering antitumor immunity but not the toxicity of CD40 agonists, while macrophages, platelets and monocytes lead to toxic events. Therefore, we designed bispecific antibodies that target CD40 activation preferentially to dendritic cells, by coupling the CD40 agonist arm with CD11c-, DEC-205- or CLEC9A-targeting arms. These bispecific reagents demonstrate a superior safety profile compared to their parental CD40 monospecific antibody while triggering potent antitumor activity. We suggest such cell-selective bispecific agonistic antibodies as a drug platform to bypass the dose-limiting toxicities of anti-CD40, and of additional types of agonistic antibodies used for cancer immunotherapy.

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Fig. 1: cDC1 mediate the efficacy of CD40 mAb.
Fig. 2: Distinct cellular pathways mediating the efficacy and toxicity of CD40 mAbs.
Fig. 3: Generation of CD40/DC bsAbs with preferential DC binding.
Fig. 4: Selective DC activation by CD40/DC bsAbs.
Fig. 5: Agonistic activity of CD40/DC bsAbs is dependent on FcγR engagement.
Fig. 6: CD40/DC bsAbs display an improved therapeutic window and superior antitumor response compared to their parental CD40 mAb.
Fig. 7: CD40/CD11c bsAb treatment activates DC and T cell responses in the TME.
Fig. 8: CD40/CLEC9A bsAb displays an improved therapeutic window and superior antitumor response compared to parental CD40 mAb.

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Data availability

Single-cell RNA-seq data supporting the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE184009, token: klsnqsikpjiphet. Refer to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE184009. Source data are provided with this paper. All numerical source data are provided as Source data files.

Code availability

MetaCell source code can be found at https://github.com/tanaylab/metacell.

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Acknowledgements

We thank J. Ravetch for fruitful discussion and for reviewing the manuscript. We also thank N. David and I. Sher for artwork, and S. Schwarzbaum for editorial assistance. Xcr1-Cre mice were kindly provided by B. Malissen and Cd11c-DTR mice by S. Jung. R.D is incumbent of the Rina Gudinski Career Development Chair. He is supported by the Rising Tide Translation Cancer Research Fund, Melanoma Research Alliance (MRA), Israel Cancer Research Fund, Israel Cancer Association, Israel Science Foundation, Teva Pharmaceutical, Moross Integrated Cancer Center, Dwek Institute for Cancer Therapy Research, Yeda Research and Development Co., Mizutani Foundation for Glycoscience, Harry J. Lloyd Charitable Trust, Emerson Collective Cancer Research Fund, Flight Attendant Medical Research Institute, Garvan-Weizmann program, Ira and Diana Riklis Fund for CAR-T Therapy, Enoch Foundation, Pearl Welinsky Merlo Foundation, Benoziyo Fund for the Advancement of Science, Fund honoring Gerty Schwarz Schaier, a research grant from Max Saad, Mexican Association of Friends of the Weizmann Institute and the Ben B. and Joyce E. Eisenberg Foundation. The research of I.A. is supported by the Seed Networks for the Human Cell Atlas of the Chan Zuckerberg Initiative, and by Merck KGaA, Darmstadt. I.A. holds the Eden and Steven Romick Professorial Chair, supported by the HHMI International Scholar Award, the European Research Council Consolidator Grant (no. 724471-HemTree2.0), an MRA Established Investigator Award (no. 509044), DFG (no. SFB/TRR167), the Ernest and Bonnie Beutler Research Program for Excellence in Genomic Medicine, the Helen and Martin Kimmel awards for innovative investigation, the SCA award of the Wolfson Foundation and Family Charitable Trust and by the Thompson Family Foundation Alzheimer’s Research Fund. Illustrations in Figs. 3a,b and Extended Data Fig. 8 were created with BioRender.com.

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  1. These authors contributed equally: Ran Salomon, Hagar Rotem.

Authors and Affiliations

  1. Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

    Ran Salomon, Hagar Rotem, Yonatan Katzenelenbogen, Assaf Weiner, Noy Cohen Saban, Tali Feferman, Ido Amit & Rony Dahan

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Contributions

Conceptualization was by R.S., H.R., Y.K., A.W., I.A. and R.D. Methodology was the responsibility of R.S., H.R., Y.K., A.W., I.A. and R.D. R.S., H.R., Y.K., A.W., N.C.S. and T.F. carried out investigations. Writing of the original draft was by R.S., H.R., Y.K., I.A. and R.D. Supervision was performed by I.A. and R.D. Funding acquisition was carried out by I.A. and R.D.

Corresponding author

Correspondence to Rony Dahan.

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Competing interests

The Weizmann Institute has filed a PCT patent application (no. WO2021/149053) related to this work, on which R.S. and R.D. are inventors. The patent application has been licensed to Teva Pharmaceuticals Industries Ltd. R.D. serves as a consultant for Teva Pharmaceuticals Industries Ltd and NucleAi Ltd, and receives research grant support from Teva Pharmaceuticals Industries Ltd. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Cell populations mediating efficacy of CD40 mAb.

(a) Expression of CD40 on the indicated cells in MC38 tumor-bearing mice. Tumor, draining lymph node (LN), spleen and liver were harvested for flow cytometry analysis. Delta geometric mean fluorescence intensities (ΔMFIs) are shown. (n = 5 mice; except tumor non-classical monocytes and LN cDC1s and cDC2s, n = 4 mice). The experiment was performed twice; representative results are shown. (*p = 0.0423, **p = 0.007, **p = 0.0016; Unpaired, two-tailed Student’s t-test). (b + c) Frequency of cDC1s, cDC2s and macrophages in tumor (b) and liver (c) of MC38 tumor bearing WT or Batf3-/- mice. The experiment was performed twice; a representative FACS analysis is shown (n = 5 mice). (d) Top: Xcr1-iDTR mice were injected with DTx 4 days following tumor inoculation and during the treatment regime. Frequency of cDC1s in LNs and spleens were evaluated at day 0. (n = 3 mice). The experiment was performed three times; representative results are shown. Bottom: tumor volume of individual mice at day 6 from treatment onset (cDC1 depletion-, n = 9 mice; cDC1 depletion + , n = 10 mice). The experiment was performed once. (***p = 0.0002, ***p = 0.0006, *p = 0.0185; Unpaired, two-tailed Student’s t-test). (e) WT mice were injected with Clodronate liposomes. After 24 hours, livers were harvested; single cell suspensions were analyzed by flow cytometry to determine the frequencies of the indicated cell populations. (n = 6 mice). The experiment was performed once. (***p = 0.0006; Unpaired, two-tailed Student’s t-test). Kupffer cells (KCs), macrophages (MFs), Dendritic cells (DCs), conventional type 1 DC (cDC1), conventional type 2 DC (cDC2). Unless otherwise indicated, each dot represents an individual mouse, and data are shown as mean ± SEM.

Source data

Extended Data Fig. 2 Cell populations mediating toxicity of CD40 mAb.

(a) WT mice were injected with Clodronate liposomes 24 hours prior to CD40 mAb injection. Blood AST and ALT levels were measured after 24 hours (n = 5 mice). The experiment was performed twice; a representative example is shown (***p = 0.0003, ***p = 0.0007, ****p < 0.0001, ***p = 0.0001; One-way ANOVA with Tukey’s post hoc test). (B + C) hCD40/FcγR mice were injected with anti-CD42b 24 hours prior to CD40 mAb injection. Platelets were measured after 24 hours (b), and livers were harvested and analyzed; (n = 4 mice). (****p < 0.0001; One-way ANOVA with Tukey’s post hoc test). (c). Representative liver H&E section; scale bar = 100μm. (b + c; the experiment was performed once). (d) hCD40/FcγR mice were injected with 2141 CD40 mAb, 0.5 mg/kg, and serum was collected after 3 hours. IL-6 and TNF-α cytokine levels were determined by ELISA. (Ctrl, n = 4 mice; CD40 mAb, n = 5 mice). The experiment was performed once (*p = 0.0249; Unpaired, two-tailed Student’s t-test). (e) Grouped IL-6 staining intensities presented in Fig. 2e. (Untreated, n = 2 mice; CD40 mAb, n = 3 mice). Data are represented as means. (f) Intracellular IL-6 expression after CD40 mAb injection. hCD40/FcγR mice were injected with 2141 CD40 mAb. After 2.5 hours, blood was analyzed by flow cytometry. The experiment was performed once. Unless otherwise indicated, each dot represents an individual mouse, and data are shown as mean ± SEM.

Source data

Extended Data Fig. 3 BsAb target selection, generation and characterization.

(a + b) Expression of CD11c and DEC-205 on the indicated cell types in MC38 tumor, draining lymph node (LN), spleen, and liver of tumor-bearing mice, and on platelets (b) of naïve mice. CD41 served as a positive control marker for platelets. DEC-205 delta geometric mean fluorescence intensities (ΔMFIs) and CD11c geometric mean fluorescence intensities (MFIs) are shown. Data are shown as mean ± SEM. Each dot represents an individual mouse (n = 5; except non-classical monocytes CD11c expression in the tumor, and DEC-205 expression in the tumor, n = 4) (a). FACS analysis of a representative mouse is shown in (b). The experiment was performed twice and a representative example is shown. (c) SDS-PAGE analysis of the indicated Abs and bsAbs. Heavy chain (HC), light chain (LC). All batches were analyzed, and representative results are shown. All batches were analyzed, and a representative example is shown. (d) Analytical size-exclusion chromatograms of monospecific and bispecific Abs; numbers indicate the estimated elution volume of the indicated molecular weights based on a run of SEC protein molecular weight standards. All batches were analyzed, and a representative example is shown. (e) Differential scanning fluorimetry stability and structure folding analysis of monospecific and bispecific Abs; numbers indicate the calculated Tm of each Ab. The experiment was performed once. (f) 2D projection (see Extended Data Fig. 7f) and density plots of mouse dLN (n = 4 per treatment). hCD40/FcγR mice were injected with CD40 mAb or CD40/CD11c bsAb. Cells were sorted 3 hours post injection using the gates as indicated on the plot. Single cell RNA-seq data for preferential binding assessment was pooled from two independent experiments.

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Extended Data Fig. 4 Gating strategy on MFs, B cells and DCs in lymph node.

(a + b) Inguinal LNs from hCD40/FcγR mice were harvested and dissociated to a single cell suspension. Live CD45+ cells were stained for the indicated surface markers for flow cytometry analysis. (a) The gating strategy shown was used to identify the indicated cell types, which were further analyzed for their cell surface expression levels of CD80, CD86 and MHC II, for the analysis shown in Fig. 4d. (b) Raw MFI graphs from the analysis shown in Fig. 4d (n = 6 mice; except CD40 mAb CD80 MFI, n = 5 mice) (*p = 0.0381, **p = 0.0068; Unpaired, two-tailed Student’s t-test). Data are shown as mean ± SEM.

Source data

Extended Data Fig. 5 Binding of human bsAb Fc variants to human FcγRIIB.

Binding of the indicated Fc variants of anti-CD40/DC bsAbs to recombinant hFcγRIIB, assessed by ELISA. The experiment was performed twice, and representative results are shown.

Source data

Extended Data Fig. 6 Improved therapeutic window of CD40/CD11c bsAb without evidence of hepatotoxicity.

(a + b) Dose dependent CRS toxicity after CD40 mAb or CD40/CD11c bsAb injections was assessed by measuring serum IL-6 and TNF-α levels. hCD40/FcγR mice were treated with the indicated doses of CD40 mAb or CD40/CD11c bsAb, and cytokine levels in the serum 3 hours following treatment were determined by ELISA. Each dot represents an individual mouse (n = 5; except untreated, n = 4 and CD40/CD11c bsAb 1.25, 2.5 and 5, n = 6). The experiment was performed once. Data are shown as the mean ± SEM. (*p = 0.0482, *p = 0.0154, *p = 0.012; Kruskal–Wallis with two-tailed Dunn’s post hoc test). (c) Liver transaminase serum levels in response to the indicated treatment. hCD40/FcγR mice were inoculated with MC38 tumor cells, and treated with CD40 mAb or CD40/CD11c bsAb at their respective MTDs (0.175 mg/kg, and 2.5 mg/kg respectively) on days 0, 2, 4, and 6. Mice were sacrificed on day 18, and liver transaminase (ALT and AST) levels were measured. Each dot represents an individual mouse (n = 4; except untreated, n = 2). The experiment was performed once. Data are shown as means. (d) H&E staining of livers from mice at day 18 after treatment onset. Representative images from each treatment are shown. Scale bars on images = 100 μm.

Source data

Extended Data Fig. 7 Effect of CD40/CD11c bsAb and CD40 mAb on tumor and dLN immune population.

(a) Gating strategy used to distinguish intratumoral cDC1s and cDC2s obtained from MC38-bearing mice. (b) hCD40/FcγR MC38 tumor-bearing mice were injected with CD40/CD11c bsAb, and after 3 hours tumors were harvested to single cell suspensions and analyzed for the bsAb in vivo binding. (n = 2 mice). The experiment was performed twice and representative results are shown. (c) Expression of maturation markers CD86, and CD80 on intratumoral cDC1s and cDC2s followed by the indicated treatment. Geometric mean fluorescence intensities (MFIs) are shown. (n = 6 mice; except CD40 mAb, n = 4 mice and CD40/CD11c bsAb cDC2s CD80 MFI, n = 5 mice) (*p = 0.0236, *p = 0.0363, **p = 0.008; One-way ANOVA with Tukey’s post hoc test). (d) Quantification of total intratumoral DCs, cDC1s, and cDC2s followed by the indicated treatment. (n = 6; except CD40 mAb, n = 4). (c + d) The experiment was performed twice and representative results are shown. Each dot represents an individual mouse, and data are graphed as mean ± SEM. (e) Characterization of the T cell compartment as a fraction of each T cell subpopulation from total CD45+ cells (X axis), and from total TCRβ+ cell (Y axis). Each dot represents one animal (n = 4). Data were generated from 9940 quality control-positive cells. (f) Two-dimensional projection of single cell RNA-seq analysis of 570 Metacells representing 33,621 intratumoral CD45+ and CD45+, TCRβ+ cells from MC38 mouse tumor model, LN and spleen. Color coding for cell type assignment is indicated on the plot. (g + h). Density plots and percentage of key cell populations in the MC38 tumor microenvironment in both CD40 mAb and CD40/CD11c bsAb treated mice (7 days post treatment). Data were generated from CD45+ and CD45+, TCRβ+ cells as indicated in the plot. Each point represents one animal (CD40 mAb, n = 5; CD40/CD11c bsAb, n = 7). Black line indicates average percentage. (*p = 0.01; Unpaired, two-tailed Student’s t-test).

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Extended Data Fig. 8 Generation of CD40/CLEC9A bispecific antibody.

Top left: Binding ELISA of the anti-CD40 monospecific and anti-CD40/CLEC9A bsAb to recombinant hCD40 protein. Top right: Binding ELISA of the anti-CLEC9A monospecific and anti-CD40/CLEC9A bsAb to recombinant CLEC9A protein. Bottom: Simultaneous binding sandwich ELISA of the indicated mAbs and bsAb to recombinant CLEC9A and hCD40 proteins. Assay configurations are illustrated to the right of the respective data. The experiment performed twice, and representative results are shown.

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Salomon, R., Rotem, H., Katzenelenbogen, Y. et al. Bispecific antibodies increase the therapeutic window of CD40 agonists through selective dendritic cell targeting. Nat Cancer 3, 287–302 (2022). https://doi.org/10.1038/s43018-022-00329-6

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