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Nanobody-based bispecific antibody engagers targeting CTLA-4 or PD-L1 for cancer immunotherapy

Nature Biomedical Engineering volume 10, pages 39–55 (2026)Cite this article

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Abstract

As immune checkpoint blockade induces durable responses in only a subset of patients, more effective immunotherapies are needed. Here we present bispecific antibody engagers, fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains (VHHkappa) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumour activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. The anti-CTLA-4 VHH-VHHkappa conjugate demonstrates superior antitumour activity compared with the conventional monoclonal anti-CTLA-4 antibody and reduces the number of intratumoural regulatory T cells in a mouse model of colorectal carcinoma. The anti-PD-L1 VHH-VHHkappa conjugate is less effective in the colorectal carcinoma model while still outperforming a conventional antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHHkappa conjugate was enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype.

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Fig. 1: Recruitment of polyclonal Igs using nanobody-based immune checkpoint inhibitor-VHHkappa conjugates: proposed mechanism of action.
Fig. 2: Affinity, specificity and antibody-recruiting capability of the anti-CTLA-4 VHH (H11)-VHHkappa and the anti-PD-L1 VHH (A12)-VHHkappa conjugates.
Fig. 3: The anti-CTLA-4 VHH (H11)-VHHkappa conjugate shows better in vivo antitumour activity than the anti-CTLA-4 antibody 9H10 or H11 VHH alone.
Fig. 4: Whole-body imaging and biodistribution of NIR dye-labelled anti-PD-L1 VHH (A12)-VHHkappa conjugate in MC38 tumour-bearing mice show its PD-L1-dependent accumulation in the tumour.
Fig. 5: The anti-PD-L1 VHH (A12)-VHHkappa conjugate has superior in vivo antitumour activity compared with anti-PD-L1 antibody 10F.9G2 or A12 VHH alone.
Fig. 6: Targeted delivery of the cytotoxicity drug maytansine enhances antitumour activity of the anti-PD-L1 VHH (A12)-VHHkappa conjugate.
Fig. 7: Targeted delivery of a STING agonist enhances antitumour activity of the anti-PD-L1 VHH (A12)-VHHkappa conjugate.

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Data availability

All data required to evaluate the conclusions in this paper are included in the main text or Supplementary Information. All unique materials used, including nanobodies and nanobody drug adducts, are available from the authors upon reasonable request. The crystal structures of mouse and human PD-L1 were obtained from the Protein Data Bank (PDB IDs 6SRU and 5XXY, respectively). Source data are provided with this paper.

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Acknowledgements

We thank the Vascular Biology Program (VBP) Bioimaging Core at Boston Children’s Hospital for training and support in the use of a PerkinElmer IVIS in vivo imaging system. We also thank the Center for Macromolecular Interactions at Harvard Medical School for their support and assistance with the SPR experiments. This research was supported by NIH grant R01AI182177 to X.L. and H.L.P., and by the NIH Director’s Pioneer Award DP1AI150593 to H.L.P. T.B. was supported by a fellowship from the Belgian American Educational Foundation and by a WBI World fellowship from Wallonie-Bruxelles International. R.K.A. was supported by the Cancer Research Institute Irvington Postdoctoral Fellowship to Promote Racial Diversity (reference number CRI3968).

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  1. These authors contributed equally: Xin Liu, Camille Le Gall, Ryan K. Alexander.

Authors and Affiliations

  1. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Xin Liu, Camille Le Gall, Ryan K. Alexander, Ella Borgman, Thomas Balligand & Hidde L. Ploegh

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  1. Xin Liu
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Contributions

X.L., R.K.A. and H.L.P. conceived the idea. X.L., C.L., R.K.A. and H.L.P. designed the studies. X.L. prepared the nanobody conjugates and their drug adducts. X.L., C.L. and E.B. characterized the nanobody conjugates in vitro. X.L., C.L., R.K.A., E.B. and T.B. tested the nanobody conjugations and their drug adducts in vivo. X.L., C.L., R.K.A., E.B., T.B. and H.L.P. interpreted the data. X.L., C.L., R.K.A. and H.L.P. wrote the paper.

Corresponding author

Correspondence to Hidde L. Ploegh.

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Competing interests

X.L. and H.L.P. have filed a patent covering the technologies described in this publication (international publication number WO2023141500A2). The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Individual tumor growth curves for the experiments presented in main Fig. 3.

a, Individual tumor growth curves for MC38 tumor-bearing mice (n = 6) treated intraperitoneally with the indicated VHHs at 5 mg/kg on day 1 post-tumor injection, followed by three times a week for 3 weeks as indicated by the arrows. b, Individual tumor growth curves for B16-F10 tumor-bearing mice (n = 6) treated intraperitoneally with the indicated VHHs at 5 mg/kg on day 1 post-tumor injection, followed by three times a week for 3 weeks as indicated by the arrows. The mice were vaccinated with GM-CSF–secreting B16 cells (GVAX) on day 0. c, Individual tumor growth curves for MC38 tumor-bearing mice (n = 6) treated intravenously with the indicated VHHs at 5 mg/kg or antibody at 12.5 mg/kg on day 7, 10, and 13 post-tumor injection as indicated by the arrows. d, Tumor growth (left) and survival curves (middle) for MC38 tumor-bearing mice (Data represent mean ± SD, n = 6) treated intravenously with the anti-CTLA-4 VHH (H11)-VHHkappa conjugate at 5 mg/kg and 2.5 mg/kg on day 7, 10, and 13 post-tumor injection as indicated by the arrows. † Mouse was sacrificed due to severe ulceration of its tumor. This is part of the study presented in Fig. 3f. Right: Individual tumor growth curves for mice treated with 2.5 mg/kg of the H11-VHHkappa conjugate. (Two-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze the tumor growth curves. The two-sided Log-rank (Mantel-Cox) test was applied to analyze the survival curves.).

Source data

Extended Data Fig. 2 The anti-CTLA-4 VHH (H11)-VHHkappa conjugate and anti-CTLA-4 monoclonal 9H10 re-polarize the tumor microenvironment.

The H11-VHHkappa conjugate and 9H10 substantially alter the TME by depleting regulatory T cells. In addition, H11-VHHkappa induces an increase of tumor-infiltrating neutrophils, and an (non-significant, P = 0.06) reduction in tumor cell proliferation. Tumor cells were defined as live CD45− while tumor-infiltrating leukocytes were gated on live CD45+, myeloid cells were defined as live CD45+CD11b+. The myeloid compartment was further analyzed for expression of CD11c, MHCII, CD86 and Ly6G. Neutrophils were gated on live CD45+CD11b+Ly6Ghi. NK cells were gated on live CD45+NK1.1+. T cells were gated on live CD45+CD3+ and further analyzed for expression of CD8, CD4 CD25 and CD69. Activated CD4+ and CD8+ T cells were selected based on CD69 expression. Regulatory T cells were gated on live CD45+CD4+CD25+FoxP3+. Proliferation was measured by staining for Ki67. Results were analyzed by one way ANOVA with Bonferoni’s correction for multiple testing (Data represent mean ± SD, n = 3). Statistical significance was plotted as follows: * P < 0.05, ** P < 0.01 *** P < 0.001 and **** P < 0.0001.

Source data

Extended Data Fig. 3 Individual tumor growth curves for the experiment presented in main Fig. 5.

a, Individual tumor growth curves for MC38 tumor-bearing mice (n = 6) treated intraperitoneally with the indicated VHHs at 5 mg/kg on day 1 post-tumor injection, followed by three times a week for 3 weeks as indicated by the arrows. b, Individual tumor growth curves for B16-F10 tumor-bearing mice (n = 6) treated intraperitoneally with the indicated VHHs at 5 mg/kg on day 1 post-tumor injection, followed by three times a week for 3 weeks as indicated by the arrows. The mice were vaccinated with GM-CSF–secreting B16 cells (GVAX) on day 0. c, Individual tumor growth curves for wild-type and PD-L1 knockout MC38 tumor-bearing mice (n = 5) treated intraperitoneally with the indicated VHHs at 5 mg/kg on day 1 post-tumor injection, followed by three times a week for 3 weeks as indicated by the arrows. d, Individual tumor growth curves for MC38 tumor-bearing wild-type and LysMcrePD-L1fl/fl (with PD-L1 deficient myeloid cells) mice (n = 5) treated intraperitoneally with the indicated VHHs at 5 mg/kg on day 1 post-tumor injection, followed by three times a week for 3 weeks as indicated by the arrows.

Source data

Extended Data Fig. 4 Individual tumor growth curves and body weight curves for the experiments presented in main Fig. 6.

a, Individual tumor growth curves for MC38 tumor-bearing mice (n = 6) treated intravenously with A12-VHHkappa-DM1 or A12-VHHkappa-DM4 at 5 mg/kg on day 7, 10,13, and 17 post-tumor injection as indicated by the arrows. b, Individual tumor growth curves for MC38 tumor-bearing mice (n = 6) treated intravenously with indicated VHH-DM4 conjugates at 5 mg/kg on day 7, 10,13, and 17 post-tumor injection as indicated by the arrows. c, Individual tumor growth curves (upper panels) and body weight curves (lower panel, data represent mean ± SD) for B16-F10 tumor-bearing mice (n = 5) treated intravenously with indicated VHH-DM4 conjugates at 5 mg/kg on day 3, 6, 9, and 13 post-tumor injection as indicated by the arrows.

Source data

Extended Data Fig. 5 Conjugation of DM4 or a STING agonist to the anti-PD-L1 VHH (A12)-VHHkappa conjugate re-polarizes the tumor microenvironment.

A12-VHHkappa-DM4 treatment led to pronounced cell death in the tumor, and halted tumor cell proliferation. Inclusion of DM4 reduced the fraction of tumor-infiltrating cells (mostly of myeloid origin), particularly CD11c+MHCII+ cells. A12-VHHkappa-DM4 also reduced CD86 expression on CD11c+ cells, while an increase in neutrophil infiltration was detected. In the T cell compartment, CD4+ and CD8+ T cell activation was increased by A12-VHHkappa-DM4 treatment. A12-VHHkappa-STING agonist surprisingly increased the proliferation of tumor cells, and, as expected, increased the activation of CD4+ and CD8+ T cells in the tumor. Tumor cells were defined as live CD45− while tumor-infiltrating leukocytes were gated on live CD45+, myeloid cells were defined as live CD45+CD11b+. The myeloid compartment was further analyzed for expression of CD11c, MHCII, CD86 and Ly6G. Neutrophils were gated on live CD45+CD11b+Ly6Ghi. NK cells were gated on live CD45+NK1.1+. T cells were gated on live CD45+CD3+ and further analyzed for expression of CD8, CD4 CD25 and CD69. Activated CD4+ and CD8+ T cells were selected based on CD69 expression. Regulatory T cells were gated on live CD45+CD4+CD25+FoxP3+. Proliferation was measured by staining for Ki67. Results were analyzed by one way ANOVA with Bonferoni’s correction for multiple testing (Data represent mean ± SD, n = 3 for PBS-, A12-VHHkappa-DM4-, and A12-VHHkappa-STING agonist–treated groups, and n = 4 for the A12-VHHkappa conjugate–treated group). Statistical significance was plotted as follows: * P < 0.05, ** P < 0.01 *** P < 0.001 and **** P < 0.0001.

Source data

Extended Data Fig. 6 Individual tumor growth curves and body weight curves for the experiments presented in main Fig. 7.

a, Individual tumor growth curves for MC38 tumor-bearing mice (n = 6) treated intravenously with A12-VHHkappa-STING agonist or A12-VHHkappa-conjugate plus free STING agonist (equimolar amount) at 5 mg/kg on day 7, 10, and 14 post-tumor injection as indicated by the arrows. b, Individual tumor growth curves (upper panels) and body weight curves (lower panel, data represent mean ± SD) for B16-F10 tumor-bearing mice (n = 5) treated intravenously with A12-VHHkappa-STING agonist or A12-VHHkappa-conjugate plus free STING agonist (equimolar amount) on day 3, 7,11, and 15 post-tumor injection as indicated by the arrows. c, Individual tumor growth curves (upper panels) and body weight curves (lower panel, data represent mean ± SD) for MC38 tumor-bearing mice (n = 6) treated intravenously with A12-VHHkappa-STING agonist or A12-VHHkappa-conjugate plus free STING agonist (equimolar amount) at 10 mg/kg on day 7, 10, and 14 post-tumor injection as indicated by the arrows.

Source data

Extended Data Fig. 7 Evaluation of toxicity of VHHkappa conjugates and their drug adducts in mice.

a, Assessment of liver and renal toxicity of VHHkappa conjugates and their drug adducts by measuring alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urea, and blood urea nitrogen (BUN) levels. BUN concentrations were calculated from urea concentrations using the formula: BUN (mg/dL) = [Urea] (mg/dL) / 2.14. Mice were intravenously treated with 5 mg/kg of VHHkappa conjugates or their drug adducts following the same dosing schedule as their therapeutic treatments: every three days for three doses (H11-VHHkappa conjugate and A12-VHHkappa-STING agonist) or four doses (A12-VHHkappa conjugate and A12-VHHkappa-DM4). Blood samples were collected three days after the final treatment (Data represent mean ± SD, n = 4, two-sided unpaired t test). b, Measurement of mouse interferon beta (IFN-β) levels in tumor homogenates and blood following treatment with A12-VHHkappa-STING agonist. Tumor-bearing mice were intravenously treated with A12-VHHkappa-STING agonist or A12-VHHkappa conjugate plus an equimolar amount of free STING agonist at 5 mg/kg on days 11 and 13. Tumors and blood samples were collected on day 14 for IFN-β measurement. IFN-β levels below the assay’s detection limit were recorded as 0 (Data represent mean ± SD, n = 3 for untreated and A12-VHHkappa-STING agonist–treated groups, and n = 4 for the A12-VHHkappa conjugate + STING agonist–treated group, two-sided unpaired t test).

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Supplementary Information

Supplementary Figs. 1–23 and Chemical synthesis.

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Supplementary data for Figs. 3, 5–8, 10, 15 and 20.

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Source Data Figs. 1–7 Source Data Extended Data Figs. 1–7

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Liu, X., Le Gall, C., Alexander, R.K. et al. Nanobody-based bispecific antibody engagers targeting CTLA-4 or PD-L1 for cancer immunotherapy. Nat. Biomed. Eng 10, 39–55 (2026). https://doi.org/10.1038/s41551-025-01447-z

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