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Small-molecule-mediated control of the anti-tumour activity and off-tumour toxicity of a supramolecular bispecific T cell engager

Nature Biomedical Engineering (2024)Cite this article

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Abstract

The broader clinical use of bispecific T cell engagers for inducing anti-tumour toxicity is hindered by their on-target off-tumour toxicity and the associated neurotoxicity and cytokine-release syndrome. Here we show that the off-tumour toxicity of a supramolecular bispecific T cell engager binding to the T cell co-receptor CD3 and to the human epidermal growth factor receptor 2 on breast tumour cells can be halted by disengaging the T cells from the tumour cells via the infusion of the small-molecule drug amantadine, which disassembles the supramolecular aggregate. In mice bearing human epidermal growth factor receptor 2-expressing tumours and with a human immune system, high intravenous doses of such a ‘switchable T cell nanoengager’ elicited strong tumour-specific adaptive immune responses that prevented tumour relapse, while the infusion of amantadine restricted off-tumour toxicity, cytokine-release syndrome and neurotoxicity. Supramolecular chemistry may be further leveraged to control the anti-tumour activity and off-tumour toxicity of bispecific antibodies.

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Fig. 1: SiTE for cancer immunotherapy.
Fig. 2: Synthesis and characterization of SiTE.
Fig. 3: Small-molecule AMD controls SiTE activity in vitro.
Fig. 4: AMD reduces the on-target off-tumour toxicity of SiTE in vivo.
Fig. 5: High doses of SiTE induce a strong tumour-specific T cell immune response.
Fig. 6: AMD reduces SiTE-induced CRS in vivo.

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Data availability

The data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

M.J.M. acknowledges support from a United States National Institutes of Health Director’s New Innovator Award (DP2 TR002776), a Burroughs Wellcome Fund Career Award at the Scientific Interface, an NSF CAREER Award (CBET-2145491) and the American Cancer Society (RSG-22-122-01-ET).

Author information

Authors and Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    Ningqiang Gong, Xuexiang Han, Lulu Xue, Margaret M. Billingsley, Xisha Huang, Rakan El-Mayta, Jingya Qin & Michael J. Mitchell

  2. Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA, USA

    Neil C. Sheppard, Carl H. June & Michael J. Mitchell

  3. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Neil C. Sheppard, Carl H. June & Michael J. Mitchell

  4. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  5. Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  6. Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  7. Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  8. Institute for RNA Innovation, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

  9. Center for Precision Engineering for Health, University of Pennsylvania, Philadelphia, PA, USA

    Michael J. Mitchell

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  1. Ningqiang Gong
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Contributions

N.G. and M.J.M. conceived and designed the experiments. N.G., L.X., J.Q. and X.H. performed the experiments. N.G., L.X., J.Q., X.H. and R.E. analysed the data. N.G., M.J.M. and M.M.B. wrote and edited the manuscript. N.C.S. and C.H.J. provided materials and were involved in discussions of the work. M.J.M. supervised the project. All authors discussed the results and commented on the manuscript.

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Correspondence to Michael J. Mitchell.

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N.G. and M.J.M. have filed a patent application related to this study. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 AMD mediates the in vivo disassembly of SiTE.

SiTE was labelled with Cy7 dye (Cy7 was labelled on CD3 Fab) and was i.v. injected to tumour-bearing mice at 0h. Then, PBS, AMD dispersed in PBS or AMD dispersed in 5% polyoxyethylene castor oil was injected at 18 h post-SiTE-Cy7 injection. 6 h later, mice were euthanized and the Cy7 signal in different organs and tumour was measured using IVIS (a-c). d, quantification of the fluorescence in tumour tissues in a-c. Data was shown as mean ± SD (n = 3), statistical differences were analysed using two-tailed unpaired Student’s t-test. e, Half-life of AMD in mouse blood when the AMD is dispersed in PBS or in 5% polyoxyethylene castor oil. When the AMD is dispersed in PBS, the half-life is about 9 hours, which is increased to 24 h when it is dispersed in 5% polyoxyethylene castor oil. f, AMD concentrations in the liver and tumour over time were investigated. g, the distribution of AMD in mouse tumours and major organs after 24h of AMD infusion. h, In order to investigate the clearance of the disassembled SiTE and the AMD, we performed another animal experiment. SiTE-Cy7 was i.v. infused into tumour bearing mice at 0 h. 4 h later, AMD was infused. After 24 h, mice were euthanized and the Cy7 signal distribution in major organs and mouse whole body was observed. Cy7 signal was detected in the spleen, kidneys and bladder. This demonstrates the potential clearance from the kidneys and urine. We then performed a kinetics study to investigate the Cy7 signal (i) and AMD level in the urine (j).

Source data

Extended Data Fig. 2 AMD controls SiTE activity in vivo.

a, 106 E0771-HER2 cells were s.c. injected into the right flank of C57/BL6 mice. When tumour sizes reached 50 mm3 (Day 7), mice were given an i.v. injection of 100 μL of their respective treatments—PBS, SiTE, SiTE + 100 μg AMD, SiTE + 5 μg AMD, or 100 μg AMD (in 5% polyoxyethylene castor oil). One group received SiTE on day 7 followed by 100 μg AMD at day 13. All mice were euthanized at day 23. b, Images of mice from all treatment groups on day 23. c, tumour growth curves for different groups. Tumour tissue was isolated on day 23 and observed for immune cell infiltration. d, repeat of the tumour growth inhibition experiment. e, mouse body weight change during the tumour inhibition experiment. f, Percentages of CD45+, CD45+CD3+, CD45+CD3+CD4+/CD8+ cells in the tumour tissues. g, Immunofluorescence images of the tumour tissues in different groups with cell nuclei labeled with DAPI (blue) and T cells labeled with anti-CD3 antibody (green). Scale bar: 100 μm. The statistical significance of tumour volume in c and e was analysed by two-tailed unpaired Student’s t-test. **P = 0.0013 ****P<0.0001. The statistical significance displayed in f was analysed by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 3 T-cell infiltration levels in the tumour tissues before and after SiTE or AMD treatment.

The mice were s.c injected with 106 E0771-HER2 cells at day 0, SiTE was injected at day 7, 9, and day 11 and AMD was injected at day 13 a, The tumour tissues at day 6, day 12 and day 14 were collected, digested, and filtered, and the immune cell infiltration in the tumour tissue was analysed using flow (flow gating strategy is shown in b). c, d, and e are flow dot plots of CD45+, CD45+CD3+, CD45+CD3+CD4+ or CD45+CD3+CD8+ cells, respectively. f-i are quantifications of c-e, respectively. The statistical significance of tumour volume in f-i was analysed by two-tailed unpaired Student’s t-test. f,***P = 0.0007, **P = 0.0027. g, ***P = 0.0005, *P = 0.0324. h, ***P = 0.0006, *P = 0.0351. i, ***P = 0.0005, *P = 0.0089.

Source data

Extended Data Fig. 4 High dose of SiTE elicited antigen-specific immune response to tumours.

C57BL/6 mice with liver expression of HER2 were injected with PBS, SiTE low dose, SiTE high dose, or AMD at days 7, 9 and 11. AMD was administered to SiTE-treated groups once severe toxicity was observed. Mice were euthanized at day 17, and the tumour tissues were collected and analysed by flow cytometry. a, mouse weight during the treatment. b, flow gating strategy. c, CD44+CD62L+ central memory cells in the tumour tissue.

Source data

Extended Data Fig. 5 High doses of SiTE enhance DC maturation and antibody production in vivo.

a-c, E0771-HER2 cells were s.c. injected to mice at day 0. E0771-HER2 tumour-bearing mice were treated with low doses (1 mg/kg) or high doses (5 mg/kg) of SiTE at days 7, 9, and 11. AMD was infused at day 13. Mice were euthanized at day 13 and the dendritic cell maturation levels in the tumour draining lymph nodes were evaluated. PBS or AMD-only were used as two control groups. d-f, In order to investigate the humoral immune response induced by SiTE, E0771-HER2 tumour-bearing mice were treated with low doses (1 mg/kg) or high doses (5 mg/kg) of SiTE at days 7, 9, and 11. AMD was infused at day 13. At day 27, the HER2-specific total IgG, IgG2c, and IgG1 levels in mouse blood were determined. P values were indicated in a-c, analysed by two-tailed unpaired Student’s t-test. n = 3.

Source data

Extended Data Fig. 6 Tumour-cell-rechallenging experiment.

Tumour-free mice from the high dose group were rechallenged with E0771-HER2 or E0771 cells and compared to mice pre-treated with either PBS or AMD challenged with E0771-HER2 as controls. Group R1: Healthy mice were treated with PBS at days −43, −41, and −39. The mice were i.v. injected with E0771-HER2 cells at day 0; Group R2: E0771-HER2 tumour-bearing mice were treated with a high dose of SiTE at days −43, −41, and −39, AMD was i.v. injected at day −37. The tumour-free mice were rechallenged with E0771-HER2 cells at day 0; Group R3, E0771-HER2 tumour-bearing mice were pre-treated with high dose of SiTE at days −43, −41, and −39. AMD was i.v. injected at day −37. The mice were rechallenged with E0771-HER2 cells at day 0. Group R4, healthy mice were treated with AMD at day −37 and the mice were i.v. injectied with E0771-HER2 cells at day 0. b-e are individual E0771-HER2 or E0771 tumour size curves in the different treatment groups. CR, complete regression (n = 8). The mice were euthanized, and the immune cell infiltration in the tumour tissue was analysed using flow. f-h, Treg percentages, CD44+CD62L+ central memory T cell percentages, and CD44+CD62L− effector memory T cells in different treatment groups, respectively. Data were shown as mean ± SD, n = 4.

Source data

Extended Data Fig. 7 High doses of SiTE generate tumour-specific T-cell immune responses.

E0771-HER2 tumour-bearing mice were treated with high doses of SiTE. 7 days-post the last dose, T cells were collected from the mice and were co-cultured with E0771-HER2 cells (express luciferase) for 24h. E0771-HER2 cell viability was determined (a). b and c, E0771-HER2 target cells (labelled with low level of CSFE) and reference cells (B16 cell line, labelled with high level of CSFE) were i.v. infused into the high dose SiTE-treated mice mentioned above. 24 later, target cell killing was determined using flow cytometry (b). c, quantification of b. Data in a and c are shown as mean ± SD, n = 4. P values in a and c was determined using two-tailed unpaired student’s t-test.

Source data

Extended Data Fig. 8 Proteomics shows that a high dose of SiTE treatment induces damage-associated molecular patterns (DAMPs) and tumour-antigen release.

E0771-HER2 cells were incubated with mouse T cells in serum-free medium and were treated with a low dose (5 ng/mL) or a high dose (20 ng/mL) of SiTE. After 24h, the supernatant was collected and the proteins in the medium were analysed. a, relative abundance of various DAMPs released to cell culture medium in the high dose SiTE, low dose SiTE or PBS-treated group. b, and c, tumour antigen HER2 and tumour neoantigen hmmr and srrm1 abundance in the cell culture medium in different groups.

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Extended Data Fig. 9 AMD reduces the on-target off-tumour toxicity of SiTE in vivo.

a, A humanized immune system mouse model was constructed by treating the mice with an i.p. injection of Busulfan at day −50 followed by an i.v. injection of 105 human CD34+ foetal liver cells at day −49. Human CD19 antigen was expressed in the livers of the mice using a piggyBac transposon system delivered via lipid nanoparticles (LNPs) encapsulating two plasmids, pCMV-hyPBase and pPB CMV-hCD19:T2A:EGFP, at day −35. At day −14, 106 Raji-Luc-GFP tumour cells were i.v. injected. Before using Blinatumomab or SiTE for cancer treatment, Tocilizumab (10 mg/kg) was administered to mice to prevent CRS-related symptoms. PBS, Blinatumomab in vivo bio-similar antibody (5 mg/kg), or SiTE (5 mg/kg) were i.v. injected every two days for three total doses. When an approximate 15% decrease in body weight (indicative of toxicity) was observed on day 7, half of the mice that had received SiTE were given an i.v. injection of AMD (in 5% polyoxyethylene castor oil). Throughout the study, mice were euthanized when body weight decreased more than 20%. IVIS was used to monitor tumour burden in vivo. b, IVIS images of the mice at days 0, 7, 10, and 30. c, and d, Mouse body weight and survival curves, respectively. n = 10 mice. The red stars in b and c indicate that mice were euthanized. e-h, Measurements for markers of toxicity and inflammation including AST, ALT, TNF-α, IFN-γ levels in mouse blood at different time points. n = 10 mice. i, In order to evaluate liver toxicity induced by various treatments, an additional animal experiment was performed and mouse livers were harvested at day 9. H&E staining was conducted to detect liver damage in different groups. Scale bar: 100 μm. j, Immunofluorescence imaging of CD3+ T cells in liver tissue. Blue: DAPI; Red, CD3+ T cells. k, tumour-free mice from the SiTE+AMD group were rechallenged with 106 Raji-Luc-GFP cells and the tumour burden post Raji-Luc-GFP cell rechallenging was monitored with IVIS. l-m, T cells in the tumour-free mice from the SiTE+AMD group were sorted and were co-cultured with Raji-Luc-GFP cells for 24 h and tumour cell viability was determined using a luciferase assay kit (l). T cells from normal humanized mice co-cultured with Raji-Luc-GFP cells were used as a control group. m, IFN-γ concentrations in the cell culture medium were determined using an ELISA kit. The data in e-h were plotted as mean ± s.d. (n = 10) from three independent experiments. P values indicated in e-h were analysed by two-tailed unpaired Student’s t-test. Pink, Blinatumomab vs SiTE + AMD at day 9; blue, SiTE vs SiTE + AMD at day 9; ***P = 0.0003, ****P<0.0001. P value in d was determined using Log-rank (Mantel-Cox) test, ****P<0.0001. P values in l and m were determined by two-tailed unpaired Student’s t-test, ****P<0.0001, ***P = 0.0006.

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Extended Data Fig. 10 AMD reduces SiTE-induced neurotoxicity in vivo.

a, NSG-SGM3 mice were treated with Busulfan at day −50 to remove mouse bone marrow. At day −49, 105 human CD34+ foetal liver cells were i.v. injected to allow the mouse develop a human immune system. 106 Raji-Luc-GFP tumour cells were i.v. injected at day −14. At days 1, 3, and 5, PBS or SiTE was i.v. injected (in the PBS group, only PBS was injected into tumour-bearing mice). When an approximate 15% decrease in body weight (indicative of toxicity) was observed on day 7, half of the mice that had received SiTE were given an i.v. injection of AMD (10 mg/kg) and half of them were given Tocilizumab (10 mg/kg) for CRS treatment. b, c, d, and e, Mouse body weight, temperature, IL-6 levels, and mouse survival curves, respectively. n = 10 mice. The red stars in b indicate that mice were euthanized. f, Measurements of IL-1 in mouse blood at different time points. n = 10 mice. At around day 33 post-SiTE injection, humanized NSG-SGM3 mice that received either PBS or Tocilizumab treatment developed paralysis (g) or experienced a seizure as indicated by movement along the red arrows (h), which are signs of lethal neurological syndrome. However, mice treated with AMD did not develop paralysis and were not observed to experience seizures indicative of lethal neurological syndrome. Brain H&E staining (i) and human CD68 immunohistochemistry (j) images of mice at day 35. The data in b-f were plotted as mean ± s.d. (n = 10) from three independent experiments. P value in f was determined using Log-rank (Mantel-Cox) test, ****P < 0.0001.

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Gong, N., Han, X., Xue, L. et al. Small-molecule-mediated control of the anti-tumour activity and off-tumour toxicity of a supramolecular bispecific T cell engager. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-023-01147-6

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