体内抗体–ADC点击连接与耐药肿瘤递送

Fig. 1 | Antibody click platform for in vivo ligation of antibody–ADC constructs via bioorthogonal chemistry. a, Co-registered EGFR and HER2 IHC images (top) from the same tumour section and corresponding segmentation masks (bottom) confirm the limited spatial overlap between regions of high protein expression. The DSC value was 0.12. DSC values range from 0 to 1 and quantify how much two regions overlap, with lower values indicating less similarity. Co-registration was repeated two times independently. b, Schematic of the antibody–ADC click strategy. Antibodies are conjugated to TCO or tetrazine (Tz) groups to enable antibody click. Details on the substituents R1 and R2 in the TCO and tetrazine chemical structures are provided in Supplementary Fig. 3. Right, SDS–PAGE confirms the increase in molecular mass after antibody click. This result was repeated for all the experiments involving antibody–ADC click. c, SDS–PAGE of non-clicked (no click; panitumumab plus trastuzumab–tetrazine) and clicked (click; panitumumab–TCO plus trastuzumab– tetrazine) antibody pairs after incubation in mouse serum (n = 2 mice per group and time). d, Cryo-TEM images of single or clicked antibodies. TEM images were obtained for n = 3 of the same conjugate. e, Western blot of EGFR and HER2 protein levels in NCIN87 cells was routinely repeated across experiments. A representative blot is shown. β-Actin was the loading control. f,g, Fluorescence quantification (f; via a plate reader) and confocal microscopy images (g) of NCIN87 cells 3 h after incubation with fluorescently labelled antibody pairs: either non-clicked (panitumumab plus trastuzumab–tetrazine) or clicked (panitumumab–TCO plus trastuzumab–tetrazine). Total fluorescence intensity was normalized to the protein amount and the no-click group (P = 0.01032, n =  5 coverslips per well per group, technical replicates). Data are the mean ± s.e.m.; significance was determined using the Welch two-sample t-test. Panitumumab or panitumumab–TCO were conjugated to Alexa Fluor 488 (green), whereas trastuzumab–tetrazine was conjugated to Alexa Fluor 594 (red). Scale bars, 2 mm (a), 28 nm (d) or 50 µm (g).

Fig. 2 | Antibody click enhances internalization in HER2– cancer cell lines. a, Schematic of immunofluorescence and live-cell imaging experiments. Cells were incubated with the first pair (panitumumab–TCO) for 30 min, followed by the second pair (trastuzumab–tetrazine) for the corresponding time of the analyses. b, Western blot of EGFR and HER2 protein levels in MIAPaCa-2 cancer cells was routinely repeated across experiments. A representative blot is shown. β-Actin was the loading control. c,d, Quantification of fluorescence (c; via a plate reader) and confocal images (d) of MIAPaCa-2 cells after incubation with fluorescently labelled antibodies: either non-clicked (panitumumab plus trastuzumab–tetrazine) or clicked (panitumumab–TCO plus trastuzumab– tetrazine) antibody pairs. Total fluorescence intensity was normalized to the protein amount and no-click group (P = 0.00216, n = 5 coverslips per well per group, technical replicates). Panitumumab and panitumumab–TCO was conjugated to Alexa Fluor 488 (green), whereas trastuzumab–tetrazine was conjugated to Alexa Fluor 594 (red). Data are the mean ± s.e.m.; significance was determined using the Welch two-sample t-test. e, Quantification of trastuzumab–tetrazine–pHrodo fluorescence in EGFRhighHER2– MIAPaCa-2 and MDA-MB-231 cells and in EGFR–HER2– CT26 cells for 24 h after incubation with either non-clicked (panitumumab plus trastuzumab–tetrazine–pHrodo) or clicked (panitumumab–TCO plus trastuzumab–tetrazine–pHrodo) antibody pairs. The results showed an increase in internalization in the click group over time (P < 1.0 × 10−6 for MIAPaCa-2 and MDA-MB-231, and P > 0.9999 for CT26, n = 3 wells per group, technical replicates). Data are the mean ± s.d.; significance between no click and click was determined using two-way analysis of variance (ANOVA). No comparison across cell lines was performed. ‘%’ represents the  phase area confluence of the cells in the well. RCU, relative calibrated units. f, Confocal fluorescence microscopy images of MIAPaCa-2 cells treated with either non-clicked (panitumumab plus trastuzumab–tetrazine) or clicked (panitumumab–TCO plus trastuzumab–tetrazine) antibody pairs and stained for LAMP1, a lysosomal marker. Panitumumab and panitumumab–TCO were conjugated to Alexa Fluor 488 (green), whereas trastuzumab–tetrazine was conjugated to Alexa Fluor 594 (red). Immunofluorescence experiments were repeated three times independently. Scale bars, 40 µm (d,f).

Fig. 3 | Antibody–ADC click enhances tumour uptake and therapeutic efficacy. a,b, PET–CT image (a) and biodistribution (b) of 64Cu-labelled trastuzumab– tetrazine in NCIN87 tumours at 24 h after injection. 64Cu-labelled trastuzumab– tetrazine was administered 24 h after injection of panitumumab–TCO (n =  3 mice, biological replicates). Data are the mean ± s.e.m. c, Left, IHC images of HER2 and EGFR in a bilateral tumour model (highlighted areas in the image to the left) inoculated with A431 (HER2ULEGFRhigh, HER2 IHC of 1+/0; green) and NCIN87 (HER2+EGFRlow, HER2 IHC of 3+; orange) xenografts. Middle, corresponding western blot analyses of HER2 and EGFR protein levels (P = 0.00765 and P = 0.00156, respectively, n = 4 tumour lysates per group, biological replicates). Data are the mean ± s.e.m.; significance was determined using the Welch two-sample t-test. Right, PET–CT analyses show uptake of 64Cu-labelled trastuzumab–tetrazine alone compared with after click with panitumumab–TCO (n = 3 mice per group, biological replicates). 64Cu-trastuzumab–tetrazine was administered 24 h after panitumumab–TCO. PET–CT images were collected 24 h after 64Cu-trastuzumab–tetrazine administration. Scale bars, 250 µm. d, 64Cu-labelled trastuzumab–tetrazine biodistribution at 24 h after injection in the bilateral A431 and NCIN87 tumour model shown in c, comparing 64Cu-trastuzumab–tetrazine single antibody versus panitumumab–TCO plus 64Cu-trastuzumab–tetrazine (click). Antibody– ADC click significantly increased tumour uptake in HER2UL A431 tumours (n = 3–4 mice per group, biological replicates). Data are the mean ±s.e.m.; significance was determined using an unpaired t-test. e, Tumour growth curves in the bilateral A431 and NCIN87 tumour model. T-DXd click (panitumumab–TCO plus T-DXd–tetrazine) improved efficacy compared with no click (panitumumab plus T-DXd) or saline. Group × day, P = 0.0138 (A431) and P = 0.00976 (NCIN87). T-DXd no click × click, P = 0.0501 (A431) and P = 0.1269 (NCIN87). T-DXd– tetrazine (5 mg kg–1, intravenously) was administered 24 h after panitumumab– TCO (5 mg kg–1, intravenously). Data are the mean ± s.e.m.; n = 4–5 mice per tumour type, biological replicates; significance was determined using type III analysis and the emmeans package (v.1.10.7) with Dunnett’s correction for multiple comparisons. f, Responses in individual NCIN87 tumours following treatment with T-DXd, no click (panitumumab plus T-DXd) or T-DXd click (panitumumab–TCO plus T-DXd–tetrazine). Bars represent the fold change in tumour volume at the study end point relative to baseline at the start of therapy (n = 9 mice per group, biological replicates). Illustrations in c adapted from Servier Medical Art (https://smart.servier.com/) under a Creative Commons licence CC BY 4.0.

Fig. 4 | Antibody–ADC click overcomes tumour resistance to T-DXd. a, IHC images (right) of the highlighted area in the PET–CT image (left) of an admixed breast cancer model demonstrating low HER2 expression (IHC 2+) and high EGFR expression. PET–CT images were obtained 24 h after injection of 64Cu-labelled trastuzumab (9.90 ± 0.65%ID ml–1 mean tumour uptake, n = 4 mice, biological replicates). b, Tumour growth curves for the admixed breast cancer model following a single intravenous dose (5 mg kg–1) of saline, panitumumab, T-DXd, T-DXd no click (panitumumab plus T-DXd) or T-DXd click (panitumumab– TCO plus T-DXd–tetrazine). T-DXd click significantly decreased tumour growth compared with monotherapies and no-click controls. Group × day, P = 7.98 × 10−7; T-DXd no click × click, P = 0.0018 (n = 10−14 mice per group, biological replicates). Data are the mean ± s.e.m. c, Kaplan–Meier survival curves of groups in b show that T-DXd click significantly prolongs survival compared with all other groups. log-rank, P = 1.87 × 10−7; T-DXd no click ×  click, P = 1.20 × 10−4 (n = 10−14 mice per group, biological replicates). Ribbons represent 95% confidence intervals. d, HER2 and EGFR IHC imaging (right) was performed routinely across experiments. Images are of the highlighted area in the left image. MIAPaCa-2 pancreatic cancer xenografts are characterized by negative HER2 expression (IHC of 0) and high EGFR levels. PET–CT imaging (left) was performed routinely across experiments 24 h after administration of 89Zr-labelled trastuzumab (9.27 ± 2.42%ID ml–1 mean tumour uptake, n = 4 mice per experiment, biological replicates). e,f, Tumour volume (e) and survival (f) in xenograft-bearing mice following a single intravenous administration (5 mg kg–1) of saline, panitumumab, T-DXd, T-DXd no click (panitumumab plus T-DXd) or T-DXd click (panitumumab–TCO plus T-DXd–tetrazine). e, T-DXd click improved tumour control relative to monotherapies and no-click therapy. Group × day, P = 3.15 × 10−13; T-DXd no click × click, P < 1.10 × 10−323 (n = 8–10 mice per group, biological replicates). Data are the mean ± s.e.m. f, Kaplan–Meier survival analysis of the treatment groups in e. Mice receiving T-DXd click showed significantly extended survival compared with all other groups. log- rank, P = 2.28 × 10−8; T-DXd no click × click, P = 5.26 × 10−5 (n = 8–10 mice per group, biological replicates). Significance was determined using type III analysis and the emmeans package (v.1.10.7) with Dunnett’s correction for multiple comparisons. For survival, the log-rank test and Bonferroni correction for multiple testing were performed. Ribbons represent 95% confidence intervals. Scale bars, 125 µm (a,d).

Fig. 5 | Site-specifically modified antibodies reduce batch variability and liver accumulation and improve biodistribution without compromising efficacy. a, Left, schematic of site-specific antibody click to obtain trastuzumab– ss-tetrazine and panitumumab–ss-TCO. Antibodies were enzymatically modified to add azide groups on Fc glycans, followed by conjugation to either Tz or TCO via azide (N3)-dibenzocyclooctyne (DBCO) reaction. Right, SDS–PAGE to confirm antibody click was repeated for all the experiments. b, Cryo-TEM images of site-specific antibody click (n = 3 of the same conjugate). Scale bars, 28 nm. c, Blot of multiple antibody–TCO conjugate (targeting EGFR, HER2, PD-L1, PSMA and VEGF) click reactions to trastuzumab–tetrazine. Results were repeated for each click conjugate. d, Left, PET–CT image of 64Cu-trastuzumab–ss-tetrazine at 24 h after injection in the bilateral A431 and NCIN87 tumour model. 64Cu-trastuzumab–ss-tetrazine was administered 24 h after panitumumab–ss-TCO. Right, liver uptake of antibody click prepared using site-specific methods was lower than for conjugates obtained through random conjugation (P = 0.0064, n = 4 mice per group). Data are the mean ±  s.e.m.; significance was determined using the Welch two-sample t-test. e, Serum chemistry of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in mice treated with saline (n = 7 mice), no click (panitumumab plus T-DXd, n = 4 mice), random click (panitumumab–TCO plus T-DXd–tetrazine, n = 3 mice) or site-specific click (panitumumab–ss-TCO plus T-DXd–ss-tetrazine, n = 4 mice). Antibodies were administered at 20 mg kg–1. Data are the mean ± s.e.m.; significance was determined using two-way ANOVA. f,g, PET–CT (f) and biodistribution (g) at 120 h after 89Zr-trastuzumab–ss-tetrazine administration in HER2–EGFRhigh MiaPaCa-2 pancreatic xenografts in click (panitumumab–ss-TCO plus trastuzumab–ss-tetrazine), pre-click (panitumumab–ss-TCO reacted with trastuzumab–ss-tetrazine before injection) or control IgG (IgG–ss-TCO plus trastuzumab–ss-tetrazine) groups. Data are the mean ± s.d. (n = 4 mice per group). Significance was determined using an unpaired t-test. h, Tumour growth curves for the MIAPaCa-2 (pancreatic cancer) and admixed breast cancer models following a single intravenous dose (5 mg kg–1) of saline (n = 10 mice per group) or site-specific T-DXd click (panitumumab–ss-TCO plus T-DXd–ss-tetrazine). Group × day, P = 4.53 × 10−14 (MIAPaCa-2) and P = 1.0 × 10−323 (admixed breast cancer) (n = 10 mice per group). Data are the mean ± s.e.m.; significance was determined using type III analysis and the emmeans package (v.1.10.7) with Dunnett’s correction for multiple comparisons.