Vaccine-induced CD8 T cells are redirected with peptide-MHC class I-IgG antibody fusion proteins to eliminate tumor cells in vivo

Figures & data

Figure 1. (a) Schematic design of the pMHCI-IgG (top) and the TCB fusion proteins (bottom). Both formats consist of a full monoclonal antibody in an IgG format (light green: antibody light chain, dark green: antibody heavy chain variable domain and CH1, gray/black: constant domain of the antibody heavy chain in the knob and hole format or with charge mutations to drive heterodimerization). A single pMHCI complex consisting of the antigenic peptide (magenta), MHC class I heavy chain lacking the transmembrane domain (purple) and beta-2-microglobulin (blue) or a single anti-CD3 binding CrossMab Fab (crossed heavy and light chain in yellow and red) are fused to one of the antibody heavy chains. (b) Frequency of IFN-γ expressing CD8 T cells measured in flow cytometry after exposure to compound-treated target cells. Incubation of MC38-FAP tumor cells loaded with MCMV-derived M38 peptide (blue), T cell bispecific TCB-αFAP (green), pMHCI-IgG M38-αFAP (red) or pMHCI-IgG IE3-αFAP (pink) and unloaded tumor cells (cyan) with splenocytes from M38 vaccinated mice. Splenocytes were isolated nine days after start of vaccination. All graphs show mean of replicates (n = 2) with error bars indicating standard deviation. (c) Specific tumor cell lysis mediated by compounds after incubation with splenocytes from M38 vaccinated mice. Number of viable cells is measured with xCELLigence as electrical impedance mediated by adherent cells on the culture dish bottom. Color code: IE3-αFAP (purple), M38 peptide (blue), TCB-αFAP (green), M38-αFAP (red). Kinetics of cell lysis at a compound concentration of 25 nM is shown (left). Tumor cell elimination after 40 hours is shown for all concentrations tested (right). All graphs show mean of replicates (n = 3) with error bars indicating standard deviation. Two-sided t-test for significance evaluation was applied. P-values from 0.01 to 0.05 were considered as significant (*), p-values from 0.001 to < 0.01 were considered as very significant (**) and p-values < 0.001 were considered as extremely significant (***). (d) Relative expression of endogenous MHC class I compared to pMHCI-IgG delivered recombinant MHC class I. FAP expressing B16 and MC38 tumor cells were loaded with Ova257-264 peptide (light green) or pMHCI-IgG Ova257-264-αFAP (dark green) and SIINFEKL peptide-MHC class I complexes were detected with the monoclonal antibody anti-Ova257-264 – mouse H-2Kb antibody (25-D1.16) in flow cytometry

Figure 2. Experimental lung metastasis model

(a) Schedule for the preventive (purple) and therapeutic (green) treatment setting: The overall timeline is depicted in blue. Intravenous injections of the compounds are shown as purple or green stars in each setting. The duration of the vaccination is shown as a red arrow. Blood sampling is illustrated with a red drop. Orange points indicate intravenous injection of 2 × 10⁵ B16-FAP melanoma cells. Orange crosses demonstrate euthanasia of mice and isolation of lungs for assessment of metastatic burden. (b) Metastatic burden after preventive treatment. Visible lung metastases were counted after harvest of lungs 21 days after injection of the tumor cells. M38 vaccinated (green) and non-vaccinated (blue) mice were either treated twice with vehicle (PBS, green triangles/blue hashes), TCB-αFAP (green squares/blue inverted triangles) or M38-αFAP (green dots) 24 hours before injection of tumor cells and 2 days after. All graphs show median per group (n = 6–10) with error bars indicating interquartile range of counted lung metastasis (left). All graphs show box plots with median of qPCR quantification of melanoma cell marker TRP-2 with whiskers indicating maximum and minimum value (right). (c) Metastatic burden after therapeutic treatment. Visible lung metastases were counted after harvest of lungs on day 21 of metastasis growth (left). TRP-2 expression was quantified by qPCR (right). M38 vaccinated (green) and non-vaccinated (blue) mice were treated twice with either vehicle (PBS, green triangles/blue hashes), TCB-αFAP (green squares/blue inverted triangles), IE3-αFAP (green hexagons) or M38-αFAP (green dots). Treatment started 9 days after injection of B16-FAP melanoma cells. All graphs show median per group (n = 8–10) with error bars indicating interquartile range of counted lung metastasis (left). All graphs show box plots with median of qPCR quantification of TRP-2 with whiskers indicating maximum and minimum value (right). For statistical analysis, Wilcoxon-test was used. P-values from 0.01 to 0.05 were considered as significant (*), p-values from 0.001 to <0.01 were considered as very significant (**) and p-values < 0.001 were considered as extremely significant (***).

Figure 3. Bright field and immunofluorescent staining of lungs containing metastases derived from either murine FAP-transfected (right) or non-transfected (left) B16 melanoma cells at day 21 after injection of tumor cells. Pigmented B16 cells can be recognized in the bright field (top row). Murine FAP in cyan blue, cell nuclei in blue (DAPI). In the top row cut ends of cryo-section tissue blocks are depicted so that metastases can be localized in the corresponding stained cryo-sections below (middle row). The close-up images (bottom row) show normal lung tissue and parts of metastases. Red arrows indicate borders of metastases

Figure 4. Solid tumor model

(a) Schedule for the subcutaneous MC38 tumor model. The purple dot indicates the subcutaneous inoculation of 10⁶ MC38-FAP colorectal cancer cells. Duration of the vaccination is depicted with a red arrow. One day after finalization of vaccination, blood was taken and the amount of M38-specific T cells was determined (red drop). Green stars show time points of treatments. The orange cross demonstrates euthanasia of mice and harvest of tumors. (b) Top: Growth kinetics of solid subcutaneous MC38 tumors after treatment with TCB-αFAP (green), M38-αFAP (red), IE3-MHCI-IgG (pink) or vehicle treatment (blue). Treatments started at an average tumor volume of 97 mm³ and were administered i.v. q3dx5 as shown by green arrows. Tumor-free mice per group are indicated on the right. All graphs show median per group (n = 8) with error bars indicating interquartile range. Bottom: Time-to-event analysis of solid subcutaneous MC38 tumors after treatment with TCB-αFAP (green), M38-αFAP (red), IE3-αFAP (pink), or vehicle treatment (blue). Treatments were administered i.v. q3dx5 as shown by green arrows. Group size was n = 8. A tumor volume > 500 mm³ was defined as event.

Figure 5. Immunofluorescent staining of murine FAP in solid tumors derived from either murine FAP-transfected (upper right, lower left, lower right) or non-transfected (upper left) MC38 colorectal cancer cells. Murine FAP-transfected tumors were treated with the TCB (lower left), the pMHCI-IgG (lower right) or untreated (upper right). Cell nuclei are stained with DAPI and depicted in blue. Murine FAP located on the cell surface is depicted in cyan blue

Figure 6. Tumor penetration and accumulation of pMHCI-IgG molecules

(a) Penetration profile of Alexa647-labeled M38-αFAP molecule. Tumors were excised 12 hours (green), 24 hours (blue), and 48 hours (red) after intravenous injection of compound or vehicle (gray). Amount of M38-αFAP molecules in the tumor is depicted in relation to distance to vessel borders. All graphs show mean of different animals per group (n = 4) with error bars indicating standard deviation. (b) Accumulation of Alexa647-labeled M38-αFAP molecules in the tumor. Total amounts of M38-αFAP molecule present in the tumor 12 (green), 24 (blue), and 48 (red) hours after injection of molecule or vehicle (gray) are shown. All graphs show mean of different animals per group (n = 4) with error bars indicating standard deviation. (c) Sections of in vivo stained tumors excised 12 hours after injection of Alexa647-labeled M38-αFAP molecule (left) or vehicle (right). Close-up image of tumor tissue with high penetration of Alexa647-labeled M38-αFAP molecules shows binding of labeled molecule to tumor cells (middle). Color code: Cell nuclei (blue), vessels (red), M38-αFAP (green).

Figure 7. CD8 T cell infiltration in solid MC38-FAP tumors after a single treatment with compounds

(a) CD8 T cells were stained by fluorescently labeled anti-mouse CD8a antibody (green) on histological sections of the MC38-FAP tumors. Cell nuclei were stained with DAPI (blue). M38 vaccination with vehicle treatment (A: upper left), M38 vaccination with TCB-αFAP treatment (A: upper right), M38 vaccination with IE3-αFAP treatment (A: middle left), M38 vaccination with M38-αFAP treatment (A: middle right), and non-vaccinated and non-treated (A: lower left) are shown. (b) Quantification of CD8 T cells in solid MC38-FAP tumors after a single treatment with compounds. Area covered by CD8 T cells in relation to tumor area is depicted for all groups. All graphs show mean of different animals per group (n = 4) with error bars indicating standard deviation.

Figure 8. Flow cytometry analysis of T cells and tumor cells from M38 vaccinated and pMHCI-IgG or TCB-treated mice

(a) CD44 (top left), CD62L (top right), CD127 (bottom left), and PD-1 (bottom right) expression of M38-specific (left column each marker) and nonspecific (right column each marker) CD8 T cells isolated from solid MC38-FAP tumors (top row each marker) and peripheral blood (bottom row each marker) 48 hours after the second treatment. Color code for treatment: Not vaccinated/vehicle (purple – markers only detectable for unspecific CD8 T cells in the blood, no T cells detectable inside the tumor mass), M38 vaccinated/vehicle (dark green), M38 vaccinated/TCB-αFAP (light green), M38 vaccinated/IE3-αFAP (orange), M38 vaccinated/M38-αFAP (blue), isotype control antibody (red). (b) Flow cytometry analysis for CD25 (left) and FoxP3 (right) of CD4 T cells in tumor samples 48 hours after the second treatment. Color code for treatment: M38 vaccinated/vehicle (dark green), M38 vaccinated/TCB-αFAP (light green), M38 vaccinated/IE3-αFAP (orange), M38 vaccinated/M38-αFAP (blue), isotype control antibody (red). (c) Flow cytometry analysis of tumors, which escaped pMHCI-IgG or TCB therapy. Tumors of the M38 vaccinated and vehicle-treated (left), M38 vaccinated and TCB-αFAP treated (middle), and M38 vaccinated and M38-αFAP treated (right) groups were analyzed for PD-L1 expression on the tumor cell surface. Color code: Autofluorescence of cells (blue), isotype control antibody (orange), anti-PD-L1 antibody (red).