Superior tumor control by bispecific AcTaferon with high safe profile

Here, we aim to investigate whether simultaneous targeting of type I IFN activity towards Clec9A and PD-L1 can induce antitumor efficacy (Fig. 1A-B). In first instance, we evaluated the antitumor potential in different mouse tumor models including B16 (Fig. 1C-D) as well as 4T1 mammary carcinoma, both subcutaneously (s.c.) (Suppl. Fig. 1A-B) and orthotopically implanted (Suppl. Fig. 1C-D). Administration of the bispecific Clec9A-PDL1-AFN didn’t affect body weight, body temperature nor analysed blood parameters in the 4T1 model (data not shown). Treatment with the Bisp-AFN resulted in B16 tumor stasis comparable to wild type (wt) IFN (Fig. 1C-D). However, wt IFN caused severe body weight loss (Fig. 1E), a decrease in body temperature although not statistically significant (Fig. 1F), and affected all blood parameters monitored (Fig. 1G, Suppl. Fig. 1E) causing anemia, lymphopenia, leukopenia and platelet destruction, which resulted in high mortality. In sharp contrast to this, treatment with Bisp-AFN was very well tolerated.

Fig. 1

Tumor control by Bispecific AcTaferon shows ample potential and is completely safe. A Design and layout of the Bispecific AcTaferon (AFN) construct. Targeting domains are N-terminally connected to the mutated cytokine. A HIS-tag was added for easy purification. B Schematic representation of the experimental setup. Mice were s.c. inoculated with 6 × 105 B16 tumor cells. When palpable tumor was detected (day 7 after tumor inoculation) mice were treated 10 times with a perilesional administration with PBS (grey), 30 μg of the Bisp-AFN Clec9A-PDL1-AFN (green) or 30 μg of the Clec9A-PDL1-IFN wild type (red) according to the indicated arrows above the timeline. C, D Figures show tumor growth (C) as well as time to reach a B16 tumor volume of 150 mm3 (D). One representative experiment out of three is shown (6/group/experiment). E–F Weight change (E) and body temperature (F) are depicted at day 14 after B16 tumor inoculation. Values are relative to day 7, representing the start of the treatment schedule. The graphs show individual values ± SEM of 3 independent experiments (6/group/experiment). G One day after the last treatment in the B16 model, blood was collected, and hematological analysis (Hemavet) was performed. Leukocyte analysis is shown as a summary of individual values ± SEM of three independent experiments (6/group/experiment). H Schematic representation of the experimental layout using a Humanized Immune System (HIS) mouse model. Mice were s.c. inoculated with RL tumor cells. When palpable tumor was detected mice were treated with a perilesional administration with PBS (grey) or 30 μg of the fully humanised Bisp-AFN Clec9A-PDL1-AFN (green) according to the indicated arrows above the timeline. Intraperitoneal administration of Flt3L is indicated by the blue arrows underneath the timeline. I-J Figures show tumor growth (I) as well as time to reach an RL tumor volume of 150 mm3 (J) in an HIS setting. K The graph shows RL tumor growth in absence of a human immune system (Non-HIS NSG mice). One representative experiment out of two is shown (5–6/group/experiment). Tumor growth (C) was analyzed using Two-way ANOVA with Tukey’s multiple comparisons test. For the HIS/non-HIS model (I, K), tumor growth was analyzed at day 24 using unpaired two-tailed student t-test. Black lines underneath the X-axis depict the treatment time. Time to reach a specific tumor size (D, J) is represented in a Kaplan Meier plot compared by log-rank (Mantel-Cox) test. Bar plots (E, F, G) were analyzed using One-way ANOVA Kruskal–Wallis test with Dunn’s multiple comparisons test. * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001

To bring the strategy closer to clinical application, we additionally tested our findings in a humanized setting. To that end, both Humanized Immune System (HIS) mice and irradiated non-HIS NSG mice were inoculated with RL cells, a human non-Hodgkin B cell lymphoma (Fig. 1H). The antitumor potential of the humanized Bisp-AFN was favorable and resulted in delayed tumor growth after therapy termination (Fig. 1I-J). Absence of a response in non-HIS NSG mice indicated the need for an active immune system, rather than the direct anti-proliferative effects of type I IFN on the tumor (Fig. 1K).

AcTaferon therapy combined with doxorubicin leads to tumor cure, therapeutic immunity and abscopal systemic effects

As evidenced in the cancer immunity cycle, establishment of durable antitumor immunity results from a cyclic process, which can be fueled at different levels using strategic interventions [20]. Combined treatment with immunogenic chemotherapeutics might be an interesting strategy (Fig. 2A), as we have shown before [10]. Combination of Bisp-AFN with a non-curative dose of doxorubicin (doxo) resulted in promising effects. In the B16 melanoma tumor model, which is considered to be non- or low-immunogenic, we observed a cure rate of 30% (6/20) (Fig. 2B, E). The outcome was even more striking in the 4T1 mammary carcinoma model. In the s.c. model, we observed a 100% cure (Fig. 2C, F). Also in an orthotopic setting, which mimics the biological and metastatic tumor cell properties observed in clinical cancer patients [21], promising cure rates were observed (70%) (Fig. 2D, G).

Fig. 2

Combination therapy with doxorubicin leads to complete tumor cure. A Schematic representation of the experimental setup. B, E B16 melanoma cells were s.c. inoculated. Results show a summary of three independent experiments (6–8/group/experiment). C, F 4T1 mammary carcinoma cells were s.c. inoculated. Shown is one experiment (n = 6). D, G Orthotopic 4T1 mammary carcinoma model. Results show a summary of two independent experiments (6/group/experiment). Tumor growth progression is depicted for the individual mice in each group (B-D). Kaplan Meier graphs (E–G) show tumor free mice. When palpable tumors were detected, mice were treated with PBS (grey), doxo (black), 30 μg of Bisp-AFN Clec9A-PDL1-AFN (green) or a combination of Clec9A-PDL1-AFN with doxo (blue). Black lines underneath the X-axis depict the treatment time, while orange arrows indicate s.c. administration of doxo. Kaplan Meier plots depicting % tumor free mice (E–G) were analyzed using log-rank (Mantel-Cox) test with * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

In clinical settings, there is often an urgent need for adequate therapies against fast growing and fully developed tumors. Hence, we analyzed the antitumor potential of AFN and doxo therapy in large established tumors (Fig. 3A). Bisp-AFN with doxo could suppress and diminish tumor growth even after the treatment was stopped, resulting in prolonged survival (Fig. 3B-C). In addition, 20% were cured upon treatment with Bisp-AFN plus doxo.

Fig. 3

Combination therapy with doxorubicin leads to therapeutic immunity and systemic effects. A Schematic representation of the experimental setup. Treatment was started when large established tumors were reached (120–150 mm3). B, C Figures show 4T1 tumor growth of individual mice in each group (B) as well as survival (C) (one experiment was performed with n = 12/group). D Schematic representation of the experimental setup for a two-site tumor model. Primary tumors were inoculated at the right flank followed by a second tumor inoculation at the contralateral flank. Primary tumors were p.l. treated whereupon systemic effects of local treatment were analyzed on disseminated non-treated tumors (left flank). E The graphs show s.c. 4T1 tumor volume of individual values ± SEM at day 18 (n = 5). F Tumor growth curves of both treated (upper panel) and non-treated (lower panel) tumors of individual mice using the s.c. 4T1 mammary carcinoma model. Mice were treated with PBS (grey), doxo (black), 30 μg of Bisp-AFN Clec9A-PDL1-AFN (green) or a combination of Clec9A-PDL1-AFN with doxo (blue). Black lines underneath the X-axis depict the treatment time, while orange arrows indicate s.c. administration of doxo. Kaplan Meier plots depicting % survival (C) were analyzed using log-rank (Mantel-Cox) test. Bar plots (E) were analysed using One-way ANOVA Kruskal–Wallis test with Dunn’s multiple comparisons test (treated tumors) or using One-way ANOVA followed by bonferroni’s multiple comparison test (non-treated tumors) dependent on Shapiro–Wilk test for normal distribution of the data. * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001

Encouraged by these results, we set up a 2-site tumor model to analyze abscopal effects and systemic therapeutic immunity (Fig. 3D). Strikingly, treatment with doxo plus Bisp-AFN resulted in complete cure of the treated tumor and prolonged growth inhibition of the non-treated 4T1 tumor (Fig. 3E-F).

These results demonstrate that antitumor immune effects elicited by local immunomodulation upon treatment with Bisp-AFN in combination with non-curative doses of doxorubicin can induce systemic effects and affect tumor growth at distant sites.

Induction of immunological memory by combined treatment of Bispecific AcTaferon with doxorubicin

Since combined therapy with doxo resulted in complete tumor eradication (Fig. 2A-G), we could evaluate the potential immunological memory of the treatments in the cured mice (Fig. 4A). Despite that only few tumors were cured in the B16 model (Fig. 2B, E), 100% (6/6) protective immunity was observed upon treatment with doxo plus the Bisp-AFN (Fig. 4B-C). In the s.c. 4T1 model, 80% (4/5) immunological memory was achieved in mice that had been cured following doxo plus Bisp-AFN treatment (Fig. 4D-E). The one individual that was not protected showed delayed tumor development and growth (Fig. 4D). Finally, in the 4T1 orthotopic model, we observed 100% (6/6) immunological memory that was achieved in mice that had been cured upon doxo plus Bisp-AFN treatment (Fig. 4F-G).

Fig. 4

Induction of immunological memory upon combined treatment with doxorubicin. A Schematic representation of the experimental layout. Tumor-free mice over the different experiments were re-challenged s.c. in the contralateral flank on day 25–30 after eradication of the primary tumor. For each tumor model, mice without prior tumor inoculation nor treatment, referred to as ‘naive mice’, were included and injected with the indicated tumor cells as a control for disease progression. B-E Mice cured from an s.c. primary tumor were s.c. re-challenged with 6 × 105 B16 cells (B-C) or 105 4T1 cells (D-E). F-G Mice in which the orthotopically implanted primary 4T1 tumor was completely cured were re-challenged s.c. with 105 4T1 cells. Figures show tumor growth measured of individual mice in each group (B, D, F) as well as % tumor free mice (C, E, G). Kaplan Meier graphs depicting % tumor free mice (C, E, G) were analyzed using log-rank (Mantel-Cox) test. * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001

These data indicate the strong curative potential of Bisp-AFN and doxorubicin treatment, independent of the histological origin of the tumor.

Role of IFN signaling and PD-L1 expression

In contrast to the bispecific Clec9A-PDL1-AFN, administration of Clec9A-PDL1-huIFN, which cannot signal in mouse cells, hardly showed any antitumor efficacy against B16 melanoma (Fig. 5A), in the 4T1 mammary carcinoma s.c. (Fig. 5B) or orthotopic (Fig. 5C) model. These data were additionally confirmed by administration of Clec9A-PDL1, a bispecific control construct without an IFN moiety (Fig. 5A-C). Altogether, these findings indicate that the pure tethering effect might be insufficient and that IFN signaling is key for the robust antitumor efficacy. Next, we analyzed antitumor responses in B16-mCD20-IFNAR−/− tumors, lacking a functional IFN-α and -β Receptor subunit 1 (IFNAR1). Potent antitumor responses were observed with the Bisp-AFN (Fig. 5D), indicating that IFN signaling in tumor cells is not needed for the antitumor effect upon administration of the BiSp-AFN. These data suggest that IFN signaling in immune cells rather than direct intrinsic and anti-proliferative effects on tumor cells is key for the observed effects. In contrast to IFNAR expression by tumor cells, PD-L1 expression by the tumor cells was needed for the antitumor efficacy of Bisp-AFN (Fig. 5E). These results were confirmed in a tumor antigen-specific proliferation assay using gp100 in a B16 melanoma model. To that end, CFSE labeled CD8+ T cells carrying a TCR specifically recognizing the melanoma differentiation antigen gp100 were adoptively transferred into B16 melanoma bearing mice. After treatment, dilution of CFSE was analyzed in draining LN. Proliferation was significantly impaired in the B16-PD-L1−/− tumor model following Bisp-AFN therapy (Fig. 5F).

Fig. 5

Role of IFN signaling and PD-L1 expression. A-C Tumor cells were s.c. or orthotopically inoculated. Tumors were p.l. treated with PBS (grey), 30 μg of the Bisp-AFN (green), 30 μg of the bispecific construct carrying an in mouse non-functional human IFN (blue) or 30 μg of the Clec9A-PDL1 construct, lacking an IFN moiety (dark grey). Figures show tumor growth in the s.c. B16 model (A) (one representative experiment out of three, 6mice/group/experiment), s.c. 4T1 tumor model (B) (n = 6) and the orthotopic 4T1 tumor model (C) (n = 6). D Tumor growth of B16-mCD20-IFNAR−/− tumors p.l. treated with PBS (grey) or Bisp-AFN (green). Shown is a summary of two independent experiments (6/group/experiment). E Tumor growth of B16-PD-L1−/− tumors p.l. treated with PBS (grey) or Bisp-AFN (green). Shown is a summary of two independent experiments (6/group/experiment). F Flow cytometry analysis of gp100-specific CD8+ T cell proliferation in draining LN of B16 or B16-PD-L1−/− tumor-bearing mice p.l. injected with 30 μg Clec9A-PDL1-AFN at days 7 and 9 after tumor inoculation. One day prior to immunization, gp100-specific CD8+ T cells (pMel) were adoptively transferred in B16 tumor-bearing mice. Data show percentages of gp100-specific CD8.+ T cells that have undergone at least one division. Tumor growth (A-C) was analyzed using ANOVA with Tukey’s multiple comparisons test. Black lines underneath the X-axis depict the treatment time. Bar charts show individual values with mean ± SEM. Two-tailed unpaired t test was performed. * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001

Besides the importance of co-localized interferon signaling in immune cell populations, we additionally suggest a role for PD-L1 expression by the tumor, which contributes to the strong and potent antitumor potential of Bisp-AFN treatment.

Single cell RNA sequencing indicates key shifts in both lymphoid and myeloid cells in the tumor microenvironment

To obtain a comprehensive understanding of the immune cell heterogeneity in the tumor microenvironment as well as effects induced by AcTaferon treatment, we performed single cell RNA sequencing (scRNAseq) on B16 tumor samples (Fig. 6A). Uniform Manifold Approximation and Projection (UMAP) for dimension reduction visualization was performed on the CD45+ living immune cell population. Cells from the various treatment conditions were pooled in a single dataset (Fig. 6B) whereupon the origin of each cell was visualized in a color-coded UMAP and linked to the different treatment conditions (Fig. 6C). Clusters were identified based on detection of differentially expressed (DE) genes (Fig. 6D, Suppl. Fig. 2, Suppl. Table 1). Based on these data, we could provide detailed information on the immune cell composition of B16 tumors (Suppl. Fig. 3). In addition, these scRNAseq data revealed key shifts in several immune cell compartments.

Fig. 6

scRNAseq reveals key shifts in both lymphoid and myeloid cells in the tumor microenvironment. A Schematic representation of the scRNA sequencing experiment. Data obtained upon administration of PBS are represented in grey while data obtained after Clec9A-PDL1-AFN administration are visualized in green. B, C UMAP visualization showing the overall CD45+ living cell population in B16 tumors (B) as well as for the individual treatment groups (C). D Annotation plot showing differentially expressed (DE) genes in the X-axis to determine the different clusters (Y-axis). The size of the dot indicates the number of cells that express the gene of interest. The color intensity reflects the expression level (red = high, blue = low). E–G Parts of whole graphs showing relative distribution of immune cell types over the different treatments

Therapy with the Bisp-AFN resulted in strong proliferation in the lymphoid compartment (NK cells, NKT cells and T cells) compared to PBS (Fig. 6E). Interestingly, tumors treated with Bisp-AFN showed less regulatory T cells compared to PBS (Fig. 6E).

On the level of APC, different DC subtypes were determined based on expression of DE genes (Suppl. Fig. 4A, Suppl. Table 1). The majority of cDC were detected in the PBS condition rather than in the Bisp-AFN condition (Fig. 6F). As cDC maturation and motility are tightly regulated, we presume that at the time-point of scRNAseq analysis (Fig. 6A) cDC in the Bisp-AFN condition already migrated from the tumor towards the draining LN. Indeed, flow cytometry analysis under the same conditions showed decreased amounts of cDC in B16 tumors after AFN treatment compared to PBS, while an increase was observed in draining LNs (Suppl. Fig. 4B). Besides cDC1, cDC2, migratory DC (migrDC) and pDC, scRNAseq data showed the presence of an additional DC population, which mainly arose in the Bisp-AFN treated condition (Fig. 6F). Based on the expression of Fcgre1, Cd300a, Mafb, Bex6, Dnajc6 and Ly6c2, this population could represent monocyte-derived cells. However, additional genes including Dpp4, Irf8, Sirpa, March1 as well as Rsad2, Iigp1, Stat1, Ifit1, Ifit3 and Ifi205 could be detected (Suppl. Table 1). Identification of these DE genes suggests that these cells could be an inflammatory cDC2 (inf-cDC2) population, as described by Bosteels et al. [22]. Inf-cDC2 arise from cDC2 upon inflammation and share phenotype, gene expression and function with both cDC1s and monocyte-derived cells [22]. Indeed, based on UMAP representation, this inf-cDC2 population is more closely related to cDC1 and cDC2 compared to the cluster of monocytes/macrophages (Fig. 6B-C). While only a negligible amount (6,4%) could be detected in the PBS condition, this inf-cDC2 population was most abundant in the Bisp-AFN condition (93,6%) (Fig. 6F).

Finally, clear differences between the different treatment conditions were detected on other cells of the myeloid compartment (Fig. 6G).

From pro-tumorigenic towards pro-immunogenic TAMs and TANs

It is well known that the tumor microenvironment is a complex heterogeneity of tumor cells, stroma and a variety of infiltrated immune cell types [23]. Among these, tumor-associated neutrophils (TAN) and macrophages (TAM) comprise two noteworthy subtypes. To underscore this, our scRNAseq data in B16 tumors revealed key changes in these subtypes.

Although neutrophils infiltrate in numerous cancer types, only low numbers were detected here. The identified neutrophils did not only almost selectively belong to the Bisp-AFN treated condition (Fig. 6G), they also showed a gene expression profile that is correlated with a pro-immunogenic TAN phenotype (Fig. 7A, Suppl. Fig. 2).

Fig. 7

scRNAseq data on myeloid cells in the B16 tumor microenvironment. A, B Heatmap plots displaying differentially expressed genes related to an immunogenic or tumorigenic phenotype within TAN (A) and TAM (B)

Depending on the immune context and the environmental stimuli, macrophages can adopt extreme phenotypes, ranging from pro-immunogenic TAM to tumorigenic TAMs [24, 25]. Indeed, our scRNAseq data of B16 tumors clearly revealed distinct TAM subsets (Fig. 6G). Remarkably, the vast majority of cells in the pro-immunogenic TAM cluster were derived from the Bisp-AFN group (96%), while most of the cells in the pro-tumorigenic TAM cluster belonged to the PBS group (94,5%). These data were visualized in an heatmap showing pooled clusters with TAM signatures. DE genes linked with pro-immunogenic TAMs are upregulated in the Bisp-AFN treatment, while DE genes specific for pro-tumorigenic TAMs are clearly upregulated in the PBS condition (Fig. 7B).

Together, these data clearly indicate the ability of Bisp-AFN treatment to reshape the suppressive tumor environment and revert the tumor-promoting activities of myeloid cells.

Bispecific AcTaferon induces migration and potent maturation of dendritic cells

cDC1 are considered as critical for antitumor immunity and their abundance within tumors has been associated with immune-mediated rejection and the success of immunotherapy [26]. We analyzed the presence and maturation status of cDC1 18 h after a single administration of either PBS or the Bisp-AFN (Fig. 8A, flow cytometry gating strategy in Suppl. Fig. 5). A significant increased presence of cDC1 in B16 tumors (Fig. 8B) as well as migratory cDC1 in draining LN (Fig. 8C), was observed upon administration of Bisp-AFN. Although not statistically significant, we observed a trend to increased presence of resident cDC1 in the draining lymph nodes (Fig. 8D) upon treatment with Bisp-AFN. In addition, these cDC1 showed a favorable matured phenotype demonstrated by their CD40 expression (Fig. 8B-D).

Fig. 8

Bisp-AFN induces migration and potent maturation of dendritic cells. A Schematic representation of the experiment. Data obtained upon administration of PBS are represented in grey while data obtained after Clec9A-PDL1-AFN administration are visualized in green. B-D Presence and maturation status as determined by CD40 expression of cDC1 in B16 tumor (B) as well as migratory cDC1 (C) and resident cDC1 (D) in draining LN. cDCs were determined as CD45+ living cells, CD11c+MHC-II+. LN migratory cDCs are CD11cintermediateMHC-IIhigh while LN resident cDCs are CD11chighMHC-IIintermediate. cDC1 were determined as CD11b−XCR1.+ cells within the described cDC population. Results show bar charts of individual values (B, C, D) with mean ± SEM. Graphs were analyzed using unpaired nonparametric Mann–Whitney t-test (migratory cDC1 within draining LN) or unpaired parametric t-test. * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001

Bispecific AcTaferon induces a shift from naive and dysfunctional T cells towards effector and reprogrammable CTLs

During cancer progression, CTLs often progress to a dysfunctional or exhausted state due to immune-related tolerance or immune-suppression within the tumor microenvironment [27, 28]. Therefore, we analyzed the status of CD8+ T cells in B16 tumors and draining LN (Fig. 9A-B). Administration of the Bisp-AFN drives T cells from a naive state into T cells with an effector phenotype in both draining LN (Fig. 9C) and tumor (Fig. 9B, D). In addition, in draining LN, CD8+ T cells with a memory phenotype, based on CD44highCD62Lhigh expression, could be increased after treatment with Bisp-AFN (Fig. 9C). Finally, in the PBS condition many tumor-resident CTLs showed a fixed dysfunctional state, while Bisp-AFN therapy resulted in presence of plastic, reprogrammable CTLs (Fig. 9B, E).

Fig. 9

Bisp-AFN induces a shift from naive and dysfunctional T cells towards effector and reprogrammable CTLs. A Schematic representation of the experiment. B Flow cytometry gating strategy to determine CD8+ T cells: CD45+ living cells, TCR-β+ T cells, CD8+CD4− T cells. Contour plots show the shift in expression of the indicated markers and cell populations over the different treatments. C, D Draining LN (C) and B16 tumors (D) were analyzed for CD44 and CD62L expression on CD8+ T cells. Naive cells were identified as CD44lowCD62Lhigh, effector T cells as CD44highCD62Llow and effector-memory T cells in draining LN as CD44highCD62Lhigh. E CD8+ T cells in B16 tumors were analyzed for CD38 and CD101 expression with dysfunctional T cells described as CD38+CD101+ and reprogrammable, plastic T cells as CD38−CD101−. Results show bar charts of individual values with mean ± SEM. Data obtained upon administration of PBS are represented in grey while data obtained after Clec9A-PDL1-AFN administration are visualized in green. Shown is one representative experiment. Graphs were analyzed using unpaired parametric t-test (draining LN naive T cells, tumor effector T cells, tumor dysfunctional and reprogrammable T cells) or unpaired nonparametric Mann–Whitney t-test (draining LN effector T cells, draining LN memory T cells, tumor naive T cells). * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001

Bispecific AcTaferon is a superior inducer of tumor-specific CTLs and promotes diversity in the TCR repertoire

To analyze the antigen-presentation skills of DCs, we performed a proliferation assay showing superior proliferation of gp100-specific T cells after Bisp-AFN treatment (Fig. 10A-C). Moreover, administration of Bisp-AFN resulted in a high fraction of gp100-specific T cells in the latest stages of proliferation (Fig. 10B-C). Induction of tumor-specific cytotoxic T cell responses is a fundamental objective in anticancer therapeutic strategies. Therefore, we analyzed the potency of the induced antigen-specific T cell response. Again, Bisp-AFN induced ample effects (Fig. 10D). In addition, the induced specific lysis inversely correlated with tumor size (Fig. 10E).

Fig. 10

Bisp-AFN is superior in the induction of tumor-specific CTLs. A-C Flow cytometry analysis of gp100-specific CD8+ T cell proliferation in draining LN of B16-bearing mice p.l. injected with PBS (grey) or Bisp-AFN (green) at days 7 and 9 after tumor inoculation. One day prior to immunization, gp100-specific CD8+ T cells (pMel) were adoptively transferred in B16 tumor-bearing mice. Data show percentages of gp100-specific CD8+ T cells that have undergone at least one division (A), stacked bar chart representing the percentage of cells per division peak (B) and representative flow cytometry proliferation profile for each treatment (C). Shown is a summary of two independent experiments (6/group/experiment). D, E B16-bearing mice were p.l. injected at days 7 and 9 after tumor inoculation with the indicated conditions. Five days after the first delivery, the killing potency of the induced tumor-specific CD8+ T cells was analyzed in draining LN by in vivo cytotoxicity assay (D). The correlation between tumor size at time of read out and % specific lysis was plotted (E). A summary of two independent experiments is shown (5–6/group/experiment). F Frequencies of the top-10 largest T cell clones within the top-100 most-abundant TCRB sequences in the draining LN (left) and tumor (right). Note that colors do not represent the same sequence in different bars. G, J Frequency distribution plots of TCRB V/J pairing usage within the top-100 most-abundant LN sequences (G) and tumors sequences that also appear in the LN (J). H, K Average TCRB V/J pairing usage frequencies within the top-100 most-abundant LN sequences (H) and tumors sequences that also appear in the LN (K). Corresponding circos plots for each treatment group are shown next to the heatmaps. I Amount of TCRB sequences from the draining LN that are also retrieved in the matched tumor (left) with relative frequencies of these sequences after treatment with Bisp-AFN. A summary of three mice per group was analysed. Results show bar charts of individual values with mean ± SEM (A, D). An unpaired nonparametric Mann–Whitney t-test (A) or parametric t-test (D) was performed depending on Shapiro Wilk normality test * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001

Next, we evaluated whether these DCs were able to establish diversity in the T cell repertoire, which we addressed via TCR analysis. To that end, B16 tumor-bearing mice were treated with either PBS or Bisp-AFN, after which CD8+ T cells were sorted from draining LNs and tumor for RNA sequencing. In both compartments, increased frequencies of several clones retrieved within the top-10 most-abundant TCRB sequences after treatment with the Bisp-AFN were noticed (Fig. 10F), which validates the findings from the abovementioned proliferation experiments (Fig. 10A-C). We further investigated this by measuring the average frequencies of TCRB V/J pairing usage per group in the LN. While CD8+ T cells isolated from control mice treated with PBS are more enriched in a selected number of relatively larger V/J pairings, the T cell response in Bisp-AFN-treated animals is spread over numerous V/J pairings that appear in lower frequencies, which is indicative of improved epitope spreading in the LN (Fig. 10G-H). Upon comparing matched tissues, we observed that more TCRB sequences initially retrieved in the draining LN also appeared and expanded in the tumor after treatment with Bisp-AFN, imposing a correlation between a durable antitumor response and T cell clone sharing between sentinel LN and tumor (Fig. 10I). In addition, average V/J pairing usage analysis for these particular shared clones reveals more oligoclonal CD8+ T cells expansion for certain TCRB V/J combinations in the tumor after treatment with Bisp-AFN compared with control (Fig. 10J-K).

Altogether, simultaneous targeting of type I interferon towards Clec9A and PD-L1 in a Bisp-AFN construct induces strong tumor-specific immune responses and increases diversity in the CD8+ T cell TCR repertoire without the need for tumor markers, as such representing a potent immunotherapeutic with broad applications.