Dual checkpoint blockade of CD47 and PD-L1 using an affinity-tuned bispecific antibody maximizes antitumor immunity

Abstract

Background: T cell checkpoint immunotherapies have shown promising results in the clinic, but most patients remain non-responsive. CD47-signal regulatory protein alpha (SIRPα) myeloid checkpoint blockade has shown early clinical activity in hematologic malignancies. However, CD47 expression on peripheral blood limits αCD47 antibody selectivity and thus efficacy in solid tumors.

Methods: To improve the antibody selectivity and therapeutic window, we developed a novel affinity-tuned bispecific antibody targeting CD47 and programmed death-ligand 1 (PD-L1) to antagonize both innate and adaptive immune checkpoint pathways. This PD-L1-targeted CD47 bispecific antibody was designed with potent affinity for PD-L1 and moderate affinity for CD47 to achieve preferential binding on tumor and myeloid cells expressing PD-L1 in the tumor microenvironment (TME).

Results: The antibody design reduced binding on red blood cells and enhanced selectivity to the TME, improving the therapeutic window compared with αCD47 and its combination with αPD-L1 in syngeneic tumor models. Mechanistically, both myeloid and T cells were activated and contributed to antitumor activity of αCD47/PD-L1 bispecific antibody. Distinct from αCD47 and αPD-L1 monotherapies or combination therapies, single-cell RNA sequencing (scRNA-seq) and gene expression analysis revealed that the bispecific treatment resulted in unique innate activation, including pattern recognition receptor-mediated induction of type I interferon pathways and antigen presentation in dendritic cells and macrophage populations. Furthermore, treatment increased the Tcf7⁺ stem-like progenitor CD8 T cell population in the TME and promoted its differentiation to an effector-like state. Consistent with mouse data, the compounds were well tolerated and demonstrated robust myeloid and T cell activation in non-human primates (NHPs). Notably, RNA-seq analysis in NHPs provided evidence that the innate activation was mainly contributed by CD47-SIRPα but not PD-L1-PD-1 blockade from the bispecific antibody.

Conclusion: These findings provide novel mechanistic insights into how myeloid and T cells can be uniquely modulated by the dual innate and adaptive checkpoint antibody and demonstrate its potential in clinical development (NCT04881045) to improve patient outcomes over current PD-(L)1 and CD47-targeted therapies.

Keywords: antibody affinity; immunity; immunotherapy; innate; tumor microenvironment.

Figure 1

Design and characterization of αCD47/PD-L1 bispecific antibody. (A) The schematic diagram of the human αCD47/PD-L1 bispecific antibody (hBisAb). (B) Workflow of common light chain antibody campaign for the generation of hBisAb. (C, D) Cell based binding of hBisAb on CHO-hCD47 (C), and CHO-hPD-L1 (D), as measured by flow cytometry. (E) In vitro blocking activity of hBisAb on the human CD47/SIRPα interaction as measured by flow cytometry. (F) In vitro blocking activity of hBisAb on the human PD-L1/PD-1 interaction using a PD-L1/PD-1 TCR blocking reporter bioassay. The blocking activity was quantified and normalized as the fold change compared with isotype control. (G) Phagocytosis of NCI-H292 human tumor cells by human monocyte-derived macrophages in the presence of human IgG isotype control, hBisAb in IgG₁ or IgG₄ format (n=2 donors/group). Phagocytosis of total tumor cells is represented as fold change compared with isotype treatment. (H) MLR assay was conducted to assess concentration of IL-2 at 72 hours in the supernatant by ELISA. A mixture of LPS matured DCs and purified CD4⁺ T cells were cocultured at a 1:4 ratio in the presence of αhCD47, αhPD-L1, the combination of αhCD47 and αhPD-L1, or hBisAb at 200 nM. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA. ADCP, antibody-dependent cellular phagocytosis; ANOVA, analysis of variance; DC, dendritic cell; IL-2, interleukin 2; MLR, mixed lymphocyte reaction; LPS, lipopolysaccharide; TCR, T cell receptor.

Figure 2

The binding selectivity of αCD47/PD-L1 bispecific antibody (A, B) The binding selectivity of hBisAb on tumor cells and RBCs in vitro. The human tumor cell line HT-1080 and RBCs were stained for CD47 and PD-L1 expression by flow cytometry (A). CSFE-labeled HT-1080 tumor cells were cocultured with RBCs in the presence of indicated antibodies and binding selectivity was measured as the percentage of antibody-bound cells per cell type by flow cytometry (B). All experiments were repeated at least twice. (C) Cell-based binding of hBisAb on human tumor cell line HT-1080 and human RBCs in vitro as measured by flow cytometry. (D) Cell-based binding of mBisAb on murine tumor cell line MC38 and murine RBCs in vitro as measured by flow cytometry. (E) The binding selectivity of the αCD47/PD-L1 mouse surrogate mBisAb in vivo. Human FcγR mice bearing subcutaneous B16F10 tumors were dosed IP on days 13 and 16 post-tumor inoculation with αmCD47, αmPD-L1, or mBisAb in human IgG isotype. Blood and tumor tissues were collected at day 18 and therapeutic antibody binding was assessed using an anti-human IgG secondary antibody by flow cytometry (n=3 mice/group).CFSE, carboxyfluorescein succinimidyl ester; hBisAb, human bispecific antibody; IP, intraperitoneally; RBC, red blood cell; TILs, tumor-infiltrating leucocytes.

Figure 3

The therapeutic effect of αCD47/PD-L1 mouse surrogates in syngeneic tumor models. (A–C) Antitumor efficacy, survival and body weight post treatment of indicated antibodies in CT26 tumor model. BALB/c mice bearing subcutaneous CT26 tumors were dosed IP with 5 mg/kg of mIgG2a isotype, 5 mg/kg of αmCD47, 5 mg/kg of αmPD-L1, the combination of 5 mg/kg of αmCD47 and 5 mg/kg of αmPD-L1, or 5 mg/kg of mBisAb on days 0, 3, and 7 post-treatment initiation (n=7–8 mice/group). (A) Tumor growth; significance was calculated by two-way ANOVA with Tukey’s multiple comparison test. (B) Kaplan-Meier survival curves post-treatment initiation. (C) Percent of initial body weight reported as a mean±SEM; significance was calculated by two-way ANOVA with Tukey’s multiple comparison test compared with isotype control. (D) BALB/c mice were dosed IP on day 0 and 3 with the same concentration in (A–C). Red blood cells from whole blood on day 5 were counted (n=6–7 mice/group). Significance was calculated by ordinary one-way ANOVA compared with control. (E) Antitumor efficacy in B16F10 tumor model. C57BL/6 mice bearing subcutaneous B16F10 tumors were treated on 9 days post-inoculation with isotype control or mBisAb three times a week for 3 weeks (n=10 mice/group). Significance was calculated by two-way ANOVA with Sidak’s multiple comparison test. (F) Dose-dependent antitumor efficacy in MC38 tumor model. C57BL/6 mice bearing subcutaneous MC38 tumors were treated 7 days postinoculation and dosed IP every 3–4 days for 3 weeks with 10, 20, and 40 mg/kg of mBisAb (n=10 mice/group). Significance was calculated by two-way ANOVA with Tukey’s multiple comparison test. (G–I) Antitumor efficacy following mBisAb treatment and immune cell depletion in MC38 model. MC38-bearing mice were dosed with vehicle (control) or mBisAb every 3–4 days for a total of six doses. Depletion antibodies were administrated IP 1 day prior to treatment with mBisAb and dosed three times a week throughout the study. (n=8–10 mice/group). Significance was calculated by two-way ANOVA with Tukey’s multiple comparison test. (J) C57BL/6 wildtype (WT) and Batf3^-/- mice bearing subcutaneous MC38 tumor were dosed IP every 3–4 days for 3 weeks with mBisAb (n=8 mice/group). Significance was calculated by two-way ANOVA with sidak’s multiple comparison test. Asterisks indicate statistical significance (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). ANOVA, analysis of variance; αmPD-L1, anti-mouse PD-L1 antibody; αmCD47, anti-mouse CD47 antibody; mBisAb, αCD47/PD-L1 mouse surrogate; IP, intraperitoneally.

Figure 4

Immunopharmacodynamics of αCD47/PD-L1 mouse surrogate treatment by CyTOF and Nanostring (A–F) MC38-bearing mice were treated with isotype (control) or mBisAb every 3–4 days starting at day 10 post-tumor implantation, and tumors and spleens were harvested at day 15, 20, or 25 and prepared for analysis by CyTOF (n=5 mice/group). (A) ViSNE analysis of total T cells from the spleen of control (left) or mBisAb (right)-treated mice at day 20 post-tumor implantation. Indicated subsets were defined by biaxial gating and position of cells in each subset on the viSNE is represented by a unique color. (B) Expression of indicated markers among total splenic T cells, with each cell in the viSNE colored according to its MMI (mean marker intensity) value of the indicated marker. (C, D) Frequency of CX3CR1⁺ (C) or KLRG1⁺ (D) cells among total CD8⁺ T cells in the spleens of control (black) or mBisAb-treated (blue) mice at the indicated time points. *P<0.05, **p<0.01, Mixed-effects analysis. (E) ViSNE analysis of total T cells from tumors of control or mBisAb-treated mice at day 20 post-tumor implantation. Indicated subsets were defined by biaxial gating and position of cells in each subset on the viSNE is represented by a unique color. (F) Expression of indicated markers among total intratumoral T cells, with each cell in the viSNE colored according to its MMI value of the indicated marker (G, H) Frequency of CD8⁺ T cells (G) or Tregs (H) among total T cells in control (black) or mBisAb-treated (blue) tumors at the indicated timepoints. *P<0.05, **p<0.01, Mixed-effects analysis. (I–M) MC38-bearing mice were treated with control or mBisAb every 3–4 days starting at day 10 post-tumor implantation. Tumors were harvested at day 20 and prepared for gene expression analysis by Nanostring. Relative RNA abundance of effector CD8⁺ T cell signature (I) and antigen processing machinery in IMvigor 210 dataset (J). Relative RNA abundance of cxcl9 (K), cxcl10 (L), and ccl5 (M). *P<0.05, ****p<0.0001, Ordinary one-way ANOVA with tukey’s multiple comparison test. ANOVA, analysis of variance; CyTOF, cytometry by time of flight.

Figure 5

αCD47/PD-L1 mouse surrogate treatment reprograms myeloid populations and drives innate activation in tumor microenvironment (A, B) MC38-bearing mice were treated with the indicated antibodies every 3–4 days starting at day 10 post-tumor implantation. Tumors were harvested at day 20 and prepared for gene expression analysis by Nanostring. Volcano plots show log2 fold change of gene expression in the indicated treatment groups with DEGs compared with the control group (A). REACTOME pathway analysis in the indicated treatment groups compared with the control group (B). (C–K) MC38 tumor-bearing mice were treated with the same condition in (A) and tumor tissues were harvested for gene expression analysis using scRNA-seq. (C) tSNE analysis of intratumoral CD45⁺ cells from all treatment conditions indicating six distinct clusters of monocyte/macrophage cells. (D) Heatmap displaying the average expressions of the selected DEGs for each cluster of monocyte/macrophage cells relative to other clusters. (E) Single-cell trajectory analysis by Monocle2 of the indicated populations. (F) Relative expression across treatment groups for selected genes that are differentially expressed among Cluster 2 Top2a⁺ TAM in mBisAb-treated vs control-treated tumors. (G) Bar graph showing selected hits from GO pathway analysis performed on genes upregulated in myeloid clusters from mBisAb-treated tumors relative to the controls. (H) tSNE analysis of intratumoral CD45⁺ cells from across all treatment conditions, cropped on 3 clusters of DCs. (I) Heatmap displaying the expression of the selected DEGs for each cluster of DC relative to other clusters. (J–K) Relative expression across treatment groups for selected genes that are differentially expressed in mBisAb-treated vs control-treated tumor DC clusters. DC, dendritic cell; DEG, differentially expressed gene; GO, gene ontology; IFN, interferon; MHC, major histocompatibility complex; scRNA-seq, single-cell RNA sequencing; TAM, tumor-associated macrophage; tSNE, t-distributed stochastic neighbor embedding.

Figure 6

Impact of αCD47/PD-L1 mouse surrogate treatment on intratumoral and splenic T cell compartments (A–K) scRNA-seq samples in figure 5C–K were analysed for CD8⁺ T cells. (A) tSNE analysis of intratumoral CD45⁺ cells from across all treatment conditions, cropped on CD8⁺ T cells, showing six distinct clusters of intratumoral CD8⁺ T cells. (B) Heatmap displaying the expression of the top 20 DEGs for each cluster of intratumoral CD8⁺ T cells relative to all other CD8⁺ T cell clusters. Each column represents an individual cell in the indicated cluster, and each row represents an individual gene. (C) Frequency of each intratumoral CD8⁺ T cell cluster among total CD45⁺ cells in each treatment condition. (D) Relative average expression of selected genes that are differentially expressed among cluster 1 CD8⁺ T cells in mBisAb-treated versus control-treated tumors, shown across all treatment conditions. (E) Selected hits from TopGO pathway analysis performed on genes differentially expressed by cluster 1 CD8⁺ T cells from mBisAb-treated tumors relative to controls, with -log(10)p value for each pathway among genes differentially expressed by αmCD47, αmPD-L1, or combination-treated tumors relative to controls shown for comparison. No bar indicates that pathway was not significant for the given gene set. (F) Violin plots depicting the relative representation of the KAECH_NAIVE_VS_DAY8_EFF_CD8_TCELL_DN geneset in cluster 1 intratumoral CD8⁺ T cells across treatment conditions. (G) Heatmap displaying the average expression of selected genes that are differentially expressed among each splenic CD8⁺ T cell clusters relative to all other CD8⁺ T cell clusters. (H) Frequency of proliferating and effector splenic CD8⁺ T cell clusters among total CD45⁺ cells in spleens from control, αmCD47, αmPD-L1, combination, and mBisAb-treated mice. *P<0.05; **P<0.01, Student’s unpaired t-test. GO, gene ontology; IFN, interferon; ns, not significant; TNF, tumor necrosis factor.

Figure 7

Non-human primate study with human αCD47/PD-L1 bispecific antibody (hBisAb) treatment. (A, B) Cynomolgus monkeys received vehicle control and 10, 30, and 100 mg/kg of hBisAb on day 1 and 8 (n=1–3 monkey/group). The peripheral blood was collected at the indicated timepoint for RBC (A) and platelet numbers (B) in response to the treatment. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test. (C, D) Representative flow cytometry plots of CD25⁺ CD4⁺ and CD69⁺ CD8⁺ T cell populations from NHP peripheral blood on day 8 after receiving vehicle control or 100 mg/kg of hBisAb. (E–H) The NHP PBMCs from peripheral blood were collected with the indicated timepoints after receiving vehicle control or 100 mg/kg of hBisAb on day 1 and 8 for RNA-seq analysis (n=2–3 monkey/group). (E) Selected DEGs associated with innate and adaptive immune pathways in vehicle-treated versus hBisAb-treated samples across all the timepoints during a 2-week period. (F) IPA pathway analysis of the RNA-seq from hBisAb-treated compared with vehicle-treated group on 6 hours post-second dose on day 8. (G, H) LM22 gene signature scores (relative to baseline) of activated DCs and M1 macrophages in vehicle (Veh) vs hBisAb (Ab)-treated group on 0, 6, and 24 hours post-second dose (2D). (I, J) The peripheral blood from cyno received 100 mg/kg of hBisAb and hBisAb2 with reduced CD47 affinity on day 1 and 8 (n=2–3 monkey/group) was collected for RNA-seq analysis. The molecule affinity and enriched IPA pathway scores (I) as well as the selected DEGs (J) expression profiles are listed for the comparison of the two antibodies. 2D, two dimensional; ANOVA, analysis of variance; DC, dendritic cell; DEG, differentially expressed gene; NHP, non-human primate; PBMC, peripheral blood mononuclear cell; RBC, red blood cell.