A highly selective humanized DDR1 mAb reverses immune exclusion by disrupting collagen fiber alignment in breast cancer

Abstract

Background: Immune exclusion (IE) where tumors deter the infiltration of immune cells into the tumor microenvironment has emerged as a key mechanism underlying immunotherapy resistance. We recently reported a novel role of discoidin domain-containing receptor 1 (DDR1) in promoting IE in breast cancer and validated its critical role in IE using neutralizing rabbit monoclonal antibodies (mAbs) in multiple mouse tumor models.

Methods: To develop a DDR1-targeting mAb as a potential cancer therapeutic, we humanized mAb9 with a complementarity-determining region grafting strategy. The humanized antibody named PRTH-101 is currently being tested in a Phase 1 clinical trial. We determined the binding epitope of PRTH-101 from the crystal structure of the complex between DDR1 extracellular domain (ECD) and the PRTH-101 Fab fragment with 3.15 Å resolution. We revealed the underlying mechanisms of action of PRTH-101 using both cell culture assays and in vivo study in a mouse tumor model.

Results: PRTH-101 has subnanomolar affinity to DDR1 and potent antitumor efficacy similar to the parental rabbit mAb after humanization. Structural information illustrated that PRTH-101 interacts with the discoidin (DS)-like domain, but not the collagen-binding DS domain of DDR1. Mechanistically, we showed that PRTH-101 inhibited DDR1 phosphorylation, decreased collagen-mediated cell attachment, and significantly blocked DDR1 shedding from the cell surface. Treatment of tumor-bearing mice with PRTH-101 in vivo disrupted collagen fiber alignment (a physical barrier) in the tumor extracellular matrix (ECM) and enhanced CD8⁺ T cell infiltration in tumors.

Conclusions: This study not only paves a pathway for the development of PRTH-101 as a cancer therapeutic, but also sheds light on a new therapeutic strategy to modulate collagen alignment in the tumor ECM for enhancing antitumor immunity.

Keywords: antibodies, neoplasm; breast neoplasms; collagen; immune reconstitution.

Figure 1

Generation and characterization of rabbit anti-hDDR1 mAbs. (A) A cartoon showing the process to generate rabbit anti-hDDR1 mAbs. Rabbits were immunized with hDDR1 ECD (catalog no. 10730-H08H, SinoBiological) five times at 3-week intervals. Individual memory B cells were isolated 2 weeks after the last boost and the B cells were cultured for antibody expression. ELISA was used to screen the individual memory B cell cultures for hDDR1 ECD targeting antibodies. The VH and VL fragments were cloned from memory B cells that were ELISA-positive. The converted rabbit IgG₁ was recombinantly expressed in HEK293F cells. (B) DDR1 mAbs screened using T cell migration assay. E0771-hDDR1 was cultured for 48 hours. Conditioned media was transferred into the bottom chamber of the transwell and co-incubated with mAbs at 37°C for 1 hour. After adding splenocytes to the upper chamber, migration was performed at 37°C for 2 hours. The migratory splenocytes were collected and quantified by flow cytometry. (C) Rabbit mAb9 was assessed by quantifying migrated CD8⁺ T cells with E0771-hDDR1 conditioned media. The results are shown as percentage normalized with isotype IgG treatment and means±SD of duplicates. (D) Binding of rabbit mAb9 (20 µg/mL) to ECDs of hDDR1, mDDR1 and hDDR2 as determined by ELISA. OD₄₅₀ values are means±SD of triplicates. (E) Binding affinity of rabbit mAb9 to hDDR1 ECD as determined by biolayer interferometry assay (BLI). (F) A cartoon showing the study design of tumor-inhibiting efficacy of the mAbs in vivo. C57BL/6 mice were injected with a mixture of rabbit mAbs and E0771-hDDR1 tumor cells on day 0, followed by intratumoral mAb injections on days 4, 6, 8, 10, 12, 14, and 16. Two mAb dose levels (5 and 10 mg/kg) were assessed. A rabbit IgG₁ isotype produced in-house was used as a control. (G) Tumor volume was measured every 4 days from day 10 after E0771-hDDR1 tumor cells inoculation. The values are shown as means±SD of duplicates. The statistical significance of the difference between 10 mg/kg of mAb treatment groups was labeled as red, purple for 5 mg/kg of mAb treatment groups. DDR1, discoidin domain-containing receptor 1; ECD, extracellular domain; mAbs, monoclonal antibodies; VH, heavy chain’s variable region; VL, light chain’s variable region.

Figure 2

Humanization of rabbit mAb9 and characterization of humanized mAb PRTH-101. (A) A cartoon showing the combined KABAT/IMGT/Paratome CDR grafting strategy to humanize rabbit mAb9. (B) The heavy chain and light chain of rabbit mAb9 are shown as mAb9_vH and mAb9_vL (blue). The heavy chain and light chain of the best-matched human antibody frameworks are shown as IGHV3-33*07 and IGKV1-12*01 (red). The humanized heavy chain and light chain are shown as PRTH-101_vH and PRTH-101_vL. (C) Binding affinity of PRTH-101 to hDDR1 ECD and mDDR1 ECD as determined by BLI. (D) Binding of rabbit mAb9 and PRTH-101 to hDDR1 ECD as determined by dose-response ELISA. The antibody was serially diluted threefold from 10 µg/mL. The values are shown as means±SD of triplicates and EC₅₀ was calculated by fitting a nonlinear regression (four parameter). (E) Binding of rabbit mAb9 and PRTH-101 at 10 µg/mL to the ECDs of hDDR1 and hDDR2 as determined by ELISA. OD₄₅₀ values are means±SD of triplicates. DDR1, discoidin domain-containing receptor 1; ECD, extracellular domain.

Figure 3

Epitope mapping of DDR1 interaction with PRTH-101 by mutagenesis and HDX-MS. (A) Schematic diagram showing the construction of hDDR1 ECD fragments fused to a His₆-tag. DS refers to the F5/8 type C domain (residues 31–185) of hDDR1 ECD. DSL refers to the DS-like (DSL) domain (residues 192–367) of hDDR1 ECD. EJXM refers to the short extracellular juxtamembrane linker (residues 368–417). (B) Binding of PRTH-101 to hDDR1 ECD, ΔDS, and ΔDSL as determined by dose-response ELISA. PRTH-101 was threefold serially diluted from 10 µg/mL. The values are shown as means±SD of triplicates and EC₅₀ was calculated by fitting a nonlinear regression (four parameter). (C) Alignment of hDDR1 and hDDR2 DSL domains by ESPript 3.0. As PRTH-101 binds to hDDR1, but not to hDDR2 ECD, nine hDDR1 ECD mutants were constructed by replacing hDDR1 regions with the corresponding hDDR2 sequences, and the mutation sites are boxed in blue. β strands are indicated by green arrows. (D) The binding of PRTH-101 to hDDR1 ECD and nine mutants in the DSL domain as determined by ELISA with a fixed concentration of PRTH-101 at 10 µg/mL. OD₄₅₀ values are means±SD of quadruplicates. The difference between two groups was calculated by two-tailed t-test. (E) Significant H/D exchange differences after PRTH-101 binding mapped to a DDR1 crystal structure (PDB 4AG4, 1 hour labeling time point). A strong reduction in exchange in presence of PRTH-101 was seen in the DSL domain (dark blue stretch in front view, main epitope). Smaller effects were observed throughout the DSL domain, suggesting additional allosteric effects caused by PRTH-101 binding (back view). No significant changes were detected in the collagen-binding DS domain (top view). Regions with no sequence coverage are colored in gray. (F) Deuterium uptake plots of representative DDR1 peptides. Error bars reflect the standard deviation (n=3). DDR1, discoidin domain-containing receptor 1; DS, discoidin; EJXM, extracellular juxtamembrane; HDX-MS, hydrogen-deuterium exchange mass spectrometry.

Figure 4

Epitope mapping of DDR1 interaction with PRTH-101 by X-ray crystallography. (A) Structure of DDR1-DSL in complex with PRTH-101 at 3.15 Å resolution. DDR1-DSL is shown in white, the Fab light chain in cyan, and the Fab heavy chain in magenta. N-linked sugars are displayed as sticks. (B) Close-up view of the DDR1 epitope, and interfacing CDRs from PRTH-101. (C, D) N-linked glycosylation sites on the DDR1 surface. Electron density for a single sugar at N211 (C) and a branched chain at N260 (D) are displayed. (E) The view of PRTH-101 CDRs from the heavy chain (HC, CDR_H1-3) and light chain (LC, CDR_L1-3) in complex with DDR1-DSL (transparent surface). Side chains are labeled and colored as in panel A. (F) Close-up of PRTH-101 CDRs from the heavy chain interacting with DDR1-DSL. The hydrogen bonds are displayed as dashed yellow lines. (G) Close-up of PRTH-101 CDRs from the light chain interacting with DDR1-DSL. The hydrogen bonds are displayed as dashed yellow lines. (H) The DDR1-DSL residues critical for binding of PRTH-101. DDR1, discoidin domain-containing receptor 1; DSL, discoidin-like.

Figure 5

Functional characterization of PRTH-101 using DDR1 phosphorylation, collagen adhesion, and DDR1 shedding assays. (A) Induction of pDDR1 in T47D cells with collagen (50 µg/mL) treatment. Vinculin and pDDR1 were detected in cell lysates by JESS technology. Individual capillaries run in the same experiment are shown. (B) Digital representation of data obtained for pDDR1 and Vinculin detection with different concentrations of PRTH-101 treatment. T47D cells were pre-incubated for 2 hours in presence of PRTH-101 or IgG control at different concentrations. Then collagen I was added at a final concentration of 50 µg/mL and pDDR1 was measured by JESS after 90 min of stimulation. Samples from the same experiment were run in individual capillaries. (C) Representation of IC₅₀ determination using data from at least two individual experiments (two replicates per condition). The IC₅₀ value was determined as best fit of data by nonlinear regression analysis. (D) Total DDR1 expression was measured by JESS with same method described in panel B. (E) PRTH-101 inhibited adhesion of HEK293 cells overexpressing hDDR1 to collagen I-coated plates. IC₅₀ was determined by nonlinear regression analysis of cell adhesion assay data from at least three individual experiments (four replicates per condition). (F) A cartoon showing the process of sandwich ELISA to detect shed DDR1. The generation of shed DDR1 from E0771-hDDR1 (G), T47D (H) and MCF-7 (I) cell cultures was inhibited by PRTH-101 in a dose-dependent manner. The IC₅₀ values were calculated as best-fit values of shed DDR1 data from three replicates. DDR1, discoidin domain-containing receptor 1.

Figure 6

The antitumor activity of PRTH-101 in C57BL/6 mice. (A) A cartoon showing the process of testing the antitumor activity of PRTH-101 in vivo. Mice were injected with E0771-hDDR1 cells in the mammary fat pad. Antibodies were intratumorally injected on day 12 every other day when the tumors reached about 100 mm³. (B) The tumor growth curve after mice were treated with IgG control and PRTH-101. The values were shown as means±SD. The difference between two groups was calculated by two-tailed Student’s t-test. n (hIgG)=7, n (PRTH-101) = 8. (C) Representative images of tumor margin and core analyzed by TO-PRO-3 staining (red), SHG (gray), and collagen fiber individualization (far right panel). Scale bar: 50 µm. (D) Quantification of collagen fiber length in tumor margin and core. The values were shown as means±SD. The difference between two groups was compared by two-tailed t-test. n (hIgG)=4, n (PRTH-101) = 4. (E) Coefficient of variation of collagen fiber in tumor margin and core are quantified. The difference between two groups was calculated with two-tailed t-test. n (hIgG)=4, n (PRTH-101) = 4. (F) Representative IHC images of CD8⁺ T cells in tumor margin and core of antibody-treated tumors. Scale bar: 50 µm, scale bar (inlet): 20 µm. An area on the tumor side with a depth of 400–600 µm from the tumor-stroma border was defined as tumor margin, which is indicated by the red dashed line. (G) Quantification of CD8⁺ T cells in tumor margin and core for antibody-treated tumors. The difference between two groups was calculated by two-tailed t-test. n (hIgG)=7, n (PRTH-101)=6. IHC, immunohistochemistry; ns, not significant; SHG, second harmonic generation.

Figure 7

A model showing multiple mechanisms of PRTH-101 contributing to antitumor activity. Top: Collagen fiber binds to DDR1 homodimer on the cell surface and induces its oligomerization. Simultaneously, downstream DDR1 signaling and shedding of DDR1 ECD are activated. The shed DDR1 facilitates collagen fiber alignment and immune exclusion. Bottom: PRTH-101 prevents oligomerization of DDR1 homodimer to block downstream signaling and DDR1 ECD shedding. The low level of shed DDR1 is captured by PRTH-101 and the collagen fiber alignment is disrupted which results in immune cell infiltration. DDR1, discoidin domain-containing receptor 1; ECD, extracellular domain.