Generation of a safe and efficacious llama single-domain antibody fragment (vHH) targeting the membrane-proximal region of 4-1BB for engineering therapeutic bispecific antibodies for cancer

Abstract

Background: The discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%-30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003).

Methods: To determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further.

Results: PM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo.

Conclusions: A unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

Keywords: antibody specificity; antigens; differentiation; tumor microenvironment.

Figure 1

Characterization of anti-4-1BB antibody (HZ-L-Yr-16-Fc). Binding activity of HZ-L-Yr-16-Fc to human 4-1BB (A) or cyno 4-1BB (B) overexpressing CHO cells as revealed by flow cytometry analysis. (C) Binding of HZ-L-Yr-16-Fc to OX40, GITR, CD40, 4-1BB determined by ELISA. (D) Effect of indicated anti-4-1BB antibodies blocking 4-1BB ligand binding to 4-1BB measured by flow cytometry. (E) Purified T cells from human PBMC were stimulated with plated anti-CD3 and indicated anti-4-1BB antibodies. Five days later, IFN-γ in the culture medium was analyzed by ELISA. (F) Purified T cells from human PBMC were stimulated with plated anti-CD3 and indicated anti-4-1BB or anti-CD28 antibodies in solution. Five days later, IFN-γ in the culture medium was analyzed by ELISA. Data are mean±SD, ***p<0.001.

Figure 2

Detailed interaction sites between the 4-1BB and HZ-L-Yr-16 and epitope comparison between different antibody agonists. (A) Overall structure of the 4-1BB: HZ-L-Yr-16 complex. 4-1BB is shown in green; HZ-L-Yr-16 is shown in cyan. (B) The interactions between 4-1BB and HZ-L-Yr-16. Hydrogen bonds are shown as black dashed lines. (C) Epitope comparison of HZ-L-Yr-16 (in cyan), Urelumab (yellow) and Utomilumab (orange). 4-1BBL (in magenta) is also aligned for comparison.

Figure 3

Detailed interaction sites between PD-L1 and HZ-C-Ye-18. (A) Overall structure of the PD-L1:HZ-C-Ye-18 complex (PDB: 7CZD). PD-L1 is shown in magenta, while HZ-C-Ye-18 is shown in purple. The CDR1, CDR2 and CDR3 domains of HZ-C-Ye-18 are colored green, cyan and yellow, respectively. (B) The interactions between PD-L1 and HZ-C-Ye-18. Hydrogen bonds are shown as black dashed lines.

Figure 4

Structure of PM1003 and the binding activity of the bispecific antibody to both 4-1BB and PD-L1. (A) Structural representation of PM1003. (B) Binding of PM1003 to human PD-L1 overexpressed on CHO cells measured by flow cytometry. (C) Binding of PM1003 to human 4-1BB overexpressed on CHO cells measured by flow cytometry. (D) PM1003 blocks the interaction between PD-1/PD-L1 determined by flow cytometry. (E) PM1003 simultaneous binding to both human PD-L1 and human 4-1BB as determined by ELISA. (F) PM1003 simultaneous binding to both human PD-L1 and human 4-1BB overexpressing cells as determined by a cell bridging flow cytometry assay.

Figure 5

4-1BB agonism via PM1003 is dependent on crosslinking via PD-L1-mediated cross-bridging. (A) PM1003 activity of Jurkat-hu4-1BB-NF-κB luciferase cells incubated with CHO-huPD-L1 cells or PD-L1-negative CHO cells. (B) PM1003 activity in a human primary T cell activation assay with non-transfected CHO cells. (C) PM1003 activity in a human primary T cell activation assay with CHO-huPD-L1 cells. (D) PM1003 activity in a mixed lymphocyte reaction with monospecific components and in combination.

Figure 6

In vivo efficacy of PM1003 in CT-26-huPD-L1 and MC38-huPD-L1 mouse model. (A) Human PD-1/PD-L1/4-1BB triple knock-in mice were injected subcutaneously with 1×10⁶ CT-26-huPD-L1 tumor cells, then molar equivalent doses of indicated antibodies were administrated on days 7, 9, 11. Tumor size was measured every other day. (B) Images of tumors (left) and tumor weight from mice (right). (C) Individual tumor growth spaghetti plots. Data are mean±SEM, *p<0.05, **p<0.01. (D) Dose-dependent efficacy and long-term immune memory induction of PM1003 were tested in a MC38-huPD-L1 mouse model. Human PD-L1/4-1BB double knock-in mice were injected subcutaneously with 1×10⁶ MC-38-huPD-L1 tumor cells, then different doses of the indicated antibodies were administrated on days 7, 9, 11. Tumor size was measured every other day after the first treatment. (E) Tumor-free mice were re-challenged by injection of 1×10⁶ MC-38-huPD-L1 tumor cells subcutaneously. Tumor size was measured every other day after the first treatment and on rechallenge.

Figure 7

PM1003 was tested for liver pathology in a 4-1BB knock-in C57 mouse model. (A) Experimental protocol for the toxicity model. Human 4-1BB knock-in C57 mice (n=4 or 5/group) were treated with 10 mg/Kg Urelumab, 6.67 mg/Kg PM1003 or PBS on days 0, 5, 10 and 15. 7 days after the final treatment, (B) liver weight, (C) spleen weight, (D) serum ALT activity, (E) liver tissue HE staining, (F) liver tissue CD8-positive T cell IHC staining and (G) percentage of CD8-positive cell infiltration, were measured. Data are mean±SEM, **p<0.01, ***p<0.001.