Discovery and Characterization of a First-in-Class LIV1-TLR7/8 Immunomodulatory Conjugate with Robust Myeloid Activation and Antitumor Activity

Abstract

Herein, we describe the discovery of a novel immunostimulatory drug conjugate (IMC) that employs TLR7/8 agonists conjugated to a tumor-targeting LIV1 antibody. Targeting TLR7/8 agonists to LIV1-expressing tumors enables localized delivery, thereby minimizing systemic toxicity while promoting inflammation and T cell recruitment within the tumor microenvironment (TME) for enhanced antitumor efficacy. Dual activation of TLR7 and TLR8 within the TME facilitates the recruitment of diverse immune cells and induces a broad spectrum of pro-inflammatory cytokines, effectively reshaping the immunosuppressive TME by upregulating costimulatory molecules. The mechanism of action of the IMC involves tumor recognition via surface antigens and Fcγ-mediated phagocytosis, followed by activation of myeloid cells to efficiently present tumor antigens to T-cells, thereby eliciting antitumor immunity. The designed IMCs demonstrate the ability to activate myeloid cells in the presence of tumor cells, display robust antitumor activity, and are well tolerated in toxicology studies.

1

Mechanism of action of the LIV1 immunomodulatory conjugate (IMC) approach.

2

Design and characterization of TLR7/8 payloads. (a) In vitro characterization of TLR7/8 dual agonist payload; (b) X-ray cocrystal structure of 1 with monkey TLR7 and TLR8 (crystal structure accession codes 9mhv and 9mhw for TLR7 and TLR8, respectively); (c) mouse PK profile of compounds 1 and 2; (d) PD effect of compounds 1 and 2 in BALB-c mice.

3

IMC optimization via structure–activity relationship study of linker payload and mode of conjugation. (a) General structure of linker payload variants designed to attach the selected payload 1 to the antibody through a cleavable linker. (b) Two modes of conjugation employed to create the IMC. A maleimide-functionalized linker was introduced to the hinge region by partial reduction of interchain disulfide bonds, while an amine-based linker payload was attached to the C-terminus of light chain via bTGase-mediated conjugation. (c) Structural features of linker payload variants and physicochemical and biochemical characterization data, including plasma stability, payload release efficiency, and hydrophobicity.

4

Characterization of binding and internalization of lead LIV1-IMC (12). (a) Structure of lead LIV1-IMC-bearing linker payload 5. (b) The LIV1 binding was tested using LIV1 + /LIV1 – cell lines by FACS analysis, demonstrating LIV1 specificity and cross-reactivity with the cynomolgus monkey antigen. (c,d) Internalization of the LIV1-TLR7/8 agonist immunomodulatory conjugate (IMC) occurs, comparable to the internalization rate of the unconjugated antibody.

5

In vitro functional characterization of the LIV1-IMCs (12). (a) PBMC cocultured with LIV1-expressing MCF7 or MDA-MB-231 cancer cells at a 10:1 ratio were treated with LIV1-TLR7/8 IMC (top concentration 50 μg/mL, 1:3 serial dilutions) or an equimolar small-molecule TLR7/8 agonist. 24 h later, PD-L1 expression on myeloid cells was assessed by flow cytometry. (b) Fresh PDX tumors cut into thin slices and placed into inserts in 6-well tissue culture plates were incubated with LIV1-TLR7/8 IMC with an Fc active backbone or an isotype control IMC with an Fc active backbone (10 μg/mL). 24 h later, supernatants were used for cytokine analysis, and tumor slices were prepared for gene analysis using a total RNA isolation kit. Graphs show fold change over the corresponding isotype control IMC. IP-10 secretion (LIV1 + tumors (blue, N = 8) vs LIV1 – tumors (black, N = 4)) and Type I IFN response (LIV1 + tumors vs LIV1 – tumors, N = 4 each) post-IMC treatment; (c) representative IHC images of tumors used for the explant study (LIV-1 positive tumor: breast, LIV-1 negative tumor: RCC). (d) PDX tumor slices were treated with LIV1-TLR7/8 IMC with an Fc active or Fc null backbone or the corresponding isotype control IMC. Pro-inflammatory cytokines were measured in the supernatant. Data is presented as fold change over isotype control. A Student’s t-test was used to calculate significance. *p < 0.05.

6

In vivo characterization of the IMCs. (a) LIV1-targeting IMCs consisting of a linker payload 5 demonstrate dose-dependent tumor growth inhibition in the LIV1-CT26 syngeneic model. The IMC was dosed at 0.5, 3, and 10 mg/kg via the IV dosing route, and tumor size was monitored. The unconjugated form of the LIV1 antibody was dosed at 10 mg/kg as a control. (b) Tissues from mice treated with 3 mg/kg LIV1-TLR7/8 IMC or naked antibody were collected at different time points after dosing. IFNα and IP-10 were measured in tumor homogenates and in serum. (c,d) For the IFNAR1 blocking experiments, mice were injected with a single dose of 10 mg/kg of LIV1 TLR7/8 IMC along with four doses of 10 mg/kg of anti-IFNAR1 antibody (Q3Dx4), the first dose prior to LIV1 IMC treatment. The tumor growth inhibitory effect was abrogated by the administration of anti-IFNAR1 antibody, demonstrating that the tumor growth inhibition of the IMC is mediated by the type I interferon pathway (c). Change in cytokine levels in the tumor at 4 h after drug administration (d). A two-way ANOVA and a Student’s t-test were used to calculate significance. ***p < 0.001.

7

Evaluation of LIV1-targeting IMC in nonhuman primates. (a–c) Pharmacokinetic data after drug administration was evaluated using a ligand-binding assay (LBA). Quantitation data for total antibody (a), total conjugate (b), and free payload (c) from two animals treated with 1.2 mg/kg of the IMC are shown. (d,e) Antidrug antibodies (ADA) were observed in all treated animals. IgG ADA titers against the payload (d) and against the IMC (e) are shown. (f,g) At doses of ≥3.6 mg/kg, marked transient increases in cytokines were observed, indicative of a pro-inflammatory response. Levels of IFNa2a and IL6 after administration of 0.6, 1.2, and 3.6 mg/kg and 7.2 mg/kg (for IL6 only) are shown.