Characterization of a novel anti-human lymphocyte activation gene 3 (LAG-3) antibody for cancer immunotherapy

Abstract

Lymphocyte activation gene 3 (LAG-3) is expressed on activated T cells, natural killer cells or B cells, and functions to negatively regulate homeostasis of these cells. Anti-LAG-3 antibodies might be useful for antitumor immunotherapy. In this study, we characterized a novel anti-LAG-3 antibody, LBL-007, which was isolated from a human antibody phage display library. LBL-007 was found to specifically bind to human LAG-3 antigen, but not to human CD4 or mouse LAG-3. LBL-007 bound activated T cells and promoted interleukin-2 secretion. LBL-007 internalization efficacy by endocytosis into different cells was better than that of another anti-LAG-3 antibody, relatlimab analog. Moreover, LBL-007 was able to block LAG-3 binding to MHC class II molecules and liver sinusoidal endothelial cell lectin, and block LAG-3-induced downstream signaling. In mice transplanted with colorectal cancer cells, treatment with either anti-PD-1 antibody or LBL-007 (10 mg/kg per mouse twice a week for three weeks) resulted in a significant delay in tumor growth compared with control IgG treatment, and their combination was even more effective. Serum LBL-007 levels were highly stable in monkeys after a single intravenous injection of LBL-007 at 3, 10, or 30 mg/kg. This study demonstrated that the combination of LBL-007 with an anti-PD-1 antibody is a promising antitumor regimen for immunotherapy of solid tumors in future that deserves further study.

Keywords: Immunotherapy; LAG-3; anti-LAG-3 antibody; anti-PD-1; solid tumor.

Figure 1.

Evaluation of LBL-007 binding specificity. LBL-007 and control antibody were evaluated for their ability to bind human LAG-3 (A) or mouse LAG-3 (B) or CD4 protein (C) by ELISA. (D) Effect of LBL-007 antibody binding to CHO-K1/hLAG-3 cells by flow cytometry. Absorbance data and mean fluorescent intensity are showed as means ± SD on triplicates.

Figure 2.

Efficacy of LBL-007 endocytosis into cells. (A, B) LBL-007 and relatlimab analog were conjugated with pHAb Reactive Dye. When antibody-pHAb conjugates track through the endosome or lysosomal system, pH sensor dye will become fluorescent, which is detected by flow cytometry. Endocytosis of the antibody was determined using Jurkat-NFAT-LAG-3 and HEK293-LAG-3 cells. Mean fluorescent intensity is showed as means ± SD on triplicates. *P < .05, **P < .01, and ***P < .001 by Student’s t-test.

Figure 3.

LBL-007 blocks binding of LAG-3 to ligands. (A) In-vitro binding assay was performed to show LBL-007 inhibition on LAG-3 binding to MHC class II in Daudi-hLAG-3 cells. Data are showed as mean fluorescent intensity and IC50. Each sample was assayed in triplicate. (B) ELISA blocking assay was used to assess the ability of the anti-LAG-3 antibodies to inhibit human LAG-3 binding to human LSECtin. Absorbance data are expressed as means ± SD on triplicates.

Figure 4.

Effects of LBL-007 on regulation of activated T cells and increase of IL-2 production. (A,B) NFAT reporter assay was used to reveal LBL-007 binding to Jurkat-NFAT-LAG-3 cells. The corresponding fluorescence intensity of serial dilutions and EC50 was detected. After activating NFAT, Supernatants were collected and IL-2 levels were measured using the Human IL-2 DuoSet ELISA Kit. Data are expressed as the mean ± SD on triplicates. (C) A series of diluted concentrations of antibodies binding to activated human T cells was assessed by flow cytometry. Data are showed as Mean Fluorescent Intensity and EC50. (D) Different concentrations of LBL-007, relatlimab analog, and IgG4 were incubated with human PBMCs for 3 days, the IL-2 level in the supernatants was measured using ELISA Kit. Data are expressed as the mean ± SD on triplicates. *P < .05 and **P < .01 by Student’s t-test.

Figure 5.

Combined effects of LBL-007 and anti-PD-1 antibody on inhibition of mouse colorectal cancer cell growth in vivo. C57BL/6-hLAG-3 mice (n = 40) were randomized (n = 8 mice/group) when MC38 colon adenocarcinoma tumor volumes reached approximately 80 mm³. (A) Tumor volumes after treatment with the vehicle (PBS), isotype control, anti-PD-1, LBL-007 or a combination of LBL-007 and anti-PD-1 antibody (10 mg/kg each, twice a week). (B) Tumor tissues were weighed. Data are presented as mean tumor volume ± SEM. **P < .01, and ***P < .001 by one-way ANOVA.

Figure 6.

Plasma levels of LBL-007 after intravenous administration into monkeys. LBL-007 was intravenously injected into cynomolgus monkeys (n = 2/group) at different concentrations, and plasma levels of LBL-007 were assessed at the 18 indicated time points.