Background
Leucine-rich repeat-containing G protein receptor 5 (LGR5) is a target gene of the oncogenic Wnt signaling pathway in colorectal cancer cells. LGR5 is overexpressed in human malignancies such as CRC, HCC, gastric and ovarian cancers, breast cancer, glioblastoma, and certain B-cell malignancies. LRG5 expression in colorectal cancer cells is required for proliferation, migration, chemosensitivity, clonogenicity, and in vivo engraftment. High LGR5 expression in tumor cells is crucial for tumor cell proliferation, making LGR5 a promising target for cancer therapy. While there is ample room for clinical application of LGR5 antibodies in immunotherapy, the lack of suitable, robustly validated antibodies has hindered therapeutic development.
On August 21, 2024, researchers published a study titled "Novel immunotherapeutics against LGR5 to target multiple cancer types" in EMBO molecular medicine, reporting highly specific monoclonal α-LGR5 antibodies that can be used in a range of experimental and therapeutic applications. The study describes the development of α- LGR5 as a therapeutic antibody, as well as its functional validation as an ADC targeting LGR5+ CRC and pre-BALL cells, and by directing cytotoxic immune cancer cell killing in BiTE and CAR models. The robust preclinical efficacy of α-LGR5 in these three treatment modes was demonstrated in a mouse model of human pre-BALL, supporting the continued development of α-LGR5-based immunotherapy for all cancer types expressing LGR5.
Preparation and validation of anti-LGR5 antibodies
LGR5 were generated by immunizing mice with the N-terminal 101 amino acids of the extracellular domain of human LGR5. The four LGR5-positive clones obtained were found to be immunoreactive only against human and cynomolgus macaque LGR5, but not against mouse LGR4 and LGR5, or the closely related human LGR4 and LGR6. Western blot analysis demonstrated that all four α-LGR5 clones specifically bound to fragment 1 of the LGR5 antigen. The N-terminal 15 amino acids of LGR5 (Frag1A) were found to contain the epitope, and the antibody clones bound to Frag1A with high affinity. Antibody binding to LGR5 did not interfere with the binding of R-spondin 1 ligands, and binding of α-LGR5 or any other reported antibodies to LGR5 did not interfere with its function in enhancing Wnt pathway activity. The α-LGR5 antibody specifically recognized overexpressed hLGR5 and cLGR5 by Western blotting, immunofluorescence in fixed cells, and flow cytometry in live cells, but did not detect other LGR family members.
Survey of LGR5 expression levels in healthy tissues and cancer
Studies have found that LGR5 is highly expressed in many cancers, with LGR5 overexpression specific to a discrete group of cancer types, such as colon and rectal cancer, liver cancer, and acute lymphoblastic leukemia. A considerable window of LGR5 expression exists between CRC, HCC, some ALL cases, and healthy tissue. In most healthy tissues, LGR5 expression levels are very low or undetectable, raising the possibility of developing therapeutic strategies targeting LGR5 that would likely have minimal impact on healthy cells. Using specific and well-validated antibodies (such as α-LGR5) is a valuable diagnostic tool.
α-LGR5 specifically detects LGR5 protein in human cancer cell lines
α-LGR5 antibody was used to detect cellular expression of LGR5 protein in three human pre-B-ALL cell lines. LGR5 was localized to intracellular punctate structures. LGR5 was primarily expressed in intracellular puncta and in small, internalized, plasma membrane-associated transient pools. Immunofluorescence detection of LGR5 in LoVo colon cancer cells using α-LGR5 revealed that expression was primarily confined to intracellular puncta, with virtually no LGR5 detected on the cell surface. This suggests that LGR5 is primarily endogenously internalized in these cells. Endogenously expressed LGR5 is rapidly internalized from the plasma membrane via the endocytic pathway, recycled to the plasma membrane, and directed to lysosomal vesicles. α-LGR5 antibody can be rapidly internalized by LGR5-overexpressing cell lines.
Validation of LGR5-based Antibody-Drug Conjugates
The high expression levels of LGR5 specific to certain malignancies, the rapid internalization kinetics of α-LGR5, and the trafficking of LGR5 to lysosomes have all created exciting prospects for targeting cancer with α-LGR5-based antibody-drug conjugates (ADCs). Studies have shown that α-LGR5-ADCs exhibited high cytotoxicity against LGR5-positive cancer cells in vitro and demonstrated significant anti-tumor effects in a human NALM6 pre-B-ALL mouse model, effectively reducing tumor growth.
In vitro and in vivo efficacy of LGR5 - based BiTEs
Based on α-LGR5, the researchers constructed a humanized α-LGR5 scFv fragment. By fusing the α-CD3ε scFv to the α-LGR5 scFv, they generated a CL-BiTE, which demonstrated highly specific and potent activation of human CD4+ and CD8+ T cells and effectively induced cancer cell killing in vitro. In vivo, the CL-BiTE, co-injected with PBMCs into NALM6 tumor-bearing mice, demonstrated an inhibitory effect on tumor growth. Following treatment, a significant reduction in tumor burden, approximately two-fold, was observed.
LGR5-based cell therapy effectively targets NALM6 cells
LGR5-CAR-T cell therapy was generated based on the α-LGR5 scFv. LGR5 scFv-CAR-T cells demonstrated specific and potent tumor cell destruction in vitro and excellent efficacy in vivo. Animals treated with LGR5 scFv-CAR-T cells showed a significant reduction in the number of tumor cells in the bone marrow, and pre-B-ALL tumor burden was reduced fourfold compared to the control group. The high specificity and efficacy demonstrated by the LGR5 scFv-CAR demonstrate its potential to effectively target multiple cancer types.
Summarize
LGR5 is an adaptable therapeutic antibody that can be used to target cancer cells in ADC, BiTE, and CAR-T cell formats. While we observed different in vivo efficacy of these three formats in targeting pre-B-ALL tumors, these compatible therapeutic strategies offer significant scope for adapting to the distinct pharmacodynamic requirements of various LGR5+ tumor types. Our studies demonstrate that LGR5 can not only be used as a research tool and biomarker but also provide a versatile building block for highly effective immunotherapy combinations against a range of LGR5-expressing cancer cells.
