LY6G6D (LY6-G): A New Member of Lymphocyte Antigen-6 Family Holds Big Opportunities in Bispecific Antibody for CRC Treatment!

1. What is Ly-6 Superfamily (Ly-6SF)?

The Ly6/uPAR/CD59/netuotoxin superfamily (Ly-6SF) belongs to the superfamily of lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) proteins ^[1-2]. Ly6/uPAR proteins were first identified in the mouse using antisera against lymphocytes in 1977 and the cDNA of Ly-6E.1 was cloned in 1986. The human Ly6 gene family located on the chromosome 8, namely Ly6-A, Ly6-C, Ly-6ThB, Ly6-F, Ly6-G, Ly6-H and Ly-6M, defined as the lymphoid antigen 6 superfamily, referred to as Ly-6SF. Most of Ly-6SF is a glycosylphosphatidylinositol (GPI) protein, which functions as an anchor to link cell membranes and protein ^[1-2].

Currently, at least 35 human and 61 mouse Ly6 proteins have been identified, such as those: CD59 expressed in human lung, kidney, tunic, and liver; Lynx1 in mouse keratinocytes; SLUPR-1 in most immune cells and bronchial epidermal cells; CD45, CD43, Sca-1/Ly-6A.2/Ly-6E.1, and CD117 in the bone marrow of mice. In addition, the discovery in higher animals of proteins from the Ly6/uPAR family, which have structural homology with snake “three-finger” neurotoxins, such as cobras and kraits, three-finger α-neurotoxins such as α-cobratoxin (α-Cbtx) and α-bungarotoxin (α-Bgtx). Available studies have shown that the Ly6 superfamily performs integral functions in different biological processes including immunity, cellular adhesion, and cell signaling ^[3-5].

2. What is LY6G6D?

2.1 LY6G6D Structure

The lymphocyte antigen 6 complex locus G6D (LY6G6D, also known as LY6-D; LY6-G; G6D; NG25; MEGT1; C6orf23) is a member of the lymphocyte antigen 6 superfamily (Ly-6SF) ^[6-7]. LY6G6D is positioned within the MHC class III region on chromosome 6, wherein it encodes a polypeptide comprising 133 amino acid residues with an approximate molecular weight of 13.7 kDa. The Ly-6SF protein family has a unique feature of containing one or more structured conserved domains known as Ly6/uPAR (LU) domains.

These domains are characterized by an N-terminal leucine or isoleucine residue and consist of around 80 amino acid residues that form four to five pairs of conserved disulfide bonds. As a result, it shapes into a three-fingered protein domain (TFPD) (Figure 1) ^[9]. Ly-6SF family members are marked by low sequence conservation and rapid evolution, reflecting a high degree of genetic diversity. This is further evidenced by the significant number of family members and their wide range of biological functions ^[7-9].

Figure 1. Ly-6SF "three-finger" structure ^[9]

2.2 LY6G6D Expression

LY6G6D is expressed in varying amounts in myeloid cells including monocytes, macrophages, granulocytes, and neutrophils during development ^[7]. Myeloid cells comprise a significant component of the innate immune response. CD11b+ Ly6G- myeloid cells have been reported to facilitate the development of mechanical inflammatory pain hypersensitivity ^[10]. LY6G6D expression is typically transient during monocyte differentiation, but persists in fully differentiated granulocytes and circulating neutrophils. Researchers have demonstrated that LY6G6D exerts a pivotal influence on the processes of neutrophil infiltration, recruitment, and migration ^[7].

3. How's the Mechanism of Action of LY6G6D and Its Family Proteins?

LY6G6D, an emerging target of the Ly-6 superfamily, has been identified as a significant player in the development of colorectal cancer. High levels of LY6G6D increase the risk of metastatic recurrence after treatment. STAT5 knockdown remarkably reduced LY6G6D levels, implying that STAT5 regulates LY6G6D expression. Furthermore, JAK/STAT inhibitors induce apoptosis in CRC cells via targeting the STAT5/LY6G6D axis ^[11].

In addition, Ly-6 families and their low structural conservatism contribute to their highly diverse functions, such as C4.4A/LYPD3 is a cancer-associated cell surface protein highly expressed on squamous cell carcinoma subtypes ^[12]; Lypd6 is present in Wnt/β-catenin signaling pathway, which is positively regulated by Wnt8 ^[13]; Lynx1 interacts with nicotinic acetylcholine receptors (nAChR) and is thought to function as a modulator of nAChR activity to prevent hyperexcitability and provide protection to neurons ^[14].

In pneumonia infection, enhanced expression of LY6G induces activation of CD69 and KRG1 of splenic natural killer cells ^{[15, 16]}. Ly6e has been reported to be involved in the regulation of viral infection like coronavirus, SARS-CoV, MERS-CoV, and SARS-CoV-2 ^[17]; CD59 regulates complement activation regulator MAC, which in turn is involved in complement-regulated apoptosis ^[18]; CD46 plays a critical role in host immunity against phage invasion by mitigating their infectivity. Moreover, CD46 helps protect the body from phages, and works together with ITG-Beta1/CD29, which functions as an adhesion molecule for retinal pigment cells in the ocular milieu ^[19].

4. The Roles of LY6G6D in Colorectal Cancer and Immune Aging

LY6G6D, belonging to the Ly-6 superfamily, has received limited global attention with its precise function remaining unclear for several years. Nonetheless, recent research have unveiled the roles of LY6G6D in colorectal carcinogenesis, while supplementary investigations have linked it to the aging process.

4.1 LY6G6D and Colorectal Cancer

Colorectal cancer (CRC) affects many people and is a common type of cancer in the digestive system. However, the cause of CRC is not yet fully understood. Research has revealed that LY6G6D is upregulated in both primary and metastatic colorectal tumor tissue as compared to normal colorectal tissue. The high expression of LY6G6D was associated with a high infiltration of immunosuppressive cells. In colorectal cancer with reduced CD8+T lymphocytes, both expression levels of LY6G6D and STAT5 were enhanced. In addition, LY6G6D-positive CRC cells restrained T cell proliferation through the expansion of myeloid-derived suppressor cells (MDSC) ^[20].

Similarly in CRC, p38α MAPK inhibitors and DNMT1 knockdown result in reduced LY6G6D expression. In the metastatic CRC group, LY6G6D hypermethylation predicts resistance to first-line treatment with FOLFOX (drugs folinic acid (LV), fluorouracil (5-FU) and oxaliplatin) ^[21]. Consequently, LY6G6D is intricately linked to the development of colorectal cancer and represents a promising prognostic indicator and therapeutic target for CRC.

4.2 LY6G6D and Immune Aging

The aging process also affects the immune system, a phenomenon commonly referred to as immune senescence or immune aging. Aging is an inevitable physiological event that exerts a pervasive impact on numerous biological processes, among which immune function is particularly susceptible.

The process of aging can prompt T cells to adopt a senescence-associated phenotype and increase the expression of co-inhibitory molecules including PD-1/CD279, TIM-3/CD366, and VISTA. These changes can result in impaired T-cell function and subsequently disrupt the delicate homeostasis of the immune system. Likewise, dysfunction of innate immune cells, notably neutrophils and macrophages, can trigger pathogen invasion and augment the potential for chronic inflammation and atherosclerosis ^[22-24].

According to a recent study, LY6G6D downregulation represents a crucial biomarker for tracking functional deterioration associated with neutrophil immune senescence. LY6G6D expression was found to be significantly downregulated in bone marrow neutrophils of aged mice compared to young mice. The study found that LY6G6D expression is closely linked to certain immune functions in neutrophils (a type of immune cell), which tend to decline with age. These include abilities such as phagocytosis, production of reactive oxygen species (ROS), and interleukin IL-1β, formation of neutrophil extracellular traps (NETs), migration, and bacterial clearance ^[8]. Immune senescence can severely impair the innate immune system. However, researchers still have much to learn about the mechanisms that control this process in certain types of immune cells. The findings suggested that LY6G6D may be able to predict immune impairment related to aging in neutrophils.

5. LY6G6D Clinical Prospects

Multiple studies have demonstrated the crucial role that LY-6 family members play in modulating immune, nervous system, and complement functions. In particular, numerous LY-6 members have been implicated in a variety of cancers such as gastric, cervical, breast, ovarian, lung, and bladder cancer. Hence, the LY-6 gene family could offer a significant value for clinical application, not only as a prognostic indicator in disease management, but also as a promising drug target.

As a novel member of the LY-6 gene family, LY6G6D has been implicated as a significant contributor to colorectal cancer pathogenesis or other disorders. Aforementioned, a dual antibody strategy based on LY6G6D has exhibited efficacy in the treatment of colorectal cancer, emphasizing its vast potential as a promising target for antibody-based therapeutic interventions, particularly treatments for CRC!

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