New B7 Family Immune Checkpoint Proteins

Targeting immune checkpoint regulators such as programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) has achieved notable benefit in a variety of cancers. Recently five new B7 family ligands, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7, were identified, all of them possibly being of high interest in immunotherapy research.

B7-H3 [CD276] has a co-inhibitory function on T cells. Its expression on either tumor cell or diffuse tumor vasculature is significantly associated with an increased risk of death and bad outcome. Targeting B7-H3 not only enhances anti-tumor immunity but also inhibits tumor angiogenesis.

B7-H4 plays a significant role in the “immune escape” of tumors and is a potential therapeutic target for the treatment of cancer. It plays an important role in the T cell-mediated immune response as a negative regulator. The ligation of B7-H4 to unidentified receptors results in the inhibition of TCR-mediated T cell proliferation, cell-cycle progression and IL-2 production.

VISTA [V-domain immunoglobulin suppressor of T cell Activation; B7-H5] is type I Ig membrane protein homologous to PD-L1. VISTA is mainly expressed on hematopoietic tissues (spleen, thymus and bone marrow) and on myeloid cells. VISTA is a new negative checkpoint regulator that potently suppresses T cell activation. It delivers a co-inhibitory impact on T cell response.  Recent publications have reported that a recombinant VISTA protein needs to be multimerized to be active as a soluble ligand. The protein VISTA (mouse):COMP (mouse) (with the extracellular domain of mouse VISTA fused to the pentamerization domain from the cartilage oligomeric matrix protein (COMP), but not VISTA-Fc, has been shown to function as an immunosuppressive agonist in vivo inhibiting the proliferation of CD4+ T cells. Additionally, a new function of the protein P-selectin glycoprotein ligand-1 (PSGL-1) as a new receptor for the immune checkpoint VISTA has been shown. Multimer VISTA binds PSGL-1 expressed on leukocytes at acidic pH, but not at the physiological pH 7. VISTA - PSGL-1 interaction shows that immune response can be regulated by acidic environments found in tumors.

B7-H6 (also known as NCR3LG1) is a ligand for NK cell activating receptor NKp30. It is expressed on tumor cells but can also be induced by inflammatory stress in healthy cells. B7-H6 has been validated as a potential target for antitumor immunotherapy strategies in tumor-bearing mice. The expression of B7-H6 is significantly correlated with post-operative prognosis in cancer patients and  with distant metastasis status.

B7-H7 is widely expressed in human malignancies and its expression is associated with poor prognostic factors. Targeting B7-H7 not only benefits antitumor immunity but also inhibits tumor angiogenesis. For B7-H7, a T cell co-inhibitory role and a co-stimulatory role have been reported, most probably based on two counter receptors, one CD28H and the other unknown.

LIT: New B7 family checkpoints in human cancers: L. Ni & C. Dong; Mol. Cancer Ther. 16, 1203 (2017) • VISTA.COMP - an engineered checkpoint receptor agonist that potently suppresses T cell-mediated immune responses: A. Prodeus, et al.; JCI Insight 2, e94308 (2017)

NEW VISTA is an acidic pH-selective Ligand for PSGL-1

PSGL-1 (human):Fc (human) (rec.) (Prod. No. AG-40B-0190) binds to its ligand VISTA only at acidic pH. Method: VISTA (human):COMP (mouse) (rec.) (His) (Prod. No. AG-40B-0183) or an unrelated protein Nampt (human) (His) (Prod. No. AG-40A-0031Y)(Control) is coated on an ELISA plate at 1μg/ml overnight at room temperature. PSGL-1 (human):Fc (human) (rec) (Prod. No. AG-40B-0190) is incubated (starting at 1000ng/ml with a twofold serial dilution) for one hour in mild acidic (pH 6.4) or neutral conditions (pH 7.4) and then detected using an anti-human Fc antibody (HRP).

Multimeric VISTA – Immunosuppressive In Vivo Agonist

The recombinant VISTA protein needs to be multimerized to be active as soluble ligand. The protein VISTA (human):COMP (mouse) (with the extracellular domain of VISTA (human) fused to the pentamerization domain from the cartilage oligomeric matrix protein (COMP)), but not VISTA-Fc, functions as an immunosuppressive agonist in vivo inhibiting the proliferation of CD4+ T cells.

Figure: Recombinant VISTA (human):COMP (mouse) (rec.) (His) (Prod. No. AG-40B-0183) inhibits anti-CD3 antibody induced TNF-α secretion of human CD4+ T lymphocytes.
Method: Human CD4+ T cells are activated with coated anti-CD3 (human) (2.5µg/ml) and incubated for 5 days with VISTA (human):COMP (mouse) (rec.) (His) or Control protein (Prod. No. AG-35B-0007) (coated at 10µg/ml). TNF-α is measured from cell supernatants using the Cymax TNF-α (human) ELISA Kit (Prod. No. YIF-LF-EK0193).

LIT: VISTA.COMP - an engineered checkpoint receptor agonist that potently suppresses T cell-mediated immune responses: A. Prodeus, et al.; JCI Insight 2, e94308 (2017)

TAPBPL (TAP Binding Protein-like) - B7 Family Related Molecule

Because of the potential clinical applications of immune checkpoint proteins, there has been intense interest in identifying additional T-cell regulators. The antigen processing (TAP) binding protein-like (TAPBPL)/TAP binding protein-related (TAPBPR) molecule has been shown to share significant sequence similarity with some known B7 family members. TAPBPL protein is expressed on the surface of T cells, on antigen-presenting cells (APCs), including resting B cells, monocytes, macrophages, and DCs, as well as on some cancer cells, including leukemia cells. TAPBPL behaves like other immune checkpoint proteins, such as B7-H5 / VISTA or PD-L1, with a soluble recombinant version TAPBPL-Fc fusion protein that inhibits the proliferation and activation of CD4 and CD8 T cells in vitro and ameliorates autoimmune disease EAE in vivo. In contrast, treatment with anti-TAPBPL blocking antibody enhances antitumor immunity and inhibits tumor growth in vivo. Therefore, TAPBPL contains typical features of B7 family members, suggesting that it is a B7 family member or a B7 family-related molecule.

LIT: Identification of TAPBPL as a novel negative regulator of T-cell function: Y. Lin, et al.; EMBO Mol. Med. 13, e13404 (2021) • Administration of Recombinant TAPBPL Protein Ameliorates Collagen-Induced Arthritis in Mice: Z. Zhang, et al.; Int. J. Mol. Sci. 24, 13772 (2023)

B7-H3 (human) ELISA Kit  - Biomarker for Immuno-Oncology Research

The B7-H3 (human) ELISA Kit (Prod. No. AG-45B-0025) is a sandwich ELISA for specific quantitative determination of soluble human B7-H3 (CD276) in serum, plasma and cell culture supernatants. Soluble human B7-H3 (CD276) is ectopically expressed in various cancers and its levels in serum of patients with cancer suggests it can be used as a noninvasive biomarker for diagnosis, prognosis and/or treatment response.

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