Post written by Czeslaw Radziejewski, Ph.D.
Antibody Engineering & Therapeutics, held in December 2022, offered many opportunities to hear exciting and informative presentations by experts in the field, including Li Peng, Ph.D., who discussed “Engineering of human Sialidase Neu2 as Novel Immunotherapy for Degrading Immunosuppressive Sialoglycans to Enhance Antitumor T-Cell Immunity”.
Glycans are the most abundant structures on the cell surface. They are involved in cell communication with immune cells, and abnormal glycans can cause immune dysfunction in cancer and inflammatory diseases. Glycans typically terminate in sialic acid, but in cancer cells, sialic acid is present at a much higher abundance. The most common sialic acid in humans is N-acetylneuraminic acid, which plays a crucial role in numerous intercellular interactions, including with immune cells in the extracellular matrix, epithelial cells, and antibodies. Many studies have shown that sialoglycans are immunosuppressive and that high levels of surface sialoglycans are linked with poor outcomes in many tumor types. Hypersialylation of the surface of cancer cells makes these cells prime ligands for sialic acid-binding immunoglobulin-type lectins (Siglecs), which are found on the surface of immune cells. Once bound to sialylated glycans, Siglecs promote immunosuppressive signaling, thus conferring protection on the tumor cell. There are 15 human Siglecs. In addition, CD-28 is also known to bind sialoglycans. Most immune cells express more than one Siglec.
In her plenary lecture at the 2022 Antibody Engineering & Therapeutics conference, Professor Carolyn Bertozzi, outlined opportunities for the development of cancer treatments based on understanding the cell-surface glycome. She favored degrading sialoglycans with the enzyme sialidase to eliminate the immunosuppression promoted by Siglecs. As proof of concept, a fusion protein was created in Bertozzi’s lab using click chemistry, linking bacterial sialidase to the C-terminus of trastuzumab. The conjugate was tested in a mouse model of a trastuzumab-resistant HER2+ breast cancer model, and the results showed that the treatment essentially abrogated tumor growth. Based on these promising findings, Bertozzi cofounded Palleon Pharmaceuticals to explore sialidase-based biotherapies for cancer treatment.
At the conference, Dr. Li Peng, Chief Scientific Officer of Palleon Pharmaceuticals, presented the company’s progress in moving this concept toward the clinic. Palleon created a set of proprietary Siglec-based reagents for immunohistological hypersialylation detection and probing its role in immunotherapy resistance. Using such reagents, Palleon examined tissues from metastatic melanoma patients treated with PD1 blockade and showed that patients with a high level of sialylation fared much worse than patients with lower levels of sialoglycans. Following Bertozzi’s line of reasoning, the company pursued a strategy of using the enzymatic functionality of sialidase to remove excessive cell-surface sialylation. To translate this idea into a human therapeutic, Palleon decided to use a genetic fusion of sialidase with human Fc.
In humans, there are four sialidases encoded by genes Neu1-Neu4. Recombinant wild-type human sialidase Sialidase-2 (cytosolic) proved to be very difficult to develop due to its aggregation propensity and rapid loss of enzymatic activity. However, through extensive protein engineering efforts, the company created a stable version (called E-602) of the fusion protein. E-602 protein could be expressed in CHO cells at 2.5 g per liter.
Palleon’s studies with E-602 led to their discovery that desialylation enhances T-cell function. Upon activation, the level of sialic acid in T cells increases. Furthermore, upon exhaustion, T cells become hypersialylated. As could be expected, sialidase treatment restored the function of T cells, as demonstrated by the rise in interferon-gamma production. Treatment with E-602 enhanced T-cell activity only in antigen-stimulated T cells; therefore, sialidase treatment should not lead to the systematic activation of these cells and to the potential release of cytokines. Although E-602 is an untargeted therapeutic, it nevertheless demonstrated activity in multiple syngeneic mouse tumor models. To address safety concerns, Dr. Peng postulated that E-602, because of its high Km (1 mM), should be most potent toward cells with abundant sialoglycans. Furthermore, desialylation of normal cells did not cause systematic immune activation, and without antigen engagement, sialidase treatment did not enhance T-cell activity. Accordingly, no adverse effects were observed at high dose levels in rats and non-human primates. Also, human peripheral blood mononuclear cell assays showed the absence of induction of cytokine release upon treatment with E-602. The effects of this molecule, administered alone and in combination with pembrolizumab, are now being evaluated in a Phase 1/2 dose escalation and dose-expansion study (NCT05259696) of patients with advanced cancers.
The company is also considering targeted approaches against immune cell and tumor-associated targets. Aiming at creating additional treatment modalities for breast cancer patients with low sialylation, Dr. Peng presented data on a bifunctional Fc fusion molecule in which one arm binds the HER2, and the other performs the sialidase function. HER2-sialidase fusion protein demonstrated robust efficacy in a mouse EMT6-HER2 tumor model with low HER2 expression. In this study, responders tended to have higher levels of desialylation of immune cells (T cells and natural killer (NK) cells) than non-responders. Respondents had higher infiltration of NK cells within the tumors and reduced levels of M2 macrophages. This program is currently in the preclinical stage.
Blockade of individual Siglecs may still have a place in overcoming the immunosuppressive signaling that hypersialylation plays in cancer pathology. At the conference, Yan Chen, CEO of Elpis Biopharmaceuticals, reported the discovery of a human anti-Siglec15 antibody (EPB-001). EPB-001 binds with picomolar affinity to a unique epitope on Siglec 15 and potentiates strong anti-tumor activity by blocking the interaction of tumor-associated macrophages with T cells. EPB-001 blocks Siglec 15 on M2 macrophages and reverses immune suppression. According to Yan Chen, EPB-001 could be a promising therapeutic for tumors that are non-responding or resistant to immune checkpoint inhibitor treatment. This molecule is currently in preclinical studies.