Trispecifics Improve Immune-Cell Engagement

With dozens of T cell–redirecting bispecific antibodies in clinical trials for a range of malignancies, drug developers have begun looking for ways to hone the precision of their off-the-shelf, cell-engaging therapeutics. For a growing number of companies, that has meant introducing a third binding domain to create trispecific antibodies with additional specificity for tumors or enhanced immune-cell activation.

The HER2 × CD3/CD28 candidate SAR443216 (Sanofi) offers a prototypical example. The trispecific T-cell engager entered human testing last year for patients with HER2-expressing tumors of the breast, stomach, and other organs. It attaches to HER2 receptors on cancer cells with one arm and binds to T cells with the other two. The inclusion of CD28-targeting—and not CD3 engagement alone, as is typical of bispecifics—provides costimulation that boosts T-cell proliferation and survival.

As scientists reported in February, CD4⁺ T-cell responses are critical to the therapy's antitumor functions—and not just in a helper capacity (Nature 2022;603:328–34).

Although CD8⁺ T cells contribute to the trispecific's activity, the researchers showed in a mouse model of breast cancer that the CD4⁺ T-cell population performs the bulk of the tumor killing, through both direct inhibition of cancer cells and indirect promotion of inflammatory pathways—a result that Sanofi's Lily Pao, PhD, a study author, describes as “unexpected.”

This immunologic action sets SAR443216 and similar trispecifics—including Sanofi's CD38 × CD3/CD28 therapy (SAR442257) now in phase I testing for patients with multiple myeloma and non-Hodgkin lymphoma—apart from many other cancer immunotherapies, which typically work through CD8⁺ T cell–mediated cell destruction. “It really points to the importance of having a deeper understanding of the proposed mechanism of action of any drug being developed,” says Valeria Fantin, PhD, global head of oncology research at Sanofi.

But that's not the only potential selling point of trispecific antibodies. The constructs can be engineered for enhanced tumor specificity or to recruit and activate natural killer (NK) cells instead of T cells. Trispecific macrophage engagers are in the works, too. “There are several different formats of trispecifics,” notes Laura Sanz Alcober, MD, PhD, of Puerta de Hierro Majadahonda University Hospital in Madrid, Spain.

Sanz Alcober and her colleagues described a trispecific arrangement that binds T cells with a CD3-specific single-chain antibody fragment and then targets two tumor-associated antigens, EGFR and EpCAM, with separate nanobody domains (Oncoimmunology 2022;11:2034355). In mouse experiments, this construct showed the greatest potency against colon cancers expressing both EGFR and EpCAM, but it still shrank tumors expressing only one of the antigens, a sign that this dual-targeting strategy may help prevent drug resistance through antigen loss and tumor escape.

Rather than focusing on T-cell engagement, many groups have deployed trispe­cific designs to bolster the potential of NK cells—and some companies are pursuing both strategies in parallel.

NK-cell engagers have “the potential for a more favorable safety profile, alleviating concerns such as cytokine release syndrome and neurotoxicity” associated with T-cell engagers, Fantin says.

“In addition, activated NK cells may also potentiate adaptive T-cell responses,” she says.

To create their trispecific NK-cell engagers, Martin Felices, PhD, and his colleagues at the University of Minnesota in Minneapolis started with antibody fragments directed against CD16, found on the surface of NK cells, and CD33, a marker of myeloid malignancies. They then incorporated a cross-linker containing a modified form of IL15, a cytokine that plays a key role in NK-cell expansion, survival, and activation (Clin Cancer Res 2016;22:3440–50).

By adding IL15, “you can increase the number of NK cells that are present while you are also driving targeting,” Felices says.

The University of Minnesota licensed the technology to GT Biopharma, which is currently evaluating the therapy, now called GTB-3550, in patients with CD33-positive acute myeloid leukemia (AML), systemic mastocytosis, or myelo­dysplastic syndrome (MDS).

Other companies with trispecific NK-cell engagers in clinical development have used the third arm of their molecules to bind another receptor on the immune-cell surface alongside CD16. The HER2-directed therapy DF1001 (Dragonfly Therapeutics) follows this playbook, targeting both CD16 and NKG2D on NK cells, as does SAR443579 (Sanofi/Innate Pharma), a CD123 × CD16/NKp46 trispecific that entered clinical testing late last year for patients with AML and MDS.

According to Innate's Chief Scientific Officer Eric Vivier, DVM, PhD, the company chose to target the NKp46-activating receptor and CD16 because tumor-infiltrating NK cells often lose expression of the latter, which can diminish the efficacy of bispecific designs binding only CD16 and tumor antigens. NKp46 has a more stable expression profile and so, as Vivier and his colleagues have shown in mice, including an antibody directed against this receptor helps bring additional, persistent NK cells to the tumor (Cell 2019;177:1701–13).

Innate is also developing a tetraspecific NK-cell engager. That construct brings an IL2 receptor–binding moiety to the same molecule. Like IL15 in GTB-3550, the extra cytokine signal delivers proliferation and activation cues to the NK cells. In preclinical experiments, scientists observed “a strong and massive accumulation of activated NK cells,” Vivier says, along with sustained B-cell depletion following treatment with a CD20-directed tetraspecific.

“Everyone is looking for strategies for next-generation immune-cell redirection,” Vivier says, and he is confident that multispecific engager platforms will constitute a growing part of the oncology toolbox. –Elie Dolgin