Novel PD-L1 Blocking Peptide with Mitochondrial Targeting Sequence: A Strategy to Counteract Resistance in T-Cell Immunotherapy for Myeloma

Background: T-cell engaging immunotherapy shows promise for relapsed refractory multiple myeloma (MM) but faces challenges due to resistance mechanisms, such as PD-1/PD-L1 activation. Traditional PD-1/PD-L1 antibodies have limitations, including poor tissue penetration, long half-lives that complicate withdrawal in case of adverse events, and limited single-agent efficacy in MM. Peptide drugs offer notable advantages over antibodies, such as superior tumor penetration, reduced toxicity, lower immunogenicity, easier chemical modification, and lower cost. We have developed a novel peptide, PP-k, which combines a PD-L1 targeting peptide with a mitochondrial-targeting peptide (klaklak)2. This construct binds to PD-L1, is internalized, and disrupts the mitochondrial membrane potential, inducing cell death. We hypothesize that combining PP-k with T-cell-engagers will significantly enhance MM cell killing and overcome resistance.

Objectives: We evaluated PP-k alone and together with teclistamab (approved BCMAxCD3 antibody) and cevostamab (FcRH5xCD3 antibody) in preclinical models of myeloma, including in vitro assays and an organ culture model where myeloma cells colonize the bone microenvironment. Our analysis focused on tumor killing, T-cell activation, and mechanisms of cell death.

Results: In a 48-hour cytotoxicity assay, PP-k demonstrated an IC50 of approximately 8 µM across various human MM cell lines. In co-cultures of NCI-H929 MM cells and human CD3 T cells, PP-k significantly enhanced cytotoxicity, achieving an IC50 of 2 µM, compared to conditions without T cells (8 uM). In contrast, the PD-L1 peptide alone and PD-L1 neutralizing antibodies inhibit PD-L1 but lack cytotoxic effects on MM cells. Importantly, PP-k does not directly impact T cell proliferation. These results suggest that PP-k enhances T cell-mediated killing of MM cells through mechanisms beyond mere PD-L1 inhibition.

In co-cultures of human CD3 T cells and MM cell lines, PP-k at sub-IC50 doses (1-2 µM), when combined with cevostamab (1 ng/ml) and teclistamab (10 nM), significantly enhanced cytotoxicity compared to each agent individually (Figure 1). This combination also promoted T cell activation, as demonstrated by an increase in CD8+CD25+ cytotoxic T cells and enhanced intracellular staining of granzyme B compared to the effects of each agent alone (Figure 2).

To assess PP-k efficacy in the bone niche, we tested it in MM cells co-cultured with T cells in mouse calvaria explants. MM cell adhesion to bone increased the IC50 of PP-k and bispecific T cell engagers, indicating resistance from the bone microenvironment. Importantly, combining PP-k with bispecific T cell engagers enhanced cytotoxicity compared to either agent alone. Additionally, PP-k augmented T cell activation seen with cevostamab, whereas PP-k alone showed limited T cell activation in the tumor:bone co-culture, potentially due to the immune-suppressive nature of the environment.

In addition to mitochondrial-mediated cell death, PP-k may also trigger immunogenic cell death (ICD), as indicated by the rapid calreticulin buildup on MM cell membranes.

Conclusions: PP-k kills MM cells directly and through T cell-mediated mechanisms, offering advantages over PD-L1 blockade alone. PP-k significantly enhances the cytotoxicity of bispecific antibodies and overcomes resistance in the bone marrow microenvironment. In the immune-suppressive tumor:bone co-cultures, PP-k significantly enhances T-cell activation when combined with bispecific antibodies. Ongoing studies with patient-derived cells and immunocompetent mouse models are investigating PP-k's efficacy, mechanism of action, and potential for future clinical trials.

No relevant conflicts of interest to declare.