May 22, 2024

Summary

The effectiveness of immune checkpoint inhibitors against metastatic melanoma has been well-established. However, their use as standalone treatments for other cancer types, such as advanced hepatocellular carcinoma, has shown limited success. As a result, recent clinical studies have increasingly focused on evaluating the effectiveness of combining immune checkpoint blockers with other treatment methods. Recently, the findings from a Phase 1/2 clinical trial evaluating the safety and immunogenicity of the anti-PD1 drug pembrolizumab, combined with a personalized cancer vaccine in patients with advanced hepatocellular carcinoma who did not respond to first-line treatments, were published and reported at ASGCT 2024.

Background

Immune checkpoint inhibition emerged as a promising cancer therapeutic strategy to essentially release the breaks limiting anti-tumor immune responses, notably T-cell mediated immunity. The first immune checkpoint inhibitor approval in 2011 for the clinical use of the antibody drug ipilimumab targeting cytotoxic–T–lymphocyte-associated–antigen–4 (CTLA4) set a new course in cancer treatment. Since then, the development of T-cell modulating antibody drugs has centered primarily on targeting programmed-cell-death-protein-1 (PD1) and its ligand, PDL1. The high efficacy of immune checkpoint inhibitors in patients with metastatic melanoma, who often achieve full remission and remain cancer-free well after treatment completion, encouraged their use for various solid tumors.¹

Immune checkpoint inhibitors have been used alone or in combination with other therapies in non-cell lung cancer, renal carcinoma, colorectal cancer, and more with variable efficacy.² For instance, Anti-PD1 antibody drugs have been implemented as therapies for advanced hepatocellular carcinoma since 2017. However, outcomes from randomized Phase 3 clinical studies have not demonstrated a significant response rate. In contrast, combination therapies with immune checkpoint inhibitors and other agents, such as tyrosine kinase inhibitors (TKIs) or anti-vascular endothelial growth factor (VEGF), have shown improved efficacy.³

More recently, a Phase 1/2 clinical trial was undertaken to evaluate the response to the anti-PD1 drug, pembrolizumab, combined with a personalized cancer vaccine in patients with advanced hepatocellular carcinoma who failed to respond to first-line treatments.⁴ Personalized cancer vaccines leverage new tumor antigens or neoantigens to drive immune responses specific to a patient’s tumor. Neoantigens are derived from new proteins arising from somatic mutations occurring during carcinogenesis. For example, insertion and deletions (INDELs) and point mutations can lead to new amino acid sequences in the tumor. The process of identifying neoantigens is laborious and complex, generally involving extensive high throughput sequencing (e.g., whole-genome or whole-exome sequencing), the use of predictive algorithms to identify potential immunogenic neoantigen peptides (i.e., MHC presented), as well as mRNA seq and/or LC-MS/MS analysis from tumor biopsies to validate the predicted peptides.⁵

Phase 1/2 clinical trial evaluating safety and immunogenicity of a personalized neoantigen vaccine and pembrolizumab.“a, Manufacturing process for GNOS-PV02 and clinical trial design.”⁴ Panel a, Figure 2, retrieved from Yarchoan et al. 2024.⁴ http://creativecommons.org/licenses/by/4.0/

Improved clinical outcomes with combined therapy

Various clinical studies have previously assessed the efficacy of immune checkpoint inhibitor monotherapy in patients with hepatocellular carcinoma. Pembrolizumab monotherapy has been shown to achieve an average objective response rate (ORR) of ~17 % in these patients.

In the recent Phase 1/2 study by Yarchoan et al., over thirty patients with advanced hepatocellular carcinoma received pembrolizumab in combination with a personalized cancer vaccine delivered as a plasmid encoding up to 40 neoantigens. Patients additionally received a second plasmid encoding interleukin-12, which served as a vaccine adjuvant.⁴

“The final assembled cassettes were codon and RNA optimized, synthesized and subcloned into the vaccine expression vector pGX0001 (GenScript).⁴

Yarchoan et al., found that such combined therapy resulted in a significant increase in efficacy when compared to pembrolizumab only, achieving an ORR of ~30.6%. Complete and partial responses were observed in 3 and 8 out of eleven patients with an objective response. Interestingly, no specific biomarker could be connected to these clinical responses, such as tumor mutational burden, immune inflammation phenotype, and alpha-ferroprotein level. However, Yarchoan and colleagues found that the number of neoantigens included in the vaccine positively correlated with objective clinical responses, with complete and partial responses occurring more frequently in patients receiving vaccines encoding over 30 neoantigen peptides.⁴ Accordingly, the team found that the expression of T cell activation and infiltration markers at the tumor site was significantly increased in responders. As shared by Dr. Renzo-Perales, Geneos Therapeutics, about this study at the recent ASGCT 20204 meeting, “more neos result in better immune responses and also clinical responses.”

Validating neoantigen-specific responses

To validate that the objective responses observed were indeed tied to neoantigen-specific immune activation, Yarchoan et al. leveraged peripheral lymphocytes collected from patients before and following vaccination. The extent and specificity of T-cell activation were evaluated in vitro through interferon-gamma secretion by ELISpot assays following stimulation with neoantigen peptide pools. A commonly used approach in such ELISpot assays is using overlapping peptide pools (e.g., overlapping 15-mers) representing sequences from single or multiple neoantigens unique to each patient.

Custom-made, recombinant, lyophilized peptides specific to each patient were produced (GenScript).⁴

Yarchoan et al. reported positive, neoantigen-specific responses in all patients, with responses increasing following vaccination. Most patients also developed immune reactivity to a greater number of neoepitopes post-vaccination.

Interferon-gamma ELISpot Assay to evaluate T cell responses following combined therapy with a personalized neoantigen vaccine and pembrolizumab.  “b, Total neoantigens (gray bars) and positive neoantigens before (black bars) and after (red bars) vaccination in each patient’s PTCV assessed by IFNγ ELISpot. c, Percentage of positive responding epitopes by groups.⁴ Figure 3, Panels b and c, retrieved from Yarchoan et al. 2024.⁴ http://creativecommons.org/licenses/by/4.0/

Beyond the interferon-gamma ELISpot Assay, neoantigen-specific activation of immune cells was evaluated by lymphocyte intracellular staining. To this end, patient-derived lymphocytes were incubated with corresponding neoantigen peptide pools and stained for various relevant markers, such as activation (CD69 and CD107a) and proliferation (Ki67) markers. Yarchoan and colleagues found neoantigen-specific polyfunctional CD4 and CD8 T cell-mediated immunity in responding patients.

Significantly, the use of a personalized neoantigen vaccine was effective at inducing T-cell clonal expansion. In many of the patients, a greater number and diversity of T cell clones were detected both in circulation and at the tumor site. In several patients, T cell clones infiltrating the tumor were shown to have specificity against neoantigens in the personalized vaccine.

Lastly, in a few patients, the team was able to identify T cell receptor (TCR) sequences from clones frequently infiltrating tumor tissues. To validate neoantigen specificity, they leveraged the identified TCR sequences to engineer autologous T-cells, which were stimulated in vitro with corresponding neoantigen peptide pools. Such a strategy not only enabled them to confirm specific reactivity but also allowed the team to zero in on epitopes inducing high immunogenicity.

Conclusions

Overall, Yarchoan and colleagues demonstrated a clinical benefit for hepatocellular carcinoma patients from combined treatment with the anti-PD1 inhibitor pembrolizumab and a personalized cancer vaccine. Their findings support that this approach could improve outcomes over those achieved with pembrolizumab monotherapy by expanding and diversifying the population of reactive T cells that can infiltrate the tumor site. Ultimately, larger randomized clinical studies are required to confirm the findings in this study.

Reference

1. Robert, C. A decade of immune-checkpoint inhibitors in cancer therapy. Nat Commun 11, 3801 (2020). https://doi.org/10.1038/s41467-020-17670-y

2. Sun, Q., Hong, Z., Zhang, C. et al. Immune checkpoint therapy for solid tumours: clinical dilemmas and future trends. Sig Transduct Target Ther 8, 320 (2023). https://doi.org/10.1038/s41392-023-01522-4

3. Zhang, N., Yang, X., Piao, M. et al. Biomarkers and prognostic factors of PD-1/PD-L1 inhibitor-based therapy in patients with advanced hepatocellular carcinoma. Biomark Res 12, 26 (2024). https://doi.org/10.1186/s40364-023-00535-z

4. Yarchoan, M., Gane, E.J., Marron, T.U. et al. Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial. Nat Med 30, 1044–1053 (2024). https://doi.org/10.1038/s41591-024-02894-y

5. Gopanenko AV, Kosobokova EN, Kosorukov VS. Main Strategies for the Identification of Neoantigens. Cancers (Basel). 2020 Oct 7;12(10):2879. doi: 10.3390/cancers12102879