Neoantigene-Specific T-Cells in TIL Therapy

In February 2024, the US Food and Drug Administration (FDA) approved lifileucel, a new type of adoptive cell transfer (ACT) immunotherapy. It is indicated for advanced melanoma in adults that does not respond to other treatments. While in Lifileucel tumor-infiltrating lymphocytes (TILs) are stimulated unspecifically, researchers are investigating the degree to which neoantigens contribute to tumor regression under TIL therapy. They are also developing methods to selectively expand neoantigen-reactive TILs, with the aim of providing treatment for common solid epithelial cancers.

The approval of lifileucel marks a milestone as the first cellular therapy for a solid cancer. [1] For this treatment, tissue samples are obtained from the growing tumor, TILs are extracted and expanded in the lab using the T cell growth factor interleukin-2 (IL-2). The reinfusion of these TILs into the patient empowers them to directly attack the tumor cells, simultaneously proliferating for a durable tumor control.

TILs have proven more effective than the immune checkpoint inhibitor ipilimumab in treating refractory melanoma, according to a recent study. [2] This efficacy is further enhanced when patients undergo lymphodepleting chemotherapy prior to cell infusion, as it eradicates inhibitory T cells that hinder TIL activity. Post-infusion, IL-2 is administered to foster the survival and proliferation of the transplanted cells. Adoptive transfer of TIL to patients naïve to anti-PD-1 therapy is particularly effective: in one study, 56% of patients with metastatic melanoma responded, with 25% achieving complete and enduring cancer regression. [3]

Neoantigens as targets of TILs

Melanoma is recognized as one of the most immunogenic types of cancer. TILs are known to react to melanoma-associated antigens, cancer-testis antigens such as PRAME and NY-ESO-1, and neoantigens, which are the result of DNA mutations. Given the high frequency of mutations in melanoma, a significant number of neoantigens is produced. The reactivity of T cells against these neoantigens is thought to play a crucial role in managing melanoma. Joost H. van den Berg and his team from the Netherlands Cancer Institute in Amsterdam investigated the correlation between patient-specific neoantigens and tumor regression during TIL therapy. [4] They analyzed the infusion products from three patients, aged 40 to 46, who showed either a partial response (patient II, female) or complete regression (patients I and III, both male) following TIL therapy. This study aimed to understand the extent to which these neoantigens contribute to the therapeutic outcomes observed.

Identification of Tumor-Specific Mutations

Van den Berg and his team identified tumor-specific mutations through the sequencing and comparison of the exome of tumor and autologous healthy peripheral blood mononuclear cells (PBMCs). They filtered potential 9-, 10-, and 11-mer peptides based on three parameters:

1. Likelihood of proteasomal processing.
2. Predicted binding affinity to patient-specific HLA alleles.
3. Predicted similarity between the mutated epitope and the wild-type counterpart.

The researchers synthesized neoantigen peptides matching these parameters and produced major histocompatibility complex (MHC) multimers with these epitopes. Using a multiplex peptide-MHC multimer staining strategy, they then assessed the reactivity of the patients' TILs and PBMCs to the neoantigens.

Neoantigen-Specific TILs and Tumor Regression

In all three patients, neoantigen-specific T-cell reactivity was detected in the TIL products, but with significant differences in the breadth and strength of responses.

• Patient I, male: CD8+ T cells responded to five different neoantigens, with 29% of the CD8+ T cells in the infusion product targeting the RBM12 S>L neoantigen. This patient has been disease-free for over nine years post-TIL therapy.
• Patient II, female: Three neoantigen-specific T-cell populations were identified in the infusion product, with frequencies of 0.007%, 1.14%, and 3.35% of total CD8+ T cells. Additionally, neoantigen-specific CD4+ T cells against one neoantigen constituted 3.8% of CD4+ T cells. The patient experienced partial remission in the first month post-TIL infusion, but the disease recurred after four months, with brain metastases developing six months later. She passed away 16 months after TIL therapy.
• Patient III, male: Showed persistent benefit from TIL therapy. T-cell reactivity was noted for only one neoantigen, accounting for 0.17% of CD8+ T cells, while overall tumor reactivity was 15% of CD8+ T cells. This indicates possible overlooked neoantigens or significant reactivity against non-mutated antigens.

The researchers also examined the expansion of various neoantigen-reactive T-cell pools by analyzing peripheral blood samples at different time points post-therapy. The results showed that in six out of nine analyzed neoantigen-specific T-cell responses, TIL therapy significantly increased the numbers of neoantigen-specific T cells in peripheral blood, with expansions ranging from 8- to 750-fold.

Neoantigen-Specific Memory T cells in peripheral blood

In addition, Gal Cafri and team from the National Cancer Institute in Bethesda, USA, were able to detect memory T cells in the peripheral blood of epithelial cancer patients. [5] These T cells targeted unique and shared somatic mutations. The researchers utilized highly sensitive in vitro stimulation and cell enrichment methods. They loaded dendritic cells with synthesized individual peptides, peptide pools, or RNA encoding tandem minigenes, then co-cultured them with peripheral blood lymphocytes. In three out of six patients with metastatic epithelial cancer, CD4+ and CD8+ memory T cells specific to the mutated variants KRAS^G12D and KRAS^G12V were successfully detected and isolated. In two additional patients with metastatic colon cancer, they identified CD8+ neoantigen-specific cells against the mutated proteins SMAD5 and MUC4. Based on these findings, the authors saw the potential for developing an effective, personalized T cell-based cancer immunotherapy, applicable to severel patients.

Targeting common epithelial cancers

The precise role of neoantigen-specific T-cell populations in shaping clinical responses following TIL therapy necessitates further investigation. Several factors point to a substantial impact:

• The robust in vivo expansion of six out of nine analyzed neoantigen-specific T-cell responses.
• A correlation between tumor mutation burden and the efficacy of TIL therapy.
• Evidence from studies utilizing neoantigen-enriched TIL infusion products.

While melanoma is one of the few cancer types that reproducibly responds to unselected TIL therapy, more than 70% of all epithelial cancers can produce TILs that react with proteins derived from random somatic mutations in the autologous carcinoma. Nevertheless, tumor-reactive TILs are scarce in epithelial cancers, with only about one in a thousand infiltrating T cells exhibiting reactivity. Extending TIL therapy to common solid carcinomas requires techniques to selectively expand tumor-reactive TILs.

Expansion of Neoantigen-Reactive TILs

Therefore, Noam Levin and colleagues, also from the National Cancer Institute in Bethesda, USA, developed the NeoExpand method, an in vitro TIL culture technique. [6] This method selectively expands neoantigen-reactive TILs using TIL fragment cultures, a pool of TIL fragment cultures, or TILs from fresh tumor digests, either directly or after culturing with IL-2 for less than a week.
The researchers tested antigen-presenting cells (APCs) like autologous dendritic cells, B cells, and HLA-modified cell lines for neoantigen-specific stimulation. Identified neoantigens were then introduced into APCs via transient transfection with mutated tandem minigene RNA or viral expression of these RNAs. Additionally, synthesized 24- to 25-mer peptides or predicted minimal epitope peptides were pulsed onto APCs. The peptides were used at concentrations between 10 ng/ml and 500 ng/ml. The TILs were then co-cultured with antigen-loaded APCs in the presence of IL-2 and IL-21.

The process of neoantigen stimulation yielded 16 unique reactivities and 42 T-cell receptors, in comparison to 9 reactivities and 14 T-cell receptors from conventional TIL expansion. The developed method successfully expanded the neoantigen-reactive CD4+ and CD8+ TIL clone repertoire, allowing for the isolation of novel neoantigen-reactive TCRs.
Neoantigen stimulation increased neoantigen-reactive TILs with stem cell-like memory phenotypes expressing IL-7R, CD62L, and KLF2 and enhanced the in vivo antitumor efficacy of TILs in p53- or KRAS-mutated xenograft mouse models.

Conclusions

Adoptive cell transfer therapy with tumor-infiltrating lymphocytes has shown promising results. To enhance response rates even further, understanding the precise role of neoantigen-reactive TILs in treatment success is crucial. Researchers have already made significant strides in developing techniques to selectively expand these potent TILs.

This advancement could potentially extend TIL therapy to less immunogenic forms of cancer, such as metastatic solid epithelial cancer - a type responsible for approximately 90% of all cancer-related deaths. By targeting these previously challenging cases, the impact of adoptive cell transfer therapy could be significantly broadened, offering new hope to cancer patients.

Literature

1. Rosenberg, Steven A. Lymphocytes as a living drug for cancer. Science 385 (2024): 25-26. DOI:10.1126/science.adp1130
2. Rohaan, Maartje W et al. “Tumor-Infiltrating Lymphocyte Therapy or Ipilimumab in Advanced Melanoma.” The New England journal of medicine vol. 387,23 (2022): 2113-2125. doi:10.1056/NEJMoa2210233
3. Seitter, Samantha J et al. “Impact of Prior Treatment on the Efficacy of Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma.” Clinical cancer research: an official journal of the American Association for Cancer Research vol. 27,19 (2021): 5289-5298. doi:10.1158/1078-0432.CCR-21-1171
4. van den Berg, Joost H et al. “Tumor infiltrating lymphocytes (TIL) therapy in metastatic melanoma: boosting of neoantigen-specific T cell reactivity and long-term follow-up.” Journal for immunotherapy of cancer vol. 8,2 (2020): e000848. doi:10.1136/jitc-2020-000848
5. Cafri, Gal et al. “Memory T cells targeting oncogenic mutations detected in peripheral blood of epithelial cancer patients.” Nature communications vol. 10,1 449. 25 Jan. 2019, doi:10.1038/s41467-019-08304-z
6. Levin, Noam et al. “Neoantigen-specific stimulation of tumor-infiltrating lymphocytes enables effective TCR isolation and expansion while preserving stem-like memory phenotypes.” Journal for immunotherapy of cancer vol. 12,5 e008645. 30 May. 2024, doi:10.1136/jitc-2023-008645