RESEARCH ARTICLE

Modulation of intracellular kinase signaling to improve TIL stemness and function for adoptive cell therapy

Hao Feng | Ling Qiu | Zixiao Shi | Yao Sheng | Peipei Zhao | Di Zhou
Fei Li | Hailin Yu | Yanan You | Hui Wang | Ming Li | Shurong Zhu

¹ Department of Gynecology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, People's Republic of China
² Grit Biotechnology Co., Ltd. Shanghai, Shanghai, People's Republic of China

Correspondence

Yarong Liu, Grit Biotechnology Co., Ltd. Shanghai, #1077 Zhangheng Road, Shanghai, 201210, People's Republic of China.

Email: yarong.liu@grit-bio.com

Hua Jiang and Xin Wu, Department of Gynecology, Obstetrics and Gynecology Hospital of Fudan University, #128 Shenyang Road, Shanghai 200090, People's Republic of China.
Email: jianghua@fudan.edu.cn and wuxin_fc@fudan.edu.cn

Funding information

Outstanding Excellence Clinical Innovation Team Project of the Obstetrics and Gynecology Hospital of Fudan University, Leading Talents Project of Huangpu District of Shanghai District Committee and Government, The Cervical Cancer Precision Treatment project of Fudan University Education Development Foundation. ZaiDing-Le Foundation from Beijing Kanghua Foundation for the Development of Traditional Chinese and Western Medicine, Grant/Award Number: KH-2020-LJJ-008; Natural Science Foundation of Shanghai, Grant/Award Number: 19ZR1407000; Pilot construction project of high level universities in Shanghai, Grant/Award Number: DGF501017-06; Shanghai Shenkang Hospital Development Center's Shenkang Promotion of Clinical Skills and Clinical Innovation in Municipal Hospitals Three-Year Action Plan (2020-2023) Major Clinical Research Project, Grant/Award Number: SHDC2020CR1048B;

Abstract

Introduction: Adoptive cellular therapy with tumor-infiltrating lymphocytes (TIL) has demonstrated promising clinical benefits in several solid tumors, but the efficacy of this therapy might be compromised by the “prone-to-exhaustion” phenotype of TIL and poor persistence in vivo. This calls for a robust expansion process to produce a large number of cells for clinical usage while at the same time maintaining favorable anti-tumor function and memory phenotype. Previous studies showed that the PI3K-AKT signaling pathway plays a key role in the regulation of T cell activation, differentiation, and memory formation.

Method: We modulated the PI3K-AKT pathway in TIL isolated from cervical and ovarian cancer by application of AKT or PI3K inhibitors or CRISPR knockout of AKT1 and/or AKT2, and characterized their effects on TIL phenotype and effector function. Mechanistic study was further performed with RNA-seq analysis of AKT1/2 KO TIL in comparison to control TIL.

Result: The inhibition of either PI3K or AKT led to an increase in the population of effector CD8 T cells with upregulation of activation markers, elevated memory T cells, and significantly enhanced cytotoxicity when cocultured with tumor cell lines and patient-derived tumor samples. Moreover, dual knockout of AKT1 and AKT2 largely phenocopies the functional impact of AKT or PI3K inhibition on TIL. This result was further validated by RNA-seq analysis indicating that AKT1/2 ablation primarily regulates T cell differentiation and function-related programs.

Conclusion: Modulation of PI3K-AKT signaling represents a promising strategy to enhance TIL stemness and cytotoxicity and improve the clinical outcome of current TIL-based therapy to treat solid tumors.

Hao Feng and Ling Qiu have equal contribution to this manuscript.

KEYWORDS

AKT inhibitor, AKT KO, PI3K inhibitor, TIL manufacture, tumor-infiltrating lymphocyte therapy

1 | INTRODUCTION

Adoptive cellular therapy with tumor-infiltrating lymphocytes has emerged as one of the most powerful therapies for late-stage solid tumors. TIL was collected from tumors of a patient through biopsy or surgery, amplified in culture with interleukin-2 (IL-2) to a clinically relevant level and then reinfused back intravenously to the patient preconditioned with a lymphodepleting regimen. Compared with CAR-T cell therapy showing remarkable clinical efficacy for certain hematological malignancies, TIL therapy holds unique advantages for treating solid tumors, including its diverse TCR clonality to recognize heterogeneous tumor antigens, superior homing capacity to the tumor site and response rates of 40%–50% have been reported in metastatic melanoma. Recently, a phase 2 trial of TIL therapy showed an objective response rate (ORR) of 44.4% in cervical cancer patients treated with multiple lines of conventional therapies. Moreover, TIL therapy demonstrated promising efficacy in patients with progressive disease following anti-PD-1 therapy: 36.4% ORR in melanoma patients and more than 20% in patients with metastatic non-small cell lung cancer (NSCLC).

The attributes of TIL are a major factor contributing to the clinical outcomes after adoptive cell transfer. In vitro cytotoxicity of TIL against autologous or HLA-matched tumor cells has previously been associated with clinical response. On the other hand, longer telomeres and higher persistence in peripheral blood 1 month after transfer is correlated with better objective responses, indicating that a memory-like phenotype might contribute to clinical efficacy. However, TIL is intrinsically prone to an exhaustion phenotype after being repeatedly stimulated by tumor antigens in the tumor site. Moreover, current manufacturing methods focus primarily on expanding TIL to large numbers, especially for tumor samples with limited T cell infiltration as starting material. This procedure could result in driving TIL to a more differentiated or exhausted phenotype. Indeed, the high dose IL-2 commonly used in the culture for TIL expansion was shown to promote TIL exhaustion. Therefore, modification of the current TIL manufacturing process in a way that would uncouple TIL expansion and differentiation would be highly desirable.

The PI3K/AKT pathway is a major signaling pathway mediating T cell proliferation and differentiation in response to T cell activation signals, including TCR, co-stimulation, and cytokine signaling. Previous studies showed that inhibition of AKT, or genetic deletion of the p110δ subunit of PI3K, had no effect on T cell expansion or survival. A less differentiated T cell phenotype and enhanced anti-tumor efficacy in a mouse model was observed in CAR-T cells after pharmacologic inhibition of AKT signaling without compromising T cell expansion. Similarly, AKT inhibition was shown to expand TIL from melanoma patients with a memory phenotype and improved persistence after adoptive transfer in vivo. Nevertheless, previous studies of AKT inhibition on cellular therapy mainly focus on memory and metabolic profile characterization. Therefore, the phenotypic and functional profile of TIL after modulation of the PI3K/AKT pathway, especially its anti-tumor cytotoxicity, remains to be fully explored to support its potential application in the large-scale TIL manufacturing process. Furthermore, it is not known if a similar effect would be seen in TIL from solid tumors apart from melanoma. Here, we studied the functional impact of modulating the PI3K/AKT pathway in TIL from cervical and ovarian cancer by applying PI3K and AKT inhibitors or dual KO of AKT1/2. Mechanistic study was subsequently performed by RNA-seq analysis.

2 | RESULTS

2.1 | Pharmacologic inhibition of PI3K or AKT signaling promotes TIL proliferation and stemness

TIL was expanded from cervical and ovarian tumors with the manufacturing procedure. In brief, tumor surgical specimens were minced into fragments, and TIL was cultured in a previous rapid expansion phase (preREP) with high dose IL-2, followed by amplification to a large number in a rapid expansion phase (REP).

We first sought to confirm whether PI3K or AKT inhibition would impact the expansion and memory phenotype of TIL. REP of TIL was performed in the continuous presence of 1 μM of AKT inhibitor (AKT Inhibitor VIII; AKTi) or 20 nM PI3K inhibitor (idelalisib; PI3Ki) with 3000 IU/ml IL-2. No significant influence of PI3K or AKT inhibition was observed on TIL expansion during REP in three independent donors. However, application of AKTi, but not PI3Ki, significantly promoted the proliferation of the final TIL product post REP stage where IL-2 had been withdrawn. This result suggests that the proliferation advantage endowed by AKTi might be overridden by high-dose IL-2 during REP stage. Moreover, both AKTi and PI3Ki could promote enrichment of the CD8 T cell population, but not the CD4 T cell population in four different donors.

The transcription factor TCF1, a marker for stem cell-like T cells, plays a critical role in inducing and maintaining T cell stemness as well as preserving CD8 T cell functionality upon exposure to tumor antigens or viral infection. Either AKTi or PI3Ki could increase TCF-1 expression in both CD4 and CD8 T cell populations. In addition, a memory-progenitor stem-like phenotype (CD39-CD69-) plays a critical role in TIL persistence and complete cancer regression in patients. Tumor-reactive CD39-CD69- TILs were capable of self-renewal and expansion and demonstrated superior antitumor response and persistence in vivo. This TIL subset was increased in both CD4 and CD8 T cell populations after PI3K or AKT inhibition compared to the control group, indicating its higher stemness and potential better in vivo efficacy and persistence. Therefore, PI3K/AKT pathway inhibition promotes TIL proliferation and stemness and facilitates the enrichment of CD8 T cells.

2.2 | AKT or PI3K inhibitor increases TIL activation and cytotoxicity

Next, we further examined the effect of PI3K and AKT inhibitors on TIL functionality. TIL with exposure of PI3K and AKT inhibitors showed increased CD25 and CD28 expression in the CD8 T cell population, suggesting a stronger TIL activation status upon inhibition. Exhaustion markers, including PD-1, LAG-3, TIM-3, CD38, and CD101, were also analyzed on the final TIL product and no significant difference was observed between the treated or non-treated group. The tumor-killing ability of TIL was performed on both HeLa cell line and autologous tumor cells (ATC) from a cervical cancer patient. TIL with AKT inhibition showed significantly stronger cytotoxicity against both HeLa and ATC compared with the control TIL group. In contrast, PI3K inhibition only showed improved TIL cytotoxicity to HeLa cells in one donor, but no effect on TIL killing against ATC, indicating AKTi may play a more important role in the regulation of TIL function compared with that of PI3Ki.

2.3 | AKT1/2 double KO enhances TIL proliferation and stemness

Given the stronger functional impact of AKTi compared to that of PI3Ki, we further studied whether AKT knockout could lead to a similar functional benefit in TIL. Through a screening experiment, guide RNAs (gRNA) with the highest knockout efficiency were selected from several candidates. AKT1 or AKT2 was knocked out independently using CRISPR/Cas9 technology with mock transfected TIL as a control. Both AKT1 and AKT2 single KO upregulated the expression of IL-7R on CD4 and CD8 TIL, but no effects were observed on TIL expansion, stemness, activation, or cytotoxicity.

As AKT Inhibitor VIII is a potent selective inhibitor of AKT1 and AKT2 with IC50 values of 58 nM and 210 nM, respectively, we hypothesized that AKT1 or AKT2 KO may not have sufficiently blocked AKT signaling in the previous experiment. Therefore, we generated AKT1 and AKT2 double knockout (AKT1/2 KO) TIL from cervical cancer and ovarian cancer. Total AKT expression in TIL was reduced by 89.8% after AKT1/2 KO, as indicated by flow cytometry.

To test the effect of AKT1/2 KO during REP stage, the final product of AKT1/2 KO, or control TIL was cultured with or without anti-CD3 restimulation. AKT1/2 KO significantly promoted TIL proliferation either with or without anti-CD3 compared to the control group. Like AKTi, AKT1/2 KO facilitated CD8 T cell expansion over that of the CD4 T cell population during REP stage in TIL from several different donors. Moreover, AKT1/2 KO increased the proportion of CD39-CD69- memory T cell and naive T cell (CD45RO-CD62L) population as well as IL-7R expression in TIL. To further determine the influence of AKT1/2 KO on TIL memory and persistence, we performed long-term culture of TIL after REP stage with no, or low, dose of IL-2. Results showed that AKT1/2 KO significantly increased the long-term survival of TIL without or with 300 IU/ml IL-2. Therefore, AKT1/2 KO showed effects consistent with those of AKT inhibition on CD8 T cell enrichment and memory phenotype preservation, as well as enhanced TIL long-term persistence in vitro.

2.4 | AKT1/2 KO enhances TIL activation, cytokine production and cytotoxicity

We further tested whether AKT1/2 KO could promote anti-tumor function of TIL in a manner similar to that seen in AKT inhibition. AKT1/2 KO increased CD25 and CD28 expression in both CD4 and CD8 TIL, which is consistent with the effect of AKTi. Further analysis showed that AKT1/2 KO promoted IFN-γ, GZMB, and TNF-α production in both CD4 and CD8 T cell subsets in response to anti-CD3 restimulation. Moreover, AKT1/2 KO enhanced tumor killing of TIL against tumor cell lines HeLa or Hey-T30 compared with the control group.

2.5 | AKT1/2 KO facilitates the transcriptional program associated with T cell function and memory in TIL

Having shown that pharmacologic inhibition or CRISPR KO of AKT in TIL promotes T cell memory and effector function, we further explored the transcriptional programs regulated by AKT in human TIL. The transcriptome of AKT1/2 KO and mock transfected TIL was analyzed by RNA-seq. To visualize the transcriptome between the two groups, we performed principal component analysis (PCA) of the RNA-seq data. It showed clear segregation between AKT1/2 KO and control groups between the two donors. Whole-transcriptome analysis revealed 582 differentially expressed genes (DEGs) out of 23,209 detected genes, and the DEGs are illustrated in volcano plot with |log2FC| > 0.25 and FDR <0.05 as cutoff. AKT1 and AKT2 were significantly downregulated in the AKT1/2 KO group, validating the efficiency of CRISPR KO in TIL. To understand the biological pathways in which the DEGs are involved in, we applied analysis of Gene Ontology Biological Processes (GO BP) and observed enrichment of these genes in lymphocyte activation, differentiation, and apoptosis, as well as cytokine-related signaling. Unsurprised clustering of the representative genes in these pathways demonstrated segregation between AKT1/2 KO and control TIL. We observed significantly upregulated CCR7 and IL2RA and downregulated PDCD1 and FASLG was observed in AKT1/2 KO TIL, which is consistent with our findings that AKT1/2 KO enhances TIL memory and function, but prevents apoptosis.

3 | DISCUSSION

Considering the promising efficacy of TIL in metastatic solid tumors, refining the current manufacturing methodologies to improve TIL function and stemness without sacrificing of its expansion potential may benefit clinical outcomes of TIL therapy in multiple late-stage solid tumors. In this study, we have shown that application of AKT or PI3K inhibitors during REP stage of TIL from cervical or ovarian cancer favors generation of the effector CD8+ T cell population with higher activation marker expression and more TCF-1+ and CD39-CD69- memory T cells in both CD4+ and CD8+ T cell populations, even while failing to impact, or even enhance TIL proliferation post-REP. On the other hand, AKTi or PI3Ki did significantly increase TIL cytotoxicity in the co-culture with tumor cell lines and autologous tumor cells. Furthermore, dual knockout of AKT1/2 in TIL during REP largely phenocopies AKT inhibition, as further confirmed by the RNA-seq analysis. These findings have positive implications in overcoming the current limitations of TIL therapy in solid tumors.

A high proportion of CD8+ T cells in the final TIL product, in vitro cytotoxicity against autologous tumor cells, and in vivo persistence of TIL post transfer have been associated with improved clinical response in metastatic melanoma. AKT inhibition or deletion increases (1) the percentage of CD8+ T cells at the end of REP, (2) cytokine production including IFN-γ, TNF-α and GZMB production upon restimulation in response to anti-CD3/anti-CD28, and (3) tumor killing ability of TIL to patient-derived tumor cells, all promising better antitumor efficacy of TIL in patients. Moreover, enhanced T cell memory phenotype and long-term survival with low dose of IL-2 and decreased apoptosis after AKT inhibition or ablation may lead to better persistence of TIL in vivo. Modulation of AKT during TIL's rapid expansion stage might contribute to better clinical outcome, which would be especially meaningful for ovarian or cervical cancer that generally has a lower response rate of TIL-based therapy compared to melanoma.

4 | METHODS

4.1 | TIL preparation

Tumor surgical specimens were obtained from cervical and ovarian cancer patients. This study was approved by the Medical Ethics Committee of Obstetrics and Gynecology Hospital of Fudan University, with the Approval number 2021– 07.

4.2 | Cell culture

Tumor cell lines (HeLa and Hey-T30, ATCC) were maintained in D10 medium. TIL was maintained in T cell media during Pre-REP and REP stage.

4.3 | Cas9/RNP nucleofection

Deletion of AKT1 and/or AKT2 in TIL was performed using CRISPR/Cas9 technology.

4.4 | Antibodies and flow cytometry

Cells were stained with fluorochrome-conjugated antibodies.

4.5 | AKT expression

TIL was harvested during REP, washed with PBS and then stained with anti-AKT antibody.

4.6 | CD107a detection, cytokine intracellular staining and apoptosis analysis

TIL was harvested during REP. Cells were then stained with surface markers and permeabilized.

4.7 | Tumor killing monitor

Target cells (HELA, HEY-T30 or autologous tumor cells) were seeded in a 96-well plate followed by addition of tumor killing substrate to each well.

4.8 | Proliferation analysis

TIL was harvested, washed with PBS and seeded in a 96-well plate with or without stimulation.

4.9 | Total RNA extraction

Total RNA was extracted from the tissues using Trizol and RNAprep pure Cell kit.

4.10 | mRNA library construction

Oligo(dT)-attached magnetic beads were used to purify mRNA.

4.11 | RNA sequencing

The sequencing data was filtered with SOAPnuke.

4.12 | Differential gene expression analysis

R and R packages were used to find and analyze differential expressed genes (DEGs).

4.13 | Quantification and statistical analysis

Statistical analyses were performed using Prism 8.4.

AUTHOR CONTRIBUTIONS

X.W., H.J., Y.L., J.S., and J.C. conceptualized and designed the study. H.Y., Y.Y., H.W., M.L., and S.Z. contributed to sample preparation. H.F., L.Q., Z.S., Y.S., P.Z., Y.D., and F.L. carried out the experiments. H.F., L.Q., Z.S., Y.S., P.Z., and D.Z. performed analyses. H.F., L.Q., Z.S., and Y.S. wrote the manuscript. X.W., H.J., Y.L., and J.S. reviewed and edited the manuscript. All authors approved the final manuscript.

CONFLICT OF INTEREST

Z.S., Y.S., P.Z., D.Z., F.L., J.C., J.S., and Y.L. were employed by Grit Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

ETHICS STATEMENT

This study was approved by the Medical Ethics Committee of Obstetrics and Gynecology Hospital of Fudan University, with the Approval number 2021– 07. Written informed consent was obtained from patients for use of tissue samples.

DATA AVAILABILITY STATEMENT

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

REFERENCES

1. Wang S, Sun J, Chen K, et al. Perspectives of tumor-infiltrating lymphocyte treatment in solid tumors. BMC Med. 2021;19:140. doi:10.1186/s12916-021-02006-4

2. Geukes Foppen MH, Donia M, Svane IM, Haanen JBAG. Tumor-infiltrating lymphocytes for the treatment of metastatic cancer. Mol Oncol. 2015;9:1918-1935. doi:10.1016/j.molonc.2015.10.018

3. Jazaeri AA, Zsiros E, Amaria RN, et al. Safety and efficacy of adoptive cell transfer using autologous tumor infiltrating lymphocytes (LN-145) for treatment of recurrent, metastatic, or persistent cervical carcinoma. J Clin Oncol. 2019;37:2538-2538. doi:10.1200/JCO.2019.37.15_suppl.2538

4. Sarnaik AA, Hamid O, Khushalani NI, et al. Lifileucel, a tumor-infiltrating lymphocyte therapy in Metastatic Melanoma. J Clin Oncol. 2021;39:2656-2666. doi:10.1200/JCO.21.00612

5. Schoenfeld AJ, Lee S, Paz-Ares L, et al. First phase 2 results of autologous tumor-infiltrating lymphocyte (LN-145) monotherapy in patients with advanced, immune checkpoint inhibitor-treated, nonsmall cell lung cancer. J Immunother Cancer. 2021;27:A1-A1054. doi:10.1136/jitc-2021-SITC2021.458.