Epi-R™ P2 Protocol Produces a Scalable Polyclonal TIL Product With a Greater Expansion Success Rate Across Hot and Cold Tumors in Shorter Culture Time
Abstract 379
Yogin Patel*, Melissa Bedard, Jaewon Yun, Audrey Garces, Saeed Azimi, Mikalya Min, Sammy Chan, Ngoc-Han Ha, Tina Ta, Ben Harris, Shobha Potluri, Melissa DeFrancesco, Carson Harms, Gary Lee, Suman Kumar Vodnala*
*Corresponding author; send inquiries to ypatel@lyell.com or svodnala@lyell.com Lyell Immunopharma, Inc., South San Francisco, CA
Objectives
TIL therapy
• Tumor-infiltrating lymphocyte (TIL) therapy has shown promising efficacy in select solid tumor types, including in patients treated with checkpoint inhibitors (CPI)1-3
• TIL products with higher proportions of stem-like T cells, tumor antigen recognition, and clonal diversity may result in improved clinical outcomes4,5
• Conventional TIL production protocols include long culture times (4-6 weeks) that may reduce TIL stemness and TCR diversity6,7
• Shorter culture duration may be correlated with longer telomere length, increased stemness, improved persistence, and positive clinical outcomes in patients with metastatic melanoma6,7
• Epi-R manufacturing protocols control T-cell activation and differentiation by optimizing cell culture media and other manufacturing steps, resulting in preservation of stem-like qualities (Table 1)8,9
Table 1: Comparison of TIL manufacturing protocols
Standard | Epi-R P1 | Epi-R P2 | |
Time | ++++ | +++ | ++ |
Media | AIM-V™/Optimizer | Epi-R™ | Epi-R™ |
Allogeneic feeder | ++++ | ++++ | + |
CD8/CD4 | ++ | ++++ | ++++ |
Stemness | + | ++++ | ++++ |
Polyclonality | ++ | ++ | +++ |
Methods
• Tissue from 21 donors across tumor types, including CPI-refractory metastatic melanoma, NSCLC, and CRC
͵ TIL were produced from these tumors using either the Epi-R P1 or Epi-R P2 protocols (Figure 1)
• Characteristics of the resulting products (Epi-R P1 and Epi-R P2) were compared using a matrix of assays including:
͵ Flow cytometry to measure markers of stemness
͵ Co-culturing with an autologous tumor cell line/tumor digest ͵ TCRβ sequencing to assess polyclonality
Figure 1: Planned manufacturing schematic for Lyell's Epi-R P2 protocol
Harvest TIL
Freeze | ||||||||
Infusion | Shipped to | product | Expansion 2 | |||||
(less than 2 weeks) | ||||||||
NSCLC | patient | |||||||
Single cell | ||||
suspension | ||||
CRC | Tumor | Expansion 1 | ||
MM | processing | + | (less than 2 weeks) | |
Tumor removed | Shipped to | |||
by surgeon | Tumor | manufacturing site | ||
sample | ||||
Stimulating | ||||
cocktail |
Results
Epi-R P2 protocol reduced the duration of culture time without compromising the favorable phenotype of the product
Epi-R P2 reduced terminally differentiated cells and preserved the number of tumor-reactive clones present in the product
• The Epi-R P2 protocol shortened TIL manufacturing time to less than 25 days
• Epi-R P2 initial expansion phase lasted less than 2 weeks and yielded significantly more TIL compared with the Epi-R P1 initial expansion phase (approximately 2 weeks) in CPI-naïve metastatic melanoma, CRLM, NSCLC (Figure 2A), and in CPI-refractory metastatic melanoma (Figure 2B)
͵ In the selected tumor types (n = 21), the Epi-R P2 initial expansion phase resulted in a median fold
• The Epi-R P2 protocol preserved polyclonality (Figure 5A) and the top 50 clone frequencies in TIL products (Figure 5B)
Figure 5: Tumor-reactive clones were preserved with the Epi-R P2 protocol
expansion of 37.44, with a median yield of 1.4 x 109 T cells
• After a second expansion step, the Epi-R P2 protocol produced an average of 6.0 x 1010 T cells in different tumor types including CPI-refractory metastatic melanoma, metastatic CRC, and NSCLC (Figure 3)
A
Polyclonality
0.5
B
clones
Top tumor clone frequency | ||
40 | in TIL product | |
Figure 2: Epi-R P2 produced more TIL in a shorter amount of time
A | Multiple | B | CPI-refractory | |
metastatic | ||||
tumor types | 150 | melanoma | ||
80 | ||||
60 | ||||
Fold expansion | Fold expansion | 100 | ||
40 | ||||
50 | ||||
Figure 3: Epi-R P2 produced more TIL across multiple tumor types
2.5x1011
2.0x1011
yield | x | 11 |
cell | 1.5 | 10 |
Projected | x | 11 |
1.0 | 10 |
Simpson clonality
0.4
0.3
0.2
0.1
0
Epi-R P1 | Epi-R P2 |
Sum of frequencies of top 50 tumor
30
20
10
0
Epi-R P1 | Epi-R P2 |
20 | |
0 | 0 |
Epi-R P1 Epi-R P2 | Epi-R P1 Epi-R P2 |
5.0x1010
0.0
CPI-refractory | Metastatic | NSCLC |
melanoma | CRC |
MHC-I blockade prevented TIL reactivity and effector functions
• Figure 6A shows representative flow cytometry of Epi-R P2 TIL incubated with or without MHC-I block and autologous NSCLC tumor cells
• CD3+ cells from four tumor samples (three NSCLC and one metastatic breast cancer) demonstrated clustering based on the expression of markers involved in T-cell differentiation, stemness, activation, and exhaustion (Figure 6B)
Epi-RP2-derived TIL demonstrated comparable product profiles compared with Epi-RP1-derived TIL
• Epi-R P1 and Epi-R P2 protocols showed a similar proportion of CD8+ T cells in TIL products derived from CPI-refractory melanoma, NSCLC, and CRC (data not shown)
• Epi-R P2 T cells had retained or improved expression of stemness markers (CD39-CD69-;Figure 4A) and reduced differentiation (data not shown) across all tumor types
• Epi-R P2 T cells retained expression of stem-like markers including CD62L and CD27 (Figure 4B-C)
• CD8+ T cells demonstrated a greater fold change in IFNγ secretion and co-stimulatory molecules (Figure 6C-D)
• Specificity of the TIL reactivity activation and effector functions were completely blockable by anti-MHC-I
antibody (Figure 6C-D).
Figure 6: MHC-I blockade prevented TIL activation and cytokine secretion
A | No MHC-I Block | MHC-I Block | C | IFNγ | D | % 4-1BB+ |
population |
Figure 4: CD8+ product profiles of Epi-R P1 and Epi-R P2 TIL were comparable
Stem-like CD8+ T cells
A | 100 | NS | B | 80 | NS | C | 100 | |
T cells | % CD62L+CD27+CD8+ T cells | |||||||
80 | 60 | % CD27+CD8+ T cells | 80 | |||||
Melanoma | CD8+ | |||||||
60 | 60 | |||||||
- | 40 | |||||||
CD69 | ||||||||
40 | 40 | |||||||
- | 20 | |||||||
% CD39 | 20 | 20 | ||||||
0 | 0 | 0 | ||||||
cells | 100 | * | cells | 50 | * | 80 | ||
NS |
NS |
77.1 | 17.5 | 97.3 | 0.089 |
CD8
5.17 | 0.27 | 2.60 | 0.030 | |
CD137/41BB | ||||
B
t-SNE 2
t-SNE 1
Fold change
80
70
60
50
40
30
20
10
0
+ MHC-I | - MHC-I |
block | block |
Fold change
60
50
40
30 | |
20 | |
10 | |
0 | - MHC-I |
+ MHC-I | |
block | block |
T | 80 | % CD62L+CD27+CD8+ T | 40 | % CD27+CD8+ Tcells | 60 | |||
CD8+ | ||||||||
NSCLC | 60 | 30 | ||||||
- | 40 | |||||||
CD69 | ||||||||
40 | 20 | |||||||
- | 20 | |||||||
% CD39 | 20 | 10 | ||||||
0 | 0 | 0 | ||||||
T cells | 80 | * | % CD62L+CD27+CD8+ T cells | 80 | NS | 80 | ||
60 | 60 | % CD27+CD8+ T cells | 60 | |||||
CD8+ | ||||||||
CRC | ||||||||
- | 40 | 40 | 40 | |||||
CD69 | ||||||||
- | 20 | 20 | 20 | |||||
% CD39 | ||||||||
0 | 0 | 0 | ||||||
Epi-R P1 | Epi-R P2 *P<0.05 | |||||||
Conclusions |
Clones retained during the Epi-R P2 protocol demonstrated cytotoxicity against autologous tumor cell lines
• The putative tumor-reactive clones preserved using the Epi-R P2 protocol exhibited strong cytotoxicity of an autologous tumor cell line (Figure 7)
Figure 7: Epi-R P2 TIL preserved tumor-reactive clones and demonstrated cytotoxicity when cultured with autologous tumor cells
NS | A | Top 50 | Top 50-100 | Confirmed tumor | B | Epi-R P1 | Epi-R P2 | Tumor only | |
100000 | reactivity | 100 | |||||||
TIL) | 10000 | ||||||||
80 | |||||||||
104 | 1000 | ||||||||
x | 60 | ||||||||
3 | 100 | ||||||||
size | Cytolysis% | ||||||||
to | |||||||||
Clone | 10 | 40 | |||||||
(normalized | 1 | 20 | |||||||
0.1 | |||||||||
0.01 | 0 | ||||||||
30 | Hours | 40 | 50 | ||||||
D0-Tumor | Epi-R P1 | Epi-R P2 | |||||||
• Epi-R P2 demonstrated successful TIL expansion from both immunologically hot (metastatic melanoma and NSCLC) and cold (CRC) tumors and comparable product profiles to Epi-R P1 using a shortened process
• Epi-R P2 retained all the improved product characteristics associated with Epi-R P1 across several tumor types, including a greater proportion of stem-like T cells and an increase in the magnitude of the most frequent clones that were present in the baseline tumor
• The Epi-R P2 protocol is a promising method for manufacturing TIL products with retained antitumor activity in both immunologically hot and cold tumors
Abbreviations
4-1BB, tumor necrosis factor ligand superfamily member 9; CD, cluster of differentiation; CRLM, colorectal cancer liver metastases; CPI, checkpoint inhibitor; CRC, colorectal cancer; IFNγ, interferon gamma; MHC, major histocompatibility complex; MM, metastatic melanoma; NS, not significant; NSCLC, non-small cell lung cancer; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte; t-SNE,t-distributed stochastic neighbor embedding.
References
- Creelan BC, et al. Nat Med. 2021;27:1410-1418.2. Seitter SJ, et al. Clin Cancer Res. 2021;27:5289-5298.3. van den Berg JH, et al. J Immunother Cancer. 2020;8:e000848. 4. Vodnala SK, et al. Science. 2019;363:eaau0135.
- Rosenberg SA, et al. Clin Cancer Res. 2011;17:4550-4557.7. Tran KQ, et al. J Immunother. 2008;31:742-51.8. Patel Y, et al. Poster presented at the SITC 37th Annual Meeting 2022, November 8-12, 2022. Abstract 370.
- Patel Y, et al. Poster presented at the AACR Special Conference: Tumor Immunology and Immunotherapy 2022, October 21-24, 2022. Abstract A54.
5. Gattinoni L, et al. Nat Med. 2017;23:18-27.
Acknowledgments
We would like to thank Smita Ghanekar, Ken Xiong, and Sahithi Cheemalamarri for flow core services. The authors would like to thank Shahin Shafiani for editorial support. Medical writing and additional editorial support were funded by Lyell Immunopharma and provided by Madison Fagan, PhD of BOLDSCIENCE Inc.
Presented at SITC Annual Meeting 2023; Nov 1-5; San Diego, CA, USA
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Lyell Immunopharma Inc. published this content on 03 November 2023 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 03 November 2023 16:19:15 UTC.