SITC 2023: Rejuvenation of tumor-infiltrating lymphocytes (TIL) through Partial Reprogramming

Rejuvenation of Tumor-Infiltrating

Lymphocytes (TIL) Through Partial Reprogramming

	Takuya Maeda1, Yin Huang1, Jessica Fioravanti1, Naritaka Tamaoki1, Burak Kutlu2, Yasuhiro Yamazaki1, Kriti Bahl1, Biao Wang1, Zheng Zhong1, Shobha Potluri1,
	Gary Lee1, Nicholas P. Restifo1, and Raul Vizcardo1
Abstract 393	1Lyell Immunopharma, Inc., South San Francisco, CA; 2Lyell Immunopharma, Inc., Seattle, WA

Abstract

• TIL therapy is a promising approach for the treatment of advanced solid

tumors; however, efficacy is limited by T-cell exhaustion and terminal

differentiation1,2

• Recent studies have highlighted the detrimental effects of aging in

T cells, including reduced T-cell function, and the efficacy of solid tumor

Figure 1: Restoring T-cell function and antitumor potential through Rejuvenation

Partially reprogrammed cells

Reprogramming

cell therapy3,4

• Reprogramming T cells to iPSCs and re-differentiating back to a T-cell

lineage has proven complex and time-consuming,5-7 as reprogramming via

iPSCs requires each TCR in the final product to be derived from an individual

iPSC clone

• To overcome these barriers, Lyell has developed a novel technology called

Rejuvenation, which does not require full reprogramming to iPSCs (Figure 1)

• Here, we report that rejuvenated TIL (TILRJ) retain polyclonality, have reduced

epigenetic age, and improved cellular function

Initial T-cell population

factors

Full reprogramming

iPSC colonies

Redirection to conventional T cells

Rejuvenated

T cellsT-cell reprogramming with persistent reprogramming factor expression results in dedifferentiated iPSCs. In contrast, Lyell's Rejuvenation technology uses partial reprogramming that maintains T-cell identity and improves T-cell function without complex iPSC re-differentiation steps.

Results

Novel T-cell Rejuvenation through partial reprogramming	Figure 4: Restoring TIL function and antitumor potential through					Figure 7: Rejuvenation improved stemness phenotype and cellular
• With Lyell's novel T-cell Rejuvenation technology, aged peripheral	Rejuvenation																											proliferation capacity in NSCLC TIL
	blood (PB) T cells transiently express transcription factors																			Prestimulation		Reprogramming
	associated with iPSC reprogramming in a non-genomic																										A											B		TCT
	integrative manner																																													91.9		4.63		1.16			0.82	1000
																																Reprogramming
• Once successfully redirected to T cells, rejuvenated T cells (TRJ)													Tumor																				TCT						92.0						800	TRJ
																							factors
	acquire the following favorable features compared to control T																	TIL	Cytokines																									39.8			58.2	change	600
	cells (TCT): (Figures 2 and 3)
																																													90.1				21.6			62.8	Fold	400
	͵ Reduced epigenetic age																																														33.9
	͵ Enhanced proliferation and metabolism																																			Partially					TRJ	CD3		CD4			62.6	CCR7					200
	͵ Preservation of stemness markers																																reprogrammed cells																0	5	10	15	20
																																																	5.82			9.77		0
• The TRJ do not require complex redifferentiation steps; thus																																										L/D		CD8			CD62L						Days
Redirection			Expansion
	reducing the time required for reprogramming to iPSC and																								TIL derived from a patient with NSCLC were subjected to Rejuvenation. (A) TRJ
	re-differentiation to conventional T cells
																																										demonstrated a better-preserved TCM phenotype (CD62L+ CCR7+) compared with TCT.
• Data demonstrate the capacity to partially "turn back" the																							Rejuvenated	Rejuvenated T cells exhibit:​				(B) TRJ demonstrated greater cellular proliferation compared with TCT.
	epigenetic clock of a T cell without returning the cell to the																								T cells		• Reduced epigenetic age
	pluripotent state associated with complete iPSC reprogramming																										•  Enhanced cell proliferation
																										and metabolism							Rejuvenation improved the antitumor efficacy of
																																		• Improved antitumor potency
																																		•  Preservation of stemness				engineered T-cell therapy in vitro and in vivo
																				Rejuvenated TILs								markers and TCR repertoire
Figure 2: Rejuvenated peripheral blood T cells from healthy donors																																• The NY-ESO-1 TCR transduced system was used as a surrogate
													(TILRJ)
showed reduced epigenetic age and increased proliferation																																									model of TIL Rejuvenation
A		Reprogramming	Redirection		Expansion	Figure 5: Rejuvenation improved cellular proliferation capacity,						• NY-ESO-1 TCR transduced T cells were rejuvenated, and their
								function was assessed by cytokine production assays and
		stemness phenotype, and epigenetic age while maintaining the
															sequential killing assays
	Reprogramming						CD4+/CD8+ population and TCR repertoire in metastatic melanoma TIL
	Partially																																											• NY-ESO-1 T		showed better proliferation, reduced epigenetic
	factors
			reprogrammed cells																																														RJ
									A																			B																				age, higher cytokine production, and better persistence upon
													106							106	21.7				106	93.2			106	3.01					106								repeated encounter with target cells (Figure 8)
													107	99.2			0.019	107						107										107	0.42			1.12
									TCT			105							105					TCT		104				104				94.2	104							• In an in vivo tumor model, NY-ESO-1 TRJ showed suppression of
											104							104
																														105										105
																																		105														tumor growth and improved probability of survival compared with
															2	0.75		4.99E-3		2			76.8				-104				103						-104	33.1			65.3
																														0										0
							Rejuvenated T cells						103							103																									NY-ESO-1 TCT (Figure 9)
												10								10
																102	103	104	105	106	107			103	104	105	106			103				102	103	104	105	106	107		-104	0	104	105	106	107
B				C										107	4.99			0.27	107	15.3						107	64.7			24.1					107	15.0			9.98
												106							106						106							106
							TRJ																	TRJ						106				68.1
50			106						105							105						105				104				105
			TCT						104							104						104									104
(y)												CD3									CD4 102						CD3					105						CCR7-104							Figure 8: Rejuvenation improved cellular proliferation capacity and
			TRJ	105							102	45.0			49.8			0.49			-104				CD4103						49.5			25.6
													103							103							0										0
Epigeneticage				Foldchange	100		TCT Donor 2	TRJ Donor 2					60																	150																1x10		CD4 TCT									60
	40				104											102	103	104	105	106	107			103	104	105	106			103	104	105	106	102	103	104	105	106	107		-104	0	104	105	106	107	epigenetic age, and enhanced the functional properties of CD4+ and
					103				C																					D																	CD8+NY-ESO-1 TCR T cells in vitro
					102							80				CD62L-CCR7+								200		CT
	30																												T
				101														CD62L+CCR7+											TRJ
								Foldchange	fromD0							CD62L+CCR7-			TRJ			Foldchange fromD0																changeFold	1x100									Epigeneticage (y)
																	TCT
							TCT Donor 1	TRJ Donor 1											CD62L-CCR7-																								A		6								B
							TCT Donor 3	TRJ Donor 3																																								1x105	CD4 TRJ
	20				10-1								40																	100																		CD8 TCT
	Donor 1	Donor 2	Donor 3	0	10	20	30																																								1x104										40			Donor age
							Days						20																	50																1x103
																																												1x102
(A) Schematic representation of T-cell Rejuvenation process. (B) PB T cells from three																																																	20
healthy donors were subjected to Rejuvenation (Donor 1, 37 yo; Donor 2, 42 yo; Donor 3,						0																	0	0	5		10		15		20			25		1x101
52 yo). Measurement of epigenetic status by Horvath's clock on D20 demonstrated a																																										0
reduced epigenetic age of TRJ compared with TCT. (C) Fold change expansion curve of																														Days									0	10		20	30			CD4		CD8	CD8
TRJ compared with TCT. TRJ exhibited increased proliferation. For reference: Average fold																																												CD4
																																											Days							TCT	TRJ		TCT	TRJ
expansion of TRJ and TCT at D26 was 61,250 and 868, respectively.			E								Reprogramming	Redirection & Expansion			F
												100					50									Donor age
Figure 3: Rejuvenated peripheral blood T cells demonstrated a					75																(y)	40															C										D NY-ESO-1 CD4+ & CD8+
																			Epigeneticage
conventional T-cell phenotype with improved metabolism and		%CD8+										Days						30															(%)+2-					****		****	cells/field		6000		TCT/TRJ cells	D3	D6	D9
stemness based on transcriptomic analyses					50																												****	****
																																						100							1/4		1/4	1/4
																																20																		CD4 TCT									A375 NLR
																																																		CD4 TRJ						12000		1st	2nd	3rd	4th
A		Bulk RNA-seq		B								25													TCT				10																80		CD8 TCT							E:T = 1:2
																								TRJ																				CD8 TRJ
	Control	Control	Rejuv	Rejuv																																																				10000
	D7	D13	D7	D13										0																	0
						D7		D13							0			5		10			15	20	25						TCT				TRJ							60									8000
																																																	No T cells
																																																40
					Fatty acid and																																										IL							#Target		2000						TCT
																																																											TRJ
					OxPhos																																																				4000
									G		4000															H		0.03																20
					metabolism				TCRsProductive																				clonalitySimpson
											3000																	0.02																0									0	0	3	6	9	12
					Glycolysis																																										No target	A375	PMA/I
										2000																																												Days
					Hypoxia						1000																	0.01
				T cell																																					CD4+ and CD8+ NY-ESO-1 TRJ and TCT were evaluated to compare (A) cellular proliferation,
														0																	0.00															(B) epigenetic age, (C) cytokine production upon co-culture with target cell line or
						-log10 (adjusted p-value)											TCT				TRJ							TCT				TRJ						PMA/I stimulation (****, P<0.0001) and (D) sequential killing assay against the A375 cell line.

C
			Gattinoni naïve score	TCT	TRJ
	TRJ	TCT
											Expressionlevel
D13				Percentageof Naivecells

1. Bulk RNA-seq heatmap analysis of previously identified conventional and unconventional genes related to T-cell identity. (B) Bulk RNA-seq showed enrichment of DEGs associated with key metabolic features in TRJ at D7 and D13 compared with TCT. (C) Single-cellRNA-seq showed elevated expression of naïve-associated markers in TRJ compared with TCT.

Rejuvenation enhanced the properties associated with the T-cell functionality of TIL

• Rejuvenated TIL (TILRJ) derived from metastatic melanoma
  (Figure 4) showed improved cellular proliferation and stem-likeproperties as well as a maintained TCR repertoire compared with control TIL (TILCT) (Figures 5 and 6)
• TILRJ derived from NSCLC demonstrated enhanced proliferative capacity and stem-like properties compared with TILCT (Figure 7)

TIL derived from a patient with metastatic melanoma (48 yo) were subjected to Rejuvenation.		Figure 9: NY-ESO-1 TRJ showed enhanced antitumor effects
(A) Partially reprogrammed TIL showed downregulation of the T-cell markers CD3, CD4,
and CD8 and temporal expression of a reprogramming marker (SSEA4). (B) Partially		compared with TCT in vivo
reprogrammed T cells redirected to CD4+ T cells and CD8+ T cells and preserved TCM
phenotype (CD62L+ CCR7+). TIL TRJ and TCT were evaluated to compare (C) phenotype,
(D) cellular proliferation, (E) %CD8+, (F) epigenetic age, and (G, H) TCR repertoire.		A	A375 tumor
		1.0E+06 cells / mouse
			SC injection
Figure 6: Rejuvenation improved cellular proliferation capacity,			NY-ESO-1 TCT /
		NY-ESO-1 TRJ
stemness phenotype, and epigenetic age in metastatic melanoma TIL			1.0E+06 cells	Monitor tumor size
		NSG MHC I/II DKO mice
			D0

A	x	4	B	25	C	70	10		B		2500	PBS				C		100
changeFold	1 10		%CD62L+CCR7+	(y)ageEpigenetic	Meanofdifferences	volumeTumor	)(mm3					Probability Survivalof	80
	3	20			2000	TCT								PBS
	x			60					1500	TRJ						60
	1 10					0				1000							40				TCT
																			**
				15
																			TRJ
	1 10	2			50					500							20
	x						-10				0							0
				10
	x	1				40					0	10	20	30	40			0	10	20	30	40
	1 10			5		-20				Days after T-cell injection			Days after T-cell injection
	1 10				30
			0
	x	0

TCT	TRJ	TCT	TRJ	TCT TRJ TRJ - TCT	(A) Tumor treatment schema. NSG MHC I/II DKO mice were injected with 1.0E+6 A375 tumor
					cells SC. ACT was given on D6. In each tumor setting, 5 mice were included in groups receiving
TIL derived from patients with metastatic melanoma (N = 3) were subjected to Rejuvenation.	PBS (Gray), 1.0E+6 NY-ESO-1 TCT (Green), or 1.0E+6 NY-ESO-1 TRJ (Gold). Tumor volumes
TIL TRJ and TCT were evaluated to compare (A) Feeder free expansion (mean of differences	were assessed every 2-3 days. (B) Tumor growth curve. Error bars indicate the
[M∆] = 2691), (B) % CD62L+ CCR7+ (M∆ = 10.6), and (C) epigenetic age (M∆ = -7.35).	M ± SD. (C) Survival curve. Survival was assessed by a log-rank test. **, P<0.05.

Conclusions

• Lyell's T-cell Rejuvenation technology utilizes a partial reprogramming process to produce T cells that are characterized by reduced epigenetic age, enhanced cellular proliferation, improved metabolism, and higher expression of stemness biomarkers. Additional research may further characterize TRJ in terms of their capacity for tumor antigen-specific polyclonality, long-term engraftment, and solid tumor eradication in vivo
• Metastatic melanoma and NSCLC TIL were successfully rejuvenated and acquired improved functionality while retaining a broad TCR repertoire
• Application of this technology has demonstrated improvements in engineered adoptive T-cell products

͵ In vitro sequential cell killing assays revealed that rejuvenated NY-ESO-1 TCR T cells exhibited improved antitumor properties compared with control T cells ͵ Rejuvenated NY-ESO-1 TCR T cells also showed improved tumor suppression and survival compared with control T cells in an in vivo setting

• Through partial reprogramming, Lyell's T-cell Rejuvenation technology has the potential to be developed as the first rejuvenated autologous polyclonal TIL therapy

Abbreviations

ACT, adoptive cell therapy; CCR7, C-C motif chemokine receptor 7; CD, cluster of differentiation; D, days; DEG, differentially expressed gene; DKO, double knock out; E:T, effector:target; LEF1, lymphoid enhancer binding factor 1; IL-2,interleukin-2; iPSC, induced pluripotent stem cells;

L/D, live/dead; M, mean; M∆, mean of differences; MHC, major histocompatibility complex; N, number of patients; NLR, NucLightRed; NSCLC, non-small cell lung cancer; NSG, NOD scid gamma; NY-ESO-1, New York esophageal squamous cell carcinoma 1; OxPhos, oxidative phosphorylation; PB, peripheral blood; PBS, phosphate-buffered saline; PMA/I, phorbol 12-myristate13-acetate/ionomycin;RNA-seq, RNA sequencing; SC, subcutaneous; SD, standard deviation; SSEA4, stage-specific embryonic antigen-4; TCR, T-cell receptor; TCM, central memory T cell; TCT, control T cell(s); TIL, tumor-infiltrating lymphocyte(s); TILCT, control tumor-infiltrating lymphocyte(s); TILRJ, rejuvenated tumor-infiltrating lymphocyte(s); TRJ, rejuvenated T cell(s); y, years; yo, years old.

References

1. Crompton JG, et al. Trends Immunol. 2014;35(4):178-185;2. Crompton JG, et al. Cell Stem Cell. 2013;12(1):6-8;3. Kishton RJ, et al. Curr Opin Immunol. 2022;74:39-45;4. Nikolich-Zugic J. Nat Rev Immunol. 2008;8(7):512-522;5. Vizcardo R, et al. Cell Stem Cell. 2013;12(1):31-36;

1. Maeda T, et al. Cancer Res. 2016;76(23):6839-6850;7. Vizcardo R, et al. Cell Rep. 2018;22(12):3175-3190.

Acknowledgments

This project was fully supported by Lyell Immunopharma, Inc. We would like to thank Lyell's T-cell Rejuvenation, in vivo, and BATT teams for their technical support and Luca Gattinoni for critical feedback. We would also like to thank the Clock Foundation for epigenetic clock analyses, Maria Romanova (Molecular House, Inc.) for graphical design support, and Madison Fagan (BOLDSCIENCE, Inc.) for medical writing support.

Presented at SITC Annual Meeting 2023; Nov 1-5; San Diego, CA, USA