Lyell Immunopharma : The Epi-R technology produces a polyclonal TIL product (LYL845) with diverse tumor-reactive clones that have stem-like qualities and anti-tumor function

Epi-R™ Technology Produces a Polyclonal TIL Product (LYL845) With Diverse Tumor-Reactive Clones That Have Stem-Like Qualities and Anti-Tumor Function

Benjamin D. Harris, Yogin Patel, Ngoc-Han Ha, Joanna Kritikou, Lora Zhao, Sheila Lou, Stefan Siebert, Emily Fu-Sum, Rigel Kishton, Purnima Sundar, Suman Kumar Vodnala, Shobha Potluri

Lyell Immunopharma, Inc., South San Francisco, CA and Seattle, WA, USA

Abstract 340

Background

• Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) is a promising method for cancer treatment

• TILs that are highly enriched with tumor-reactiveT-cell clones have been shown to mediate treatment response in advanced cancers1-3

• Conventional TIL products comprise a mixture of extensively differentiated T cells and low levels of stem-like T cells

• Higher proportions of stem-like T cells have been associated with improved outcomes in patients treated with TIL therapies4

• LYL845 is an autologous TIL product enhanced with Epi-R™️ technology, a proprietary epigenetic reprogramming protocol designed to improve T-cell stemness and anti-tumor functionality in ACT products

•

Methods

Production of LYL845 Using Epi-R Technology

• LYL845 was prepared with Epi-R technology designed to intentionally and reproducibly generate TILs with durable stemness that can proliferate, persist, and provide prolonged anti-tumor functionality

• The Epi-R protocol is composed of a specifically formulated cell culture media with optimized cytokine composition and cell activation that induces metabolic reprogramming via reduced glycolysis and hypoxia

• LYL845 was produced at research scale using a total of 15 tumor samples (melanoma, lung, and colon) and was compared with control TIL products generated without Epi-R

• LYL845 produced at large scale were generated from nine tumor samples and compared with corresponding research-scale products

Identifying and Tracking Putative Tumor-Reactive Clones (Figure 1)

• Using previously validated methods,4-5 high-frequency and exhausted clones in the dissociated tumor suspension of Day 0 tumor samples were identified as putative tumor-reactive clones and used to:

͵ Identify the top 100 high-frequency TCR clones using bulk TCR-seq

͵ Identify exhausted cell TCR clones using single-cellRNA-seq and single-cellTCR-seq

͵ Identify a subset of 100 high-frequency and exhausted clones as putative tumor-reactive clones

• Bulk TCR-seq was used to check the presence and proportion of putative tumor-reactive clones in the T-cell products

• Single-cellRNA/TCR-seq was used to evaluate the phenotype of putative tumor-reactive clones in the T-cell products

Experimental Testing of Tumor-Reactive Clones in LYL845 (Figure 1)

• Putative tumor-reactive TCRs were transduced into healthy donor CD8+ T cells and then co-cultured individually against autologous tumor cells. Tumor reactivity was measured by cytolysis and IFNγ secretion 24 hours post co-culture

• TIL products were co-cultured with either patient-dissociated tumor suspension or autologous tumor cells, and tumor reactivity was measured by 4-1BB and IFNγ expression in single-cellRNA/TCR-seq

Figure 1: Methodology for Identifying, Tracking, and Testing Putative Tumor-Reactive Clones

Results

Key Findings

Preclinical analysis of the Epi-R-produced TIL product demonstrated that LYL845 exhibited: • High polyclonality and anti-tumor activity

• Preservation of putative tumor-reactive clones

• Increased stemness and reduced exhaustion-associated genes in putative tumor-reactive clones

LYL845 Products Are Highly Polyclonal and Demonstrate Potent Anti-Tumor Activity

• LYL845 expanded from three tumor types using Epi-R technology demonstrated a high degree of polyclonality (based on the Simpson Clonality Index); LYL845 manufactured at research scale indicated that clonal diversity of source tumor tissue is preserved (Figure 2A)

• LYL845 large-scale products demonstrated higher polyclonality compared with research-scale products (Figure 2B)

• LYL845 demonstrated potent anti-tumor function through 1) dose-dependent cytolytic activities during co-culture with an autologous tumor cell line, 2) pro-inflammatory cytokine secretion after CD3/CD28 stimulation

Figure 2: LYL845 Retained Polyclonality With Preserved TCR Diversity of Source Tumor Tissue

A													B
Tumor																					LYL845 Research Scale
Control
																	LYL845 Large Scale
LYL845 Research Scale
0.0							0.1	0.2	0.3	0.4	0.0	0.1		0.2	0.3	0.4
													Simpson Clonality										Simpson Clonality

(A) Simpson clonality of dissociated tumor suspension, LYL845 manufactured at research scale, and product from control process at research scale. (B) Simpson clonality of large-scale and research-scale LYL845 products.

Figure 3: LYL845 Demonstrated Dose-Dependent Cytolytic Activity and Pro-Inflammatory Cytokine Secretion

A					B		IFNγ		IL-2			TNFα
	100			E:T	Concentration (pg/ml)	50000		Concentration (pg/ml)	40000		Concentration (pg/ml)	40000
	80			10:1	40000
% Cytolysis					30000		30000
60			5:1	30000
				20000		20000
				20000
40
20			1:1	10000		10000		10000
0			0:1	0		0		0
		40
		30	50		Unstimulated	anti-CD3/CD28		Unstimulated	anti-CD3/CD28		Unstimulated	anti-CD3/CD28
			Hours

(A) Tumor killing by LYL845 as percent cytolysis with an autologous tumor cell line (metastatic melanoma) with tumor cell killing observed in all E:T ratios compared with control tumor-only (0:1) in a dose-dependent manner. (B) Increased IFNγ, IL-2, and TNFα cytokine secretion in LYL845 cells after 24 hours of CD3/CD28 stimulation as compared with unstimulated cells.

Epi-R Created LYL845 Product That Preserved Putative Tumor-Reactive Clones From Initial Tumor Sources

• Putative tumor-reactive clones for each tumor were identified from a dissociated tumor suspension using bulk TCR-seq to identify high-frequency clones and using single-cellRNA/TCR-seq to identify clones with an exhausted phenotype (Figure 4)

Figure 5: Testing of Three TCRs With an Autologous Tumor Cell Line Validated Tumor-Reactive Clones

A		5:1			B								5:1
100			TR TCR 1	1000
	80			TR TCR 2		800
Cytolysis%			TR TCR 3	pg/mLIFNγ,
60			600
		Tumor only
	40					400
	20					200
	0	40	50			0
	30			only	only	1	only		2	only	3
						TCR		TCR		TCR
							TIL		TIL		TIL
	Hours (post tumor plating)				Tumor	TR	TR		TR

The top three putative tumor-reactive TCRs were cloned into healthy donor T cells and co-cultured with the patient's autologous tumor cell line. At an E:T ratio of 5:1, two of the three TCRs showed (A) 100% cytolysis which (B) correlated with specific IFNγ secretions.

Figure 6: Putative Tumor-Reactive Clones Tested at High Frequencies Were Verified as Tumor Reactive

								Autologous
Tumor Reactive		True		False	Dissociated Tumor Suspension		Tumor Cell Line

	530D144 Research Scale		LY224 Research Scale	30	LY258 Large Scale	1000	LY222 Large Scale	LY130 Research Scale
Culture-	19/44 (43%)	1000	16/32 (50%)	14/41 (34%)	61/68 (90%)	7/21 (33%)
clones validated	clones validated	10	clones validated	100	clones validated	clones validated
	100							30
Co		100						10
in
10
Cells	10		3		10		3
# of	1	1		1		1		1

Validation of putative tumor-reactive clones by co-culturing LYL845 with a target autologous tumor cell line or dissociated tumor suspension, then measuring activation (4-1BB+/IFNγ+) showed that putative tumor-reactive clones are tumor-reactive when captured at higher frequencies.

Figure 7: Identified Putative Tumor-Reactive Clones Were Preserved in LYL845 and Control Products

A				B
Putativeof# Tumor- ClonesReactivePreserved	75			Productof% with Putative -TumorReactive Cells	0
					40
	50
					20
	25
	Control	LYL845 Research	LYL845 Large		Control	LYL845 Research	LYL845 Large
		Scale	Scale			Scale	Scale

Bulk TCR-seq of research-scale products showed that LYL845 and T cells prepared with the control protocol preserved a comparable number of putative tumor-reactive clones. (A) Large-scale production of LYL845 preserved 94% of the identified putative tumor-reactive clones versus 57% in research-scale products. (B) Both research-scale and large-scale preparations of LYL845 retained a large percentage of putative tumor-reactive cells.

Putative Tumor-Reactive Clones in LYL845 Demonstrated Increased Stem-Like Characteristics With Reduced Expression of Exhaustion Related Genes

• Single-cellRNA/TCR-seq of LYL845 and control TILs demonstrated enrichment of putative tumor-reactive clones in non-stem-like compartments (Figure 8)

• Putative tumor-reactive clones in LYL845 demonstrated up-regulation of genes associated with

Figure 9: LYL845 Putative Tumor-Reactive Cells Had Up-Regulation of Stemness Genes and Down-Regulation of Exhaustion Genes Compared With Control TIL

Stemness-Associated Genes			Exhaustion-Associated Genes
			Control	LYL845	LYL845
A		B	Research Scale	Large Scale
			2	Condition
							Control
					1		LYL845 Research Scale
					0		LYL845 Large Scale
					-1
					-2

530D144	LY222 LY193 LY170 LY158 LY142 LY137 P33556 LY224 LY193 LY170 LY163 LY158 LY142 LY137 530D144 P33556 LY224 LY193 LY170 LY158 LY142 LY137										LY222 LY193 LY170 LY158 LY142 LY137 P33556 LY224 LY193 LY170 LY163 LY158 LY142 LY137 530D144 P33556 LY224 LY193 LY170 LY158 LY142 LY137 530D144
C															D
Geneset	Krishna ACT Stem Like														Krishna ACT Stem Like																	-log10(padj)
Caushi CD8 Stem-like memory													Caushi CD8 Stem-like memory
																							4.0
Oliveira TTE													Oliveira TTE
	REACTOME_G2_M_CHECKPOINTS														REACTOME_G2_M_CHECKPOINTS
																										3.5
	Zhang_TEX_onlyColon														Zhang_TEX_onlyColon													3.0
				-2	-1	0	1	2				-2	-1	0	1	2
	LYL845 Research Scale vs Control NES	LYL845 Research Scale vs Control NES

(A, B) Pseudobulk differential expression between putative tumor-reactivecells in LYL845 research-scaleand control products indicated up-regulationof stemness- and down-regulationof exhaustion-associatedgenes and gene sets from literature. The large-scalepreparation of LYL845 putative tumor-reactivecells had a comparable phenotype to the research-scalepreparation. (C, D) GSEA identified up-regulationof stemness-associatedgene sets from literature and down-regulationof exhaustion-associatedgenes in both research- and large-scalepreparations of LYL845 compared with control.4-5,9-10

Conclusion

• Preclinical analysis of LYL845 revealed a highly polyclonal product with potent anti-tumor activity • LYL845 demonstrated dose-dependent cytolytic activity and cytokine secretion when co-cultured

with an autologous melanoma cell line

• Preclinical data showed that LYL845 has preserved tumor-reactive clones with stem-like qualities • These promising preclinical data led to the further development of LYL845, an investigational

autologous TIL therapy enhanced with Epi-R

• An Investigational New Drug application for LYL845 was accepted by the FDA in October 2022, and it will be evaluated for safety, tolerability, and anti-tumor activity in an upcoming Phase 1 clinical trial

	Enzymes
	added
	Dissociation
	by gMACS
Malignant		Dissociated
tumor		tumor suspension
tissue		(tumor + T cells)

Identifying and Tracking Putative Tumor-Reactive Clones

Identify high frequency	Identify subset of TCR
clones from "exhausted"	Check presence
TCR clones in	cells (single-cell	of putative
dissociated	RNA/TCR-seq on	tumor-reactive
tumor suspension	dissociated	clones in LYL845	LYL845
(top 100 from
tumor suspension) as		(bulk TCR-seq)
bulk TCR-seq)	putatively tumor reactive

Evaluate tumor reactivity

of putative clones

Experimental Testing of Tumor-Reactive Clones in LYL845

	Co-culture individual		24 hrs	Identify true tumor-reactive
	TCR clones with cell line				TCR clones from cytolysis
	OR				and IFNγ secretion
	Co-culture TIL product		Single-cellRNA-seq	Identify true
Autologous	with cell line/dissociated				tumor-reactive TCR clones
tumor cell line	tumor suspension	Single-cellTCR-seq (4 hrs)	from 4-1BB+/IFNγ+ cells

• Culturing individual putative tumor-reactive TCRs with an autologous tumor cell line confirmed tumor reactivity with cytolysis and IFNγ secretion (Figure 5)

• Culturing LYL845 with a dissociated tumor suspension or an autologous cell line validated tumor reactivity of putative tumor-reactive clones (Figure 6)

• Using the validated putative tumor reactivity approach, large-scale LYL845 products preserved an average of 94% of putative tumor-reactive clones (Figure 7A)

• Both research-scale and large-scale LYL845 products retained a comparable percentage of putative tumor-reactive cells (Figure 7B)

Figure 4: Putative Tumor-Reactive Clones Were Identified From Sequencing Thirteen Day-0 Tumors

A		B							C	D
		Putative Tumor-Reactive Clones			CXCL13			CD137(4-1BB)			Tumor-Reactive Phenotype			Tumor-Reactive Phenotype +
6		2	6		2	6				6			6		Frequency (bulk TCR)
	3		3			High
	UMAP			UMAP
		0			0
3		-3			-3				3			3
		-6				-6

2						-8	-4	0	4		-8	-4	0	4	2					2
_	0				CD4		UMAP_1				UMAP_1		_	0				_	0
UMAP				3		2	3		UMAP				UMAP
-3				2							-3				-3
					CD8	CD279(PD-1)		TIGIT(VSTM3)
	-6				UMAP	6				UMAP	6					-6					-6
				0				0
						-3					-3
	-8	-4	0	4		-6	-4	0	4		-6	-4	0	4	Low	-8	-4	0	4		-8	-4	0	4
		-8		-8
			UMAP_1				UMAP_1				UMAP_1					UMAP_1					UMAP_1

(A) Analysis by single-cellRNA/TCR-seq of 203,389 cells from 11 melanomas, three lung, and two colon cancers identified putative tumor-reactive clones. (B, C)​Tumor-reactive phenotypes were identified by CXCL13, 4-1BB,PD-1, and TIGIT expression.4-7

(D) Cells identified as having a tumor-reactive phenotype and high frequency in bulk TCR-seq were defined as being putatively tumor-reactive and biased towards CD8+ T cells.

stemness and down-regulation of genes associated with exhaustion when compared with TIL products manufactured using the control process, a trend also seen in large-scale

LYL845 production (Figure 9)

Figure 8: Putative Tumor-Reactive Cells Were Largely Enriched in

Non-Stem-Like Compartments

High

Low

C	Stem-Like Cells

(A) Analysis by single-cellRNA-seq/single-cellTCR-seq of 25 products (nine control, ten LYL845 research-scale, and six LYL845 large-scale) enabled phenotypic characterization of putative tumor-reactive clones. (B) Stem-like clusters were identified by expression of SELL, CD39, CD69, and expression of stemness gene sets from literature.8 (C) Putative tumor-reactive clones were enriched in non-stem-like clusters.

Abbreviations

4-1BB, tumor necrosis factor ligand superfamily member 9; ACT, adoptive cell therapy; CD, cluster of differentiation; CXCL, chemokine ligand; E:T, effector:target; gMACS, gentle magnetic-activated cell sorting; GSEA, gene set enrichment analysis; IFNγ, interferon gamma; IL-2,interleukin-2; NES, normalized enrichment score; PD-1, programmed cell death protein 1; RNA, ribonucleic acid; RNA-seq, ribonucleic acid sequencing; TCR, T-cell receptor; TCR-seq,T-cell receptor sequencing; TIGIT,

T-cell immune receptor with immunoglobin and ITIM domains; TIL, tumor-infiltrating lymphocyte; TNFα, tumor necrosis factor alpha;

TR, tumor-reactive; UMAP, uniform manifold approximation and projection.

Acknowledgments

We thank members of Lyell Immunopharma's flow cytometry core (Andrew Jimena, Elizabeth Pedrosa, Ken Xiong), process development team (Meri Galindo Casas, Carson Harms, Melissa DeFrancesco), and Queenie Vong for their experimental contributions. Medical writing and editorial support were funded by Lyell Immunopharma and provided by Madison Fagan of

BOLDSCIENCE Inc.

References

1. van den Berg JH, Heemskerk B, van Rooij N, et al. J Immunother Cancer. 2020;8:e000848. 2. Tran E, Turcotte S, Gros A, et al. Science. 2014;344:641-645. 3. Zacharakis N, Chinnasamy H, Black M, et al. Nat Med. 2018;24:724-730. 4. Krishna S, Lowery FJ, Copeland AR. Science. 2020;370:1328-1334. 5. Oliviera G, Stromhaug K, Klaeger S, et al. Nature. 2021;596:119-125. 6. Pasetto A, Gros A, Robbins PF, et al. Cancer Immunol Res. 2016;4:734-743. 7. Lowery FJ, Krishna S, Yossef R, et al. Science. 2022; 875:877-884. 8. Caushi JX, Zhang J, Ji Z, et al. Nature. 2021;596:126-32.Erratum in: Nature. 2021;598:E1. 9. Zhang L, Yu X, Zheng L. Nature. 2018;564:268-272. 10. Sade-FeldmanM, Yizhak K, Bjorgaard SL, et al. Cell. 2018;175:998-1013.

Presented at SITC Annual Meeting 2022; November 8-12, 2022; Boston, MA