Personalized gene editing of T cells to express neoantigen-specific TCRs isolated

from peripheral blood of patients on PD-1 blockade therapyCristina Puig-Saus1, Barbara Sennino2, Bhamini Purandare2, Duo An2, Boi Quach2, Songming Peng2, Huiming Xia3, Sidi Zhao3, Zheng Pan2, Yan Ma2, Justin Saco1, Sameeha Jilani1, Jia Pang1, Ivan Perez-Garcilazo1, Agustin Vega-Crespo1,

Ignacio Baselga1, Christine Shieh2, Michael Yi2, Katharine Heeringa2, Corey Meadows2, Olivier Dalmas2, Robert Moot2, Diana Nguyen2, William Lu2, Kyle Jacoby2, Andrew Conroy2, Adam Litterman2, John Gagnon2, Beata Berent-Maoz1,

Jasreet Hundal3, Malachi Griffith3, Stefanie Mandl2, Alex Franzusoff2, Antoni Ribas1

1 Division of Hematology-Oncology, Department of Medicine, David Geffen School of Medicine (DGSOM), University of California, Los Angeles (UCLA), Los Angeles, California, 2PACT Pharma, South San Francisco, California and 3McDonnel Institute, Washington University School of Medicine, St Louis, Missouri

Neoepitopes (neoE) derived from nonsynonymous mutations are the primary target of T cell responses induced by immunecheckpoint blockade therapy and could serve as personalized cancer targets for adoptive cell transfer (ACT) therapy. We used an

ultra-sensitive and high-throughput technology (imPACT Isolation Technology®) to isolate neoE-recognizing TCRs from patientson anti-PD-1 therapy and studied their functionality. Briefly, based on computational prediction of patient-specific neoEs,hundreds of capture reagents were made consisting of the patient HLA class I subtypes loaded with the corresponding predictedneoE (Peng et al. AACR 2019); neoE-specific T cells were then isolated and the TCR alpha and beta sequenced. Isolated neoTCRswere studied functionally by generation of primary human T cells expressing the neoTCRs using non-viral precision genomeengineering to replace the endogenous TCRs (Jacoby et al., AACR 2019, Sennino et al., AACR 2019).We analyzed T cell responses in two patients with metastatic melanoma receiving anti-PD-1 therapy. NeoE-specific T cells wereisolated from peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL) at different time points.Patient PT476 had a durable response; tumor mutational burden (TMB) was 2556; 243 neoE-HLA complexes were producedacross 3 HLA types, HLA-A03:01, A24:01 and C12:03. This resulted in isolation of 17 TCRs specific for 5 neoE-HLAs. T cells specificfor neoE’s were present at baseline in TILs and expanded during treatment in TILs and PBMCs. Patient PT461 had rapid diseaseprogression on anti-PD-1; TMB was 61; 78 neoE-HLA-complexes covering HLA-A02:01, A03:01, B07:02, C05:01 and C07:02 wereproduced, resulting in isolation of 2 TCRs to 1 neoE-HLA.To further characterize the T cell responses, we gene edited T cells to express 14 different TCRs isolated from patient PT476,specific for neoEs in the mutated IL8, PUM1 and TPP2 genes. All 14 T cell preparations displayed specific cytotoxicity against amatched autologous melanoma cell line established from a biopsy of patient PT476 (50-75% tumor growth inhibition comparedto melanoma cell line growth in co-culture with a mismatched control TCR, 96 hour assay using P:T 1:1, p < 0.000001 for eachcomparison), and had no cytotoxic effect against an unmatched control human melanoma cell line. Upon co-culture with thematched autologous melanoma cell line, neoE TCR T cells upregulated 4-1BB and OX-40, secreted IFNγ, IL-2 and TNFα, andinduced T cell proliferation and degranulation. No responses were seen when T cells were co-cultured with unmatched targets.Our results show that anti-PD-1 therapy induces focused neoE-specific T cell responses to a restricted number of neoE’s, andthat non-viral precision genome engineering can successfully redirect T cells to neoE expressing tumors which can be used as anapproach for personalized ACT therapy.

imPACT Isolation Technology®: diagram illustrating the capturing of NeoE-specific T cells from the patient blood

Gene editing: CD8 and CD4 T cells from healthy donor were precision genome engineered to express the neoTCR. Briefly, neoE-specific TCR sequencesare cloned into homologous recombination (HR) DNA templates. These HR templates are used with site-specific nucleases to engineer primary human Tcells. The single-step (non-viral) precision genome engineering results in the seamless replacement of the endogenous TCR with the patient’s neoE-specific TCR (of native sequence), whose expression is under endogenous regulation.

Co-Culture assay: NeoTCR-P1 T cells were co-cultured with a melanoma cell line derived from the baseline biopsy of the same patient (M489) or amismatched melanoma tumor cell line at a final Product to Target (P:T) ratio of 1:1 or 5:1. Target cell killing was evaluated over 6 days using the IncuCytesystem. Expression of the proliferation marker Ki67 was assessed by flow cytometry at 48h. Expression of activation marker was assessed by flowcytometry at 24h. Cytokine secretion was measured in the cell supernatant at 48h using the BD Cytokine Bead Array (CBA) Human Th1/Th2 Cytokine KitII

Figure 1. Biopsies and PBMCs were collected at multiple time-points after anti-PD-1 treatment (A: PT476responder B: PT461 non-responder). TILs and cell lines were established from the patient’s biopsies. TheimPACT Isolation Technology® was used to isolate NeoE-specific T cells and monitor their evolution overtime. NeoE derived from non-synonymous mutations were predicted using the Whole Exome Sequencing(WES) and RNAseq from the baseline cell lines and ranked according to the predicted HLA-binding affinity,the truncality of the mutation and the level of expression. HLA-NeoE capture reagents for the top-rankedNeoE were used to isolate the NeoE- specific T cells. A. PT476: 243 neoE-HLA complexes were producedacross 3 HLA types, HLA-A03:01, A24:01 and C12:03 and 17 TCRs specific for 5 neoE-HLAs were isolated.B. Patient PT461: 78 neoE-HLA-complexes covering HLA-A02:01, A03:01, B07:02, C05:01 and C07:02 wereproduced, resulting in isolation of 2 TCRs to 1 neoE-HLA.

Gene neoE peptide HLA

IL8 KTYF(S)KPFHPK A03:01

IL8 YF(S)KPFHPKF A24:02

PSMC6 KIHAGSITK A03:01

PUM1 AMMDYFFQR A03:01

TPP2 CFSEVSAKF A24:02

A

B

Gene neoE peptide HLA

ACER3 RLYTRTLYL A03:01

Figure 2. A. Time-lapse microscopy of tumor cell death and T cell proliferation. NeoTCR-T cells were co-cultured with autologous or mismatched melanoma cell lines expressing a red fluorescent protein(nuclear RFP) in a stable manner. Images shown here were collected at time 0 (left panels), 2 days (middlepanels) and 5 days (right panels). T cells were not labeled in this experiment, but antigen-specificproliferation can be appreciated visually by increased numbers of T cells over the course of 5 days (topimages). B. Quantification of tumor cell confluency (percentage of nuclear RFP) over 6 days. * p<0.05compared to neo12 T cells (t test with Holm-Sidak method for multiple comparison correction).

MockT cells

T cells

TCR219(IL8 HLA-A03)

Autologous tumor cells

Day 0 Day 2 Day 5

Engineered neoTCR-T cells kill autologous melanoma tumor cells

PACT magnetic nanoparticles -barcoded with 105 HLA-peptide

tetramers

‘High avidity (sub-nM) CAPTURE'

T

C

R

T

C

R

Nanoparticle

PACT HLA-peptide tetramer

‘Modest avidity (nM)

DETECTION’

HLA-peptide monomer

‘Low affinity (µM) BINDING’

For low mutation burden tumor

For high mutation burden tumor

Peptide-HLA: recognition/ stimulationPACT

neoTCRT cells

PACTneoTCRT cells

Target cell killingProliferation

Activation markersCytokine secretion

Evolution of neoE-specific T cells in a patient with melanoma and clinical response on anti-PD-1

NeoE-specific T cells in a patient with melanoma and rapid disease progression on anti-PD-1

NeoTCR-T cells express activation markers upon co-culture with autologous tumor cells

A

TIL day -37

IL8-KTYFKPFHPK

IL8-YFKPFHPKF

PBMC day 84PUM1

IL8-KTYFKPFHPK

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PUM1PBMC day 14

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PBMC day 43PSMC6

IL8-YFKPFHPKF

IL8-KTYFKPFHPK

TIL Day 82

IL8-KTYFKPFHPK

PSMC6

IL8-YFKPFHPKF

PUM1

TPP2

0.00

0.25

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NeoE Predicted Binding Affinity Ranking from Highest to Lowest

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NeoE Predicted Binding Affinity Ranking from Highest to Lowest

ResultsAbstract

Methods

Conclusions

B

Figure 4. NeoTCR-T cells were co-cultured with autologous (dark grey bars) or mismatched tumor cellline (white bars) and after 24h the percentage of CD8 neoTCR T cells expressing the activationmarkers 4-1BB (top) or the percentage of CD4 neoTCR T cells expressing the activation marker OX40(bottom bar graph) was assessed by flow cytometry. * p<0.05 compared to mismatched melanomatumor cells (t test with Holm-Sidak method for multiple comparison correction). T cells expressing theNeoTCR neo12 were used as negative control. To measure OX-40 upregulation in the CD4 neoTCR Tcells, melanoma cells were pre-treated with IFN for 24h prior to the co-culture with T cells.

• Using the ultra-sensitive and high-throughput imPACT Isolation Technology® neoepitope-specific TCRs (neoTCRs) were successfully isolated from PBMCs and TILs of patients with melanoma.

• Analysis of T cell receptor diversity in a patient with clinical response on anti-PD1 therapy demonstrated dynamic T cell responses detectable in the tumor and in the blood. Responses were detected to 5 NeoE restricted to 2 HLA (A24 and A3).

• To study functional activities mediated by the isolated neoE-specific TCRs, non-viral precision genome engineering was used to replace the endogenous TCR of primary human T cells with the isolated neoTCRs to generate neoepitope-specific T cells (NeoTCR-T cells).

• Newly generated NeoTCR-T cells expressing the TCRs isolated using the imPACT technology upregulated markers of activation and proliferation upon co-culture with autologous tumor cells. More importantly, all NeoTCR-T cells specifically kill patient derived autologous melanoma cells.

Figure 3. NeoTCR-T cells were co-cultured with autologous (dark grey bars) or mismatched melanomatumor cells (white bars) and after 48h the percentage of Ki67 (proliferation marker) expressing CD8neoTCR T cells was assessed by flow cytometry. * p<0.05 compared to mismatched melanoma tumorcells (t test with Holm-Sidak method for multiple comparison correction). T cells expressing the NeoTCRneo12 were used as negative control

Engineered CD8 neoTCR-T cells proliferate upon co-culture with autologous tumor cells

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22

(P

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23

(I L

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TC

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24

(I L

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TC

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25

(I L

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TC

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28

(I L

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TC

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29

(I L

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TC

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(I L

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HLA-A*03:01 HLA-A*24:02

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Autologous tumor cells

Mismatched tumor cells

Abstract: B14

Baseline

PBMCs

Figure 5. NeoTCR-T cells were co-cultured with autologous melanoma tumor cells and after 48h IFNsecretion was assessed by flow cytometry (CBA). mock T cells were used as negative control. * p<0.05(t test with Holm-Sidak method for multiple comparison correction).

NeoTCR-T cells secrete interferon-gamma (IFN) upon co-culture with autologous tumor cells

Cell-based assay:M489

M489

PACTneoTCRT cells

PACTneoTCRT cells

PACTneoTCRT cells

PACTneoTCRT cells

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T C R 2 2 8

T C R 2 2 9 T C R 2 3 2 T C R 2 4 0

T C R 2 4 1

N e o 1 2R P M I

( M o c k T C R T a r g e t s )

( I L 8 - H L A - A * 0 3 : 0 1 )

( I L 8 - H L A - A * 2 4 : 0 2 )

( P U M 1 - H L A - A * 0 3 : 0 1 )

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T C R 2 2 7

T C R 2 2 8

T C R 2 2 9 T C R 2 3 2 T C R 2 4 0

T C R 2 4 1

N e o 1 2R P M I

( M o c k T C R T a r g e t s )

( I L 8 - H L A - A * 0 3 : 0 1 )

( I L 8 - H L A - A * 2 4 : 0 2 )

( P U M 1 - H L A - A * 0 3 : 0 1 )

( T P P 2 - H L A - A * 2 4 : 0 2 )

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*

TCR22

2 (P

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TCR21

9 (IL

8)

TCR22

0 (IL

8)

TCR22

3 (IL

8)

TCR22

4 (IL

8)

TCR22

5 (IL

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TCR22

8 (IL

8)

TCR22

9 (IL

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TCR23

2 (IL

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TCR24

0 (IL

8)

TCR24

1 (IL

8)

TCR22

1 (IL

8)

TCR22

7 (IL

8)

TCR21

8 (T

PP2)

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12

0

20

40

60

80

Neo

TC

R+ C

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cells

% K

i67

HLA-A*03:01 HLA-A*24:02

*

*

*

*

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*

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* *

*

*

*

Autologous tumor cell line Mismatched tumor cell line

M489: Melanoma tumor cell line from patient biopsy

14 neoTCR-T candidates from imPACT:

12 IL-8 (HLA-A24:02 & HLA-A03:01)PUM1 (A3:01)TPP2 (A24:02)

Autologous tumor cells

Mismatched tumor cells

Autologous tumor cells

TCR22

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UM

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TCR21

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TCR22

0 (IL

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TCR22

3 (IL

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TCR22

4 (IL

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TCR22

5 (IL

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TCR22

8 (IL

8)

TCR22

9 (IL

8)

TCR23

2 (IL

8)

TCR24

0 (IL

8)

TCR24

1 (IL

8)

TCR22

1 (IL

8)

TCR22

7 (IL

8)

TCR21

8 (T

PP2)

mock

0

10

20

30

40

50

60

70

IFN (

pg

/10

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ited

cells) *

* ** * * * *

*

*

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