Noninvasive positron emission tomography (PET) imaging of Sleeping Beauty (SB) modified CD19-specific T cells expressing HSV1-Thymidine kinase

Pallavi R.Manuri, PhD. Department of Pediatrics Research

Adoptive Cell Therapy

• Development of engineered T cells using – T-cell receptors– Chimeric antigen

receptors (CARs)• Methods of genetic

moditication– Viral – No-viral

CD19-specific CAR

Rationale

However, to improve the design, application and evaluation of adoptive T-cell therapy requires monitoring methods that can

• Detect

• Locate and

• Serially quantify the cell-mediated immune responses

Rationale

Currently monitoring methods are chiefly invasive techniques

•Histology

•Flow cytometry

•Q-PCR and/cytokine analysis

In contrast, Positron emission tomography (PET) is a•Noninvasive, accurate, and

•Sensitive whole-body imaging technology allowing

•Repetitive measurement in vivo

Herpes Simplex Virus 1- thymidine kinase (HSV1-tk)

• Expression of reporter genes and use of corresponding reporter probes (radiotracers) labeled with positron-emitting radionuclides.

18F-FEAU18F-FHBG

TK

TK

TKTK

TK

TK

P

P

P

P

The specificity and/or sensitivity of HSV1-tk were altered by•Mutations in the nucleoside binding region (HSV1-sr39tk)

•Inactivation of the nuclear localization signal (NLS) of HSV1-tk Arg25-26 were replaced by Gly25-26

•Addition of the nuclear export sequence (NES)

Generation of CD19-specific T cells capable of being imaged non-invasively by PET

Cytoplasm

NucleusTransposaseTransposase

TransposonTransposon

Gene XCAR #1

mRNA

SB11 protein

TransposonTransposon

Gene XCAR #2

CD19RCD28mZ(CoOp)/pSBSO

Kan/R

CD19RCD28mZ(CoOp)BGH

hEF-1alpha/p

Kan/p

CoIE1

IR/DR

IR/DR

Flag-NES-NLSm-TK(CoOp)-Hygro/pSBSO

Kan/R

NES-NLSm-TK (CoOp)

Hygro

3(flag)

LinkerBGH

hEF-1alpha/pKan/p

EM7

CoIE1

IR/DR(L)

IR/DR(R)pCMV-SB11

4732bp

APr

Transposase

SV40 polyA

CMV promoter/enhancer

Scheme of expansion of T cells on artificial antigen presenting cells

Co-expression of CD19-specific CAR and TK

CAR+TK- CAR+TK+

FLAG

CA

R

98.9 0.44

0.000.63 0.01

2.85 96.8

0.37

Redirected specificity of CD19CAR+TK+ T cells

20:1

10:1 5:1 2.5:1

1.25:1

0

20

40

60

80Nalm-6Daudi--2mU251TtCD19U251T

Effector:Target ratio

% S

peci

fic L

ysis

] CD19+

Specificity towards nucleoside analogs

0.0 uM

0.01u

M0.1

uM1.0

uM10

.0 uM

0

50

100

150TK negativeNES-(NLMm)TKHy

Ganciclovir (M)

% T

cel

l Sur

viva

l

Sensitivity towards Ganciclovir

CAR+TK-

CAR+TK+

In vitro accumulation of 3H-FEAU

TK negati

ve

NES-(NLS)m

-TKHy

0

10

20

30

40

50

Cel

l/Med

ia ra

tio

CAR+TK+

CAR+ TK-

In Vivo Imaging of CD19CAR+TK+ T cells with PET

7.5x 106 T cells subcutaneously and 100Ci of 18F-FEAU intravenously

Axial view Coronal view Saggital view

In Vivo Imaging of CD19CAR+TK+ T cells with PET

15x 106 T cells subcutaneously

Axial view Coronal view Saggital view

3 dimensional reconstruction of CD19CAR+TK+T cells imaged by PET

Summary

• Co-expression of a CD19-specific CAR and HSV1-tk by SB transposition.

• SB modified CD19CAR+TK+ T cells – visualized spatio-temporally by PET using

18F-FEAU – ablated in the presence of ganciclovir and

also– Have the ability to kill CD19+ tumor targets.

Implication

Clinical Significance: in non-invasively monitoring the persistence and trafficking SB modified adoptively transferred CD19-specific T cells.

Ongoing work: in vivo tumor model is being worked out

Thanks

Cooper labKirsten SwitzerTiejuan MiLing ZhangHarjeet SinghHelen Huls

• Carl June• Bruce Levine

Gelovani labAmer NajjarLeo Flores II

Perry Hackett

David Largaespada