Developing TCR gene therapy
for multiple myeloma
Gavin Bendle
LLR Bennett Senior Fellow
School of Cancer Sciences
University of Birmingham
Overview
•Introduction to TCR gene therapy of cancer
•Assessing the value of TCR gene therapy in a
autochthonous mouse cancer model
•Pre-clinical development of TCR gene therapy of MM
Multiple myeloma
•Plasma cell malignancy that is the second most common
haematological malignancy worldwide
•Novel agents (e.g. lenalidomide, bortezomib) have contributed to
increase in average survival times from 4 to 8 years in the last decade
• Despite these advances MM remains largely incurable
Need for new therapeutic approaches that not only
increase survival times but are also ultimately curative
Adoptive T cell therapy
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T cell immunity to tumours can be induced by adoptive T cell therapy:
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Transfer of T cells
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T cell replete allogeneic-HSCT & DLI for haematological malignancies
Mortality/morbidity due to GVHD limits use
Transfer of genes encoding the TCR
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Ag-specificity of a T cell determined by TCR
Endow patient T cells with tumour-reactivity of a defined Agspecificity
Destroy tumours without damaging normal tissues
TCR gene therapy
Cancer patient
Removal of peripheral
blood lymphocytes
Infusion of autologous
TCR gene modified T cells
Ex vivo transduction process
•
TCR gene transfer is conceptually attractive:
– Generate large numbers of defined antigen-specific patient T cells
– Generate tumor-reactive specificities not present in pre-existing T cell repertoire
Clinical trials shown feasibility and potential of TCR gene
therapy
CT scan of liver metastasis in patient
treated with TCR-modified T cells
Clinical testing of TCR gene therapy for MM has commenced
Safety risks associated with TCR gene therapy
• Most targets of TCR gene therapy are tumor-associated self-antigens
• Toxicity may occur if target antigen is also expressed by some normal tissues
• If vital normal tissues express target antigen toxicity can be severe or even fatal
Expression profile of target antigen in normal tissues:
the critical parameter determining the safety of TCR gene therapy
Additional genetic modification of T cells
as a strategy to enhance TCR gene therapy efficacy
Can additional genetic modification of TCR transduced T cells be
used to obtain durable clinical responses with TCR gene therapy?
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TCR gene transfer endows patient T cells with desired antigenspecificity
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Can additional genetic modification endow these cells with
optimal functional properties?
Used a mouse model of prostate carcinoma to assess if
additional genetic modification of T cells enhances TCR gene therapy efficacy
TRAMP mice: prostate tumour model
TRansgenic Adenocarcinoma of the Mouse Prostate
(Greenberg et al, PNAS 1995)
SV40 large T antigen under control of a rat probasin promoter
3
8-12
16
24
Does TGF- signalling blockade in TCR transduced T cells
promote tumour regression in TRAMP mice?
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Block by co-transducing TCR Td T cells with dnTGFRII
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Rationale for blockade of TGF- signalling in TCR transduced T cells:
• TGF- signalling inhibits CTL proliferation & effector functions
• Elevated TGF-expression in human malignancies including prostate cancer
• Elevated levels of TGF- in prostate of TRAMP mice with advanced prostate cancer
Does blockade of TGF- signalling in TCR transduced T cells
lead to tumour regression in TRAMP mice?
Histopathology
Week
3
24
8-12
Group I:
16
24
Pre-conditioning (5Gy TBI) + non Td T cells
Group II: Pre-conditioning (5Gy TBI) + SV40 TCR Td T cells
Group III: Pre-conditioning (5Gy TBI) + dnTGFRII Td T cells
Group IV: Pre-conditioning (5Gy TBI) + SV40 TCR & dnTGFRII co-Td T cells
28
Regression of advanced prostate cancer in TRAMP mice after
TCR gene therapy and blockade of TGF- signalling
Tumour regression in TRAMP mice after TCR gene therapy & TGF- blockade
Non-Td
SV40 TCR Td
SV40 TCR &
dnTGFRII co-Td
SV40 Large T Ag
Immunostaining
Ki67
Immunostaining
TGF-1
Immunostaining
Is the tumour regression observed at 28 wks of age sustained?
Sustained regression of advanced prostate cancer in TRAMP mice
after TCR gene therapy and blockade of TGF- signalling
Does the observed tumor regression lead to enhanced survival?
Enhanced survival of TRAMP mice after TCR gene therapy & TGF- blockade
Summary
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Blockade of TGF- signalling in TCR transduced T cells promotes tumour
regression in TRAMP mice with advanced prostate cancer
•
Demonstrates potential of additional genetic modification of TCR
transduced T cells to enhance TCR gene therapy efficacy
•
Elevated expression of TGF- in many human malignancies including MM
suggest that this approach warrants clinical testing
Pre-clinical development of
TCR gene therapy for MM
TCR a- & -chains are generated by genetic rearrangement
TCR α-chain
TCR β-chain
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Sequence information in TCR a- & -chain genes that defines TCR specificity:
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V element used
J element used
Nucleotide sequence at the junction between V-J elements
Cloning of TCR genes is a bottleneck in production of new TCRs
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Developed high throughput strategy to isolate TCR gene sequences for TCR gene therapy
TCR gene capture technology
Identify potentially clinically relevant T cell specificities
in biological material using multiplexing technology
FACS sort single T cells of desired specificity,
expand in vitro for 14 days and extract gDNA
Reconstruction of
TCR a- & -chain genes
Align sequence pairs to
reference genome to identify
rearrangements at TCR a &  loci
Shear gDNA & enrich for
gDNA fragments
encoding TCR a &  loci
Paired-end deep sequencing
Linnemann et al. Nature Med in press
A library of cancer-testis antigen-specific TCRs
assembled using TCR gene capture
Target antigen
Restriction
Peptide
# TCRs
Material
MAGE-C2
A2
ALKDVEERV
2
PBMC
MAGE-C2
A2
LLFGLALIEV
1
TIL
MAGE-C2
A2
KVLEFLAKL
3
PBMC
MAGE-A10
A2
GLYDGMEHL
2
TIL
SSX-2
A2
MLAVISCAV
3
PBMC
MAGE A2
A2
YLQLVFGIEV
3
PBMC
NY-ESO-1
A2
SLLMWITQA
1
PBMC
LAGE-1
A2
MLMAQEALAFL
1
TIL
MAGE-A1
B7
RVRFFFPSL
1
TIL
HERV-Kmel
A2
MLAVISCAV
2
TIL
TAG
A3
RLSNRLLLR
2
TIL
Rapidly assembled a panel of tumor-reactive TCRs with the potential
to be used for TCR gene therapy of a variety of malignancies
Cancer-testis antigens
•Cancer-testis (C/T) antigens:
•
Expressed in human germ line & variety of human malignancies
•
Attractive targets for TCR gene therapy of MM:
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Expression in MM cells in a high frequency of patients
• Examples:
• MAGE C1: ~ 70%
• MAGE C2: ~ 50%
•
Absent from normal tissues accessible to immune system
Validation and characterisation of C/T Ag-specific TCRs
C/T Ag-specific TCRs isolated to date
Target antigen
Restriction
Peptide
# TCRs
Material
MAGE-C2
A2
ALKDVEERV
2
PBMC
MAGE-C2
A2
LLFGLALIEV
1
TIL
MAGE-C2
A2
KVLEFLAKL
3
PBMC
MAGE-A10
A2
GLYDGMEHL
2
TIL
SSX-2
A2
MLAVISCAV
3
PBMC
MAGE A2
A2
YLQLVFGIEV
3
PBMC
NY-ESO-1
A2
SLLMWITQA
1
PBMC
LAGE-1
A2
MLMAQEALAFL
1
TIL
MAGE-A1
B7
RVRFFFPSL
1
TIL
TAG
A3
RLSNRLLLR
2
TIL
•Assess which C/T Ag-specific TCRs are best suited to clinical translation of TCR gene
therapy for MM:
– C/T Ag expression frequency in MM patient population
– Efficacy & safety of T cells transduced with different C/T Ag-specific TCRs
Blockade of TGF- signalling in C/T Ag-specific TCR transduced T cells
•Additional genetic modification of TCR transduced T cells to tailor their
activity can enhance TCR gene therapy efficacy
•TGF- in MM
dysregulation
microenvironment
leads
to
myeloma-induced
T
cell
•Selectively block TGF- signalling in C/T Ag-specific TCR transduced T cells
– Generate and validate a retroviral construct encoding the relevant C/T
Ag-specific TCR and a dominant-negative TGF- receptor
Primary benefit:
novel clinical trials of TCR gene therapy in MM patients in B’ham
Summary
•TCR gene therapy holds promise as a treatment for cancer
•Additional genetic modification of TCR transduced T cells to tailor their
activity and enhance therapeutic efficacy
•TCR gene capture utilized to assemble a library of C/T Ag-specific TCRs
that can be used for TCR gene therapy of various cancers
•Assessing which TCRs are best suited to clinical translation of TCR gene
therapy for multiple myeloma
•Pre-clinical development will be followed by clinical testing in B’ham
Acknowledgements
•NKI, Amsterdam
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Carsten Linnemann
Laura Bies
Kaspar Bresser
Bianca Heemskerk
Pia Kvistborg
Roel Kluin
Ron Kerkhoven
Marja Nieuwland
Ji-Ying Song
John Haanen
Ton Schumacher
•Cancer Sciences, B’ham
– Guy Pratt
– Paul Moss
•Shemyakin & Ovchinnikov Institute,
Moscow
– Dmitriy Chudakov
– Dmitriy Bolotin
•MDC, Berlin
– Xioajing Chen
– Thomas Blankenstein