Developing TCR gene therapy for multiple myeloma

Gavin BendleLLR Bennett Senior FellowSchool of Cancer SciencesUniversity of Birmingham

•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

Overview

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

• T cell immunity to tumours can be induced by adoptive T cell therapy:

• Transfer of T cells

• T cell replete allogeneic-HSCT & DLI for haematological malignancies• Mortality/morbidity due to GVHD limits use

• Transfer of genes encoding the TCR

• Ag-specificity of a T cell determined by TCR• Endow patient T cells with tumour-reactivity of a defined Ag-specificity• Destroy tumours without damaging normal tissues

Adoptive T cell therapy

Cancer patient

Removal of peripheralblood lymphocytes

Infusion of autologous TCR gene modified T cells

Ex vivo transduction process

TCR gene therapy

• 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

CT scan of liver metastasis in patienttreated with TCR-modified T cells

Clinical trials shown feasibility and potential of TCR gene therapy

Clinical testing of TCR gene therapy for MM has commenced

Safety risks associated with TCR gene therapy

Expression profile of target antigen in normal tissues: the critical parameter determining the safety of 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

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?

• TCR gene transfer endows patient T cells with desired antigen-specificity

• 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

expression Large T

intraepithelial

neoplasia (PIN)

Microinvasivecarcinoma

3 8-12 16

Invasivecarcinoma

24

• Block by co-transducing TCR Td T cells with dnTGFRII

• 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 TGF- signalling blockade in TCR transduced T cells promote tumour regression in TRAMP mice?

Group I: 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) + dnTGFRII Td T cells

Group IV: Pre-conditioning (5Gy TBI) + SV40 TCR & dnTGFRII co-Td T cells

Does blockade of TGF- signalling in TCR transduced T cells lead to tumour regression in TRAMP mice?

expression Large T

intraepithelial

neoplasia (PIN)

Microinvasivecarcinoma

3 8-12 16

Invasivecarcinoma

24

Week 24 28

Histopathology

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 TdSV40 TCR &

dnTGFRII co-Td

SV40 Large T Ag Immunostaining

TGF-1Immunostaining

Ki67Immunostaining

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

• 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

Summary

Pre-clinical development ofTCR gene therapy for MM

TCR α-chain

TCR β-chain

TCR a- & -chains are generated by genetic rearrangement

• Sequence information in TCR - & -chain genes that defines TCR specificity:– 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– Developed high throughput strategy to isolate TCR gene sequences for TCR gene therapy

TCR gene capture technology

Shear gDNA & enrich for gDNA fragments

encoding TCR & loci

Paired-end deep sequencing

Align sequence pairs to reference genome to identify

rearrangements at TCR & loci

Reconstruction of TCR - & -chain genesIdentify 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

Linnemann et al. Nature Med in press

A library of cancer-testis antigen-specific TCRs assembled using TCR gene capture

Rapidly assembled a panel of tumor-reactive TCRs with the potentialto be used for TCR gene therapy of a variety of malignancies

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

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:

• 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

•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

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 PBMCMAGE-A10 A2 GLYDGMEHL 2 TIL

SSX-2 A2 MLAVISCAV 3 PBMCMAGE A2 A2 YLQLVFGIEV 3 PBMCNY-ESO-1 A2 SLLMWITQA 1 PBMC

LAGE-1 A2 MLMAQEALAFL 1  TILMAGE-A1 B7 RVRFFFPSL 1 TIL

TAG A3 RLSNRLLLR  2 TIL

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 microenvironment leads to myeloma-induced T cell dysregulation

•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

•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

Summary

Acknowledgements

•NKI, Amsterdam– Carsten Linnemann– Laura Bies– Kaspar Bresser– Bianca Heemskerk– Pia Kvistborg– Roel Kluin– Ron Kerkhoven– Marja Nieuwland– Ji-Ying Song– John Haanen– Ton Schumacher

•Shemyakin & Ovchinnikov Institute, Moscow

– Dmitriy Chudakov– Dmitriy Bolotin

•MDC, Berlin– Xioajing Chen– Thomas Blankenstein

•Cancer Sciences, B’ham– Guy Pratt– Paul Moss