HERV-E TCR Transduced Autologous T Cells for patients with clear cell RCC Rosa Nadal, M.D. Molecular Therapeutic Branch National Heart, Lung, and Blood Institute National Institutes of Health, Bethesda, MD, USA Kidney Cancer Research Summit 2019
HERV-E TCR Transduced Autologous T Cells for patients with clear cell RCC
Rosa Nadal, M.D.Molecular Therapeutic BranchNational Heart, Lung, and Blood InstituteNational Institutes of Health, Bethesda, MD, USA
Kidney Cancer Research Summit 2019
1998- February1998 April
1 month post transplant
Childs R, et al. N Engl J Med. 2000;343:750-8
Takahashi et al. J Clin Invest. 2008;118:1099-1109
(CD3+CD8+:95%, CD3+CD4+:3%)
HLA-A*11:01 positive RCC panel
2019 Follow-up CT scan
Screening of cDNA library by gene expression cloning to identify the transcripts encoding for RCC specific antigen
RCC cell linerecognized by HLA-A11
restricted CTL
cDNA library
Pools of 100 bacterial colonies
Plasmid DNA extractionCo-transfection intoCOS-7-HLA-A11 cells
Amplification of plasmids for 24h
Addition of CTL clone for 24h
Measurement of GM-CSF productionIn supernatant by ELISA
Identification of two transcripts(called CT-RCC-8 and CT-RCC-9)
Takahashi et al. J Clin Invest. 2008;118:1099-1109
CT-RCC HERV-E provirus and known transcripts
Cherkasova, E, et al. Cancer Research. 2016. 76:2177-85.
Common RegionCT-RCC -1
SU1 and TM1 env-derived peptides
HLA-A 11:01Phase 1
HLA-A 02:01
Chr 6q
Retrovirus
Germ cell
Reverse transcription and integration into germ cell
genome
gag pol envLTR pro LTR
Mutation accumulationMethylation of promoters
The making of HERVs
Mechanisms of regulation CT-RCC HERV-E expression in ccRCC
Functional pVHL absent in clear cell RCC
HIF2pVHL
HIF2 HIF2
HIF2
HIF2
gag pol envLTR pro LTR6q
HIF2
HERV-Eantigen display
• HERV-E only expressed in ccRCC with absence of functional VHL protein
• HIF-2 alpha level correlates with HERV-E expression
• HIF-2 alpha binds HRE in HERV-E LTRs• Demethylated HERV-E LTRs
CT-RCC HERV-E expression is restricted to the clear cell histology
No CT-RCC HERV-E expression in Normal Tissues
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4000
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Clear cell lines Clear cell fresh tumors
Non Clear Cell Kidney Tumors
HERV-E expression
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500
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1500
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Ad
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Cherkasova et al. Oncogene 2011; 30:4697-4706
Clear Cell RCC
HERV-E as a tumor-associated antigen in ccRCC
▪ Regression of metastatic ccRCC after HSCT is associated with recognition of an HERV-E -
antigen by donor T cells without GVHD
▪ Antigens derived from this HERV-E provirus are immunogenic, stimulating cytotoxic T-cells
and killing of ccRCC cells in vitro and in vivo
▪ HERV-E is selectively expressed in most of ccRCC but not in normal tissues
▪ Restricted expression of HERV-E in ccRCC is consequence of VHL gene inactivation
Takahashi, Y et al. J.Clin.Invest. 2008; 118:1099-1109.Cherkasova, E et al. Oncogene. 2011; 30:4697-706.Cherkasova, E et al. Cancer Research. 2016; 76:2177-85HERVs: human endogenous retrovirus; VHL, Von Hippel-Lindau
TCR from a HERV-E Reactive T-Cell Line Cloned into a Retroviral Expression Vector
0
200
400
600
800
1000
1200
1400
Concentration of peptides (nM)
Pro
du
ctio
n o
f IF
Ny
(pg/
ml)
PATWLGSKTWK
ATWLGSKTWK
TWLGSLTWKR
TWLGSKTWKR
Identification of peptide recognized by HERV-E reactive CD8+ T-cells
Cloned Vb7.1 TCR recognizingHERV-E peptide ATWLGSKTWK
RV vector encoding Vb7.1 TCR sequence
T-cells expressing the HERV-E specific TCR Kill RCC tumor cells
Transduction of cancer patient
T-cells
HERV- E TCR Transduced T-cells Recognize and Kill RCC Cells
0
200
400
600
800
1000
1200
1400
1600
HERVhigh/A11+
HERVhigh/A11-
HERVlow/A11-
HERVlow/A11+
No target
IFN
-y, p
g/m
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ccRCC cell lines
0
5
10
15
20
25
30
35
40
45
50
0 5 10 15 20 25
HERV-E high/HLA-A11+ RCC
HERV-E high/HLA-A11 negRCC
HLA-A11 T cells
Act. HLA-A11 T cells
E:T ratio
% o
f sp
ecif
ic ly
sis
ELISA, IFN gamma Cr release cytotoxicity assay
R. Nadal. ESMO 2018 Congress. Annals of Oncology. 2018; 29 (suppl_8)
Time Procedures and Testing
Day 0Sterility TestingPerform Ficoll PBMCs IsolationActivate 2x109 PBMCs x OKT3, IL-2 and IL-15
Day 2CD4 DepletionTransduction of CD8 cells
Day 6CD34t+ Selection on CliniMACsVector Copy Number Assay and RCR Detection Assay
Day 8 Sterility Testing
Day 10REP Day 0
Rapid Expansion Program (REP)Vector Copy Number Assay and RCR Detection Assay
Day 15REP Day 5
Transfer to WAVEElisaSterility Testing
Day 20REP Day 10
Package and Cryopreserve Infusion ProductSterility testsFull RCR testing
Day 21REP Day 11
Ship Cryopreserved Infusion product to NIH
CD34t expression
RETROVIRAL VECTOR: MMLV
HERV-E TCR gene CD34 truncated Cassette
Clinical-Grade Manufacturing HERV-E transduced T cells
MMLV, Moloney murine leukemia virus.
Post-thaw of clinical-grade TCR transduced CD8+ T-cellsTest Method Acceptance Criteria
Viability at time of Packaging
Trypan Blue Exclusion
> 70%
Total Viable Cell NumberVisual
microscopic count
Minimum Treatment
Dose Cell #
CD34+ FACS analysis >25%
CD8+ FACS analysis >80%
CD8+/CD34+ FACS analysis > 25%
Sterility Testing - Negative
Endotoxin Limulus assay <5 EU/Kg
Vector Copy # PCR-based assay <5 copies/cell
RCR by PCR PCR-based assay Negative
Interferon Gamma Release
ELISA 2X over background
93.4% CD8+
99.0% CD34t+ and tetramer+
A Phase I Study of HERV-E TCR Transduced Autologous T Cells in Patients with
Metastatic Clear Cell Renal Cell Carcinoma
https://clinicaltrials.gov. NCT03354390
Study Design and Key Eligibility Criteria
• Phase I ‘3+3’ design
• End-points:
• Primary: Safety
• Secondary:
• ORR, time to response, duration of response,
PFS and OS
• Immunologic correlates
• Key eligibility criteria:
▪ RCC with clear cell component
▪ HLA-A*11:01 positive
▪ Measurable disease
▪ Age < 70 years old
▪ ECOG PS 0-1
▪ Prior antiangiogenic and immune-checkpoint inhibitors
D 0
C+F F F
D+14 D+21
Tumor
Assessment
D+1 D+7
ApheresisIn vitro Generation
HERV-E TCR T-cells
Cryopreserve Cells at
specified dose
HERV-E TCR transduced T-cells
IL-2
Lymphodeplecting chemotherapy:
C: Cyclophosphamide 1000 mg/m2/day I.V.
F: Fludarabine 30 mg/m2/day I.V.
IL-2: 2,000,000 IU/m2 I.V. q12h x 14 doses
Investigational Plan
Dose Escalation Plan
Escalation Plan
Dose Level Dose of HERV-E TCR transduced T-cells
Level 1 1 x106 HERV-E TCR transduced CD8+/CD34+ enriched T-cells per kg body weight
Level 2 5 x 106 HERV-E TCR transduced CD8+/CD34+ enriched T-cells per kg body weight
Level 3 1 x 107 HERV-E TCR transduced CD8+/CD34+ enriched T-cells per kg body weight
Level 4 5 x 107 HERV-E TCR transduced CD8+/CD34+ enriched T-cells per kg body weight
✓
1
Challenges and Future
• Establish safety of HERV-E TCR transduced T cells
• Confirm HERV-E as a target for cellular therapy
• Widen the HLA restriction of targets• Identification of new HERV-E derived peptides
• Development of CAR-T cells
▪ Dr. Michael I. NishimuraProfessor of SurgeryVice Chair for Surgery ResearchCancer Immunology Program Leader
▪ Gina ScurtiLab Director, Nishimura Lab
Cardinal Bernardin Cancer CenterStritch School of MedicineLoyola University Chicago
Childs lab:▪ Elena Cherkasova▪ Robert Reger▪ Stefan Barisic▪ Long Cheng▪ George Aue▪ Kristen Wood▪ Tatyana Worthy
Our Collaborators:
▪ Julie Erb-Alvarez▪ Steve Highfill▪ Adriana Byrnes▪ Sasha Morehouse▪ Lisa Cook▪ Brian WellsDr. Richard W. Childs. MD
Assistant United States Surgeon GeneralRear Admiral Commissioned Corps USPHSChief Section of Transplantation Immunotherapy. NHLBI. NIH