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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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

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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

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HERV-E high/HLA-A11 negRCC

HLA-A11 T cells

Act. HLA-A11 T cells

E:T ratio

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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

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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