A Phase I Study of HERV-E TCR Transduced Autologous T Cells in Patients With Metastatic Clear Cell Renal Cell Carcinoma
Phase: PHASE1
Conditions: Kidney Cancer
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

 
 Protocol with Statistical Analysis Plan Cover Page:   
 
Official Title: 
A Phase I Study of HERV-E TCR Transduced Autologous T Cells in Patients with 
Metastatic Clear Cell Renal Cell Carcinoma 
NCT number: 
 
NCT03354390 
Document Type:  
Protocol with Statistical Analysis Plan (SAP)  
Document Date: 
May 11, 2023 
 

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Richard Childs, MD 
 
 
CLINICAL RESEARCH PROJECT 
 
 
 
 
Protocol #18-H-0012  
 
 
 
 
 
 
 
 
IND #17690 
 
Date:    May 11, 2023 
 
Title: A Phase I Study of HERV-E TCR Transduced Autologous T Cells in Patients with Metastatic 
Clear Cell Renal Cell Carcinoma 
 
Other identifying words: kidney cancer, genetically modified lymphocytes, tumor antigens, T cell 
receptor immunotherapy, genomic retroviral elements. 
 
Principal Investigator:   
Richard W. Childs, M.D., 
Cellular and Molecular Therapeutics Branch (CMTB) 
National Heart Lung and Blood Institute 
Building 10, Room CRC 3-5330 
9000 Rockville Pike,  
Bethesda, MD 20892 
Phone: 301-451-7128 
Email: childsr@nhlbi.nih.gov 
 
 
 
 
Loyola University Site Investigators: 
Michael I. Nishimura, PhD.  Site Principal Investigator 
Gina Scurti, PhD.  
Annika Dalheim  
 
Subjects: 
 
 
Number 
 
                             Sex 
 
Age range 
Recipients: 
 
 
up to 24 
 
 
 
Either  
18-75  
 
 
Project involves ionizing radiation?  
Yes (medically and research indicated) 
Off-site project? 
 
 
Yes (for cell processing and TCR transduction only) 
Multi-institutional project? 
 
Yes 
DSMB Involved? 
 
 
Yes 
Tech Transfer?  
 
 
Yes 
 
Investigational Agent 
Drug Name: 
HERV-E TCR transduced CD8+/CD34+-enriched T cells 
IND Number: 
17690 
Sponsor: 
NHLBI OCD 
Manufacturer: 
Dr. Michael Nishimura’s Laboratory 
 
 

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Richard Childs, MD 
 
 
1. PRECIS  
Metastatic renal cell carcinoma (RCC) is an incurable condition. Current therapy for this disease consists 
of the serial administration of agents such as VEGF, mTOR inhibitors and immunotherapy (high-dose 
(HD) IL-2 or immune-checkpoint inhibitors).  Long-term survival can be achieved with high-doses IL-2 
or immune-checkpoint inhibitors. However, of those patients treated with immunotherapy, three quarters 
will not respond at all and only 5-8% will achieve a complete and durable response.   
 
Allogeneic hematopoietic stem cell transplantation is also capable of inducing prolonged disease 
regression in patients with metastatic clear cell RCC (ccRCC). In vitro studies have established that 
transplanted donor T-cells targeting antigens expressed on RCC cells mediate these anti-tumor effects. 
However, hematopoietic stem cell transplantation can be toxic and associated with a 10-20% risk of 
procedure-related mortality. The observation that transplanted donor T-cells have the potential to cure a 
subset of patients with metastatic disease forms the basis for continued efforts in our laboratory to harness 
the power of T-cells to cure this disorder.  
 
Our team isolated a tumor-specific cytotoxic T lymphocyte (CTL) line from peripheral blood 
mononuclear cells (PBMCs) obtained after an allogeneic transplant from a patient who showed prolonged 
tumor regression. Using limiting dilution cloning, we identified an allogeneic (derived from the stem cell 
donor) CD8+ T-cell clone that killed ccRCC cells in an HLA A11 restricted fashion. Using cDNA 
expression cloning, we identified a HERV-E derived antigen expressed in the patient’s ccRCC cells to be 
the target of this T-cell clone. Remarkably, we found this HERV-E was expressed in the majority of 
ccRCC cells with no expression in normal tissues. Based on the identification of the antigenicity of the 
HERV-E transcripts in ccRCC, our team in collaboration with Dr. Nishimura’s laboratory (Loyola 
University Cardinal Bernardin Cancer Center) has cloned, expressed and characterized the TCR from this 
CD8+ T-cell clone that recognizes an HLA A11 restricted HERV-E antigen. 
 
This research protocol is therefore designed to evaluate the safety and effectiveness of infusion of HERV-
E TCR transduced CD8+/CD34+ enriched T cells in HLA-A*11:01 positive patients with metastatic clear 
cell RCC. Subjects will receive a novel non-myeloablative immunosuppressive conditioning regimen of 
cyclophosphamide and fludarabine followed by an infusion of HERV-E TCR transduced CD8+/CD34+ 
enriched T cells. To mediate T cell survival and sustain function, moderate-doses of IL-2 (aldesleukin) 
will be administered intravenously twice a day for 14 doses.  
 
The primary endpoint is safety by day 21. Secondary endpoints will include overall response rate, 
progression-free survival and overall survival. Exploratory studies will include persistence of circulating 
HERV-E TCR transduced CD8+/CD34+ enriched T cells, changes in immune cell subsets and activation 
status of T cells, as well as, other immunologic determinants with clinical outcomes at baseline, at 
different time points during treatment and at the time of disease progression. 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
 
1. 
PRECIS .................................................................................................................................... 2 
2. 
BACKGROUND ..................................................................................................................... 5 
3. 
SCIENTIFIC AND CLINICAL JUSTIFICATION .............................................................. 21 
3.1 Study Design.......................................................................................................................... 21 
3.2 Dose-limiting toxicities ......................................................................................................... 22 
3.3 Product manufacturing and testing ........................................................................................ 23 
4. 
ELIGIBILITY CRITERIA .................................................................................................... 28 
4.1 
Inclusion Criteria ............................................................................................................ 28 
4.2 
Exclusion Criteria ........................................................................................................... 29 
5. 
CLINICAL EVALUATION OF THE PARTICIPANT ........................................................ 30 
6. 
TREATMENT PLAN............................................................................................................ 35 
6.1 
Drug Administration ...................................................................................................... 35 
6.2 
Concomitant Medications/Measures .............................................................................. 36 
6.3 
Detailed Treatment Plan ................................................................................................. 38 
6.4 
Blood Product Support ................................................................................................... 39 
6.5 
Anti-Emetics................................................................................................................... 39 
6.6 
Granulocyte Colony-Stimulating Factor ........................................................................ 39 
6.7 
Avoidance of Corticosteroids ......................................................................................... 39 
7. 
MANAGEMENT OF COMPLICATIONS ........................................................................... 40 
7.1 Dose Modification/Delay .................................................................................................... 40 
7.2 Cytokine release syndrome ................................................................................................. 40 
7.3 
IL-2 Toxicities ................................................................................................................ 40 
8. 
BIOSPECIMEN COLLECTION .......................................................................................... 40 
8.1 Tumor Tissue ...................................................................................................................... 40 
8.2 Immunological Testing ....................................................................................................... 41 
8.3 Monitoring Gene Therapy Trials: Persistence and Replication Competent Retrovirus 
(RCR) ........................................................................................................................................ 42 
8.4 Post Infusion and Tumor Tissue Correlative Studies ......................................................... 42 
9. 
BIOSTATISTICAL CONSIDERATIONS ........................................................................... 45 
10. DATA and SAFETY MONITORING .................................................................................. 48 
10.1 Definitions......................................................................................................................... 48 
10.2 Adverse Event Management ............................................................................................. 48 

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10.3 NIH-Intramural IRB and CD reporting ............................................................................. 49 
10.5 Reporting of Pregnancy .................................................................................................... 49 
10.6 IND Sponsor Reporting Criteria ....................................................................................... 50 
10.7 FDA Reporting Criteria .................................................................................................... 50 
10.8 FDA Annual Reports (Refer to 21 CFR 312.33) .............................................................. 50 
10.9 Reports to the DSMB ........................................................................................................ 50 
10.10 Routine Adverse Event Recording .................................................................................. 51 
10.11 Clinical trial monitoring .................................................................................................. 51 
11. DATA COLLECTION AND EVALUATION ..................................................................... 51 
Research Sample Collection ..................................................................................................... 51 
12. BIOSPECIMEN AND DATA MANAGEMENT PLAN ..................................................... 51 
13. RESPONSE CRITERIA ........................................................................................................ 53 
14. HUMAN SUBJECT PROTECTIONS .................................................................................. 56 
15. RISK and DISCOMFORTS .................................................................................................. 58 
15.1 Related to cyclophosphamide ........................................................................................... 58 
15.2 Related to fludarabine ....................................................................................................... 58 
15.3 Related to Interleukin-2 (IL-2, aldesleukin) ..................................................................... 59 
15.4 Related to the apheresis procedure ................................................................................... 60 
15.5 Related to blood draws ...................................................................................................... 61 
15.6 Related to central line placement ...................................................................................... 61 
15.7 Related to metastatic site biopsy (when indicated) ........................................................... 61 
15.8 Related to Thoracentesis ................................................................................................... 61 
15.9 Related to Paracentesis ..................................................................................................... 62 
15.10 Related to imaging studies (when indicated) .................................................................. 62 
15.11 Related to cardiac function ............................................................................................. 64 
15.12 Safety Considerations for HERV-E TCR transduced CD8+/CD34+ enriched T cell .... 64 
15.13 Risks in Relation to Benefit ............................................................................................ 65 
16. INFORMED CONSENT PROCESSES AND PROCEDURES ........................................... 66 
17. PHARMACEUTICALS ........................................................................................................ 66 
18. APPENDICES ....................................................................................................................... 69 
19. REFERENCES ...................................................................................................................... 87 
 
 
 

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OBJECTIVES   
Primary Objectives:  
 
- 
To determine the safety of infusing escalating doses of HERV-E TCR transduced CD8+/CD34+ 
enriched T cells in patients who are HLA-A*11:01 positive who have progressive metastatic clear 
cell renal cell carcinoma. 
 
Secondary Objectives: 
 
- 
To assess for tumor response using Response Evaluation Criteria in Solid Tumors (RECIST) 
version 1.1 criteria, as well as progression-free survival (PFS) and overall survival (OS). 
- 
To evaluate biologic and immunologic parameters to understand and improve the safety and 
efficacy of these TCR transduced T cells in metastatic ccRCC patients. 
 
2. BACKGROUND 
 
The worldwide incidence of renal cell carcinoma (RCC) is increasing. Each year >300,000 new diagnoses 
and more than 140,000 deaths are recorded. RCC is also the most frequent form of kidney cancer accounting 
for 2.4% of all adult cancers and 90% of all kidney cancers.1 The most common subtypes are clear cell 
(cc)RCC, accounting for 70–75% of cases.2 Sixty to seventy percent of patients present with localized 
disease (stages I or II), for which radical or partial nephrectomy is largely curative with 5-year survival 
rates greater than 70%. Patients diagnosed with stage III and IV RCC have poorer prognosis, with 5-year 
survival rates of approximately 50% and 10%, respectively.3 Metastatic RCC (mRCC) is resistant to 
chemotherapy and radiotherapies and is in most cases incurable. 
 
The genetics of clear cell RCC 
A modest proportion (2–4%) of RCC is associated with the von Hippel–Lindau (VHL) syndrome. This 
familial form of RCC is caused by germ line mutations in the VHL tumors suppressor gene on chromosome 
3p.25.4 Pioneering investigations of VHL in sporadic ccRCC, the most common form of RCC, showed that 
VHL is inactivated by mutation or epigenetic changes in >90% of ccRCC, establishing this gene as a major 
driver of ccRCC. 5-7 Inactivating genetic alterations in VHL leads to constitutive stabilization of HIF-α and 
the formation of a HIF-α–HIF-β complex. This complex translocates to the nucleus and stimulates the 
expression of HIF target genes, which are involved in angiogenesis, glycolysis, cell proliferation, invasion 
and metastasis.3 
 
Genomic studies have also revealed that the PI3K–mTOR pathway, which has a regulatory role in hypoxia 
signaling and affects the activity of HIF-1α, is widely disrupted in ccRCC.8 In fact, the functional relevance 
of aberrant mTOR signaling was first demonstrated by the observation that deficiency of PTEN,9,10  and 
activation of AKT,11 were associated with poor prognosis. Subsequently, 27–28% of ccRCC tumors were 
found to be affected by genetic alterations in the PI3K–mTOR pathway.12,13 
 
Overall, 80% of ccRCC tumors harbor non-synonymous mutations in epigenetic regulatory pathways, 
emphasizing the important role of epigenome aberrations in this disease. These genes include the SWI/SNF 
chromatin remodeling gene PBRM1(mutated in 40% of patients); SETD2, (mutated in 19% of patients); 
and the BRCA1-interacting deubiquitinase gene BAP1 (mutated in 12% of patients).12,14,15 These three 
genes are near VHL on chromosome 3p and are affected by LOH in 90% of patients. Other epigenetic 
modifiers also harbor somatic mutations in ccRCC, including the X-linked genes KDM5C (also known as 
JARID1C)16 and KDM6A (also known as UTX).17 

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Furthermore, increased levels of tumor cell dedifferentiation and other sarcomatoid features are present in 
1–8% of RCC tumours,18 of which 65% exhibit clear cell histology.19 Patients with sarcomatoid ccRCC 
have a much worse prognosis than those with non-sarcomatoid tumours;20 however, little is known about 
the etiology of these tumors and the mechanisms underlying their aggressive behavior.21 Although 
sarcomatoid and carcinomatous components within the same tumors harbor equivalent mutations in genes 
known to drive ccRCC, the available sequence data suggest that sarcomatoid elements also harbor unique 
mutations in other cancer- related genes such mutations in TP53, and have a higher overall somatic mutation 
rate.22,23 
 
Treatment of clear cell RCC 
The treatment of RCC has been transformed by recognition of its immunogenic properties and pathogenesis 
(described previous paragraph). Prior to 2005, treatment options for mRCC were limited, but evidence 
existed that this tumor is particularly sensitive to manipulations of the immune system. Everson and Cole 
were among the first to describe spontaneous remissions in patients with mRCC when they reported cases 
of tumors exhibiting shrinkage without any substantial treatment in the 1960s.24,25 IL-2, a cytokine 
important for regulating circulating lymphocytes, was found to result in durable complete remissions in 
about 5–7% of clear cell mRCC patients, leading to the approval of aldesleukin by the US FDA for this 
population in 1992. Unfortunately, most patients did not respond and this treatment was extremely toxic, 
limiting its use to experienced, high volume centers and reserved for patients with a good performance 
status and acceptable comorbid conditions.26,27 A second cytokine, IFN-α, was also used for this disease, 
with a modest response rate (RR) of approximately 15% as a single agent. Overall, the initial era of 
immunotherapy for mRCC was characterized by low rates of disease control and high rates of toxicity.  
 
In 2005 and 2006, the Food and Drug Administration (FDA) approved sorafenib and sunitinib and 
subsequently five other antiangiogenic drugs were approved (pazopanib, axitinib, bevacizumab, 
cabozantinib, and lenvatinib). Two mTOR inhibitors, temsirolimus and everolimus also showed benefit in 
randomized, phase 3 trials and were also approved by the FDA.28 Second-line trials of FDA-approved 
agents are summarized in Table 1.  
 
Second-line or later treatment for advanced clear cell RCC 
Agents 
Patient 
no 
Median 
PFS 
month 
HR for Disease 
Progression 
(95%CI) 
P 
value 
Median 
OS 
HR for Death 
(95%CI) 
P 
value 
Objective 
Response Rate 
percent 
Motzer et al. 
Everolimus 
Placebo 
 
272 
138 
 
4.9 
1.9 
 
 
0.33(0.25-0.43) 
 
 
<0.001 
 
14.8 
14.4 
 
 
0.87(0.65-1.15) 
 
 
0.16 
 
1.8 
0 
Escudier et al. 
Sorafenib 
Placebo 
 
451 
452 
 
5.5 
2.8 
 
 
0.44(0.35-0.55) 
 
 
<0.01 
 
17.8 
15.2 
 
 
0.88(0.74-1.04) 
 
 
0.15 
 
2 
0 
Rini et al. 
Axitinib 
Sorafenib 
 
361 
362 
 
6.7 
4.7 
 
 
0.66(0.55-0.81) 
 
 
<0.001 
 
20.1 
19.2 
 
 
0.97(0.80-1.17) 
 
 
0.37 
 
19 
9 
Motzer et al. 
Lenvatinib/everolimus 
Lenvatinib 
Everolimus 
 
51 
52 
50 
 
12.8 
9.0 
5.6 
 
0.45(0.22-0.79) 
0.62(0.37-1.04) 
- 
 0.003 
0.12 
 
25.5 
19.1 
15.4 
 
0.51(0.3-0.88) 
0.68(0.41-1.14) 
- 
  0.02 
0.12 
 
35 
39 
0 
Choueiri et al. 
Cabozantinib 
Everolimus 
 
330 
328 
 
7.4 
3.9 
 
 
0.51(0.41-0.62) 
 
 
<0.001 
 
21.4 
16.5 
 
 
0.66(0.53-0.83) 
 
 
0.003 
 
17 
3 

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Motzer et al. 
Nivolumab 
Everolimus 
 
406 
397 
 
4.6 
4.4 
 
 
0.88(0.75-1.03) 
 
 
0.11 
 
25.0 
19.6 
 
 
0.73(0.57-0.93) 
 
 
0.002 
 
25 
5 
 
The response of some patients to immunomodulatory cytokines and the rare spontaneous regressions of 
metastatic disease provided the rationale for other immunologic approaches for the treatment of mRCC.  
PD-1 is an immune-inhibitory receptor that belongs to the CD28/CTLA-4 family and is inducibly expressed 
on CD4+ and CD8+ T cells, natural killer (NK) cells, B cells and monocytes within 24 hours from their 
immunological activation. 29 Usually, activated T cells, B-cells, NK cells, dendritic cells (DCs) 
and monocytes express PD-1 to restrict autoimmunity during inflammatory states such as infections. 
However, tumors have evolved the ability to express the main PD-1 ligand (PD-L1) to exploit this 
mechanism, thus downregulating the antitumor T cell response.30 Therefore, inhibiting this pathway with 
monoclonal antibodies (mAbs) targeting either PD-1 or PD-L1 can reenergize exhausted T cells 
downregulated by tumor-directed PD-L1 expression, culminating in innate antitumor detection and 
coordinated tumor cell death.  
 
After promising results in early-phases clinical trials on nivolumab, the Phase III CheckMate-025 trial 
demonstrated superior median overall survival (OS) with nivolumab (n = 406) in patients with advanced 
RCC who progressed on antiangiogenic therapy compared with patients receiving everolimus (n = 397; 25 
vs 19.6 months, respectively; HR: 0.73; p = 0.002).31 Nivolumab also showed a trend toward improved 
progression-free survival (PFS); (HR: 0.88; p = 0.11) and fewer patients experienced grade ≥ 3 adverse 
events (19 vs 37%, respectively). Based on this trial, nivolumab earned approval by the FDA as a second-
line therapy following antiangiogenic treatment failure in patients with advanced RCC [20].  
 
Another immunomodulatory approach studied for the treatment of mRCC is allogeneic hematopoietic stem 
cell transplantation (allo-SCT) with the hope to induce immune-mediated graft versus-tumor (GVT) effects. 
In 1999, Childs et al. reported the first successful use of non-myeloablative allogeneic transplantation for 
the treatment of RCC.32 In 2000, we reported findings from a series of 19 patients with advanced RCC 
treated with this approach.33 All 19 patients had had progressive disease before the transplantation, and 
prior cytokine therapy had failed to produce a response in 17 patients. Stem cells from human leukocyte 
antigen (HLA)-matched sibling donors were used for 17 patients, and the other two were given cells from 
a sibling donor mismatched at a single HLA locus. The overall response rate (ORR) was 53%, with three 
complete and seven partial responses. Responses were seen only among patients with clear-cell carcinomas. 
The onset of tumor response was delayed by a median of 4 months after transplantation; responses typically 
appeared after complete donor chimerism had been achieved, after withdrawal of immunosuppressive 
therapy, or after the development of GVHD. Based on our experience and other investigators 
experiences,33,34 allogeneic hematopoietic stem cell transplantation for metastatic RCC was established to 
be capable of inducing disease regression and sometimes disease remission in patients with advanced RCC, 
suggesting the presence of GVT effects. Hematopoietic stem cell transplantation can be toxic and associated 
with a 10-20% risk of procedure-related mortality which greatly limits its application. However, transplant 
trials showing GVT effects against this malignancy again highlight that ccRCC is an immune responsive 
tumor where long-term responses can be observed in a small fraction of patients. 
 
Tumor-associated antigen in humans 
Tumor cells can express certain molecules, termed tumor-associated antigens (TAAs), which can be used 
to discriminate tumor cells from most normal tissues. These antigens can be intact on the cell surface or, in 
the case of proteins, peptide fragments bound to HLA. Among the first HLA-restricted TAAs identified 
were the melanoma-associated antigen recognized by T cells 1 (MART1), tyrosinase complex and GP100 
(also known as PMEL), and T cells reactive with these antigens could be found in tumor infiltrating 

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Richard Childs, MD 
 
 
lymphocytes (TILs) that were used successfully in adoptive cell transfer (ACT) for patients with 
melanoma.35,36 In these studies, effective TILs could not be isolated from all patients, but tumor-reactive T 
cells could be generated from peripheral blood lymphocytes (PBLs) through genetic modification of TCR 
genes that were isolated from effective TILs.  
 
Relevant to our study is that some cancers associated with transforming viruses can express viral products, 
which are attractive targets because they are not expressed by normal tissues. Prior studies indicated that 
oncogenic proteins encoded by cancer-associated viruses, such as those derived from Epstein–Barr virus37 
and human papillomavirus (HPV)38 can be recognized by TCR Gene Engineered T Cells. Pre-clinical data 
also support that the successful targeting of mutant tumor-associated epitopes, especially those derived 
from driver mutations or transforming oncogenes, can induce regression of cancers.39  
 
The goal of adoptive T-cell therapy is to generate a robust immune-mediated antitumor response through 
the ex vivo manipulation of T cells. This aim can be accomplished through the selection and expansion of 
tumor-infiltrating lymphocytes (TIL), or through gene transfer of a synthetic TCR (TCR) or a chimeric 
antigen receptor (CAR) into T cells. 
 
Clinical experience with TCR-modified T cells 
T cells genetically engineered to express novel receptors can have enhanced tumor specificity and advances 
in ex vivo expansion enable the production of clinically relevant doses of these therapeutic cells. Several 
TCR clinical trials have been initiated in the US and Europe for the treatment of hematologic and solid 
cancers.  For example, a TCR trial completed in 2015 targeted a human leukocyte antigen HLA-A2–
restricted epitope derived from NY-ESO-1, a CGA located on the X chromosome.40 In this study, more 
than half of the patients with metastatic melanoma had evidence of cancer regression. Four patients 
achieved complete remissions, three of which were ongoing after >36 months. An additional 18 patients 
with synovial cell sarcoma were treated in this same study with also favorable clinical results.  
 
Other trials in this fast-growing field have subsequently demonstrated significant and prolonged tumor 
regression in patients with melanoma or sarcoma using genetically modified TCRs directed against 
MART1, melanoma-associated antigen 3 (MAGE-A3), gp100 and carcinoembryonic antigen (CEA). 41-44  
Similarly, a TCR trial targeting an A2-restricted epitope derived from the E6 oncoprotein of the high-risk 
HPV-16 serotype has also been completed in patients with HPV-16+ malignancies (NCT02280811).  
 
Chimeric antigen receptor (CAR) T cells for solid tumors  
The demonstration of clinical efficacy in trials using CAR-T-cell therapy is, at present, limited to 
hematological malignancies, but this modality is being explored clinically in the treatment of solid tumors. 
Solid tumors present three unique challenges not seen in, for example, B-cell acute lymphoblastic leukemia 
(B-ALL). Firstly, when compared with B-ALL, their microenvironment can be considerably more 
immunosuppressive. Secondly, antigen selection is, in general, more difficult because the antigen 
heterogeneity across the same malignancy is generally higher in solid tumors 45,46. Thirdly, 'on-target, off-
tumor' toxicity is more problematic because potential target antigens in solid tumors are more likely to be 
expressed in other essential organs. New targets for solid tumors that are beginning to enter clinical studies 
include mesothelin for the treatment of mesothelioma 47,48, pancreatic49,50 and ovarian cancer50; 
disialoganglioside GD251,52 and EGFRvIII53 for CNS malignancies; and mucin-1654 for the treatment of 
ovarian cancer.  
 
 
Risk of T-cell therapy 

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The infusion of T cells is generally well tolerated. Infusional adverse events are infrequent and mild, and 
are mostly due to the cryoprotectant, dimethyl sulphoxide, or concomitant medication.55 The main concern 
of T-cell therapy is the potential for delayed side effects. This became evident from the early days of 
allogeneic bone marrow transplantation when T cells were recognized as the central mediators of graft-
versus-host disease (GVHD).56,57 Donor T-cell infusion in patients with post-transplant relapse can bring 
about disease remission through a graft-versus-leukemia effect but this is generally associated with the 
development of GVHD as a result of alloimmunity against non-hematopoietic tissues.58,59 Although the 
antigenic targets in adoptive T cell therapy are much better defined, the potential for adverse effects, both 
on-target and off-target, remains. 
 
• 
On-target but off-tumor adverse effects 
T cells targeting differentiation antigens can be expected to also recognize nonmalignant cells that express 
the same antigens, resulting in adverse events. For example, melanoma patients treated with T cells 
targeting melanocyte differentiation antigens, such as MART-1 and gp100, often developed vitiligo and 
uveitis. These on-target toxicities have been observed across all forms of  T-cell therapeutic approaches, 
including tumor-infiltrating cells,60 in vitro-expanded T-cell clones,61 and TCR-transgenic cells.42 In 
general, on-target autoimmunity is associated with tumor regression,60,61  and is more prominent in 
treatment approaches that are more efficacious. Melanoma patients treated with T cells transduced with a 
high-avidity MART-1(27-35) TCR and murine-derived gp100 TCR had a significantly higher rate of tumor 
response than an earlier cohort of patients treated with T cells transduced with a lower-avidity MART-1(27-
35) TCR.42  T cells with gene-engineered TCRs specific for PMEL or MART1 mediated the destruction of 
normal melanocytes in the skin, eyes and ears of treated patients, and these detrimental effects sometimes 
required intervention with corticosteroid treatment.42 Not unexpectedly, the rate of autoimmunity was also 
correspondingly higher: of the 20 patients treated with the high-avidity TCR transgenic T cells, 11 (55%) 
developed uveitis and 10 (50%) developed a transient steroid-responsive hearing loss; the latter was not 
observed in previous studies and was attributed to the presence of melanocytes in the striae vascularis of 
the inner ear.42  
 
A similar pattern was seen in CD19 CAR T-cell therapy, where better disease response was associated with 
long-term B-cell depletion and hypogammaglobulinemia.62,63 On-target but off-tumor toxicities can be 
immediately life-threatening. A patient with colorectal cancer with lung and liver metastases developed 
respiratory distress within 15 min of HER2-specific CAR T-cell infusion and subsequently died from 
multiorgan failure 5 days later.64 It was postulated that the T cells recognized HER2 expressed by normal 
lung tissues, leading to the release of inflammatory cytokines, pulmonary toxicity and a cascading cytokine 
storm that cumulated in multiorgan failure. This adverse event was not foreseeable as it has not been 
observed in HER2 vaccine trials or the many breast cancer patients treated with the HER2 monoclonal 
antibody, trastuzumab. It is thought that the fatal toxicity was a function of the high potency of the CAR 
construct that contained CD28 and 4-1BB co-stimulatory molecules, and the use of prior nonmyeloablative 
chemotherapy that further enhanced treatment effect. 
 
On-target toxicities that are not immediately life-threatening can still be treatment-limiting. Carbonic 
anhydrase-IX (CAIX)-specific CAR T cells, which were studied in patients with metastatic RCC, were 
associated with dose-limiting liver toxicity because of low-level CAIX expression in bile duct 
epithelium.65,66  Similarly, a study using T cells transduced with a high-avidity murine TCR against human 
carcinoembryonic antigen (CEA) in patients with metastatic colorectal carcinoma was halted after all three 
patients developed severe transient colitis caused by the recognition of normal levels of CEA in the colonic 
mucosa.41 The hepatitis and colitis in both studies were either self-limiting or responsive to corticosteroids 
and there were no treatment-related deaths.41,65,66 More recently, however, fatal on-target toxicities were 

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reported in patients treated with anti-MAGE-A3 TCR-transduced T cells.44 In this study, HLA-A*0201 
transgenic mice were immunized with a MAGE-A3 peptide epitope to generate a high-avidity TCR that 
recognized not only MAGE-A3 but also MAGE-A9 and MAGE-A12. Nine patients with various 
malignancies were treated on this protocol and three developed altered mental status within a few days, two 
of whom became comatose and died. Autopsy showed necrotizing leukoencephalopathy with extensive 
white matter defects associated with CD8+ T-cell infiltration, caused by the previously unrecognized low-
level expression of MAGE-A12 in human brain.44 In each of these cases, the adverse effects occurred 
despite relatively low levels of antigen expression in the off-tumor sites, thus highlighting the potential for 
harm in using redirected T cells with high avidity and potency. 
 
• 
Cytokine release syndrome 
Cytokine release syndrome (CRS), which is characterized by fevers, rigors, hypotension and hypoxia, has 
been observed in a number of CD19 CAR T-cell studies as a result of large-scale T-cell activation upon the 
recognition of CD19+ malignant cells.62,63,67,68,69 The symptoms usually begin a few days following T-cell 
infusion but can be as early as 24 h, depending on the co-stimulatory domains, and coincide with the in vivo 
expansion of CD19 CAR T cells and the elevation of a number of serum cytokine levels, including 
interferon-γ, soluble interleukin-2 receptor α, interleukin-2, interleukin-6 and tumor necrosis factor. 67-70   
Some patients also develop features of macrophage activation syndrome, including very high ferritin levels, 
histological features of hemophagocytic lymphohistiocytosis, hepatosplenomegaly and disseminated 
intravascular coagulation.71A significant proportion of patients develop alarming but reversible 
neurological symptoms, including delirium and seizure-like activity, the reason for which is not fully 
understood but thought to be related to generalized T cell-mediated inflammation rather than direct toxicity 
of CAR T cells on the brain.62,67-69  In general, patients with evidence of persistent disease at the time of T-
cell infusion are more likely to develop CRS.69,72 
 
• 
Off-target adverse effects 
In mid-2011, a patient with metastatic melanoma suffered a sudden cardiac death 4 days after an infusion 
of autologous T cells transduced with an affinity-enhanced HLA-A1-restricted MAGE-A3 TCR.73 This 
high-affinity TCR was generated by introducing mutations into the α chain of a MAGE-A3 TCR that was 
isolated from another patient from a previous vaccination study. The α chain mutations increased the 
potency of the T cells against MAGE-A3-expressing targets in vitro and in vivo while maintaining a high 
level of specificity in vitro.73 Investigations into the death did not identify any evidence of direct T cell-
mediated toxicity and a second patient was enrolled the following year. This patient also suffered a cardiac 
death 5 days after T-cell infusion and was found to have extensive myocardial necrosis on autopsy.73 Further 
investigation using a combination of amino acid substitution and in silico screening showed that the MAGE-
A3 TCR also recognized a peptide from an unrelated muscle protein, Titin, which is important for the 
contraction of striated muscles.74 This off-target toxicity would have been very difficult to predict: Titin 
expression was undetectable in cardiac-derived primary cell lines and could only be detected in a more 
elaborate beating myocyte culture system,74 and the toxicity would not have been detected in HLA-A1 
transgenic mouse models either because there was no reactivity against the equivalent mouse Titin 
peptide.74 Although this remains the only example of off-target toxicity, the difficulties in predicting such 
toxicity and the fatal outcome are very concerning. Another potential source of off-target toxicity is the 
mispairing of transgenic α or β TCR chains with endogenous TCR, which can potentially give rise to TCRs 
with new specificities and autoreactivity.75 This has been demonstrated in murine models76 but has not been 
observed in human clinical studies encompassing >150 patients to date.77  
 

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There have been few completed clinical trials published utilizing CARs in solid tumor malignancies. While 
early trials did not demonstrate long-term cell engraftment nor anti-tumor efficacy, the CAIX trial did lead 
to reports of unexpected biliary tract toxicity, illustrating the potential risk for acute in vivo on-target, off-
tumor effects in patients.66 This study was stopped due to the advent of competing treatments before 
reaching therapeutic or maximum tolerated dose. 
 
Two case reports and results of a 19-patient trial have also been published. The first case by Morgan, et al. 
reported on the first in human use of a third generation CAR in solid tumors to treat a patient with refractory 
metastatic melanoma.64 Unfortunately, this patient experienced an immediate lethal severe adverse event 
following infusion of a large number (1011) of HER2 CAR T cells, attributed to off-tumor, on-target toxicity 
in normal tissues. Another report using the second-generation CAR targeting the same HER2 antigen in 
patients with refractory metastatic sarcomas was recently published.78 This trial started at a much lower cell 
dose, with as few as 104 T cells, working up to a maximum dose of 108 cells, still 3 logs below the dosage 
given in the NCI trial. While this trial showed no overt toxicity, it did have some indication of efficacy, 
with one patient experiencing a partial tumor response revealed by positron emission tomography (PET). 
Table 2 shows characteristics of selected published trials on TCRs and CARs. 
Table 2. 
TCRs Studies 
TCR Target 
HLA 
restriction 
Tumor histology 
Patient 
number 
RR 
Toxicity 
Potential mechanism of 
toxicity 
MART-1 
HLA-A2 
Metastatic Melanoma 
17 
12% (2PR) 
None 
N/A 
MART-1 
(+vaccine) 
HLA-A2 
Metastatic Melanoma 
20 
30%  (6PR) 
G3 skin 
G3 hearing 
G3 sight 
On-target, 
off-tumor 
toxicity on melanocytes in 
skin, eye and inner ear 
Gp100 
HLA-A2 
Metastatic Melanoma 
16 
19% (1CR) 
G3 skin 
G3 hearing 
G3 sight 
On-target, 
off-tumor 
toxicity on melanocytes in 
skin, eye and inner ear 
CEA 
HLA-A2 
Metastatic colorectal cancer 
3 
33% 
G3 colitis 
On-target, off-tumor in 
normal colon 
NY-ESO-1* 
HLA-A2 
Metastatic 
melanoma/Synovial 
cell 
sarcoma 
20/18 
55%(3CR)/61% 
(1CR) 
None 
N/A 
NY-ESO-1 
HLA-A2 
Multiple Myeloma 
20 
80% 
None 
NA 
MAGE-A3* 
HLA-A2 
Metastatic 
melanoma/Synovial 
cell 
sarcoma/Esophageal 
carcinoma 
9 
55% 
Mental status 
changes 
and 
fatalities (2/3) 
Neurotoxicity 
due 
to 
cross-recognition 
of 
MAGE-A12 in normal 
brain 
MAGE-A3 
HLA-A1 
Metastatic melanoma/ 
Multiple myeloma 
2 
0% 
Fatalities 
Cardiac toxicity due to 
cross-recognition 
of 
Titin-1 in heart tissue 
MAGE-A4* 
HLA-A2 
Esophageal cancer 
10 
0% 
None 
N/A 
HPV E6* 
HLA-A2 
Cervical cancer 
9 
33% (2CR) 
None 
N/A 
CARs Studies 
CAR Target 
HLA 
restriction 
Tumor histology 
Patient 
number 
RR 
Toxicity 
Potential mechanism of 
toxicity 
Alpha folate 
receptor 
- 
Ovarian cancer 
14 
0% 
None 
N/A 
CD171/L1-
CAM 
- 
Neuroblastoma 
6 
0% 
None 
N/A 

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Richard Childs, MD 
 
 
CAIX 
- 
Renal cell carcinoma 
3 
0% 
Biliary 
toxicity 
On-target, off-tumor 
toxicity in bile duct 
GD2 
- 
Neuroblastoma 
11 
27% 
None 
N/A 
IL13Rα2 
- 
Glioblastoma multiforme 
3 
0% 
None 
N/A 
Mesothelin 
- 
Mesothelioma/Pancreatic 
cancer 
1/1 
0% 
None 
N/A 
HER2/ERBB2 
- 
Sarcoma 
19 
0% 
None 
N/A 
HER2/ERBB2 
- 
Metastatic melanoma 
1 
NA 
Fatality 
On-target, off-tumor 
toxicity in lung and 
other normal tissues 
 
• 
Insertional mutagenesis 
The integration of viral vectors proximate to growth-promoting genes can result in the transactivation of 
proto-oncogenes and malignant transformation. Acute leukemia as a result of insertional mutagenesis (IM) 
has plagued a number of gene therapy studies for primary immunodeficiency disorders, including X-linked 
severe combined immunodeficiency,79,80 chronic granulomatous disease81 and Wiskott–Aldrich 
syndrome.82 IM was also reported in a β-thalassemia patient who was treated with a self-inactivating HIV-
1-based vector containing the β-globin gene controlled by its wild-type promoter. The patient was treated 
with autologous CD34+ cells transduced with a lentiviral vector.83 Clonal population analysis demonstrated 
a bias for one hematopoietic clone derived from transduced cells with the proviral cassette integrated into 
the HMGA2 proto-oncogene sequence causing a benign cell expansion. Interestingly, HMGA2 was 
overexpressed in myeloid cells, but the deregulation was not found in granulocyte-monocyte cells sharing 
the same vector integration pattern.  A recent report in one patient with chronic lymphocytic leukemia 
(CLL) treated with CARs showed that there may be IM of T cells via lentiviral integration  that impact on 
behavior CAR-T cells, with a TET2 insertion resulting in clonal expansion, direct tumor cell recognition 
and killing, and persistence of the T cell clone.84 
 
In contrast, there has not been any report of insertional mutagenesis arising from gene-modified T cells that 
have been administered to hundreds of patients. This vast difference in genotoxicity profile may be related 
to the nature of the transgenes involved and the pluripotency of hematopoietic stem cells, which may render 
them much more susceptible to malignant transformation than mature T cells.85 
 
Targeting endogenous retroviral elements 
Approximately 8% of the human genome consists of human endogenous retroviruses (HERVs) that are 
remnants of ancient infections and incorporation of viral sequences into the germline.86  The HERVs are 
mostly transcriptionally inactive due to epigenetic changes,87 but may be re-activated in tumors and can 
elicit anti-tumor immunity.88  There is pre-clinical evidence that TLR7 or RAG knockouts in mice develop 
uncontrolled endogenous retrovirus expression, endogenous retrovirus infectivity and endogenous 
retrovirus insertion-driven tumors.89,90 HERVs have been evolutionary inactivated by accumulation of 
indels and promoter methylation, but some HERVs have been shown to be transcriptionally active in human 
malignancies including melanoma91,92, breast cancer93, prostate adenocarcinoma94, ovarian cancer95, and 
most recently by our group in ccRCC.96,97  
 
Discovery and characterization of CT-RCC HERV-E expressed in ccRCC 
Childs and colleagues examined peripheral blood mononuclear cell (PBMC) from ccRCC patients treated 
with allo‐HSCT, who experienced tumor response following allo‐HSCT.  The aim was to identify tumor-

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Richard Childs, MD 
 
 
restricted antigens that may be involved in T cell-mediated antitumoral activity. As result of this pre-clinical 
work, cytotoxic T lymphocytes (CTLs) that killed patient tumor cells in vitro were isolated from patient’s 
PBMC collected after transplant. These RCC-reactive CD8+ T cells were undetectable in the PBMCs of 
both the donor and the patient before transplantation.96 Utilizing cDNA expression cloning, an HLA-A11–
restricted 10-mer peptide (named CT-RCC-1) was found as a target antigen for RCC-specific T cells in a 
number of patients. This peptide (ATFLGSLTWK) was found to be encoded by a novel human endogenous 
retrovirus (HERV) type E (named CT-RCC HERV-E).96 At present, three different HERV-E transcripts 
have been identified, named CT-RCC-8, CT-RCC-9 and CT-RCC-Env (e.g., GenBank Accession Nos. 
EU137846, EU137847, and JQ733905). Importantly, these transcripts were found to be expressed only in 
ccRCC cells, but not in other tumors or normal tissues.7, 9   
 
A recent report by Rooney et al. of the molecular and genetic properties of tumors that had associated local 
immune cytolytic activity using solid tissue tumor biopsies revealed numerous instances of endogenous 
retrovirus being reactivated in tumors. In the same study, three tumor–specific endogenous retroviruses, all 
with minimal to undetectable expression in normal tissues and elevated expression in tumor tissues, were 
described. One of them was observed in ccRCC tumors (referred as ERVE-4) in strong correlation with 
local high immune cytolytic activity.98 The ERVE-4 locus is the same as CT-RCC HERV-E described 
previously in our lab, but it was named in accordance with other nomenclature in Rooney et al. paper. In 
the following link https://www.ncbi.nlm.nih.gov/gene/?term=ERVE%E2%88%924 you will find evidence 
that ERVE-4 and CT-RCC HERV-E  represent the same HERV-E locus on chromosome 6q. Therefore, the 
findings by Rooney et al. represent confirmation of our group’s findings on the potential of this HERV-E 
to serve as an excellent target for T cell based immunotherapy trials. 
 
The CT-RCC HERV-E transcripts represent spliced variants sharing common region from 5’LTR and 
sequences encoding for parts of the protease and polymerase as well as the entire envelope genes, 
respectively (GenBank accession numbers EU137846.1, EU137847.1, and JQ733905).99 Sequence analysis 
of the envelope transcript revealed long open reading frames partially encoding putative surface and 
transmembrane envelope proteins.  
 Figure 1. 
 
Inactivation of the VHL tumor suppressor leads to selective expression of a human endogenous retrovirus 
in ccRCC 
 
In an attempt to further understand the mechanisms leading to specific expression of the CT-RCC HERV-
E in ccRCC, a total of 50 fresh (primary) ccRCC tumors (17 patients with sporadic ccRCC and 23 patients 

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Richard Childs, MD 
 
 
with VHL disease) and 17 non- ccRCC tumor samples and cell lines were analyzed for CT-RCC HERV-E 
expression and status of VHL tumor suppressor gene.  
 
Although expression levels varied among tumors, most ccRCCs (80%) were found to express CT-RCC 
transcripts. HERV-E expression was found to be restricted to clear cell histological type of RCC and caused 
by inactivation of the VHL tumor suppressor gene and HIF-2α transcriptional factor stabilization. 
Cherkasova, et al.  demonstrated that HIF-2α may serve as transcriptional factor for the CT-RCC HERV-
E by binding with HIF response elements (HRE) localized in proviral long terminal repeat (LTR). 
Remarkably, all ccRCC tumors expressing CT-RCC HERV-E possessed hypomethylated LTR. In contrast, 
non-ccRCC tumors and normal tissues had a highly methylated proviral LTR preventing CT-RCC HERV-
E expression. These findings suggest loss of function of the VHL tumor suppressor and HIF-2α stabilization 
together with epigenetic modifications lead to the selective expression of this endogenous retrovirus 
encoding antigens that serve as targets for T cells in ccRCC.97 (Figure 2) 
 
Our group also demonstrated that exposure of HERV-E-negative ccRCC lines containing a 
hypermethylated 5’LTR region to the demethylating agent DAC and the histone deacetylase inhibitor DP 
could enhance the provirus expression in vitro. 97 Although the mechanisms involved in deregulation of 
DNA methylation are not totally understood, our data suggest that treatment of ccRCC patients with 
demethylating agents would potentially lead to higher levels of CT-RCC HERV-E expression in kidney 
tumors. 
 
Figure. 2 
 
Our findings also concur with current evidence that cellular transformation by endogenous retroviruses-
encoded antigens is typically accompanied by major alteration of the epigenetic landscape that is normally 
found in healthy cells. As result, endogenous retrovirus elements are no longer fully transcriptionally 
repressed and adaptive immune responses to epitopes derived from endogenous retroviruses are frequently 
detected in the cancers.100,101  
 

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In summary, we consider CT-RCC HERV-E to be an exciting new target for ACT for several reasons. First, 
HERV-E is expressed by ~80% of the ccRCC fresh tumors and established cell lines.96  Second, CT-RCC 
HERV-E expression confined to the ccRCC cells with no expression found in other tumors or normal 
tissues.96,57,58 Finally, its expression is regulated by inactivation of VHL tumor suppressor gene which is 
inactivated by mutation or epigenetic changes in the vast majority of ccRCC. 12,102 
 
Two decades ago, it was demonstrated that it is possible to redirect the specificity of human T cells by 
engineering them to express TCR genes.103 However, there were no TCRs cloned that were specific for an 
antigen expressed by ccRCC and thus, no ACT options existed for such patients.  Based on the identification 
of the antigenicity of the HERV-E transcripts in ccRCC, our team in collaboration with Dr. Nishimura’s 
laboratory at Loyola University Cardinal Bernardin Cancer Center has cloned and characterized a TCR that 
recognizes HLA-A11-restricted HERV-E-derived  antigen CT-RCC-1. To bring this discovery from bench 
to bedside, our team has already generated two high titer retroviral producer clones which make virus 
capable of transferring the HERV-E TCR to normal human T cells. We have also created two Master Cell 
Banks and completed the Good Manufacturing Process (GMP) qualification process. Results of the pre-
clinical studies are described in detail in next section. Figure 3 below  summarizes the steps taken to develop 
this immunotherapy.  
Figure 3. 
 
HERV-E TCR transduced autologous T cells 
 

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Richard Childs, MD 
 
 
➢ Identification of the HLA-A11 restricted TCR sequence specific for CT-RCC-1 antigen and 
construction of retroviral vectors capable of transferring this TCR to normal PBL-derived CD8+ 
T cells  
Utilizing a RACE-PCR of total RNA extracted from a CTL clone followed by sequencing, we have 
identified the sequence of TCR recognizing CT-RCC-1 antigen. Full length cDNAs for α and β chains were 
cloned into SAMEN retroviral vector (Figure 4). The SAMEN CMV/SR retroviral vector was designed 
specifically for introducing TCR genes into alternate T cells. The 5’ long terminal repeat (LTR) in the 
SAMEN SR backbone was replaced with a hybrid LTR consisting of human CMV enhancer and promoter 
fused to the MMLV 5’ LTR. This modification permits production of transient retroviral supernatants in 
293 GP cells. Other key elements of the SAMEN CMV/SR vector include an internal SR promoter to 
permit the expression of multiple genes, unique SalI and XhoI restriction sites for ease of inserting TCR 
chains, and an internal ribosome entry site/neor cassette for G418 resistance.104 Our collaborators and others 
in the Surgery Branch, NCI used it for in vitro studies and clinical trials.  However, Dr. Nishimura modified 
it to remove the IRES/neor cassette since we no longer use G418 for selection. 
 
The retroviral vector, HERV-E TCR CD34t 7G1, consists of 7,948 bps including the 5' LTR from the 
murine stem cell virus (promoter), packaging signal including the splicing donor (SD), splicing acceptor 
sites, alpha chain and beta chain genes of the anti-HERV-E TCR. The HERV-E TCR is also linked to a 
truncated CD34 chain (CD34t) which was obtained from Dr. Timothy Clay when he was at Duke 
University.  Its origin is human. We used PCR to link the  and  TCR chains together with a T2A self-
cleavage sequence to facilitate high and equal expression of both TCR chains. The HERV-E TCR is linked 
to a CD34t by a different self-cleavage sequence (P2A) resulting in a final transgene cassette consisting of 
TCR  -TCR - CD34t.  The CD34t protein includes the extracellular and transmembrane regions of CD34, 
and as a result, it is expressed on the cell surface of transduced cells, but does not affect activity of cells 
expressing the truncated protein which is normally expressed on hematopoietic progenitor cells. T-cells 
expressing CD34t can be identified with an anti-CD34 antibody, and can be isolated using flow cytometry 
or immuno-magnetic methods to purify these transduced T cells and also for subsequent immune 
monitoring of the genetically modified T cells in vivo following adoptive transfer.105,106  Using this 
approach, our collaborators routinely isolate >98% pure transduced T cells.  
 
Multiple approaches for inserting TCR genes into T cells by using gammaretroviruses43,44,60 and lentiviruses 
74,107 have been assessed. The backbone of the retrovirus used here is also being used in an ongoing phase 
Ib gene therapy trial of TIL 1383I TCR transduced T cells in melanoma patients lead by our collaborator 
Dr. Nishimura at Loyola University (NCT02870244).  
 
Figure 4. 
 
➢ Generation of a panel high titer PG13 HERV-E TCR retroviral producer clones and selection of 
appropriate clones for use in clinical trials 

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Richard Childs, MD 
 
 
Once it was established that we successfully cloned a functional HERV-E TCR, the retroviral vector 
containing this TCR was introduced into the PG13 packaging cell line to isolate high titer retroviral 
producer clones for clinical use as previously reported.108 The bulk PG13 producer line was cloned in 
limiting dilution to generate high titer clones and clones were screened for transduction efficiency based on 
CD34 expression of the transduced T cells.  Then, the clones were screened for their ability to efficiently 
transduce and transfer anti-HERV-E reactivity to human T cells. Two clones were selected (7G1 and 27A7) 
for further testing.  
 
➢ Evaluation of transduction efficiency, the function of the transduced T cells, and the FDA 
mandated copy number testing.  
 
For this purpose, retrovirus was prepared from each clone and two separate full scale validation runs were 
performed. The Standard Operating Procedures (SOPs) for development of our clinical trial were developed 
and tested using these two full scale production runs.  
 
An example of one full scale production run is shown in Figure 4. T cells from 2 healthy donors were 
transduced with retrovirus from clone 7G1 or 27A7. They were 63.2% and 67.5% (X̅=65.4%) or 65.2% and 
61.6% (X̅=63.4%) CD34+ respectively indicating both clones transduced T cells with equal efficiency. 
Following CD34 purification of the transduced cells using immunomagnetic beads, the cultures were 99.3% 
and 98.4% (X̅=98.9%) or 99.4% and 99.7% (X̅=99.6%) CD34+ respectively indicating we had pure TCR 
transduced T cell cultures. (Figure 5) 
 
Figure 5. Efficiency of Purifying 
HERV-E TCR Transduced T 
Cells. 
A 
representative 
CD34 
purification 
of 
HERV-E 
TCR 
transduced 
T 
cells 
using 
the 
CliniMACS is shown. Supernatants 
from the PG13 retroviral producer 
clone 7G1 were used to transduce 
normal PBL-derived T cells using our patient treatment SOP’s that will be used on treated patients. Three 
days post-transduction, the cells were incubated with anti-CD34 coated immunomagnetic particles and 
purified using the CliniMACS and stained for CD34 expression. The log fluorescence of >104 live cells 
were measured by flow cytometry. Unstained (panel A), pre-selection (panel B), the positive fraction (panel 
C), and the negative fraction are shown. 
 
➢ Production and GMP qualification of the Master Cell Banks  
We generated a 212 vial PG13HERV-E Master Cell bank from clone 7G1 of 5x106 cells per vial for both 
clones (212 vials for 7G1 and 220 vials 27A7) in the event one fails GMP qualification. A large 6-liter 
batch of virus was prepared from the 7G1 clone and both the 7G1 clone and 7G1 virus were sent to the 
Vector Production Facility/National Gene Vector Biorepository at Indiana University for GMP 
qualification. Six liters of virus is adequate to complete all GMP qualification studies, the shipping 
validation runs to the National Institute of Health (NIH) for the clinical trial, and treatment runs for at least 
20 patients.  
 
➢ Characterization of the reactivity and specificity of HERV-E TCR transduced T cells and 
determination of the relative affinity of the HERV-E TCR 

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Richard Childs, MD 
 
 
We also confirmed that the HERV-E TCR transduced T cells function as predicted. We assessed antigen 
specificity of HERV-E TCR transduced T cells measuring interferon-y (INF-) secretion by enzyme-linked 
immunosorbent assay (ELISA) using a panel of ccRCC cell lines as targets. Figure below shows that 
transduced T cells produce INF- only when coculturing with both HLA-A11-positive and CT-RCC 
HERV-E-expressing ccRCC cells (>twice background and >1 ng/ml). 
 
HERV-E TCR transduced T cells also lyse an HLA-A11:01+/HERV-E+ tumor and an HLA-
A11:01+/HERV-E- tumor cells loaded with the CT-RCC-1 peptide but not an HLA-A11:01+/HERV-E- or 
an HLA-A11:01-/HERV-E- tumor cells. The killing was not NK cell-mediated because K562 cells were not 
lysed. These results support the ability of our HLA-A11-restricted HERV-E-specific TCR to transfer anti-
tumor reactivity against ccRCC via targeting of a HERV-E antigen to normal PBL-derived T cells. (Figure 
7). 
 
An FDA mandated assay required to be performed for our clinical trial is to determine the average number 
of copies of the integrated virus per cell. The FDA mandates the copy number to be less than 5 for any gene 
0
400
800
1200
1600
HERV-E+/HLA-A11+
HERV-E+/HLA-A11-neg
HERV-E-neg/HLA-A11+
HERV-E-neg/HLA-A11-neg
K562
Figure 7. Killing of RCC Cells by HERV-E TCR Transduced T Cells. 
A representative CTL assay shows the ability of HERV-E TCR 
transduced T cells to selectively lyse targets expressing the HERV-E 
antigen . 51Cr labeled RCC lines were incubated at various effector to 
target ratios with HERV-E TCR transduced T cells. The amount of 51Cr 
released was measured in a -counter and the percent specific lysis 
was determined. As a positive control, an HLA-A11:01+ HERV-E target 
loaded with the HERV-E peptide was included (green). K562 cells 
were included as targets to control for NK lytic activity (black). 
Figure 6. INF- secretion levels after coculture of HERV-E TCR transduced T cells with cancer cells. A 
representative ELISA assay shows the ability of HERV-E TCR transduced T cells to selectively target ccRCC cells 
expressing both HLA-A11 allele and the CT-RCC HERV-E provirus. ccRCC cells were incubated at 1:10 effector to 
target ratio with HERV-E TCR transduced T cells for 18 hours. K562 cells were included as targets to control for NK 
reactivity. Axis Y shows IFN- secretion levels in pg/ml. 

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Richard Childs, MD 
 
 
therapy product being used to treat a patient. We used a PCR-based assay to measure the copy number on 
days 6 and 10 of culture of ~99% pure HERV-E TCR transduced T cells. On day 6 and 10, the two full 
scale production runs of T cells transduced with retrovirus from clone 7G1 or 27A7 had less than 5 copies 
of the integrated virus per cell. These results indicate our cell production SOPs will generate cell products 
that conform to the FDA copy number requirements. 
 
The frequency of the HLA-A11:01 gene in various U.S. populations is as follows 109 (number of patients 
with the allele is approx. 2x the frequency minus frequency of homozygosity at the A locus): 
 
Gene frequencies of HLA-A alleles in US populations 
HLA-A 
African Americans 
(n = 252) 
 
Caucasians 
(n = 265) 
 
 
Hispanics 
(n = 234) 
 
 
North American 
Natives  
(n = 187) 
 
Asians 
(n = 358) 
 
11:01 
0.0238 
0.0698 
0.0577 
0.0267 
0.2312 
 
Although HLA-A*10:01 is present in around 15% of the population, there are over 15,000 patients in the 
U.S. who will die of kidney cancer, so we expect grossly 2,000 patients per year in the U.S. to have HLA-
A11:01 and ccRCC for which we have identified a specific TCR. Numbers would be much larger should 
efforts be made to recruit patients with ccRCC from Asia. 
 
Rationale for immunosuppressive chemotherapy and selection of lymphocyte-depleting chemotherapy 
regimen 
 
Conditioning chemotherapy regimen of cyclophosphamide and fludarabine is planned to be administered 
before infusions of HERV-E TCR transduced T-cells because substantial evidence demonstrates an 
improvement of the anti-malignancy activity of ATC when chemotherapy or radiotherapy are administered 
before the T cell infusions.110-112 In mice, administering chemotherapy or radiotherapy prior to infusions of 
tumor-antigen-specific T cells dramatically enhanced the anti-tumor efficacy of a variety of different tumor-
reactive T cells.37,110,111,113,114 
 
Studies in murine models and humans have defined the mechanisms of action of the lymphodepleting 
chemotherapy before cell transfer.115,116 Lymphodepletion has a variety of positive effects including the 
elimination of T‐regulatory cells and/or myeloid‐derived suppressor cells and the elimination of 
endogenous lymphocytes that provide a sink for growth promoting cytokines such as IL‐7 and IL‐
15.110,111,113,117 In summary, elimination of endogenous “cytokine sinks” by depleting endogenous T cells 
and natural killer cells induced an increase of serum levels of important T-cell stimulating cytokines such 
as IL-15 and IL-7, and the enhancement in T-cell function and anti-tumor activity were dependent on IL-
15 and IL-7.110 The lymphodepleting regimen can also lead to activation of antigen-presenting cells, in part 
by increased susceptibility to toll-like receptor stimulation.118  
 
Although pre-clinical and clinical data initially suggested that adding total body irradiation (TBI) to the 
non-myeloablating chemotherapy preparative regimen for adoptive transfer of tumor infiltrating 
lymphocytes (TIL) may lead to more favorable clinical outcomes, a randomized study in metastatic 
melanoma patients receiving adoptive transfer of TILs could not confirm this initial hypothesis.119 This 
randomized study tested the impact of the addition of TBI to the preparative lymphodepleting chemotherapy 
regimen with similar results in clinical outcomes in both arms. Complete responses rates were 24% in both 
groups (12 of 50 versus 12 of 51), and OS was also similar (median OS of 38.2 months versus 36.6 months 

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Richard Childs, MD 
 
 
with a hazard ratio, 1.11; 95% CI, 0.65 to 1.91; P = .71). Based on these results, we opted to use only 
lymphodepleting chemotherapy without total body irradiation (TBI) as a conditioning regimen in our 
current protocol. 
 
The chemotherapy regimen that best increases the anti-malignancy efficacy of TCR gene engineered T cells 
is not known, but the most commonly used chemotherapy regimens that have been used in clinical trials 
and that convincingly demonstrate persistence and in vivo activity of adoptively transferred T cells have 
included cyclophosphamide and/or fludarabine.38,40,120 We will use cyclophosphamide 1,000 mg/m2 IV x 1 
day (day-4) and fludarabine 30mg/m2 IV daily x 3 days (days -4 through-2) with the aim of achieving 
maximum lymphodepletion at the day of the HERV-E TCR transduced T-cells infusion. 
 
Rationale for dose-escalation of HERV-E TCR transduced T-cells 
 
The clinical trial described in this protocol is designed as a dose escalation study in which the number of 
HERV-E TCR transduced T-cells administered to patients will be increased with sequential dose levels. 
The rationale for conducting a dose-escalation trial of a cellular therapy is based on two main sources of 
evidence. First, the anti-tumor efficacy of adoptively-transferred T cell treatments increases as the dose of 
T cells administered to mice increases.121-123  Second, in the setting of allogeneic transplantation, relapsed 
malignancy is often treated with infusions of allogeneic donor lymphocytes (DLIs).124,125 The incidence of 
graft-versus-host disease, which is caused by T cells attacking allogeneic antigens on host tissues, increases 
as the dose of T cells administered in DLIs increases.124,125 In this trial, the first dose level has been set at 1 
x106 HERV-E TCR transduced CD8+/CD34+ enriched T-cells/kilogram, which is less than a third of the 
dose that was established to be tolerable on protocol NCT02870244. 
 
Rationale for IL-2 regimen after HERV-E TCR transduced T-cells infusion 
 
Additional improvements can be achieved by the systemic administration of interleukin (IL)-2 or other 
cytokines to support the transferred cells.122,126  The use of IL-2 to grow T cells in vitro that can be used for 
ACT, as well as the administration of IL-2 to support the growth and survival of antitumor cells infused 
into patients, has been a major translation from basic studies of IL-2 to the clinic.127-133 Most TIL-ACT 
protocols have used HD bolus IL-2 regimen and the efficacy of this three-step treatment (lymphodepleting 
chemotherapy, T cell infusion and HD-IL2 administration) has been proven in several phase II trials. 
41,43,44,134-137  The toxicities associated with high-dose (HD) bolus IL-2 classically administered after T cell 
infusion are severe and represents a major limitation for a more widespread use of ACT. Because of this, 
various doses and schedules of IL-2 administration were evaluated in the previous decades in an attempt to 
reduce toxicity without compromising efficacy. Different doses and schedules of IL-2 administered in this 
context have been explored resulting also in long-lasting complete responses in patients with treatment-
refractory melanoma.138,139 Although transient, the toxicities associated with HD IL-2 classically 
administered together with TILs and TCRs therapies are often severe leading to premature discontinuation 
of the exogenous administration of IL-2 .38,134,140 For example, of the 15 planned doses of HD IL-2 to be 
administered to patients being treated with human papillomavirus–targeted tumor-infiltrating T cells, 
patients were only able to receive a median 5 (range 0-9) doses due to IL-2 toxicities . 38 Furthermore, no 
convincing evidence exists to date between the number of doses of IL-2 administered and the efficacy of 
most adoptive T-cell based immunotherapies. 134,140 Therefore, in the current study, we have opted to utilize 
an intermediate dose of IL-2 of 2 million units/M2 IV q 12 hours x 14 doses following the T-cell infusion. 
Prior research conducted by our group in patients receiving the same dose of IL-2 given subcutaneously 
followed adoptive infusion of ex vivo expanded NK cells (NIH Protocol 08-H-186) have established this 
dose to be well tolerated, to not be associated with any grade 4 toxicities, and capable of maintaining IL-2 
serum level troughs of at least 100 pg/ml (Figure 8 below- data from Protocol 08-H-0186) 

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Richard Childs, MD 
 
 
 
Figure 8. Serum levels of IL-2 based on daily (a) versus every 12-hour (b) dosing 
 
3. SCIENTIFIC AND CLINICAL JUSTIFICATION 
3.1 Study Design 
This is a single-arm, phase 1 trial of HERV-E TCR transduced CD8+/CD34+ enriched T cells in HLA-
A*11:01 positive patients with metastatic ccRCC. The study is planned based on a Phase 1 “3+3” dose 
escalation design. The maximum tolerated dose (MTD) is defined as the highest dose at which 0 or 1 patient 
in six has experienced a dose limiting toxicity (DLT). Patients with evaluable advanced/metastatic ccRCC 
will be recruited in up to 4 dose levels. The decision to escalate, de-escalate or suspend the dose escalations 
in the study will follow the rules outlined in the Table 3.  
 
Table 3. 
 
Outcome: No of DLTs out of No. of Patients at a Given 
Dose Level 
 
Decision Rule 
0 DLT out of 3 patients 
Enter up to 3 patients at the next dose level 
2 DLTs out of 2-3 patients 
Stop dose escalation: Enter up to 3 additional patients at the 
previous dose level if only 3 patients have been treated at that 
dose. 
1 DLT out of 3 patients 
Enter up to 3 more patients at the same dose level . 
1 DLT out of 6 patients 
Enter up to 3 patients at the next dose level 
2 DLTs out of 4-6 patients 
Stop dose escalation: Enter up to 3 additional patients at the 
previous dose level if only 3 patients have been treated at that 
dose. 
 
Since adverse events associated with adoptive T cell transfer generally occur within 21 days after the T cell 
infusion, each subject will be observed for 21 days post T cell infusion before the next subject in the cohort 
or the first subject in the next cohort is treated.  Therefore, there will be a minimum of 21 days between the 
HERV-E TCR transduced T-cell infusion for each patient before the next patient enrolled starts 
conditioning chemotherapy.  

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Richard Childs, MD 
 
 
 
This study will enroll a minimum of 15 patients if no DLTs are observed. If sufficient cells cannot be grown 
for a patient to meet the criteria for the first dose of cells for the assigned dose level, treatment for that 
patient on the clinical trial will be aborted. A second attempt will be made to prepare cells for the patient if 
the patient agrees and if the patient still meets all eligibility criteria. If cell growth limitations still preclude 
administration of the cohort specified dose, the patient will be taken off study and not receive treatment due 
to technical failure. If it proves to be technically impossible or impractical to achieve the higher dose levels 
due to cell production constraints and a maximum tolerated dose has not been defined, the highest 
achievable dose level will be declared the maximum feasible dose.  
 
3.2 Dose-limiting toxicities  
➢ Because cyclophosphamide, fludarabine and IL-2 are FDA approved drugs with known toxicity profiles 
at the doses utilized in this protocol, any observed or volunteered grade 3 or 4 adverse events that are 
as listed on the drug labels will not be counted toward DLT unless the grade 3 or 4 toxicity is previously 
unknown. Because subjects enrolled on this study may have disease-related or prior treatment-related 
hematologic toxicity, hematologic DLTs will not be based on CTCAE version 5.0 grading, rather they 
will be based on recovery of baseline hematologic indices.  
 
➢ Dose-limiting toxicity is defined as all grade 3 and 4 toxicities judged probably or definitely associated 
to the HERV-E TCR transduced CD8+/CD34+ enriched T-cells infusion except for:  
▪ 
Myelosuppression, defined as lymphopenia, neutropenia and thrombocytopenia.  
▪ 
IL-2 expected toxicities. (described section 14.3)  
▪ 
Immediate hypersensitivity reactions (excluding symptomatic bronchospasm and grade 4 
hypotension) occurring within 2 hours of cell infusion (related to cell infusion) that are resolved 
within 24 hours of cell administration with standard supportive treatments. 
▪ 
Grade 3 Fever. 
▪ 
Grade 3 autoimmunity, that resolves to less than or equal to a grade 2 autoimmune toxicity after 
medical management (e.g., immunosuppressant treatment) within 10 days. 
▪ 
Grade 3 metabolic laboratory abnormalities without significant clinical sequela that resolve to 
grade 2 within 7 days. 
▪ 
Hypotension requiring treatment with vasopressors (norepinephrine dose of >2 mcg/minute or 
equivalent) for 72 hours or less. The 72 hours is measured from the first institution of vasopressors 
even if vasopressors are temporarily discontinued and then re-started. Norepinephrine doses of <2 
mcg/minute or equivalent doses are not a DLT. 
 
➢ Any adverse event that leads to a discontinuation of the T-cell infusion will be considered a DLT.  
 
If a patient experiences a grade 3-4 toxicity during the cell infusion (reaction to cellular product or infusion 
reaction) the patient will receive no further cells but may continue to receive IL-2 if the toxicity returns to 
grade 2 or less within 48 hours. If a patient develops an acute infusion reaction, we will follow a supportive 
care plan as outlined in Appendix D. 
 
Protocol Schema 

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Richard Childs, MD 
 
 
 
All subjects who meet the enrollment criteria and sign the informed consent will undergo a 15-liter (3-4 
hour) apheresis at the National Institutes of Health (NIH) Blood Bank to obtain the large number of T cells 
needed for transduction. The apheresis product will be shipped to the Cellular Therapy Center for 
processing and TCR transduction to the Cellular Therapy Center at Loyola University Medical Center 
(LUMC) or another ORSC-approved contracted manufacturing facility for processing and TCR 
transduction (by World Courier). 
 
3.3 Product manufacturing and testing 
After cells are obtained by apheresis at the NIH, the product will be packaged and shipped to the Cellular 
Therapy Center at Loyola University Chicago, HSD in Maywood IL or another ORSC-approved contracted 
manufacturing facility for further cell processing to generate HERV-E TCR transduced T cells.  
 
The process to generate the HERV-E TCR transduced T cells is briefly described herein. The PBMCs are 
isolated from the apheresis product using the cGMP density gradient separation solution Ficoll Paque 
Premium. Once the PBMCs are isolated, they are washed a minimum of three times with a 0.9% Sodium 
Chloride solution or DPBS. A sample of the washed PBMCs is inoculated into BD aerobic and anaerobic 
culture bottles and submitted to the clinical microbiology laboratory for sterility testing.  
 
2x109 PBMCs are diluted to a concentration of 1x106/mL and activated with 50 ng/mL of soluble anti-
hCD3 monoclonal antibody (OKT3) in AIM-V culture media containing 5% human AB Serum, 300 IU/mL 
rhIL2 and 100 ng/mL rhIL15. A maximum of 200 mLs of cell suspension per one T175cm2 flask is added 
to ten (10) T175cm2 flasks then placed in a humidified 5% CO2 incubator for 2-3 days.  
 
A large lot of HERV-E retrovirus was previously prepared and cGMP qualified.  Briefly, the retrovirus was 
prepared by seeding T175cm2 flasks each with 8x106 PG13 HERV-E TCR CD34 T cells from the cGMP 
qualified PG13 HERV-E TCR CD34t 7G1 Master Cell Bank. The following day, each of the seeded flasks 
were treated with media containing Sodium Butyrate for 8-10 hours, rinsed with DPBS then 20 mL of 
Outpatient follow-up
Inpatient administration of IL-2 
Infusion of recipient with HERV-E TCR transduced T-cells (inpatient for 7-days minimum 
post-infusion)
Outpatient or Inpatient administration cyclophosphamide plus fludarabine conditioning 
chemotherapy 
In vitro generation of HERV-E TCR transduced CD8+/CD34+T-cells (21 days)
Leukapheresis or Frozen Cells
Pretreatment staging
Protocol enrollment screening

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Richard Childs, MD 
 
 
IDMEM+10%FBS media was added for overnight retrovirus production. The retrovirus was collected and 
filtered with a 0.45μm cellulose acetate filter to remove debris, aliquoted into labeled conical tubes, samples 
removed for testing and the aliquots snap frozen and stored in the -80oC freezer located within the Cellular 
Therapy Center. 
 
On the day of transduction, the activated T cells will be collected, counted and CD4 depleted using the 
CliniMACS Cell Selector instrument. Post CD4 depletion, 200x106 cells will be transferred to a separate 
conical tube, pelleted and re-suspended in AIM-V media with 5% human AB serum to a concentration of 
4x106 cells/ml. Cytokines (300IU/mL rhIL2, 100ng/mL rhIL15) are added to the cell suspension based on 
the final total volume of 240mL. 0.5mL of the cell suspension will be added to each well of 24-well non-
tissue culture treated plates which have been pre-coated with 30ug/mL clinical grade Retronectin. Aliquots 
of the cGMP qualified retrovirus will be thawed in a 37oC water bath and 2mL will be added to each well 
containing the cell suspension. Each plate is sealed with parafilm, centrifuged at 2000xg, at 32oC for 2 
hours with no brake to bind the retroviral particles and the cells to the retronectin facilitating interaction of 
the viral particle with the activated T cells. When the spin is complete, the parafilm is removed and the 
plates are placed in a humidified 5% CO2 incubator overnight.  
 
The day after transduction, the cells are collected from each well of the plates, transferred to a sterile conical 
tube, counted, pelleted by centrifugation and re-suspended in fresh complete media containing 300IU/mL-
rhIL2 and 100ng/mL-rhIL15 to a concentration of 0.5x106/mL. A maximum of 200mLs of the cell 
suspension is transferred to each T175cm2 flask and placed in a humidified 5% CO2 incubator for culture. 
 
Approximately 3-5 days after the transduction, the transduced T cells are collected and counted. A sample 
is removed for analysis by flow cytometry. The cells are pelleted and re-suspended in Miltenyi buffer in 
preparation for CD34 purification. The transduced T cells, which express CD34+ on the cell surface as a 
transduction marker, are incubated and tagged with Miltenyi GMP biodegradable magnetic micro-particles 
coated with CD34+ antibody. The magnetic particles bind to the CD34+ molecule present on the cell surface 
of transduced T cells. The CD34+ cells are positively selected by magnetic separation using the CliniMACs 
Cell Selector. The CD34+ enriched positive fraction and the CD34 depleted negative fraction is collected 
and individually transferred to sterile tubes and counted. Samples from both groups are removed for 
analysis by flow cytometry. The negative fraction is cryopreserved in a 10% DMSO solution. The positive 
fraction is re-suspended to a concentration of 0.5x106/mL in complete media containing 300IU/mL-rhIL2 
and 100ng/mL-rhIL15 and plated in T175cm2 flask (maximum of 200mLs per flask). The flask(s) are placed 
in a humidified 5% CO2 incubator for culture. The positive fraction cells are cultured for several days until 
the start of Rapid Expansion Protocol (REP).  
 
The positive selected CD34+ T cells are placed into a REP to rapidly generate the number of cells needed 
for infusion. The number of transduced cells that are placed in the REP is determined by the patient’s weight 
and the infusion cohort. To initiate the REP, 1x106 HERV-E TCR transduced cells will be combined with 
200x106 feeder cells in an upright T175cm2 flask containing 150mLs complete cytokine media 
(rhIL2/rhIL15) with 30ng/mL of soluble anti hCD3 antibody (OKT3). The feeder cells consist of PBMC 
from a minimum of 3 separate normal donors which are mixed together and irradiated with 5000 RAD of 
irradiation. The flasks are placed upright in a humidified 5% CO2 incubator for 5-6 days.  The flasks are 
removed from the incubator, the cells collected, counted, and re-suspended in fresh media containing 
300IU/mL-rhIL2 and 100ng/mL-rhIL15. The cell suspension is transferred into Wave bioreactor bag(s) for 
further expansion. The culture media is replenished daily with fresh medium containing cytokines by 
utilizing the Wave bioreactor perfusion system.  
 

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Richard Childs, MD 
 
 
Periodically during the expansion process, samples are removed for microbiology sterility testing and 
analysis by flow cytometry. The microbiology sterility samples are submitted to the clinical microbiology 
laboratory in the Loyola Medical University Hospital or another ORSC-approved contracted manufacturing 
facility in aerobic and anaerobic sterility bottles for 7-14 day sterility testing. The sample removed for flow 
cytometry is stained for the appropriate markers with fluorochrome conjugated monoclonal antibodies to 
determine the percent transduction and transgene expressions levels on the transduced T cells. 
 
Approximately 10 days (+/- 2 days) after initiation of the REP, the final infusion product is prepared for 
packaging and cryopreservation.   The HERV-E TCR transduced T cells are collected from the Wave bag, 
washed, resuspended, counted and a sample removed for flow cytometry to determine the percent 
CD8+CD34+ enriched T cells. The number of CD8+CD34+ enriched T cells needed for infusion, a test 
sample vial and product testing are transferred to a separate conical tube. The cells are pelleted, the 
supernatant aspirated and the cells are resuspended in cold cell suspension media to ½ the total packaging 
volume. ½ the total packaging volume of cold 2x DMSO freeze media is slowly added dropwise to the cell 
suspension. The cells are well mixed and transferred to the labeled cryopreservation bag. Samples are 
removed from the final product bag for gram stain testing, endotoxin testing and full sterility testing. The 
product is cryopreserved to -80oC then transferred to a LN2 freezer for storage in the vapor phase of LN2.  
 
Ms. Gina Scurti from Loyola or designee at another ORSC-approved contracted manufacturing facility will 
be shipping the cryopreserved final product after gram stain, endotoxin and preliminary sterility results are 
obtained and the QA representative releases the product will be placed in a Princeton Cryo dry shipper 
(MVE CryoShipper with 4R9953 Blood Bag Rack) and shipped overnight to the NIH in Bethesda, MD 
using World Courier. These shippers are designed to safely ship cells while maintaining temperatures of -
150oC for up to 10 days. The final products will be stored at the NIH Department of Transfusion Medicine 
(DTM). Infusion of the HERV-E TCR transduced T cells will occur after the 7-day sterility results are 
obtained and provided to the NIH DTM.  The patient materials being sent to Loyola another ORSC-
approved contracted manufacturing facility will be identified with patient name and other identifying 
information to maintain proper chain of custody.   
Time 
Procedures and Testing 
Day 0 
Receive apheresis from NIH 
Sterility Testing 
Perform Ficoll peripheral blood mononuclear cell (PBMC) 
Isolation 
Stimulate 1.2x109 PBMCs 
Day 1 
Coat Plates with Retronectin 
Day 2 
CD4 Depletion 
Transduction of CD8 cells 
Day 3 
Transfer cells to flasks 
Day 6 
CD34+ Selection on CliniMACs 
Flow Analysis pre/post selection 
Cryopreserve Negative Fraction 
Isolate germline DNA 
Vector Copy Number Assay 
Replication Competent Retrovirus Detection Assay 

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Richard Childs, MD 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Release criteria for the final HERV-E TCR transduced CD8+/CD34+ enriched T cells product are described in 
Table 4. 
 
Table 4. Test performed on final product prior to release for use from the NIH DTM (frozen product) 
 
Test 
Method 
Acceptance Criteria 
Viability at time of 
Packaging  
Trypan Blue Exclusion 
> 70% 
Total Viable Cell 
Number 
Visual microscopic count 
Minimum Treatment 
Dose Cell # 
CD34+ 
FACS analysis 
>25% 
CD8+ 
FACS analysis 
>80% 
CD8+/CD34+ 
FACS analysis 
> 25% 
Sterility  
 
Gram Stain 
Negative 
Day 8 
Sterility Testing 
Mycoplasma Testing 
Day 10 
REP Day 0 
Rapid Expansion Program (REP) 
FACs Analysis 
Isolate germline DNA 
Vector Copy Number Assay 
Replication Competent Retrovirus Detection Assay 
Day 15 
REP Day 5 
Transfer to WAVE 
Elisa 
Sterility Testing 
Mycoplasma Testing 
Day 18 
REP Day 8 
FACs analysis 
Day 20 
REP Day 10 
FACs analysis 
Package Infusion Product 
Cryopreserve Infusion Product 
Sterility, Gram Stain 
Mycoplasma 
Endotoxin 
Isolate germline DNA 
Replication Competent Retrovirus Detection Assay 
Sent sample for Full RCR Testing 
Day 21 
REP Day 11 
Ship Cryopreserved Infusion product to NIH 

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Richard Childs, MD 
 
 
 
BACTEC Aerobic culture 
No growth 
 
BACTEC Anaerobic culture 
No growth 
 
Mycoplasma test 
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 
 
Once the T cells have been transduced and shown to have met interim lot release criteria, so they are 
projected to be ready for infusion, patients will receive non-myeloablative lymphocytic-depleting 
chemotherapy.  Cells must be on site at the NIH before conditioning begins with the combination of 
cyclophosphamide 1,000 mg/m2 IV x 1 day (day -4) and fludarabine 30mg/m2 IV daily x 3 days (days -4 
through -2). On day 0, subjects will then receive a single infusion of thawed, autologous HERV-E TCR 
transduced CD8 enriched T cells supported with moderate-dose IL-2 to begin as soon as is feasible after 
the cell infusion. Although the autologous HERV-E TCR transduced T cells will be enriched for CD8+ T 
cells, they will consist of a polyclonal mixture of both CD4+ and CD8+ T cells expressing the transduced 
HLA-A11-restricted HERV-E-specific TCR. 
 
Four cohorts of 3 patients will be treated with increasing doses of HERV-E TCR transduced T cells as 
shown in Table 5. Since the lot release criteria will require a minimum of 25% of HERV-E TCR transduced 
CD8+/CD34+ enriched T cells, the actual number of cells infused will be based on patient weight in 
kilograms with cohort 1 receiving 1 x 106 HERV-E TCR transduced T cells per kg body weight, cohort 2 
– 5 x 106 HERV-E TCR transduced T cells per kg body weight, cohort 3 – 1 x 107 HERV-E TCR transduced 
T cells per kg body weight, and cohort 4 – 5 x 107 HERV-E TCR transduced T cells per kg body weight. If 
the patient has a body mass index (BMI) >35 (i.e. meets criteria for morbid obesity), the weight used to 
calculate HERV-E TCR transduced T cells doses will utilize a weight derived from the subject's BMI 
capped at 35. A deviation of  10% in cells from the listed doses are acceptable. The weight used to calculate 
the number of HERV-E cells infused will be the weight on the day of the consent.   
 
Patients will receive intravenous infusions of moderate dose IL-2 (2,000,000 IU/m2) IV every 12 hours for 
14 doses for 7 days post-infusion. Patients will be monitored clinically and immunologically for a year after 
infusion. 
 
 
 

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Table 5. 
First-dose 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 
 
4. ELIGIBILITY CRITERIA 
4.1 Inclusion Criteria 
 
• 
Patients must have histologically confirmed RCC with clear-cell component by the Laboratory of 
Pathology of the NIH and/or outside Pathology Department prior to entering this study. 
• 
Patients must be HLA-A 11:01 positive (confirmed by HLA typing at the NIH DTM)  
• 
Patients must have measurable disease per RECIST version 1.1 and have disease progression during or 
after the last treatment regimen and within 6 months before study enrollment 
• 
Patients must have received at least one antiangiogenic drug and an immune-checkpoint inhibitor (i.e. 
nivolumab unless the patient has contraindications to receiving these medications, the agents are not 
available to the patient, or the patient declines to receive these drugs due to personal preference.  
• 
Patients must agree to use adequate contraception (hormonal or barrier method of birth control; 
abstinence) prior to study entry, the duration of study participation and 180 days (female patients) or 
90 days (male patients) after the end of the treatment if sexually active and able to bear or beget children. 
In addition, male patients must refrain from sperm donation for 90 days after the final dose of 
investigational product. Female patients must refrain from egg cell donation for 180 days after the final 
dose of investigational product. 
• 
Patients must be between the ages of 18 and 75 years. 
• 
Patient must have an anticipated life expectancy of at least 3 months. 
• 
Patients must have a performance status of 0 or 1 ECOG performance status (PS) scale (Appendix B). 
• 
Patients must have a caregiver willing to stay with them during the first month of treatment (30 days 
+/- 7 days). 
• 
Patients receiving treatment with bisphosphonates or denosumab are eligible for enrollment if on a 
stable dose for ≥4 weeks  
• 
Serology:  
o Seronegative for HIV antibody. (The experimental treatment being evaluated in this protocol 
depends on an intact immune system. Patients who are HIV seropositive can have decreased 
immune-competence and thus be less responsive to the experimental treatment and more 
susceptible to its toxicities.)  
o Seronegative for hepatitis B antigen, and seronegative for hepatitis C antibody. If hepatitis C 
antibody test is positive, then patient must be tested for the presence of antigen by RT-PCR and be 
HCV RNA negative.   

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Richard Childs, MD 
 
 
• 
Organ Function 
Hematology 
Absolute neutrophil count 
500/ µL 
WBC 
1500/µL 
Platelet count 
75.000/µL (without transfusional support) 
Chemistry 
Serum AST/ALT 
 2.5 x upper limit of normal (ULN) 
Total bilirubin 
 1.5 mg/dl except for patients with Gilbert’s 
syndrome who must have a total bilirubin  3 
mg/dl  
creatinine clearance  
 
 50 ml/min/1.73m2 by the method of CKI-EPI 
or  
 50 ml/min by the method of 24h Clearence of 
Creatinine Calculation 
INR 
< 1.5 
Cardiology 
Estimated left ventricular ejection fraction by 
echocardiography  
 45%  
Respiratory 
 
Predicted DLCO/Alveolar Volume Adjusted by 
PFT 
> 45%  
 
 
4.2 Exclusion Criteria  
 
• 
Patients that require immediate therapy due to tumor mass effects or spinal cord compression. 
• 
Patients must not have had standard of care anti-VEGFR therapy (mean half-life around 30 hours), 
mTOR inhibitors (mean half-life around 30 hours) at least for the last 7 days prior to T-cell infusion, 
and radiotherapy or major surgery within the last 2 weeks prior to T-cell infusion. For PD-1/PD-
L1 inhibitors or CTLA-4 inhibitors, a 4-week period must have elapsed before T-cell infusion. For 
recent experimental therapies a 28-day period must have elapsed before infusing expanded T-cells.  
• 
Patients with active CNS involvement by malignancy either by imaging or cerebrospinal fluid 
involvement or biopsy-proven (due to poor prognosis and potential for neurological dysfunction 
that would confound evaluation of neurological and other adverse events) except for: 
a) Patients with 3 or fewer brain metastases of <1cm treated with either stereotactic or gamma knife 
radiotherapy and remained stable on MRI for 2 weeks are eligible. 
b) Patients with surgically resected brain metastases and no evidence of active disease in the CNS at 
the time of screening evaluation are eligible. 
• 
Patients with hypercalcemia (>10 mg/dL) of malignancy. 
• 
Any prior Grade ≥ 3 immune-related adverse event (irAE) while receiving immunotherapy, including 
anti-CTLA4 treatment that requires long-term immunosuppressive therapy. Note: Active or history of 
vitiligo or hypothyroidism will not be a basis for exclusion. 

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Richard Childs, MD 
 
 
• 
Patients with second malignancies in addition to their clear cell RCC are not eligible if the second 
malignancy has required systemic treatment within the past 4 years or is not in complete remission. 
There are exceptions to this criterion: successfully treated non-metastatic basal cell, squamous cell skin 
carcinoma, in situ non-invasive cervical cancer and in situ non-invasive breast cancer.  
• 
Women of child-bearing potential who are pregnant or breastfeeding because of the potentially 
dangerous effects of the preparative chemotherapy on the fetus or infant.  
• 
Active coagulation disorders or other major uncontrolled medical illnesses of the respiratory, endocrine, 
renal, gastrointestinal, genitourinary or immune system, uncontrolled systemic infection, active 
obstructive or restrictive pulmonary disease. 
• 
Patients who have recent history of cerebrovascular accident, transient ischemic attack should be 
cleared by the neurology consult service before enrolling this study. 
• 
Patients who have recent history of  coronary artery disease or cardiac arrhythmia should be cleared by 
the cardiology consult service before enrolling this study. 
• 
Any form of primary immunodeficiency (such as Severe Combined Immunodeficiency Disease).  
• 
Patients with autoimmune diseases such as Crohn’s disease, ulcerative colitis, rheumatoid arthritis, 
autoimmune hepatitis or pancreatitis, and systemic lupus erythematosus that requires treatment with 
chronic immunosuppressive therapy. 
• 
Systemic corticosteroid steroid therapy of any dose is not allowed within  2 days prior to enrollment.   
The following are exceptions to this criterion: 
o Intranasal, inhaled, and topical steroids 
o Systemic corticosteroids at physiologic doses not to exceed 10 mg/day of prednisone or equivalent 
o Steroids as premedication for hypersensitivity reactions (e.g., CT scan premedication).   
• 
History of severe immediate hypersensitivity reaction to any of the agents used in this study.  
• 
Unable to understand the investigational nature of the study or give informed consent and does not have 
a legally authorized representative or surrogate that can provide informed consent per section 14. 
• 
Any condition that, in the opinion of the investigator, would interfere with evaluation of the 
investigational product or interpretation of subject safety or study results.  
 
5. CLINICAL EVALUATION OF THE PARTICIPANT 
Study eligibility is based on meeting all of the study inclusion criteria and none of the exclusion criteria 
at screening before study treatment administration. Screening evaluations will be performed as part of 
an NIH Screening protocol. Laboratory results from other NIH IRB approved protocols that subjects may 
have participated in within 30 days of enrollment in this study may also be used as part of the pre-study 
evaluation.  
 
Protocol evaluation (on 97-H-0041) 
▪ 
Complete history and physical examination, including, weight, height, vital signs and ECOG 
performance status. 
▪ 
Patients with favorable, intermediate and poor risk categories will be eligible for the study. We will 
include poor prognostic factors in the baseline H&P the following as described per International 
Metastatic RCC Database Consortium (IMDC):  
o KPS equal to 70 or less 
o Less than 1 year from diagnosis to initial systemic treatment  
o Hemoglobin less than the LLN  
o Corrected calcium concentration greater than 10 mg/dL  
o Absolute neutrophil count greater than the ULN  
o Platelet count greater than the ULN  

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Richard Childs, MD 
 
 
▪ 
ECG 
▪ 
Confirmation of diagnosis of a clear cell renal cell carcinoma by the NIH Laboratory of Pathology. The 
sample used for the confirmation of the diagnosis can come from any time prior to enrollment on the 
protocol. The sample can be a fresh biopsy or paraffin-fixed slides. 
▪ 
Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine, Glucose, Urea 
nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline 
Phosphatase, ALT, AST, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid. 
▪ 
CBC with differential.  
▪ 
Pre-albumin and lipid panel 
▪ 
PT/PTT. 
▪ 
TBNK studies 
▪ 
High resolution HLA typing to confirm HLA-A* 11:01 typing.  
▪ 
Antibody screen for Hepatitis B, Hepatitis C and HIV.  
▪ 
Anti-HTLV-I/II, Anti-HBcAntibody, West Nile Virus, HIV-1/HCV/HBV NAT, T. Cruzi Antibody, 
Toxoplasma Antibody. Syphilis (RPR testing) (DTM blood screening panel) 
▪ 
Anti-CMV antibody titer, HSV serology and EVB panel.  
▪ 
Chest radiograph (Posterior-Anterior and Lateral views). 
▪ 
CT of the neck  with IV contrast in patients with known or suspected metastatic disease in the neck (or 
MRI of the neck with IV gadolinium contrast if clinically indicated) 
▪ 
CT with IV contrast of the chest/abdomen/pelvis (or CT chest without contrast plus MRI 
abdomen/pelvis with IV gadolinium contrast if clinically indicated) to document measurable 
malignancy.  
Note: studies are to be performed with contrast unless renal impairment or allergy prohibits the use of 
contrast.  
▪ 
Tc99-bone scintography (and/or whole-body MRI if clinically indicated) in patients with known or 
suspected bone metastases.  
▪ 
MRI of the brain with IV gadolinium contrast (or CT of the brain with IV contrast if indicated) Note: 
Studies are to be performed with contrast unless renal impairment or allergy prohibits the use of 
contrast. 
▪ 
Cardiac function: echocardiogram  
▪ 
Pulmonary function tests. 
▪ 
β-HCG pregnancy test (serum or urine) on all women of child-bearing potential. 
▪ 
Urine analysis/random Urine Protein-Creatinine Ratio or 24-hour urine collection if clinically 
indicated. 
▪ 
Type and screen + DAT 
▪ 
Venous access assessment. Placement of peripheral IV catheter versus Central Venous Catheter 
Placement (when indicated) will be performed by the NIH Procedures, Vascular Access and Conscious 
Sedation (PVCS) Service.  A separate consent will be obtained at the time of the procedure. 
• 
Research blood for enumeration of absolute # of circulating CD34+/CD8+ T-cells [30 ml of blood 
collected in a green tube] will be collected in patients that are screened for re-treatment with HERV-E 
TCR transduced CD8+/CD34+ enriched T-cells and a second apheresis is needed. 
 
 
Baseline evaluation and procedures under this protocol (within 8 weeks of initiating chemotherapy)  
The following assessments must be completed within 4 weeks prior to starting the leukapheresis and/or 8 
weeks of initiating chemotherapy unless otherwise noted (if not, then the evaluation must be repeated). Any 
of these procedures done during screening do not need to be repeated for baseline analysis. 
▪ 
Interim history and physical examination, including, weight, height, vital signs and ECOG 
performance status. Note in detail the exact size and location of any lesions that exist. 

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Richard Childs, MD 
 
 
▪ 
Sodium, Potassium, Chloride, Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), 
Albumin, Calcium total, Magnesium total, Inorganic Phosphorus, Alkaline Phosphatase, ALT, 
AST, Total Bilirubin, Direct Bilirubin, LDH, Total Protein. 
▪ 
CBC with differential. 
▪ 
PT/PTT and D-dimer 
▪ 
TBNK studies 
▪ 
C-reactive protein 
▪ 
Urine analysis and random Urine Protein-Creatinine Ratio (or 24-hour urine collection if clinically 
indicated) 
▪ 
Glucose-6- phosphate dehydrogenase (G6PD) deficiency screening. 
▪ 
Thyroid panel (TSH, if TSH is abnormal, then T4). 
▪ 
Serum cortisol 
▪ 
β-HCG pregnancy test (serum or urine) on all women of child-bearing potential.  
▪ 
RCR testing  
▪ 
Research blood as per Table 8 and SOE 
▪ 
Durable Power of Attorney Form Completed 
▪ 
F18-FDG-PET CT –obtained within 2 weeks of initiating chemotherapy as a baseline procedure  
▪ 
CT of the neck  with IV contrast in patients with known or suspected metastatic disease in the neck. 
(or MRI of the neck with IV gadolinium contrast if clinically indicated) 
▪ 
CT with IV contrast of the chest/abdomen/pelvis (or CT chest without contrast plus MRI 
abdomen/pelvis with IV gadolinium contrast if clinically indicated) to document measurable 
malignancy will be obtained within 2 weeks of initiating chemotherapy as a baseline procedure.  
Note: studies are to be performed with contrast unless renal impairment or allergy prohibits the use 
of contrast.  
▪ 
Imaging tests will be obtained within 2 weeks of initiating chemotherapy as a baseline procedure. 
Note: studies are to be performed with contrast unless renal impairment or allergy prohibits the use 
of contrast.  
 
Optional Biopsies or Malignant Effusions 
Selection of lesion amenable for biopsy and target lesions in consultation with radiology within 4 weeks 
after leukapheresis but prior to conditioning chemotherapy. Tumor biopsies (nodal or visceral) will be 
optional for analyses of HERV-E expression, tumor line establishment, VHL mutation status and other 
molecular analysis and will be obtained at baseline and may be obtained at different time points after the 
HERV-E TCR transduced CD8+/CD34+ enriched T-cells infusion and at the time of disease progression. 
If patient happens to have a malignant effusion, we may request a sample of the effusion via pleural 
aspiration or paracentesis for research purposes such as tumor line establishment. 
 
During the preparative chemotherapy regimen: (mandatory only D-4 prior to chemotherapy unless 
noted) 
• 
Daily vital signs (Daily) 
• 
Weight 
• 
Complete Blood Count with differential.  
• 
Sodium, Potassium, Chloride, Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), 
Albumin, Calcium total, Magnesium total, Inorganic Phosphorus, Alkaline Phosphatase, ALT, AST, 
Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid 
• 
C-reactive protein 
• 
Research blood (on days specified in Table 8). 

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18-H-0012 
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Richard Childs, MD 
 
 
 
Immediately after Cell Infusion: 
• 
Vital signs will be monitored hourly (+/- 15 minutes) until stable and then routinely (every 4-6 
hours) unless otherwise clinically indicated. 
 
Studies to be performed during inpatient admission after cell infusion  
• 
Daily interim history and physical examination, including, weight, and vital signs  
• 
Vital signs including pulse oximetry will be monitored every 4-6 hours unless otherwise clinically 
indicated. 
• 
CBC once daily until discharge 
• 
TBNK studies on the day of HERV-E TCR transduced T-cells infusion on day 0 (cell infusion) and 
every other day through day 6. On D0, D+2, D+4 and D+6 
• 
Sodium, Potassium, Chloride, Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), 
Albumin, Calcium total, Magnesium total, Inorganic Phosphorus, Alkaline Phosphatase, ALT, 
AST, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid, once daily until 
discharge.  
• 
PT/PTT once daily until discharge.  
• 
C-reactive peptide on D0, D+2, D+4 and D+7 
• 
Thyroid panel (TSH, if TSH is abnormal, thenT4) and antithyroid panel (THY2) only on Day 0 
and D+7 
• 
Type and screen every 4 days 
• 
Research blood (as specified in Table 8). 
 
Post-infusion outpatient evaluation  
Patients will be seen at the NIH (OP7) in follow-up to evaluate disease status and late problems related to 
HERV-E TCR transduced T-cells infusion at the following time-points: after hospital discharge patient may 
be seen twice a week (+/- 2 days) for clinical and safety evaluation up to day +14 (+/- 2 days) , then on days 
+21 (+/- 3 days), +30 (+/- 5 days), +60 (+/- 7 days), +90 (+/- 7 days), +120 (+/- 14 days), +180 (+/- 14 
days), +240(+/- 14 days), +300 (+/- 30 days) and +360 (+/- 30 days) after HERV-E TCR transduced 
CD8+/CD34+ enriched T-cells infusion and when clinically indicated.  
 
At all outpatient follow-up visits unless otherwise noted, patients will have the following tests performed 
to determine clinical response and toxicities. Health status data will be obtained from surviving patients for 
a total of 15 years after cell infusion. 
- 
Interim history and physical examination/SOAP note, including, weight, vital signs and ECOG 
performance status.  
- 
CBC with differential. 
- 
Sodium, Potassium, Chloride, Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), 
Albumin, Calcium total, Magnesium total, Inorganic Phosphorus, Alkaline Phosphatase, ALT, 
AST, Total Bilirubin, Direct Bilirubin, LDH, Total Protein, Total CK, Uric Acid. 
- 
PT/PTT. 
- 
C-reactive peptide 
- 
TBNK cell analysis on day +14 and then every follow-up appointment post HERV-E TCR 
transduced T-cells infusion. 
- 
Blood for serum quantitative immunoglobulins on day +21 and then only as clinically indicated. 

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18-H-0012 
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Richard Childs, MD 
 
 
- 
Thyroid panel (TSH, if TSH is abnormal, thenT4) and antithyroid panel (THY2) on Day+14 and 
then only if prior abnormal as dictated by PI. 
- 
CT scans of the neck (if applicable based on baseline disease), chest, abdomen, and pelvis with IV 
contrast (or CT scan of the chest without contrast and MRI of the neck, abdomen and pelvis with 
IV gadolinium contrast if clinically indicated) at outpatient follow-up appointments starting day 
+30 after infusion and then as indicated in SOE (D+90, D+120, D+180, D+240, D+300 and 
D+360). CT scans will not be obtained at D+60 unless tumor assessment at D+30 is consistent with 
disease progression.  Note: studies are to be performed with contrast unless renal impairment or 
allergy prohibits the use of contrast. 
- 
Tc99-bone scintigraphy and/or whole-body MRI will be obtained only in those patients with known 
bone metastasis stating on day +30 after infusion and then as indicated in SOE.  (D+90, D+120, 
D+180, D+240, D+300 and D+360). Tc99-bone scintigraphy and/or whole-body MRI will not be 
obtained at D+60 unless tumor assessment at D+30 is consistent with disease progression. 
- 
MRI of the brain with IV gadolinium will be obtained only in those patients with known brain 
metastasis starting on day +30 after infusion and then as indicated in SOE (D+90, D+120, D+180, 
D+240, D+300 and D+360) or when clinically indicated. MRI of the brain will not be obtained at 
D+60 unless tumor assessment at D+30 is consistent with disease progression 
- 
All patients should get a F18-FDG-PET CT scan at day +60, +120, +180, +240, +300  and +360 
follow-up visits and anytime a F18-FDG-PET CT is clinically indicated.  
- 
MRI for specific body area if metastasis present will be performed only if clinically indicated 
- 
Research blood for correlative studies as described in SOE and Table 8. 
- 
Detection of RCR and persistence of TCR gene transduced cells: (3mo, 6mo, 1yr. see Follow-up 
SOE). 
- 
Long-term follow up of patients receiving gene transfer: Patients will be followed as described 
below. Physical examinations will be performed and documented annually at least for 5 years 
following cell infusion to evaluate long-term safety. After 5 years, health status data will be 
obtained from surviving patients via telephone contact or mailed questionnaires. The long-term 
follow up period for retroviral vectors is 15 years.  
 
NOTE: All CT scan with IV contrast of the abdomen and pelvis must be ordered with artherial phase.  Other tests 
will be performed as clinically indicated. 
 
Subjects will begin long term follow up one year following the last gene therapy administration, or at the 
time of disease progression (if occurring <12 months after gene therapy), as described below: 
 
➢ Years 1 -5: Annual history and physical examination from a health care provider. It is preferable but 
not necessary for this to be performed at the Clinical Center. If the patient elects not to return, a copy 
of the physical exam should be obtained from the patient’s referring health care provider and the health 
status information should be obtained via phone or e-mail with the patient.  
 
➢ Years 6 -15 (if required): Annual health status check via telephone or e-mail contact with the patient.  
 
• 
Patients who are treated with gene therapy which includes a retroviral vector (including gamma-
retroviral vectors and lentiviral vectors), will be requested to send a blood sample to the NIH at 3, 6, 
and 12 months post-treatment, and annually thereafter for 4 more years. These blood samples will be 
used along with a pre-treatment sample to assay for replication-competent retroviruses. If any indication 
of gene therapy-related toxicity develops, additional blood samples will be required to define and 
investigate the toxicity. 
 

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Richard Childs, MD 
 
 
• 
Patients who are treated with gene therapy and persistence of HERV-E TCR transduced CD8+/CD34+ 
T-cells in peripheral blood at the 12-month time-point will be requested to send a blood sample (Table 
8) to NIH every 6 months up to 5 years and then yearly up to 15 years to 1 year for: 
▪ 
Quantification of HERV-E TCR transduced CD8+/CD34+ enriched T cells.  
▪ 
Cytolytic potency/activity of HERV-E TCR transduced CD8+/CD34+ enriched T cells.  
▪ 
HERV-E TCR transduced CD8+/CD34+ enriched T cell surface phenotype and immune subsets. 
▪ 
Comprehensive cytokine/Chemokine studies. 
 
Please Note: Patients who are unable or unwilling to return for follow up evaluations will be followed via 
phone or e-mail contacts. A request will be made to send laboratory, imaging and physician exam reports 
performed by their treating physician; and any outstanding toxicities will be reviewed with the patient. 
 
6. TREATMENT PLAN  
Drug Administration 
 
• 
Leukapheresis  
The patient will undergo a 12-15 (+/- 500 ml) liters leukapheresis in the DTM Dowling Apheresis Clinic 
according to DTM standard operating procedures. The procedure requires dual venous access and takes 
approximately 3-4 hours to complete. A central line will be placed if peripheral venous access is not 
sufficient. The apheresis product will be processed to purify the PBMC, transduced to express the HERV-
E TCR, and prepared for patient infusion.  
 
NOTE: Dr. Nishimura’s team may retain excess PBMCs from initial apheresis collection. PBMCs will be 
cryopreserved under GMP conditions and stored at Loyola University or another ORSC-approved 
contracted manufacturing facility. Frozen PBMCs may be used to manufacture CD8+ T cells transduced 
with HERV-E TCR.  
 
 
• 
Preparation of the HERV-E T cell receptor transduced T cells for adoptive cell transfer 
After cells are obtained by apheresis, they will be shipped to LUC or another ORSC-approved 
manufacturing facility for processing and TCR transduction per SOPs. Freshly-collected cells will be used 
to initiate the cell-preparation process as described in section 3.2.  HERV-E TCR- expressing T cells will 
be cryopreserved at LUC or another ORSC-approved contracted manufacturing facility  on day 21 and 
shipped to NIH. Cells will be released for infusion after 7 days of negative cultures are confirmed on the 
day 21 expanded T-cell product. 
 
• 
Conditioning therapy and autologous HERV-E TCR transduced T-cell administration. 
All patients will receive a non-myeloablative lymphocyte depleting preparative regimen of fludarabine (30 
mg/m2/day IV) on days -4 through -2 and cyclophosphamide (1,000 mg/m2/day IV) on day -4 (Table 6).  
The non-myeloablative lymphocyte depleting preparative regimen can be administered as an inpatient or 
outpatient.  Patients will be admitted to NIH inpatient ward on day -1, if not before, for administration of 
HERV-E TCR transduced T-cells (Table 6). 
 
The day of the cell infusion is day 0. It will be a single infusion according to the CC nursing standards of 
practice guidelines. During cell infusions, the subjects will be evaluated for side effects and possible 
toxicities of the infusion, which include flu-like symptoms. Patients will remain on the inpatient service 

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18-H-0012 
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Richard Childs, MD 
 
 
as needed (at a minimum days 0 through 7). Patients will start moderate -dose IL-2 2,000,000 IU/m2 
given every 12 hours by intravenous infusion for 14 doses beginning as soon as practical after completing 
the cell infusion (Table 6).  
 
• 
Line Placement 
Subjects will have inserted a midline catheter, a peripherally inserted central catheter (PICC) or another 
type of central venous catheter placed prior to chemotherapy administration to aid with blood draws and 
ensure infusion of the expanded T-cells and IL-2 administration.  
 
Table 6. Overall summary of the treatment plan  
Day 
-4  
-3 
-2 
-1 
0 
1 
2 
3 
4 
5 
6 
7 
Therapy 
  
  
  
  
  
  
  
  
  
  
 
Cyclophosphamide IV 1000 
mg/m2  
X 
 
  
  
  
  
  
  
  
  
 
Fludarabine IV 30 mg/m2  
X 
X 
X 
 
  
  
  
  
  
  
 
Infusion of HERV-E TCR 
transduced CD8+/CD34+ T-cells 
  
  
  
  
X 
  
  
  
  
  
 
IL-2 IV 2,000,000 IU/m2 q12h  
for 14 doses 
  
  
  
  
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant Medications/Measures 
 
INFECTION PROPHYLAXIS  
Note: Appropriate anti-infective agents may be substituted at the discretion of the treating physician. 
 
▪ 
Pneumocystis Jirovecii Pneumonia 
All patients will receive the fixed combination of trimethoprim and sulfamethoxazole (TMP/SMX) as 
double strength (DS) tab (DS tabs = TMP 160 mg/tab, and SMX 800 mg/tab) P.O. daily three times a week 
on non-consecutive days, beginning on day -4. Pentamidine will be substituted for TMP/SMX-DS in 
patients with sulfa allergies. It will be administered aerosolized at 300 mg per nebulizer within one week 
prior to admission and monthly thereafter. PCP prophylaxis will be stopped after 2 consecutive CD4 counts 
greater than 200/μL are documented, first tested at least 30 days following the conditioning regimen or 
when clinically indicated 
 
▪ 
Herpes Virus Prophylaxis 
Patients will be given acyclovir orally at a dose of 800 mg twice daily, beginning on day -4, or acyclovir, 
250 mg/m2 IV every 12 hours if the patient is not able to take medication by mouth. Reversible renal 
insufficiency has been reported with IV but not oral acyclovir. Neurologic toxicity including delirium, 
tremors, coma, acute psychiatric disturbances, and abnormal EEGs have been reported with higher doses 
of acyclovir. Should this occur, a dosage adjustment will be made, or the drug will be discontinued. 
Acyclovir will not be used concomitantly with other nucleoside analogs which interfere with DNA 
synthesis, e.g. ganciclovir. In renal disease, the dose is adjusted as per product labeling. Prophylaxis for 
Herpes will continue until the CD4 count is greater than 200 for 2 consecutive measures or when clinically 
indicated 
▪ 
Fungal Prophylaxis 
Primary antifungal prophylaxis for cryptococcal disease is not mandated. Patients will start Fluconazole 
200 mg p.o. starting on the day of cell infusion and continue until clinically indicated only at the PI 

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Richard Childs, MD 
 
 
discretion. The drug may be given IV at the same dose in 0.9% sodium chloride USP daily in patients 
unable to take it orally.  
 
▪ 
Neutropenia 
A CBC with differential will be obtained daily during inpatient admission. If the absolute neutrophil 
count becomes less than 500/microliter, filgrastim or filgrastim-sndz may be initiated at a dose of 300 
micrograms daily subcutaneously for patients under 70 kg in weight and at a dose of 480 micrograms daily 
for patients 70 kg and over in weight. Filgrastim or filgrastim-sndz will be given daily until absolute 
neutrophil count recovers to > 1500/μL.  
▪ 
Empiric Antibiotics 
Patients will start on broad-spectrum antibiotics, in accordance with current institutional guidelines for 
fever of 38.3°C once or two temperatures of 38.0°C or above at least one hour apart, AND an ANC 
<500/mm3. Infectious disease consultation will be obtained for all patients with unexplained fever or any 
infectious complications. 
 
Table 7. Prophylactic Treatments 
Prophylactic treatment 
Dosage 
Administration 
TMP/SMX - DS 
160mg/800mg PO daily 
Every Monday-Wednesday-Friday 
beginning on day -4. 
PCP prophylaxis will be stopped after 
2 consecutive CD4 counts greater than 
200/μL are documented or when 
clinically indicated 
Pentamidine* 
(Pentamidine will be 
substituted for TMP/SMX
in patients with sulfa alle
Aerosolized at 300 mg per 
nebulizer 
PCP prophylaxis will be started within 
one week of chemotherapy start date 
and then monthly. 
PCP prophylaxis will be stopped after 
2 consecutive CD4 counts greater than 
200/μL are documented or when 
clinically indicated 
Fluconazole 
200 mg PO daily 
If unable to tolerate PO: 
Fluconazole 100 mg in 0.9% 
sodium chloride USP daily IV 
daily 
Primary untifungal prophylaxis for 
cryptococcal disease is not mandated. 
Antifungal Prophylaxis will start on 
day 0 and will be administered only at 
the discretion of the PI and until 
clinically indicated. 
Acyclovir 
800mg PO twice daily (preferred) 
If unable to tolerate PO: acyclovir 
250mg/m2 IV every 12 hour 
HSV and VZV prophylaxis will be 
started days -4 and stopped after 2 
consecutive CD4 counts greater than 
200/μL are documented or when 
clinically indicated 
Filgrastim 
300 micrograms daily for patients 
under 70 kg in weight and a dose 
of 480 micrograms daily for 
Until absolute neutrophil count 
recovers to > 1500/μL 

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Richard Childs, MD 
 
 
patients over 70 kg in weight may 
be started as per PI discretion 
Detailed Treatment Plan 
 
Conditioning chemotherapy and HERV-E TCR transduced T-cells -this can be either inpatient or 
outpatient 
Note: No subjects will be treated at Loyola University. 
  
Day -4, -3, and -2: Patients will receive pre-hydration with 1000 mL 0.9% sodium chloride I.V. over 1 to 
3 hours on day -4. Patients will receive anti-emetics following NIH Clinical Center guidelines, but 
dexamethasone will not be administered. One suggested regimen is ondansetron 16 to 24 mg orally on 
days -4, -3, and -2 one hour before chemotherapy (I.V. ondansetron can be substituted). Patients will be 
provided with anti-emetics such as lorazepam and prochlorperazine to use at home.  
On day -4, cyclophosphamide at a dose of 1000 mg/m2 I.V. will be diluted in 100 ml 5% dextrose solution 
and infused. After the cyclophosphamide on day -4, patients will receive 30 mg/m2 I.V. fludarabine in 100 
mL 0.9% sodium chloride. Following the fludarabine infusion, patients will receive 1000 mL 0.9% sodium 
chloride I.V. over 1-2 hours. Furosemide will be given if needed.  
 
Next, on days -3, and -2, patients will receive 30 mg/m2 I.V. fludarabine in 100 mL 0.9% sodium chloride.  
 
Note: in patients with a creatinine clearance calculated by the CKD-EPI equation less than 80 
ml/minute/1.73 m2 of body surface are, the daily dose of fludarabine will be reduced by 20%.  
 
Note: The dose of fludarabine can be rounded to the nearest vial size to conserve medication as long 
as the dose reduction is ≤10% of the calculated dose. 
 
Day -1: No interventions except as needed for general supportive care such as anti-emetics. To minimize 
bladder toxicity, patients should increase normal oral fluid intake to at least 2 liters/day.  
 
Day 0: HERV-E TCR transduced CD8+/CD34+ enriched T cells will be thawed in the DTM and following 
confirmation that they meet release criteria, will be administered to the patient. The pre-medications are 
acetaminophen 650 mg orally and diphenhydramine 25mg IV. Cells will be delivered to the patient care 
unit from DTM. Prior to infusion, the cell product identity label is double-checked by two authorized staff 
(MD or RN), and identification of the product and documentation of administration are entered in the 
patient’s chart as is done for blood banking protocols. The cells are to be infused intravenously according 
to the CC nursing standards of practice guidelines.  
 
Days 0 to 7: Mandatory hospitalization for observation and treatment as necessary. IL-2 2,000,000 
IU/m2 given every 12 hours IV infusion for 14 doses beginning as soon as practical after completing the 
cell infusion. One dose of IL-2 may be started on Day 0, and then every 12 hours for a total of 14 doses. 
Doses will be preferentially administered every twelve hours; however, up to 24 may elapse between 
doses depending on patient tolerance. Dosing time adjustment may be performed at the investigator’s 
discretion. 
 

39 
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6.4 Blood Product Support 
▪ 
Leukocyte filters will be utilized for all blood and platelet transfusions except for the HERV-E 
TCR transduced T-cells to decrease sensitization to transfused WBC and decrease the risk of CMV 
infection.  
▪ 
Using daily CBC’s as a guide, the patient will receive platelets and packed red blood cells (PRBC’s) 
as needed. Attempts will be made to keep Hgb >8.0 gm/dl, and platelets >10,000/μL.  
▪ 
All blood products except for the HERV-E TCR transduced T-cells will be irradiated.  
6.5 Anti-Emetics 
Anti-emetics Guidelines (except that corticosteroids will be avoided).  
 
6.6 Granulocyte Colony-Stimulating Factor 
If the absolute neutrophil count becomes less than 500/microliter, filgrastim or filgrastim-sndz may be 
initiated at a dose of 300 micrograms daily for patients under 70 kg in weight and a dose of 480 micrograms 
daily for patients over 70 kg in weight. Filgrastim or filgrastim-sndz may be discontinued as soon as the 
absolute neutrophil count recovers to 1500/μL.  
 
6.7 Avoidance of Corticosteroids 
Patients should not take systemic corticosteroids including prednisone, dexamethasone or any other 
corticosteroid at any dose for any purpose without approval of the Principal Investigator.  
 
 
RE-TREATMENT 
 
Patients experiencing a partial or complete response or stable disease for at least 3 months (+/- 1 week) (SD 
measured at the tumor assessment on D+90 associated with relief of one or more tumor-associated 
symptoms) may receive one additional retreatment at a higher dose level (i.e. a higher cell dose) when 
progression by RECIST criteria is documented after evaluation by the Principal Investigator.  Such patients 
would be retreated at the currently enrolling dose level, which must be a higher dose level than the initial 
treatment. Patients retreated at a higher dose level will be counted for the primary study endpoint in both 
dose levels to determine the maximum tolerated dose, as the use of conditioning lymphodepletion 
chemotherapy given before the second T-cell infusion should remove any remaining HERV-E TCR 
transduced T cells from circulation or tissues, and patients will be only enrolled if there is no evidence for 
any active immune-mediate toxicities related to prior treatment. 
 
Available frozen apheresis cells used for the initial TCR transduction that were collected prior to the first 
T cell infusion will be the preferred cell source for manufacturing the 2nd cell product to be used at the 
higher dose level.  
 
The collection of a fresh apheresis product can be used to manufacture the 2nd cell product when a prior 
frozen apheresis sample is not available for further T-cell transduction. Fresh 2nd apheresis product will not 
be collected, if  any circulating CD34 + /CD8+ T-cells (these are the transduced T-cells) are detectable in 
the circulation from the first infusion. This will exclude any possibility of toxicity  being caused by any T-
cells in the 2nd collection that were remaining transduced T-cells from the original transduction. 
 
Any patients who developed a grade 3 or grade 4 toxicity possibly or definitely related to the HERV-E TCR 
T-cell product will not be eligible for retreatment at a higher dose level. Patients must continue to meet the 

40 
18-H-0012 
May 11, 2023 (Amendment T) 
Richard Childs, MD 
 
 
original eligibility criteria to be considered for retreatment. Retreatment benefits and risks will be carefully 
explained to the patient. A maximum of 1 retreatment course may occur. 
  
 
 
7. MANAGEMENT OF COMPLICATIONS  
7.1 Dose Modification/Delay 
For patients who have yet to receive treatment, patients may be removed from treatment or have their 
treatment delayed if they have active infections defined as infections causing fevers or infections requiring 
intravenous anti-microbial therapy and intravenous anti-microbial therapy occurring less than 72 hours 
before the start of chemotherapy. This means that patients are on-study but have not yet started treatment 
could have treatment cancelled or delayed; but, such patients are eligible for treatment after the infection 
resolves. If a patient experiences a grade 3 or greater toxicity (except for cytopenias or nausea or other 
toxicities as per investigator’s discretion) while on-study before the HERV-E TCR transduced T-cells 
infusion, the HERV-E TCR transduced CD8+/CD34+ enriched T-cells infusion must be delayed until the 
toxicity improves to a grade 2 or less or cancelled.  
 
7.2 Cytokine release syndrome 
Cytokine release syndrome (CRS), which will be graded according to the CRS grading scale of Lee et al.141 
Please see section Appendix C. These are guidelines only. It is understood that treatment of these toxicities 
must be individualized for each patient. Not following the exact recommendations in Appendix C is not a 
protocol deviation. 
7.3 IL-2 Toxicities 
Guidelines for dealing with toxicities that often occur after HERV-E TCR- expressing T cells infusions and 
IL-2 treatment are given in Appendix D. In addition, patients are required to stay within 60 minutes driving 
distance from the Clinical Center until day 30 (+/- 7 days) after the HERV-E TCR-expressing T cells 
infusion. 
 
8. BIOSPECIMEN COLLECTION 
8.1 Tumor Tissue 
 
Blood and tissue are tracked at the patient level and can be linked to all protocols on which the patient has 
been enrolled. Samples will be used to support the specific objectives listed in the treatment protocol(s), 
e.g., immunologic monitoring, cytokine levels, persistence, as well as to support long term research efforts 
within the NHLBI, Laboratory of Transplantation Immunotherapy, and with collaborators.  
 
The amount of blood that may be drawn from adult patients for research purposes shall not exceed 10.5 
mL/kg or 550 mL, whichever is smaller, over any eight-week period. Samples will be ordered and tracked 
through the CRIS Screens. Should a CRIS screen not be available, the NIH form 2803-1 will be completed 
and will accompany the specimen and be filed in the medical record. Samples will not be sent outside NIH 
without IRB notification and an executed MTA. 
 
Archival Tissue 
Archival tissue (Recommended: 15 unstained slides representative of the tumor for diagnosis confirmation 
and 15 unstained slides or 1 tumor block to be used for research purposes), will be required for enrollment 

41 
18-H-0012 
May 11, 2023 (Amendment T) 
Richard Childs, MD 
 
 
onto the study for diagnosis confirmation. The patient will have the option to undergo a pre- treatment 
biopsy if archival tissue is not available for diagnosis confirmation. (preferably of metastatic disease).  
 
Fresh Tissue and Malignant Effusions 
Optional tumor biopsies or sampling of malignant effusions as described below are obtained before the 
initiation of study therapy, on treatment and at the time of progression (all of them are optional tumor 
samples). In the case of autopsy, we may request samples from different organs and tumor for research 
purposes. 
 
Fresh tissue samples should be collected into empty, unused barcoded cryovials (Nunc catalog #5001-0012, 
5001-1020 or equivalent) and stored on dry ice or liquid nitrogen. Tissue may be obtained by core biopsy, 
surgical dissection, or aspiration in the case of malignant effusions. The interval between sample acquisition 
and freezing on dry ice must not exceed 5 minutes. Frozen, vialed samples should be transferred from dry 
ice to -80°C (minimum) or liquid nitrogen (preferred) frozen storage within 2-3 hours.  
 
The tumor tissue will be divided among two laboratories: 
o Laboratory of pathology (Maria Merino or designee) will perform diagnosis confirmation 
(Recommended: 15 unstained slides representative of the tumor). 
o Laboratory of Transplantation Immunotherapy (Recommended: 15 unstained slides or 1 tumor 
block, fresh tissue, and malignant effusions). The recommended slides received will be first 
evaluated by Dr. Merino or designee for pathology assessment and then stored at the Laboratory of 
Transplantation Immunotherapy. 
 
▪ 
Samples sent to the Laboratory of Transplantation Immunotherapy, Dr. Childs’ Lab 
Venous blood samples will be collected to be processed for serum if appropriate and stored for future 
research. Record the date and exact time of draw on the tube. Tissue will be processed as described and 
stored for future research. 
• 
For questions regarding sample processing or for immediate help, contact Robert Reger by email 
(robert.reger@nih.gov) 
or 
at 
301.594.8004, 
or 
Elena 
Cherkasova 
by 
email 
(cherkasovae@nhlbi.nih.gov) or at 301-827-3958. 
• 
The samples will be processed, barcoded, and stored in Dr. Childs’s lab until used. 
8.2 Immunological Testing (Exploratory studies) 
• 
Samples of all infused cell products will be cryopreserved, and extensive retrospective analysis of 
infused cell phenotype and function will be performed to attempt to find in vitro characteristics of 
the infused cells which correlate with in vivo antitumor activity. Analyses of peripheral blood 
lymphocytes (PBL) samples will include evaluation of the activity and specificity of infused 
CD8+/CD34+ enriched T cells. 
• 
Lymphocytes will be tested directly and following in vitro culture using some or all of the following 
tests. Direct immunological monitoring will consist of quantifying T cells reactive with targets 
FACS analysis using mouse V-beta antibody. Ex vivo immunological assays will consist of 
cytokine release by bulk PBL (+/- peptide stimulation) and by other experimental studies such as 
cytolysis if sufficient cells are available. If cell numbers are limiting, preference will be given to 
the direct analysis of immunological activity. Immunological assays will be standardized by the 
inclusion of 1) pre-infusion PBMC and 2) an aliquot of the transduced PBMC cryopreserved at the 
time of freezing the product. In general, differences of 2 to 3 fold in these assays are indicative of 
true biologic differences. 

42 
18-H-0012 
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Richard Childs, MD 
 
 
• 
Research apheresis may be performed after the treatment.  Apheresis product will be transferred 
to Dr. Childs’ lab, Section of Transplantation Immunotherapy, attention Robert Reger 
(robert.reger@nih.gov), building 10/CRC, room  5-5264.  Possible laboratory research studies on 
PBMC and PBL are as follows: Specific cytolysis determined by calcein AM assay, quantity of 
cytokine production as determined by coculture assay with cytokine quantification, cytokine 
production by intracellular flow cytometry, phenotypic analysis by flow cytometry. 
 
 
8.3 Monitoring Gene Therapy Trials: Persistence and Replication Competent Retrovirus 
(RCR) 
• 
Engineered cell survival. TCR and vector presence will be quantitated in PBMC samples using 
established PCR techniques. Immunological monitoring using both tetramer analysis and staining 
for the TCR will be used to augment PCR-based analysis. This will provide data to estimate the in 
vivo survival of lymphocytes derived from the infused cells. In addition, measurement of CD4 and 
CD8 T cells will be conducted and studies of these T cell subsets in the circulation will be 
determined by using specific PCR assays capable of detecting the unique DNA sequence for each 
retroviral vector engineered T cell. Note: samples will be batched and assayed at the conclusion of 
the study. 
• 
Patients’ blood samples will be obtained and undergo analysis for detection of RCR by PCR prior 
to cell infusion approximately at 3 and 6 months, and at one-year (+/- 30 days) post cell 
administration**. Blood samples will be archived annually thereafter if all previous testing has 
been negative with a brief clinical history. If a patient dies or develops neoplasms during or after 
this trial, efforts will be made to assay a biopsy sample for RCR. If any post-treatment samples are 
positive, further analysis of the RCR and more extensive patient follow-up will be undertaken, in 
consultation with the FDA. RCR PCR assays detect the GaLV envelop gene and are performed 
under contract by the Indiana University Vector Production Facility. The results of these tests are 
maintained by the contractor performing the RCR tests and by the NHLBI research team. 
 
**NOTE: We will obtain 1 EDTA tube with 4-5 ml of peripheral blood.  These samples need to 
be sent on ice packs priority overnight to the attention of: 
Lisa Duffy 
IU Vector Production Facility 
980 W. Walnut St. 
R3-C668 
Indianapolis, IN  46202 
 
RCR testing sample will need to be shipped the day drawn if at all possible.  IU Vector 
Production Facility is not open on the weekends, if a sample must be drawn on a Friday, it will be 
placed at 4ºC over the weekend and shipped on Monday. 
8.4 Post Infusion Blood  Samples and Optional Tumor Tissue for  Exploratory Studies 
Correlatives studies including peripheral blood (mandatory) and fresh tumor tissue or malignant 
effusions (recommended but optional) will be collected at baseline, on-treatment (pre-determined time 
points) and at the time of progressive disease. If available, samples collected under protocol 04-H-0012 
can be used for the research purposes of this protocol as described below.  
 

43 
18-H-0012 
May 11, 2023 (Amendment T) 
Richard Childs, MD 
 
 
▪ 
CT-RCC HERV-E expression.  Determine target tumor-associated antigen expression levels in 
tumor tissues (fresh and/or FFPE-fixed). This will be done by qRT-PCR of total RNA extracted 
from tumor material using CT-RCC HERV-E specific primers. 
▪ 
Quantification of HERV-E TCR transduced CD8+/CD34+ enriched T cells. Evaluate the in vivo 
persistence and peak blood levels of HERV-E TCR transduced CD8+/CD34+ T cells. This will be 
measured by staining with anti-CD8 and anti-CD34 antibodies. The methods for 
immunofluorescence staining of T cells have been published.142 
▪ 
Cytolytic potency/activity of HERV-E TCR transduced CD8+/CD34+ enriched T cells. Ex vivo 
immunological assays will be used to measure the antigen-specific functional activity of the HERV-
E TCR transduced CD8+/CD34+ enriched T cells and will consist of intracellular cytokine staining, 
chemokines release assays and cytotoxicity assays. Immunological assays will be standardized by 
the inclusion of pre-infusion recipient PBMC and in some cases an aliquot of the engineered T cells 
cryopreserved at the time of infusion. 
▪ 
HERV-E TCR transduced CD8+/CD34+ enriched T cell surface phenotype and immune 
subsets. T cell subset analysis will be performed to evaluate markers of T cell activation and 
exhaustion in HERV-E TCR transduced CD8+/CD34+ enriched T cell over time and to measure 
lymphoid reconstitution. 
▪ 
Comprehensive cytokine/Chemokine studies. Multiple cytokines and chemokines are necessary 
for effective host immunity against tumor cells. Correlate these chemical signals that attract T cells 
to infiltrate the tumor and activate targeted cytotoxic responses with presence of HERV-E TCR 
transduced CD8+/CD34+ enriched T cells in tumor and response. 
▪ 
Presence of HERV-E TCR transduced CD8+/CD34+ enriched T cells in tumor tissue. 
Determine the infiltration of HERV-E TCR transduced CD8+/CD34+ enriched T cells in tumor 
tissue. 
▪ 
Tumor Immune Subsets. Determine recruitment and activation of immune effector cells in tumor 
tissue. 
▪ 
Establishment of cell lines from patient tumor cell biopsies if possible. 
 
Table 8. Summary of biospecimen collection, handling, distribution and storage 
Test Description 
Amount/Type/ 
Tube/Handling 
Collection 
Timing 
Contact 
Special Instruction 
Diagnostic 
Confirmation 
 
Recommended 15 
unstained slides 
representative of 
the tumor  
Baseline 
pre-treatment 
 
Kristen Gunn 
Email: kristen.gunn@nih.gov 
Phone #: 301-827-2977  
 
Tissue will be submitted 
to Pathologist . 
HERV-E expression 
in paraffin-
embedded tumors 
Recommended 15 
unstained slides or 
1 block of tumor 
Baseline 
pre-treatment 
Kristen Gunn 
Email: kristen.gunn@nih.gov 
Phone #: 301-827-2977  
 
Tissue will be submitted 
to Pathologist for initial 
evaluation and then 
stored at the Laboratory 
of Transplantation 
Immunotherapy. 
Baseline pre-
treatment: 
- Immune subsets 
characterization 
Optional Fresh 
Tumor Biopsy (1-
5) or malignant 
effusions at 
NIH  
Baseline 
pre-treatment, 
on treatment 
and at the time 
of disease 
Email: Robert Reger by e-mail 
(robert.reger@nih.gov) or at 
301.594.8004, 24 hours prior to 
anticipated 
collection 
1-5 cores will be flash 
frozen at the time of 
biopsy and malignant 
effusion in a vacuum 
bottle. 

44 
18-H-0012 
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Richard Childs, MD 
 
 
-Generate tumor cell 
lines 
 
On-Treatment: 
- Presence of 
HERV-E TCR 
transduced 
CD8+/CD34+ T-
cells and generate 
cell cultures 
 
Disease 
Progression: 
-Presence of HERV-
E TCR transduced 
CD8+/CD34+ T-
cells and generate 
cell cultures 
 
progression or 
other time 
points if 
clinically 
indicated. i.e. 
drainage of 
malignant 
effusions 
 
Then contact Robert Reger by e-
mail (robert.reger@nih.gov) or 
at 301.594.8004, or Elena 
Cherkasova by email 
(cherkasovae@nhlbi.nih.gov) or 
at 301-827-3958 for questions 
with regards sample collections 
instructions and immediate pick-
up. 
 
When the patient is 
scheduled for biopsy will 
call Robert Reger 
(301.594.8004) and cores 
will be distributed 
accordingly. 
 
1-5 cores will be sent to 
Dr. Child’s laboratory for 
further processing and 
experiments. 
Enumeration of 
absolute number of 
circulating HERV-E 
TCR transduced 
CD8+/CD34+ T-
cells by Flow 
cytometry* 
30 ml of peripheral 
blood collected in 
green top tubes  
 
D-4, D0, D+2, 
D+4 and D+7 
after T cell 
infusion and 
then on day 
+14, and then 
every planned 
follow-up 
appointment  
Email: Robert Reger, 
robert.reger@nih.gov 
24 hours prior to anticipated 
collection 
 
Please contact Robert Reger by 
e-mail (robert.reger@nih.gov) 
or at 301.594.8004, or at   240-
750-4660 or Elena Cherkasova 
by email 
(cherkasovae@nhlbi.nih.gov) or 
at 301-827-3958 for questions 
about sample collections 
instructions. 
 
After the tube has been 
filled with blood, 
immediately invert the 
tube several times to 
prevent coagulation. 
 
Interferon-γ release 
and cytotoxicity 
assays* 
 
D-4, D0, D+2, 
D+4 and D+7 
after T cell 
infusion and 
then on day 
+14 and then 
every planned 
follow-up 
appointment 
- 
- 
Intracellular 
cytokine staining of 
HERV-E TCR 
transduced 
CD8+/CD34+ T-
cell* 
 
D-4, D0, D+2, 
D+4 and D+7 
after T cell 
infusion and 
then on day 
+14 and then 
every planned 
follow-up 
appointment 
- 
- 

45 
18-H-0012 
May 11, 2023 (Amendment T) 
Richard Childs, MD 
 
 
HERV-E TCR 
transduced 
CD8+/CD34+ T-cell 
surface phenotype 
and activation 
status* 
 
 
D-4 , D0, D+2, 
D+4 and D+7 
after T cell 
infusion and 
then on day 
+14 and then 
every planned 
follow-up 
appointment 
- 
- 
Cytokine/chemokine 
study 
IL-2, IL-4, IL-5, IL-
10, IL-12, IL-13, 
GM-CSF, IFN-y, 
and TNFalpha* 
 
10 cc of peripheral 
blood in a 
SST tube 
D-4, D0, D+2, 
D+4 and D+7 
and then D+14 
and then every 
planned 
follow-up 
appointment  
Email: Robert Reger, 
robert.reger@nih.gov 
24 hours prior to anticipated 
collection 
 
Please contact Robert Reger by 
e-mail (robert.reger@nih.gov) 
or at 301.594.8004, or Elena 
Cherkasova by email 
(cherkasovae@nhlbi.nih.gov) or 
at 301-827-3958 for questions 
about sample collections 
instructions. 
- 
Research Apheresis 
4-5 L 
- 
Please contact Robert Reger by 
e-mail (robert.reger@nih.gov) 
or at 301.594.8004. 
 
*Research Blood can be collected +/- 1 day except for D-4 and D0 which samples should be collected prior 
chemotherapy and HERV-E TCR transduced T-cell infusion. Additional research blood may be requested at the 
discretion of the investigators as long as the amount of blood drawn does not exceed 10.5 mL/kg or 550 mL, 
whichever is smaller, over any eight-week period. Obtaining blood samples may be discontinued for patients 
who had a disease progression at any time as per PI’s discretion. 
 
 
 
 
9. BIOSTATISTICAL CONSIDERATIONS 
 
Primary Objective: The primary objective of this study is to evaluate the safety of infusing escalating 
doses of HERV-E TCR transduced CD8+/CD34+ enriched T cells for the treatment of metastatic ccRCC. 
 
Primary End-point: The primary endpoint will be the toxicity profile at each dose level captured using 
the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 except for hematological 
toxicities (Section 3.2). 
 
Secondary End-points: 
- 
To assess for tumor response using Response Evaluation Criteria in Solid Tumors (RECIST) version 
1.1 criteria  
- 
To estimate the PFS and OS in such population. 
 
Exploratory Studies: 

46 
18-H-0012 
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Richard Childs, MD 
 
 
- 
To determine the persistence, phenotype (including expression of checkpoint inhibitors) and function 
of HERV-E TCR transduced CD8+/CD34+ enriched T-cells circulating in patients post-infusion and to 
correlate findings with safety and efficacy (tumor response) of this therapy at each dose level.  
- 
To study peripheral and tumor immune subsets and correlate findings with safety and efficacy (tumor 
response) of this therapy at each dose level. 
- 
To evaluate changes in serum-soluble factors associated to T cell activation and migration and correlate 
findings with safety and efficacy (tumor response) of this therapy at each dose level. 
- 
To determine the ex-vivo antigen-specific functional activity of HERV-E TCR transduced CD8+/CD34+ 
T-cells and correlate findings with safety and efficacy (tumor response) of this therapy at each dose 
level. 
- 
To investigate the presence of HERV-E TCR transduced CD8+/CD34+ enriched T-cell in tumor tissue 
and its correlation with response to therapy. 
 
Sample size 
A standard ‘3+3’ design will be used for this trial where 3 patients are treated at the first dose level. At a 
given dose level, if there are no DLT, the dose will be escalated to the next level. If there is one DLT in the 
first 3 patients treated at a dose level, an additional 3 patients will be treated at that level. If no additional 
DLTs are observed (for a total of 1 DLT in 6 patients), then the dose will be escalated. 
 
If a level with 2 or more DLTs in 3-6 patients has been identified, 3 additional patients will be accrued at 
the next-lowest dose for a total of 6, in order to further characterize the safety of the maximum tolerated 
dose. The MTD is defined as the highest dose at which 0 or 1 patient in six has experienced a DLT. We 
will report adverse events using the Common Terminology Criteria for Adverse Events version 4.0. If no 
DLTs are observed, we expect to treat up to 15 patients. If DLTs are observed, up to 6 patients may need 
to be treated at each cohort to establish the MTD. Thus, up to 24 patients may need to be enrolled onto the 
trial. 
 
Statistical Methods 
The planned analyses will include descriptive statistics on the incidence and severity of adverse events, 
such as the percentage of subjects with hematological or immunological toxicities. Since the risk of toxicity 
is assumed to increase with dosage, the isotonic regression method will be used to estimate probabilities of 
toxicity for different dose levels. A 21-days safety assessment period following cell administration will be 
conducted between each subject before the next subject in the cohort or the first subject in the next cohort 
would be treated. 
 
The proportions of local adverse events, systemic adverse events and TRSAEs will be estimated using the 
sample proportions, with 95% confidence intervals. The time to adverse events will be analyzed using 
appropriate survival analysis methods such as Kaplan-Meier estimates. 
 
The secondary endpoints will be plotted over time for each subject. The mean change of each variable over 
time will be estimated and compared among different dose cohorts. Ideally there will be a decrease in tumor 
mass on average when the cohorts are compared with respect to each of their doses of HERV-E TCR 
transduced CD8+/CD34+ enriched T-cells. Exploratory correlative analysis will be performed among 
biomarkers in peripheral blood and tumor tissue and tumor response.   
 

47 
18-H-0012 
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Richard Childs, MD 
 
 
If a subject fails to complete a cycle of protocol therapy for reasons other than toxicity, he/she will be 
deemed non-evaluable for determining the maximum tolerated dose and will be replaced. However, these 
subjects will be included in the evaluation of safety/toxicity. Toxicities will be tabulated by dose and grade.  
 
Response will be first assessed at the day +30 time point following the HERV-E TCR transduced 
CD8+/CD34+ enriched T-cells infusion. 
 
Protocol Stopping Rules 
The study will be halted pending discussions with the FDA and NIH IRB regarding safety and the need for 
protocol revisions if any of the following conditions are met: 
▪ 
If two DLTs, as described in Section DLT, occur in the first cohort of this study. 
▪ 
If 2 or more patients develop a grade 3 or greater toxicity related to the cell product, except for: 
o Grade 3 metabolic laboratory abnormalities without significant clinical sequela that resolve to 
grade 2 or less within 7 days 
o Grade 3 fever 
▪ 
If 1 of the first 3 patients (OR 2 of the first 6 patients, OR 3 of the first 9 patients, OR 4 of the first 12 
patients) develop grade 3 autoimmunity, that cannot be resolved to less than or equal to a grade 2 
autoimmune toxicity within 10 days, or any grade 4 or greater autoimmune toxicity. 
▪ 
If one or more treatment related deaths occur due to the cell infusion, we will immediately stop study 
enrollment and promptly initiate discussions with the NIH IRB and FDA 
 
Criteria for removal from protocol therapy and off study criteria 
 
Off Treatment Criteria (Note: patients taken off-treatment will remain on study for the purpose of long-
term follow-up data collection, RCR testing (recommended by FDA guidelines for patients treated with 
Retroviral Vector-Based Human Gene Therapy Products) and research sample at the discretion of the PI). 
• 
Rapid disease progression prior to cell infusion that it would preclude the patient’s ability to tolerate 
protocol therapy, 
• 
Disease progression after HERV-E TCR transduced T cells infusion treatment 
• 
Intercurrent illness that prevents administration of HERV-E TCR transduced CD8+/CD34+ 
enriched T-cells infusion, 
• 
Unacceptable adverse event(s) that prevents administration of HERV-E TCR transduced 
CD8+/CD34+ enriched T-cells infusion, 
• 
General or specific changes in the patient’s condition render the patient unacceptable for further 
treatment in the judgment of the investigator, or 
• 
Positive pregnancy test 
 
Patients who are off treatment are to be followed until they meet the criteria for Off Study (see below). 
When off treatment, long-term follow-up data on clinical data related to time-endpoints or FDA-mandated 
evaluation for patients treated with Retroviral Vector-Based Human Gene Therapy Products will be 
collected during this period will be collected unless consent is withdrawn. 
RCR testing will be performed as per FDA guidelines for patients treated with Retroviral Vector-Based 
Human Gene therapy Products and research sample will be collected at the discretion of the PI. 
 
Off Study Criteria  
• 
The patient voluntarily withdraws, 
• 
There is significant noncompliance, 

48 
18-H-0012 
May 11, 2023 (Amendment T) 
Richard Childs, MD 
 
 
• 
General or specific changes in the patient’s condition render continued participation the patient 
unacceptable for further follow up in the judgment of the investigator, or 
• 
Death 
• 
Completion of protocol 
 
10. DATA and SAFETY MONITORING 
 
Safety Monitoring Principal Investigator:  Accrual, efficacy and safety data will be monitored by the PI  
 
NIH IRB: Prior to implementation of this study, the protocol and the proposed patient consent forms will 
be reviewed and approved by the properly constituted IRB operating according Title 21 Code of Federal 
Regulations (CFR) Part 56. This committee will also approve all amendments to the protocol or informed 
consent, and conduct continuing annual review so long as the protocol is open to accrual or follow up of 
subjects.  
 
DSMB: The NHLBI Data Safety and Monitoring Board (DSMB) will review the protocol at least annually. 
A progress report will be forwarded to the DSMB at these times and their recommendations will be 
expeditiously implemented. The DSMB may recommend early termination of the study for considerations 
of safety and efficacy.  
 
FDA: (IND 17690) An annual progress report, any amendments to the protocol, and any change in the 
status of the protocol will be forwarded to FDA. 
 
10.1 Definitions 
The PI or designee will refer to HRPP Policy 801 “Reporting Research Events” for definitions. 
 
10.2 Adverse Event Management 
All abnormal physical exam findings, laboratory events and other events defined in CTCAE v5.0 will be 
collected.  The AEs will be attributed (unrelated, unlikely, possibly, probably or definitely) to study 
medication and/or disease. This study will utilize the CTCAE version 5.0 for toxicity and adverse event 
reporting. 
A 
copy 
of 
the 
CTCAE 
version 
5.0 
can 
be 
downloaded 
from 
the 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm  
 
Baseline symptoms will be collected starting on the day the subject signs the screening protocol informed 
consent. However, we will use baseline symptoms and laboratory results collected during the clinical visit 
and laboratory tests prior to starting chemotherapy for the purpose of reporting treatment-related adverse 
event.  
Adverse events of any grade will be collected during the first year after cell infusion or until a subject is 
determined to have disease progression, at which time, the subject will remain on study only for gene 
therapy related long-term follow up. After one year, or after a subject is determined to have disease 
progression, no adverse events will be collected, unless they are thought to be at least possibly related to 
cell infusion.  
 
 
 

49 
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Richard Childs, MD 
 
 
10.2.1 Grading of Adverse Events 
 
1 Mild 
Asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated.  
2 Moderate 
Moderate; minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental ADL*  
3 Severe 
Severe or medically significant but not immediately life-threatening; hospitalization 
or prolongation of hospitalization indicated; disabling; limiting self-care ADL**.  
4 Life-
threatening 
Life-threatening consequences; urgent intervention indicated.  
5 Death 
Death related to AE  
Hospitalization (overnight admission) for routine supportive care (platelet or red blood cell transfusions) or 
admission from the NIH inpatient unit to the NIH ICU for routine monitoring will not be reported as a 
serious adverse event.  
 
10.3 NIH-Intramural IRB and CD reporting 
Expedited Reporting 
Events requiring expedited reporting will be submitted to the IRB per Policy 801 “Reporting 
Research Events”. 
 
Reports to the IRB at the time of Continuing Review: 
The PI or designee will refer to HRPP Policy 801 “Reporting Research Events” to determine 
IRB reporting requirements. 
 
Reports to the CD: 
The PI or designee will refer to NHLBI DIR Policy to determine CD reporting requirements and 
timelines.  
 
10.5 Reporting of Pregnancy 
 
Subjects who become pregnant during the first year on study should discontinue any study drugs that may 
adversely affect the pregnancy. The investigator, or his/her designee, will collect pregnancy information on 
any subject who becomes pregnant while participating in this study. 
The sponsor-investigator, or his/her designee, will submit pregnancy information to the NHLBI Clinical 
Director and NIH IRB within two weeks of learning of a subject’s pregnancy. Information on the status of 
the mother and child will also be reported to the CD and NIH IRB no longer than 6 to 8 weeks following 
the estimated delivery date. Any premature termination of the pregnancy will be reported. While pregnancy 
itself is not considered to be an AE or SAE, any pregnancy complication or elective termination of a 
pregnancy for medical reasons will be recorded and reported as an AE or SAE. 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the 
telephone, managing money, etc.  
**Self-care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden. 

50 
18-H-0012 
May 11, 2023 (Amendment T) 
Richard Childs, MD 
 
 
 
10.6 IND Sponsor Reporting Criteria 
 
Events will be submitted to Dr. Richard Childs, IND Sponsor Authorized Representative at: 
Richard Childs, M.D.,  
Bldg 10, CRC, Room CRC 3-5332 
Phone: (301) 594-8008 
 
10.7 FDA Reporting Criteria 
 
IND Safety Reports to the FDA (Refer to 21 CFR 312.32) 
 
The Sponsor will notify the FDA of any unexpected fatal or life-threatening suspected adverse reactions as 
soon as possible but no later than 7 calendar days of initial receipt of the information using the MedWatch 
Form 3500a.  
 
The Sponsor is also responsible for reporting any:  
• 
suspected adverse reaction that is both serious and unexpected 
• 
any findings from clinical, epidemiological, or pooled analysis of multiple studies or any findings 
from animal or in vitro testing that suggest a significant risk in humans exposed to the drug 
• 
clinically important increase in the rate of a serious suspected adverse reaction over that listed in 
the protocol or investigator brochure to the FDA and to all investigators no later than 15 calendar 
days after determining that the information qualifies for reporting using the MedWatch Form 
3500a.  If FDA requests any additional data or information, the sponsor must submit it to the FDA 
as soon as possible, but no later than 15 calendars days after receiving the request.  
 
Copies of all IND Safety Reports submitted to the FDA per 21 CFR 312.32 will also be submitted to CTEP: 
CTEPSupportAE@tech-res.com.  
 
10.8 FDA Annual Reports (Refer to 21 CFR 312.33) 
 
The study Sponsor will submit a brief report annually of the progress of the trial within 60 days of the 
anniversary date that the IND went into effect as indicated in 21CFR 312.33, and any associated FDA 
correspondences regarding the IND annual report.  
 
10.9 Reports to the DSMB 
 
Reports of serious adverse events that are unexpected and suspected will also be forwarded as soon as 
possible and no later than seven (7) days in the case of death or life-threatening serious adverse events or 
within fifteen (15) days after the occurrence of all other forms of serious adverse events to the Data and 
Safety Monitoring Board (DSMB).  A summary of all SAEs will be included for review semiannually or 
annually by the DSMB.  
If the serious adverse event is thought to be due to the experimental component of the protocol, accession 
to the protocol will be stopped until a full discussion with the IRB has been held.   
 

51 
18-H-0012 
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Richard Childs, MD 
 
 
10.10 Routine Adverse Event Recording 
 
All adverse events will be recorded in the patient’s medical record. Following enrollment, adverse events 
will be reviewed by the research nurse and principal investigator and captured into an in-house password 
protected electronic system and ensuring data accuracy, consistency and timeliness. All events occurring 
during the treatment phase of the study will be followed until resolution or stabilization. 
 
10.11 Clinical trial monitoring  
 
As per ICH-GCP 5.18 and FDA 21 CFR 312.50 clinical protocols are required to be adequately monitored 
by the study sponsor.  
The monitoring of this study will be conducted by Clinical Research Associates (CRAs)/monitors employed 
by an independent contract organization working under an agreement with NHLBI to monitor aspects of 
the study in accordance with the appropriate regulations and the approved protocol.  
 
The objectives of a monitoring visit will be: 1) to verify the existence of signed informed consent form 
(ICF) and documentation of the ICF process for each monitored subject; 2) to verify the prompt and accurate 
recording of all monitored data points, and prompt reporting of all SAEs; 3) to compare abstracted 
information with individual subjects’ records and source documents (subject’s charts, laboratory analyses 
and test results, physicians’ progress notes, nurses’ notes, and any other relevant original subject 
information); and 4) to help ensure investigators are in compliance with the protocol. The monitors also 
will inspect the clinical site regulatory files to ensure that regulatory requirements (Office for Human 
Research Protections-OHRP), FDA and applicable guidelines (ICH-GCP) are being followed. During the 
monitoring visits, the investigator (and/or designee) and other study personnel will be available to discuss 
the study progress and monitoring visit. The investigator (and/or designee) will make study documents 
(e.g., consent forms and pertinent hospital or clinical records readily available for inspection by the local 
IRB, the FDA, the site monitors, and the NHLBI staff for confirmation of the study data.  
 
11. DATA COLLECTION AND EVALUATION 
During the course of study participation, specimens will be collected for correlative studies. 
 
Research Sample Collection: The quantities of blood to be drawn for research purposes will be consistent 
with the Clinical Center policy as provided in Medical Administrative Series (MAS) 95-9.  The amount of 
blood that may be drawn from adult patients and volunteers for research purposes shall not exceed 10.5 
mL/kg or 550 mL, whichever is smaller, over any eight week period.  For pediatric patients, no more than 
5 mL/kg may be drawn for research purposes in a single day, and no more than 9.5 mL/kg may be drawn 
over any eight-week period. 
 
12. BIOSPECIMEN AND DATA MANAGEMENT PLAN 
Biospecimen Tracking and Management 
Samples will be ordered and tracked through the CRIS research screens.  Should a CRIS screen not be 
available, the NIH form 2803-1 will be completed and will accompany the specimen and be filed in the 
medical record.  Specimens and their derivatives (e.g., genomic material, cell lines) will be coded and stored 
in conformity with DIR Policy (e.g., BSI). Coded biospecimens may be sent to collaborators outside of the 
NIH with IRB approval in accordance with applicable NIH and DIR Policy for sharing research resources, 
including an executed material transfer agreement (MTA).  Biospecimens with subject personal identifiers 

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Richard Childs, MD 
 
 
may be sent to associate investigators and collaborators outside of the NIH only after approvals of both 
NIH and local IRBs, an executed reliance agreement with NIH IRB, or an extension of the NIH’s FWA 
through an Individual Investigator Agreement. 
 
Loss or destruction of samples:  Should we become aware that a major breech in our plan for tracking and 
storage of samples has occurred, the IRB will be notified. 
 
Data management  
The PI will be responsible for overseeing entry of data into an in-house password protected electronic 
system and ensuring data accuracy, consistency and timeliness. Laboratory values from referring home 
physicians will be entered into the system. The principal investigator, associate investigators/research 
nurses and/or a contracted data manager will assist with the data management efforts to ensure that data is 
verifiable and evaluable.  
 
Research data will be prospectively collected by authorized Investigator personnel and entered into an 
NHLBI-approved database. The database will consist of the study specific set of electronic CRFs (e-CRFs) 
used for capturing, managing and reporting clinical research data.  
 
The database will maintain complete data records on each research subject. Subjective and objective patient 
experiences during the duration of the study will be documented in the patient medical record notes. These 
protocol notes will serve as the primary source material from which data will be collected and research 
analyses will be performed. Any pertinent supplementary information obtained from outside laboratories, 
outside hospitals, radiology reports, laboratory reports, or other patient records will be used as additional 
source for data collection. 
 
Identifiable data will not be sent outside NIH without prior IRB approval or appropriate conditions for 
disclosure outlined in the executed CDA or MTA. 
 
CDUS-Abbreviated reporting/monitoring to CTEP 
 
This study will be monitored by the Clinical Data Update System (CDUS) Version 3.0.  Cumulative 
protocol- and  
patient-specific CDUS data will be submitted electronically to CTEP on a quarterly basis by FTP burst of 
data.  Reports are due January 31, April 30, July 31, and October 31.  Instructions for submitting data using 
the CDUS can be found on the CTEP Web site (http://ctep.cancer.gov/reporting/cdus.html). 
 
CDUS-Abbreviated reporting: no adverse event reporting (routine or expedited) is required to be reported 
via CDUS, but expedited adverse events are still required to be submitted via CTEP-AERS. 
 
End of study procedures:  Data will be stored in locked cabinets and in a password protected database 
until it is no longer of scientific value.                               
 
Loss or destruction of data:  Should we become aware that a major breech in the plan to protect subject 
confidentiality and trial data has occurred, the IRB will be notified. 
 
Publication policy: Given the research mandate of the NIH, subject data including the results of testing 
and responses to treatment will be entered into an NIH-authorized and controlled research database. Any 

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Richard Childs, MD 
 
 
future research use will occur only after appropriate human subject protection institutional approval such 
as prospective NIH IRB review and approval or an exemption from the NIH Office of Human Subjects 
Research Protections (OHSRP). 
 
Data sharing and future use of data  
Research data may be shared with qualified non-collaborator recipients following publication of the primary 
research results after removal of PII and IRB approval.  Future research use of data not defined in the 
research protocol may occur only after IRB review and approval or an exemption from the NIH OHSRP.  
Refusal of a research subject participant to permit future use of data--other than required in the protocol or 
by the FDA--will be honored.  Limitations in data sharing and future use of data due to contractual 
obligations (e.g., CRADAs) or intellectual property proceedings (such as patent filings) will be honored. 
 
Future use of biospecimens  
Following analyses of biospecimens for primary research purposes, remaining samples suitable for future 
research will be stored in the Principal Investigator’s laboratory freezers in a coded manner to ensure data 
protection.  
 
Any future research use of biospecimens not defined in the protocol in which NHLBI investigators are 
engaged in research (e.g., they are undertaking research activities and hold the key that identifies research 
subjects) requires IRB review and approval.  Coded biospecimens (NHLBI investigators hold the key that 
identifies research subjects) to be shared outside of NIH for future research use where results will not be 
returned to the Principal Investigator does not require IRB review or approval.  
 
13. RESPONSE CRITERIA 
Response will be documented by physical exam and/or noninvasive imaging using techniques that 
demonstrate lesions. Response and progression will be evaluated in this study using the new international 
criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 
1.1).143 Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest 
diameter in the case of malignant lymph nodes are used in the RECIST criteria. For the purposes of this 
study, patients should be re-evaluated for response every 6-8 weeks. In addition to a baseline scan, 
confirmatory scans should also be obtained at least 4 weeks following initial documentation of objective 
response. 
 
If a patient is classified as having PD at the first post-baseline tumor assessment (D+30), then confirmation 
of PD by a second scan in the absence of rapid clinical deterioration is required on D+60. The definition of 
confirmation of progression at the first post-baseline tumor assessment represents an increase in tumor 
burden ≥20% compared to the baseline tumor size confirmed at two consecutive time points at least 4 weeks 
apart. It is recommended that this be done at the discretion of the investigator because follow-up with 
observation alone may not be appropriate for patients with a rapid decline in performance status. However, 
we anticipate that confirmation of PD will allow for the capture of all observed responses, as late-responses 
were observed in patients with RCC months after transplantation (median of 129 days), and often followed 
the withdrawal of cyclosporine and the establishment of complete donor–T-cell chimerism.33 Otherwise, 
the definition of disease progression represents an increase in tumor burden of ≥20% compared with the 
nadir. Confirmation of progression as describe above will be performed at the PI’s discretion after D+90 
time point. 
If a patient is classified as having CR, PR or SD at the first post-baseline tumor assessment (D+30), then; 
next tumor assessment will take place on D+90 unless otherwise clinically indicated.  
 

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Evaluation of Target Solid Lesions 
Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether 
target or non-target) must have reduction in short axis to <10 mm. 
Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as 
reference the baseline sum of diameters. 
 
Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions,  
taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). 
In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 
5 mm. (Note: the appearance of one or more new lesions is also considered progressions). 
 
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for 
PD, taking as reference the smallest sum of diameters while on study. 
 
Evaluation of Bony Lesions in Patients with Isolated Bone Metastases 
Complete Response (CR): Normalization of all bony lesions (target and nontarget) to SUL less than mean 
liver SUL and equal to normal surrounding tissue SUL. Verification with follow-up study in 1 month if 
anatomic criteria indicate disease progression. 
 
Partial Response (PR): > 30% decrease in SUL peak; minimum 0.8 unit decrease. Verification with 
follow-up study if anatomic criteria indicate disease progression.  
 
Progressive Disease (PD): > 30% increase in SUL peak (minimum 0.8 unit increase in SUL peak), > 75% 
increase in TLG of the 5 most active lesions, Visible increase in extent of FDG uptake, or new lesions. 
Verification with follow-up study if anatomic criteria indicate complete or partial response. 
 
Stable Disease (SD): Does not meet other criteria. 
 
Evaluation of Non-Target Lesions 
Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker 
level. All lymph nodes must be non-pathological in size (<10 mm short axis). Note: If tumor markers are 
initially above the upper normal limit, they must normalize for a patient to be considered in complete 
clinical response. 
 
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker 
level above the normal limits. 
 
Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal progression of 
existing non-target lesions.  
Unequivocal progression should not normally trump target lesion status. It must be representative of overall 
disease status change, not a single lesion increase. Although a clear progression of “non-target” lesions only 
is exceptional, the opinion of the treating physician should prevail in such circumstances, and the 
progression status should be confirmed at a later time by the review panel (or Principal Investigator). 
 
Evaluation of Best Overall Response 

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The best overall response is the best response recorded from the start of the treatment until disease 
progression/recurrence (taking as reference for progressive disease the smallest measurements recorded 
since the treatment started). The patient's best response assignment will depend on the achievement of both 
measurement and confirmation criteria. 
 
For Patients with Measurable Tumors (i.e., Target Disease) 
Target 
Lesions 
Non-Target Lesion 
New 
lesions 
Overall 
Response 
Best Overall Response when 
Confirmations is Required* 
CR 
CR 
No 
CR 
 4 weeks. Confirmation* 
CR 
Non-CR/Non-PD 
No 
PR 
 4 weeks. Confirmation** 
CR 
Not evaluated 
No 
PR 
PR 
Non-CR/Non-PD/not 
evaluated 
No 
PR 
SD 
Non-CR/Non-PD/not 
evaluated 
No 
SD 
Documented at least once  4 weeks from 
baseline** 
PD 
Any 
Yes or No 
PD 
No prior SD,PR or CR 
Any 
PD*** 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
For Patients with Non-Measurable Disease (i.e. Non-Target Disease) 
 
Non-Target Lesions 
New lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
No-CR/non-PD* 
Not all evaluated 
No 
Not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
 
Duration of Response 
 
Duration of overall response: The duration of overall response is measured from the time measurement 
criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive 
disease is objectively documented (taking as reference for progressive disease the smallest measurements 
recorded since the treatment started). 
 
The duration of overall CR is measured from the time measurement criteria are first met for CR until the 
first date that progressive disease is objectively documented. 
 
Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for 
progression are met, taking as reference the smallest measurements recorded since the treatment started, 
including the baseline measurements.  
 
* See RECIST 1.1 manuscript for further details on what is evidence of a new lesions 
**Only for non-randomized trials with response as primary response 
***In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as disease progression. 
Note: Patients with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease 
progression at that time should be reported as “ symptomatic deterioration”. Every effort should be made to document the objective 
progression even after discontinuation of treatment.  
*’Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since SD is increasingly used as a endpoint for assessment of 
efficacy in some trials so to assign this category when no lesions can be measured is not advised 

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14. HUMAN SUBJECT PROTECTIONS 
 
Rationale for Subject Selection 
 
All patients between the ages of 18 and 75 years with eligible diseases will be considered for the protocol. 
No subjects will be excluded from participation based on gender, race or ethnicity. Due to the experimental 
nature of the study and the potential serious risk and limited information of its effects in elderly patients, 
subjects greater than 75 years will not be eligible.  
 
We anticipate distribution by race, gender, and age will be similar to our concurrent solid tumor transplant 
protocol (99-H-0064) as follows: 
▪ 
By gender: females (48%), and males (52%).  
▪ 
By age: 24 to 67 years of age (median 47),  
▪ 
By race: Asian (2%), Black (7%), Hispanic (3%), and white (88%).  
 
HIV-positive patients are excluded because the experimental treatment being evaluated in this protocol 
depends on an intact immune system. Patient who are HIV seropositive can have decreased immune 
competence and thus be less responsive to the experimental treatment and more susceptible to its toxicities. 
Palliative radiation therapy to areas of metastatic tumors is commonly used to treat patients with metastatic 
cancer. Such patients often maintain a good performance status and sufficient hematopoietic reserve after 
such radiation making them candidates for subsequent investigational trials or other standard treatment 
modalities.  
 
Pregnant or nursing mothers are excluded because effects of chemotherapy, IL-2 and HERV-E TCR 
transduced CD8+/CD34+ enriched T-cells on the developing human fetus are unknown. For this reason, 
women of child-bearing potential should agree to use adequate contraception prior to study entry and for 
the duration of study participation. Should a woman become pregnant or suspect she is pregnant while 
participating in this study, she should inform her treating physician immediately.  
 
Exclusion of Pregnant Women 
Pregnant women are excluded from accrual to eliminate the possibility of drug effects on a developing 
fetus.  Subjects are required to use effective contraceptive measures to prevent pregnancy within one year 
of participation in the study. 
 
Participation of Children 
Patients less than 18 years of age will not be eligible in this study due to the rarity of the eligible diseases 
in the pediatric population and because of the potential serious risks and limited information of the protocol 
regimen’s effects in children. Should the results of this trial demonstrate efficacy, consideration will be 
given to expanding accrual to this patient population.  
 
Participation of subjects unable to give consent 
Informed consent for adult research participants unable to provide consent 
If there is an unexpected enrolment of a research participant unable to provide informed consent, we will 
follow the procedure for obtaining informed consent from a legally authorized representative (LAR) per 
NIH HRPP Policy 403 (Appendix D). 
 

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Justification for inclusion: 
This research provides the prospect of direct benefit; therefore, inclusion is justified. The benefits to the 
participants could be regression of metastatic kidney cancer resulting in improved quality of life and also 
decreased morbidity and mortality from oncological complications (i.e. cord compression, dyspnea, pain). 
Potentially, oncological complications and treatment with other toxic therapies could also be avoided or 
postponed. Not allowing participants who cannot provide consent would deny them the potential benefits 
this protocol offers for their disease. There are no plans to include institutionalized participants. 
 
 
Consent and Assent: 
Procedures to determine capacity: If documentation of decision making capacity is not present in the 
medical record or the investigator questions the decision making capacity of the individual, then the Ability 
to Consent Assessment Team (ACAT) (301-496-9675 or 301-496-2429) will be contacted to make the 
determination. 
 
Procedures to obtain assent and documentation of assent or dissent: 
The informed consent discussion will include the individual unable to provide informed consent along with 
LAR. The individual unable to provide informed consent will be asked if they agree to participate in the 
research and this will be documented in the medical record. Assent from decisionally impaired subjects will 
be obtained orally and documented in the patient’s file. Subject dissent will be honored. 
 
For Patients that become cognitively impaired during treatment 
Re-consent may be necessary and there is a possibility, though unlikely, that subjects could become 
decisionally impaired. For this reason and because there is a prospect of direct benefit from research 
participation (section 4.2), all subjects will be offered the opportunity to fill in their wishes for research and 
care, and assign a substitute decision maker on the “NIH Advance Directive for Health Care and Medical 
Research Participation” form so that another person can make decisions about their medical care in the 
event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI 
or AI will contact the NIH ACAT for evaluation. For those subjects that become incapacitated and do not 
have pre-determined substitute decision maker, the procedures described in MEC Policy 87-4 for 
appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and (b) who do 
not have a legal guardian or durable power of attorney, will be followed. 
 
No Competition among Branch Protocols 
 
Strategies for patient recruitment at the NIH: The study may use the following strategies of recruitment 
including:  
• 
ClinicalTrials.gov website 
• 
Clinical Center Research Studies (“Search the Studies”) website 
• 
Engagement with patient advocacy groups such as Kidney Cancer Association, American Cancer 
Society, Action to Cure Kidney Cancer, Inspire Kidney Cancer Association Support Group, and 
the American Cancer Fund  
• 
National Heart, Lung and Blood Institute (NHLBI) patient recruitment website 
• 
Twitter messages and chats with study investigators 
• 
Facebook Posts through official NIH accounts 
• 
Google Ad Words 

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Richard Childs, MD 
 
 
• 
Use of Clinical Center Office of Patient Recruitment services including creation and distribution 
of study flyers and information through pre-existing recruitment avenues such as the NIH 
recruitment listserv. 
 
The DIR Patient Recruitment Office (PRO) will work with study investigators to ensure accrual goals are 
being met.   
▪ 
Biannual reviews will be performed by investigators and the PRO.   
▪ 
If recruitment goals are lagging, an enhanced plan of recruitment will be triggered, which may 
include a communication blitz to known and newly contacted hematologists and oncologists 
throughout the country; additional Twitter feeds and chats, Facebook posts and recirculation of 
updated and/or new patient recruitment flyers.  All recruitment materials and tools will use IRB-
approved language and information to include standard recruitment contacts. 
 
Oncologists and Renal Cancer specialists throughout the country will be actively informed about the 
protocol by letter. 
 
No subjects will be recruited at Loyola University, Chicago. 
 
Reimbursement for protocol participation, travel, food, and lodging: There is no payment or 
compensation to subjects for their participation in this protocol. Reimbursement for travel, food, and 
lodging will be consistent with NHLBI DIR Travel and Lodging Compensation of Clinical Research 
Subjects policy or institutional guidelines. 
 
15. RISK and DISCOMFORTS  
15.1 Related to cyclophosphamide 
Cyclophosphamide toxicities includes: 
1) Nausea and vomiting-variable; symptomatically improved with standard anti-emetics and/or 
benzodiazepines [e.g., lorazepam]. 
2)  Water retention–cyclophosphamide may rarely provoke the syndrome of inappropriate antidiuretic 
hormone secretion and resultant hyponatremia, usually manifested 12-48 hours after IV administration. 
3) Cardiomyopathy-cyclophosphamide may cause severe, sometimes lethal, hemorrhagic myocardial 
necrosis or congestive cardiomyopathy. Patients may present with congestive cardiomyopathy as late as 2 
weeks after the last dose of cyclophosphamide. In an attempt to minimize this complication, patients with 
significant cardiac dysfunction are excluded from this protocol [see patient eligibility]. Congestive failure 
is managed according to standard medical therapeutics. Cardiac toxicity with the dose of cyclophosphamide  
administered on this protocol is extremely unlikely. 
4) Hemorrhagic cystitis–this is a serious, potentially life-threatening complication related to injury of the 
bladder epithelium by cyclophosphamide metabolites. 
5) Less common but serious complications include pulmonary fibrosis and secondary malignancies. Less 
common but reversible toxicities include alopecia and skin rash. 
 
15.2 Related to fludarabine 
Fludarabine toxicities include myelosuppression (dose limiting toxicity), fever, nausea, vomiting, 
stomatitis, diarrhea, gastrointestinal bleeding, anorexia, edema, skin rashes, myalgia, headache, agitation, 
hearing loss, transient episodes of somnolence and fatigue, anemia, auto-immune thrombocytopenia, 
paresthesias, peripheral neuropathy, renal, and pulmonary toxicity (interstitial pneumonitis). Severe fatal 
CNS toxicity presenting with loss of vision and progressive deterioration of mental status were encountered 

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Richard Childs, MD 
 
 
almost exclusively after very high doses of fludarabine monophosphate. Such toxicity has only rarely been 
demonstrated at the 25-30 mg/m2/day dosage of fludarabine. Very rarely described complications include 
transfusion-associated graft-versus-host disease, thrombotic thrombocytopenic purpura, and liver failure.  
 
Tumor lysis syndrome following fludarabine administration has been observed, especially in patients with 
advanced bulky disease. Opportunistic infections (protozoan, viral, fungal, and bacterial) have been 
observed post-fludarabine, especially in heavily pre-treated individuals, and in individuals receiving 
fludarabine combined with another agent. 
 
15.3 Related to Interleukin-2 (IL-2, aldesleukin) 
Interleukin-2 boxed warnings. The boxed warnings are for high dose interleukin-2 (600,000 IU/kg every 8 
hours). For the average adult (70 kg) the total daily dose would be 126 x106IU/day. This protocol calls for 
moderate dose interleukin-2 (2,000,000 IU/m2 q 12 hours on day 0 through day +6). For the average adult 
(1.73 m2) the total daily dose would be 7 x106 IU/day or roughly 6% of the high dose amount, therefore, 
we do not expect any of the boxed warnings as detailed below:  
 
15.3.1 Boxed Warnings related to interleukin-2:  
Cardiac and pulmonary: Therapy with interleukin-2 should be restricted to patients with normal cardiac 
and pulmonary functions as defined by thallium stress testing and formal pulmonary function testing. 
Extreme caution should be used in patients with a normal thallium stress test and a normal pulmonary 
function test who have a history of cardiac or pulmonary disease.  
 
Hospital setting: Interleukin-2 should be administered in a hospital setting under the supervision of a 
qualified physician experienced in the use of anti-cancer agents. An intensive care facility and specialists 
skilled in cardiopulmonary or intensive care medicine must be available.  
 
Capillary Leak Syndrome: Interleukin-2 administration has been associated with capillary leak syndrome 
(CLS) which is characterized by a loss of vascular tone and extravasation of plasma proteins and fluid into 
the extravascular space. CLS results in hypotension and reduced organ perfusion which may be severe and 
can result in death. CLS may be associated with cardiac arrhythmias (supraventricular and ventricular), 
angina, myocardial infarction, respiratory insufficiency requiring intubation, gastrointestinal bleeding or 
infarction, renal insufficiency, edema, and mental status changes.  
 
Neutropenia: Interleukin-2 treatment is associated with impaired neutrophil function (reduced chemotaxis) 
and with an increased risk of disseminated infection, including sepsis and bacterial endocarditis. 
Consequently, preexisting bacterial infections should be adequately treated prior to initiation of interleukin-
2 therapy. Patients with indwelling central lines are particularly at risk for infection with gram-positive 
microorganisms. Antibiotic prophylaxis with oxacillin, nafcillin, ciprofloxacin, or vancomycin has been 
associated with a reduced incidence of staphylococcus infections.  
 
CNS Toxicity (Lethargy, somnolence, coma): Interleukin-2 administration should be withheld in patients 
developing moderate to severe lethargy or somnolence; continued administration may result in coma. 
 
15.3.2 Adverse Events related to interleukin-2:  
Adverse events associated with interleukin-2, while potentially serious, are generally predictable, 
manageable, reversible and more commonly occur with high dose therapy. Patients treated with interleukin-
2 may experience significant side effects; but interleukin-2 is acute therapy, with most patients receiving 

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Richard Childs, MD 
 
 
only 1 or 2 courses of treatment. Nearly all toxicities are self-limiting, rapidly reversible or improve within 
2 to 3 days of discontinuing therapy. Long-term sequelae associated with interleukin-2 are extremely rare.  
 
The following common adverse events are based on 525 patients (255 with metastatic renal cell carcinoma 
and 270 with metastatic melanoma) treated with the recommended interleukin-2 infusion dosing regimen: 
 
Event 
Grade 1-4 Seen in  30% of 
Patients (n=525) 
Grade 4 Seen in > 1% of 
Patients (n=525) 
Hypotension 
71% 
3% 
Diarrhea 
67% 
2% 
Oliguria 
63% 
6% 
Chills 
52% 
- 
Vomiting 
50% 
1% 
Dyspnea 
43% 
1% 
Rash 
42% 
- 
Bilirubinemia 
40% 
2% 
Thrombocytopenia 
37% 
1% 
Nausea 
35% 
- 
Confusion 
34% 
1% 
Creatinine increase 
33% 
1% 
Anuria 
- 
5% 
Respiratory 
- 
3% 
Coma 
- 
2% 
Infection 
- 
1% 
Sepsis 
- 
1% 
Cardiovascular disorder 
- 
1% 
Myocardial Infarct 
- 
1% 
Psychosis 
- 
1% 
 
Other adverse events that could occur because of IL-2 are muscle and joint pain.  
 
15.4 Related to the apheresis procedure 
The apheresis procedures will be performed in accordance with standard apheresis donation policies and 
procedures operative in the Department of Transfusion Medicine and will be following the Blood Donor 
Standards of the American Association of Blood Banks and the rules and regulations of the Food and Drug 
Administration. Adverse reactions to apheresis procedures are rare, but include: 
• 
Pain and hematoma at the needle placement site. 
• 
Vasovagal episodes, characterized by transient hypotension, dizziness, nausea and rarely, syncope 
are seen in less than 2% of the procedures. Hypotension secondary to volume depletion is known 
for the rare potential for a cerebrovascular or cardiovascular event.  

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• 
Cutaneous or circumoral parasthesias, chills, nausea, heartburn and rarely muscle spasms may 
result from the use of citrate anticoagulant used to prevent clotting in the extracorporeal circuit. 
Citrate reactions are usually relieved by slowing the rate of the anticoagulant infusion and by 
administering oral calcium carbonate tablets or with intravenous calcium gluconate. 
 
Prior to each apheresis, the potential risks associated with the procedure will be explained to the subject 
and a separate informed consent obtained. 
15.5 Related to blood draws  
No major risks are involved with blood draws. Minor complications including bleeding, pain, and 
hematoma formation at the site of the blood draw. Infection may rarely occur. 
 
15.6 Related to central line placement  
It is estimated that about 50% of subjects will require intravenous line placement, typically in the femoral 
vein to successfully complete apheresis. All subjects who do not have a chronic indwelling central venous 
line will have a single-lumen PICC intravenous line (or other central line if arm veins are sub-optimal) 
placed by the Venous Access Device (VAD) service of the Clinical Center. This line will aid with blood 
draws, medication infusion and ensure IV infusion of the expanded HERV-E TCR-expressing CD8+/CD34+ 
enriched T cells. If a PICC line cannot be placed, subjects may have a midline catheter placed as an 
alternative to a central venous catheter. 
 
Intravenous line placement carries a small risk of bleeding, bruising or pain and a very low risk of accidental 
injury to the adjacent artery and nerve. Some subjects may experience a vasovagal reaction 
(lightheadedness, or rarely, fainting due to temporary lowering of blood pressure). Using only trained 
experienced MICU/VAD staff for the procedure minimizes these risks. 
 
15.7 Related to metastatic site biopsy (when indicated)  
A fine-needle or core needle aspiration biopsy puts a thin needle into the lymph node or other visceral 
metastatic sites and removes cells to look at. Subjects will feel only a quick sting from the local anesthesia 
used to numb the skin and may feel some pressure from the biopsy needle. After a fine-needle aspiration 
biopsy or core needle biopsy, the site may be tender for 2 to 3 days. An open biopsy of a lymph node makes 
a cut in the skin and removes the lymph node.  
 
15.8 Related to Thoracentesis 
Potential complications from an ultrasound-guided thoracentesis,  include pain at the puncture site (2.7%), 
bleeding (hematoma, hemothorax, or hemoperitoneum) (0.4%), pneumothorax (2.5%), shortness of breath 
(1.0%) empyema, soft-tissue infection, spleen or liver puncture, re-expansion pulmonary edema when a 
large amount of fluid is removed (0.2%), vasovagal events (0.6%), and adverse reactions to local 
anesthetic or topical antiseptic solutions. The most common clinically important complication is 
pneumothorax, with an incidence of 2.5%. When a pneumothorax occurs, it is usually small and often 
requires no additional therapy. Tube thoracostomy is required in only 0.8% of patients. Liver or spleen 
puncture may occur when the patient is not sitting absolutely upright, because movement towards 
recumbency causes cephalad migration of the abdominal viscera. Nonetheless, even if the liver or spleen 
is punctured by a small-bore needle, the outcome is generally favorable if the patient is not receiving 
anticoagulants and does not have a bleeding diathesis.  There is also a risk for infection, anaphylaxis, and 
sustained hypotension (mean arterial pressure < 60 mm Hg unresponsive to intravenous fluids). 

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15.9 Related to Paracentesis 
The risks of an abdominal paracentesis include pain at the puncture site, bleeding (hematoma or 
hemoperitoneum), pneumothorax bacterial peritonitis, soft-tissue infection, sepsis, spleen or liver 
puncture, vasovagal events and adverse reactions to local anesthetic or topical antiseptic 
solutions, anaphylaxis, and sustained hypotension (mean arterial pressure < 60 mm Hg 
unresponsive to intravenous fluids). 
15.10 Related to imaging studies (when indicated)  
15.10.1 Chest X-ray: The risks associated with x-rays are very small. For example, one chest x-ray has the 
same increased risk of death (due to cancer) as smoking 1.4 cigarettes or drinking ½ liter of wine. On 
average, all of us receive an equivalent dose of x-rays every 30 days from nature. However, the more 
radiation you receive over the course of your life, the greater the risk of developing cancerous tumors or of 
inducing changes in genes. The changes in genes could cause abnormalities or disease in your future 
offspring. We do not expect the radiation in this study to greatly increase these risks, but we do not know 
the exact increase in such risks.  Additionally, we do not stipulate x-rays to be done as routine monitoring 
and they would only be done at the PI discretion as medically indicated. 
 
15.10.2 CT scan: CT (computed tomography), sometimes called CAT scan, uses special x-ray equipment 
to obtain image data from different angles around the body and then uses computer processing of the 
information to show a cross-section of body tissues and organs. Oral and/or intravenous contrast agents will 
be used and are usually well tolerated. However, some subjects will experience allergic reactions to 
intravenous contrast. To lower the risk of allergic reactions, low allergenic contrast agents are administered 
at NIH Clinical Center. In addition, subjects will be advised that approximately 2-7% of patients who 
receive contrast agents will experience a temporary reduction in kidney function lasting up to 2 weeks 
following infusion and that in rare instances, permanent renal damage can result from the use of the IV 
contrasting agent. Therefore, in subjects with impaired kidney function, we will not use intravenous 
contrast. 
 
15.10.3 F18-FDG-PET CT: Positron emission tomography or “PET scan” is an alternative approach to 
monitoring disease activity. Rapidly growing tissue, such as cancer, take up and use more sugar than does 
normal tissue. To detect areas of rapidly growing tissue we will inject a radioactive sugar (FDG) into the 
body through a vein. Then by means of a special scanner which senses the uptake of the radioactive sugar, 
images will be produced identifying areas of rapidly growing tissue in the body.  
 
The subject will be asked to come to the NIH 2 hours before the PET scan. We will place one small plastic 
tube (called a catheter) into a vein into the arm. We will use this tube for injections of the radioactive sugar. 
Subjects will be asked to wait for 60 min in the Nuclear Medicine Department.  After 60 minutes, we will 
ask the subject to lie down on the scanner bed. The scanning procedure will take in total 2 – 3 hours to 
complete. He/she will be asked to lie still during the scan. We will monitor the subject throughout the 
procedure. When the scanning is completed, the subject will be asked to go to the bathroom to void which 
will minimize radiation exposure. All women of child bearing potential will be required to sign and have a 
pregnancy test no more than 24 hours before the PET scan tracer injection. 
 
Subjects may feel a quick sting or pinch when the IV is inserted. Most of the tracer will be flushed from the 
body within 6 to 24 hours. Allergic reactions to the tracer are very rare. In rare cases, some soreness or 
swelling may develop at the IV site where the radioactive tracer was put in. Subjects will be advised to 
apply a moist, warm compress to his/her arm. 
 

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15.10.4 Magnetic resonance imaging (MRI): Magnetic resonance imaging (MRI) uses a strong magnetic 
field and radio waves to obtain images of body organs and tissues. The MRI scanner is a metal cylinder 
surrounded by a strong magnetic field. During the MRI, the subject lies on a table that can slide in and out 
of the cylinder. Scanning time varies, with most scans lasting between 45 and 90 minutes. While in the 
scanner subjects will hear loud knocking noises, and may request earplugs to muffle the sound. Subjects 
with a fear of confined spaces may become anxious during this procedure. MRI staff and patients are able 
to communicate at all times during the scan, and may ask to be moved out of the machine at anytime they 
become uncomfortable. If contrast is ordered, Gadolinium, an FDA approved medication will be used to 
improve MRI images. About 98% of patients receiving gadolinium have no symptoms related to the 
injection of this medication. Mild symptoms that may occur include: coldness in the arm at injection, a 
metallic taste, headache, and nausea. In an extremely small number of patients, more severe symptoms have 
been reported including: shortness of breath, wheezing, and lowering of blood pressure. The FDA has 
issued a warning that administration of gadolinium, the contrast imaging agent that may be used in this 
protocol, has been associated with development of a disease called nephrogenic systemic fibrosis (NSF). 
The syndrome is rare (approximately 200 cases reported worldwide as of December 2006 out or several 
million administrations of gadolinium), but disabling and in some cases, fatal. All cases to date have 
occurred in patients with severe renal disease, including patients on dialysis. We will ask the patient whether 
they have or have had kidney disease or diabetes, whether they take diuretics (water pills) for any medical 
condition and whether they have received x-ray dye or drugs recently that might affect their kidney function. 
Depending on their history and creatinine levels, it will be determined whether they may receive 
gadolinium.  
 
On December 19, 2017, the FDA is requiring a new class warning and other safety measures for all 
gadolinium-based contrast agents (GBCAs) for magnetic resonance imaging (MRI) concerning gadolinium 
remaining in patients’ bodies, including the brain, for months to years after receiving these drugs. 
Gadolinium retention has not been directly linked to adverse health effects in patients with normal kidney 
function, and FDA has concluded that the benefit of all approved GBCAs continues to outweigh any 
potential risks. However, after additional review and consultation with the Medical Imaging Drugs 
Advisory Committee, FDA is requiring several actions to alert health care professionals and patients about 
gadolinium retention after an MRI using a GBCA, and actions that can help minimize problems. These 
include requiring a new patient Medication Guide, providing educational information that every patient will 
be asked to read before receiving a GBCA. FDA is also requiring manufacturers of GBCAs to conduct 
human and animal studies to further assess the safety of these contrast agents. 
 
In response to the December 2017 MedWatch warning on gadolinium accumulation from the FDA, the 
healthy subjects may undergo no more than two gadolinium exposures during a 12-month period, per 
NHLBI policy. 
 
In accordance with the FDA Drug Safety Communication of 05/16/2018, the Medication Guide for 
gadobutrol (or other macrocyclic gadolinium contrast agent if applicable) will be made available to all 
subjects with scans that will involve gadolinium-based contrast agent administration. 
 
15.10.5 Bone scan: A bone scan is a nuclear scanning test that identifies new areas of bone growth or 
breakdown. It can be done to evaluate damage to the bones, detect cancer that has spread (metastasized) to 
the bones, and monitor conditions that can affect the bones (including infection and trauma). A bone scan 
can often detect a problem days to months earlier than a regular X-ray test. For a bone scan, a radioactive 
tracer substance is injected into a vein in the arm. The tracer then travels through the bloodstream and into 
the bones. A special camera (gamma) takes pictures of the tracer in the bones. This helps show cell activity 
and function in the bones. Areas that absorb little or no amount of tracer appear as dark or "cold" spots, 

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which may indicate a lack of blood supply to the bone (bone infarction) or the presence of certain types of 
cancer. Areas of rapid bone growth or repair absorb increased amounts of the tracer and show up as bright 
or "hot" spots in the pictures. Hot spots may indicate the presence of a tumor, a fracture, or an infection. 
 
15.10.6 Radiation Risks: The amount of radiation subjects may receive from the research scans in this 
study is 21 rem of radiation annually.  All female subjects will receive pregnancy testing prior to radiation 
exposure. 
 
15.11 Related to cardiac function  
 
15.11.1 Electrocardiogram (EKG): EKG is a test that measures the electrical activity of the heartbeat. 
With each beat, an electrical impulse (or “wave”) travels through the heart. This wave causes the muscle to 
squeeze and pump blood from the heart. A technician will put patches (electrodes) on the chest, arms and 
legs. 
 
The electrodes are soft and don’t cause any discomfort when they’re put on or taken off by the technician. 
The machine only records the EKG. It doesn’t send electricity into the body. There’s no pain or risk 
associated with having an electrocardiogram. 
 
15.11.2 Echocardiogram: This test involves holding a small probe against the subject’s chest wall to obtain 
pictures of the heart. The probe uses sound waves to detect the structures of the heart and does not involve 
any radiation. It has no known side effects. 
 
15.12 Safety Considerations for HERV-E TCR transduced CD8+/CD34+ enriched T cell 
The risks of the infusion of large numbers of retrovirally modified tumor reactive T-cells fall into 3 general 
categories. The first category of toxicity is cytokine-release-type toxicities such as high fevers, tachycardia, 
transient abnormalities of liver function, and hypotension. These cytokine-release-type toxicities have been 
detected rarely in other clinical trials of TCR T cells during the first 2 weeks after modified tumor reactive 
T-cells are infused.  
 
The second possible category of toxicity is direct damage to normal tissues by the retrovirally modified 
tumor reactive T-cells. This could happen because of unexpected cross-reactivity of the anti-HERV-E TCR 
with proteins other than HERV-E in vivo. This trial will be the first to target an antigen derived from a 
HERV-E, so cross-reactivity with normal proteins is not inconceivable. We have performed extensive 
testing of HERV-E expression by RT-PCR of cells derived from a variety of human tissues and other 
malignant cell lines and we have not seen expression of this HERV-E in these specimens. Rooney et al. 
performed extensive testing of ERV-4 (also called HERV-E) by RNA-Seq-derived with minimal to 
undetectable expression in normal tissues or other malignancies and elevated expression in clear cell RCC 
samples.98 Amino acid scanning approach also was performed that demonstrated no known naturally 
occurring peptides with high sequence homology to CT-RCC-1 making alternative target peptides for the 
HERV-E A11 TCR unlikely (Table 9).  
 
Other potential type of toxicities that can occur in a clinical trial of T-cell gene therapies include occurrence 
of accidental exposure to the agent, replication-competent retrovirus or transformation of T cells caused by 
insertional mutagenesis. The transgenes expressed by the HERV-E/CD34t SAMEN retroviral vector 
integrate stably into the genome of cells infected with the GALV pseudotyped retrovirus.  Given the CD34 
selection, the T cell cultures produced are highly enriched for transduced T cells. The transduction 

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conditions were developed to limit copy number to an average of <5 integrated copies per cell as mandated 
by the FDA (usually between 1 and 2 copies per cell).  Despite the low copy number, there are potential 
risks of using the retroviral vector and the transduced T cells which are as follows: a) Accidental exposure 
to the agent by contact with the skin, inhalation, injection or through a puncture could result in inflammation 
or an immune reaction to the virus or other components in the virus preparation, b) insertion of the provirus 
into cells of the host at a point in genome where it can disrupt normal cell function leading to clonal 
expansion or malignant transformation, and/or c) recombination in the host cells between the virus and an 
defective endogenous retrovirus in the host cell leading to replication competent virus.  
 
While these are formal possibilities, the risks are considered to be small. In the case of accidental exposure 
to the agent, appropriate blood borne pathogen precautions will be adopted by NIH staff.  Although the risk 
of insertional mutagenesis is a known possibility using retroviral vectors, this has only been observed in 
the setting of infants treated for XSCID, WAS and X-CGD using retroviral vector-mediated gene transfer 
into CD34+ bone marrow cells. Although continued follow-up of all gene therapy patients will be required, 
data suggest that administration of retrovirally transduced mature T cells is a safe procedure. While the risk 
of insertional mutagenesis is extremely low, the proposed protocol follows all current FDA guidelines 
regarding testing and follow up of patients receiving gene transduced cells. Experience at the NIH Clinical 
Center and other centers treating patients with advanced cancers with genetically engineered T cells 
modified using retroviral vectors indicates that these cells do not have a significant risk of malignant 
transformation in this setting. 
 
Table 9. 
 
In summary, this protocol is designed with a dose escalation design with the hope that off target toxicities 
related to TCR transduced T-cells will be unlikely to occur because 1) the TCR targets a foreign viral 
protein that are not expressed in healthy tissues, so there should be a low chance of toxicity from TCR 
transduced T-cells targeting healthy tissues that unexpectedly expresses the target, 2) the TCR came from 
a human who did not manifest any symptoms of autoimmunity 3) the TCR came from a human and therefore 
has been selected in thymus to not have autoreactivity, so autoimmunity is unlikely and 4) the TCR 
specificity has not been altered by affinity enhancement making it less likely that cross reactivity against 
normal human proteins has been introduced. 
15.13 Risks in Relation to Benefit 
Clinically, the approach is ethically acceptable because we are targeting a patient group with incurable 
metastatic cancers or hematological malignancies, which have become refractory to conventional therapy.  
 
The risk of death due to complication from the protocol therapy is justified by the anticipated benefit of 
potentially eradicating the subject’s underlying metastatic disease or hematologic malignancy.  
 
BLAST search for peptides with at least 6 (or at least 5 identical + 1 conservative change) amino acids shared with CT-RCC-1 
peptide sequence (ATFLGSKTWK). Capital/Underlined = amino acid identical to CT-RCC-1 peptide Capital/Not Underlined = 
conservative change. 

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16. INFORMED CONSENT PROCESSES AND PROCEDURES 
Informed consent will be conducted following OHSRP Policy 301- Informed Consent. 
An IRB-approved consent form will be provided to the participant electronically or by hard copy for review 
prior to consenting. The initial consent process as well as re-consent, when required, may take place in 
person or remotely (e.g., via telephone or other NIH approved platforms). The investigational nature and 
objectives of this trial, the procedures, and their attendant risks and discomforts and potential benefits will 
be carefully explained to the participant in a private setting. The participant will be given as much time as 
they need to review the document and to consult with their family, friends, and personal health care 
providers. In addition, a study team member will be available to answer any questions. 
A signed and dated informed consent document will be obtained by any investigator authorized to consent 
(See Key Study Personnel Page) prior to entry onto the study. Consent may be obtained with required 
signatures on the hard copy of the consent  or on the electronic document.   
When a document that is in electronic format is used for obtaining  consent, this study may use the iMed 
platform which is 21 CFR, Part 11 compliant, to obtain the required signatures. 
During the consent process, participants and investigators may view the same approved consent document 
simultaneously when participant is being consented in person at the Clinical Center or both may view 
individual copies of the approved consent document on screens in their respective locations 
remotely. Signatures may be obtained either by both directly signing on the device that the consenting 
investigator is using (when in person) or through iMed Mobile Signature Capture  (remotely) which allows 
texting or emailing a link to the participant. That link allows the participant to review the consent, then 
proceed to sign on the device they are using. 
Whether hard copy or electronic consent document, both the investigator and the participant will sign the 
document with a hand signature using a pen, finger, stylus, or mouse. 
When done remotely, if the participant prefers to sign a hard copy, they may be instructed to sign and date 
the consent document during the discussion and mail, secure email or fax the signed document to the 
consenting investigator. 
Whether in person or remotely, the privacy of the participant will be maintained. 
Finally, the fully signed informed consent document will be stored in the electronic medical record, and the 
participant will receive a copy of the signed informed consent document.  
 
At any time during participation in the protocol that new information becomes available relating to risks, 
adverse events, or toxicities, this information will be provided orally or in writing to all enrolled or 
prospective subject participants.  Documentation will be provided to the IRB and if necessary, the informed 
consent amended to reflect relevant information. 
 
A patent application on the HERV-E TCR sequence developed in our lab at the NHLBI was filed with the 
U.S Patent and Trademark Office. Because Dr. Childs is listed in the patent application, he will not be 
allowed to consent any subjects and will leave the room during the consent process. 
 
17. PHARMACEUTICALS 
 
T-CELL PRODUCT 
Cell preparation (anti- HERV-E transduced PBL) 

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The procedure for expanding the human PBL and the certificate of analysis are similar to those approved 
by the food and drug administration and used at Loyola University Chicago in ongoing protocols evaluating 
cell therapy. The PBL will be transduced with retroviral supernatant containing the alpha chain and beta 
chain genes of the Anti-HERV-E.  Note: Penicillin, Streptomycin, and gentamycin will not be used in the 
manufacture of products for patients.  
 
Retroviral vector containing the anti-HERV-E gene 
The retroviral vector supernatant encoding a T cell receptor directed against HERV-E was prepared and 
preserved following cGMP conditions in the Cellular Therapy Center at Loyola University Chicago. The 
batch of virus generated was cGMP qualified by Indiana University Vector Production Facility located in 
Indianapolis, IN.  
 
The retroviral vector, HERV-E TCR CD34t 7G1, consists of 7,948 bps including the 5' LTR from the 
murine stem cell virus (promoter), packaging signal including the splicing donor (SD), splicing acceptor 
sites, alpha chain and beta chain genes of the anti-HERV-E TCR. The HERV-E TCR is also linked to a 
truncated CD34 chain (CD34t). We used PCR to link the  and  TCR chains together with a P2A self-
cleavage sequence to facilitate high and equal expression of both TCR chains. The HERV-E TCR is linked 
to a CD34t by a different self-cleavage sequence (T2A) resulting in a final transgene cassette consisting of 
TCR  -TCR - CD43t. This CD34t cassette lacks its intracellular signaling domain so it does not function 
in cells.  The physical titer of the clinical vector will be measured in transducing units per mL following a 
titration on human PBL.  The viral supernatant was aliquoted, snap frozen at –80 °C and is stored in a secure 
–80 °C freezer within the Cellular Therapy Center at Loyola University Chicago. There will be no re-use 
of the same unit of supernatant for different patients. Retroviral titer has been shown to be stable after 
immediate thawing and immediate administration (coating the tissue culture wells previously coated with 
Retronectin). Handling of the vector should follow the guidelines of Biosafety Level-2 (BSL-2).   The 
specific guidelines for Biosafety Level-2 (BSL-2) can be viewed at http://bmbl.od.nih.gov/sect3bsl2.htm. 
 
The SAMEN retroviral backbone can only produce infectious virus when introduced into a retroviral 
packaging cell line which contains the gag, pol, and env genes. The SAMEN vector containing the HERV-
E TCR and CD34t cassette has been introduced into the PG13 retroviral producer cell line. PG13 uses the 
GALV envelope which has a host range of human and primate cells only. A PG13 clone was isolated that 
has high transduction efficiency. It was expanded in the GMP laboratory and a Master cell bank of 212 
vials of 5 million cells per vial was established and cryopreserved. 6 liters of retroviral supernatant was 
generated from a vial from the Master cell bank and cryopreserved in 40 cc aliquots then frozen. The Master 
cell bank is stored in the liquid nitrogen freezer within the GMP facility and the cGMP qualified retroviral 
supernatant is stored in a locked -80oC freezer also in the GMP facility. The virus titer or the retroviral 
supernatant was measured to be 7.4 x 10^5 TU/ml. The Master cell bank and retrovirus was sent in the Fall 
of 2016 to the Indiana University Vector Production Facility for GMP qualification (see attached COA for 
the Master cell bank or testing results for the virus). More specifically, 28 vials of Master cell bank and 822 
cc of the virus supernatant (taken at the time of packaging) were sent to Indiana for GMP qualification.  
 
CYCLOPHOSPHAMIDE  
(Refer to FDA-approved package insert for complete product information) 
Supply: Commercially available 
Product Description: Cyclophosphamide is a nitrogen mustard-derivative alkylating agent. Following 
conversion to active metabolites in the liver, cyclophosphamide functions as an alkylating agent; the drug 
also possesses potent immunosuppressive activity. The serum half-life after IV administration ranges from 

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3 to 12 hours; the drug and/or its metabolites can be detected in the serum for up to 72 hours after 
administration. 
Preparation: Reconstitute Cyclophosphamide with 0.9% Sodium Chloride Injection, USP only, using the 
volumes listed below in Table 10. Gently swirl the vial to dissolve the drug completely. Do not use Sterile 
Water for Injection, USP because it results in a hypotonic solution and should not be injected directly. 
Storage and Stability: Following reconstitution as directed with sterile water for injection, 
cyclophosphamide is stable for 24 hours at room temperature or 6 days when kept at 2 to 8oC. 
Administration: It will be diluted in 100 mL D5W and infused. The dose will be based on the patient’s 
actual weight for all subjects except those who meet criteria for morbid obesity (i.e. BMI > 35); for morbidly 
obese subjects, cyclophosphamide dosing will be based on the practical weight. For definition of practical 
weight, refer to NHLBI Supportive Care Weight Based Dosing Guidelines.  
 
Table 10. 
Reconstitution for Direct Intravenous Injection 
Strength 
Volume of 0,9% Sodium 
Chloride 
Cyclophosphamide 
concentration 
500 mg 
25 mL 
20 mg per ml 
1 gram 
50 mL 
2 grams 
100 mL 
FLUDARABINE 
(Refer to FDA-approved package insert for complete product information) 
Supply: Commercially available 
Product description: Fludarabine phosphate is commercially available as both a lyophilized powder for  
injection in vials containing 50 mg of fludarabine phosphate with mannitol 50 mg and sodium hydroxide 
for pH adjustment and a solution for injection in 2 mL vials containing 50 mg of fludarabine phosphate (25  
mg/mL of fludarabine) with 25 mg/mL mannitol and sodium hydroxide for pH adjustment. 
Preparation: Fludarabine lyophilized powder for injection should be reconstituted with 2 mL of sterile 
water  
for injection, up to a concentration of 25 mg/mL. The prescribed dose of fludarabine should be diluted in 
100 mL of either 0.9% sodium chloride or 5% dextrose in water for intravenous administration. 
Storage and Stability: Fludarabine vials should be stored under refrigeration between 2-8o C (36-46oF). 
Reconstituted fludarabine phosphate is chemically and physically stable for 24 hours at room temperature 
or if refrigerated. The manufacturer recommends use of either the reconstituted powder for injection or the 
solution for injection (once diluted for administration) within 8 hours because neither product contains an 
antimicrobial preservative.  
Administration: The prescribed dose of fludarabine should be diluted in 100 mL of either 0.9% sodium  
chloride or 5% dextrose in water for intravenous administration. The dose will be based on the patient’s 
actual weight for all subjects except those who meet criteria for morbid obesity (i.e. BMI > 35); for morbidly 
obese subjects, fludarabine dosing will be based on the practical weight. For definition of practical weight, 
refer to NHLBI Supportive Care Weight Based Dosing Guidelines.   
 
INTERLEUKIN-2 (Aldesleukin, Proleukin®, IL-2)  
(Refer to FDA-approved package insert for complete product information) 
Supply: Commercially available 
Product description: Interleukin-2 is available as a parenteral formulation. It is supplied in individually 
boxed single-use vials. Each vial contains 22 x 106 IU (22 million IU) of interleukin-2 as a sterile, white to 
off-white lyophilized cake plus 50 mg mannitol and 0.18 mg sodium dodecyl sulfate, buffered with 
approximately 0.17 mg monobasic and 0.89 mg dibasic sodium phosphate to a pH of 7.5 (range 7.2 to 7.8).  

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Interleukin-2 vials contain a sterile, white to off-white, preservative free, lyophilized powder that requires 
reconstitution. 
Preparation: Reconstitute each vial aseptically with 1.2 mL of sterile water for injection, USP. When 
reconstituted as directed, each milliliter (mL) contains 18 million IU of interleukin-2 (18 million IU per 1 
mL). The resulting solution should be clear, colorless to slightly yellow liquid. The vial is for single-use 
only and any unused portion should be discarded. During reconstitution, the sterile water for injection, USP 
should be directed at the side of the vial and the contents gently swirled to avoid excess foaming. DO NOT 
SHAKE. Reconstitution with bacteriostatic water for injection, USP or 0.9% sodium chloride injection, 
USP should be avoided because of increased aggregation.  
Storage and stability: Before and after reconstitution store in a refrigerator at 2o to 8o C (36O to 46O F). 
Do not freeze. Following reconstitution with sterile water for injection, USP, interleukin-2 reconstituted 
vials should be used within 24 hours. 
Administration: The dosage will be calculated based on total body weight. The final dilution of IL-2 will 
be administered as an inpatient.  
 
Note: None of the intravenous drugs used in this protocol will not be stored longer than 24 hours. 
18. APPENDICES 
A. ECOG Performance Status 
GRADE 
 
ECOG PERFORMANCE STATUS 
 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a 
light or sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all selfcare but unable to carry out any work activities; up and 
about more than 50% of waking hours 
3 
Capable of only limited selfcare; confined to bed or chair more than 50% of waking hours 
4 
Completely disabled; cannot carry on any selfcare; totally confined to bed or chair 
5 
Dead 
 

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B. Clinical management recommendations for IL-2 therapy 
 
Issue 
Considerations 
Management 
Venous Access 
Central line (for possible 
vasopressors) 
Double or triple lumen 
Power inject and large volume 
capacity 
Minimize catheter associated 
infection 
Typical 
PICC line placement 
Remove temporary lines at 
end of cycle 
Variations 
Broviac/Hickman catheter 
Subclavian/IJ catheter 
IV Fluids 
Maintenance of volume with CLS 
Boluses for blood pressure support 
Administration of drugs 
Replacement of electrolytes 
IL-2 only compatible with D5W 
Typical 
D5NS or D5LR 10 ml – 125 
ml/hr 
PRN KCL, HCO3, Mg 
replacement 
Variations 
D5W, NS, 0.45% NaCl 
Infections 
No active infections  
IV catheter likeliest source  
Avoid unnecessary in-dwelling 
catheters  
 
Typical 
Prevention Gram + 
prophylactic antibiotic 
Variations 
Expanded coverage per 
hospital 
Chills/rigors  
 
 
Fever  
 
 
 
Constitutional  
symptoms 
Chills and rigors occur 1–2 hours 
after IL-2 
 
 
Fever is common 2–4 hours after 
IL-2 
 
 
 
Muscle joint aches continuous and 
progressive during IL-2 treatment 
Fever-Typical 
Prophylaxis 
 
Acetaminophen 650 mg 30 
min pre-dose, 
q 4–6 hrs and prn 
Indomethacin 25 mg q 6–8 
hrs 
 
Fever-Variation 
Naproxen 
Ibuprofen 
Chills-Typical 
Meperidine 25 mg IV q 15 
min PRN 
Morphine 2-4 mg IV q 15 
min PRN 
Nausea/vomiting 
Episodic occurrence throughout 
therapy 
Nausea/Vomiting 
Typical-Prophylaxis 
Ondansetron 0.15 mg/Kg q 
8 hours 
 
Variations 
Granisetron 1 mg daily 
Ondansetron at longer 
interval 

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Compazine 10 mg PO q 6 
hours 
Use the antinausea  agents 
PRN 
Epigastric distress 
Gastritis induced by stress, 
medications 
Typical 
H2 blocker prophylaxis 
Variation 
PPI prophylaxis 
Mucositis/stomatitis 
Progressive with continued 
treatment 
Typical 
No prophylaxis 
Oncology mouthwash 
Diarrhea 
Can be profuse and increase with 
therapy 
5HT-3 antagonist anti-emetic 
prophylaxis may have positively 
impacted. 
Typical 
Loperamide 
Lomotil (diphenoxylate 
hydrochloride and atropine 
sulfate) 
Narcotic (Opium Tincture) 
Break between IL-2 doses 
5HT-3 antagonist 
prophylaxis 
Patient monitoring 
I&O, Weight 
Blood pressure, pulse, respirations, 
temperature 
Blood work 
EKG 
O2 saturation 
Mental status examination 
Per shift and daily 
Q 2-4 hours 
Daily 
Continuous cardiac 
monitoring 
Q 2-4 hours 
Q 8 hours 
Increase frequency as 
needed 
IL-2 dose and administration 
- 
IL-2 incompatible with salt 
solutions 
- 
Dissolve in sterile water for 
injection. Dilute in 50 cc D5W 
- 
Stop infusion, flush IV tubing 
with 50 cc D5W before and 
after each dose.  
 
Hypotension 
Maintain systolic BP 80-90 mmHg 
 
Blood pressure nadirs 4-6 hrs each 
dose with diminished recovery 
with cumulative dosing 
 
 
Prior to each dose anticipate ability 
to respond to next nadir 
 
Progressive refractoriness to 
support measures 
Fluid boluses, 250-500 ml 
NS 
2xday 
Increase maintenance fluid 
rate  
Phenylephrine  0.1-0.4 
mcg/Kg/min  
Hold next dose 
Discontinue IL-2 
Variations: 
Dopamine 1-6 g/Kg/min 
Pressors with minimal fluids 

72 
18-H-0012 
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Richard Childs, MD 
 
 
Fluids with pressors 
Cardiac arrhythmias 
Sinus tachycardia 
- 
Common and progresses 
over a cycle 
- 
Peaks 2-4 hours after dose 
with fever and hypotension 
- 
Must resolve prior to next 
dose 
 
Troponin elevation 
 
 
 
Supraventricular tachycardia, 
atrial fibrillation 
Less common 
 
Atrial fibrillation 
Ventricular tachycardia 
Manage BP and fever 
 
 
 
 
 
Must stop Il- and will need 
an ECHO before next cycle 
of IL2 to rule out 
myocardial dysfunction.  
Future IL-2 may be 
considered if the ECHO is 
normal 
 
Medical Conversion 
Diltiazem as needed 
 
 
Digoxin 
Medical Conversion 
Acute treatment 
Discontinue IL-2 
Renal function 
 
Oliguria 
 
 
 
Rising creatinine 
 
 
Urine output and creatinine 
resolve after discontinuation of 
IL-2 
 
If only one kidney always consider 
obstruction of ureter 
Typical 
Output less than 50-100 
cc/8hours: Fluid bolus. 
If no improvement within 8 
hours will hold IL-2 dose 
Consider to add Dopamine 
after 1-1.5 L of fluid boluses 
 
Creatinine >3-4 
Stop NSAIDS and 
nephrotoxic antibiotics 
 
Hold overnight dose 
If morning creatinine 
improved continue 
 
Variations 
Dopamine 1-6 mcg/Kg/min 
Furosemide 

73 
18-H-0012 
May 11, 2023 (Amendment T) 
Richard Childs, MD 
 
 
Pulmonary 
Tachypnea/Dyspnea 
Diagnose etiology and treat 
 
Hypoxic causes-Fluid overload, 
capillary leak, bronchospasm 
 
Non hypoxic causes 
 
Anxiety, fever, acidosis 
 
Maintain O2 sat > 92-95% 
Typical 
Oxygen 2-4 L nasal cannula, 
increasing up to 35% 
rebreather 
 
Reassurance or sedative for 
anxiety, treat broncospasm 
or acidosis if appropriate  
 
Hold IL-2 dose if O2 sat < 
95% 
 
Variation 
Furosemide 
Bronchodilators 
Monitor bicarbonate 
Peripheral Edema 
Expect to gain 5-10% body weight 
 
Treat edema symptomatically 
 
Entrapment of peripheral nerves in 
upper extremity may need therapy 
Elevation, compression, 
limit fluid support in 
subsequent cycles 
Diuretics upon conclusion 
of IL-2 dosing are not 
necessary but may speed 
process 
Treat peripheral nerve pain 
Neurotoxicity 
 
Protean manifestations  
Gradual onset with sudden 
worsening near end of cycle 
May persist after cessation of 
therapy  
Delusions, Visual hallucinations  
Typical 
Formal neurologic checks 
Enlist family evaluation 
 
Lorazepam and Haloperidol 
Hold IL-2 liberally for 
suspected neurotoxicity 
Warn patient of vivid 
dreams after discharge 
 
Dermatologic 
 
Rash, erythema, dry desquamation  
Pruritus  
Moist dermatitis  
 
Typical 
Emollient lotions and 
creams Oatmeal bath 
Antihistamines 
Hold IL-2 dose 
Variations 
Gabapentin 
Naloxone 
Narcotics 
Non-alcohol, no steroid 
topicals 
Metabolic 
Hypomagnesemia, hypocalcemia 
(but low albumin – so corrected 
may be WNL), Hypokalemia. 
 
Daily electrolyte panels 
 
 
 

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Richard Childs, MD 
 
 
Acidosis due to diarrhea, 
hypoperfusion  
 
Hypothyroidism a slow onset 
problem  
 
Correct electrolytes 
cautiously prn 
 
Magnesium and HCO3, 
particularly if diarrhea a 
problem 
HCO3 < 18 meq/L hold 
dose of IL-2 
Check TSH at beginning of 
cycle 
RL as support fluids may 
decrease need for HCO3 
Hepatic 
↑Bilirubin (up to 10)  
↓Albumin (down to 1.8)  
↑Hepatic aminotransferases  
Monitor daily 
No intervention except if 
SGOT SGPT are >5x 
Resolves spontaneously 
Stop acetaminophen if 
bilirubin > 5 
Hematologic 
↓Platelets  
Lymphs ↓during IL-2, ↑post 
therapy  
Eosinophils progressively ↑with 
several cycles  
Transfuse platelets if < 
20,000/uL 
Other abnormalities require 
no intervention 
Significant anemia needs 
evaluation for cause 
Endocrine 
Hypothyroidism – slow onset after 
completion of treatment 
 
Requires serial monitoring 
Check TFTs at beginning of 
cycle and monitor 
Thyroid function test with 
subsequent visits 
 
 
Criteria for holding and stopping IL2 (see guidance below for when to delay or stop IL-2*) 
 
System 
Relative Criteria 
Absolute Criteria 
Supportive Measures  
Cardiovascular 
Sinus tachycardia 
100–130 BPM 
 
(Persistent at the time 
of dosing after 
correcting 
hypotension, fever, 
and tachycardia and 
discontinuing 
dopamine) 
Sinus tachycardia > 130 
BPM 
ECG indications of 
ischemia 
Atrial fibrillation 
Supraventricular 
tachycardia 
Ventricular arrhythmias 
Elevated CKB-MB 
isoenzyme of troponin 
levels 
Correction of fluid and 
electrolyte imbalances; 
chemical conversion or 
electrical conversion 
therapy.  
Dermatologic 
Moist desquamation, 
Rash, Erythema, 
Pruritus 
 
Hydroxyzine 10-20mg 
po q6, prn; 
Diphenhydramine HCL 
25-50mg, po, q4h, prn. 

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Richard Childs, MD 
 
 
Gastrointestinal 
Diarrhea, 1000 
mL/8hours 
 
Ileus/abdominal 
distention 
Bilirubin > 7 mg/dL 
Diarrhea 1000 mL/8 hours 
x 2 
Vomiting not responsive to 
medication 
Severe abdominal 
distention affecting 
breathing 
Severe, unrelenting 
abdominal pain 
Loperamide 2mg, po, 
q3h, prn; Diphenoxylate 
HCL 2.5mg and atropine 
sulfate 25mcg, po, q3h, 
prn.  
Hemodynamic 
Maximum 
Phenylephrine 1–1.5 
mcg/kg/min  
Minimum 
Phenylephrine > 0.5 
mcg/kg/min  
 
(To maintain 
acceptable BP and 
pulse criteria) 
Maximum Phenylephrine 
1.5-2.0 mcg/kg/min 
Minimum Phenylephrine > 
0.8 mcg/kg/min 
Fluid resuscitation  
Vasopressor support 
Hemorrhagic 
Sputum, emesis, or 
stool heme-positive 
Platelets 30,000-
50,000/mm3 
Frank blood in sputum, 
emesis, or stool 
Platelets < 30,000 mm3 
Transfusion with 
platelets  
Infectious/Fever 
 
Strong clinical suspicion or 
documented 
Acetaminophen 650mg, 
po, q4-6h 
Musculoskeletal 
Weight gain > 15% 
Extreme tightness  
 
Extreme paresthesias 
Bedrest interspersed 
with activity. 
Diuretics prn.  
Neurologic 
Vivid dreams 
Mild anxiety 
Emotional lability 
Hallucinations 
Persistent crying 
Mental status changes not 
reversible in 2 hours 
Unable to subtract serial “7 
s” or spell “WORLD” 
backward 
Disorientation 
Observation 
Pulmonary 
Resting shortness of 
breath  
3–4 L O2 by nasal 
cannula for O2 
saturation ≥ 95% 
Rales 1/3 up chest  
> 4 L O2 by nasal cannula 
or 40% by mask to 
maintain ≥ 95% O2 
saturation 
Endotracheal Intubation 
Moist rales 1/2 up chest 
Pleural effusion requiring 
tap or chest tube while on 
therapy 
Stop Il-2 administration 
if ventilatory support is 
required.  
Renal 
Urine output 80–160 
mL/8 hours 
Urine output 10-20 
mL/hour 
Urine < 80 mL/8 hours 
Urine output < 10 ml/hour 
Creatinine ≥ 3 mg/dL 
HCO3 ≤ 18 meq/dL 
Fluid boluses or 
dopamine at renal doses. 
Dialysis for renal 
failure. 

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18-H-0012 
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Richard Childs, MD 
 
 
Creatinine 2.5-2.9 
mg/dL  
 
* When to delay, or discontinue interleukin-2 
 
OBSERVATION CATEGORY 
ACTION 
Any relative criteria 
Initiate corrective measure ± delay IL-2 
 3 relative criteria 
Initiate corrective measures, delay IL-2 
Stop IL-2 if not easily reversible 
Any absolute criteria 
Initiate corrective measure, delay IL-2 
Stop IL-2 if not easily reversible 
 
 
 
 

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Richard Childs, MD 
 
 
C. GUIDELINES FOR MANAGEMENT OF COMMON TOXICITIES THAT OCCUR AFTER 
T-CELL INFUSIONS 
 
Infusions of TCRs T cells are rarely complicated by significant acute toxicities in the first 2 to 3 weeks after 
the infusion. In many cases the toxicities correlate with serum inflammatory cytokine levels.144 
 
Note these are guidelines that might require modification based on clinical circumstances of each patient, 
and failure to exactly follow these guidelines is not a protocol deviation or violation. 
 
For acute infusion reactions, the following guideline is recommended to be followed: 
 
Acute Infusion Reactions PRN supportive care: 
• 
Stop infusion 
• 
If patient develops symptoms consistent with acute infusional, infusion of HERV-E TCR 
Transduced Autologous T Cells will be stopped. 
• 
0.9% Sodium Chloride Injection, by intravenous infusion at 500ml/hour, one time dose as needed 
for reaction with T-cell product. Infuse over 30 minutes. Give fluid bolus upon verbal order of 
provider (MD, NP or PA) for infusion reaction. 
• 
Acetaminophen-325 mg tablet. Dose: 650mg (two tablets) by mouth one time dose as needed for 
febrile infusion reaction with HERV-E TCR Transduced Autologous T Cells. Given upon verbal 
order of provider (MD, NP or PA) for febrile infusion reaction.(Maximum adult acetaminophen 
daily dose: 3 grams/Day). 
• 
Diphenhydramine Inj-50mg/ml. Dose: 50 mg, by intravenous push, one time dose as needed for 
PRN infusion reaction with HERV-E TCR Transduced Autologous T Cells.  
• 
Hydrocortisone Inj. Dose 100 mg by intravenous push, one time dose as needed for sever infusion 
reaction (grade 4 infusion reaction by CTCAE 5.0 or when clinically indicated) with HERV-E TCR 
Transduced Autologous T Cells. Give upon verbal order of provider (MD, NP or PA) for infusion 
reaction. 
• 
Albuterol Nebulization Solution-0.083% (2.5mg/3ml vial). Dose:2.5 mg by nebulizer one time 
dose as needed for infusion reaction with shortness of breath or wheezing with HERV-E TCR 
Transduced Autologous T Cells. 
• 
Epinephrine Auto-Injection 0.3 mg (EpiPen). Dose 0.3 mg by intramuscular injection as needed for 
severe infusion reaction (grade 4 infusion reaction by CTCAE 5.0 or when clinically indicated) 
with HERV-E TCR Transduced Autologous T Cells. Give upon verbal order of provider (MD, NP 
or PA) for infusion reaction. 
• 
HERV-E TCR Transduced Autologous T Cell infusion will be resumed at PI or AI discretion. 
 
For all other reactions the following guidance is recommended to be followed (Note: Administration of 
corticosteroids should be avoided if at all possible to avoid killing or impairing the function of the infused 
TCR T-cells. 
 
1. All patients with significant malignancy burdens and without a contradiction such as allergy should be 
started on allopurinol at the time of the start of the chemotherapy conditioning regimen or 1 day before 
the TCR T-cell infusion. The suggested allopurinol dose is 200 to 300 mg/day with a loading dose of 
300 to 400 mg.  
2. Vital signs should be checked a minimum of every 4 hours during hospitalization. Increasing the time 
interval between vital sign checks for patient convenience or other reasons should be avoided.  
3. Strict ins and outs should be recorded on all patients.  
4. As a minimum, keep hemoglobin greater than 8.0 g/dL and platelets greater than 20K/microliter. 

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18-H-0012 
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Richard Childs, MD 
 
 
5. Administer fresh frozen plasma (FFP) for a PTT 1.5-fold or more above the upper limit of normal.  
6. For patients with an increased PTT, check the fibrinogen level and keep the fibrinogen level above 100 
mg/dL with cryoprecipitate. 
7. Fevers should be treated with acetaminophen and comfort measures. NSAIDs and corticosteroids 
should be avoided. 
8. Patients with a heart rate persistently higher than 115/minute and fever should have vital signs checked 
every 2 hours.  
9. Patients who are neutropenic and febrile should be receiving broad-spectrum antibiotics as per 
institutional guidelines 
10. If the absolute neutrophil count becomes less than 500/microliter, Filgrastim  or filgrastim-sndz may 
be initiated at a dose of 300 micrograms daily for patients under 70 kg in weight and a dose of 480 
micrograms daily for patients over 70 kg in weight only in patients with absolute neutrophil counts less 
than 500/microliter. Filgrastim or filgrastim-sndz will be discontinued as soon as the absolute 
neutrophil count recovers to 1500/microliter. 
11. Hypotension is a common toxicity requiring intensive care unit (ICU) admission. In general patients 
should be kept well-hydrated. Maintenance I.V. fluids (normal saline (NS) should be started on most 
patients with high fevers especially if oral intake is poor or the patient has tachycardia. I.V. fluids are 
not necessary for patients with good oral intake and mild fevers. For patients who are not having 
hypotension or tumor lysis syndrome, a generally even fluid balance should be strived for after allowing 
for insensible fluid losses in patients with high fevers. The baseline systolic blood pressure is defined 
for this protocol as the average of all systolic blood pressure readings obtained during the 24 hours 
prior to the TCR T-cell infusion. The first treatment for hypotension is administration of IV NS boluses.  
• 
Patients with a systolic blood pressure that is less than 80% of their baseline blood pressure and 
less than 100 mm Hg should receive a 1 L NS bolus.  
• 
Patients with a systolic blood pressure less than 85 mm Hg should receive a 1 L NS bolus regardless 
of baseline blood pressure.  
 
These I.V. fluid management suggestions may need to be modified based on the clinical characteristics of 
individual patients such as pulmonary status, cardiac function, edema and other factors. 
 
12. Patients receiving more than 1 fluid bolus for hypotension should have a stat EKG and troponin, and a 
cardiac echocardiogram within 24 hours of the second fluid bolus.  
13. Patients should be transferred to the ICU under these circumstances. Patients not meeting these criteria 
could also require ICU admission at the discretion of the clinical team caring for the patient.  
• 
Systolic blood pressure less than 75% the patient’s baseline blood pressure and less than 100 mm 
Hg after administration of a 1L NS bolus.  
• 
Anytime the systolic blood pressure is less than 90 mm Hg after a 1L NS bolus if 90 mm Hg is less 
than the patient’s baseline systolic blood pressure. 
• 
Continuous tachycardia with a heart rate higher than 125 beats per minute on at least 2 occasions 
separated by 4 hours.  
• 
Oxygen requirement of more than a 4L standard nasal cannula 
• 
Greater than grade 2 neurological toxicity 
 
14. All patients transferred to the ICU for hypotension or tachycardia should have a stat EKG and a cardiac 
echocardiogram within 24 hours of the time of transfer. 
15. Patients with hypotension not responding to IV fluid resuscitation should be started on norepinephrine 
at doses called for by standard ICU guidelines.* 
16. Patients should have a cardiac echocardiogram and an EKG within 12 hours of starting norepinephrine. 

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18-H-0012 
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Richard Childs, MD 
 
 
17. Patients in the ICU should get twice-daily labs (CBC with differential, acute care panel, mineral panel, 
hepatic panel, uric acid, LDH. Patients in the ICU should also get a daily troponin level).  
18. Anecdotal evidence suggests that the IL-6 receptor blocker Tocilizumab can be an effective treatment 
for cytokine-release syndrome toxicities after TCR T-cell infusions. Tocilizumab may be administered 
under the following circumstances if the listed disorders are thought to be due to cytokine release from 
TCR T cells. Tocilizumab is administered at a dose of 4 mg/kg infused IV over 1 hour (dose should not 
exceed 800 mg). 
• 
Left ventricular ejection fraction less than 45% by echocardiogram 
• 
Creatinine greater than 2. 0- fold higher than the most recent level prior to TCR T-cell infusion 
• 
Norepinephrine requirement >3 micrograms/minute for 36 hours since the first administration of 
norepinephrine even if norepinephrine administration was not continuous.  
• 
Systolic blood pressure of 90 mm Hg cannot be maintained with norepinephrine.  
• 
Oxygen requirement 60% or greater fraction of inspired oxygen (FIO2) for more than 2 continuous 
hours.  
• 
Dyspnea that is severe enough to potentially require mechanical ventilation.  
• 
PTT or INR>2x upper limit of normal 
• 
Clinically-significant bleeding  
• 
Creatine kinase greater than 5x upper limit of normal for greater than 2 days 20. 
 
*NOTE: In case of diagnosis of Cytokine Release Syndrome, we will consider high-dose of vasopressors 
if patient receives (all doses are required for  3 hours):141 
- Norepinephrine monotherapy  20g/Kg/min  
- Dopamine monotherapy  10g/Kg/min 
- Phenylephrine monotherapy  200g/Kg/min 
- Epinephrine monotherapy  10g/Kg/min 
- If on vasopressin: Vasopressin + norepinephrine equivalent of  10g/Kg/min 
- If on combination vasopressors (not vasopressin): norepinephrine equivalent of  20g/Kg/min 
 
19. THERE IS NO EVIDENCE THAT TOCILIZUMAB HELPS NEUROLOGICAL TOXICITY, SO IT 
SHOULD NOT BE ADMINISTERED FOR THIS PURPOSE. 
20. If no improvement in hypotension or tachycardia occurs within 6 hours of Tocilizumab infusion, 
consider other agents such as methylprednisolone 1 mg/kg every 12 hours or etanercept. 
21. Avoid meperidine due to seizure risk.  
22. All patients with grade 2 or greater neurological toxicities should get a neurology consult. 
23. The following patients should receive dexamethasone 10 mg intravenously every 6 hours until the 
toxicities improve to Grade 1 or resolve or until at least 8 doses of dexamethasone have been given. 
Note: for seizures administer standard seizure therapies in addition to dexamethasone. 
1. Patients with Grade 3 or 4 neurological toxicities except that dexamethasone is not recommended 
for isolated Grade 3 headaches.  
2. Any generalized seizure. 
24. In any patient with a cardiac left ventricular ejection fraction of 30% or less start methylprednisolone 
100 mg IV every 6 hours. 
25. In any patient likely to need mechanical ventilation within the next 4 hours, start methylprednisolone 
100 mg IV every 6 hours. 
 
 
 
 
 

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Richard Childs, MD 
 
 
D. For Cognitive Impaired subjects: Policy 403 
REQUIREMENTS FOR THE DETERMINATION OF AN LAR’S APPROPRIATENESS TO CONSENT 
TO RESEARCH NOT INVOLVING GREATER THAN MINIMAL RISK (CATEGORY A) AND FOR 
RESEARCH INVOLVING GREATER THAN MINIMAL RISK BUT PRESENTING THE PROSPECT OF 
DIRECT BENEFIT TO THE INDIVIDUAL SUBJECTS (CATEGORY B) 
 
First preference is #1. If not possible, go to option #2. If #2 is not possible, go to option #3.) 
 
Cognitively Impaired Adults and 
Identification of a LAR  
Requirements for Determining 
Appropriateness of LAR to Consent 
to Research at Clinical Center (CC) 
and non- CC sites  
Role of the LAR at 
all sites  
1. Adults who cannot consent and have a 
court-appointed guardian from a state that 
allows iti or a DPAii for healthcare and/or 
research participation.  
PI/designeeiii, unless the IRB designates 
an independent person(s) to perform this 
role (e.g., ACATiv if the protocol is 
taking place at the CC), must assess 
appropriateness of LAR to consent to 
research.  
 
An appropriate LAR is one who at least:  
(a) Understands that the protocol 
involves research;  
(b) Understands the risks, potential 
benefits (if any), and alternatives to the 
study; and  
(c) Has sufficient reason to believe 
participation in the study is consistent 
with the subject’s preferences and 
values.  
LAR may give 
permission for the 
research and sign 
the consent form for 
the protocol on 
behalf of the 
subject.  
2. Adults who cannot consent and who do 
not have a DPA or court-appointed 
guardian, but who are capable of 
understanding the DPA process and can 
assign a DPAv . 
3. Adults who cannot consent, who do not 
have a DPA or court-appointed guardian, 
and cannot appoint a DPA:  
 
At the CC: A person at the highest level of 
the following may consent to research 
participation if found to be appropriate: 1) 
spouse or domestic partnervi, 2) adult child, 
3) parent, 4) sibling, 5) other close relative  
 
At non-CC sites: Consult with OGC to 
identify applicable state law.  
i A court appointed guardian may only consent to enroll a subject in research if the guardian has authority to do so under the laws of the state that 
issued the guardianship order and the terms of the guardianship order. The Office of the General Counsel (OGC) should be asked to review 
guardianship orders to determine if the guardian has legal authority to consent to the subject’s participation in the research. PIs are encouraged to 
seek an OGC consultation in advance of a potential subject with a guardianship order coming to an NIH research site to enroll on a study.  
ii 
DPA means the individual holding the durable power of attorney for healthcare. Consult with OGC if concerned about the authority provided in 
a DPA.  
iii 
If the protocol is taking place at the CC, the PI’s designee may be someone on the research team or a member of ACAT. If not at the CC, the 
PI’s designee may be someone on the research team or an independent person outside of the research team if it is felt that the team does not have 
the required competencies to undertake the evaluation.  
iv 
NIH Ability to Consent Team. For definition please see Policy 403. 
 
 
 

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Richard Childs, MD 
 
 
E. Schedule of Events 
 
SCREENING AND BASELINE ASSESSMENT  
 
Screening 
  
Baseline 
PROTOCOL TIMEPOINT 
1 
  
2 
+/- days or months 
  
  
  
DAY 
  
  
<60 
DAYS 
REGISTRATION 
  
  
  
Screening consent 97-H-0041 
X 
  
  
Eligibility criteria 
 X 
  
Consent 18-H-0012 
  
  
X 
Durable Power of Attorney 
  
  
X 
High resolution HLA typing  
X 
  
  
Confirmation of diagnosis of a clear cell renal cell carcinoma  
X 
  
  
TREATMENT 
  
  
  
Recipient apheresis (venous access assessment) 
  
  
X 
Central line placement (3 Lumen IJ or PICC)  
  
  
O 
EVALUATIONS 
  
  
  
History and physical or progress note 
X 
  
X 
ECOG  
X 
  
X 
Vital signs (Temp, Pulse, BP, RR, Wt, O2 Sat) 
X 
  
X 
Chemistry labs 
  
  
  
Acute care/mineral panels 
X 
  
  
Hepatic Panel, Total Protein, LDH 
X 
  
  
Prealbumin and lipid panel 
X  
  
O 
Uric acid and CK 
 X 
  
O 
C-reactive protein 
 
 
X 
Hematology 
  
  
  
CBC with differential 
X 
  
X 
PT, PTT,  
X 
  
X 
Lymphocyte Phenotyping (TBNK, CD4) 
 X 
  
X 
DTM Blood Screening panel 
X 
 
 
Antibody screen 
  
  
  
HIV 
X 
  
  
HBV 
X 
  
  
HVC 
X 
  
  
Endocrine labs 
  
  
  
Thyroid panel (TSH, if TSH is abnormal, thenT4) 
  
  
X 
Cortisol Serum 
  
  
X 
Human Chorionic Gonadotropin/ Preg. test urine 
X 
  
X 
Research blood/ biopsies 
  
  
  
Replication Competent Retrovirus Testing  
(1 EDTA tube with 4-5 ml).  
Ship to Indiana University Vector Production Facility 
  
  
X 
 
  
Tumor biopsy (Accessible lesions) up to 3 biopsies 
  
  
O 
Enumeration of absolute # of circulating CD34+/CD8+ T-cells [30 ml of blood collected in a green 
tube]. Only for patients screened for retreatment and a second apheresis is needed 
X 
 
 
Pre-T cell product infusion Radiology tests  
  
  
  

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May 11, 2023 (Amendment T) 
Richard Childs, MD 
 
 
Chest X-ray 
 X 
  
CT scan of the chest, abdomen and pelvis with IV contrast 
 X 
  
X  
MRI abdomen/pelvis with IV gadolinium* 
X 
  
MRI brain with IV gadolinium 
 X 
  
CT of the brain with IV contrast 
 O 
  
CT neck with IV contrast 
 O 
  
PET scan (F-18 FDG PET-CT) 
  
  
X 
Bone scan (TC99 bone) and/or whole-body MRI  
 O 
  
Whole-body MRI  
  
  
O 
MRI for specific body area if metastasis present and clinically indicated 
  
  
O 
Pre-T cell product infusion tests 
  
  
  
Pulmonary function Test 
X  
  
EKG 
X 
  
2 D echo 
X  
  
X 
Sesti Mibi 
  
  
O 
G6PD deficiency screen 
  
  
X 
24 hour creatinine clearance 
O  
  
Type and Screen 
X  
  
DAT 
 
 
X 
Pre-T cell product Infusion Consults 
  
  
  
DTM: vein assessment 
  
  
X 
Radiology Consult 
  
  
X 
  
  
  
  
X mandatory 
  
  
  
O Optional  
  
  
  
• 
If patient cannot receive IV iodine contrast or for better characterization 
of metastatic lesions 
 
 
 
 
 
 
 

83 
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May 11, 2023 (Amendment T) 
Richard Childs, MD 
 
 
TREATMENT PHASE  
 
 
Inpatient or 
Outpatient 
 
 
Inpatient 
PROTOCOL TIMEPOINT 
3 
4 
5 
6 
 
7 
8 
9 
10 
11 
12 
13 
14 
+/- days or months 
 
 
 
 
 
 
 
 
 
 
 
 
 
DAY 
-4 
-3 
-2 
-1 
 
0 
1 
2 
3 
4 
5 
6 
7 
TREATMENT 
 
 
 
 
 
 
 
 
 
 
 
 
 
Central line placement (3 Lumen IJ or PICC) 
O 
 
 
 
 
 
 
 
 
 
 
 
 
Immunoablative regimen 
 
 
 
 
 
 
 
 
 
 
 
 
 
     Cyclophosphamide 1000 mg/m2 
X 
 
 
 
 
 
 
 
 
 
 
 
 
     Fludarabine 30mg/m2 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
Admission to the inpatient ward 
 
 
 
O 
 
 
 
 
 
 
 
 
 
HERV-E TCR transduced CD8+/CD34+ 
enriched T cells 
 
 
 
 
 
X 
 
 
 
 
 
 
 
Supportive Medications 
 
 
 
 
 
 
 
 
 
 
 
 
 
IL2 2 million U/M2 q 12 IV** 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
PCP and Toxo prophylaxis (Bactrim or others) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Antifungal (Fluconazole or others) 
 
 
 
 
 
O 
 
 
 
 
 
 
 
Antiviral prophylaxis (Acyclovir or others) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Filgrastim 
 
 
 
 
 
O 
O 
O 
O 
O 
O 
O 
O 
EVALUATIONS 
 
 
 
 
 
 
 
 
 
 
 
 
 
History and physical or progress note 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
ECOG  
O 
 
 
 
 
O 
 
 
 
 
 
 
O 
Vital signs (Temp, Pulse, BP, RR, Wt, O2 Sat) 
X 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Chemistry labs 
 
 
 
 
 
 
 
 
 
 
 
 
 
Acute care/mineral panels 
X 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Hepatic Panel, Total Protein, LDH 
X 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Quantitative immunoglobulins (IGG, IGA, IGM) 
 
 
 
 
 
 
 
 
 
 
 
 
 
Uric acid and CK 
O 
 
 
 
 
X 
O 
O 
O 
O 
O 
O 
O 
C-reactive protein 
X 
 
 
 
 
X 
 
X 
 
X 
 
 
X 
Hematology 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with differential 
X 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
PT, PTT  
X 
 
 
 
 
X 
x 
x 
x 
x 
x 
x 
x 
Lymphocyte Phenotyping (TBNK, CD4) 
 
 
 
 
 
X 
 
X 
 
X 
 
x  
 
Type and Screen 
O 
O 
 
 
 
X 
 
 
 
X 
 
 
 
Endocrine Labs  
 
 
 
 
 
 
 
 
 
 
 
 
 
Thyroid panel (TSH, if TSH is abnormal, 
thenT4) and antithyroid panel only if prior 
abnormal as dictated by PI (THY2). 
 
 
 
 
 
x 
 
 
 
 
 
 
x 
Research blood/ biopsies 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enumeration of absolute # of circulating 
HERV-E TCR T-cells [30 ml of blood collected 
in a green tube] 
X 
 
 
 
 
X** 
 
X 
 
X 
 
 
X 
Cytokine studies (1 SST Red Serum tube 
(10cc) tubes to order) 
X 
 
 
 
 
X** 
 
x 
 
x 
 
 
x 
Tumor biopsy up to 3 biopsies 
 
 
 
 
 
 
O 
O 
O 
O 
O 
O 
O 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
X mandatory 
 
 
 
 
 
 
 
 
 
 
 
 
 
O Optional 
 
 
 
 
 
 
 
 
 
 
 
 
 
* patient can be discharged after completion 
of IL-2 if clinically stable. 
 
 
 
 
 
 
 
 
 
 
 
 
 

84 
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Richard Childs, MD 
 
 
** Prior to chemotherapy and prior to T-cell 
infusion respectively 
 
FOLLOW-UP  
 
INPATIENT or OUTPATIENT 
 
OUTPATIENT 
PROTOCOL TIMEPOINT 
  
15 
16 
17 
18 
19 
20 
21 
 
22 
23 
24 
25 
26 
27 
28 
29 
30 
+/- days or 
months 
  
 
 
 
 
 
 
+/- 
2 
 
+/-
3 
+/-
5 
+/- 
7 
+/-
7 
+/-
7 
+/-
14 
+/-
14 
+/-
30 
+/-
30 
DAY 
  
8 
9 
10 
11 
12 
13 
14 
 
21 
30 
60 
90 
120 
180 
240 
300 
360 
EVALUATIONS 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
History and physical or progress 
note 
  
O 
O 
O 
O 
O 
O 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ECOG  
  
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs (Temp, Pulse, BP, RR, 
Wt, O2 Sat) 
  
O 
O 
O 
O 
O 
O 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Chemistry labs 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Acute care/mineral panels 
  
O 
O 
O 
O 
O 
O 
X 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Hepatic Panel, Total Protein, LDH 
  
O 
O 
O 
O 
O 
O 
X 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Prealbumin and lipid panel 
  
 
 
 
 
 
 
O 
 
O 
O 
O 
O 
O 
O 
O 
O 
O 
Quantitative immunoglobulins 
(IGG, IGA, IGM) 
  
 
 
 
 
 
 
 
 
X 
O 
O 
O 
O 
O 
O 
O 
O 
Uric acid and CK 
  
O 
O 
O 
O 
O 
O 
X 
 
 
 
 
 
 
 
 
 
 
C-reactive protein 
 
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Hematology 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with differential 
  
O 
O 
O 
O 
O 
O 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
PT, PTT,  
  
O 
O 
O 
O 
O 
O 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Lymphocyte Phenotyping (TBNK, 
CD4) 
  
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Endocrine labs 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Thyroid panel (TSH, if TSH is 
abnormal, thenT4) and 
antithyroid panel (THY2) 
 
 
 
 
 
 
 
X 
 
O 
O 
O 
O 
O 
O 
O 
O 
O 
Research blood/ biopsies 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enumeration of absolute number 
of circulating HERV-E TCR 
transduced CD8+/CD34+ enriched 
T-cells [30 ml of peripheral blood 
collected in a green to tubes] 
  
O 
O 
O 
O 
O 
O 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Cytokine studies  
(1 SST Red Serum tube (10cc) 
tubes to order) 
  
O 
O 
O 
O 
O 
O 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Replication Competent 
Retrovirus Testing (1 EDTA tube 
with 4-5 ml). Ship to Indiana 
University Vector Production 
Facility 
  
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
 
X 
Tumor biopsy (Accessible 
lesions) up to 3 biopsies 
  
O 
O 
O 
O 
O 
O 
O 
 
O 
O 
O 
O 
O 
O 
O 
O 
O 
Post-T cell product infusion 
Radiology tests  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CT scan of the abdomen and 
pelvis with IV contrast 
  
 
 
 
 
 
 
 
 
 
X 
Oƭ 
X 
X 
X 
X 
X 
X 
MRI abdomen/pelvis with IV 
gadolinium** 
  
 
 
 
 
 
 
 
 
 
X 
O 
X 
X 
X 
X 
X 
X 
MRI brain with IV gadolinium# 
  
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
CT of the brain with IV contrast# 
  
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 

85 
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Richard Childs, MD 
 
 
CT neck with IV contrast# 
  
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
PET scan (F-18 FDG PET-CT) 
  
 
 
 
 
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
Bone scan (TC99 bone)#  
  
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Whole-body MRI#  
  
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
MRI for specific body area if 
metastasis present and clinically 
indicated 
  
 
 
 
 
 
 
 
 
 
O 
O 
O 
O 
O 
O 
O 
O 
Clarifications 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X mandatory 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
O Optional 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
* patient can be discharged after 
completion of IL-2 if clinically 
stable. 
** If patient cannot receive IV 
iodine contrast or for better 
characterization of metastatic 
lesions 
ƭ Scan only needed if progression 
found at D+30.  If disease 
progression is confirmed, no 
additional scans are required for 
that subject. 
# Perform only when baseline 
imaging test positive for 
metastatic disease 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

86 
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Richard Childs, MD 
 
 
F. Interleukin-15 (NSC 745101) 
 
Classification: Recombinant human interleukin-15 (rhIL-15) is a cytokine of the 4-alpha helix bundle 
family of cytokines whose mature form consists of 115 amino acids.  It has two cysteine disulfide cross 
linkages at positions Cys 42-Cys 88 and Cys 35-Cys 85. 
 
M.W.: 12,898.8 Daltons 
 
Mode of Action: IL-15 interacts with a private receptor subunit IL- 15R alpha as well as the IL-2/IL-15R 
beta chain shared with IL-2 and the common gamma chain shared with IL-2, IL-4, IL-7, IL-9 and IL-21.  
IL-15 shares a number of biological activities with IL-2, including stimulation of the proliferation of 
activated CD4+, CD8+ as well as gamma-delta subsets of T cells.  IL-15 also stimulates the proliferation 
of NK cells and acts as a co-stimulator with IL-12 to facilitate the production of Interferon-gamma and 
TNF-alpha. 
 
How Supplied:  IL-15 is supplied by the Pharmaceutical Management Branch, DCTD, NCI as a sterile, 
frozen liquid product in single use vials containing no preservatives. IL-15 is supplied as a 0.49 mg/mL, 
0.3 mL vial formulated in 25 mM sodium phosphate containing 0.5 M sodium chloride at a pH of 7.4. 
 
IL-15 supply will be provided by the NCI to Michael I. Nishimura, Loyola University, Chicago for the 
purposes of improving HERV-E TCR transduction of T cells according to applicable procedures. 
 
Preparation:  Vials of frozen IL-15 should be thawed at ambient room temperature and used within 5 hours 
of thawing.  Upon thawing, the solution should be clear and colorless with no evidence of particulates or foreign 
matter.   
 
Storage:IL-15 vials should be stored at or below (– 70°C). 
 
If a storage temperature excursion is identified, promptly return IL-15 vials to – 70°C and quarantine the 
supplies.  Provide a detailed report of the excursion (including documentation of temperature monitoring 
and duration of the excursion) to PMBAfterHours@mail.nih.gov for determination of suitability. 
 
Stability:Stability studies are ongoing. 
 
Agent Inventory Records – The investigator, or a responsible party designated by the investigator, must 
maintain a careful record of the inventory and disposition of all agents received from DCTD using the 
NCI Drug Accountability Record Form (DARF). 
 
 

87 
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Richard Childs, MD 
 
 
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3. 
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