A Pilot Study to Evaluate the Safety and Tolerability of Escalating Doses of Autologous CD4 T-Cells Modified With Lentiviral Vector Expressing an HR2, C34-peptide Conjugated to the CXCR4 N-terminus in HIV-infected Subjects
Phase: EARLY_PHASE1
Conditions: Hiv
Sponsor: University of Pennsylvania

NCT03020524

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TABLE OF CONTENTS 
PROTOCOL TEAM ROSTER ...................................................................................................... 5 
ACRONYMS ................................................................................................................................. 7 
STUDY SUMMARY ...................................................................................................................... 9 
1.0 
INTRODUCTION ............................................................................................................. 12 
1.1 
Background ......................................................................................................... 12 
1.1.1 
Current Status of “the cure” of HIV Infection ........................................................ 12 
1.1.2 
Lentiviral Vectors for Gene Therapy .................................................................... 13 
1.1.3 
Adoptive T-cell Therapy for HIV ........................................................................... 14 
1.1.4 
Rationale for testing C34-CXCR4 in T-cells in HIV-infected subjects ................. 16 
1.2 
Investigational Agent ........................................................................................... 17 
1.2.1 
C34-CXCR4 fusion .............................................................................................. 17 
1.2.2 
HIV-derived Lentiviral Vector Expressing C34-CXCR4 ....................................... 17 
1.2.3 
Autologous CD3/28 Bead Expanded T-cells ....................................................... 19 
1.3 
Pre-Clinical Data ................................................................................................. 19 
1.3.1 
Antiviral activity of C34-CXCR4 against laboratory HIV strains ........................... 19 
1.3.2 
Selective advantage and enrichment of C34-CXCR4 cells ................................. 22 
1.3.3 
C34-CXCR4 inhibition of enfirvutide (T-20)-resistant HIV-1 ................................ 23 
1.3.4 
In vivo data in humanized NSG mice ................................................................... 24 
1.4 
Clinical Data to Date ............................................................................................ 26 
1.5 
Rationale and Risks/Benefits............................................................................... 26 
1.5.1 
Study Population Rationale .................................................................................. 26 
1.5.2 
Dose Rationale..................................................................................................... 27 
1.5.3 
Risks of Analytical Treatment Interruption ........................................................... 27 
1.5.4 
Examination of Lymphoid Tissue by Rectal Mucosal Biopsy .............................. 30 
2.0 
STUDY OBJECTIVES .................................................................................................... 32 
2.1 
Primary Objectives .............................................................................................. 32 
2.2 
Secondary Objectives ......................................................................................... 32 
2.3 
Exploratory Objectives ........................................................................................ 34 
3.0 
STUDY DESIGN ............................................................................................................. 34 
3.1 
Monitoring for delayed adverse events associated with lentiviral vector 
gene transfer ....................................................................................................... 36 
4.0 
SELECTION, ENROLLMENT, AND WITHDRAWAL OF SUBJECTS ........................... 36 
4.1 
Inclusion Criteria .................................................................................................. 36 
4.2 
Exclusion Criteria ................................................................................................ 38 
4.3 
Study Enrollment Procedures .............................................................................. 39 
4.4 
Co-enrollment Guidelines .................................................................................... 40 
4.5 
Premature Subject Discontinuation ..................................................................... 40 
5.0 
STUDY DRUGS .............................................................................................................. 41 
5.1 
Description of Study Drugs .................................................................................. 41 
5.2 
Treatment Regimen ............................................................................................. 41 
5.3 
Preparation of Autologous CD4+ T genetically modified with C34-CXCR4 ........ 42 
5.4 
Administration of Study Drugs ............................................................................. 43 

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5.4.1 
Administration of autologous T-cells genetically modified with 
C34-CXCR4 ......................................................................................................... 43 
5.5 
Concomitant Medications .................................................................................... 46 
5.5.1 
Recommended Medications ................................................................................ 46 
5.5.2 
Prohibited Medications ......................................................................................... 46 
5.6 
Withdrawal of HAART Medication ....................................................................... 46 
6.0 
CLINICAL AND LABORATORY EVALUATIONS- STUDY PROCEDURES ................. 46 
6.1 
Schedule of Events ............................................................................................. 46 
6.2 
Study Visit Procedures ........................................................................................ 52 
6.2.1 
Screening Evaluations Visit (~Week -15 to -11) .................................................. 52 
6.2.2 
Apheresis 1 Visit (~Week -9 to -7 prior to dosing) ............................................... 53 
6.2.3 
Rectal Biopsy Procedure and Visit....................................................................... 54 
6.2.4 
Safety Evaluations (within 14 days (+/- 3 days) of T-cell Infusion) ...................... 54 
6.2.5 
Infusion of C34-CXCR4 modified CD4+ T-cells (Day 0) ...................................... 55 
6.2.6 
Post Infusion Visits ............................................................................................... 55 
6.3 
Protocol Definitions and Evaluations ................................................................... 55 
6.3.1 
Medical History and Physical Examination .......................................................... 55 
6.3.2 
Clinical Laboratory Evaluations ............................................................................ 57 
6.3.3 
Research Laboratory Evaluations ........................................................................ 58 
7.0 
CLINICAL MANAGEMENT ISSUES .............................................................................. 59 
7.1 
Toxicity ................................................................................................................ 59 
7.2 
Criteria for pausing or stopping the study ............................................................ 61 
7.3 
Special Procedures for Subjects Developing Cancer or Dying ........................... 62 
7.4 
Pregnancies ........................................................................................................ 62 
7.5 
Long-Term Follow-up .......................................................................................... 63 
7.6 
RCL testing, patient monitoring, and procedure for dealing with positive 
test results in subjects ......................................................................................... 63 
8.0 
STATISTICAL CONSIDERATIONS ............................................................................... 63 
8.1 
General Design Issues ........................................................................................ 63 
8.2 
Primary Objective Analysis .................................................................................. 64 
8.3 
Secondary Objectives Analysis ........................................................................... 65 
8.4 
Sample Size and Accrual .................................................................................... 66 
9.0 
DATA COLLECTION, MONITORING, AND ADVERSE EVENT REPORTING ............. 67 
9.1 
Records to Be Kept ............................................................................................. 67 
9.2 
Role of Data Management .................................................................................. 67 
9.3 
Clinical Site Monitoring and Record Availability .................................................. 67 
9.4 
Safety and Adverse Events ................................................................................. 68 
9.4.1 
Definitions ............................................................................................................ 68 
9.4.2 
Recording of Adverse Events .............................................................................. 70 
9.4.3 
Reporting of Serious Adverse Events .................................................................. 71 
9.4.4 
Investigator Reporting:  Local Regulatory Review Committees .......................... 71 
9.4.5 
Sponsor reporting: Notifying the FDA .................................................................. 71 
9.4.6 
Reporting to the IBC ............................................................................................ 72 
9.4.7 
Reporting to the Division of AIDS ........................................................................ 72 
9.5 
Protocol Exceptions and Deviations .................................................................... 72 

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10.0 
HUMAN SUBJECTS ....................................................................................................... 73 
10.1 
Institutional Review Board (IRB) Review and Informed Consent ........................ 73 
10.2 
Subject Confidentiality ......................................................................................... 73 
10.3 
Source Documents .............................................................................................. 74 
10.4 
Case Report Forms ............................................................................................. 74 
10.5 
Records Retention ............................................................................................... 74 
10.6 
Study Discontinuation .......................................................................................... 74 
11.0  STUDY MONITORING, AUDITING, AND INSPECTING .................................................. 74 
11.1 
Study Monitoring Plan ......................................................................................... 74 
11.2 
Auditing and Inspecting ....................................................................................... 75 
12.0 
ETHICAL CONSIDERATIONS ....................................................................................... 75 
12.1 
Rationale for Exposure to Risks .......................................................................... 75 
13.0 
STUDY FINANCES ......................................................................................................... 76 
13.1 
Funding Source ................................................................................................... 76 
13.2 
Conflict of Interest ............................................................................................... 76 
13.3 
Subject Stipends or Payments ............................................................................ 77 
14.0 
BIOHAZARD CONTAINMENT ....................................................................................... 77 
15.0 
PUBLICATION PLAN ..................................................................................................... 77 
16.0 
REFERENCE LIST ......................................................................................................... 78 
 
 

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NIH / NIAID 
The below team members are involved in protocol oversight on behalf of the NIH based on the 
grant funding requirements.  Please see Section 9.4.7 for additional details. 
 
 
DAIDS Medical Officer 
 
 
 
 
 
 
 
 
 
DAIDS Protocol Officer 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 

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ACRONYMS 
AE 
adverse event/experience 
AIDS 
acquired immunodeficiency syndrome 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase (SGPT) 
ANC 
absolute neutrophil count 
ARS 
acute retroviral syndrome 
ART 
antiretroviral therapy 
AST 
aspartate aminotransferase (SGOT) 
ATI 
analytical treatment interruption (removal from HAART) 
BLQ 
below level of quantification 
CAR 
chimeric antigen receptor 
CBC 
complete blood count 
CCI 
Center for Cellular Immunotherapies 
CDC 
Centers for Disease Control and Prevention  
CHPS 
Center for Human Phenomic Science  
CRF 
case report form 
CVPF 
Clinical Cell and Vaccine and Production Facility 
DLT 
dose limiting toxicity 
DSMB 
Data and Safety Monitoring Board 
ELISA 
enzyme-linked immunosorbent assay 
ENV 
HIV envelope 
EAE 
expedited adverse event 
FDA 
Food and Drug Administration 
HAART 
Highly Active Antiretroviral Therapy 
HBsAg 
hepatitis B surface antigen 
HCV  
hepatitis C virus 
HIC 
Human Immunology Core 
HIV 
human immunodeficiency virus 
HR2 
Heptad Repeat 2 
ICS 
intracellular cytokine staining 
IRB 
institutional review board 
IUD 
intrauterine device 
MRN 
medical record number 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
NNRTI 
non-nucleoside reverse transcriptase inhibitor 
OHRP 
Office for Human Research Protections (formerly OPRR) 
PBMC 
peripheral blood mononuclear cell 
PCR 
polymerase chain reaction 
PID 
patient identification number  
RCL 
replication-competent lentivirus 
RNA 
ribonucleic acid 
RNAi 
RNA interference 
SAE 
serious adverse event 
SADR 
serious adverse drug reaction 
SCID 
severe combined immunodeficiency 
SOE 
schedule of events 
STI 
Structured treatment interruption (removal from HAART) 

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TCSL 
Translational and Correlative Studies Laboratory 
ULN 
upper limit of normal 
UPenn 
University of Pennsylvania 
VL 
viral load 
Wt 
wild-type 
 
 

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STUDY SUMMARY 
Title 
A Pilot Study to Evaluate the Safety and Tolerability of Escalating 
Doses of Autologous CD4 T-Cells Modified with Lentiviral Vector 
Expressing an HR2, C34-peptide conjugated to the CXCR4 N-terminus 
in HIV-infected Subjects 
Short Title 
C34-CXCR4-modified CD4+ T-cells in HIV Therapy 
Protocol Number 
Sponsor #C34-CXCR4; IRB #826035 
Phase 
Pilot 
Methodology 
This is a single cohort, open-label pilot study of the safety and 
tolerability of a single infusion of autologous CD4+ T-cells genetically 
modified with an HR2, C34-peptide conjugated to the CXCR4 N-
terminus using a lentiviral vector in HIV-infected subjects.  This is a first 
in human study of C34-CXCR4-T cells. 
 
There will be a single cohort in this study, which consists of subjects 
with well-controlled HIV replication on HAART. Within this cohort will be 
3 escalating doses of T-cell infusions. A modified 3+3+3 dose-
escalation design will be followed, in which the standard dose-
escalation algorithm is stopped when a maximum of 9 evaluable 
subjects or a DLT stopping point has been reached, whichever comes 
first. At each dose level, three patients are treated. For dose levels 1 
and 2, if 0/3 subjects have a dose limiting toxicity (DLT), then the dose 
is escalated. If 1/3 has a DLT (grade 3 or higher unexpected, related 
adverse event [AE]) at a dose level then 3 additional patients are 
treated at that dosage before escalating, and if <2/6 have DLT (i.e. no 
additional DLT is observed) then the dose is escalated to the next 
planned dose level and patients treated until a maximum of 9 evaluable 
subjects has been reached.  The study will comprise of 5 steps: 
 
In Step 1, all participants will undergo leukapheresis to obtain CD4 
positive T-cells that will be genetically modified.  A second 
leukapheresis and an optional rectal biopsy will provide baseline 
specimens to evaluate the size of the HIV reservoir 
 
In Step 2, all participants will receive a single infusion of C34-CXCR4-
modified CD4+ T-cells at one of 3 dose levels.  The first 3 subjects will 
receive Dose Level 1 of 0.8-1x109 transduced CD4+T-cells.  Provided 
no dose limiting toxicity (DLT) is seen at the first dose level, the next 3 
subjects will receive infusion at Dose Level 2 of 2.4-3x109 transduced 
CD4+ T-cells.  If no DLT occurs at that dose, the final 3 subjects will 
receive an infusion at Dose Level 3 of 0.8-1x1010 transduced CD4+ T-
cells.  In the event of a DLT (grade 3 or higher unexpected, related AE) 
recruitment will be paused pending DSMB decision.   
 
At the end of step 2 all participants will undergo mini-leukapheresis and 
an optional rectal biopsy. 
 
In Step 3 all participants will participate in a 16-week analytical 

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treatment interruption beginning 4 weeks after T-cell infusion.   
 
At the end of step 3 all participants will undergo mini-leukapheresis and 
an optional rectal biopsy 
 
In Step 4 all participants will be advised to resume antiretroviral therapy 
and will be followed until plasma HIV RNA falls below the limit of 
detection. 
 
In Step 5 all participants will undergo leukapheresis and an optional 
rectal biopsy at 52 weeks post infusion.  At the completion of the study, 
participants will be asked to participate in a long-term follow-up study 
as required by regulatory authorities. 
Study Duration 
 
Approximately 1 year and 3 months from screening visit. At the 
completion of the study participants will roll over into a long-term gene 
therapy safety follow-up study. 
Study Center(s) 
University of Pennsylvania Medical Center 
Objectives 
The primary objective of this clinical study is to evaluate the safety and 
tolerability of a single dose of this novel biological drug in HIV positive 
subjects.  Secondary objectives focus mainly on persistence and 
enrichment 
of 
C34-CXCR4-modified 
autologous 
CD4+T-cells, 
correlating the number of transduced T-cells infused with effects on 
CD4+ T-cell count and HIV viral load, trafficking of vector modified cells 
in mucosal tissue, immunogenicity of C34-CXCR4, and effects on 
immune function.  
Number of Subjects 
Maximum of 9 evaluable subjects. Evaluable subjects are defined as 
those who have received T-cell infusion and undergo analytical 
treatment interruption. 
Diagnosis and Main 
Inclusion Criteria 
HIV-1-positive women and men, ≥18 years old with HIV-1 RNA levels 
undetectable by ultrasensitive HIV PCR assay, CD4+ T-cells counts 
≥450 cells/ mm3, with a documented CD4 nadir of not lower than 200 
cells/ mm3, and who have a recorded viral load set point prior to starting 
therapy.   
Study Product, 
Dose, Route, 
Regimen 
Autologous CD4+ T-cells modified with lentiviral vector expressing an 
HR2, C34-peptide conjugated to the CXCR4 N-terminus (C34-CXCR4-
T), given at a single dose of either: 1) 0.8-1x109, 2) 2.4-3x109, or 3) 0.8-
1x1010 transduced T-cells via intravenous infusion.  
Duration of 
administration 
Based on the total volume to be infused and the recommended infusion 
rate of approximately 10mL-20mL/minute. Study subjects will be treated 
with only a single infusion of modified CD4+T-cells. 
Statistical 
Methodology 
The primary focus of this trial is establishing safety.  As such, with three 
evaluable subjects in each dose level, we can be reasonably assured of 
observing an adverse event that occurs in the population at a rate of at 
least 80%.   
 
As this is a pilot study, there will be limited statistical power to evaluate 
efficacy and related biological endpoints.  Therefore, analyses will be 
primarily descriptive.  In general, an evaluable sample size of three 

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subjects will provide 80% power, at a two-sided significance level of 
5%, to identify an effect size of 3.3 (corresponding to 3.3 standard 
deviations) for the within person change for normally distributed 
outcomes. 
 
In this exploratory study we will evaluate the impact of C34-CXCR4-
modified CD4+ T-cells in HIV-positive subjects.  In order to have an 
evaluable sample size, subjects who prematurely discontinue the study 
prior to the infusion or analytical treatment interruption will be replaced 
with another subject. 
 
 
 
 
 
 
 
 
Figure 1: Study Schema  
 
 
 
 

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1.0 
INTRODUCTION 
1.1 
Background  
1.1.1 Current Status of “the cure” of HIV Infection  
There are an estimated 36.9 million people living with HIV and a corresponding 1.2 million 
deaths annually worldwide (UNAIDS, 2015). In North America and Western and Central Europe, 
it is estimated that 2.4 million people are infected with HIV, almost 26,000 people die each year, 
and more than 85,000 become infected annually. The use of highly active antiretroviral therapy 
(HAART) in HIV infected individuals has been associated with prolonged suppression of HIV 
viremia. However the persistence of HIV in latently infected resting CD4+ T-cells creates an 
almost insurmountable challenge in the long-term control of HIV infection in the absence of 
antiretroviral therapy and in the prospects for eradication and the “cure” of HIV infection. 
 
The recent case of a German patient who underwent an allogeneic stem cell transplantation 
using CD34+ selected peripheral blood progenitor cells from a donor identified to be 
homozygous for CCR5-Δ32, after conditioning with the FLAMSA regimen has challenged the 
widespread perception that HIV eradication, or at least a “functional” cure was not possible 
(Hutter, Nowak et al. 2009). That particular individual had been treated with TDF/FTC/EFV for 4 
years before the acute myeloid leukemia appeared. After relapse of his leukemia he underwent 
a bone marrow transplant from an HLA-identical donor who had been screened for 
homozygosity for the CCR5 Δ 32 allele. Antiretroviral therapy was discontinued on the day of 
the procedure. GVHD prophylaxis followed standard regimens and engraftment was achieved 
on day +13. Complete chimerism as detected by competitive PCR was observed on day +60. 
The virus load was measured both by RNA-PCR and proviral DNA-PCR. DNA-PCR was 
negative from day +68. Although HAART has now been discontinued for over 5 years, HIV-1 
has not been detected, as determined by RNA and proviral DNA PCR assays of peripheral 
blood, bone marrow, and rectal mucosa. This patient has raised the interest in treatment 
strategies aimed at eradication (either biological or functional) of HIV infection (Allers, Hutter et 
al. 2010). In the field of HIV gene therapy, particular interest surrounds the idea of making cells 
refractory, or protected, from HIV infection. 
 
Recently two Boston patients received an allogeneic bone marrow transplant. As with the Berlin 
patient, they also received aggressive conditioning chemotherapy and developed graft versus 
host disease, however they did not receive delta 32 bone marrow. Both patients discontinued 
antiretroviral therapy after no evidence of residual HIV infection could be found. In both cases 
the virus returned after several weeks. These two cases demonstrate that protecting cells in 
order to prevent the return of viremia is critical in any strategy of eradicating HIV infection 
(Henrich, Hanhauser et al. 2014).    
 
One potential factor believed to have contributed to the “functional cure” of HIV infection in the 
case of the Berlin patient was the transplantation of CCR5 Δ32 cells, which due to a lack of a 
functional CCR5 coreceptor were unable to be HIV infected.  The ultimate goal of our approach 
is similar in that it also intends to protect CD4 cells from HIV infection.  Our hypothesis is that 
adoptive cell transfer of autologous CD4 T-cells genetically modified to express C34 peptide 
conjugated to the CXCR4 N-terminus in HIV-infected individuals will preserve CD4 cells that will 
be immunologically functional and can help control viremia, and that these cells will survive and 
be enriched in the absence of antiretroviral drugs, potentially contributing to a delay in the return 
of viral load.  
 

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Curing HIV will take a combinatorial approach involving activation of the HIV from its reservoir, 
targeting the activated reservoir by the immune system, and protecting the uninfected cells 
during the process. Although these strategies will most likely be used in the future in 
combination, it will require that each strategy is tested separately before being used in a 
combined approach. Our study aims to protect the HIV uninfected cells during the process of 
activating the reservoir by making the CD4+ T-cells resistant to HIV, thus preserving the 
immunological response against HIV. 
  
Gene therapy for HIV-1 infection has been proposed as an alternative to antiretroviral drug 
regimens (Sarver and Rossi 1993, Dropulic and June 2006, Leibman and Riley 2015).  A 
number of different genetic vectors with antiviral payloads have been utilized to combat HIV-1, 
including antisense RNA, transdominant proteins, ribozymes, RNA decoys, single chain 
antibodies, and RNAi (RNA-interference) (Dropulic and June 2006).  Payloads targeting entry of 
HIV have also been investigated both in preclinical studies and in human trials (Li, Kim et al. 
2005, van Lunzen, Glaunsinger et al. 2007).  Blocking HIV at an early step may be important for 
conferring a selective advantage to genetically modified cells in the body, and hence allow 
outgrowth of HIV–resistant T-cells in the body (von Laer, Hasselmann et al. 2006).  Our 
proposed study will investigate the inhibition of HIV entry using CD4 T-cells expressing the C34 
peptide of gp41 conjugated to CXCR4 N terminus and the ability of those modified cells to 
confer such a selective advantage. 
 
1.1.2 Lentiviral Vectors for Gene Therapy 
The early vectors used for human gene transfer research have included murine oncoretrovirus-
derived and adenovirus-derived vectors, and the more recent vectors are those derived from 
adeno-associated virus, herpes simplex virus, and now lentivirus (Kootstra, Matsumura et al. 
2003, Nathwani, Davidoff et al. 2005, Verma and Weitzman 2005).  Murine oncoretrovirus-
derived vectors were initially found to be useful for long-term gene expression because of their 
ability to integrate into host DNA.  However, with the evidence that this vector could produce 
lymphoma due to insertional mutagenesis (Hacein-Bey-Abina, Von Kalle et al. 2003, Hacein-
Bey-Abina 2006), oncoretroviruses have been used less as a vehicle for cell transduction.   
 
Lentiviral vectors are an alternative to oncoretroviral vectors and are known to have higher 
transduction efficiencies.  However, an important question has been whether lentivirus vectors 
are safe, and the use of T-cell-based approaches has been a useful step in vector evaluation.  
Murine retrovirus vectors, although used in hundreds of persons and found to be generally safe, 
were known to induce lymphomas in animals, principally by means of insertional mutagenesis 
(Tsichlis 1987).  The T-cell proliferation actually seen in two trials of human retrovirus gene 
therapy for severe combined immunodeficiency disease is likely due not only to insertional 
mutagenesis but also to the effects of a ‘growth’ promoting transgene used in this treatment 
(Hacein-Bey-Abina et al., 2003) (unpublished data on www.asgt.org).  Lentivirus vectors 
integrate into sites of active gene transcription (Bushman 2002, Mitchell, Beitzel et al. 2004), but 
the relevance of this process to oncogenesis is unknown.  The lentiviral vector poly-A signal 
more strongly inhibits transcriptional read through than oncoretroviral vectors (Zaiss, Son et al. 
2002).  Additionally, a study in tumor prone mice comparing the tumorigenicity of retroviral 
vectors to lentiviral vectors demonstrated that lentiviral vector gene transfer into hematopoietic 
stem cells of up to an average of 6 copies per cell was not tumorigenic in contrast to retroviral 
vectors which were tumorigenic at an average copy number of only 3 per cell (Montini, Cesana 
et al. 2006). Lentivirus vectors can result in multiple integrations per cell and can even integrate 
in known oncogenes (Woods, Muessig et al. 2003).  Yet, there is little association between HIV-
1 infection and T-cell leukemia, and the increased rate of B cell lymphoma in AIDS is commonly 

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known to result from immunodeficiency rather than from HIV-1 integration events (Kaplan, 
Shiramizu et al. 1995, Sandler and Kaplan 1996). 
 
Recent evidence suggests that variations in cellular sites of integration account for varying 
levels of HIV expression from such sites (Lewinski, Bisgrove et al. 2005).  Provirus integrations 
occur in HIV-1 infected persons at rates that vary with progression of infection, and for 
advanced disease, provirus integration is seen in 1 per 700-3500 peripheral blood cells 
(Simmonds, Balfe et al. 1990).  Yet, considering the long, high, and continuous exposure to 
HIV-1 in infected patients, the relative lack of significant clinical problems due to insertional 
mutagenesis, per se, is supported by laboratory evidence suggesting that lentivirus vectors may 
be safe in this regard (Mitchell, Chiang et al. 2003, Mitchell, Beitzel et al. 2004). 
 
1.1.3 Adoptive T-cell Therapy for HIV 
The objectives of T-cell immunotherapy have been to augment HIV-specific T-cells and to 
reverse or decrease the progressive destruction of CD4 T-cells that leads to clinical AIDS. 
Levine and colleagues have developed a bead based system for stimulating T-cells using the 
anti-CD3 and CD28 specific monoclonal antibodies (Levine, Bernstein et al. 2002). In our 2002 
study, polyclonal peripheral blood CD4 T-cells from HIV patients were co-stimulated with anti-
CD3/CD28 ex vivo and were infused into patients at doses of up to 3  1010 activated T-cells. In 
the single infusion dose-escalation portion of this study, CD4 T-cell counts were increased for 
2–3 months. With three repeat infusions, CD4 T-cells were increased for approximately 1 year.  
 
Extensive safety data indicate that genetically engineered T-cells are safe in patients with 
congenital immunodeficiency and AIDS. Probably the most illuminating case of safety is a 
patient who has adenine deaminase (ADA) deficiency. Beginning in 1990, she was treated by 
Dr. R. Blaese and colleagues with 11 infusions comprising a total of 1.4x1011 retrovirally 
modified T-cells (Blaese, Culver et al. 1995). More than twenty years after her first infusion, she 
has sustained correction of her immunodeficiency, and 25% of her T-cells still express the 
transgene, illustrating that high level persistence can occur following infusions of retroviral-
modified T-cells (Muul, Tuschong et al. 2003). In collaboration with Gary Nabel (Ranga, 
Woffendin et al. 1998) and scientists at Cell Genesys (Mitsuyasu, Anton et al. 2000, Walker, 
Bechtel et al. 2000, Deeks, Wagner et al. 2002) we first began infusions of retrovirally modified 
CD4 cells in the early 1990s.  In collaboration with ViRxSys Corp, our group has treated 22 
patients with lentiviral engineered T-cells at the University of Pennsylvania (Table 1.1.3). The 
extensive experience with retroviral and lentiviral gene modified T-cells indicates that at 
least 497 T-cell infusions (at least 205 patients) have been given, and importantly, there 
has not been a single SAE due to the use of retroviral or lentiviral vector engineered T-
cells. 
 
Table 1.1.3.  Patients infused with retroviral or lentiviral modified T-cells for immunodeficiency 
Indication 
Transgene 
Retro / 
Lenti 
Sponsor 
# Patients 
infused  
(# infusions) 
Comments 
Congenital 
immunodefi
ciency 
(SCID) 
Adenosine 
deaminase 
Murine 
retrovirus 
NIH 
2 
(23 infusions) 
Persistence at high levels in 1 of 2 patients for at 
least 10 years. 
No SAE.(Blaese, Culver et al. 1995, Muul, 
Tuschong et al. 2003) 
HIV 
neoR CTL 
Murine 
retrovirus 
Fred Hutchinson Cancer 
Research Center 
3 
(6 infusions) 
Persistence for up to 1 month. 
No SAE.(Brodie, Lewinsohn et al. 1999) 

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Table 1.1.3.  Patients infused with retroviral or lentiviral modified T-cells for immunodeficiency 
Indication 
Transgene 
Retro / 
Lenti 
Sponsor 
# Patients 
infused  
(# infusions) 
Comments 
HIV 
HyTK CTL 
Murine 
retrovirus 
Fred Hutchinson Cancer 
Research Center 
6 
(24 infusions) 
Host immune response limited persistence to 6 
weeks in 5 of 6 patients. 
No SAE (Riddell, Elliott et al. 1996) 
HIV 
RevM10 
Murine 
retrovirus 
University of Michigan 
3 
(3 infusions) 
No SAE reported (Ranga, Woffendin et al. 1998) 
HIV 
CD4 CAR 
coupled with 
the CD3  
Murine 
retrovirus 
Cell Genesys 
70  
(306 infusions) 
3 trials reported (Mitsuyasu, Anton et al. 2000, 
Walker, Bechtel et al. 2000, Deeks, Wagner et al. 
2002).  
No SAE.  
HIV 
Anti-HIV-1 tat 
ribozyme (Rz2) 
Murine 
retrovirus 
Johnson and Johnson 
4  
(4 infusions) 
Transgene persistence detected in all patients for 
4 years (Macpherson, Boyd et al. 2005). 
No SAE 
HIV 
Trans-
dominant rev 
and/or 
transdominant 
rev with TAR 
antisense 
Murine 
retrovirus 
NIH 
10 
(19 infusions) 
6 of 6 patients studied had transgene persistence 
detected for at least 2 years (Morgan, Walker et 
al. 2005). 
No SAE 
HIV 
CD4 CAR 
coupled with 
the CD3  
Murine 
retrovirus 
University Pennsylvania 
10 
(10 infusions) 
9 of 9 patients studied had transgene persistence 
detected for at least 4 years (Aronson, Benstein 
et al. 2008). 
No SAE 
HIV 
gp41 fusion 
peptide 
inhibitor 
Murine 
retrovirus 
Fresenius AG, Germany 
10 
(10 infusions) 
4 of 10 patients had transgene persistence for at 
least 9 months (van Lunzen, Glaunsinger et al. 
2007). 
No SAE 
HIV 
Anti HIV-1 
antisense 
against the 
envelope gene 
HIV-derived 
lentivirus, 
conditionally 
replicating 
University Pennsylvania 
22 
(92 infusions) 
14 of 22 had transgene persistence detected for 
at least 1 year (Levine, Humeau et al. 2006, 
Collman, Shaheen et al. 2009, Wang, Levine et 
al. 2009). 
No SAE 
HIV 
Anti HIV-1 
antisense 
against the 
envelope gene 
HIV-derived 
lentivirus, 
conditionally 
replicating 
ViRxSys 
65 
 
Patients given 4 or 8 repeat infusions  
ClinicalTrials.gov 
NCT00131560.  
No SAE 
HIV 
SL9 gag TCR HIV-derived 
lentivirus 
Adaptimmune, NIH (at 
University of Pennsylvania) 
2 
2 of 2 subjects had transgene persistence 
detected for at least 6 months. 
No SAE 
HIV 
MazF 
endoribonuclease 
Murine 
retrovirus 
Takara Bio (at University of 
Pennsylvania) 
10 
8 subjects have completed main study to date 
with persistence out to 90 days. 
 
 
Our investigation plans to utilize the CD3/CD28 stimulation of CD4 T-cells which has been 
shown to be safe in HIV patients for single or multiple doses (Mitsuyasu, Anton et al. 2000, 
Walker, Bechtel et al. 2000, Deeks, Wagner et al. 2002, Levine, Bernstein et al. 2002, Levine, 
Humeau et al. 2006). Long term persistence of genetically altered autologous T-cells has also 
been observed for cells manufactured using the CD3/28 protocol at the University of 
Pennsylvania (Deeks, Wagner et al. 2002, Levine, Humeau et al. 2006). In the Deeks protocol, 

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gene-modified cell persistence in peripheral blood was seen in all patients for at least 24 weeks 
(0.1%–10%). There were no treatment-related serious adverse events in over 120 infusions, 
and T-cell-related adverse events were mild and included fever (10%), chills (17%), asthenia 
(10%), headache (10%), and nausea (10%). In the Levine study, 5 patients were administered 6 
doses of gene modified cells and 2 of the 5 patients had circulating levels of gene modified cells 
at one year post-infusion.  No serious adverse events related to the treatment were observed. 
 
In a follow up study testing the lentivirally transduced antisense VRX496-modified T-cells, we 
observed antiviral effects during analytic treatment interruption in a subset of 13 patients. 
VRX496-T was associated with significant decreases in the viral load set point. In addition, A-to-
G transitions were enriched in HIV sequences post infusion, consistent with a model where 
transduced CD4 T-cells exert anti-sense mediated genetic pressure on HIV during infection. 
The engraftment half–life in the blood was approximately 5 weeks, with stable persistence in 
some patients for up to five years. No evidence of clonal selection of lentiviral vector transduced 
T-cells or integration enrichment near oncogenes could be detected. This was the first 
demonstration that gene modified cells can exert genetic pressure on HIV (Tebas, Stein et al. 
2013). Thus gene modified T-cells have the potential to decrease the fitness of HIV-1.   
 
Recently our group published results from a study using zinc finger nucleases to permanently 
disrupt CCR5 expression in autologous CD4 T-cells of HIV-1 infected subjects.  Six subjects in 
this study entered into an analytical treatment interruption intended to last 12 weeks.  In the 4 
subjects who completed the 12 week ATI, viral load decreased by an average of 1.2 log10 during 
the interruption from the peak level without HAART.  Notably, HIV RNA became undetectable in 
1 of those 4 evaluable subjects.  CCR5-modified CD4 cells had an estimated half-life of 48 
weeks, and could be detected in all subjects at all subsequent long-term follow up time points.  
Additionally, CCR5-modified cells could be detected in the rectal biopsies of all 11 subjects that 
consented, indicating trafficking to gut mucosa. This first in human study of gene editing 
demonstrated that the protection of CD4 T-cells is an important component for the development 
of a “functional cure” (Tebas, Stein et al. 2014).      
 
Our group has also reported long-term persistence of genetically modified T-cells from three 
clinical trials evaluating gammaretroviral vector-engineered T-cells for HIV. The vector encoded 
a chimeric antigen receptor (CAR) composed of CD4 linked to the CD3ζ signaling chain (CD4ζ). 
CAR T-cells were detected in 98% of samples tested for at least 11 years after infusion at 
frequencies that exceeded average T-cell levels after most vaccine approaches. The CD4ζ 
transgene retained expression and function. The CD4ζ T-cells had stable levels of engraftment, 
with decay half-lives that exceeded 16 years, in marked contrast to previous trials testing 
engineered T-cells. These findings indicate that host immunosuppression before T-cell transfer 
is not required to achieve long-term persistence of gene-modified T-cells (Scholler, Brady et al. 
2012). These previous human studies in HIV patients support the manufacturing methodology 
for, and the safety and long-term persistence of, adoptively transferred anti-CD3/CD28-
stimulated T-cells for immunotherapy in HIV.  
 
1.1.4 Rationale for testing C34-CXCR4 in T-cells in HIV-infected subjects 
In collaboration with the Hoxie laboratory at UPENN, Sangamo Therapeutics, Inc. has 
developed a novel approach to render primary CD4+ T-cells highly resistant to infection by 
diverse HIV-1 clades.  CXCR4, when edited to contain a 34 amino acid peptide from the gp41 
HR2 domain (C34) conjugated to the coreceptor amino terminus, potently inhibits HIV-1 entry 
(Leslie, et al. submitted). This effect occurs with high specificity, is effective against HIVs that 
are resistant to soluble T20, and remarkably, occurs irrespective of tropism when stably 

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transduced (i.e., C34-conjugated CXCR4 will inhibit both R5- and X4-tropic viruses). This 
inhibition is highly dependent on the positioning of the peptide on the coreceptor (CXCR4) and 
occurs at molar ratios of approximately one C34-CXCR4 to 10-20 unconjugated CXCR4. T-cell 
proliferation and cytokine expression were similar to non-transduced cells, and C34-CXCR4 
expressing cells migrated in response to CXCL12 or beta chemokines in chemotaxis assays. 
The breadth and potency of HIV inhibition was demonstrated in vitro on primary PBMCs where 
cells expressing C34-CXCR4 showed a clear survival advantage during HIV-1 infection. By 
inhibiting both R5 and X4 viruses this novel strategy offers advantages to approaches that 
simply delete the CCR5 gene. In addition, by tethering the HR2 peptide directly to molecules 
required for HIV-1 entry, the strategic location of this inhibitory peptide can be expected to 
exhibit greater potency than when delivered non-specifically on the cell surface on constructs 
where high levels of expression are required for antiviral effects. 
 
In order to assess the protective ability of these C34-CXCR4 modified CD4+ T-cells against 
HIV-1, subjects will undergo a 16-week analytical treatment interruption.  Risks of ATI are 
discussed in Section 1.5.3, Risks of Analytical Treatment Interruption.  
 
1.2 
Investigational Agent 
The investigational agent tested under this protocol is autologous CD4+ T-cells genetically 
modified with lentiviral vector to express the C34-peptide conjugated to the CXCR4 N-terminus. 
The investigational cell therapy will be administered to HIV positive patients. 
 
1.2.1 C34-CXCR4 fusion 
C34 is a peptide from the HIV-1 heptad repeat (HR2) domain of gp41 that inhibits formation of 
the gp41 6-helix bundle during viral entry.  In this study, C34 is conjugated to the N-terminus of 
CXCR4, a coreceptor for HIV-1 entry.  In vitro, the C34 peptide conjugated to CXCR4 (C34-
CXCR4) is inhibitory for HIV-1 entry irrespective of HIV-1 tropism and clade. 
   
1.2.2 HIV-derived Lentiviral Vector Expressing C34-CXCR4 
The plasmids used in virus production include pTRPE C34-CXCR4 (the vector plasmid), pC-
GP2 (the plasmid encoding gag/pol), pC-Rev2 (the plasmid encoding HIV rev), and pVSV-G 
(the plasmid encoding the pseudotyping-envelope VSV-G-protein of the vesicular-stomatitis 
virus).   
 
The gene transfer vector pTRPE C34-CXCR4 is an HIV derived self inactivating (SIN) vector 
that comprises a 5’ LTR and a 3’ U3 deleted LTR.  Transgene transcription is driven off of the 
mammalian ef-1α promoter (Kim, Uetsuki et al. 1990).  The vector also contains the central 
polypurine tract and central termination sequence (cppt/CTS) (Sirven, Pflumio et al. 2000) for 
improved transduction efficiency, the rev response element (RRE) for RNA transport, the WPRE 
element for improved RNA translation (Zufferey, Nagy et al. 1997, Donello, Loeb et al. 1998), 
and the packaging sequence.  A schematic representation is presented in Figure 1.2.2. 
 
The clinical grade vector, pTRPE C34-CXCR4, will be provided by Sangamo Therapeutics, Inc.  
Frozen aliquots of vector will be shipped to the CVPF at the University of Pennsylvania for use 
in the production of the cell products for this study. 
 
 
 
 
 

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Figure 1.2.2. C34-CXCR4 lentivirus components 
 
   
 
This protocol will use a vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped 
replication defective HIV-based lentiviral vector to insert the C34-CXCR4 fusion construct into 
CD4 cells. The basis of using retroviruses as vehicles for efficient therapeutic gene delivery into 
mammalian cells was established in 1981 when it was shown that a replication competent 
murine oncoretrovirus could incorporate a herpes simplex virus thymidine kinase (tk) gene into 
the genome of a mouse cell, and to convert NIH-3T3 TK- cells into TK+ transformants. Today, 
HIV based lentiviral vectors are more advantageous, and may have less leukemogenic 
potential, since natural disease associated with HIV infection does not lead to leukemia. High-
titer concentrated lentiviral vectors (>10e9 infectious units [IU]/mL) can now be generated. The 
ability to pseudotype retrovirus vectors with a variety of envelope proteins, including the VSV-G, 
significantly broadens the tropism of replication-defective lentiviral vectors.  
 
VSV is a negative sense, single stranded RNA virus that replicates entirely in the cytoplasm 
(Lichty, Power et al. 2004).  VSV consists of five genes that encode the five major viral proteins: 
the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and 
the large polymerase protein (L). The glycoprotein of VSV serves both to bind the surface of the 
host T-cell and to fuse viral and cellular membranes, enabling the release of the viral genome 
and replicase into the cytoplasm. The VSV glycoprotein binds to phosphatidylserine, a near-
universal component of cell-surface membranes, enabling VSV to infect virtually all animal cells. 
 
The cell product infused in this protocol is tested for residual VSV-G DNA, and must be negative 
for infusion to proceed.  However, it is possible that low levels of VSV-G DNA may be present at 
levels below the limit of detection of the assay.  In this case, it is possible the patient may 
become seropositive for VSV-G, while such a conversion would not indicate VSV infection.  
Seropositivity for VSV-G has no known adverse health consequences.  In prior gene therapy 
protocols, where lentiviral vectors pseudotyped with VSV-G were also used for gene transfer, 
monitoring for VSV-G antibody was used as a surrogate for RCL detection.  In those trials, 
patients enrolled on the study were tested at baseline for VSV-G seropositivity and only those 
who were seronegative were enrolled.  It is important to note that in our previous VRX496 study, 
7/13 subjects did become seropositive for VSV-G.  Although positive for VSV-G antibodies, 
there were no adverse infusion reactions associated with the seropositivity, and the presence of 
VSV-G antibody did not correlate with VRX496-T persistence (Tebas, Stein et al. 2013). In this 
trial, RCL is monitored by PCR for VSV-G DNA, which is an acceptable alternative method for 
RCL monitoring in accordance with current FDA regulations on RCR/RCL testing (November 
2006; 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGene
Figure 1.2.2 Schematic representation 
of the lentiviral vector and packaging 
plasmids.  The C34-CXCR4 transgene is 
inserted into a lentiviral vector backbone 
derived from the Dull vector (Dull, Zufferey 
et al. 1998), and is expressed by the ef-1a 
promoter which has efficient expression 
with low levels of gene silencing.  The 
packaging 
system 
consists 
of 
three 
plasmids (gag/pol, rev, and envelope), 
similar 
to 
that 
previously 
published 
(Zufferey et al, 1997).  

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Therapy/ucm072961.htm). Therefore VSV-G seronegativity is not a requirement for inclusion in this 
this trial, and there are no plans to monitor for antibodies to VSV-G. 
 
1.2.3 Autologous CD3/28 Bead Expanded T-cells 
As in previous studies, autologous CD4 selected cells will be prepared at University of 
Pennsylvania (UPENN), and transduction with pTRPE C34-CXCR4 will be done at the Clinical 
Cell and Vaccine Production Facility (CVPF) at UPENN.  These cells will be expanded in a closed 
system using CD3/CD28 polystyrene beads for ~8-14 days and then cryopreserved.  Quality 
control assays will also be performed for the transduced T-cells prior to release for patient 
infusion. 
 
1.3 
Pre-Clinical Data 
In collaboration with Sangamo Therapeutics, Inc., our group has developed a novel approach to 
render primary CD4+ T-cells highly resistant to infection by diverse clades of HIV-1.  By 
conjugating a 34 amino acid peptide from the gp41 HR2 domain (C34) to the amino terminus of 
CXCR4, we have observed potent inhibition of HIV entry.  This inhibition is highly specific, is 
effective on viruses resistant to soluble enfuvirtide (Fuzeon®, T20), and inhibits infection by both 
X4 and R5-tropic HIV-1 when CD4 cells are stably transduced by lentiviral vector.  
 
This approach to protecting CD4 T-cells is based, in part, on our success in giving CD4 cells a 
selective advantage in HIV infection using CCR5 zinc finger nucleases.  In that study 
(NCT00842634), which was recently published in the New England Journal of Medicine, 12 
aviremic, chronic HIV-positive subjects on HAART were given a single infusion of 10 billion 
autologous CD4 T-cells where between 11 and 28% of the cells were genetically modified with 
CCR5 ZFN.  Six of these subjects underwent an analytical treatment interruption beginning 4 
weeks after infusion of modified cells.  CD4 T-cell counts increased in all 12 subjects infused, 
from a median of 448 per cubic millimeter at baseline to 1517 per cubic millimeter at week 1.  
CCR5-modified cells were detected in the gut mucosa of the 11 subjects that assented to 
biopsy, demonstrating homing and persistence in the rectal mucosal tissue. In the 4 subjects 
who completed the 12 week treatment interruption, the viral load decreased by an average of 
1.2 log10 from the peak level during the treatment interruption.  In one subject who was 
determined to be a delta 32 heterozygote, increase in viral load did not occur until week 6 of the 
ATI, at which time the viral load peaked at 6247 copies/ml (pretreatment setpoint was 165,000 
copies/ml).  The modified cells were long-lived, with an estimated half-life of 48 weeks, and 
CCR5-modified T-cells could be detected in all subjects at all subsequent long-term follow up 
visits (Tebas, Stein et al. 2014).  In brief, the collective data indicate that CCR5-modified CD4 
cells can be safely infused in patients and that those cells persist long-term and have a survival 
advantage in the presence of HIV infection.  This data serves as a partial proof-of-concept that 
approaches which render patients intrinsically resistant to HIV infection so that they do not 
require HAART are feasible and promising.  Therefore, this type of approach which generates 
HIV resistant T-cell populations warrants further investigation as a potential therapeutic 
intervention in the treatment of HIV/AIDS and as a part of a combination strategy for a functional 
cure of HIV infection.  Altogether, these data support the rationale for this protocol to test C34-
CXCR4-modified CD4+ T-cells in HIV infected subjects for safety, as well as secondary 
objectives such as immune resonstitution and survival in the presence of HIV. 
 
1.3.1 Antiviral activity of C34-CXCR4 against laboratory HIV strains 
Preclinical in vitro studies have been performed to assess the ability of C34-CXCR4 cells to 
permit normal coreceptor function, while also being non-permissive to HIV entry in CD4+ T-

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Fig. 1.3.1.2  C34-CXCR4 inhibits both X4- and R5- tropic HIV in the presence of each respective 
coreceptor. (A) C34-CXCR4 plasmids were transfected at the indicated ratios with unlabeled CXCR4 in 
transfected CF2-Luc cells and inoculated with HIV-1 HxB2. (B) C34-CXCR4 plasmids were transfected at 
the indicated ratios with unlabeled CCR5 in transfected CF2-Luc cells and inoculated with HIV-1 BaL.  Both 
homologous (A) and heterologous (B) trans-inhibition was observed up to a ratio of 1:10 conjugated to 
unconjugated coreceptors. 
 
 
To evaluate the inhibitory effect of C34-CXCR4 in primary cells, CD4+ T cells from healthy 
donors, stimulated with CD3/CD28 beads and maintained in media containing IL-2, were 
transduced with C34-CXCR4 or GFP control, inoculated or not inoculated with R5-tropic HIV-1 
JRFL, and monitored using flow cytometry with an anti-C34 monoclonal antibody to assess 
expression over time.  HIV infection was also monitored via intracellular p24-Gag expression 
(Figure 1.3.1.3). Uninfected CD4 T cells transduced with C34-CXCR4 maintained expression 
levels 95% of total T cells for up to 14 days (Figure 1.3.1.3, A).  In cells inoculated with JRFL 
(Figure 1.3.1.3 B), 57.1% and 45.4% p24-Gag cells (ie, HIV+ cells), respectively, were seen for 
non-transduced and GFP-transduced cells. In contrast, in C34-CXCR4 transduced cells, p24-
Gag expression was markedly reduced to 1.3%, and over time this inhibition persisted with<1% 
p24-Gag+ cells at Day 14 post-inoculation. Consistent with the results in cell lines, CD4 T-cells 
transduced with C34-CXCR4 failed to become HIV infected, as compared to untransduced 
primary CD4+ controls. 
 
These primary CD4 cell assays were repeated with various strains of HIV, including -X4, -R5, 
and R5/X4 dual tropic viruses.  As shown in Table 1.3, primary CD4 T-cells transduced with 
C34-CXCR4 were able to inhibit HIV infection, regardless of HIV tropism by 98-99%. 
 

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Figure 1.3.1.3 C34-CXCR4 expression inhibits HIV infection in transduced primary CD4 cells.  Primary 
human CD4 T cells were transduced with C34-CXCR4 or GFP control, inoculated or not inoculated with HIV-
1 JRFL, and monitored by flow cytometry with an anti-C34 peptide antibody.  HIV infection was assessed by 
intracellular p24-Gag expression. (A) Expression of C34-CXCR4 in cells not inoculated with HIV-1 at Days 5 
and 14. (B) Cells inoculated with HIV-1 JRFL show stable expression of C34-CXCR4 at Days 5 and 14 with 
marked inhibition of p24-Gag expression in that culture relative to GFP-transduced cells.   
 
 
1.3.2 Selective advantage and enrichment of C34-CXCR4 cells 
As previously observed with cells protected from HIV infection via CCR5 ZFN treatment, we 
aimed to assess the ability of C34-CXCR4 cells to be selected for in the presence of an actively 
replicating HIV infection.  To assess the survival advantage of C34-CXCR4-modified primary 
CD4 cells, modified cells were diluted with untransduced CD4 T-cells at a ratio of 1:3 for a final 
percentage of 25% transduced cells and challenged with the JRFL (R5-tropic) strain of HIV-1.  
As a control, GFP-transduced CD4 cells were also diluted with untransduced CD4 cells.  C34-
CXCR4-modified CD4+ T-cells were assessed by C34 surface staining, while HIV infection was 
monitored concurrently over time by intracellular Gag co-staining.  The course of infection was 
monitored for a period of 14 days.  As shown in Figure 1.3.1.4, C34-CXCR4 cells enriched from 
approximately 25% of the culture to 66% of the culture at Day 14.  Correspondingly, GFP+ cells 
declined to 10.6% of the culture at Day 14. Results of the entire timecourse are graphed in 
panel B.  
 
The HIV infection peaked at Day 5 in the culture with 29.5% Gag-positive cells.  In comparison, 
GFP transduced CD4 T-cells reached their maximum level of HIV infection also at Day 5 with 
59% of cells staining Gag-positive.  This experiment was repeated with multiple HIV strains of 
various tropisms.  Those results are provided in tabular form in Figure 1.3.1.4, panel C. 

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Collectively, these data suggest that C34-CXCR4 can inhibit both R5 and X4 tropic HIV-1 
isolates, and that C34-CXCR4-modified CD4+ T-cells may have a selective advantage in the 
presence of active HIV replication.  
 
Figure 1.3.1.4 
 
 
Figure 1.3.1.4 Selective Enrichment of C34:CXCR4 T-cells. A) Cells transduced with C34-CXCR4 or 
GFP were added to untransduced cells to reach a final percentage of 25% transduced cells and inoculated 
with JRFL. Cells were monitored for enrichment using flow cytometry for GFP or C34, and also monitored for 
HIV infection by intracellular gag staining. Expansion of C34-CXCR4 cells is shown over time.  No 
expansion of GFP-transduced cells was seen.  B) Enrichment over time is shown graphically. C) This 
experiment was replicated with multiple strains of HIV with various tropisms, each resulting in dramatic 
enrichment of the C34:CXCR4-modified cells. 
 
 
1.3.3 C34-CXCR4 inhibition of enfirvutide (T-20)-resistant HIV-1 
Acquired HIV-1 resistance to the fusion inhibitor enfirvutide (T-20) has been observed both in 
vitro and in vivo, and is generally associated with mutations within a 10 amino acid motif (aa 26-
45) in the viral gp41 (Perez-Alvarez, Carmona et al. 2006, Melikyan 2008).  Three mutations 
associated with T-20 resistance in HIV-infected patients receiving antiretroviral therapy were 
tested to determine whether infection by these mutated strains was susceptible to inhibition by 
C34-CXCR4.  As shown in Figure 1.3.3, all T-20 resistant strains tested were highly susceptible 
to C34-CXCR4 inhibition. 

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1.3.4 In vivo data in humanized NSG mice  
To evaluate the ability of C34-linked chemokine co-receptors to protect CD4 T cells in vivo, 
experiments were performed using a previously described NSG model that has been used to 
evaluate the ability of a wide range of antiviral agents to protect T cells from HIV-1 infection  
(Perez, Wang et al. 2008, Richardson, Carroll et al. 2008, Mukherjee, Plesa et al. 2010, Wilen, 
Wang et al. 2011, Didigu, Wilen et al. 2014, Richardson, Guo et al. 2014). We infused 10 million 
untransduced, GFP-transduced, or C34-CXCR4 transduced T cells into cohorts of mice.  Once 
T cell engraftment was confirmed and enumerated (Fig 1.3.4 A), the mice were challenged with 
HIV.  After eight days, viral load was measured at a time when CD4 T cell levels were 
comparable (Fig 1.3.4 B).  We observed that mice engrafted with C34-CXCR4 expressing T 
cells had significantly lower viral loads than mice engrafted with either untransduced or GFP-
transduced T cells (Fig 1.3.4 C).  After an additional 20 days, the mice were sacrificed and the 
number of human CD4 T cells in the spleens was measured (Fig 1.3.4 D).  The spleens were 
chosen because they contain the majority of human T cells in this model.  Mice engrafted with 
C34-CXCR4 expressing T cells had remarkably more T cells than mice infused with 
untransduced or GFP-transduced T cells.  The in vitro data indicated that C34-CXCR4 was 
superior in protecting T cells from HIV infection when compared to C34-CCR5. In a second 
experiment, we wished to directly compare whether C34-CCR5 or C34-CXCR4 transduced T 
cells had a survival advantage in vivo following HIV-1 infection over control T cells transduced 
with GFP.  After infusion of T cells and HIV infection, mice were then bled at 10-day intervals to 
assess T cell survival and expansion (Figure 1.3.4 E).  Remarkably, CD4 T cells expressing 
C34-CCR5 survived poorly, similar to T cells expressing GFP.  However, in marked contrast, 
C34-CXCR4 transduced cells persisted throughout the period of HIV-1 infection in both the 
peripheral blood and in the spleens (Fig 1.3.4 F, G).  Together, this data demonstrates that T 
cells expressing C34-CXCR4 are highly resistant to HIV-1 infection in vivo. 

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sponsor. 
Figure 1.3.4 
 
Figure 1.3.4.  C34-CXCR4 expressing cells are protected from HIV mediated destruction in vivo.  A) 
NSG mice were infused with 10 million CD4 T cells that were either untransduced, GFP expressing, or C34-
CXCR4 expressing CD4 T cells. ~85% of each transduced population expressed the indicated marker. 
Engraftment was assessed (Day -7) and one week later all mice were infected with a viral mix of HIV-1 US1 
(R5) and CMU-02 (X4).  8 days later mice were bled and both B) Number of modified T cells C) and viral 
load were determined.  D) 28 days post HIV-1 infection the mice were sacrificed and the number of T cells in 
the spleen were enumerated.  In separate experiment, NSG mice were infused with 10 million CD4 T cells 
that were either GFP, C34-CCR5 or C34-CXCR4 expressing CD4 T cells.  Engraftment was checked (Day 
0) and two days later mice were infected with a viral mix of HIV-1 US1 (R5) and CMU-02 (X4).  After 3 
weeks of HIV infection, the mice were sacrificed and the number of T cells in the peripheral blood (F) and 
spleen (G) were enumerated. 

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1.4 
Clinical Data to Date 
There are no available clinical research data to date on the investigational product. However 
there are extensive early phase clinical trial data regarding the use of autologous T-cell 
infusions for HIV as described earlier in the background section of this protocol. 
 
1.5 
Rationale and Risks/Benefits 
1.5.1 Study Population Rationale 
This protocol is designed to determine the safety of a single administration of C34-CXCR4 
modified cells in individuals with well controlled HIV replication.  This patient population was 
selected because it is ultimately the target population in which these types of interventions will 
be used if they prove to be effective.  It is also the population that we have successfully enrolled 
in our prior trials.  Additionally from our experience, individuals with well controlled HIV 
replication that are willing to participate in gene therapy studies are highly motivated, are willing 
to take the risk(s) associated with this type of study, and have the commitment to adhere to the 
close clinical monitoring required. We believe that selecting this particular population is what 
makes the study feasible. 
 
Immune activation has long been correlated with HIV disease progression (Fahey, Taylor et al. 
1998), and the etiology of immune activation and its role in HIV pathogenesis is an active area 
of investigation which suggests that reducing immune activation may be a critical adjunct 
approach to preventing HIV disease progression (Brenchley, Price et al. 2006, Chehimi, Azzoni 
et al. 2007, Meier, Alter et al. 2007, Milush, Reeves et al. 2007).  Chronic immune activation is 
thought to be the cause of the increased susceptibility of PBMCs to apoptosis in HIV infected 
individuals.  In HIV-1 and HIV-2 infected subjects, there is a statistically increased rate of 
apoptosis in PBMCs that is positively correlated with both disease stage (3/4, 2, or normal 
donor) and CD4 counts (>500, 200-500, and <200) (Gougeon 1996, Michel, Balde et al. 2000). 
Taking these data into consideration, it follows that later stage patients or patients with more 
advanced HIV disease are less likely to have healthy immune cells capable of supporting cell-
based immunotherapy over the long-term.  A primary endpoint of this study is to evaluate the 
long-term safety of C34-CXCR4-modified CD4+ T-cells, which will require long-term persistence 
of these cells. Therefore, subjects with a current or prior AIDS diagnosis, or those with a CD4 
nadir lower than 200 cells/mm3 will be excluded from the study.   
 
We will enroll one cohort in this study.  Within the cohort will be a maximum of 3 escalated 
doses of C34-CXCR4-modified CD4+ T-cells:  Dose Level 1 = 0.8-1x109; Dose Level 2 = 2.4-
3x109; and Dose Level 3 =0.8-1x1010.  If no DLT are observed, three subjects will be treated at 
each dose, for a total of 9 subjects in the study. For dose levels 1 and 2, if 0/3 has a dose 
limiting toxicity (DLT), then the dose will be escalated. If 1/3 has a DLT at a dose level then 3 
additional patients will be treated at that dosage before escalating, and if <2/6 have DLT (i.e. no 
additional DLT is observed) then the dose will be escalated to the next planned dose level until 
the maximum of 9 evaluable subjects has been reached. 
 
In Step 3, all participants will participate in a 16-week analytical treatment interruption beginning 
4 weeks after T-cell infusion. After the analytical treatment interruption (ATI), resumption of 
antiretroviral therapy will be recommended and participants will be closely followed until their 
HIV RNA falls below the limit of quantification. One year after T-cell infusion subjects will 
undergo leukapheresis to evaluate the long-term persistence of C34-CXCR4-modified CD4+ T-
cells.  After completing this study the patients will be transferred to a destination protocol for the 
long term follow-up of individuals that have been exposed to gene therapies. 

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Risks associated with C34-CXCR4-T are in Section 12.1. Risks associated with treatment 
interruption are discussed in Section 1.5.3. 
 
1.5.2 Dose Rationale  
The starting dose was established based on: 1) Guidance for Industry: S6 Preclinical Safety 
Evaluation of Biotechnology-derived Pharmaceuticals; 2) Joint taskforce report from the 
BioIndustry Association (BIA) and the Association of the British Pharmaceutical Industry (ABPI) 
and 3) the available literature on phase I trials to date conducted with autologous T-cells in 
patients with HIV.  The FDA guidance on safe starting doses (FDA Guidance for Industry: 
Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult 
Healthy Volunteers) does not apply well for cell and gene therapies, and as such was not used 
to guide the selection of the starting dose for this study.  
  
Engraftment of 1-2% of genetically modified cells in the peripheral circulation has been 
observed following infusions of about 10 billion cells (Levine, Humeau et al. 2006), and higher 
cell doses result in higher levels of engraftment (Ranga, Woffendin et al. 1998, van Lunzen, 
Glaunsinger et al. 2007). In order to effectively detect any potential toxicity or tolerability issues, 
achieving measurable engraftment levels is necessary.  However, exposure should be limited 
so as to reduce any potential C34-CXCR4 associated toxicities.  Therefore, in the present study, 
we are proposing a dose escalation approach whereby participants receive a single dose of 
cells (either 0.8-1 x109, 2.4-3x109, or 0.8-1x1010 cells; three subjects per dose), as this dose 
range is known to result in measurable but limited engraftment levels based upon our prior 
experience.  
 
Safety.  Safety is enhanced by using a 3+3+3 dose escalation.  If there are no safety concerns 
regarding the 0.8-1x109 dose by completion of the Day 21 study visit of the 3rd subject, then this 
dose will be increased to 2.4-3x109 in the next 3 subjects.  If no safety concerns are raised at 
that dose by the completion of the Day 21 study visit of the 3rd subject (6th subject overall) to 
receive the 2.4-3x109 dose, the dose will be increased once more to 0.8-1x1010 in the final three 
subjects. 
 
1.5.3 Risks of Analytical Treatment Interruption 
Discontinuation of ART may result in viral rebound, immune de-compensation, increase in the 
size of the latent reservoir, and clinical progression. In HIV-infected subjects, an ART 
interruption is not recommended unless it is done in a clinical trial setting. Interruptions in a 
clinical trial setting have been termed “analytical treatment interruptions” (ATI) and are accepted 
tools in the evaluation of immunological interventions, gene therapy or therapeutic vaccines for 
the treatment of HIV infection (Kutzler and Jacobson 2008). In order to minimize the risk 
associated with treatment interruptions, the duration of these interruptions is kept to a maximum 
of 16 weeks. This duration allows the subject to reach a new viral load (VL) “set point,” which is 
defined by the AIDS Clinical Trial Group as the mean of week 12 and week 16 post treatment 
interruption values (Henry, Katzenstein et al. 2006). VL setpoint changes are the usual endpoint 
evaluated in these types of studies. Another frequently used endpoint is time to return of 
viremia, which uniformly occurs within 2 to 3 weeks of discontinuation of antiretroviral therapy, 
with kinetics that are well characterized.  In most cases viral replication is re-supressed with 
resumption of ART (Davey, Bhat et al. 1999, Harrigan, Whaley et al. 1999, Papasavvas, 
Kostman et al. 2004, Henry, Katzenstein et al. 2006, Jacobson, Pat Bucy et al. 2006, Strategies 
for Management of Antiretroviral Therapy Study, El-Sadr et al. 2006).  
 

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Temporary treatment interruption to reduce inconvenience, potential long-term toxicity, and/or 
overall treatment cost was considered as a strategy for subjects on ART who had maintained 
high CD4+ counts. Several clinical trials were designed to determine the safety of such 
interruptions, in which reinitiation is triggered by predetermined CD4+ count thresholds 
(Maggiolo, Ripamonti et al. 2004, Ananworanich, Siangphoe et al. 2005, Cardiello, Hassink et 
al. 2005, Strategies for Management of Antiretroviral Therapy Study, El-Sadr et al. 2006, 
Holkmann Olsen, Mocroft et al. 2007, Pogany, van Valkengoed et al. 2007, Skiest, Su et al. 
2007, Walmsley, Thorne et al. 2007). In these trials, various CD4+ count levels have been set to 
guide both treatment interruption and reinitiation. Several separate, randomized clinical trials of 
CD4+ count-guided treatment interruption have been reported. In the SMART study (Strategies 
for Management of Antiretroviral Therapy Study, El-Sadr et al. 2006), the largest of such trials 
with over 5,000 subjects, interrupting treatment with CD4+ counts >350 cells/μL and reinitiating 
when <250 cells/μL was associated with an increased risk of disease progression and death 
compared with the trial arm of continuous ART. However, most of these events tended to occur 
more than 16 weeks after the treatment interruption (Figure 2.1). 
 
 
Figure 2.1 Cumulative probabilities of AEs following CD4-guided ARV interruption (Copied 
from El-Sadr et al, 2006).  Data were collected from the SMART trial and show AEs in subjects on 
continual ARV (blue) or subjects with CD4+ guided ARV (red).  AEs include opportunistic infection 
or death from any cause (A), death from any cause (B), major cardiovascular, renal, or hepatic 
disease (C), or grade 4 AEs (D).  Of note, at 16 weeks (shown as 4 months) there is little difference 
between the two groups.   
 
The participants in the SMART study had more advanced disease than the subjects we will 
include in our trial, and the treatment interruption was much more prolonged. We believe the 
subjects in this study will be at very low risk of developing clinical events during the brief 
treatment interruption proposed. For additional subject safety, both CD4+ T-cell counts and HIV-
1 VLs will be monitored closely in the ATI period and subjects with a confirmed CD4 T-cell count 
≤350 cells/ mm3, will be advised to reinstitute ART. The threshold of 350 cells/μL, is higher than 

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250 cells/ mm3 in the SMART study to provide a greater level of safety. Additionally, if viremia 
resumes at or above a threshold of 100,000 copies/ml, ART will be resumed. It is also possible 
that some subjects could experience symptoms compatible with retroviral rebound syndrome or 
rarely, other clinical events that occur before resumption of ART (eg, immune 
thrombocytopenia). 
 
Although in vitro studies allow assessment of the antiviral activity of C34-CXCR4, the best way 
to evaluate the in vivo activity of these cells is to discontinue ART for a brief period of time in 
individuals with well-controlled viral replication. This then allows investigation of the ability of 
these gene modified cells to control or prevent the initiation of viral replication and to 
characterize the survival of C34-CXCR4-modified CD4+ T-cells (C34-CXCR4-T) in the presence 
of HIV infection. 
 
A single ATI, as proposed in our study, remains appropriate to evaluate the antiviral activity of 
C34-CXCR4-modified CD4+ T-cells as a treatment intervention for three reasons: 
 
1. Upon entering into the trial, all subjects will have CD4+ counts ≥450 cells/ mm3 and will 
have a nadir CD4+ cell count of not lower than 200 cells/ mm3. The subject will be 
advised to reinitiate HAART treatment as soon as the CD4+ T-cell count drops below 
350 cells/ mm3 or the HIV-1 VL increases to ≥100,000 copies/mL and remains as such 
for 3 successive weeks (see Section 7.1). Selecting subjects who were started on ART 
with CD4+ counts of not lower than 200 cells/ mm3 will increase the likelihood that 
subjects will have reconstituted immune responses.  
 
2. All subjects will receive C34-CXCR4-modified CD4+ T-cells; an intervention that we 
hope will improve the outcome of treatment interruption. In this proof of concept study 
we will not utilize a placebo group, as the kinetics and the frequency of reactivation 
during an ATI in chronically infected individuals are well known. 
 
3. All subjects will be followed very closely after the treatment interruption, and therapy will 
be restarted if any problems appear. It is very unlikely that the participants will develop 
resistance to any of the antiretrovirals that they have been receiving. 
 
Importantly, in our previous CCR5 ZFN Trial (NCT00842634) the ATI was for 12 weeks.  At the 
end of the 12 weeks, although the viral load was declining, the protocol required that all subjects 
re-initiate ART.  This may have prevented us from fully documenting the antiviral effects of the 
CCR5 ZFN treatment.  By extending the ATI to 16 weeks, we believe that we can better define a 
new set point as well as results of the treatment, while not compromising subject safety. 
 
In this protocol, CD4+ cell counts will be monitored closely in the treatment interruption period 
and patients in whom CD4+ cell counts drop to 350 cells/mm3 or below, and who are confirmed 
to remain at or below 350 cells/ mm3 with a second reading a week later, will be advised to 
reinstitute ART.  Additionally, if a subject’s viral load increases to ≥100,000 copies, and remains 
as such for 3 successive weeks, the subject will be advised to restart ART.  Those subjects who 
fail to complete the 16 week ATI due to viral rebound will be tested for antiretroviral therapy 
resistance.  The purpose of the treatment interruption is to serve as an analytic tool to evaluate 
both the effects of the study drug on the subject’s virus and the virus on the genetically modified 
T-cells.  The most stringent test of an immunologic intervention like the one proposed in this 
study would be its ability to lead to sustained suppression of plasma HIV-1 RNA after the 

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discontinuation of antiretroviral therapy, and/ or the protection of the genetically modified CD4 in 
the presence of ongoing viral replication.  
 
During the treatment interruption, it is very likely subjects will have detectable viral loads.  Given 
the positive correlation between viral load and HIV transmission (Quinn, Wawer et al. 2000), use 
of condoms during sexual activity will be stressed to subjects undergoing ATI to minimize the 
risk transmission.   
 
It is also possible subjects may develop Acute Retroviral Syndrome (ARS), during which the HIV 
virus is duplicating at a rapid rate.  Acute Retroviral Syndrome is a diagnosis that must be made 
by a site investigator or primary care provider.  Signs and symptoms that may support the 
diagnosis of ARS include: 
 
Fever 
 
Sore throat 
 
Headache 
 
Rash 
 
Diarrhea 
 
Swollen lymph nodes 
 
Fatigue 
 
Joint or other body aches 
 
Antiretroviral therapy will be restarted if the symptoms persist for more than one week, or affect 
the normal activities of daily living for more than one week. 
 
1.5.4 Examination of Lymphoid Tissue by Rectal Mucosal Biopsy 
It has been documented that HIV preferentially infects activated, memory CD4 cells.  Memory 
cells are defined physiologically as those lymphocytes that have seen antigen, and 
phenotypically by CD45RO expression and high levels of CD62L expression. Generally these 
cells migrate to tertiary lymphatic sites such as the lamina propria and skin. The reason for the 
preferential infection by HIV-1 is probably related to the high density on these cells of the 
chemokine receptor CCR5, the co-receptor used by HIV throughout most of the course of 
infection.  However, the cell culture process that is used in this study results in downregulation 
and undetectable expression of CCR5 for at least 6 weeks in vivo (Levine, Bernstein et al. 
2002).  In terms of survival of CD4 cells transduced with a retroviral vector, studies in a rhesus 
model showed persistence of marked cells in lymph nodes for 100 days (Bunnell et al., 1997; 
Donahue et al., 1998).  
 
Lymphoid tissue, particularly gut-associated lymphoid tissue (GALT), rather than the blood is 
the dominant site of HIV-1 replication, and lymphoid tissue serves as the major reservoir of 
productively infected cells in the body. Only 2% of the body’s total T cells circulate in the 
peripheral blood while the remainder are found in lymphoid tissue such as the lymph nodes, 
tonsils, and gut mucosa. The colon is known to have a large number of lymphoid nodules. In 
addition, the colonic mucosa has high concentrations of intra-epithelial lymphocytes (IEL), which 
are predominately CD8+T-cells and lamina propria lymphocytes (LPL) which are predominately 
CD4+ T-cells. Thus, the GALT may serve as an important and accessible reservoir of HIV-1 
infected lymphocytes and C34-CXCR4 gene modified T-cells. The advantages of performing 
rectal mucosal biopsies as opposed to lymph node or tonsillar biopsies include greater patient 
acceptance as the procedure is less painful; relative ease of repetitions; and fewer associated 
complications (Rodriguez-Rosado, Jimenez-Nacher et al. 1998).  

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Following infection with HIV, depletion of CD4 T-cells occurs very rapidly and predominantly in 
the gastrointestinal tract (Lim, Condez et al. 1993, Brenchley, Schacker et al. 2004). 
Reconstitution of the GALT typically takes 8-12 months in chronically infected patients, although 
early treatment with HAART during primary infection avoids this problem (Guadalupe et al., 
2003). The depletion is accompanied by an increased overall immune activation which is 
thought to contribute to the inability of CD4 T-cells to repopulate the GALT (Guadalupe, 
Sankaran et al. 2006).  Chronic immune activation resulting from a breakdown of the gut 
immune system has been cited as a possible cause of chronic immune activation and thus 
disease progression in HIV-infected patients (Brenchley, Price et al. 2006).  Notably, one study 
discovered that in a small group (3) of long term non progressors, such patients maintain their 
GALT CD4 population (Guadalupe, Reay et al. 2003).  Reconstitution of the GALT appears to 
occur by homing of circulating cells to the GALT as opposed to local expansion of endogenous 
CD4 cells.  Thus, enhancing or blocking the ability of circulating CD4 T-cells to home to the gut 
may have implications for overall disease progression.  In kind, establishing a baseline effect on 
gut trafficking will be important in evaluating the overall potential of the proposed HIV cell based 
therapy. 
 
Results from a phase II trial of CD4-zeta modified autologous T-cells demonstrate trafficking of 
CD4-zeta modified cells and quantifiable HIV-1 in the rectal mucosa of subjects with HIV-1 
infection and undetectable viral replication in plasma (Deeks, Hoh et al. 2002).  Others have 
successfully measured HIV proviral load in the GALT to evaluate HIV burden and its possible 
relationship with clinical outcome (Anton, Mitsuyasu et al. 2003).  Similarly, in our ongoing HIV 
gene therapy trial utilizing lentiviral vector transduced CD4 T-cells, we have observed trafficking 
of gene modified cells to the GALT and are performing ongoing measurements on proviral load 
that reflect the data published in the Anton studies.  In that study, we also worked out the 
procedures necessary to perform quantitative analysis in the compartment.  In this protocol, we 
plan on performing sequential rectal mucosal biopsies on all patients in accordance with the 
Schedule of Events, and will analyze the mucosal T-cell viral load, C34-CXCR4 CD4 cell 
engraftment and, if enough cells are available, cellular responses as described previously 
(Shacklett, Yang et al. 2003). 
 
The primary risks associated with rectal mucosal biopsies include mild rectal irritation and 
urgency and limited rectal bleeding for 2-3 days following the procedure. Infection or bowel 
perforations are extremely rare complications that may require antibiotics and/or surgical repair. 
Subjects will be followed in clinic for any complications. Although four rectal mucosal biopsies 
are planned for all subjects, the subject may decide at any point to have no further biopsy 
procedures. Prophylactic antibiotics should be given to subjects who would routinely be given 
such treatment prior to invasive procedures (e.g. cardiac valvular disease). 
 
1.5.4.1 Viral Persistence in Mucosal Tissues and Its Clinical Relevance 
Given the concentrated nature of susceptible CD4+ T-cells in the gut mucosa, most HIV 
replication is thought to occur in this compartment.  This is particularly true during acute HIV 
(and SIV) infection in which high-level viral replication results in depletion of 60% to 90% of 
CD4+ T-cells during the first few weeks (Veazey, DeMaria et al. 1998, Guadalupe, Reay et al. 
2003, Brenchley, Schacker et al. 2004, Mehandru, Poles et al. 2004, Li, Duan et al. 2005, 
Mattapallil, Douek et al. 2005).  This process involves both activated and resting T-cells (at least 
initially) (Mehandru, Poles et al. 2007), and may even result in destruction of epithelial cells (Li, 
Duan et al. 2005) as well as the irreversible fibrotic changes to the lymphoid architecture 
(Brenchley, Schacker et al. 2004, Estes, Baker et al. 2008).  The degree to which this process is 
reversed by HAART is controversial, with some studies suggesting irreversible damage to the 

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gut mucosa (Brenchley, Schacker et al. 2004, Mehandru, Poles et al. 2004, Mehandru, Poles et 
al. 2006, Brenchley and Douek 2008) while other studies suggesting that if therapy is started 
early enough (Estes, Baker et al. 2008),or if therapy is given for many years (Sheth, Chege et 
al. 2008), then normalization of the certain mucosal T-cell populations may occur.   
 
The relationship between viral persistence and gut immune reconstitution during HAART is also 
poorly defined.  HIV RNA and DNA can often be readily detected in gut tissue, even after years 
of HAART (Anton, Mitsuyasu et al. 2003).  Viral DNA levels are typically higher in gut tissues 
than peripheral blood (as measured on a per CD4+ T-cell basis) (Chun, Nickle et al. 2008, 
Macal, Sankaran et al. 2008). As has been observed in peripheral blood, viral burden in the gut 
mucosa appears to remain stable during long-term therapy (Poles, Boscardin et al. 2006). The 
degree to which viral persistence in gut mucosa reflects ongoing viral replication is not known, 
but the consistent association between measures of virus burden and either T-cell reconstitution 
or T-cell activation (Macal, Sankaran et al. 2008, Sheth, Chege et al. 2008) suggests that viral 
expression in these tissues may have clinical consequences (Sheth, Chege et al. 2008).  
 
These observations indicate (1) that antiretroviral therapy does not fully restore normal immunity 
(Alexander, Ortiz et al. 2003), (2) that suboptimal CD4 gains during therapy may predict 
premature morbidity and mortality, (3) that suboptimal CD4 gains during therapy is common in 
clinical practice, especially as patients age and (4) that virus production persists during therapy, 
with much of this occurring in gut mucosa. Although the association between virus production in 
gut and immune reconstitution remains undefined, it is clear that many treated patients with 
poor immune reconstitution have elevated levels of markers of inflammation and of persistent 
microbial translocation. Indeed, in a series of recent papers, the inability to restore peripheral 
blood CD4 was predicted by persistent markers of microbial translocation of gut microbes 
across the gut mucosa (Macal, Sankaran et al. 2008, Jiang, Lederman et al. 2009, Nies-Kraske, 
Schacker et al. 2009).  It is hypothesized that reconstitution of effective CD4 T-cell function in 
gut mucosa will be needed to restore long-term health in the antiretroviral treated patients. 
 
 
2.0 
STUDY OBJECTIVES  
The study objectives were defined according to the hypothesis that forms the basis of this study. 
Our hypothesis is that autologous CD4+ T-cells genetically modified with lentiviral vector 
expressing an HR2, C34-peptide conjugated to the CXCR4 N-terminus will be safe and 
tolerable in HIV-1 positive subjects and will have a survival advantage in the presence of 
viremia. 
 
2.1 
Primary Objectives 
The primary objective of this study is to evaluate the safety and tolerability of a single infusion 
ranging from 0.8x109 to a maximum of 1x1010 of autologous CD4+ T-cells genetically modified 
with C34-CXCR4 in HIV-1-positive subjects.  
 
2.2 
Secondary Objectives 
The secondary objectives of this study are: 
 
1. To evaluate the enrichment of C34-CXCR4-modified CD4+ T-cells during 
analytical treatment interruption by comparing the percentage of modified cells in 
subjects prior to ATI (week 2 post infusion) to the percentage of modified cells just 

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prior to the reinitiation of antiretroviral therapy or at weeks 16 and 20 after the 
administration of modified CD4 positive T-cells (weeks 12 and 16 of the ATI), 
whichever comes first, using flow cytometry,  and determine the half-life of modified 
cells compared to unmodified CD4 cells in the presence of HIV viremia during an 
analytical treatment interruption.  Modified cells from both the peripheral blood and 
gut mucosa will be assessed. 
 
2. To evaluate the effect of C34-CXCR4 modified CD4 cells on CD4+ T-cell count : 
 
a) Comparing the change between baseline CD4+ T-cell count (as above) and 
the average of two consecutive CD4+ T-cell count values after dosing and just 
prior to the analytical treatment interruption (Step 2) and prior to the reinitiation 
of antiretroviral therapy or at weeks 16 and 20 after the administration of the 
modified CD4 positive T-cells (weeks 12 and 16 of the analytical treatment 
interruption, Step 3), whichever comes first. 
 
3. To evaluate the antiviral effect of C34-CXCR4-modified CD4+ T-cells by: 
 
a) Comparing the viral set point log10 HIV-1 RNA level (defined as the median of 
values available before the initiation of antiretroviral therapy) and the set point 
after the intervention, during the analytical treatment interruption. The new viral 
set point will be defined as the average of two consecutive viral load 
measurements of log10 HIV-1 RNA levels after dosing and just prior to the 
reinitiation of antiretroviral therapy or at weeks 16 and 20 after the 
administration of the modified CD4 positive T-cells (weeks 12 and 16 of the 
analytical treatment interruption), whichever comes first. 
b) Comparing the time and the frequency to recrudescence to >200 copies/ml 
viral load with historical controls. 
 
4. To evaluate the effects on immune function of C34-CXCR4- modified CD4 cells by 
comparing the CD4+ and CD8+ T-cells responses following stimulation by HIV-
specific and other viral antigens, at baseline (defined as the average of the Step 1 
values), and after the infusion of C34-CXCR4-modified autologous T-cells. The 
responses will be evaluated by intracellular cytokine staining for cytokines and/or 
CD107a in response to stimulation with HIV peptide pools of 15 mers overlapping by 
11aa, as well as peptide pools for Flu, EBV and CMV. In addition, proliferative 
capacity will be assessed. 
 
 
The immune response comparisons will be made using descriptive statistics and 
graphical methods for both phases of the study. Immune response measures will be 
correlated with dose level/treatment arm and participant outcome (i.e., adverse 
events) and with each other as well.  
 
For continuous immune response values, the actual and % change in the level of 
each of the biomarkers from baseline to post‐baseline time points will be explored 
within each dose level/arm. For all translational endpoints, any notable statistical 
result will be viewed as an impetus for further study rather than as a definitive 
finding. 
 
5. To evaluate the immunogenicity of C34-CXCR4-modified CD4 T-cells, analysis will 

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be done in parallel on cryopreserved patient sera from baseline and various post-
infusion timepoints with CD4 T-cells transduced with lentiviral C34-CXCR4. The flow 
cytometry assay will utilize A66 SupT1 cells transduced with either the lentiviral C34-
CXCR4 construct or a CXCR4 construct.  Human antibodies binding to the former 
but not the latter will be considered C34-specific.  The SupT1 cells will be incubated 
with dilutions of patient-derived sera to be empirically determined, washed, and cell 
aliquots then incubated separately with anti-human IgG, IgM, IgA and IgE reagents.  
The advantage of this cell-based assay over ELISA using plates coated with purified 
C34-CXCR4 is that antibodies to conformational epitopes are more likely to be 
detected.  This flow assay will be based on an assay developed and qualified in the 
UPenn Translational and Correlative Studies Laboratory to evaluate human-anti 
chimeric antibodies (HACA) to mesothelin-specific chimeric antigen receptor mRNA-
engineered T-cells (Beatty, Haas et al. 2014).  
 
2.3 
Exploratory Objectives 
1. Studies will be initiated to evaluate the effects of C34-CXCR4-modified CD4+ T-cells 
on the HIV reservoir. The size of the reservoir will be measured using the following 
techniques: 
 
a) Number of latently-infected resting CD4 T-cells quantified using a limiting 
dilution co-culture assay (comparing Step 1 to Step 2). 
b) Frequency of integrated provirus as measured by Alu-PCR (comparing Step 1 
to Step 2). (Liszewski, Yu et al. 2009)  
 
 
3.0 
STUDY DESIGN 
This is a single cohort, open-label pilot study of the safety and antiviral activity of a single 
infusion of autologous CD4+ T-cells genetically modified with lentiviral vector expressing an 
HR2, C34-peptide conjugated to the CXCR4 N-terminus in HIV-infected subjects. There will be 
1 cohort in this study consisting of subjects with well-controlled HIV replication on HAART.  
Infusions will be staggered by at least 21 days, to allow for the previous subject to be sufficiently 
evaluated for adverse events before infusion of the next subject.  This 21 day inter-subject 
interval will allow for the capture of any delayed adverse events.  If any serious adverse sevents 
arise that pose a safety concern, or fall under the criteria for pausing or stopping the study, a 
DSMB meeting will be convened to review study safety data.This study will have five steps: 
 
In Step 1, all participants will undergo 2 leukaphereses and an optional rectal biopsy to obtain 
CD4 positive T-cells that will be genetically modified and to have baseline specimens to 
evaluate the size of the HIV reservoir. 
 
In Step 2, all participants will receive a single infusion of C34-CXCR4-modified CD4+ T-cells at 
one of 3 dose levels.  The first 3 subjects will receive dose level 1 of 0.8-1x109 transduced 
CD4+T-cells.  Provided no dose limiting toxicity (DLT) is seen at the first dose level at the 
completion of the Day 21 study visit of the 3rd subject, the next 3 subjects will receive infusion at 
the 2nd dose level of 2.4-3x109 transduced CD4+ T-cells.  If no DLT occurs at that dose by the 
completion of the Day 21 study visit of the 6th overall subject infused, the final 3 subjects will 
receive the 3rd dose level of 0.8-1x1010 transduced CD4+ T-cells.  In the event of a DLT (grade 

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3 or higher unexpected, related AE) recruitment will be paused pending DSMB review and 
decision to proceed.   
 
At the end of Step 2 all participants will undergo mini-leukapheresis and optional rectal biopsy. 
 
In Step 3 all participants will participate in a 16 week analytical treatment interruption beginning 
4 weeks after T-cell infusion. At the end of Step 3 all participants will undergo mini-
leukapheresis and optional rectal biopsy. 
 
In Step 4 all participants will be advised to resume antiretroviral therapy and be followed until 
plasma HIV RNA fall below the limit of detection. 
 
Note:  For subjects with viral loads ≤1000 copies/ml at the end of 16 weeks, re-initiation 
of antiretroviral therapy will be recommended.  However, resumption of ART medications 
will be left to the discretion of the subject and their physician.  If a subject declines to 
restart antiretroviral medications, the subject will be followed in Step 3 with monthly visits 
until ART is resumed, in an effort to fully capture the impact of the intervention.  These 
monthly visits should capture all procedures outlined in the Week 16 visit of the schedule 
of events.  The study team will discuss re-initiation of ART with the subject at each visit.  
The subject may choose to restart ART at any time, and thus would move to Step 4 of 
the study when antiretroviral medication is resumed.  All procedures outlined in the ATI 
Discontinuation visit should be performed just prior to restarting antiretroviral 
medications (Step 4), if possible. 
 
In Step 5 all participants will undergo leukapheresis and an optional rectal biopsy at 52 weeks 
post infusion and then be followed long-term as required by regulatory authorities. 
 
This study will consist of subjects who are well controlled on HAART.  Each will receive a single 
dose of C34-CXCR4-modified CD4+ T-cells, followed by analytical treatment interruption of 16 
weeks. Dosing of C34-CXCR4-modified cells will occur in a 3+ 3 +3 dose escalation format, 
capped at 9 evaluable subjects.  Dose level 1 consists of 0.8-1x109 cells, dose level 2 consists 
of 2.4-3x109 cells, and dose level 3 contains 0.8-1x1010 cells.  At each dose level, given no 
observed DLT, three patients will be treated. For dose levels 1 and 2, if 0/3 has a dose limiting 
toxicity (DLT), then the dose is escalated. If 1/3 has a DLT at a dose level then 3 additional 
patients will be treated at that dosage before escalating, and if <2/6 experience DLT (i.e. no 
additional DLT is observed) then the dose will be escalated to the next planned dose level, until 
the 9 evaluable subjects have been reached.   
 
After enrollment, subjects will undergo leukapheresis (approximately a 10L volume) to obtain 
large numbers of PBMCs.  To ensure sufficient T-cells in the event of a manufacturing failure, a 
second leukapheresis will be performed.  If the second leukapheresis is not needed for 
manufacturing, a small scale apheresis (approximately a 5L volume) will be performed instead 
to serve as a cell bank for baseline controls for immunological evaluations. The second 
leukapheresis will be scheduled approximately 3 weeks after the first leukapheresis.  CD4+ T-
cells will be purified from the PBMCs, genetically modified using lentiviral transduction with the 
C34-peptide conjugated to the CXCR4 N-terminus, expanded in vitro and then frozen for future 
administration. Within approximately 15 weeks after enrollment confirmation, subjects will 
receive a single infusion of C34-CXCR4-modified CD4+ T-cells.   
 

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Subjects will have clinical blood tests to assess safety. Research tests such as HIV-1 RNA 
levels and CD4+ T-cell counts, HIV-1-specific CD4+ and CD8+ T-cell responses, and the 
proportion of circulating CD4+ T-cells that contain the genetically modified cells will be assessed 
at regular intervals as detailed in the schedule of events section.  Subjects will be enrolled in the 
study in order to obtain a maximum of 9 evaluable subjects who complete the infusion of C34-
CXCR4-modified CD4+ T-cells and undergo treatment interruption.  Any subject who 
prematurely discontinues the study prior to the infusion will be replaced with another subject.  
Evaluable subjects are defined as those who receive infusion of C34-CXCR4 modified CD4 T-
cells and undergo treatment interruption.  Although it is standard across our protocols to allow 
for a 20% dropout rate, there were no such discontinuations in our previous CCR5 ZFN study 
(NCT00842634).  Subjects who discontinue ATI prior to the 16 week duration will not be 
replaced. 
 
3.1 
Monitoring for delayed adverse events associated with lentiviral 
vector gene transfer 
After the completion of the analytical treatment interruption, subjects will have their white blood 
cells monitored for vector sequences to evaluate whether there is an increase in marking, which 
may indicate a growth advantage, possibly from a transforming event associated with 
integration. In addition, patients will be monitored for evidence of a vector derived RCL. 
Monitoring for these events will occur for 12 months under this protocol, and then for up to 15 
years under a separate long-term follow-up protocol, as described under the Clinical 
Management Issues (Section 7) section of this protocol.  
 
 
4.0 
SELECTION, ENROLLMENT, AND WITHDRAWAL OF SUBJECTS 
4.1 
Inclusion Criteria  
4.1.1 HIV-1 infection, documented by any licensed rapid HIV test or HIV enzyme or 
chemiluminescence immunoassay (E/CIA) test kit at any time prior to enrollment 
and confirmed by a licensed Western blot or a second antibody test by a method 
other than the initial rapid HIV and/or E/CIA, or by HIV-1 antigen, plasma HIV-1 
RNA VL. 
 
NOTE: The term “licensed” refers to a US FDA-approved kit.  
 
WHO (World Health Organization) and CDC (Centers for Disease Control and 
Prevention) guidelines mandate that confirmation of the initial test result must 
use a test that is different from the one used for the initial assessment. A reactive 
initial rapid test should be confirmed by either another type of rapid assay or by 
an E/CIA that is based on a different antigen preparation and/or different test 
principle (e.g., indirect versus competitive), or a Western blot or a plasma HIV-1 
RNA assay.  Alternatively, if a rapid HIV test or any FDA-approved HIV-1 
Enzyme or Chemiluminescence Immunoassay (E/CIA) test kit is not available, 
two HIV-1 RNA values ≥ 2000 copies/mL at least 24 hours apart performed by 
any laboratory that has CLIA certification, or its equivalent, may be used to 
document infection. 
 
4.1.2 Ability and willingness of subject to provide informed consent.  
 

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4.1.3 Men and women ages ≥18 years. 
 
4.1.4 Clinically stable on their first or second HAART regimen. Changes while the 
patient HIV viral load is undetectable does not count toward the number of ART 
regimens used, only changes made for virologic failure (for example an individual 
switching from an NNRTI-based regimen to an integrase inhibitor based regimen 
while the HIV viral load is undetectable will still be in their first regimen). Site 
investigator anticipates that a fully active alternative ART regimen could be 
constructed in the event of virologic failure on the current ART regimen.  
 
The current regimen should have no changes within 4 weeks of enrollment. 
Subjects must be willing to continue on current antiretroviral therapy for the 
duration of the study except for the duration of the 16 week analytical treatment 
interruption. (NOTE: changes to safely begin the treatment interruption –see 
section 5.6– are permitted).  
 
4.1.5 Screening HIV-1 RNA that is ≤50 copies/mL using a FDA-approved assay 
performed by any laboratory that has a CLIA certification or its equivalent within 
30 days prior to enrollment. 
 
4.1.6 HIV-1 RNA ≤50 copies/mL using a FDA-approved assay for at least 24 weeks 
prior to enrollment performed by any laboratory that has a CLIA certification or its 
equivalent. 
 
NOTE: HIV-RNA must be measured at least once in the last 24 weeks and 
at least 3 days before the screening measure.  Single determinations that 
are between >50 and <400 copies/mL (ie, blips) are allowed as long as the 
preceding and subsequent determinations are ≤50 copies/mL.  The 
screening value may serve as the subsequent determination ≤50 copies/mL 
following a blip. 
 
NOTE: subjects who have participated in other trials using ATI’s will be 
permitted since detectable virus during the interruption does not represent 
virologic failure. These subjects should have at least 24 weeks of VL <50 
copies/mL. 
 
4.1.7 Screening CD4+ T-cell count ≥450 cells/ mm3 within 30 days of enrollment. 
 
4.1.8 Started ART with nadir CD4+ ≥200 cells/ mm3.  
 
4.1.9 The following laboratory values obtained within 30 days prior to enrollment 
meeting the following criteria: 
 
Absolute neutrophil count (ANC)  ≥1000 cells/mm3 
 
Hemoglobin:≥10.0(males); ≥9.5 (females) g/dL  
 
Platelet count  100,000/mm3 
 
Calculated creatinine clearance ≥50 mL/min estimated by the Cockcroft-
Gault equation  
NOTE:  A program for calculating creatinine clearance by the Cockcroft-
Gault method is available at 

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http://reference.medscape.com/calculator/creatinine-clearance-cockcroft-
gault  
 
Alanine aminotransferase (ALT) ≤  2.0 x ULN 
 
4.1.10 Negative HBsAg within 6 months prior to enrollment. 
 
4.1.11 Negative HCV serology, or if positive, negative HCV RNA within 6 months prior 
to enrollment. 
 
4.1.12 Adequate venous access and no other contraindications for leukapheresis. 
 
4.1.13 Have a Karnofsky Performance Score of 70 or higher. 
 
4.1.14 Have a recorded viral load set point prior to starting antiretroviral therapy 
 
4.2 
Exclusion Criteria  
4.2.1. Acute or chronic hepatitis B or hepatitis C infection  
4.2.2. Current or prior AIDS diagnosis. 
4.2.3. History of cancer or malignancy, with the exception of successfully treated basal 
cell or squamous cell carcinoma of the skin 
 
4.2.4. History or any features on physical examination indicative of active or unstable 
cardiac disease or hemodynamic instability. 
 
NOTE: Subjects with a history of cardiac disease may participate with a 
physician’s approval.  
 
4.2.5 History or any features on physical examination indicative of a bleeding diathesis. 
 
4.2.6 Have been previously treated with any HIV experimental vaccine within 6 months 
prior to enrollment, or any previous gene therapy using an integrating vector. 
 
NOTE:  Subjects treated with placebo in an HIV vaccine study will not be 
excluded if documentation that they received placebo is provided.  
 
4.2.7 Use of chronic systemic corticosteroids, hydroxyurea, or immunomodulating 
agents (e.g., interleukin-2, interferon-alpha or gamma, granulocyte colony 
stimulating factors, etc.) within 30 days prior to enrollment.  
 
NOTE:  Recent or current use of inhaled steroids is not exclusionary. If 
subjects are prescribed a brief course of oral corticosteroids, the use 
should be limited to less than 7 days.  Use of steroids before apheresis 
and immune assessment blood draws should be discouraged as it will 
affect white blood cell function. 
 
4.2.8 Breast-feeding, pregnant, or unwilling to use acceptable methods of birth control. 
 

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4.2.9 Anticipated use of aspirin, dipyridamole, warfarin or any other medication that is 
likely to affect platelet function or other aspects of blood coagulation during the 2-
week period prior to leukapheresis. 
 
4.2.10 Active drug or alcohol use or dependence that, in the opinion of the site 
investigator, would interfere with adherence to study requirements.  
 
4.2.11 Serious illness requiring systemic treatment and/or hospitalization within 30 days 
prior to enrollment. 
 
4.2.12 Asymptomatic baseline serum chemistry elevations in LFTs, bilirubin, and serum 
creatinine due to HAART medication are not exclusionary, when in the opinion of 
the investigator, the abnormalities are not attributable to intrinsic hepatorenal 
disease.  Such baseline elevations must be due to HAART. 
 
4.2.13 Receipt of vaccination within 30 days prior to enrollment.  
 
NOTE: It is recommended that subjects enrolling into this study should have 
completed their routine vaccinations (hepatitis A, hepatitis B, pneumococcus, and 
tetanus diphtheria booster) at least 30 days prior to enrollment. 
 
4.2.14 Have an allergy or hypersensitivity to study product excipients (human serum 
albumin, DMSO and Dextran 40).  
 
4.3 
Study Enrollment Procedures 
4.3.1 Prior to implementation of this protocol, the site must have the protocol and 
consent form approved by their local institutional review board (IRB). 
 
Once a candidate has been identified, details will be carefully discussed with the 
subject. The subject will be asked to read and sign the approved consent form 
before any study-specific procedures have been performed.  
 
Note: For the purposes of this study, enrollment is defined as the date the 
Investigator confirms subject eligibility. 
 
4.3.1.1 
Prisoner Participation 
 
 
This protocol does not meet Federal requirements governing prisoner 
participation in clinical trials and prisoners should not be considered for 
recruitment purposes.  
 
4.3.1.2 
Birth Control 
 
Female subjects of reproductive potential (women who have reached menarche 
or women who have not been post-menopausal for at least 24 consecutive 
months, i.e., who have had menses within the preceding 24 months, or have not 
undergone a sterilization procedure [hysterectomy or bilateral oophorectomy]) 
must have a negative serum pregnancy test at the time of enrollment and the 
Safety Evaluation Visit.  A urine pregnancy test must also be performed on Day 0 
(T cell infusion day). 

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Due to the high risk level of this study, while enrolled, all subjects must agree not 
to participate in a conception process (e.g., active attempt to become pregnant or 
to impregnate, sperm donation, in vitro fertilization). Additionally, if participating in 
sexual activity that could lead to pregnancy, the study subject must agree to use 
at least two reliable methods of contraception during the follow-up period of the 
protocol, including at least one barrier method of contraception (diaphragm, 
cervical cap with spermicide, or condom). 
 
Acceptable birth control includes a combination of two of the following methods, 
where at least one must be barrier contraception: 
 
Condoms* (male or female) with or without a spermicidal agent (barrier 
contraception). 
 
Diaphragm or cervical cap with spermicide (barrier contraception) 
 
Intrauterine device (IUD) 
 
Hormonal-based contraception 
 
* Condoms are recommended because their appropriate use is the only 
contraception method effective for preventing HIV transmission. 
 
 
Subjects who are not of reproductive potential (women who have been post 
menopausal for at least 24 consecutive months or have undergone 
hysterectomy, salpingotomy, and/or bilateral oophorectomy or men who have 
documented azoospermia) are eligible without requiring the use of contraception.  
Acceptable documentation of sterilization, azoospermia, and menopause is 
specified below. 
 
 
Written or oral communication by clinician or clinician’s staff of one of the 
following: 
 
 
Physician report/letter   
 
Operative report or other source documentation in the subject record (a   
laboratory report of azoospermia is required to document successful 
vasectomy)  
 
Discharge summary 
 
Laboratory report of azoospermia  
 
Follicle stimulating hormone measurement elevated into the menopausal 
range 
 
4.4 
Co-enrollment Guidelines  
For specific questions and approval for co-enrollment in other studies, site must contact the 
Sponsor Project Manager at 
. 
 
4.5 
Premature Subject Discontinuation 
Subjects may be withdrawn for the following reasons:  
 
 
Request by the subject to withdraw. 
 
Request of the primary care provider if he or she thinks the study is no longer in the 
best interest of the subject. 

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 
Subject judged by the investigator to be at significant risk of failing to comply with the 
provisions of the protocol as to cause harm to self or seriously interfere with the 
validity of the study results. 
 
Decision to stop further study visits by the Data Safety Monitoring Board (DSMB), or 
an independent review board or regulatory review committee. 
 
At the discretion of the IRB, Office for Clinical Research (OCR), Food and Drug 
Administration (FDA), NIH/NIAID, DAIDS, investigator, or pharmaceutical supporter. 
 
 
5.0 
STUDY DRUGS 
5.1 
Description of Study Drugs 
The study drug in this protocol is autologous CD4+ T cells genetically modified by lentiviral 
vector expressing the C34-peptide conjugated to the CXCR4 N-terminus. Study treatment is 
defined as infusion of subject specific autologous CD4+ T cells genetically modified by lentiviral 
vector expressing the C34-peptide conjugated to the CXCR4 N-terminus. 
 
5.2 
Treatment Regimen 
 
 
 
Step 1  
 
Subject is screened, undergoes the first and second leukapheresis and rectal biopsy, 
and undergoes safety evaluations before dosing. 
 
The University of Pennsylvania’s Clinical Cell and Vaccine Production Facility (CVPF) 
manufactures autologous CD4 T-cells genetically modified by lentiviral vector expressing 
the C34-peptide conjugated to the CXCR4 N-terminus. 
Step 2 
 
All participants will receive a single infusion of C34-CXCR4-modified CD4+ T-cells at 
one of 3 dose levels.  The first 3 subjects will receive dose level 1 of 0.8-1x109 
transduced CD4+T-cells.  Provided no dose limiting toxicity (DLT) is seen at the first 
dose level at the completion of the Day 21 study visit of the 3rd subject, the next 3 
subjects will receive infusion at the second dose level of 2.4-3x109 transduced CD4+ T-
cells.  If no DLT occurs at that dose by the completion of the Day 21 study visit of the 6th 
overall subject infused, the final 3 subjects will receive the third dose level of 0.8-1x1010 
transduced CD4+ T-cells.  In the event of a DLT (grade 3 or higher unexpected, related 
AE) recruitment will be paused pending DSMB decision.  

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 
At the end of Step 2 all participants will undergo mini-leukapheresis and an optional 
rectal biopsy. 
 
Step 3  
 
HAART interruption for 16 weeks (or less if patients viral load is sustained ≥100,000 or 
CD4 count <350). 
 
All participants undergo a mini-leukapheresis and an optional rectal biopsy at the end of 
the Step 3. 
 
Step 4 
 
All participants will be advised to resume antiretroviral therapy and be followed until 
plasma HIV RNA fall below the limit of detection. 
 
Note:  For subjects with viral loads ≤1000 copies/ml at the end of 16 weeks, re-
initiation of antiretroviral therapy will be recommended.  However, resumption of ART 
medications will be left to the discretion of the subject and their physician.  If a 
subject declines to restart antiretroviral medications, the subject will be followed in 
Step 3 with monthly visits until ART is resumed, in an effort to fully capture the 
impact of the intervention.  These monthly visits should capture all procedures 
outlined in the Week 16 visit of the schedule of events.  The study team will discuss 
re-initiation of ART with the subject at each visit.  The subject may choose to restart 
ART at any time, and thus would move to Step 4 of the study when antiretroviral 
medication is resumed.  All procedures outlined in the ATI Discontinuation visit 
should be performed just prior to restarting antiretroviral medications (Step 4), if 
possible. 
 
Step 5 
 
All participants will undergo leukapheresis and an optional rectal biopsy 52 weeks post 
infusion and then be enrolled onto a separate long-term follow-up study as required by 
regulatory authorities. 
 
5.3 
Preparation of Autologous CD4+ T genetically modified with C34-
CXCR4  
The manufacture and release of C34-CXCR4 modified CD4 T-cells will be performed by the 
Clinical Cell and Vaccine Production (CVPF) at the University of Pennsylvania.  CD4+ 
lymphocytes will be isolated from leukapheresis product using negative selection for monocytes 
and CD8+ T-cells.  The resulting enriched CD4+ T-cells are activated with anti-CD3/CD28 mAb 
coated magnetic beads and transduced with lentiviral vector expressing an HR2, C34-peptide 
conjugated to the CXCR4 N-terminus.  The cell expansion process will initiate in a static tissue 
culture bag. Cells will be transferred to a WAVE bioreactor if needed for additional expansion.  
At the end of the culture, cells will be depleted of the magnetic beads, washed, concentrated 
using the Haemonetics Cell Saver system, and then formulated in infusible cryopreservation 
media. Each dose will be packed and cryopreserved in infusion bag(s) in a volume dependent 
on the total cell number which is a function of the transduction efficiency; the minimum volume 
will be 10mL per bag.  
 
 

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The cell product will be ready to be released after 14 days post-formulation and 
cryopreservation. Modified cells are not released from the CVPF until release criteria for the 
infused cells (e.g., cell purity, sterility, transduction efficiency, viability, cell number) are met.  
 
Note:  In cases where a product may need to be remanufactured, use of frozen apheresis as 
starting material is permitted.  However, the apheresis may not be frozen longer than 6 months 
prior to use in manufacturing. 
 
Additionally, several vials of cells will be cryopreserved and retained as sentinel vials, for 
performing an endotoxin gel clot and viability count at the time of the infusion. Remaining vials 
will be used to conduct the “for information only (FIO)” functional assays. All cryopreserved cells 
will be stored in a monitored freezer at ≤ -130°C. 
 
5.4 
Administration of Study Drugs 
5.4.1 Administration of autologous T-cells genetically modified with 
C34-CXCR4 
Procedure for Study Agent Handling and Preparation 
The autologous T-cells genetically modified by lentiviral vector expressing the C34-peptide 
conjugated to the CXCR4 N-terminus will not be released from the manufacturer (University of 
Pennsylvania Clinical Cell and Vaccine Production Facility, CVPF) until the release criteria for 
the infused cells (e.g., cell purity, sterility, identity, HIV-1 gag level) are met. On the day of T-cell 
infusion, the genetically modified T-cells will be transported on dry ice from CVPF to the 
subject’s bedside in the Center for Human Phenomic Science (CHPS) according to CVPF SOP 
for thaw and infusion.  Each bag of cryopreserved genetically modified CD4+ T-cells will contain 
approximately 1 x 109 to 1 x 1010 cells at a concentration of approximately 1 x 108 cells/mL of 
infusible cryomedia. 
 
Package and Labeling 
The infusion bag will be affixed with a label containing information regarding the dose, the 
method of manipulation, the vector and the following statements “FOR AUTOLOGOUS USE 
ONLY” And “Caution: New Drug- Limited by Federal Law to Investigational Use”.    In addition 
the label will have at least two unique identifiers.  
 
Premedication 
Side effects following T-cell infusions include transient fever, chills, and/or nausea.  It is 
recommended that the subject be pre-medicated with acetaminophen (650 mg) by mouth and 
diphenhydramine hydrochloride by mouth or IV, prior to the infusion of C34-CXCR4-modified 
CD4+ T-cells. If Benadryl is contraindicated, an H2-blocker such as ranitidine will be 
administered. These medications may be repeated every six hours as needed.  A course of 
non-steroidal anti-inflammatory medication may be prescribed if the subject continues to have 
fever not relieved by acetaminophen. It is recommended that subjects not receive systemic 
corticosteroids 
such 
as 
hydrocortisone, 
prednisone, 
prednisolone 
(Solu-Medrol) 
or 
dexamethasone (Decadron) at any time, except in the case of a life-threatening emergency, 
since this may have an adverse effect on T-cells. If corticosteroids are required for an acute 
infusional reaction, an initial dose of hydrocortisone (100 mg) is recommended. 
 
Administration of genetically modified T-cells 
The cells will be thawed by trained personnel using a water bath maintained between 36°C to 
38°C.  The bag will be gently massaged until the cells have just thawed. There should be 

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no frozen clumps left in the container at the time of infusion.  If the cell product appears to have 
a damaged or leaking bag, or otherwise appears to be compromised, it should not be 
infused, and should be returned to the CVPF.  Prior to the infusion, two individuals from the 
study staff will independently verify all information in the presence of the subject to confirm that 
the information is correctly matched to the subject.   
 
A licensed nurse, in the presence of the Principal Investigator or Sub-Investigator, will infuse the 
cells into the subject within approximately 10-15 minutes after thaw to ensure that the cells are 
still cold. The genetically modified T-cells will be administered to subjects by intravenous 
infusion at a flow rate of approximately 10-20 mL per minute. There should be no frozen clumps 
left in the bag at the time of the infusion.  The cells will be infused into an intravenous catheter, 
either through a peripheral vein (preferred) or central vein.  A macrodrip intravenous tubing will 
be used to infuse the cells by gravity (i.e., no infusion pump).  The macrodrip intravenous tubing 
will be connected to a “Y” adapter with one end of the adapter spiked to the product bag(s) and 
the other to a normal saline solution bag. A leukoreduction filter must not be used for the 
infusion of the cell product.  At the end of infusion, the bag will be rinsed twice with saline to 
maximize removal of cells from the bag.   
 
Subjects will be monitored before the infusion and for 2 hours after the infusion of the 
transduced T-cells.  Vital signs (temperature, pulse/heart rate, blood pressure, and respiratory 
rate) and pulse oximetry will be obtained and recorded within 10 minutes before the infusion and 
within 15 minutes after the infusion.  Thereafter, vital signs will be measured every 15 minutes 
(+/ - 5 min) after the infusion for the first hour, and then every 30 minutes (+/- 5 minutes) for the 
next hour. A crash cart must be available for emergency situations. Emergency medical 
equipment will be available during the infusion in case the subject has an allergic response, 
severe hypotensive crisis, or any other reaction to the infusion. The subject will be discharged 
when medically stable and in accordance with hospital policy. 
 
Product Return/Disposition 
C34-CXCR4 CD4+-modified T-cells may require return to the CVPF for a variety of reasons, 
including but not limited to: 1) Mislabeled product; 2) Condition of patient prohibits 
infusion/injection, and 3) Subject refuses infusion/injection.  Any unused product that has not yet 
been thawed will be returned for disposal as per CVPF SOP.  
 
Any unused product will be disposed of according to local institutional procedures for the 
disposal of hazardous medical waste/blood product.  Final disposition of the investigational 
product will also be documented. 
 
Research Tests 
A blood sample will be drawn before the infusion of C34-CXCR4 CD4+ T-cells and at 20 min (±5 
min) and 2 hours (±5 min) post-infusion. It is recommended that post-infusion blood draws on 
Day 0 be obtained from the arm not used for infusion. 
 
Reactions following infusion 
The following reactions are possible during and/or after infusion: 
 
Febrile reactions 
In the unlikely event that the subject develops sepsis or systemic bacteremia following T-
cell infusion, appropriate cultures and medical management should be initiated. If a 

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contaminated T-cell product is suspected, the product can be retested for sterility using 
archived samples that are stored in the CVPF.  
 
Flu-like Symptoms 
 
Fever/arthralgia/myalgia: The onset of these symptoms usually occurs 2 to 4 hours after 
the cell product administration. Administration of acetaminophen or non-steroidal anti-
inflammatory agents (NSAIDS) is effective in controlling and preventing these 
symptoms.  
 
 
Rigors/chills: These symptoms may occur during and/or after cell infusion administration. 
The agent of choice to improve severe chills or rigors is an opioid such as dilaudid.  If 
subjects develop rigors consistently after cell product administration, prophylactic 
administration of dilaudid may prevent these reactions.  
 
 
Hemodynamic Effects 
 
Edema: Adequate hydration is necessary to ensure renal perfusion, but over hydration 
should be avoided. Subjects should be encouraged to drink electrolyte-containing fluids 
such as sport drinks and soups. If diuretics are to be used, they should be used with 
caution to avoid decreasing intravascular volume. If the participants develop 
hypotension, IV fluids and observation in an acute hospital bed is advisable. 
 
 
Hypotension: Organ dysfunction, oliguria, and increases in BUN and creatinine are 
almost always reversible upon discontinuation of cell product and 24 to 48 hours of 
supportive treatment. On rare occasions, low doses of dopamine and fluid support may 
be warranted. Educating the subject to slowly rise from a supine to a sitting position and 
then to a standing position can prevent orthostatic hypotension. Quick evaluation in the 
ER if symptoms occur is advisable. 
 
Dermatologic Reactions 
Dry skin, pruritus, erythema, and sloughing commonly occur following cell infusions. No 
interruption of therapy is usually needed.  Antihistamines, water or oil-based lotions, and 
oatmeal baths may help control the symptoms. Diphenhydramine hydrochloride 
(Benadryl) should be given before the administration of the T-cell product to prevent this 
from happening. 
 
Cytokine release syndrome  
Cytokine release syndrome is caused by a release of inflammatory cytokines such as 
IL2, IFN-γ, and TNFα.  This type of reaction is common in cancer immunotherapy where 
antibodies bind T-cells that release large amounts of cytokines, or cause rapid 
destruction of tumor/target cells.  This causes a systemic inflammatory response similar 
to sepsis and includes fever, nausea, chills, rash or flushing, rigors, hypotension, 
tachycardia, headache, rash, throat tightness, and dyspnea.  Capillary leak with fluid 
retention is worsened by hydration commonly given to treat hypotension.  The syndrome 
can be associated with pulmonary infiltrates, pulmonary edema, arrhythmias and cardiac 
arrest.  The syndrome is also associated with an elevation in LFTs, d-dimers, LDH, CR, 
uric acid, and phosphorus from immune-mediated cytolysis of targeted cells, with 
release of intracellular contents as well as a possible bystander effect (on neighboring, 
nontargeted cells). We have not had this type of reaction in any of the HIV positive 
subjects treated to date at the University of Pennsylvania.  
  

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5.5 
Concomitant Medications 
Below are lists of selected concomitant medications. These lists are only current as of the date 
of this protocol. Therefore, whenever a concomitant medication or study agent is initiated or a 
dose changed, investigators must review the concomitant medications’ and study agents' most 
recent package inserts, or updated information from DAIDS to obtain the most current 
information on drug interactions, contraindications, and precautions. 
 
5.5.1 Recommended Medications 
Each subject will receive acetaminophen (650 mg) and diphenhydramine hydrochloride 
(25-50 mg) between 30 minutes to approximately 1 hour before cell infusion. If Benadryl is 
contraindicated, an H2-blocker, such as ranitidine, will be administered. 
 
5.5.2 Prohibited Medications 
Aspirin, dipyridamole, warfarin, or any other medication likely to affect platelet function or other 
aspects of blood coagulation is prohibited during the 2-week period prior to leukapheresis. Oral 
corticosteroids, hydroxyurea, and immunomodulating agents are prohibited until completion of 
study follow-up.  A brief course (< 1 week) of oral corticosteroids is allowed during Steps 2 and 
3. 
 
Routine vaccines are also prohibited prior to completion of study follow-up unless medically 
indicated. 
 
NOTE:   It is recommended that enrolled subjects should not receive their influenza vaccine 
during the ATI (Step 3) as it may cause a transient increase in viral load in some subjects.  If 
medically indicated while on study, attempts should be made to receive influenza vaccine in the 
window between screening (Weeks -15 to -11) and Day 28.  Influenza vaccines may also be 
administered at any time after the subject restarts antiretroviral medications (Step 4). 
 
5.6 
Withdrawal of HAART Medication  
Subjects will discontinue HAART regimens at week 4 post-infusion in consultation with primary 
care providers.  NNRTIs (e.g. Rescriptor, Sustiva, Viramune and rilpivirine) have been reported 
to have longer half lives than other ARV classes. Therefore, based on the preference of the 
subject in consultation with the referring physician, subjects on an NNRTI containing regimen 
should either (a) discontinue the NNRTI 7 days prior to discontinuation of the other 
antiretrovirals in their regimens or (b) initiate a potent PI-based regimen (with discontinuation of 
the NNRTI) without altering the nucleosides of the regimen for the last two weeks of HAART 
prior to simultaneous interruption of all antiretrovirals.The time of medication interruption will be 
noted in medication records and CRFs.  
 
 
6.0 
CLINICAL AND LABORATORY EVALUATIONS- STUDY 
PROCEDURES 
6.1 
Schedule of Events  
 

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1. Peripheral Blood processed at TCSL and transferred to appropriate labs for analysis; Remaining PBMC to be archived (TCSL or Riley Lab).  
In the event something unexpected occurs during the subject’s participation in the protocol, the research team may request an additional blood 
draw be performed to collect additional blood samples for research analysis such as viral sequencing, antiviral resistance testing, etc. This is 
being done with the intention of evaluating the likely effects from the investigational product. The maximum amount of extra blood that will be 
collected will be 3 tablespoons of blood twice in one week. 
2. Rectal biopsy processed at UPenn HIC and analyzed or transferred to appropriate labs for analysis (TCSL for qPCR). Any remaining MMC to 
be archived (TCSL or Riley Lab). 
3. Blood draw for research samples and viral load to be performed on Day 0 before T-cell infusion and at 20 min (±5 min) and 2 hours (±5 min) 
post-infusion.  It is recommended that post-infusion blood draws on Day 0 be obtained from the arm not used for infusion. 
4. If the apheresis is collected at this time-point, additional peripheral blood (purple top tubes) should still be drawn for use in the C34-CXCR4 
detection; the apheresis products can be used for other research analysis.  Serum collection (red top tube) is still required where indicated in 
Schedule of Events. 
5. Optional rectal biopsies will be performed during Step 1, at end of the Step 2 and just prior to restarting HAART/end of Step 3 if possible.  
Rectal biopsy will also be performed during the Step 5 window. Windows for rectal biopsies are as follows:  Baseline (Apheresis 2 study visit) 
should be done between Week -5 and Week-3.  Step 2 rectal biopsy (Day 28 visit) should occur within 2 weeks prior to Analytical Treatment 
Interruption/entry into Step 3, and the Step 3 biopsy (Week 20 visit) should be done within 2 weeks prior to re-starting antiretroviral therapy. If 
a subject must restart HAART early, best efforts should be made to schedule a rectal biopsy prior to the reinstatement of HAART. In no cases 
should the restarting of HAART for safety reasons be delayed for the purpose of obtaining a rectal biopsy. 
6. HIV co-receptor usage assay at screening is a tropism
 
DNA assay recommended for patients with undetectable viral loads. Draw 4 mL of 
whole blood into an EDTA (lavender-top) test tube; DO NOT CENTRIFUGE; place tube and test request form in specimen bag; freeze sample 
immediately at -20˚C; ship to Quest.  Note:  This tube should be separate from blood designated for TCSL. 
7. For subjects restarting ART before week 20 due to viral rebound, ART resistance samples and all other required research/clinical lab samples 
will be collected on the last visit before ART re-initiation.  
8. Consent may be obtained at any time prior to research procedures being performed. 
9. Hepatitis B Surface Antigen (HBsAg):  Subjects must have documentation of a negative HBsAg test within 6 months prior to screening. 
Subjects who have not previously had the test or subjects who were negative more than 60 days prior will have a test at screening for Step 1. 
HCV Antibody:  Subjects must have documentation of a negative HCV antibody test within 6 months prior to screening for step 1. Subjects 
who have not previously had the test or subjects who were negative more than 60 days prior to screening will have a HCV antibody test at 
screening for step 1. If the HCV antibody is positive, the subject may return for a HCV RNA test. If the HCV RNA test is negative, the subject 
may be enrolled in the study. 
HCV RNA:  If the HCV antibody is positive, a screening HCV RNA by any RT-PCR or bDNA assay must be performed at screening by a local 
laboratory with a CLIA certification or its equivalent. Eligibility will be determined based on the screening value. The test is not required if 
documentation of a negative result of a HCV RNA test performed within 6 months prior to screening is provided. 
10. 2-3ml frozen serum to be delivered to Hoxie lab (from TCSL) for pre-existing antibody detection. 
11. CBC with diff/platelets and CD4+ T cell counts should come from the same 4 mL lavender tube. 

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12. If a subject experiences a decrease in CD4+ T-cell count to <350 cells/mm3, the test will be repeated the following week. If the CD4+ T-cell 
counts stay equal to or below 350 cells/mm3, the subject will be advised to reinstitute HAART. The HAART will not be provided by the study.  If 
the CD4+ decrease is not sustained the subject will continue in the study. 
13. If prior to initiating treatment interruption the subject experiences an increase in viral load from undetectable to greater than or equal to 5,000 
copies/ml, a viral load test will be repeated once a week for up to two additional weeks or until the viral load is below 5,000 copies/ml 
(whichever occurs first).  If the viral load increase is sustained over this 3-week period, this event will be reported as an SAE and will be 
managed as medically appropriate, and the subject will not undergo ATI. If during ATI the subject experiences a viral load ≥100,000 copies/ml, 
this test will be repeated every week for up to 3 weeks.  If the subject experiences a sustained viral load ≥100,000 copies/ml consistently over 
a period of 3 weeks, the subject will be advised to restart HAART.  The HAART will not be provided by the study.  If the viral load is not 
sustained (i.e. as indicated by a single test < 100,000 copies/ml), the subject will be advised not to reinstate HAART, and continue the study 
as planned, until week 16 after the treatment interruption to allow for evaluation of the subject’s viral load set point. 
14. HIV co-receptor usage assay at Weeks 6 and 20 is a tropism assay recommended for patients with viral load ≥ 1,000 copies/mL.  Using 2 
lavender-top (EDTA) tubes, draw enough whole blood to provide at least 3 mL plasma; transfer to a plastic screw-cap tube; centrifuge 
immediately at 1,000 to 1,200g for 10 to 15 minutes (Don’t leave samples in centrifuge after spinning.); place tube(s) and test request form in 
specimen bag; freeze sample immediately at -20˚C; ship to contracted laboratory. For subjects restarting ART before week 20, the coreceptor 
tropism samples will be collected at the last visit before ART re-initiation. 
15. Complete Physical Exam with Karnofsky performance status should be obtained at least every 8 weeks, unless symptoms require more 
frequently. 
16. Test for VSV-G (RCL) and pre-existing antibody should be performed at the 6 month visit.  These tests are not required again until the Week 
52 visit. 
17. First monthly visit only 
18. Apheresis 1 is a full apheresis (~10L).  Apheresis 2, if not needed for cell processing is a mini-apheresis (~5L).  Mini-apheresis (~5L) to be 
done at the end of Steps 2 and 3.  Full ~10L apheresis should be performed at the end of Step 5.  Final apheresis should be >16 weeks after 
subject’s viral load becomes undetectable. 
19. Apheresis product (if not used for cell manufacturing) will be processed by TCSL.  Cells from apheresis product will be processed and used for 
assays including, but not limited to, immune analysis, and to extract DNA for integration analysis.  C34-CXCR4 detection should be done 
using peripheral blood draw.  Remaining cells should be viably frozen in TCSL freezing media to be archived. 
20. To monitor compliance to ATI, random testing of blood and/or urine for antiretroviral drug levels can be done during Step 3 at the discretion of 
the Primary Investigator.  This is not required at each Step 3 visit. 
21. If a subject will restart antiretroviral therapy prior to completing 16 week of treatment interruption, the ATI Discontinuation Study Visit should be 
performed prior to restarting antiretroviral medication, and all procedures outlined for that visit should be captured prior to restarting ART, if 
possible  
22. For subjects extending the treatment interruption beyond a total of 16 weeks, subjects should be seen every 30 days (±7 days) with visits 
capturing the procedures outlined in the Step 3 Monthly visit.  Before restarting ART, all procedures outlined in the Schedule of Events at the 
ATI Discontinuation Study Visit should be captured prior to restarting ART, if possible.   
23. It is possible that the ATI discontinuation visit may be combined with another study visit.  In this situation, all procedures captured in both visits 
should be performed, however, they do not need to be completed in duplicate. 

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24. The month 6 post-infusion visit is a required visit, even if the subjects has already reached undetectable viral load as part of Step 4.  If a 
subject is still in Step 3 at the 6 month post-infusion time point, this visit may be combined with a Step 3 monthly visit.  If this month 6 visit is 
being combined with a Step 3 Monthly or Step 4 Monthly visit, all procedures captured in the schedule of events for both visits should be 
performed, however they do not need to be completed in duplicate. 
 

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6.2 
Study Visit Procedures 
 
STEP 1 
 
6.2.1 Screening Evaluations Visit (~Week -15 to -11) 
Before an individual undergoes screening, informed consent must be obtained by means of a 
written subject information document. The information will be provided in the language of the 
subject’s choice and written so that the subject can easily understand it. The subject will be 
given sufficient time to read the document and to ask questions before deciding to participate. If 
the person would like to participate, he or she must indicate this by signing the document. The 
document must also be dated with the date of signature. The subject will receive a copy of the 
signed and dated informed consent document. All potential subjects screened for eligibility will 
be listed on a subject screening and enrollment log. This log will include documentation of 
reasons for ineligibility and for non-participation of eligible subjects. 
 
The following screening evaluations to determine eligibility must be completed within 15 weeks 
of dosing (Step 2) unless a protocol exception has been granted: 
 
Obtain a signed subject informed consent form- may be performed at any time prior to 
research procedures being performed 
 
Review inclusion/exclusion criteria 
 
History and physical exam (see section 6.3) 
 
Karnofsky performance status 
 
Concomitant medications (see section 6.3) 
 
EKG 
 
Leukapheresis screening (assess for adequate iv access) 
 
Hepatitis B surface antigen and hepatitis C antibody (see section 6.3) 
 
Serum pregnancy test (see section 6.3) 
 
Chemistry/LFTs (see section 6.3) 
 
CBC with differential and platelets 
 
Urinalysis 
 
CD4+ T-cell counts 
 
Viral load (HIV-1 RNA ultrasensitive PCR assay) 
 
A documented CD4+ nadir and documented HIV viral load set point 
 
Once required screening tests have been completed and the subject has been determined 
eligible by the physician-investigator, provide the documents listed below to:  
 
Protocol Monitor and Sponsor Project Manager 
 
  
 
Documents required:  
 
Completed Eligibility Checklist 
 
Redacted copies of any source documentation to confirm eligibility 
 
Redacted copies of signed subject informed consent and HIPAA Authorization 
 
Upon informed consent completion and receipt of screening and eligibility documentation, the 
Sponsor Protocol Monitor will review and provide documentation that the monitoring visit for 

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eligibility has been completed.  This documentation must be received prior to apheresis and cell 
product manufacturing.  
 
A Subject Screening and Enrollment Log will be maintained by the site. 
 
6.2.2 Apheresis 1 Visit (~Week -9 to -7 prior to dosing) 
6.2.2.1 Leukapheresis Procedure 
For subjects who have successfully passed screening and who wish to continue to participate in 
the study, a visit date for a ~10L leukapheresis will be scheduled. This will be done at the 
University of Pennsylvania Apheresis Unit according to standard clinical procedures. From a 
single ~10L volume leukapheresis, at least 5 x 109 white blood cells will be harvested to 
manufacture autologous CD4+ T genetically modified by lentiviral vector expressing the C34 
peptide conjugated to the CXCR4 N-terminus. At the time of leukapheresis, PBMCs for baseline 
immunologic studies (CD4+ and CD8+ ICS) will also be obtained. 
 
At this visit, subjects will have vital signs (temperature, respiration, pulse and  blood pressure) 
recorded at baseline and as per hospital practice until the procedure has been completed and 
the vital signs are found to be satisfactory and stable. Blood for research is also obtained.   
Subjects are monitored for adverse events. In addition to the screening testing requirements 
provided in the protocol, blood from all apheresis donors undergoes testing according to 
Apheresis Unit SOP.  
 
Leukapheresis takes ~2 to 3 hours. This procedure removes only the white blood cells as blood 
flows out of the body through a needle inserted into one arm and returns to the body through a 
second needle inserted in the other arm. The cells are separated into blood components. The 
white cells are collected and the rest of the blood components (including the red blood cells) are 
returned. There are risks associated with leukapheresis; however, all precautions are taken to 
avoid complications. The leukapheresis procedure can occasionally cause local infections, pain 
or bleeding from the needle stick, bruising of the skin, or inflammation or irritation of the vein 
(phlebitis). There is a possibility that due to technical failure (breakage in equipment) the 
subject’s blood circuit through the leukapheresis machine may be interrupted resulting in blood 
loss. If this should occur, the blood loss would not be more than that occurring during donation 
of whole blood and the procedure will be stopped. Anemia may occur. With the infusion of ACD 
during leukapheresis which can lower the calcium, the subject may experience tingling around 
the mouth, hands or feet, or slight twitching of muscles. Any signs and symptoms of citrate 
toxicity due to apheresis (such as perioral paresthesia and muscle cramps) will be treated 
according to the COH Donor/Apheresis Center procedure.  However, these symptoms can be 
reduced by slowing down the rate of infusion of the blood being returned or by calcium 
administration. Subjects may also experience chilling, nausea, vomiting, lightheadedness, or 
migraine headache, which can be treated with appropriate measures, including warm 
compresses and slowing or stopping the procedure. 
 
A sample aliquot from the leukapheresis product (leukapheresis 1 or 2) will be used for baseline 
viral and immune response analysis.  In the event that the manufacturing of the cellular product 
fails using the first leukapheresis, then this second leukapheresis will be used for a second 
manufacturing run. In this case, this second leukapheresis will be a larger scale (≥10L) run. If 
the second leukapheresis is not needed for manufacturing, a small scale apheresis 
(approximately a 5L volume) will be performed instead to serve as a cell bank for baseline 
controls for immunological evaluations. If a mini-apheresis is collected at this timepoint, no 

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additional research blood will be drawn; the mini-apheresis products will be used for research 
analysis. Excess leukapheresis product will be archived for the look-back purposes mentioned 
above. A 3rd and 4th mini-apheresis (~5L) will be performed to obtain cells for research purposes 
(week 4 and 20). A 5th leukapheresis will be performed at the end of study (week 52). 
6.2.3 Rectal Biopsy Procedure and Visit  
All subjects enrolled in the study are asked to undergo four optional rectal mucosal biopsies pre 
and post gene-modified T-cell infusion to evaluate the GALT for viral burden and the presence 
of gene-modified T-cells. These data will be compared to concurrent data of viral load obtained 
from peripheral blood. Refer to the Schedule of Events, for the timing of these procedures 
(Apheresis 2 Visit, Day 28 Visit, Week 20 Visit, and Week 52 Visit).  
 
The biopsy procedure takes approximately 30 minutes to complete, is performed in the 
outpatient setting, and does not require anesthesia. A trained gastroenterologist at the GI clinic 
will perform the rectal biopsy. Biopsy tissue will be collected in medium and delivered to the 
UPenn Human Immunology Core (HIC) laboratory for preparation of mucosal mononuclear cells 
(MMCs) and analysis; aliquots of MMCs will be transferred to the TCSL for storage, analysis, 
and distribution to other labs (HIV reservoir). The primary risks associated with rectal mucosal 
biopsies include mild rectal irritation and urgency and limited rectal bleeding for 2-3 days 
following the procedure. Infection and bowel perforation are extremely rare complications that 
may require antibiotics and/or surgical repair. Study volunteers will be followed in clinic for any 
complications. Even if a participant consents to these optional rectal biopsies, they may decide 
at any point to have no further biopsy procedures. Prophylactic antibiotics should be given to 
patients who would routinely be given such treatment prior to invasive procedures (e.g., cardiac 
valvular disease).  Subjects should not have taken antithrombotic medication (except ASA and 
NSAIDS) for 5 days prior to the procedure.  If a participant is taking these medications for 
medical reasons, biopsies should not be collected and the medications should not be 
interrupted. 
 
Subjects should be instructed that they should not have rectal sex and/or insert any foreign 
object or substance into the rectum for 5 days after biopsy samples have been collected. 
 
At one week following the second rectal biopsy, the study team will contact the subject by phone 
call to follow up and monitor possible adverse effects related to the procedure such as bleeding, 
infection, or discomfort.   
 
The first rectal biopsy is scheduled during the Apheresis 2 Visit (~Week -5 to -3 prior to dosing): 
 
Leukapheresis 
 
Assessment of adverse events related to rectal biopsy 
 
Research Blood  
NOTE: if the mini-apheresis is collected at this timepoint, no additional research 
blood is drawn; the mini-apheresis product to be used for research analysis.  
 
Rectal Biopsy - to be performed approximately between Week -5 and Week -3 
 
6.2.4 Safety Evaluations (within 14 days (+/- 3 days) of T-cell Infusion) 
The following entry evaluations must be completed within 14 days (+/-3 days) of entering into 
step 2.  Subjects must have all pre-dosing evaluations completed and enter Step 2 within 15 
weeks (~100 days) after entry into Step 1 (see study schema Figure 1).  Results for 
hemoglobin, ANC, platelets, creatinine, AST, ALT, and pregnancy test must be known prior to 

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dosing and must continue to meet the protocol defined limits for inclusion into the study.  If any 
of the test results are outside the limits used to determine eligibility, they may be repeated x 1.  
If any test result continues to be outside the protocol defined limits, the subject may not be 
infused and will terminate from the study. 
 
Concomitant medications 
 
Complete Physical exam and Karnofsky performance status 
 
Assessment of adverse events 
 
Pregnancy test -serum 
 
Chemistry/LFTs 
 
Cholesterol/LDL, HDL, triglycerides 
 
CBC with differential and platelets 
 
Urinalysis 
 
CD4+ T-cell count 
 
Viral load (HIV-1 RNA ultrasensitive PCR assay) 
 
Research blood  
 
STEP 2 
 
6.2.5 Infusion of C34-CXCR4 modified CD4+ T-cells (Day 0) 
All assessments and tests to be performed prior to infusion of C34-CXCR4 modified CD4+ T-
cells and results of pregnancy test must be known 
 
Concomitant medications 
 
Targeted physical Exam (assessments directed at causes of complaints 
or adverse events) 
 
Assessment of adverse events 
 
Pregnancy test (urine)  
 
CD4+ T-cell count 
 
Blood draw for research samples and viral load to be performed before T-
cell infusion and at 20 minutes (±5 min) and 2 hours (±5 min) post-
infusion. 
 
If for any reason a subject is not able to enter Step 2 (infusion of CD4+ modified T-cells) within 
15 weeks of enrollment, contact the regulatory sponsor to discuss granting a protocol exception. 
However, all subjects must be enrolled in Step 2 within 16 weeks of enrollment (enrollment is 
defined as the date eligibility is confirmed by the Principal Investigator). 
  
6.2.6 Post Infusion Visits  
Post Infusion visits will take place per the Schedule of Events in Section 6.1. 
 
6.3 
Protocol Definitions and Evaluations 
All clinical and laboratory information required by this protocol is to be present in the source 
documents. All evaluations are to be recorded on the CRF and keyed into the database unless 
otherwise specified.  
 
6.3.1 Medical History and Physical Examination 
Medical History. The medical history must include all diagnoses identified by the ACTG criteria 
for clinical events and other diagnoses. The medical history must include all diagnoses and 

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surgical procedures of major organ systems. Surgical procedures must be entered into all 
source documents only. Any allergies to any medications and their formulations must be 
documented using Medra guidelines. 
 
Medication History. All prescription and nonprescription medication, vitamins, herbal, and 
nutritional supplements, taken by the subject within 90 days prior to study enrollment will be 
recorded at the screening visit. A complete antiretroviral medication history including start and 
stop dates will be recorded in the medical records and on the appropriate CRF.   
 
Concomitant Medications. At screening, all prescription and nonprescription medications, 
vitamins, herbal and nutritional supplements, taken by the subject within 90 days prior to study 
enrollment will be recorded in the medical record and on the appropriate CRF.  At each study 
visit, any additions, deletions, or changes of these medications will be documented. All study-
mandated pre-medications taken by the subject during the study will be recorded in the medical 
records and on the appropriate CRF. 
 
Karnofsky Performance. Karnofsky Performance Score will be assessed at screening using 
the Karnofsky Performance Status Scale presented by using the DAIDS Clinical Trial Toxicity 
Criteria of November 2014.  
 
Complete Physical Exam. This physical exam is performed by a physician or nurse practitioner 
listed on the 1572 form for this study.  A complete physical examination will be conducted at the 
screening visit, safety evaluation, and at least every 8 weeks, unless symptoms require more 
frequently. This examination must include at a minimum an examination of the skin, head, 
mouth, and neck; auscultation of the chest; cardiac exam; abdominal exam; and examination of 
the lower extremities for edema. The complete physical exam will also include signs and 
symptoms, diagnoses, Karnofsky Performance Status and vital signs (temperature, pulse, and 
blood pressure). 
 
Targeted Physical Exam. A targeted physical exam can be performed by a licensed 
professional delegated this task (Physician, Nurse Practitioner, Physician Assistant, Registered 
Nurse, etc).  A targeted PE consists of vital signs and assessment of the subject including 
symptom complaints and adverse event (AE) assessment. 
 
-If a subject reports no AEs or symptoms then no additional components of the physical 
exam will be performed 
 
 
-If a subject reports any AEs or symptoms then targeted components of the physical 
exam will be performed to further evaluate causes of those AEs and/or symptoms.  If a nurse is 
completing the targeted PE and notes a finding, a physician will evaluate the subject.  The 
physician may then complete a complete physical exam if indicated.   
 
Height: Height will be measured and recorded at the screening visit only. 
 
Diagnoses. Record all diagnoses identified by the Medra criteria for clinical events and other 
diseases.  
 
Study Intervention Modifications. Record all study intervention modifications, subject-initiated 
and/or protocol-mandated modifications, and inadvertent and deliberate interruptions at each 
visit. Record any permanent discontinuation of treatment. 
 

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Clinical Adverse Event. Sites must refer to the Division of AIDS Table for Grading the Severity 
of Adult and Pediatric Adverse Events (DAIDS AE Grading Table), Version 2.0, November 
2014, which can be found on the Regulatory Support Center Web site: http://rsc.tech-
res.com/docs/default-source/safety/daids ae grading table v2 nov2014.pdf   
 
6.3.2 Clinical Laboratory Evaluations 
All laboratory results collected at screening and enrollment must be recorded on the CRFs. 
 
Documentation of HIV-1. HIV-1 infection, as documented by any licensed ELISA test kit and 
confirmed by Western blot at screening. HIV-1 culture, HIV-1 antigen, plasma HIV-1 RNA, or a 
second antibody test by a method other than ELISA is acceptable as an alternative confirmatory 
test. 
 
Plasma HIV-1 RNA. Screening HIV-1 RNA must be performed as indicated in the Schedule of 
Events. Eligibility will be determined based on the screening value. The screening HIV-1 RNA 
must be done with an Ultrasensitive HIV-1 PCR assay at a laboratory with a CLIA certification or 
equivalent. This assay will be used to assess the anti-viral effect of the investigational cell 
therapy by comparing the viral set point log10 HIV-1 RNA level (defined as the median of values 
available before the initiation of antiretroviral therapy) and the set point after the intervention, 
during the analytical treatment interruption as part of the secondary objectives. The new viral set 
point will be defined as the average of two consecutive viral load measurements of log10 HIV-1 
RNA levels after dosing and just prior to the reinitiation of antiretroviral therapy or at weeks 20 
and 24 after the administration of the modified CD4 positive T-cells (weeks 12 and 16 of the 
analytical treatment interruption), whichever comes first. 
 
Hepatitis B Surface Antigen (HBsAg). Subjects must have documentation of a negative 
HBsAg test within 6 months prior to study enrollment. Subjects who have not previously had the 
test or subjects who were negative more than 60 days prior will have a test at screening for 
Step 1. 
 
HCV Antibody. Subjects must have documentation of a negative HCV antibody test within 6 
months prior to screening for Step 1. Subjects who have not previously had the test or subjects 
who were negative more than 60 days prior to screening will have a HCV antibody test at 
screening for step 1. If the HCV antibody is positive, the subject may return for a HCV RNA test. 
If the HCV RNA test is negative, the subject may be enrolled in the study. 
 
HCV RNA. If the HCV antibody is positive, a screening HCV RNA by any RT-PCR or bDNA 
assay must be performed at screening to Step 1 by a local laboratory with a CLIA certification or 
its equivalent. Eligibility will be determined based on the screening value. The test is not 
required if documentation of a negative result of a HCV RNA test performed within 6 months 
prior to screening is provided. 
 
Complete blood count (CBC)/differential. Hemoglobin, hematocrit, platelet count, red cell 
count, and white cell count with differential count.  
 
Liver Function Tests/Chemistries. Bilirubin, alanine aminotransferase (ALT), aspartate 
aminotransferase (AST), alkaline phosphatase, sodium, potassium, chloride, carbon dioxide, 
urea nitrogen, creatinine, total protein, albumin, calcium, and phosphorus. 
 

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Total cholesterol, LDL, HDL, triglycerides. Periodic measurements will be made to monitor 
patients for HIV treatment related metabolic abnormalities that may pose safety concerns. 
 
Pregnancy Test. For women with reproductive potential (see definition in section 4.3.3): Serum 
or urine -HCG (urine test must have a sensitivity of 25-50 mIU/mL). 
 
Absolute CD4 T-cell counts. Absolute CD4+ T-cells count, and CD3, CD4, and CD8 
percentage and absolute counts must be obtained from a laboratory with a CLIA certification or 
equivalent.  
 
Urinalysis or Urine Dip Stick Analysis will include measurements of pH, glucose, protein and 
ketones and assessment by microscopy if urinalysis is abnormal (i.e., WBC, RBC, epithelial 
cells, casts, and crystals). 
 
ART Resistance Testing for those subjects that fail to complete the 16 week analytical 
treatment interruption (ATI) due to viral rebound.   A PCR/sequencing test will be performed to 
ensure that subjects resume ART using a regimen to which their HIV is most likely susceptible.  
GenoSURE PRIme and GenoSURE MG are examples of acceptable ART resistance tests. 
 
6.3.3 Research Laboratory Evaluations 
C34-CXCR4 Detection assay. This flow cytometry assay employing an anti-C34 monoclonal 
antibody will be used to detect T-cells expressing the C34-CXCR4 protein. This assay will be 
used for release of manufactured T-cells from the CVPF, and also for monitoring the 
persistence of C34-CXCR4 modified T-cells in subjects’ peripheral blood and gut mucosa as 
part of the secondary objectives.  As a supportive assay, qPCR for C34-CXCR4 will be 
conducted to track persistence of modified cells post-infusion.  Subjects will also be assessed 
for the presence of C34-CXCR4-modified cells in peripheral blood prior to and after initiating the 
analytical treatment interruption using these assays. 
 
Immunologic Studies. These include activation of T-cells and intracellular cytokine staining 
and /or CD107 degranulation of T-cells in response to HIV, EBV, Flu and CMV peptides. The 
intracellular cytokine staining will be done on frozen/thawed cells. The specific assays to be 
done include staining of CD4+ and CD8+ cells for the following cytokines: interferon-gamma, 
interleukin-2, and tumor-necrosis factor-alpha. The stimulating HIV antigens will be pools of 
peptides spanning HIV gag, env, and nef. The peptides will match the HIV clade B consensus 
sequence and consist of 15-mers with 11aa overlaps.  
 
Virologic Studies. These studies are designed to measure the size of the HIV reservoir using 
the available methodologies 
 
The effects of C34-CXCR4-modified CD4 T-cells on the HIV reservoir will be measured in 
subjects with undetectable viral loads at the end of the 16 week analytical treatment interruption 
using the following assays: 
1) Number of latently-infected resting CD4 T-cells quantified using a limiting dilution co-
culture assay (Comparing Step 1 to Step 2). 
2) Frequency of integrated provirus as measured by Alu-PCR (Comparing Step 1 to Step 
2). (Liszewski, Yu et al. 2009)  
 
Storage of PBMC, serum, plasma. Samples will be stored for future use to assess the kinetics 
of the response and/or other research questions and for future virology and immunology testing.  

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Samples will be collected and processed by the Translational and Correlative Studies 
Laboratory (TCSL) at the University of Pennsylvania prior to archiving in the laboratory of James 
L. Riley. 
 
Rectal Biopsies. All subjects enrolled in the study are asked to undergo four optional rectal 
mucosal biopsies. The biopsy procedure takes approximately 30 minutes to complete, is 
performed in the outpatient setting and does not require anesthesia. A trained gastroenterologist 
at the UPenn GI clinic will perform each rectal biopsy.  Please see Section 6.2.3 for additional 
details.  
 
VSV-G DNA. VSV-G DNA in PBMC will be measured by DNA-TaqMan PCR assay using 
primers and a probe that is specific for the VSV-G sequence. This assay will have specificity 
equal to or greater than 50 copies per μg of genomic DNA, in accordance with FDA Guidance. 
 
Transcriptional Profiling. These studies are designed to define the transcriptional program 
and differentiation state of C34-CXCR4 modified CD4 T cells in subjects before, during and after 
treatment interruption.  
 
 
7.0 
CLINICAL MANAGEMENT ISSUES 
7.1 
Toxicity  
 
 
Infusion related adverse events may develop as described in Section 5.4.2. The 
premedication helps to mitigate the symptoms; if they develop, the symptoms resolve 
spontaneously within 1-2 days and the clinical investigator will follow the subject until 
these events resolve.  
 
If a subject experiences a new grade 3 or 4 treatment related toxicity (ACTG table for 
grading severity of adult adverse experiences) that is confirmed by further tests, and 
fails to normalize or return to baseline within 1 month, the subject will be followed 
until the toxicity resolves or until no further improvement is anticipated by the 
investigator. 
 
Cytokine Release Syndrome (CRS). CRS is managed in the same fashion as septic 
shock following infection with gram negative bacteria, with the exception that 
antibiotics are not required. Capillary leak with fluid retention is worsened by 
hydration commonly given to treat hypotension.  The syndrome can be associated 
with pulmonary infiltrates, pulmonary edema, arrhythmias and cardiac arrest.  The 
syndrome is also associated with an elevation in LFTs, d-dimers, LDH, Creatinine, 
uric acid, and phosphorus from immune-mediated cytolysis of targeted cells, with 
release of intracellular contents as well as a possible bystander effect (on 
neighboring, nontargeted cells). Prophylactic measures to prevent CRS include 
correction of overhydration/volume overload before infusion with diuresis if 
necessary and pre-medication. Patients are pre-medicated with acetaminophen and 
an antihistamine before T-cell infusion. As a routine, after infusion the vital signs 
(including pulse oximetry) will be monitored pre- and post-infusion per section 5.4.1. 
In the event of CRS, patients with rigors will be treated symptomatically with hot 
pack, blankets, and Dilaudid (0.5 mg IVP prn). An MD will be called immediately if 
subject experiences a decrease in blood pressure. If a subject experiences 

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hypotension, this will be managed by administering intravenous fluids in boluses as 
needed to maintain an adequate blood pressure.  Should the hypotension not 
respond to intravenous fluid boluses, admission to the CHPS or inpatient hospital will 
be performed to provide the appropriate level of care.  Should hypoxia or dyspnea 
develop that results in oxygen desaturation as detected by standard pulse oximetry, 
the patient will be administered nasal cannula oxygen and have a chest X-ray 
performed as deemed appropriate by the investigator. The use of ACE inhibitors and 
angiotensin-II receptor blockade may have benefits for CRS. Corticosteroids have 
been reported to block systemic effects of T-cell toxicity.  
 
If a subject develops CRS, all standard of care will be offered to assure subject 
wellbeing including treatment with steroids. According to the protocol, the serum 
cytokine levels will be tested from subjects’ blood draws pre-infusion (baseline), and 
20min and 2h post-infusion; however, in case of a CRS, blood will be collected as 
soon as possible and sent to the TCSL for real time cytokine analysis.  Our 
experience to date on other research protocols treating oncologic subjects with CAR 
modified T-cells has indicated that CRS is associated with high levels of serum IL-6 
among other cytokines. Consequently, some of our subjects developing CRS have 
been offered Tocilizumab as treatment for CRS following laboratory confirmation of 
elevated levels of serum IL-6 post-infusion. This approach has proved successful; 
however it is at the discretion of the clinical PI as to its usage. 
 
If a subject develops CRS prior to initiating treatment interruption the subject will not 
undergo ATI. 
  
 
Sustained (confirmed) virologic failure without an alternative explanation: 
Before ATI: If prior to initiating treatment interruption the subject experiences an 
increase in viral load from undetectable to greater than or equal to 5000 copies/ml, a 
viral load test will be repeated once a week for two additional weeks.  If the viral load 
increase is sustained over this period, this event will be reported as an SAE and will 
be managed as medically appropriate, and the subject will not undergo ATI.  
 
During ATI:  If the subject experiences a viral load ≥100,000 copies/ml, this test will 
be repeated every week for up to 3 weeks. If the subject experiences a sustained 
viral load to ≥100,000 copies/ml over a period of 3 weeks, it will be recommended to 
the subject that HAART should be reinstated.  The HAART will not be provided by 
the study.  If the viral load is not sustained (i.e. as indicated by a single test <100,000 
copies/ml), the subject will be advised not to reinstate HAART until week 16 after the 
treatment interruption to allow for evaluation of the subject’s viral load set point.  
 
 
Sustained (confirmed) decrease in CD4+ T-cell count:  
If a subject experiences a decrease in CD4+ T-cell count to <350 cells/mm3, the test 
will be repeated the following week. If the CD4+ T-cell counts stay equal to or below 
350 cells/mm3, the subject will be advised to reinstitute HAART. The HAART will not 
be provided by the study.  If the CD4+ decrease is not sustained the subject will 
continue in the study. 
 

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 
Monitoring for C34-CXCR4-Mediated Clonal Outgrowth 
Patients will be monitored for total lymphocyte count at each study visit.  If a patient 
experiences a total lymphocyte count greater than 10,000, then their lymphocytes 
will be analyzed for monoclonality by Vβ TCR analysis and for C34-CXCR4 marking 
by flow cytometry and/or PCR.  If monoclonality is observed and the cells are 
confirmed to be modified by C34-CXCR4, then best attempts will be made to 
determine the etiology of the cancer and the relationship to the treatment (C34-
CXCR4-T cells). In parallel, the patient will be referred to a hematologist for a 
leukemia/lymphoma work-up.  
 
Monitoring for aberrant T cell trafficking 
Patients will be monitored for any new incidence of pulmonary, cardiovascular, or 
gastrointestinal disorders (considered to be atypical) following the infusion of 
autologous C34-CXCR4 gene-modified cells. The C34-CXCR4 transgene that will be 
expressed in autologous CD4 T cells under control of the lentivirus promoter does 
retain competence to mediate migration of cells (i.e., chemotaxis) in response to the 
chemokine ligand (CXCL12) for CXCR4. Thus, any new clinical problem in which 
organ infiltration by lymphocytes could be taking place will be thoroughly 
investigated. 
7.2 
Criteria for pausing or stopping the study 
The study will be paused if: 
 
 
There are excessive or unexpected toxicities associated with the protocol.  
Specifically, we will pause the study and re-evaluate the approach if it is determined 
that two or more of the subjects have one of the following:  grade 3 toxicity 
determined by using the DAIDS Clinical Trial Toxicity Criteria of November 2014. 
SAEs will be reported to the DSMB in accordance to the DSMB Charter.  
 
A patient experiences an absolute lymphocyte count greater than 10,000 during 
visits spaced 1 week apart, until the evaluation of the nature of the lymphocyte 
increase is determined.  If it is found to be unrelated to the study treatment, then the 
study will resume. 
 
If a positive VSV-G DNA signal is detected and confirmed by a second PCR test 
(taken from a fresh blood sample). The study will be paused and no new patients will 
be infused with the study drug until the patient who has the positive test is evaluated 
for RCL. If an RCL is confirmed the study will be stopped. If an RCL is not confirmed, 
the study can be restarted after the source of the positive signal in the VSV-G DNA 
test is understood by the investigators 
 
The study will be stopped if: 
 
 
The Investigator, Study Funder, Sponsor or any independent review board or 
regulatory body decides for any reason that subject safety may be compromised by 
continuing the study.   
 
The Sponsor or Study Funder decides to discontinue the development of the 
intervention to be used in this study. 

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 
An analysis of clonal outgrowth of T-cells determines that it is a result of C34-CXCR4 
lentiviral-mediated oncogenesis.  
 
Any subject develops an increase from baseline in viral load to > 5000 and is 
sustained for at least 3 weeks during Step 2 of the protocol, and has no alternative 
explanation (such as non-compliance) other than the study treatment.   
 
Any subject develops a decrease in CD4 T-cell count of 350 or below which is 
sustained for at least 2 weeks during Step 2 of the protocol, and has no alternative 
explanation (such as non-compliance) other than the study treatment. 
Futility assessment has been included in the responsibilities of the DSMB.  Futility will be 
assessed at each DSMB meeting and will be evaluated using guidelines stated in the DSMB 
charter. 
If stopping rules are invoked, accrual of additional subjects to the study will be precluded.  
7.3 
Special Procedures for Subjects Developing Cancer or Dying  
For subjects who develop cancer and who have previously given their consent, an attempt will 
be made to obtain a sample of the biopsy of the cancerous tissue and a blood sample will be 
obtained for evaluation.  
 
For subjects who die and who have previously given their consent, the subject’s family will be 
asked to consent to an autopsy procedure in order to obtain appropriate tissue samples for 
evaluation during a scheduled autopsy. 
 
7.4 
Pregnancies 
It is not known whether the study intervention carries increased risks for pregnant women or 
their unborn children.  Therefore, pregnant women cannot participate in the study. Women 
should be informed through the informed consent process that if, while in the study, they 
become pregnant or miss a menstrual period or for other reasons suspect that they might be 
pregnant, they should immediately notify the investigator who should then notify the regulatory 
sponsor. If the subject is confirmed to be pregnant, the investigator will immediately discontinue 
the subject from the study treatment. Counseling will also be provided to the women to help 
them decide how to proceed.   
 
To ensure patient safety, each pregnancy occurring while the patient is on study treatment must 
be reported to the protocol sponsor within 24 hours of learning of its occurrence.  The 
pregnancy should be followed up to determine outcome, including spontaneous or voluntary 
termination, details of the birth, and the presence or absence of any birth defects, congenital 
abnormalities, or maternal and/or newborn complications.   
 
Pregnancy should be recorded on a Clinical Trial Pregnancy Form and reported by the 
investigator to the protocol sponsor.  Pregnancy follow-up should be recorded on the same form 
and should include an assessment of the possible relationship to the study drug for any 
pregnancy outcome.  Any SAE experienced during pregnancy must be reported on the SAE 
Report form.  
 

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Pregnancy outcomes must be collected for the female partners of any males who took study 
treatment in this trial.  Consent to report information regarding these pregnancy outcomes 
should be obtained from the mother. 
 
Pregnancies will also be reported to the Antiretroviral Pregnancy Registry. 
 
7.5 
Long-Term Follow-up 
Long-term follow-up for this study will be conducted under a separate long-term follow-up 
protocol.  This destination protocol describes long term follow up of patients for up to 15 years in 
accordance with guidelines set forth by the FDA.  At the time of informed consent for this 
protocol, subjects will be made aware of the long term follow-up requirement. 
 
7.6 
RCL testing, patient monitoring, and procedure for dealing with 
positive test results in subjects 
RCL may be generated during the C34-CXCR4-T cell manufacturing phase or subsequently 
after introduction of vector transduced cells into the patient. However, an RCL resulting from the 
production phase is highly unlikely since elements are incorporated in the design of the vector 
system that minimize vector recombination and generation of RCL. Furthermore, the vector 
used to transduce the product undergoes sensitive assays for detection of RCL before it can be 
released to a patient. Nevertheless, generation of an RCL following infusion of the vector 
product remains a theoretical possibility. For ongoing patient monitoring for RCL, PBMC 
samples obtained from subjects at 3, 6, and 12 months post-treatment will be tested for VSV-G 
DNA sequences.  
 
If a positive VSV-G DNA signal is obtained, the PI will be informed and the patient will be 
scheduled for a retest. If the second test is positive, the patient with the confirmed positive VSV-
G signal will be scheduled for apheresis and a biological RCL will be performed on the 
apheresed product. If the biological RCL is positive, the study will be stopped. If the test is 
negative, infusions for all patients can resume.  
 
 
8.0 
STATISTICAL CONSIDERATIONS 
8.1 
General Design Issues 
This is a single cohort, open-label pilot study of the safety and antiviral activity of a single 
infusion of autologous CD4+ T-cells in HIV-infected subjects. Participants will have well 
controlled viral replication on HAART. Participants will be HIV-1-positive men and women > 18 
years of age with HIV-1-RNA levels ≤50 copies/ml for at least 2 years who have peripheral 
CD4+ T-cell counts ≥450 cells/mm3 and a documented CD4 nadir of not lower than 200 
cells/mm3, and who have a recorded viral load set point prior to starting therapy.  Following a 
leukapheresis at Step 1 (study enrollment), eligible subjects will enroll into Step 2 of the study 
and receive one infusion of thawed, autologous T-cells genetically modified by lentiviral vector 
expressing the C34-peptide conjugated to the CXCR4 N-terminus. All subjects will undergo a 16 
week analytical treatment interruption starting at week 4 (Step 3). 
 
At enrollment to Step 1, subjects will undergo 2 leukaphereses and an optional rectal biopsy. 
Within ~15 weeks after enrollment to Step 1, subjects will enter Step 2 and receive one infusion 

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modified by C34-CXCR4 throughout the duration of the study and attempt to correlate with 
viremia values during the analytical treatment interruption.  
 
To evaluate the effects on immune function, we will compare the percent of CD4+ and CD8+ T-
cells that secrete cytokines as a response to stimulation by HIV-specific or other viral antigens 
(Flu, EBV, CMV) at baseline (defined as the average of the Step 1 and 2 values) and after the 
infusion of genetically modified CD4+ T-cells. Summary statistics for time points at baseline and 
after the infusion will be shown. Additionally, proliferative capacity will also be assessed. 
 
The assay for CTL Phenotyping will employ a 6-hour in vitro stimulation of unfractionated patient 
PBMCs using antigens as described above.  Specifically, the CTL Phenotyping assay will 
examine the following external cellular markers: CD3, CD4, CD8 (T cell identification), CD45RO, 
CD27, CCR7 (memory subset identification), CD107a, (marker of lytic degranulation and 
cytotoxic potential).  The CTL Phenotyping assay will additionally analyze the following 
intracellular markers: Interferon Gamma (Th1 biasing cytokine), Tumor Necrosis Factor Alpha, 
Perforin (proteins involved in lytic degranulation and cytotoxic potential). 
 
The ability to proliferate in response to a specific antigen is a central part of immune 
assessment.  We will utilize carboxyfluorescein diacetate succinimidyl ester (CFSE), and 
surface staining for CD4 and CD8 to monitor T cell proliferative responses to antigen-specific 
stimulation. 
 
Statistics 
The immune response comparisons will be made using descriptive statistics and graphical 
methods for both phases of the study. Immune response measures will be correlated with dose 
level/treatment arm and participant outcome (i.e., adverse events) and with each other as well. 
Descriptive statistics and simple scatter plots will be generated to review the continuous 
immune response data. In addition, for continuous immune response values, the actual and % 
change in the level of each of the biomarkers from baseline to post‐baseline time points will be 
explored within each dose level/arm using Wilcoxon signed rank tests, and paired sample t-
tests. All categorical variables will be analyzed using chi‐square tests or Fisher’s exact test (the 
latter if warranted). Confidence intervals for both continuous and categorical data will be 
provided as well, as appropriate. For all translational endpoints, any notable statistical result will 
be viewed as an impetus for further study rather than as a definitive finding in and of itself. 
 
To evaluate immunogenicity of C34-CXCR4-modified CD4 T-cells, the ability of modified cells to 
persist, protect CD4 cell decay and control viremia as described above will be important 
indicators. Additionally, the development of a humoral anti-C34-CXCR4 response post-infusion 
will also be monitored as a marker of immunogenicity.    
 
8.4 
Sample Size and Accrual 
This is a pilot study with one cohort that completes infusions of C34-CXCR4-modified CD4 cells 
(Step 2) and undergoes an analytical treatment interruption (Step 3). In order to have an 
evaluable sample size, any subject who prematurely discontinues the study prior to the infusion 
will be replaced with another subject. Subjects who discontinue ATI prior to completion of the 
16-week ATI duration will not be replaced. 
 
 
Accrual is anticipated to take up to 72 weeks.  
 

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9.0 
DATA COLLECTION, MONITORING, AND ADVERSE EVENT 
REPORTING 
9.1 
Records to Be Kept 
Electronic CRFs will be provided for each subject. Subjects must not be identified by name on 
any eCRFs. Subjects will be identified by the subject identification number. 
 
9.2 
Role of Data Management 
Instructions concerning the recording of study data on eCRFs will be provided by the sponsor 
(eCRF Completion Guidelines). 
 
It is the responsibility of the principal investigator to assure the quality of computerized data for 
the study. This role extends to generation of the final study databases. 
 
9.3 
Clinical Site Monitoring and Record Availability 
This study will be monitored according to the Sponsor Data and Safety Monitoring Plan. 
 
Interim Monitoring Visits will be conducted during the course of the study. The Monitors will 
assure that submitted data are accurate and in agreement with source documentation; verify 
that investigational products are properly stored and accounted for; verify that subject consent 
for study participation has been properly obtained and documented; confirm that research 
subjects entered into the study meet inclusion and exclusion criteria; and assure that all 
essential documentation required by Good Clinical Practices (GCP) guidelines are appropriately 
filed.  At the end of the study, Monitors will conduct a close-out visit and will advise on storage 
of study records and disposition of unused investigational products. 
 
The investigator will allocate adequate time for such monitoring activities.  The Investigator will 
also ensure that the monitor or other compliance or quality assurance reviewer is given access 
to all the above noted study-related documents and study related facilities and has adequate 
space to conduct the monitoring visit. 
 
Independent Data and Safety Monitoring Board (DSMB) 
An Independent DSMB comprised of at least three individuals including physicians with 
experience in HIV infection and/or gene transfer therapy will be assembled and will work under 
a charter specifically developed for safety oversight of this study. They will provide advice to the 
Sponsor. The DSMB will evaluate subject-subject safety as specified in the DSMB Charter.  
 
The initial responsibility of the DSMB will be to approve the initiation of this clinical trial. 
Thereafter, the DSMB will meet approximately every 6 months. If necessary, additional meeting 
of the DSMB may be held if safety issues arise in between scheduled meetings. 
 
Meetings will also be convened in the event of any DLT, or any grade 3 or higher serious 
adverse event that is possibly related to the study drug and unexpected. These meetings can be 
initiated by the Sponsor, PI of the study, the protocol team, or the DAIDS Medical Officer to 
address specific safety issues and/or pausing of dosing and/or stopping the study. 
 

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If a Grade 4 event or death is observed that is determined to be at least possibly related to 
study treatment (based on the core team assessment), accrual will be halted and the DSMB will 
convene to review the study data.  
 
A sponsor representative will share the outcome of the DSMB meeting with the PI via email for 
submission to the IRB and other local regulatory review committees per institutional 
requirements. 
 
9.4 
Safety and Adverse Events 
9.4.1 Definitions 
 
Adverse Event 
An adverse event (AE) is any untoward medical occurrence associated with the use of a drug 
in humans, whether or not considered drug related.  Intercurrent illnesses or injuries should be 
regarded as adverse events.   
 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  A serious adverse event is any AE 
that is:  
 
fatal 
 
life-threatening 
 
requires or prolongs hospital stay: 
 
leads to a persistent or significant disability or incapacity or substantial disruption of the 
ability to conduct normal life functions 
 
a congenital anomaly or birth defect 
 
an important medical event 
 
Note that hospitalizations that meet the following criteria should not be reported as serious 
adverse events: 
 
Routine treatment or monitoring of the studied indication, not associated with any 
deterioration in condition, such as preplanned study visits and preplanned 
hospitalizations for study procedures or treatment administration 
 
Elective or pre-planned treatment for a pre-existing condition that is unrelated to the 
indication under study and has not worsened since signing the informed consent 
 
Social reasons and respite care in the absence of any deterioration in the patient’s 
general condition 
 
Note that treatment on an emergency outpatient basis that does not result in hospital 
admission and involves an event not fulfilling any of the definitions of a SAE given above is 
not a serious adverse event. 
 
Important medical events are those that may not be immediately life threatening, but are 
clearly of major clinical significance.  They may jeopardize the patient, and may require 
intervention to prevent one of the other serious outcomes noted above.  For example, drug 
overdose or abuse, a seizure that did not result in patient hospitalization, or intensive treatment 
of bronchospasm in an emergency department would typically be considered serious. 
 
All adverse events that do not meet any of the criteria for serious should be regarded as non-
serious adverse events. 

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Unexpected adverse events 
An adverse event is considered unexpected if the event severity and/or frequency is not 
described in the investigator brochure or protocol.  As this investigational product is first in 
humans the severity/frequency of events are not yet known, an investigator brochure is not 
available.  
 
Related adverse events 
An adverse event is considered related to participation in the research if there is a reasonable 
possibility that an event was caused by an investigational product, intervention, or research-
required procedures.  For the purposes of this study, "reasonable possibility" means there is 
evidence to suggest a causal relationship.  
 
Dose Limiting Toxicity (DLT) 
Dose limiting toxicity is a grade 3 or higher new AE within 21 days which is at least possibly 
related to study treatment and not manageable or reversible.   A formal DLT assessment will be 
performed prior to dose level advancement or expansion.  
 
Adverse Event Reporting Period 
Adverse events will be collected and reported from the time of informed consent to the 
completion of the scheduled study visits following the administration of study treatment.  
 
If a subject is taken off study within 30 days of receiving C34-CXCR4-modified autologous T-
cells, all SAEs experienced within 30 days after the T-cell infusion should be reported to the 
sponsor. Any SAEs experienced after this 30 day period should be reported to the sponsor if the 
investigator suspects a causal relationship to the study treatment.  
 
Preexisting Condition/General Physical Examination Findings 
 
A preexisting condition is one that is present at the start of the study.  At screening, any 
clinically significant abnormality should be recorded as a preexisting condition on the medical 
history eCRF.  During the course of the study, a preexisting condition should be recorded as an 
adverse event if the frequency, intensity, or the character of the condition worsens. Preexisting 
conditions that improve should also be recorded appropriately. 
 
Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse event if any one of the 
following conditions is met:  
 
The laboratory abnormality is not otherwise refuted by a repeat test to confirm the 
abnormality 
 
The abnormality suggests a disease and/or organ toxicity 
 
The abnormality is of a degree that requires active management; (e.g. change of dose, 
discontinuation of the drug, more frequent follow-up assessments, further diagnostic 
investigation, etc.) 
 
Laboratory abnormalities that meet the criteria for Adverse Events should be followed until they 
have returned to normal or an adequate explanation of the abnormality is found. When an 
abnormal laboratory or test result corresponds to a sign/symptom of an already reported 
adverse event, it is not necessary to separately record the lab/test result as an additional event. 
Laboratory abnormalities that do not meet the definition of an adverse event, should not be 

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reported as adverse events.  A Grade 3 or 4 event (severe) as per the DAIDS AE Grading Table 
Version 2.0 does not automatically indicate a SAE unless it meets the definition of serious 
defined above and/or as per investigator’s discretion.  Whenever possible, a diagnosis, rather 
than a symptom should be provided (i.e. anemia instead of low hemoglobin). 
 
9.4.2 Recording of Adverse Events 
Safety will be assessed by monitoring and recording potential adverse effects of the treatment  
using the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, 
Version 2.0, November 2014.  This table can be found at:  
http://rsc.tech-res.com/docs/default-source/safety/daids ae grading table v2 nov2014.pdf 
 
Patients will be monitored by medical histories, physical examinations, and blood studies to 
detect potential toxicities from the treatment.  If grading does not exist for an adverse event, the 
severity of mild, moderate, severe, life-threatening, and death, corresponding to Grades 1-5, will 
be used whenever possible. 
 
At each contact with the subject, the investigator must seek information on adverse events by 
non-directive questioning and, as appropriate, by examination. Adverse events also may be 
detected when they are volunteered by the subject during the screening process or between 
visits, or through physical examination, laboratory test, or other assessments.  Information on all 
adverse events should be recorded in the source documentation.  All clearly related signs, 
symptoms, and abnormal diagnostic procedure results should be recorded in the source 
document, though should be grouped under one diagnosis.  To the extent possible, adverse 
events should be recorded as a diagnosis and symptoms used to make the diagnosis recorded 
within the diagnosis event.  Do not list symptoms separately if a diagnosis can be assigned. 
 
All adverse events occurring during the adverse event reporting period (defined in Section 9.4.1) 
must be recorded.   
 
As much as possible, each adverse event should be evaluated to determine: 
1. The severity grade (Grade 1-5) 
2. Its duration (Start and end dates) 
3. Its relationship to the study treatment- [Reasonable possibility that AE is related: No 
(unrelated/ not suspected) or Yes (a suspected adverse reaction)].  If yes (suspected)- is 
the event possibly, probably or definitely related to the investigational treatment?  
4. Expectedness to study treatment- [Unexpected- if the event severity and/or frequency is 
not described in the investigator brochure (if applicable) or protocol]. 
5. Action taken with respect to study or investigational treatment (none, dose adjusted, 
temporarily interrupted, permanently discontinued, unknown, not applicable) 
6. Whether medication or therapy taken (no concomitant medication/non-drug therapy, 
concomitant medication/non-drug therapy) 
7. Whether it is serious, where a serious adverse event (SAE) is defined as in Section 
9.4.1. 
 
All adverse events should be treated appropriately. If a concomitant medication or non-drug 
therapy is given, this action should be recorded.  Once an adverse event is detected, it should 
be followed until its resolution or until it is judged to be permanent, and assessment should be 
made at each visit (or more frequently, if necessary) of any changes in severity, the suspected 
relationship to the study treatment, the interventions required to treat it, and the outcome.   
 

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Serious adverse events that are still ongoing at the end of the adverse event reporting period 
must be followed to determine the final outcome.  Any serious adverse event that occurs after 
the adverse event reporting period and is considered to be possibly related to the study 
treatment or study participation, should be recorded and reported. 
 
9.4.3 Reporting of Serious Adverse Events  
Every SAE, regardless of suspected causality, occurring during the adverse event reporting 
period defined in Section 9.4.1 must be reported to the sponsor within 24 hours of learning of 
its occurrence. The original SAE notification may take place by email to meet the 24 hour 
reporting window.  However within 3 business days of knowledge of the event, the investigator 
must submit a complete SAE form to the Sponsor along with any other diagnostic information 
that will assist the understanding of the event.  The Investigator will keep a copy of this SAE 
Form on file at the study site.   
 
Follow-up information on SAEs should be reported when updates are available, as a follow-up 
to the initial SAE form, and should include both the follow-up number and report date.  New 
information on ongoing serious adverse events should be provided promptly to the sponsor.  
The follow-up information should describe whether the event has resolved or continues, if there 
are any changes in assessment, if and how it was treated, and whether the patient continued or 
withdrew from study participation.  
 
Report serious adverse events by email to: 
 
Attention: Sponsor Clinical Safety Manager or designee 
 
 
 
At the time of the initial notification, the following information should be provided: 
1. Study identifier 
2. Subject number 
3. A description of the event 
4. Date of onset 
5. Current status 
6. Whether study treatment was discontinued 
7. The reason the event is classified as serious 
8. Investigator assessment of the association 
between the event and study treatment 
9. Expectedness relative to investigational 
product(s) 
 
9.4.4 Investigator Reporting:  Local Regulatory Review Committees 
Report events to local regulatory review committees per institutional requirements. 
 
9.4.5 Sponsor reporting: Notifying the FDA 
The study sponsor is required to report certain study events in an expedited fashion to the FDA.  
These written notifications of adverse events are referred to as IND safety reports. The following 
describes the safety reporting requirements by timeline for reporting an associated type of 
event: 
 
 Within 7 calendar days 
Any study event that is: 
– associated with the use of the study drug 
– unexpected, and 

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– fatal or life-threatening 
 
 Within 15 calendar days 
Any study event that is: 
– associated with the use of the study drug, 
– unexpected, and 
– serious, but not fatal or life-threatening 
-or- 
– a previous adverse event that was not initially deemed reportable but is later found to 
fit the criteria for reporting (reporting within 15 calendar days from when event was 
deemed reportable). 
 
Any finding from tests in laboratory animals that:  
– suggest a significant risk for human subjects including reports of mutagenicity, 
teratogenicity, or carcinogenicity. 
 
Additional reporting requirements 
Sponsors are also required to identify in IND safety reports all previous reports concerning 
similar adverse events and to analyze the significance of the current event in light of the 
previous reports. 
 
 9.4.6 Reporting to the IBC 
Notify the Institutional Biosafety Committee of serious adverse events according to institutional 
requirements. 
 
9.4.7 Reporting to the Division of AIDS 
The DAIDS Medical Officer and Project Officer will receive copies of all serious adverse drug 
reactions (SADRs) and all expedited adverse events (EAEs) from the Sponsor.  They should 
also receive study progress updates and be informed of all unanticipated problems involving risk 
to subjects or others, as well as quarterly reports of all adverse events (AEs) from the Sponsor. 
 
9.5 
Protocol Exceptions and Deviations 
Exception 
A one time, intentional action or process that departs from the IRB approved study protocol, 
intended for one occurrence. If the action disrupts the study progress, such that the study 
design or outcome (endpoints) may be compromised, or the action compromises the safety and 
welfare of study subjects, advance documented approval from the Regulatory Sponsor and 
other local regulatory review committees per institutional guidelines is required.  Approval from 
the Regulatory Sponsor must be received prior to submission to local regulatory review 
committees for approval.  
 
Deviation 
A one time, unintentional action or process that departs from the IRB approved study protocol, 
involving one incident and identified retrospectively, after the event occurred. If the impact on 
the protocol disrupts the study design, may affect the outcome (endpoints) or compromises the 
safety and welfare of the subjects, the deviation must be reported to the Regulatory Sponsor 
within 10 business days of PI knowledge and to local regulatory review committees per 
institutional guidelines.  Acknowledgement from the Regulatory Sponsor must be received prior 
to submission to local regulatory review committees. 

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Other deviations that do not meet the reporting criteria above should be documented in a memo 
to file or on a deviation log. 
 
Include the following information on the Sponsor supplied exception/deviation form: protocol 
number, subject study number, description of protocol deviation or exception request and 
rationale.  Ensure all completed exception/deviation forms are signed by the Principal 
Investigator (or sub-investigator) and submitted to the Sponsor Project Manager for review.  
 
Attention: Sponsor Project Manager  
 
  
 
Once approval of the exception request or acknowledgement of the deviation, has been granted 
by the Regulatory Sponsor, the exception or deviation will be submitted to all other applicable 
committees for review and approval as required per institutional guidelines.  
 
 
10.0 HUMAN SUBJECTS 
10.1 Institutional Review Board (IRB) Review and Informed Consent 
This protocol and the informed consent document and any subsequent modifications will be 
reviewed and approved by the IRB, IBC or ethics committee responsible for oversight of the 
study. A signed consent form will be obtained from the subject. The consent form will describe 
the purpose of the study, the procedures to be followed, and the risks and benefits of 
participation. A copy of the consent form will be given to the subject and this fact will be 
documented in the subject’s record. 
 
10.2 Subject Confidentiality 
All laboratory specimens, evaluation forms, reports, and other records that leave the site will be 
identified by coded number only to maintain subject confidentiality. All records will be kept 
locked.  All computer entry and networking programs will be done with coded numbers only. 
Clinical information will not be released without written permission of the subject, except as 
necessary for monitoring/auditing activities. 
 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). Those 
regulations require a signed subject authorization informing the subject of the following:  
 
• What protected health information (PHI) will be collected from subjects in this study  
• Who will have access to that information and why  
• Who will use or disclose that information  
• The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization. For subjects that have revoked authorization to collect or use PHI, attempts 
should be made to obtain permission to collect at least vital status (i.e. that the subject is alive) 
at the end of their scheduled study period. 
 

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10.3 Source Documents 
Source data are all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial. Source 
data are contained in source documents. Examples of these original documents, and data 
records include: hospital records, clinical and office charts, laboratory notes, memoranda, 
subjects’ diaries or evaluation checklists, pharmacy dispensing records, recorded data from 
automated instruments, copies or transcriptions certified after verification as being accurate and 
complete, microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject 
files, and records kept at the pharmacy, at the laboratories, and at medico-technical 
departments involved in the clinical trial. 
 
10.4 Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the study.  All 
data requested on the CRF must be recorded. All missing data must be explained. All entries 
will be entered into an electronic data capture system (EDC) via Oracle.  The Principal 
Investigator is responsible for assuring that the data entered into eCRF is complete, accurate, 
and that entry and updates are performed in a timely manner. 
 
10.5 Records Retention 
It is the investigator’s responsibility to retain study essential documents for at least 2 years after 
the last approval of a marketing application in their country and until there are no pending or 
contemplated marketing applications in their country or at least 2 years have elapsed since the 
formal discontinuation of clinical development of the investigational product. These documents 
should be retained for a longer period if required by an agreement with the sponsor. In such an 
instance, it is the responsibility of the sponsor to inform the investigator/institution as to when 
these documents no longer need to be retained. 
 
10.6 Study Discontinuation 
The study may be discontinued at any time by the Sponsor, IRB, NIAID, DAIDS, the financial 
supporter, the FDA, OCR or other government agencies as part of their duties to ensure that 
research subjects are protected. 
 
 
11.0  STUDY MONITORING, AUDITING, AND INSPECTING 
11.1 Study Monitoring Plan 
This study will be monitored according to the Sponsor Data and Safety Monitoring Plan.   
 
Interim Monitoring Visits will be conducted during the course of the study. The Monitors will 
assure that submitted data are accurate and in agreement with source documentation; verify 
that investigational products are properly stored and accounted for, verify that subjects’ consent 
for study participation has been properly obtained and documented, confirm that research 
subjects entered into the study meet inclusion and exclusion criteria, and assure that all 
essential documentation required by Good Clinical Practices (GCP) guidelines are appropriately 
filed. 
 

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At the end of the study, Monitors will conduct a close-out visit and will advise on storage of 
study records and disposition of unused investigational products. 
 
The investigator will allocate adequate time for such monitoring activities. The Investigator will 
also ensure that the monitor or other compliance reviewer is given access to all the above noted 
study-related documents and study related facilities (e.g. pharmacy, diagnostic laboratory, etc.), 
and has adequate space to conduct the monitoring visit. 
 
11.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, the 
sponsor, government regulatory bodies, and University compliance groups of all study related 
documents (e.g. source documents, regulatory documents, data collection instruments, study 
data etc.).  The investigator will ensure the capability for inspections of applicable study-related 
facilities (e.g. pharmacy, diagnostic laboratory, etc.).  
 
Participation as an investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable University compliance offices. 
 
The Principal Investigator must notify the Sponsor in real-time if an audit/inspection notification 
is received. 
 
 
12.0 ETHICAL CONSIDERATIONS 
This study is to be conducted according to US and international standards of Good Clinical 
Practice (FDA Title 21 part 312 and International Conference on Harmonization guidelines), 
applicable government regulations and Institutional research policies and procedures.  
 
This protocol and any amendments will be submitted to a properly constituted Institutional 
Review Board (IRB), in agreement with local legal prescriptions, for formal approval of the study 
conduct. The decision of the IRB concerning the conduct of the study will be made in writing to 
the investigator and a copy of this decision will be provided to the sponsor before 
commencement of this study.  
 
All subjects for this study will be provided a consent form describing this study and providing 
sufficient information for subjects to make an informed decision about their participation in this 
study. This consent form will be submitted with the protocol for review and approval by the IRB 
for the study. The formal consent of a subject, using the IRB-approved consent form, must be 
obtained before that subject undergoes any study procedure. The consent form must be signed 
by the subject and by the investigator-designated research professional obtaining the consent. 
 
12.1 Rationale for Exposure to Risks 
As this is a Pilot study to establish safety and tolerability, there are no direct benefits expected 
for study participants.  There are, however, some risks associated with C34-CXCR4 modified T-
cells. 
 

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Risks associated with C34-CXCR4-modified cells include: 
 
Chills and fever 
 
Headache, myalgia, arthralgia 
 
Increase in blood pressure 
 
Low heart rate 
 
Allergic reaction (itching, swelling of the tongue) 
 
Seizures 
 
Nausea and vomiting 
 
Injection site reactions such as bruising, swelling, black and blue marks, fainting and/or 
infection at the site 
 
A decrease in hemoglobin and hematocrit (red blood cell number) 
 
Worsening of HIV infection (increase in HIV-1 viral load or decrease in T-cell count) 
 
Exclusion from future gene therapy or vaccine trials as a result of participation in this study. 
 
Possible restriction from future HIV trials based on the timing of HIV viral rebound in this 
study. 
 
Antibody formation that could lead to allergic reaction (skin rash, itching, fever) 
 
There is a theoretical risk of blood cancer in gene therapy studies which use integrating vectors.  
However, to date, no malignancies have been identified with the lentiviral vector used in this study. 
 
In an effort to minimize the above-mentioned risks, patients will be closely monitored, have 
frequent visits, and have supportive care.  Acetaminophen and Diphenhydramine hydrochloride 
will be used to prevent side effects (flu-like symptoms) associated with infusion of the C34-
CXCR4-modified cells. 
 
Although there are risks to the individual subject, as outlined above, the majority of the side 
effects associated with autologous T-cell infusions are transient, spontaneously solved in 24-48 
hours, or can be managed medically.  There is a great potential for benefit to both general 
scientific understanding and knowledge, and to society on the whole.  The individuals who 
participate in the completion of this study will potentially provide new and important information 
on the safety of controlling HIV through co-receptor modification.  Because both quality of life 
and economic burden on the health care system would be improved by obviating the need for 
patients with HIV infection to take daily and life-long antiviral medication, one goal for the future 
is to build an HIV-reisistant immune system.  This study will be a step along the path to reaching 
that goal, and will further support the concept of using a cell-based therapy to advance work 
towards improving HIV control/cure. 
 
 
13.0 STUDY FINANCES 
13.1 Funding Source 
This study is financed through a U19 grant awarded by the NIH NIAID.   
 
13.2 Conflict of Interest  
All University of Pennsylvania Investigators will follow the University of Pennsylvania Policy on 
Conflicts of Interest Related to Research. 
 

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13.3 Subject Stipends or Payments 
Subjects will be compensated for their time in participation of the study.  Compensation will be paid 
via ClinCard (a secure, reloadable debit card).  Compensation for the apheresis procedure is 
commensurate with that offered in other trials.  The reimbursement will be $25 per study visit, with 
the following exceptions: 
 
STEP 1 
Completion of Apheresis 1........................................................ $75  
Completion of Apheresis 2........................................................ $75  
Completion of baseline rectal biopsy ........................................ $75 
 
STEP 2 
Completion of Infusion .............................................................. $75  
Completion of rectal biopsy/leukapheresis Day 28 .................. $75 each (total $150) 
 
STEP 3 
Completion of rectal biopsy/leukapheresis week 20 ................ $75 each (total $150) 
 
 
 
STEP 5 
Completion of rectal biopsy/leukapheresis Week 52 ............... $75 each (total $150) 
 
Estimated Total compensation for the trial ......................... $1150 
 
 
14.0 BIOHAZARD CONTAINMENT 
As the transmission of HIV and other blood-borne pathogens can occur through contact with 
contaminated needles, blood, and blood products, appropriate blood and secretion precautions 
will be employed by all personnel in the drawing of blood and shipping and handling of all 
specimens for this study, as currently recommended by the Centers for Disease Control and 
Prevention and the National Institutes of Health. 
 
All dangerous goods materials, including diagnostic specimens and infectious substances, must 
be transported according to the instructions detailed in the International Air Transport 
Association (IATA) Dangerous Goods Regulations. 
 
 
15.0 PUBLICATION PLAN 
Publication of the results of this trial will be governed by University of Pennsylvania policies. 
Neither the complete nor any part of the results of the study carried out under this protocol, nor 
any of the information provided by the sponsor for the purposes of performing the study, will be 
published or passed on to any third party without the consent of the study sponsor.   Any 
investigator involved with this study is obligated to provide the sponsor with complete test 
results and all data derived from the study. 

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16.0 REFERENCE LIST  
Alexander, T. H., G. M. Ortiz, M. F. Wellons, A. Allen, E. J. Grace, 2nd, B. Schweighardt, J. 
Brancato, J. K. Sandberg, S. N. Furlan, G. D. Miralles, D. F. Nixon and J. A. Bartlett (2003). 
"Changes in CD4+ T-cell differentiation phenotype during structured treatment interruption in 
patients with chronic HIV-1 infection." J Acquir Immune Defic Syndr 34(5): 475-481. 
Allers, K., G. Hutter, J. Hofmann, C. Loddenkemper, K. Rieger, E. Thiel and T. Schneider 
(2010). "Evidence for the cure of HIV infection by CCR5{Delta}32/{Delta}32 stem cell 
transplantation." Blood. 
Ananworanich, J., U. Siangphoe, A. Hill, P. Cardiello, W. Apateerapong, B. Hirschel, A. 
Mahanontharit, S. Ubolyam, D. Cooper, P. Phanuphak and K. Ruxrungtham (2005). "Highly 
active antiretroviral therapy (HAART) retreatment in patients on CD4-guided therapy achieved 
similar virologic suppression compared with patients on continuous HAART: the HIV 
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Syndr 39(5): 523-529. 
Anton, P. A., R. T. Mitsuyasu, S. G. Deeks, D. T. Scadden, B. Wagner, C. Huang, C. Macken, 
D. D. Richman, C. Christopherson, F. Borellini, R. Lazar and K. M. Hege (2003). "Multiple 
measures of HIV burden in blood and tissue are correlated with each other but not with clinical 
parameters in aviremic subjects." AIDS 17(1): 53-63. 
Aronson, N., W. Benstein, V. Polonis, B. Gibbs, H. Flaks, B. Mulhall, A. Weintrob and C. June 
(2008). "A clinical trial of CD4 zeta gene-modified T cell infusion with and without IL-2 in HIV-
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Beatty, G. L., A. R. Haas, M. V. Maus, D. A. Torigian, M. C. Soulen, G. Plesa, A. Chew, Y. 
Zhao, B. L. Levine, S. M. Albelda, M. Kalos and C. H. June (2014). "Mesothelin-specific 
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malignancies." Cancer Immunol Res 2(2): 112-120. 
Blaese, R. M., K. W. Culver, A. D. Miller, C. S. Carter, T. Fleisher, M. Clerici, G. Shearer, L. 
Chang, Y. Chiang and P. Tolstoshev (1995). "T lymphocyte-directed gene therapy for ADA- 
SCID: initial trial results after 4 years." Science 270(5235): 475-480. 
Brenchley, J. M. and D. C. Douek (2008). "The mucosal barrier and immune activation in HIV 
pathogenesis." Curr Opin HIV AIDS 3(3): 356-361. 
Brenchley, J. M., D. A. Price, T. W. Schacker, T. E. Asher, G. Silvestri, S. Rao, Z. Kazzaz, E. 
Bornstein, O. Lambotte, D. Altmann, B. R. Blazar, B. Rodriguez, L. Teixeira-Johnson, A. 
Landay, J. N. Martin, F. M. Hecht, L. J. Picker, M. M. Lederman, S. G. Deeks and D. C. Douek 
(2006). "Microbial translocation is a cause of systemic immune activation in chronic HIV 
infection." Nat Med 12(12): 1365-1371. 

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sponsor. 
Brenchley, J. M., T. W. Schacker, L. E. Ruff, D. A. Price, J. H. Taylor, G. J. Beilman, P. L. 
Nguyen, A. Khoruts, M. Larson, A. T. Haase and D. C. Douek (2004). "CD4+ T cell depletion 
during all stages of HIV disease occurs predominantly in the gastrointestinal tract." The Journal 
of experimental medicine 200(6): 749-759. 
Brodie, S. J., D. A. Lewinsohn, B. K. Patterson, D. Jiyamapa, J. Krieger, L. Corey, P. D. 
Greenberg and S. R. Riddell (1999). "In vivo migration and function of transferred HIV-1-specific 
cytotoxic T cells." Nat.Med 5(1): 34-41. 
Bushman, F. (2002). "Targeting retroviral integration?" Mol Ther 6(5): 570-571. 
Cardiello, P. G., E. Hassink, J. Ananworanich, P. Srasuebkul, T. Samor, A. Mahanontharit, K. 
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