Conditions: Human Immunodeficiency Virus (HIV)
Keywords: Antiretroviral Therapy (ART)
Allocation: NON_RANDOMIZED | Intervention model: SEQUENTIAL
Enrollment: 17
Healthy volunteers: no

Brief summary:
The primary purpose of the trial is to determine the safety and tolerability of ixazomib in HIV infected patients on antiretroviral therapy. The secondary purpose is to determine the effect of ixazomib on the size of the HIV reservoir.

Detailed description:

Arms:
arm 1: Cohort A: Patients will receive ixazomib 1mg 3 times monthly for 12 weeks, then weekly for 12 weeks. arm 2: Cohort B: Patients will receive ixazomib 2mg 3 times monthly for 12 weeks, then weekly for 12 weeks. arm 3: Cohort C: Patients will receive ixazomib 3 mg 3 times monthly for 12 weeks, then weekly for 12 weeks. arm 4: Cohort D: Patients will receive ixazomib 4mg 3 times monthly for 12 weeks, then weekly for 12 weeks.

Interventions: intervention 1: Ixazomib 1 MG intervention 2: Ixazomib 2 MG intervention 3: Ixazomib 3 MG intervention 4: Ixazomib 4 MG
Intervention descriptions: intervention 1: 1 mg on days 1, 8 and 15 for three 28 days cycles, then be reviewed by DSMB and based upon their recommendation patients will receive ixazomib once weekly for 12 weeks or ixazomib 1 mg on days 1, 8 and 15 for three 28 days cycles. Visits 3-15 will have a window of ± 3 days. intervention 2: 2mg on days 1, 8 and 15 for three 28 days cycles, then be reviewed by DSMB and based upon their recommendation patients will receive ixazomib once weekly for 12 weeks or ixazomib 2 mg on days 1, 8 and 15 for three 28 days cycles. Visits 3-15 will have a window of ± 3 days. intervention 3: 3 mg on days 1, 8 and 15 for three 28 days cycles, then be reviewed by DSMB and based upon their recommendation patients will receive ixazomib once weekly for 12 weeks or ixazomib 3 mg on days 1, 8 and 15 for three 28 days cycles. Visits 3-15 will have a window of ± 3 days. intervention 4: 4 mg on days 1, 8 and 15 for three 28 days cycles, then be reviewed by DSMB and based upon their recommendation patients will receive ixazomib once weekly for 12 weeks or ixazomib 4 mg on days 1, 8 and 15 for three 28 days cycles. Visits 3-15 will have a window of ± 3 days.

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
PILOT STUDY OF IXAZOMIB TO REDUCE THE 
NUMBER OF 
HIV DNA POSITIVE LYMPHOID CELLS 
 
NCT02946047 
 
November 16, 2018 


Ixazomib for HIV Cure 
 
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PILOT STUDY OF IXAZOMIB TO REDUCE THE NUMBER OF 
HIV DNA POSITIVE LYMPHOID CELLS 
 
 
 
 
Regulatory Sponsor: 
Andrew Badley, Division of Infectious Diseases, Mayo 
Clinic, Rochester, MN 
Principal Investigator: 
Nathan Cummins, Division of Infectious Diseases, Mayo 
Clinic, Rochester, MN 
 
Co-Investigators 
Shaji Kumar, Division of Hematology, Mayo Clinic, 
Rochester, MN 
Stacey Rizza, Division of Infectious Diseases, Mayo Clinic, 
Rochester, MN 
Aaron Tande, Division of Infectious Diseases, Mayo Clinic, 
Rochester, MN 
John Zeuli, Medication Therapy Management, Mayo Clinic, 
Rochester, MN 
 
DSMB Members 
Zelalem Temesgen, Division of Infectious Diseases, Mayo 
Clinic, Rochester, MN 
Jennifer Whitaker, Division of Infectious Diseases, Mayo 
Clinic, Rochester, MN 
Vincent Rajkumar, Division of Hematology, Mayo Clinic, 
Rochester, MN 
Study Product 
Ixazomib 
Protocol Number: (IRBe) 
16-001938 
IND Number: 
 IND 130299 
 
 
 
 
 
Initial version: 03/16/2016 Version (1.0)  
Revision# 1: 07/22/2016 Version 2.0 
Revision#2: 08/31/2016 Version 3.0 
Revision#3: 02/02/2017 Version 4.0 
Revision#4: 04/26/2017 Version 5.0 


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Revision#5: 06/07/17 Version 6.0 
Revision#6: 09/18/17 Version 7.0 
Revision#7: 10/18/17 Version 8.0 
Revision#8: 1/15/18 Version 9.0 
Revision#9 1/26/18 Version 10.0 
Revision#10 11/26/18 Version 11.0 
 
 


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Table of Contents 
 
STUDY SUMMARY ...................................................................................................................................... 7 
1 
INTRODUCTION ................................................................................................................................. 9 
1.1 
BACKGROUND .................................................................................................................................... 9 
1.2 
INVESTIGATIONAL AGENT .................................................................................................................. 27 
1.3 
PRECLINICAL DATA ........................................................................................................................... 30 
1.4 
CLINICAL DATA TO DATE................................................................................................................... 31 
1.5 
DOSE RATIONALE AND RISK/BENEFITS ................................................................................................ 32 
2 
STUDY OBJECTIVES ........................................................................................................................ 35 
3 
STUDY DESIGN ................................................................................................................................. 35 
3.1 
GENERAL DESIGN ............................................................................................................................. 35 
3.2 
PRIMARY STUDY ENDPOINTS .............................................................................................................. 35 
3.3 
SECONDARY STUDY ENDPOINTS ......................................................................................................... 35 
4 
SUBJECT SELECTION ENROLLMENT AND WITHDRAWAL ...................................................... 35 
4.1 
INCLUSION CRITERIA ......................................................................................................................... 35 
4.2 
EXCLUSION CRITERIA ........................................................................................................................ 36 
4.3 
SUBJECT RECRUITMENT, ENROLLMENT AND SCREENING ....................................................................... 38 
4.4 
EARLY WITHDRAWAL OF SUBJECTS .................................................................................................... 38 
4.4.1 
When and How to Withdraw Subjects ........................................................................................ 38 
4.4.2 
Data Collection and Follow-up for Withdrawn Subjects .............................................................. 39 
5 
STUDY DRUG .................................................................................................................................... 40 
5.1 
DESCRIPTION .................................................................................................................................... 40 
5.2 
TREATMENT REGIMEN ....................................................................................................................... 40 
5.3 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ................................................................ 42 
5.4 
PREPARATION AND ADMINISTRATION OF STUDY DRUG ......................................................................... 43 
5.5 
SUBJECT COMPLIANCE MONITORING ................................................................................................... 45 
5.6 
PRIOR AND CONCOMITANT THERAPY .................................................................................................. 45 
5.7 
PACKAGING ...................................................................................................................................... 46 
5.8 
RECEIVING, STORAGE, DISPENSING AND RETURN ................................................................................. 46 
5.8.1 
Receipt of Drug Supplies .......................................................................................................... 46 
5.8.2 
Storage ................................................................................................................................... 46 
5.8.3 
Return or Destruction of Study Drug ......................................................................................... 47 
6 
STUDY PROCEDURES ...................................................................................................................... 47 
6.1 
VISIT 1 (SCREENING AND ENROLLMENT) ............................................................................................. 49 
6.2 
VISIT 2  ............................................................................................................................................ 49 
6.3 
VISIT 3 (STUDY ENTRY, TIME 0 WEEKS) .............................................................................................. 49 
6.4 
VISIT 4 (TREATMENT PHASE, TIME 1 WEEK ±3 DAYS) ............................................................................ 49 
6.5 
VISIT 5 (TREATMENT PHASE, TIME 2 WEEKS ±3 DAYS) .......................................................................... 50 
6.6 
VISIT 6 (TREATMENT PHASE, TIME 3 WEEKS ±3 DAYS) .......................................................................... 50 
6.7 
VISIT 7 (TREATMENT PHASE, TIME 4 WEEKS ±3 DAYS) .......................................................................... 50 
6.8 
VISIT 8 (TREATMENT PHASE, TIME 6 WEEKS ±3 DAYS) .......................................................................... 50 
6.9 
VISIT 9 (TREATMENT PHASE, TIME 8 WEEKS ±3 DAYS) .......................................................................... 51 
6.10 
VISIT 10 (TREATMENT PHASE, TIME 12 WEEKS ±3 DAYS) .................................................................. 51 
6.11 
VISIT 11 (TREATMENT PHASE, TIME 16 WEEKS ±3 DAYS) .................................................................. 51 
6.12 
VISIT 12 (TREATMENT PHASE, TIME 20 WEEKS ±3 DAYS) ................................................................. 51 
6.13 
VISIT 13 (LEUKAPHERESIS, TIME 22 WEEKS ±3 DAYS) ....................................................................... 52 
6.14 
VISIT 14 (TREATMENT COMPLETION, TIME 24 WEEKS ±3 DAYS) ........................................................ 52 
6.15 
VISIT 15 (FINAL STUDY VISIT, TIME 28 WEEKS ±3 DAYS) .................................................................. 52 


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6.16 
TABLE 1.  TABLE OF STUDY ACTIVITIES .......................................................................................... 53 
6.17 
TABLE 2: INSTRUCTIONS FOR TREATMENT DELAYS AND DOSE REDUCTIONS PRIOR TO IXAZOMIB 
ADMINISTRATION ....................................................................................................................................... 53 
6.18 
EXCLUDED CONCOMITANT MEDICATIONS AND PROCEDURES ............................................................ 55 
6.19 
PERMITTED CONCOMITANT MEDICATIONS AND PROCEDURES ........................................................... 56 
6.20 
PRECAUTIONS AND RESTRICTIONS .................................................................................................. 56 
6.21 
MANAGEMENT OF CLINICAL EVENTS .............................................................................................. 57 
7 
STATISTICAL PLAN ......................................................................................................................... 60 
7.1 
SAMPLE SIZE DETERMINATION ........................................................................................................... 60 
7.2 
STATISTICAL METHODS ..................................................................................................................... 60 
7.3 
SUBJECT POPULATION(S) FOR ANALYSIS ............................................................................................. 61 
8 
SAFETY AND ADVERSE EVENTS ................................................................................................... 61 
8.1 
DEFINITIONS ..................................................................................................................................... 61 
8.2 
RECORDING OF ADVERSE EVENTS ....................................................................................................... 64 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS .......................................... 65 
8.3.1 
Sponsor-Investigator reporting: notifying the Mayo IRB ............................................................. 65 
8.3.2 
Sponsor-Investigator reporting: Notifying the FDA ..................................................................... 66 
8.4 
PROCEDURES FOR REPORTING DRUG EXPOSURE DURING PREGNANCY AND BIRTH EVENTS ...................... 68 
9 
ADMINISTRATIVE REQUIREMENTS ............................................................................................ 69 
9.1 
PRODUCT COMPLAINTS ...................................................................................................................... 69 
9.2 
UNMASKING/UNBLINDING PROCEDURES ............................................................................................. 69 
9.3 
STOPPING RULES ............................................................................................................................... 69 
9.4 
MEDICAL MONITORING ..................................................................................................................... 70 
9.4.1 
Internal Data and Safety Monitoring Board ............................................................................... 70 
10 
DATA HANDLING AND RECORD KEEPING .................................................................................. 70 
10.1 
CONFIDENTIALITY ........................................................................................................................ 70 
10.2 
SOURCE DOCUMENTS .................................................................................................................... 70 
10.3 
DATA MANAGEMENT .................................................................................................................... 71 
10.4 
DATA PROCESSING ........................................................................................................................ 71 
10.5 
RECORDS RETENTION .................................................................................................................... 71 
11 
STUDY MONITORING, AUDITING, AND INSPECTING ................................................................ 72 
11.1 
STUDY MONITORING PLAN ............................................................................................................ 72 
11.2 
AUDITING AND INSPECTING ........................................................................................................... 72 
12 
ETHICAL CONSIDERATIONS ......................................................................................................... 72 
13 
STUDY FINANCES ............................................................................................................................ 73 
13.1 
FUNDING SOURCE ......................................................................................................................... 73 
13.2 
CONFLICT OF INTEREST ................................................................................................................. 73 
13.3 
SUBJECT STIPENDS OR PAYMENTS .................................................................................................. 73 
14 
PUBLICATION PLAN ....................................................................................................................... 73 
15 
REFERENCES ................................................................................................................................... 73 
16 
APPENDIX I ....................................................................................................................................... 78 
 
 
 
 
 
 


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List of Abbreviations 
 
 
LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
ANC 
Absolute neutrophil count 
ART 
Antiretroviral therapy 
ARV 
Antiretroviral 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
DLT 
Dose limiting toxicity 
DRESS Syndrome 
Drug Reaction (or Rash) with Eosinophilia and Systemic 
Symptoms 
DSMB 
Data and Safety Monitoring Board 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
hERG 
Human ether-à-go-go related gene 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
IB 
Investigator’s Brochure 
IC50 
Half-maximal inhibitory concentration 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
IV 
Intravenous 
LLN 
Lower limit of normal 
MLN2238 
Ixazomib/NinLaro® 
MM 
Multiple myeloma 
NCI CTCAE 
National Cancer Institute Common Terminology Criteria for 
Adverse Events 
NNRTIs 
Non-nucleoside reverse transcriptase inhibitors 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PK 
Pharmacokinetic 
PN 
Peripheral Neuropathy 
PO 
Oral administration 
RBC 
Red blood cell 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SCID 
Severe combined immunodeficient 
SOP 
Standard Operating Procedure 
TEN 
Toxic epidermal necrolysis 
ULN 
Upper limit of normal 
UPP 
Ubiquitin-proteasome Pathway 
 


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Study Summary 
Title 
PILOT STUDY OF IXAZOMIB TO REDUCE THE NUMBER OF 
HIV DNA POSITIVE LYMPHOID CELLS 
Running Title 
Ixazomib for HIV cure 
Protocol Number 
16-001938 
Phase 
Phase I/II 
Methodology 
Dose escalation, open-label clinical trial. 
Overall Study 
Duration 
24 months 
Subject Participation 
Duration 
7 months 
Single or Multi-Site  
Multi-Site 
Objectives 
The primary objective of the trial is to determine the safety and 
tolerability of ixazomib in HIV positive persons on antiretroviral 
therapy. The secondary objective is to determine the effect of ixazomib 
on HIV reservoir size. 
Number of Subjects 
Maximum of 17 
Diagnosis and Main 
Inclusion Criteria 
Virologically suppressed HIV disease on at least 3 active drug initial or 
salvage antiretroviral regimens for at least 6 months 


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Study Product, Dose, 
Route, Regimen 
Patients will be treated in sequential groups after the safety evaluation 
of each group by the DSMB as follows: 
 
Group 1: 3 patients with HIV infection, who meet inclusion criteria, will 
receive ixazomib 1 mg on days 1, 8 and 15 for three 28 days cycles, 
then be reviewed by DSMB and based upon their recommendation 
patients will receive either (i) ixazomib once weekly for 12 weeks or (ii) 
ixazomib 1 mg on days 1, 8 and 15 for three 28 days cycles. Visits 3-15 
will have a window of ± 3 days. 
 
Group 2: 3 patients with HIV infection, who meet inclusion criteria, will 
receive ixazomib 2mg on days 1, 8 and 15 for three 28 days cycles, then 
be reviewed by DSMB and based upon their recommendation patients 
will receive either (i) ixazomib  once weekly for 12 weeks or (ii) 
ixazomib 2 mg on days 1, 8 and 15 for three 28 days cycles. Visits 3-15 
will have a window of ± 3 days. 
 
Group 3: 3 patients with HIV infection, who meet inclusion criteria, will 
receive ixazomib 3 mg on days 1, 8 and 15 for three 28 days cycles, 
then be reviewed by DSMB and based upon their recommendation 
patients will receive either (i) ixazomib once weekly for 12 weeks or (ii) 
ixazomib 3 mg on days 1, 8 and 15 for three 28 days cycles. Visits 3-15 
will have a window of ± 3 days. 
 
Group 4: 3 patients with HIV infection, who meet inclusion criteria, will 
receive ixazomib 4 mg on days 1, 8 and 15 for three 28 days cycles, 
then be reviewed by DSMB and based upon their recommendation 
patients will receive either (i) ixazomib  once weekly for 12 weeks or 
(ii) ixazomib 4 mg on days 1, 8 and 15 for three 28 days cycles. Visits 
3-15 will have a window of ± 3 days. 
 
 
Group 5: 5 patients with HIV infection, who meet inclusion criteria will 
receive the maximum tolerated dose of ixazomib per DSMB 
recommendations. Visits 3-15 will have a window of ± 3 days. 
Duration of  
Treatment 
Administration for 
Each Patient 
24 weeks 
Reference therapy 
None 


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Statistical 
Methodology 
This study is designed to assess the safety and tolerability of ixazomib 
in an HIV infected patient population, side effects will be graded 
according to The NCI CTCAE v4.0 
Secondary endpoints are  
1) Change in HIV DNA in CD4 T cell subsets from Week -1 to Week 
24;  
2) Change in culturable HIV from Week -1 to Week 24;  
3) Change in absolute CD4 T cell count and percentage, CD8 T cell 
count and percentage, and CD4/CD8 ratio from Week -1 to 24; 
 
1 Introduction 
 
This document is a protocol for a human research study. This study will be carried out in 
accordance with the applicable United States government regulations and Mayo Clinic research 
policies and procedures.  
1.1 
Background 
 
Intense activity is focused on identifying a clinical intervention that results in a long term, 
drug free remission of HIV-1 infection. Latently infected CD4 T cells harbor transcriptionally 
silent, replication competent HIV.  Because these cells persist long term and are unaffected by 
current therapies, they represent an HIV reservoir that remains undiminished by current 
approaches. Pilot clinical trials have tested whether reactivation of HIV-1 from latency will 
decrease the number of cells containing HIV DNA due to viral cytopathic effect or immune 
mediated clearance. The latency reversal agents vorinostat, panobinostat and romidepsin result in 
HIV reactivation as measured by increases in cell associated HIV RNA, yet no change in cell-
associated HIV DNA, indicating that the reactivating cells do not die (1-3). Multiple ongoing 
studies are testing augmentation of the anti-HIV immune response in combination with viral 
reactivation as a strategy for HIV eradication. We propose that designing strategies to reduce 
HIV reservoir size will be informed by a mechanistic understanding of how HIV can kill 
CD4 T cells in different circumstances of activation, and HIV replication. 
 
Viral infection is often detected by intrinsic host defense mechanisms that activate cell 
suicide programs in order to limit the production of progeny virions. In response, viruses have 
evolved mechanisms to inhibit cell death pathways. Prototypic examples include the human 
papilloma virus E6 and E7 proteins (4), Baculovirus inhibitor of apoptosis (IAP) and p35 
proteins (5), and Adenovirus E1B-55K (6). The role of these viral death inhibitors in viral 
persistence is well established. In cervical epithelial cells with integrated HPV, for example, 
chromosomal disruption of either E6 or E7 with CRISPR/Cas9 results in the activation of p53 or 
retinoblastoma (Rb), respectively, thereby activating cell death cascades (4) and disrupting 
persistence of the virally infected cell. In baculovirus infected cells, p35 and IAP inhibit 
apoptosis; and baculoviruses deficient in either of these proteins will induce premature death 
after infection, resulting in fewer progeny viruses (7). Similarly, deletion of anti-apoptotic EBV 
BALF1, BHRF1 or both results in premature cell death during EBV infection of primary B cells 
(8) and deletion of the adenoviral BCL2 homolog E1B-19K results in premature cell death and 
loss of persistence of adenoviral infection (9).  Thus, virally encoded apoptosis inhibitors are 
necessary for infected cells to persist, and enable the persisting cells to produce progeny 


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viruses. 
 
Although HIV does not encode an apoptosis inhibitory protein per se, HIV-infected cells 
are nonetheless resistant to apoptosis (reviewed in (10, 11)). This apoptosis resistance reflects the 
ability of various HIV encoded proteins to upregulate endogenous apoptosis inhibitory proteins 
and down-regulate pro-apoptotic proteins. For example, Tat upregulates anti-apoptotic Bcl-2, c-
FLIP, XIAP and C-IAP2 (12), while Nef causes phosphorylation and inactivation of pro-
apoptotic Bad (13), thereby conferring apoptosis resistance (14).  This shift in apoptotic balance 
affords an HIV-infected cell the ability to survive and produce more virions. Experimentally, 
addition of the pan-caspase inhibitor z-VAD-fmk to primary T cells infected with HIV reduces 
cell death and augments HIV production because fewer HIV infected cells die (15). Similarly, 
when primary CD4 T cells transduced and selected for  Bcl-2 expression, and then  infected with 
GFP-HIV, some cells revert to a quiescent (GFP low) state, which recapitulates many features of 
latency, including lack of basal HIV transcription and induction of GFP/HIV by latency 
reversing agents (16). Thus there is an established association between apoptosis resistance, 
Bcl-2 expression and 
latency 
during 
in 
vitro HIV infection.  
 
While 
numerous 
pathways 
may contribute to the 
decline of uninfected 
CD4 T cells during 
uncontrolled 
HIV 
infection (17), fewer 
pathways have been 
implicated 
in 
the 
demise 
of 
cells 
directly infected by 
HIV.  Following HIV 
attachment, at least 
three 
distinct 
pathways can initiate 
the death of infected 
cells: RIG-I mediated 
sensing of HIV RNA 
(18, 
19); 
IFI-16 
sensing 
of 
unintegrated HIV DNA (20, 21), and DNA-PK-sensing of HIV integrase nicking of host DNA 
(22). Once integrated into host DNA, HIV can remain in a latent state for years, or reactivate and 
replicate. When HIV reactivation and replication occur, HIV protease is active within the cytosol 
(23), where it cleaves both viral and host substrates, leading to apoptotic cell death (24) HIV 
protease induced death is dependent upon protease cleaving procaspase 8 to generate the 
fragment Casp8p41(25-27), which contains an α-helical  BH3-like domain that binds and 
activates the pro-apoptotic Bcl-2 family member Bak, triggering mitochondrial outer 


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membrane permeabilization (MOMP) and apoptosis (28, 29) (Figure 1).  
 
HIV is currently incurable due to the presence of HIV integrated into the genome of 
resting cells, predominantly 
central memory CD4 T cells 
(TCM), which constitute the 
major reservoir for HIV in 
patients on suppressive ART 
(30). 
In 
the 
context 
of 
Casp8p41 biology, there are 
two possible reasons why 
reactivating cells might not 
die:  Either Casp8p41 is not 
generated, 
or 
the 
pro-
apoptotic effects of Casp8p41 
are antagonized by intrinsic 
apoptosis 
resistance 
mechanisms. 
 
Since TCM 
express detectable levels of 
Caspase 8 (Figure 2A), we 
analyzed whether TCM from 
HIV-infected 
persons 
generate Casp8p41 in vivo 
(Figure 2B).  Flow cytometry 
readily detected Casp8p41 
in TCM, suggesting that the 
failure of TCM to die when 
HIV reactivates is not due to 
a 
failure 
to 
generate 
Casp8p41, 
but 
instead 
perhaps due to antagonism 
of 
Casp8p41-mediated 
killing. 
 
TCM cells are long 
lived 
and 
proliferate 
in 
response 
to 
antigenic 
re-
stimulation.  By contrast, 
effector memory CD4 T cells 
(TEM), which are a lesser HIV 
reservoir (31), are found in areas of inflammation and have a shorter half-life than TCM (32). The 
longer life span of TCM prompted us to speculate that these cells might be intrinsically resistant to 
death stimuli. To assess this possibility, we treated primary PBMCs from 3 uninfected donors 
with a variety of pro-apoptotic stimuli and analyzed cell death in TCM and TEM. TCM were 
significantly less susceptible than TEM to both Fas ligation and various triggers of the 
mitochondrial apoptotic pathway (Figure 2C,D). Not only did TCM die less in response to the 


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Fas-agonist antibody CH11 compared to TEM (absolute difference -19.0 ± 3.7%, P=0.006), but 
TCM also died less in response to chemotherapy agents such as etoposide and camptothecin, 
suggesting a global death resistance. These findings extend prior results showing that 
phosphorylation mediated inactivation of Foxo3a results in the resistance of TCM to 
spontaneous, Fas ligation-induced and TCR-ligation induced cell death (33). 
 
To understand the mechanism underlying apoptosis resistance of TCM compared to TEM, 
we performed RNAseq using cells from 2 HIV-infected donors with long term suppressed HIV 
infection. In comparison to TEM, TCM up-regulated 6 proliferation genes and down-regulated 46 
cell death genes by at least 2-fold (P=6.5x10-6 for enrichment within the death and proliferation 
gene sets, Figure 2E). These results were validated by querying previously published (34), 
publicly available gene expression data (GSE61697, Gene Expression Omnibus), comparing 
expression of 4 genes differentially expressed in our dataset (TNFRSF9, TNFRSF11A, PRF1 
and GZMA, Figure 2F). Therefore TCM upregulate antiapoptotic genes, and down regulate 
proapoptotic genes consistent with prior reports that during the development of memory T 
cells upregulate anti-apoptotic molecules such as Bcl2, Bcl-xL and c-IAPs (35).  
 
Since Bcl-2 antagonizes Bak activation in lymphoid cells (36), we assessed the relative 
expression of Bcl-2 and Procaspase 8 protein in TCM versus TEM and observed that the Bcl-
2:Procaspase 8 ratio is 60% higher in TCM compared to TEM (Figure 2A), suggesting that TCM 
might resist the pro-death effects of Casp8p41 by virtue of elevated Bcl-2 expression.  This 
relative increase in the Bcl-2:Procaspase 8 ratio in TCM cells was further supported by gene 
expression data showing increased BCL2 gene expression in TCM compared to TEM 
(P=0.031, Figure 2F).   
 
Whether Bcl-2 alters the ability of Casp8p41 to kill cells is unknown. Accordingly, we 
compared the responses of parental Jurkat T cells to those of Jurkat cells stably overexpressing 
Bcl-2 (Jurkat-Bcl-2) to transfection with GFP-Casp8p41 or empty vector.  Over 24 hours, 
significantly more parental Jurkat T cells expressing Casp8p41 became apoptotic as measured by  
TUNEL positivity (Figure 3A), compared to Jurkat-Bcl-2 cells expressing Casp8p41 (Mean 
AUC 1500 vs 870 [95% CI of difference -790, -480; P =0.004]) despite similar degrees of 
Casp8p41 expression (Figure 3B).  Thus, cells overexpressing Bcl-2 die less in response to 
Casp8p41, indicating that Bcl-2 antagonizes Casp8p41 induced killing and suggesting that 
Bcl-2 overexpression might also alter the outcome of HIV infection. 


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To experimentally test this hypothesis, Jurkat and Jurkat-Bcl-2 cells were infected with 
HIV IIIb and analyzed over time for cell viability, HIV p24 production, and cell associated HIV 
DNA content.  Bcl-2 overexpression was accompanied by increased cell survival following HIV 
infection (mean 36% vs 11% viability at day 9 post infection, P<0.001, Figure 3C), increased 
cell associated HIV DNA (5.5 log increase, P=0.001, Figure 3D), and increased HIV p24 
production in culture supernatants (2.5 log increase, P=0.023, Figure 3E). Consequently Bcl-2 
reduces HIV induced cell death and increases the number of HIV-infected cells (reflected 
by increased HIV DNA), thereby allowing more cells to produce progeny virions (reflected 
by increased p24). 
 
To assess whether Bcl-2 overexpression impacts Casp8p41-mediated Bak activation, 
Jurkat or Jurkat-Bcl-2 cells were transfected with GFP or GFP-Casp8p41 and we used 
conformational specific antibodies which recognize activated Bak, but not inactive Bak, to assess 
the state of Bak activation in the GFP+ cells.  As expected, GFP-Casp8p41+ parental Jurkat cells 
contained more activated Bak than parental cells expressing GFP alone (MFI difference 52+/-16, 
P=0.029; Figure 3F,G).  Also and consistent with our hypothesis, GFP-Casp8p41+ Jurkat-Bcl-2 
cells did not exhibit increased Bak activation compared to GFP alone (MFI difference 3.5+/-16, 
P=0.834), indicating that Bcl-2 prevents Bak activation in response to Casp8p41 expression.  
Furthermore, the overall number of activated Bak positive cells was lower in Jurkat-Bcl-2 cells 
compared to Jurkat cells (P=0.045).  Thus, Bcl-2, which is increased in TCM relative to other 
T cell subsets, inhibits Casp8p41-induced Bak activation.  
 
We next investigated how Bcl-2 antagonizes Casp8p41 killing. Bcl-2 family members are 
classified into three groups based on their structure and function(37): the pro-apoptotic proteins 
Bax and Bak, which contain BH1, BH2, and BH3 domains, permeabilize the outer mitochondrial 


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1 4  
 
me m bra ne  w he n  acti vat e d;  t he  a nti -a p o pt otic  pr otei ns,  e. g.,  Bcl -2,  Bcl - xL  a n d  Mcl -1,  w hic h  
c o ntai n B H 1, B H 2, B H 3 a n d B H 4 d o mai ns, bi n d a n d ne utralize acti vate d Ba x a n d Ba k as well as 
ot her pr otei ns t hat c o ntai n B H 3 d o mai ns; a n d t he pr o -a p o pt otic B H 3 -o nl y pr otei ns, e. g., Bi m, 
Bi d a n d Ba d, w hic h c o nt ai n o nl y a B H 3 d o mai n, ser ve as se ns ors of vari o us cell ular stress es a n d 
tri g ger a p o pt osis b y i) directl y acti vati n g Ba x a n d Ba k or ii) dis placi n g t hese pr otei ns fr o m a nti-
a p o pt otic ne utralizers.  
 
 
Beca use t he α -h elical B H 3 -li ke d o mai n of Cas p 8 p 4 1 directl y acti vat es Ba k b y bi n di n g t o 
t he Ba k B H 3 bi n di n g gr o o ve (2 9 ), we i n vesti gat e d w het her Cas p 8 p 4 1 als o bi n ds a nti-a p o pt otic 
Bcl -2, Bcl - xL  a n d Mcl -1 t hr o u g h t heir B H 3 bi n di n g gr o o v es (3 8 , 3 9 ). I n i nitial e x peri me nts, 
l ysates  fr o m  2 9 3  T  cells  tra nsfecte d  wit h  e m pt y  v ect or,  H A-Cas p 8 p 4 1,  or  H A - Cas p 8 p 4 1 
V 1 5 0 E/ L 1 5 7 K ( Cas p 8 p 4 1- E K, a v aria nt wit h decreas e d affi nit y f or Ba k d ue t o disr u pti o n of 
critical bi n di n g i nter acti o ns (2 9 )) were i m m u n o preci pitate d f or Bcl-2 a n d pr o be d f or ass ociate d 
H A t a g ge d pr otei ns. I m m u n o preci pitati o n of Bcl -2 de m o nstrat e d i nteracti o n wit h H A Cas p 8 p 4 1 
( Fi g ure 4 A) t hat was re d uce d b y t he V 1 5 0 E/ L 1 5 7 K s u bstit uti o ns. S urface plas m o n res o na n ce 
( S P R), a wi del y us e d tec h ni q ue t o assess t he affi nit y of pr otei n: pr ot ei n i nteracti o ns, was us e d f or 
m ore detaile d st u d y of Cas p 8 p 4 1 i nteracti o ns wit h Bcl -2, Bcl - xL , a n d M cl-1, t he a nti -a p o pt otic 
Bcl -2  f a mil y  me m bers  e x presse d  i n  T  cells.  T hese  e x peri me nts  i n dicate d  e q uili bri u m  
diss ociati o n c o nsta nts ( K ds) of 1 3 ± 4 n M f or Cas p 8 p 4 1 bi n di n g t o Bcl-2, 1 1 ± 8 n M t o Bcl - xL , 
a n d 8 n M ± 6 n M t o M cl- 1 ( Fi g ure 4 B a n d C), w hic h are si milar t o affi nities of t he sa me  pr otei ns 
f or  B H 3-o nl y  pr otei ns  s uc h  as  Bi m  a n d  P u ma  (8 ).   We  als o  c o nfir me d  t hat  t he  pe pti de  
c orres p o n di n g t o t he α -helical  B H 3 -li ke d o m ai n of Cas p 8 p 4 1 als o b o u n d Bcl-2, pr o vi di n g 


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evidence that the same Casp8p41 domain is responsible for binding both Bak and Bcl-2 (data not 
shown).  
 
Using multiple low-mass molecular dynamic simulations (40, 41), we constructed a 
three-dimensional model of Casp8p41 bound in the BH3 binding groove of Bcl-2 (Figure 4D). 
This model predicts that Arg146 of Bcl-2 is critical for binding electrostatically with Glu147 and 
Glu154 of Casp8p41. Mutation of Bcl-2 Arg146 to Ala (42) decreased the affinity of Bcl-2 for 
the Casp8p41 activator peptide (Supplementary Figure 3) or full length Casp8p41 250-fold 
(Figure 3E and F), confirming that Casp8p41 is binding to the BH3 binding groove of Bcl-2. The 
model also suggests that Val150 and Leu157 of Casp8p41 contribute to binding in two 
hydrophobic regions of the Bcl-2 BH3 binding groove that are similar to the two hydrophobic 
holes of the Bak BH3 binding groove (43). Consistent with this prediction, the affinity of 
Casp8p41 for Bcl-2 was reduced over 300-fold by introduction of the Val150Glu/ Leu157Lys 
mutations in Casp8p41 (Figure 4G and H). Thus, Casp8p41 favors cell death both by de-
repressing Bak (by inhibiting Bcl-2, Bcl-xL and Mcl-1) as well as by directly activating Bak 
(29).  
 
We propose a unifying model wherein activated HIV infected CD4 cells replicate 
virus, and HIV protease cleaves the abundantly available procaspase 8 to generate 
Casp8p41 which is minimally antagonized by the less prevalent Bcl-2; thus cell death 
ensues. Conversely HIV reactivation in TCM, where less procaspase 8 is present, results in 
less Casp8p41 being produced, and what little Casp8p41 is produced is effectively 
antagonized by the abundant Bcl-2. However, the fate of Casp8p41 after it has been bound 
and inhibited by Bcl-2 remains unknown.   
 
 
 
Within a cell, unneeded, misfolded, damaged and potentially toxic proteins are 
sometimes generated, in which case they are removed through the ubiquitin–proteasome 
pathway. This involves the reversible conjugation of the protein with ubiquitin chains, which 
target the protein to the proteasome for degradation.  As proteasomal degradation has been 
shown to occur following neutralization of other pro-apoptotic proteins (e.g. when the pro-
apoptotic NOXA is inhibited by the anti-apoptotic protein Mcl1 (44), the complex is degraded by 
the proteasome), we postulated that Casp8p41 bound and neutralized by Bcl2 might be 
ubiquitinated and degraded by the proteasome.  
 
 
First, we sought to confirm that Casp8p41 is indeed ubiquitinated, by expressing HA-
tagged Casp8p41 in 293T T cells. We did not detect ubiquitinated proteins in the Anti-HA 
pulldowns from control-transfected cells. By contrast anti HA pulldown from HA-Casp8p41-
expressing cells revealed an abundance of ubiquitinated proteins, migrating at high molecular 
weights, corresponding to polyubiquitinated HA-Casp8p41 (Figure 5A).  


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Covalent 
modification of 
proteins with 
ubiquitin can 
drive a number of 
outcomes ranging 
from degradative 
(in which the 
protein is tagged 
by ubiquitin 
chains at their 
Lys48 linkages 
resulting is 
proteasome 
degradation) to 
non-degradative 
(in which the 
protein is tagged 
by ubiquitin 
chains at their 
Lys63 linkages 
which results in 
modified 
protein:protein 
interactions and 
alteration of 
signal 
transduction 
pathways) (45). 
(46-50). To 
assess whether 
ubiquitinated 
Casp8p41 is 
targeted for 
degradation, we 
reversed this 
post-translational modification using specific de-ubiqitinating enzymes (DUBs), which 
distinguish between Lys48 and Lys63 ubiquitin linkages (51). The ubiquitination of HA-
Casp8p41 was completely reversed  by the Lys48-specific DUB USP2 and partially reversed by 
another Lys48-specific DUB YOD-1, but not other DUB’s (52, 53), (Figure 5B), and this effect 
was abrogated by inhibiting DUB activity with the de-ubiquitinase inhibitor N-ethylmaleimide 
(NEM, Figure 5C) (54). This indicates that Casp8p41 is ubiquitinated in a manner that is 
consistent with it being targeted to the proteasome for degradation.  
 


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To confirm that Casp8p41 complexed with Bcl2 is targeted for ubiquitination and 
degradation, we co-expressed HA-Casp8p41 and Bcl2 in 293T cells, and performed reciprocal 
immunoprecipitation (IP). IP of Bcl2 and blotting for HA confirmed the association of Bcl2 with 
HA-Casp8p41 (Fig 5D, left panel) as a band at the expected size of ~43kDa. However, a longer 
exposure revealed additional HA immunoreactive bands that were detected in the IP of Bcl2 that 
were effectively absent in cells treated with the DUB USP2, and weakened relative to the control 
in cells treated with YOD1, suggesting that these bands were ubiquitinated forms of Casp8p41 in 
complex with Bcl2. The effects of YOD1 and USP2 were reversed by treatment with NEM, as 
expected. These results were confirmed by ubiquitin blotting of the immunoprecipitates (Fig 5D, 
right panel). Both Bcl2 and HA IP brought down ubiquitinated proteins, which completely 
disappeared following treatment with the DUB USP2 and partially disappeared with YOD1, and 
the de-ubiquitination was reversed by NEM. These data suggest that the Casp8p41–Bcl2 
complex is ubiquitinated and targeted to the proteasome for degradation. 
 
Proteasome inhibition increases Casp8p41 expression levels. 
Altering the activity of the proteasome impacts a variety of biologic activities, and this 
characteristic has been successfully exploited with the clinical development of proteasome 
inhibitors. For example multiple myeloma is a disease in which plasma cells produce too much 
immunoglobulin protein, and blocking the ubiquitin mediated degradation of these 
immunoglobulin proteins using proteasome inhibitors results in the intracellular accumulation of 
immunoglobulin, and cell death through activation of the unfolded protein response.(55). 
Based on our data above, we predict that treatment of Casp8p41 expressing cells with 
proteasome inhibitors will increase expression levels of Casp8p41, and since Casp8p41 is only 
physiologically expressed in HIV infected cells, that increase should facilitate killing of the HIV 
infected cells. We therefore tested whether the currently used (and approved) proteasome 
inhibitor bortezomib (which is widely used clinically for the treatment of hematologic 
malignancies) and ixazomib (which is an orally bioavailable proteasome inhibitor recently 
approved by FDA for the treatment of refractory multiple myeloma) would also block the 
degradation of Casp8p41 following ectopic expression. We first performed dose-ranging toxicity 


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studies of these agents on primary CD4+ T cells from healthy donors. As ixazomib is 
administered as a citrate ester prodrug (ixazomib citrate) that is hydrolyzed under physiological 
conditions to its biologically active form, ixazomib,  in vivo, we treated cells with ixazomib  
(MLN2238; hereafter referred to as ixazomib).  In these experiments, toxicity as determined by 
active Caspase 3 staining was negligible at doses below 10nM for Bortezomib, and toxicity of 
Ixazomib was negligible at doses below 100nM (Figure 6A). Consequently, we used bortezomib 
at 5 and 10 nM and Ixazomib at 50 and 100nM for the remaining experiments. These doses also 
closely reflect peak plasma levels seen in patients (56, 57).  
 
We next  treated Jurkat CD4+ T cells with either bortezomib or ixazomib overnight, 
transfected them with GFP-tagged Casp8p41, and analyzed GFP expression 6 hours later. 
Consistent with our expectations, pretreatment of cells with either bortezomib or with ixazomib 
significantly increased the proportion of cells expressing GFP-tagged Casp8p41 – 10nM 
bortezomib resulted in a 2.5-fold increase in Casp8p41 relative to control cells (p=0.009), and 
100nM ixazomib resulted in a 2.4-fold increase (p=0.045; Figure 6B,C).   
 
Proteasome inhibitors independently decrease HDAC, activate NF-κB, and induce 
HIV reactivation. 
In treating J-Lat-10.6 cells with bortezomib or ixazomib we observed enhanced 
expression of GFP by J-Lat-10.6 cells over time, suggesting that these agents increase HIV 
replication (Figure 7A), consistent with a prior report suggesting that bortezomib activates latent 
provirus yet paradoxically decrease HIV replication(58). Since HIV reactivation is necessary for 
production of Casp8p41, and is also a preferred approach to effecting HIV cure strategies, this 
finding is of great potential relevance, and therefore we further assessed that possibility. 
Treatment of J Lat 10.6 cells with ixazomib/bortezomib was accompanied by an increase in the 
levels of HIV p55 gag protein (Figure 7B), which was detectable 48 hours after treatment. Next 
we sought to determine the mechanism underlying this reactivation. 


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Histone deacetylases are critical in inducing and maintaining HIV latency, and HDAC 
inhibitors are potent inducers of HIV reactivation (59) (60), (61) (62) . Since Bortezomib 
functions as a de facto HDAC inhibitor by transcriptional repression of class 1  HDAC 


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expression in myeloma cells (63) (64), we assessed whether treatment of primary CD4 T cells 
with bortezomib or Ixazomib also altered expression of different HDAC. Reproducibly, over 48 
hours of treatment, HDAC 2 and HDAC 6 were reduced by treatment with either Bortezomib or 
Ixazomib (Figure 7C). Thus proteasome inhibitor mediated downregulation of these HDAC 
might contribute to enhanced HIV replication.  
 
Bortezomib also directly activates the canonical NF-κB axis in multiple myeloma cells  
(65), raising the possibility that bortezomib and Ixazomib do similarly in other cell types 
including CD4 T cells. We therefore evaluated whether bortezomib or ixazomib affects the 
NFKB mediated HIV LTR activation in CD4+ T cells. Jurkat CD4+ T cells were transfected 
with luciferase reporter constructs in which either the HIV LTR was linked to luciferase (HIV-
luc) or the HIV LTR missing the NF-κB-binding sites was linked to luciferase (HIVdeltaκB-
Luc), as previously described (66). Treatment with bortezomib or ixazomib resulted in a dose-
dependent increase in luciferase expression (normalized to Renilla in cells containing the HIV-
Luc reporter construct), demonstrating a direct effect on HIV LTR-mediated replication in cells 
containing HIV-luc (Figure 7D). However, as expected, this effect was abrogated in the cells 
containing HIVdeltaκB-Luc reporter, indicating that proteasome activation of the HIV LTR is 
mediated through NF-κB (Figure 7E). Internally consistent with these findings the negative 
repressor of NFKB, IκB, was inactivated (through Ser32 phosphorylation(67).) and the p65 
subunit of NF-κB was activated (through Ser536 phosphorylation) within 6 hours of treating J-
Lat-10.6 cells with bortezomib or ixazomib,  (Figure 7F)  
 
Proteasome inhibitors preferentially kill HIV-infected T cells  
As we have established that bortezomib and ixazomib independently induce HIV 
reactivation – one of the key requirements for the prime shock and kill model of HIV eradication 
(68) – we next assessed whether these agents also favor the death of the HIV-positive cells. We 
treated J-Lat-10.6 cells or parental Jurkat CD4+ T cells separately with vehicle control, 
bortezomib or ixazomib, and measured caspase 3 expression over time by flow cytometry. Both 
bortezomib and ixazomib increased caspase 3 expression in J-Lat-10.6 cells in a dose- and time-
dependent manner compared with control-treated J-Lat-10.6 cells (Figure 8A and B) without 
significant toxicity to uninfected Jurkats at the same doses. 


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Our model posits that HIV reactivation induced by bortezomib or ixazomib initiates a 
mitochondrial death pathway ultimately causing activation of caspase 3, cleavage of structural 
and regulatory proteins including PARP and cell death. Therefore, we performed western blot 
analyses of treated J-Lat-10.6 cells to assess HIV reactivation (as measured by p24 expression), 
and cleavage of both caspase 3 and PARP (Figure 8C). Consistent with our previous data (Figure 
7A and B), treatment with bortezomib or ixazomib resulted in reactivation of HIV as evidenced 
by intracellular p24 expression in a dose dependent manner (Figure 8C, top panel). Consistent 
with the flow cytometry data (Figure 8A), treatment with bortezomib or ixazomib resulted in 
expression of cleaved Caspase 3 (Figure 8C, second panel), with associated cleavage of PARP 
(Figure 8C, third panel), indicated the cells were undergoing apoptosis.  
 
Any therapy which kills HIV infected cells will need to kill infected cells preferentially 
over uninfected cells. Our data indicate that proteasome inhibitors kill HIV-infected cells by 
promoting HIV reactivation and prolonging the half-life of Casp8p41 (which is only present in 
HIV-infected cells) and so should preferentially target HIV-infected cells. Thus, we assessed 
whether cell death occurs preferentially in latently infected J-Lat-10.6 cells over uninfected 
Jurkat T cells.  After confirming that both cell lines have a similar propensity to undergo 
apoptosis (Figure 9A and 9B), we performed clonotypic assays to assess which cells survive 


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following coincubation of the cells with bortezomib or ixazomib. To distinguish the 
phenotypically indistinguishable cell types, we first labelled chronically HIV-infected J-Lat cells 
with the lipophilic dye CellTracker Orange (CTO), and mixed these cells 50:50 with uninfected, 
unlabeled Jurkat T cells. These cell mixtures were then treated with vehicle control, bortezomib 
or ixazomib. Conceptually, if treatments cause non-specific killing, then the proportion of 
labelled CTO+ J-Lat cells should remain at ~50 percent over time, whereas if killing is selective 
for one cell type, then the proportion of labelled CTO+ J-Lat cells should change accordingly. 
Strikingly, at all doses tested, bortezomib and ixazomib resulted in a decrease in the proportion 
of CTO-positive J-Lat-10.6 cells (Figure 9C), indicating that the HIV-infected cells are 
selectively killed. Indeed, by 48 hours after treatment only approximately 25% of remaining live 
cells were chronically HIV-infected CTO+ J-Lat cells.  
 
 
 
 
 
 
 
 
 
 


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Likewise, in the presence of 10nM bortezomib, 79% of J-Lat-10.6 and 30% of Jurkat 
cells were stained for caspase 3 (P<0.0001), while in the presence of 100nM ixazomib 76% of J-
Lat-10.6 and 33% of Jurkat cells were positive for caspase 3 (P=0.001) (Figure 9D and 9E).  
Taken together, in this in vitro system, HIV-infected cells die preferentially in response to 
bortezomib or ixazomib treatment. 
 
Casp8p41 is expressed in central memory T cells which reactivate HIV, is increased 
by proteasome inhibitors, resulting in killing of the reactivating cells. 
 
Taken together our cumulative results indicate that proteasome inhibitors reactivate HIV 
in vitro, and preferentially induce the death of HIV infected cells over uninfected cells. Therefore 
we next assessed the effects of proteasome inhibitors in cells from HIV infected patients, treated 
ex vivo with proteasome inhibitors.  In these cells however, it is not possible to identify which 
cells are latently infected and which ones are not. Therefore, it is impossible to compare rates of 
cell death in latently HIV-infected cells with death in cells which are not latently infected. 
Instead, it is only possible to make this comparison by: (i) assessing death in cells which begin to 
express HIV specific markers such as p24 after reactivation treatment (which would suggest that 
they are latently infected); or (ii) assess the amount of cell-associated HIV DNA pre versus post 
treatment as these values normally do not change over a several day treatment window (69)).  
First, we looked for Casp8p41 expression in latently infected cells that had been induced 
to reactivate HIV. Using primary CD4+ T cells from two ART-suppressed, HIV-infected 
patients, we induced reactivation using αCD3/αCD28, and assessed for intracellular Casp8p41, 
using our previously validated neo-epitope-specific Casp8p41 antibody (70, 71). Consistent with 
our previous findings, reactivated cells contained Casp8p41 at detectable levels, and the 
frequency of Casp8p41 positivity was reflective of the relative frequency of HIV latency in each 
cell subset (Figure 10A); for instance, up to 1% of sorted resting memory CD4 T cells have 
detectable levels of HIV Gag DNA (72), and up to 2% of central memory CD4 T cells are 
positive for p24, even in the setting of ART (73).  
Next, we wanted to test whether the reactivating cells were in fact the cells that died in 
response to treatment. We treated cells from two ART-suppressed HIV-infected patients with 
bortezomib or ixazomib in the presence of anti-retroviral drugs to prevent spreading infection, 
and analyzed cells for the co-expression of caspase 3 (as a marker of apoptosis) and p24 (as a 
marker of reactivation) (Figure 10B and 10C). In cells that stained positive for p24, low and high 
dose bortezomib increased the proportion of caspase 3-positive cells from 17% (control) to 24% 
(5 nM bortezomib) and 60% (10 nM bortezomib); however bortezomib also increased the 
proportion of HIV p24 positive cells that were caspase 3 positive, suggesting nonspecific 
toxicity, consistent with bortezomib causing leukopenia in a significant proportion of patients 
(74).  In the case of ixazomib, treatment with 100 nM of the drug resulted in a significant 
increase in the proportion of caspase 3-expressing p24-positive cells (27% vs 17% in controls, 
p=0.023), while not significantly increasing the proportion of p24 negative cells that stain for 
active caspase 3, suggesting limited nonspecific toxicity, and consistent with the recent phase 
three trial data using ixazomib in myeloma patients, in which the rate of all side effects including 
leukopenia were not different between placebo and Ixazomib treatment arms.  (Figure 10B and 
C). 
 


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There is considerable debate about which is the best technique available to estimate HIV 
reservoir size (75). Highly sensitive nucleic acid amplification techniques have been developed 
which can accurately detect low copy number HIV genes with a very low coefficient of variance, 
such a digital droplet PCR (ddPCR) (76). However, such approaches cannot distinguish 
replication-competent from replication-incompetent (defective) virus (77), the latter being 
estimated to represent ~80 % of detectable viral sequences. On the other hand, functional assays 
such as the quantitative viral outgrowth assay (QVOA) are specific for replication-competent 
virus, but they underestimate the size of the latent pool (69, 78, 79), and are infrequently positive 
in cells from ART-suppressed patients. Thus, we opted to estimate HIV reservoir size ex vivo 
using the more sensitive approach – ddPCR – with the understanding that any reduction in HIV 
DNA would confer at least a similar degree of reduction in replication competent virus. 
 In our first series of experiments, we established and validated ddPCR, as follows. Cell-
associated HIV polymerase copies were measured repeatedly over 10 different runs in positive 
control samples containing a fixed ratio of uninfected Jurkat T cells and chronically HIV-
infected 8E5 T cells, which contain one integrated HIV provirus per cell. We were able to obtain 
a coefficient of variance of ddPCR of 3.8% (Figure 10D), thereby enabling us to interpret 
changes of greater that 3.8% as being greater than the noise of the assay. Next, 37 HIV-negative 
samples were assayed for cell-associated HIV polymerase, establishing a detection limit of 45 
HIV DNA copies per million cells (Figure 10E).  
Next we assessed changes in cell-associated HIV DNA in primary CD4+ T cells from 
ART-suppressed HIV patients, following maximal HIV reactivation by αCD3/αCD28 in the 
presence or absence of bortezomib or ixazomib. First, we isolated CD4+ T cells from 
cryopreserved leukapheresis PBMC samples from 6 individual ART-suppressed HIV patients,  
and pre-treated with bortezomib or ixazomib for 24 hours prior to viral reactivation with 
αCD3/CD28 stimulation.  Total cell-associated HIV DNA was measured by ddPCR 72 hours 
after reactivation.   Treatment with bortezomib followed by reactivation did not reduce HIV 
DNA in the leukapheresis samples (Figure 10F left panel), whereas ixazomib followed by viral 
reactivation resulted in a geometric mean 31% reduction (P=0.06) in HIV DNA when compared 
with reactivation alone (Figure 10F right panel).  We next treated freshly obtained primary CD4 
T cells from 12 ART suppressed patients with ixazomib or vehicle control prior to reactivation 
with αCD3/28 as above. Cells from 8 of 12 patients treated with ixazomib had a decrease in cell 
associated HIV DNA of ≥10% compared to control, and were considered treatment responders. 
In these responders (N=8), ixazomib reduced cell associated HIV DNA by a geometric mean of 
44% (P=0.002) compared to control treated cells (Figure 10G). We next focused on whether 
treatment of cells from an additional 11 HIV-infected, ART-suppressed patients with ixazomib 
alone would be sufficient to both reactivate HIV and prime the cells towards an apoptosis-prone 
phenotype, so as to result in a reduction in cell-associated HIV DNA content when used as 
monotherapy, in the setting of continued ART.  Treatment of CD4+ T cells with ixazomib 
(100nM) alone for 96 hours without additional reactivation stimuli reduced cell associated HIV 
DNA by a geometric mean 37% compared to vehicle treated cells (P=0.02, data not shown).   
 


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Summary of Hypothesis 
Current ART effectively reduces HIV replication (measured by HIV RNA levels) to 
undetectable levels within several months of starting therapy. Thereafter HIV persists primarily 
as integrated HIV DNA within central memory CD4 T cells. The half-life of these latently HIV 
infected CD4 T cells is ~44 months(80), meaning that ~70 years of ARV suppression would be 
required for eradication of all latently infected cells. To date, no therapy has been identified that 
reduces HIV DNA levels from latently infected cells. For the reasons outlined above, and 
supported by the accompanying preliminary data, we propose that using proteasome inhibitors 
will stabilize Casp8p41 in cells which reactivate HIV, thereby causing apoptosis of those cells, 
and promoting eradication of HIV infected cells. 
 
 
Choice of Bortezomib versus Ixazomib 
Dr Anthony Fauci, Director of the National Institute of Allergy and Infectious Diseases 
(NIAID) is a long time champion of HIV cure. Due to the global nature of HIV, and the fact that 
persons infected with HIV do not always have access to tertiary medical care, he espouses that 
any curative regimen must be effective, simple, safe, and scalable (81) . Thus concerning the 
choice between bortezomib and ixazomib, both of which show activity in vitro for HIV 
eradication, it would be preferable from the standpoint of simplicity and scalability to use 
ixazomib as it is oral, and has permissible storage condition excursion range is 8°C to 40°C for a 
maximum period of 30 days Moreover since HIV infection and older HIV treatments are 
commonly associated with peripheral neuropathy (PN), many HIV infected patients are either 
predisposed to PN, or have ongoing PN, thus as ixazomib is associated with less peripheral 
neuropathy than bortezomib, it will be preferable to use ixazomib. Ixazomib is also associated 
with better overall volume of distribution given its faster dissociation and greater tissue 
penetration. This would be of great relevance in terms of its ability to get into lymph nodes, 
which are critical sites of HIV persistence and replication. Finally the use of oral ixazomib would 
be more acceptable compared to IV bortezomib, from a patient perspective as well. Finally the 
superior toxicity profile of ixazomib compared to bortezomib (82) would be favorable for this 
patient population.  


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1.2 
Investigational Agent  
Ixazomib, which has been formulated for both intravenous (IV) and oral (PO) 
administration, is a small molecule proteasome inhibitor.  The proteasome is a large protein 
complex that degrades ubiquitinated proteins via the ubiquitin-proteasome pathway (UPP), 
which is responsible for the degradation of the majority of intracellular proteins.  Due to the 
accumulation of many different proteasome substrates, proteasome inhibition affects multiple 
signaling cascades within cells, resulting in downstream effects including antitumor activity, 
promotion of apoptosis, and antiangiogenic and antiproliferative activities.  These consequences 
of proteasome inhibition are of particular importance in plasma cells and multiple myeloma cells, 
which produce high levels of secreted Ig proteins.  Ixazomib is a small molecule proteasome 
inhibitor.   
 
Ixazomib citrate is the citrate ester of the biologically active form, ixazomib (MLN2238), 
a modified dipeptide boronic acid with structural differences from bortezomib.  In water or 
aqueous systems, ixazomib citrate rapidly hydrolyzes to ixazomib (MLN2238), therefore all 
doses and concentrations are expressed as ixazomib (MLN2238).  Nonclinical studies were 
conducted with a solution of either ixazomib (MLN2238) or ixazomib (MLN2238) in 
equilibrium with ixazomib citrate.  Similar to bortezomib, ixazomib (MLN2238) potently, 
reversibly, and selectively inhibits the 20S proteasome.  However in contrast to bortezomib, it 
has a shorter dissociation half-life (t1/2) that may contribute to increased tissue distribution.  
Bortezomib has a slowly reversible dissociation rate from the red blood cell proteasome, while 
ixazomib (MLN2238) demonstrates a more rapidly reversible dissociation rate from the blood 
but sustained effects on bone marrow and tumor proteasomes suggesting better tissue 
distribution.  The pharmacologic implications of this difference in binding kinetics and tissue 
distribution may in turn result in differences in safety and efficacy profiles in a broader range of 
tumors.  In xenograft-bearing mice, the more rapid dissociation rate correlates with an increased 
ratio of tumor proteasome inhibition to blood proteasome inhibition, and ixazomib shows greater 
antitumor activity in several xenograft models, both solid tumor and bortezomib-resistant 
xenografts, than bortezomib.   
 
In Vitro Pharmacology: MLN2238 preferentially binds the β5 site of the 20S proteasome; 
at higher concentrations, it also inhibits the activity of the β1 and β2 sites.  MLN2238 inhibits ß5 
site 20S proteasome activity in vitro, with a half-maximal inhibitory concentration (IC50) of 3.4 
nM.  Potency is reduced roughly 10-fold versus ß1 (IC50  31 nM) and 1,000-fold versus ß2 
(IC50 =3500 nM).  MLN2238 was also tested for inhibition against a panel of 103 kinases, 18 
receptors (neurotransmitter, ion channel, brain and gut receptors), and 9 serine proteases.  In all 
cases, the IC50 values were > 10 μM.  MLN2238 and bortezomib have different β5 proteasome 
dissociation half-lives (t1/2), reflecting differences in their on-off binding kinetics (the β5 
proteasome dissociation t1/2 for MLN2238 and bortezomib are 18 and 110 minutes, 
respectively).  Based on these favorable characteristics, ixazomib is anticipated to be effective 
against multiple myeloma. (ixazomib Investigator’s Brochure (IB). 
Proteasome inhibition results in the accumulation of poly-ubiquitinated substrates within the cell 
and leads to cell cycle disruption, with concomitant activation of apoptotic pathways and cell 
death.  Consistent with inhibition of ß5 20S activity, MLN2238 demonstrated potent activity 
against cultured MDA-MB 231 human breast cancer cells in the WST cell viability assay.  In 
nonclinical models MLN2238 has activity against both solid tumor and bortezomib-resistant 
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In Vivo Pharmacology: To determine the activity of MLN2238 in vivo, 
pharmacodynamic studies were performed in immunocompromised mice bearing either CWR22 
human prostate or WSU-DLCL2 (human diffuse large B-cell lymphoma [DLBCL]) tumors.  
Pharmacodynamic responses in xenograft tumors were analyzed by assessing 20S proteasome 
inhibition and by evaluating levels of accumulated protein markers such as deoxyribonucleic 
acid (DNA) damage-inducible protein 34 (GADD34) and activating transcription factor-3 (ATF-
3) as well as measuring growth arrest.  Increased expression of GADD34 and ATF-3 is 
indicative of a downstream biological response to proteasome inhibition.  After a single dose of 
MLN2238, a clear dose response was observed in CWR22 xenografts as seen in both tumor 20S 
proteasome inhibition and in changes in GADD34 and ATF-3 expression.  In WSU-DLCL2 
xenografts, greater tumor proteasome inhibition was observed with MLN2238 compared to 
bortezomib and resulted in increased expression of GADD34 and ATF-3. 
 
MLN2238 efficacy experiments demonstrated strong antitumor activity in 4 xenograft 
models: CWR22 (a human prostate cancer cell line) and 3 human lymphoma cell lines (WSU-
DLCL2, OCI-Ly7-7D1-luc, and PHTX-22L).  In the case of the CWR22 xenograft model, 
significant antitumor activity was seen with both IV and PO dosing, demonstrating that this 
molecule has antitumor activity when administered via different dosing routes.  In all 3 
lymphoma lines, MLN2238 demonstrated stronger antitumor activity than did bortezomib. 
 
In summary, MLN2238, similar to bortezomib, is a dipeptide boronic acid proteasome 
inhibitor that potently, reversibly, and selectively inhibits the proteasome.  There are several 
features, such as sustained pharmacodynamic effects and activity in a bortezomib-refractory 
lymphoma xenograft model, that suggest that it may have activity that extends beyond that seen 
with bortezomib. 
 
Nonclinical Pharmacokinetics and Pharmacodynamics: The pharmacokinetic (PK) 
properties of MLN2238 were studied in severe combined immunodeficient (SCID) mice bearing 
human CWR22 tumor xenografts, Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys.  
Because of the extensive red blood cell (RBC) partitioning of MLN2238, both blood and plasma 
PK parameters were determined in these studies.  MLN2238 had a very low blood clearance 
(CLb) and a moderate blood volume of distribution at steady-state (Vss,b) after IV 
administration.  The concentration-versus-time curve of MLN2238 displayed a distinct bi-
exponential profile with a steep initial distribution phase and a long terminal t1/2 (> 24 hr) in all 
species tested.  MLN2238 had higher plasma clearance (CLp) and a larger plasma volume of 
distribution at steady-state (Vss,p) than in blood, largely because of the extensive RBC 
partitioning. 
 
The PK properties of MLN2238 after oral administration were studied in rats and dogs.  
The plasma oral bioavailability (F) was 41% in rats and nearly 100% in dogs.  A clinical 
prototype formulation of the ixazomib capsule demonstrated that MLN2238 had excellent oral F 
and an excellent absorption profile in dogs.  In addition, interindividual variability, as measured 
by %CV, in Cmax and AUC0-24hr after oral administration was low to moderate, similar to that 
after IV administration.  The terminal t1/2 after oral administration was also similar to that after 
IV administration.  Comparison of the PK profiles after IV or PO administration in the dog is 
reported in further detail in the IB. 


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MLN2238 is predicted to have very low CLb (0.0045 L/hr/kg) and a moderate Vss,b 
(0.79 L/kg) with a long terminal t1/2 (> 24 hours) in humans.  The human efficacious IV dose of 
MLN2238 is predicted to be 2.0 mg/m2 (0.054 mg/kg) twice weekly. 
 
The human efficacious oral dose is predicted to be between 2 and 5 mg/m2 twice weekly, 
based on a predicted oral F of between 41% (as seen in rats) and 100% (as seen in dogs).  The 
efficacious dose projection for once weekly oral would be higher than twice weekly oral (data 
not provided). 
 
Metabolism appears to be a major route of elimination for MLN2238 and urinary 
excretion of the parent drug was negligible (< 5% of dose).  In vitro in liver microsomes, the 
metabolism of MLN2238 was high in mice and low to moderate in all other species studied.  
Metabolism by multiple CYP enzymes and non-CYP proteins is expected to be the major 
clearance mechanism for ixazomib (MLN2238).  At clinically relevant ixazomib concentrations, 
in vitro studies using human cDNA-expressed cytochrome P450 isozymes showed that no 
specific CYP isozyme predominantly contributes to ixazomib metabolism.  At higher than 
clinical concentrations, ixazomib was metabolized by multiple CYP isoforms with estimated 
relative contributions of 3A4 (42%), 1A2 (26%), 2B6 (16%), 2C8 (6%), 2D6 (5%), 2C19 (5%) 
and 2C9 (<1%).   
 
Ixazomib (MLN2238) is neither a reversible nor a time-dependent inhibitor of CYPs 
1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4/5. Ixazomib did not induce CYP1A2, CYP2B6, and 
CYP3A4/5 activity or corresponding immunoreactive protein levels.  Ixazomib is not expected to 
produce drug-drug interactions via CYP inhibition or induction. 
 
In a Caco-2 cell assay, MLN2238 showed medium permeability with a B-to-A/A-to-B 
permeability ratio of 2.9.  MLN2238 may be a low-affinity substrate of para-glycoprotein (P-gp), 
breast cancer resistance protein (BCRP), and multidrug resistance protein 2 (MRP2) efflux pump 
transporters.  MLN2238 is not an inhibitor of P-gp, BCRP, and MRP2 (IC50 > 100 μM).  
Consequently, the potential for MLN2238 to cause DDIs with substrates or inhibitors of P-gp, 
BCRP, and MRP2 is low.   
 
Safety Pharmacology: In exploratory safety pharmacology studies, MLN2238 was a 
weak inhibitor of the cloned cardiac potassium (K+) human ether à-go-go related gene (hERG) 
channel, with an IC50 of 59.6 μM, which exceeds, by approximately 200-fold, the plasma Cmax 
(111 ng/mL [0.3 μM]) predicted to occur in humans at the optimally efficacious dose after IV 
administration. 
 
In the GLP-compliant, 1-cycle, repeat-dose, PO toxicology study in beagle dogs, an 
increase in QTc was seen in male dogs at non-tolerated doses, and a potential increase in QTc 
was seen in male dogs at tolerated doses.  However, increased QTc was not seen in female dogs 
at any dose, despite the fact that female dogs had plasma Cmax values similar to those of male 
dogs.  Additionally, in a GLP-compliant, 2-cycle, repeat-dose, IV toxicology study in beagle 
dogs, no increase in QTc was seen in either male or female dogs at any dose, even though dogs 
in the IV study had higher MLN2238 plasma Cmax values than did the male dogs in the PO 
study.  These data suggest that MLN2238 has a low potential for prolonging the QT interval in 
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1.3 
Preclinical Data  
Toxicology: All studies discussed in this section were conducted with a solution of either 
MLN2238 or MLN2238 in equilibrium with ixazomib citrate.  Because ixazomib citrate was 
shown to dissociate immediately to MLN2238 upon exposure to plasma in vitro and therefore 
could not be detected in plasma samples in vitro all doses, concentrations, and PK parameters 
noted, here and in the IB, are expressed as the boronic acid, MLN2238. 
 
The toxicology studies of MLN2238 were studied in SCID mice bearing human CWR22 
tumor xenografts, Sprague-Dawley rats, beagle dogs,  and cynomolgus monkeys.  Details of 
these studies are included in the IB. 
 
In Vitro Toxicology: MLN2238 was not mutagenic in a Good Laboratory Practice 
(GLP)-compliant bacterial reverse mutation assay (Ames assay).  
 
In Vivo Toxicology: Details of the in vivo toxicology IV dosing and oral dosing studies 
are provided in the IB.  To summarize, the toxicologic effects seen in the IV and PO studies are 
qualitatively similar to what was previously observed in rodents dosed with bortezomib, for 
which ixazomib is the next-generation molecule.  MLN2238 did not cause significant toxicities 
that have not been previously observed after dosing with bortezomib.  Therefore, on the basis of 
the similarity in the toxicity profile in rats between MLN2238 and bortezomib, MLN2238 is not 
known to present any additional safety risks beyond those that occur after treatment with 
bortezomib.  In addition, there were no significant findings at tolerated exposures in dogs 
observed after PO administration that were not seen after IV administration, and similar 
exposures were tolerated regardless of the route of administration. 
 
The potential risks identified from nonclinical studies in dogs and rats include: 
 
• GI toxicity that could result in nausea, vomiting, diarrhea, dehydration, electrolyte 
imbalance, bleeding, bowel obstruction including ileus and intussusception, and sepsis. 
• Reduced blood counts manifest as thrombocytopenia, neutropenia, and anemia.  
Reticulocytopenia was described in animals and may be associated with anemia.  
Reductions in blood counts may predispose to an increased susceptibility to infection, 
bleeding, and anemia. 
• Peripheral nerve ganglia effects that may be associated with peripheral neuropathy that 
includes pain, burning sensation, and numbness.  Autonomic and motor neuropathy may 
be observed, as both have been reported for bortezomib. 
• Lymphoid cell depletion that may be associated with increased risk of infection, 
including re-activation of herpes zoster. 
• Acute phase response that may result in fever and metabolic changes. 
 
All of the effects seen in the GLP-compliant PO toxicology studies in both dogs and rats 
at tolerated doses were reversible/reversing and can be monitored in the clinic with routine 
clinical observations (GI disturbances and infections secondary to lymphoid compromise), 
clinical pathology assessments (inhibition of erythropoiesis, thrombocytopenia, and 
inflammatory leukogram), and neurologic assessment, as are commonly done for patients treated 
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after treatment with bortezomib and are believed to be the cause of the peripheral neuropathy 
observed in patients treated with bortezomib. 
 
Further details are presented in the current version of the ixazomib IB. 
1.4 
Clinical Data to Date  
 
Clinical Experience: To date, there is no published experience with ixazomib in HIV 
positive persons.   
 
Ixazomib has been evaluated as an oral single agent in phase 1 studies that have included 
patients with advanced solid tumors, lymphoma, relapse/refractory MM (RRMM), and relapsed 
or refractory light-chain (AL) amyloidosis and demonstrated early signs of activity. Ongoing 
studies continue to investigate both single-agent ixazomib and ixazomib in combination with 
standard treatments. Based on encouraging preliminary data observed in patients with MM 
requiring systemic treatment, 2 phase 3 trials in newly diagnosed MM (NDMM) (C16014) and 
RRMM (C16010) patient populations are currently evaluating ixazomib in combination with 
Revlimid and Dexamethasone (RevDex) versus placebo/RevDex. Both trials are combining 
ixazomib at a weekly dose of 4.0 mg on Days 1, 8, and 15 in a 28-day cycle to a standard dose of 
lenalidomide with a weekly dexamethasone dose of 40 mg. In addition, clinical pharmacology 
studies have evaluated  drug-drug interactions with ketoconazole, clarithromycin, and rifampin, 
as well as the effect of food, renal impairment, and hepatic impairment on the PK of ixazomib.  
Studies evaluating the safety and pharmacokinetic (PK) of ixazomib alone (in Japanese patients) 
and in combination with lenalidomide and dexamethasone in Asian adult patients (including 
Japanese patients) with a diagnosis of RRMM are ongoing. 
 
As of 27 March 2013, preliminary clinical data is available for a total of 653 patients 
across 13 studies. The emerging safety profile indicates that ixazomib is generally well tolerated. 
The adverse events (AEs) are consistent with the class-based effects of proteasome inhibition 
and are similar to what has been previously reported with VELCADE though the severity of 
some, for example peripheral neuropathy, is less. While some of these potential toxicities may be 
severe, they can be managed by clinical monitoring and standard medical intervention, or, as 
needed, dose modification or discontinuation. 
 
Fatigue was the most common AE reported among 384 patients treated in the oral (PO) 
studies (47%). Other common AEs reported in the pooled intravenous (IV) and PO safety 
populations include nausea, thrombocytopenia, diarrhea, and vomiting. Rash is also a commonly 
reported treatment-emergent event; however, there is some variety in its characterization and 
causality resulting in different preferred terms to describe it. A high-level term outline of rash 
events includes rashes, eruptions and exanthems NEC; pruritus NEC; erythemas; 
papulosquamous conditions; and exfoliative conditions. The dose escalation phases of most trials 
reported in the IB have now completed enrollment, and gastrointestinal (GI) symptoms were the 
common dose-limiting toxicities (DLTs) when the use of prophylactic anti-emetics was not 
permitted per protocol. In the expansion cohorts or phase 2 cohorts (as per each study), the 
incidence and severity of GI symptoms was mitigated by the use of the lower maximum tolerated 
dose (MTD)/recommended phase 2 dose (RP2D) (as per each study) and standard clinical usage 
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emetics has not been required as with other agents but (as outlined in Section 6.7) has been used 
according to standard practice and are effective. 
 
The most frequent (at least 20%) treatment-emergent adverse events (TEAEs) reported 
with the PO formulation pooled from single-agent studies (n = 201) irrespective of causality to 
ixazomib, include nausea (53%), fatigue (51%), diarrhea (44%), thrombocytopenia (34%), 
vomiting (38%), decreased appetite (32%), fever (21%), and anemia (21%). The most frequent 
(at least 20%) TEAEs reported with the PO formulation pooled from combination trials 
(irrespective of the combination) (n = 173), irrespective of causality to ixazomib, include 
diarrhea (47%), fatigue (44%), nausea (38%), peripheral edema (35%), constipation (33%), 
insomnia (29%), thrombocytopenia (28%), anemia (26%), vomiting (26%), neutropenia (25%), 
back pain (24%), pyrexia (23%), peripheral edema (21%, each), fever (20%), cough (20%), 
hypokalemia (20%), neutropenia (20%), and upper respiratory tract infection (20%). Overall rash 
of all grades is reported in approximately 50% of patients and is more common when ixazomib is 
given in combination with lenalidomide where rash is an overlapping toxicity. 
 
Additional detailed and more recent information regarding the clinical experience of 
ixazomib may be found in the IB, including information on the IV formulation.   
1.5 
Dose Rationale and Risk/Benefits 
 
Safety is our first concern, therefore we have chosen a dose escalation study, starting at a 
low dose with regular and frequent DSMB safety review. The lowest starting dose in this study is 
1 mg weekly, which is expected to achieve <30% 20S proteasome inhibition based on previously 
published pharmacodynamics data in advanced non-hematologic malignancies (Invest New 
Drugs. 2015; 33(3): 652–663). The maximum possible dose of ixazomib to be administered in 
this trial is similar to the doses SK Kumar et al. have previously used in a Phase I study of 
weekly ixazomib for relapsed/refractory multiple myeloma (60).  However, during that study, 
dosing was BSA based, and the maximum tolerated dose was 2.97 mg/m2 (translating to a fixed 
dose of 5.5 mg).  The estimated half-life of ixazomib was 3.6 to 11.3 days, supporting weekly 
dosing, which is the dosing schedule studied in Phase 2 and 3 trials, and ultimately approved by 
the FDA for treatment of multiple myeloma. 
 
Furthermore, the doses of ixazomib to be administered in this trial are expected to 
correlate with the concentrations used in the preliminary data noted above.  Maximum biologic 
effects were noted in the in vitro and ex vivo experiments above with a single treatment of 100 
nM ixazomib, which corresponds to a peak in vitro concentration of 36.1 ng/ml.  In Study 
C16009, Arm 3, adult patients with a diagnosis of an advanced non-hematologic malignancy or 
lymphoma were treated with a standard 4 mg oral dose of ixazomib, and geometic mean Cmax in 
the fasted state was 77 ng/ml with a 57% CV. Therefore, the proposed dose range for this study 
(from 1 to 4 mg) is expected to result in plasma Cmax values that approximate the doses used in 
the in vitro studies. Cmin and Cmax levels before and after the second dose of ixazomib on 
Day+15 will be measured.   
 
Potential Risks of ixazomib:  Because ixazomib has not been previously studied in HIV infected 
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will have in otherwise healthy HIV infected patients. In the absence of healthy volunteer data, 
the best indication of likely side effects is best informed from the phase three trial that has 
recently been reported (Moreau et al. NEJM 374;17:1621-1634) . In that phase three trial 
refractory multiple myeloma patients were randomly assigned to receive Dexamethasone and 
Lenalidomide along with either placebo , or Ixazomib 4 mg once weekly on days 1,8, and 15 of a 
four week cycle.  The incidence of side effects was no different between study arms, and 
therefore additional toxicities (including peripheral neuropathy) directly attributable to ixazomib 
were concluded to be limited (see below for details).  Based on observations from nonclinical 
toxicology studies of ixazomib and the  clinical observations from the ongoing  and completed 
clinical studies, the following potential safety risks of ixazomib  are noted in the ixazomib IB: 
Anemia, neutropenia, thrombocytopenia, fatigue, nausea, vomiting, constipation, diarrhea, 
peripheral edema, pyrexia, decreased appetite, dizziness, dysgeusia, peripheral neuropathy, 
peripheral sensory neuropathy, insomnia, progressive multifocal leukoencephalopathy and  rash 
(erythematous, generalized, macular, macular-papular, and papular) with or without pruritus 
have been listed in the ixazomib IB as adverse drug reactions reported with ixazomib use.  It is 
also possible that ixazomib will have toxicities that were not previously observed in or predicted 
from such sources.  Patients will be monitored closely for anticipated and unanticipated 
toxicities.   
 
While the above listed adverse drug reactions have been reported in association with 
ixazomib use in previous clinical trials, definitive attribution of causation of these adverse events 
to ixazomib is difficult. First, participants in these trials often have underlying conditions that 
predispose to the development of these adverse events – i.e. up to 20% of multiple myeloma 
patients have peripheral neuropathy at the time of diagnosis prior to any treatment (Handb Clin 
Neurol. 2013;115:443-59).  Second, many trial participants have received prior therapies, or 
receive concomitant therapies during the trail, that have overlapping toxicities.  Therefore, 
careful examination of toxicities observed in a phase 3 trial when ixazomib is compared to 
placebo, in addition to an optimized background regimen is instructive.. 
 
The TOURMALINE-MM1 trial was a phase 3, double blind, placebo-controlled, 
randomized trial comparing ixazomib 4 mg plus lenalidomide-dexamethasone to placebo plus 
lenalidomide-dexamethasone in patients with relapsed and/or refractory multiple myeloma 
(Moreau et al. NEJM 374;17:1621-1634). Importantly, 70% of participants had prior treatment 
with bortezomib, which has a known association with the development of peripheral neuropathy, 
and other adverse events, and as noted above approximately 20% of patients with multiple 
myeloma have peripheral neuropathy at the time of diagnosis, prior to any treatments. Notably, 
the rates of serious adverse events were similar in the placebo and in the ixazomib groups (47% 
in the ixazomib group versus 49% in the placebo group). The incidence of any peripheral 
neuropathy was 29% in the ixazomib group and 24% in the placebo group (P>0.05); the 
prevalence of peripheral neuropathy at baseline prior to treatment was not reported.  2% of 
participants developed grade 3 peripheral neuropathy in both groups, and no participants 
developed grade 4 peripheral neuropathy. These data suggest that the attributable risk of 
peripheral neuropathy (or any other toxicity) due to ixazomib in multiple myeloma patients is 
low.  Furthermore, previous studies have demonstrated that the peripheral neuropathy associated 
with bortezomib use is reversible in the majority of patients (Br J Haematol. 2009 


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Mar;144(6):895-903.).  Preliminary clinical data with ixazomib suggest that associated 
peripheral neuropathy is mild and reversible (see current IB page 153).   
 
Ixazomib has principally been studied in combinations and in patients with multiple 
myeloma.  There are two ongoing studies evaluating ixazomib monotherapy in patients with 
diseases other than myeloma:  
• Phase 2, open-label, dose escalation trial of ixazomib in adult patients with non-
Hodgkin lymphoma.  The dose and schedule of ixazomib (monotherapy) during 
the dose-escalation phase was either 4 mg, 5.3 mg or 7 mg administered on Days 
1, 8 and 15 of a 28-day cycle (ClinicalTrials.gov Identifier: NCT01939899).   
• Phase 1b, randomized placebo controlled trial of ixazomib in patients with ISN / 
RPS Class III or IV Lupus Nephritis (ClinicalTrials.gov Identifier: 
NCT02176486).   
These studies will be informative as to the association of peripheral neuropathy (or lack thereof) 
with ixazomib monotherapy in patients with diseases other than multiple myeloma. 
 
Additional details on the potential risks of ixazomib may be found in the current IB. 
 
Potential Benefits of ixazomib: It is likely that individual participants in this phase I/IIa study 
will derive no direct clinical benefit from treatment with ixazomib, as specified in the informed 
consent form. 
 
The hypothesis we are exploring based on in vitro and ex vivo data is that therapy with 
ixazomib decreases cell-associated HIV DNA by 1) inducing HIV reactivation, 2) increasing 
expression of pro-apoptotic Casp8p41 in HIV-infected cells, and 3) inducing cell death 
specifically in HIV-infected cells while sparing HIV-uninfected cells. If these positive effects are 
recapitulated when treating patients with HIV on antiretroviral therapy with ixazomib, this would 
represent the first clinical intervention found to decrease the size of the HIV reservoir, which is 
the main barrier to HIV cure.   
There is an urgent need for an HIV cure. While patients with HIV on antiretroviral 
therapy live longer and have reduced rates of opportunistic infections and malignancies 
compared to untreated HIV patients, data suggest that life expectancy for patients with treated 
HIV in the current treatment era are still nearly 14 years shorter than age and sex-matched 
uninfected controls (Marcus et al. J Acquir Immune Defic Syndr. 2016 Mar 29), indicating 
persistent increased mortality even in treated patients. Chronic, treated HIV infection is 
associated with abnormal chronic inflammation, cardiovascular disease, metabolic syndrome, 
bone disease, neurocognitive defects and age-related non-AIDS malignancies.  Furthermore, 
current HIV treatments require lifelong therapy with antiretroviral medications, which have 
substantial associated burdens of costs and adverse drug effects themselves.  
 
Therefore, establishment of a maximal tolerated dose, and obtaining preliminary efficacy 
data at reducing HIV reservoir size, will inform future therapeutic study designs with the intent 
of establishing whether ixazomib has beneficial effects in patients with HIV infection. 
 


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2 Study Objectives 
Primary Objective 
 
To determine the safety and tolerability of ixazomib in HIV infected patients who are on 
a stable regimen of ART that suppresses HIV replication (HIV RNA to undetectable levels using 
clinical assays). 
 
Secondary Objectives 
 
To determine the effect of ixazomib on: 
1) Change in cell associated HIV DNA in CD4 T cell subsets from Week -1 to Week 24;   
2) Change in culturable HIV from Week -1 to Week 24; 
3) Change in absolute CD4 T cell, CD8 T cell count and percentage as well as CD4/CD8 ratio 
from Week -1 to 24 
3 Study Design 
3.1 
General Design 
This study is an open label, phase I/IIa pilot trial of the safety and tolerability of 
ixazomib, in the treatment of HIV.  Subjects will be screened at outpatient clinic visit 
appointments and interested qualified subjects will be offered and consented for participation in 
this trial. All study visits except the screening visit will be conducted at the Mayo Clinic. The 
screening visit may be completed at the Mayo Clinic, Hennepin County Medical Center or any 
other site that has been approved by the regulatory sponsor.  Once consent has been obtained, 
baseline values will be established and subjects will begin treatment and follow-up for the next 6 
months.  A final visit for evaluation and collection of lab samples will be conducted at the end of 
the study. 
3.2 
Primary Study Endpoints 
The primary endpoint to be analyzed in the study is the safety and tolerability of 
ixazomib in HIV positive patients on antiretroviral therapy. 
 
(See pages 39 and 49 for a list of adverse events to be assessed).   
3.3 
Secondary Study Endpoints 
Secondary endpoints will include change from baseline in measures of HIV infection 
(cell associated HIV DNA and culturable HIV), and immune parameters (CD4 and CD8 T cell 
count, as well as CD4/CD8 Ratio). 
 
4 Subject Selection Enrollment and Withdrawal 
4.1 
Inclusion Criteria 
• The following laboratory values obtained <=14 days prior to registration. 
• 
ANC ≥ LLN (lower limit of normal) and ≤ULN (upper limit of normal), Hgb 
≥ 12.0 g/dL and ≤ULN, PLT ≥ LLN and ≤ULN 


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• 
Total bilirubin ≤ULN and the direct bilirubin must be ≤ ULN; AST <1.5 x 
ULN and ALT <1.5 x ULN  
• 
Creatinine <2.0 x ULN and an estimated creatinine clearance > 60 ml/min 
(patients on dolutegravir based regimens will have cystatin C measured for a 
more accurate estimation of creatinine clearance) 
• HIV infection with suppressed viral replication on at least 3 active drug ART for at least 
6 months  
• 
Suppressed viral replication is defined by plasma HIV viral load <20copies/mL.  
• 
Patient must have HIV viral load <20 copies/ml on two occasions at least 3 
months apart.   
• 
In the opinion of the treating physician, patients must have available other 
regimens likely to suppress HIV should their current regimen fail.   
• Male or female patients age >=18 years 
• A plasma HIV RNA viral load demonstrating a measure of <20 copies/mL within 30 
days prior to study initiation.  
• CD4 count >500 cells/mm3 within 30 days prior to study enrollment 
• Females must have a negative urine pregnancy test prior to receiving the 1st dose of 
ixazomib and be postmenopausal for at least 1 year before the screen visit, or surgically 
sterile,  
• Male patients, even if surgically sterilized (ie, status post-vasectomy), must agree to one 
of the following: 
o Agree to practice effective barrier contraception AND a second method of 
contraception for female partners of childbearing potential during the entire study 
treatment period and through 90 days after the last dose of ixazomib,  
o OR 
o Agree to practice true abstinence when this is in line with the preferred and usual 
lifestyle of the subject.  (Periodic abstinence (eg, calendar, ovulation, 
symptothermal, post-ovulation methods] and withdrawal are not acceptable 
methods of contraception.) 
o AND 
o Agree to forego sperm donation for the same period as above.  
4.2 
Exclusion Criteria 
• The following laboratory values obtained <=14 days prior to registration. 
o ANC < LLN or >ULN, Hgb < 12.0 g/dL or >ULN, PLT < LLN or >ULN 
o Total bilirubin >ULN or the direct bilirubin is > ULN; AST >1.5 x ULN or AST 
>1.5 x ULN  
o Creatinine >=2.0 x ULN or an estimated creatinine clearance <=60mL/min 
• Diagnosed and treated for a malignancy within 5 years before randomization, or 
previously diagnosed with a malignancy and have any evidence of residual disease. 


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Patients with nonmelanoma skin cancer or carcinoma in situ of any type are not excluded 
if they have undergone complete resection 
• Any infection except HIV (excluding benign conditions that is unlikely to be affected or 
modulated by treatment with ixazomib, e.g. stye or furuncle), or treatment with anti-
infective agents within 14 days of enrollment. 
• Pregnant women  
• Women of childbearing potential and Nursing women 
• Men who are unwilling to use a condom (even if they have undergone a prior vasectomy) 
while having intercourse, while taking the drug and for 90 days after stopping ixazomib. 
• Any history of peripheral neuropathy, or peripheral neuropathy detected during the 
screening period. Screening for peripheral neuropathy will be performed using the the 
European Organization for Research and Treatment of Cancer QLQ-CIPN20 
Questionnaire (see section 6 and Appendix II). 
• Major surgery within 14 days before study registration 
• Systemic 
treatment 
with 
strong 
CYP3A 
inducers 
(rifampin, 
rifapentine, 
rifabutin,carbamazepine, phenytoin, phenobarbital), or use of St. John’s wort.  
• Evidence of current uncontrolled cardiovascular conditions, including serious cardiac 
arrhythmias, congestive heart failure, angina, or myocardial infarction within the past 6 
months.  Note: Prior to study entry, any ECG abnormality at screening must be 
documented by the investigator as not medically relevant IF the ECG abnormality is 
judged NOT to be clinically relevant. 
• QTc > 450 milliseconds (msec) for men and >470 milliseconds for women (83) on a 12 
lead ECG obtained during the Screening period.  Note: If a machine reading is above this 
value, the ECG should be reviewed by a qualified cardiologist. 
• Known hepatitis B DNA positive status and/or HBsAg positive and/or HBeAg positive, 
or active hepatitis C replication (HCV RNA positive) or currently on hepatitis C 
treatment. 
• Known history of cirrhosis or active liver inflammation, including “fatty liver” or non-
alcohol steatohepatitis (NASH). 
• Any serious medical or psychiatric illness that could, in the investigator’s opinion, 
potentially interfere with the completion of treatment according to this protocol. This 
includes a recent history of substance abuse with less than 6 months of sobriety.  
• Known allergy to any of the study medications, their analogues or excipients in the 
various formulations. 
• Any other recent or concurrent medical condition that, in the Investigator’s opinion, 
would impose any risk to the patient 
• Known GI disease or GI procedure that could interfere with the oral absorption or 
tolerance of ixazomib including difficulty swallowing. 
• Participation in other clinical trials, including those with other investigational agents not 
included in this trial, within 30 days of the start of this trial and throughout the duration 
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4.3 
Subject Recruitment, Enrollment and Screening 
A maximum total of 17 subjects will be recruited from patients from the Mayo Clinic 
HIV Clinic and the Hennepin County Medical Center (HCMC).  Potential study subjects will be 
identified and screened for inclusion and exclusion criteria by a member of the study team. 
Furthermore, other treating clinicians, including residents, fellows and staff physicians, in the 
Mayo Clinic HIV clinic and HCMC continuity clinic may refer potential study subjects to a 
study team member.  Evaluation and documentation of inclusion and exclusion criteria will be 
obtained and recorded by an investigator prior to enrollment. Informed consent will be obtained 
by an MD investigator.  Patients will be remunerated for participation in the study (see section 
12.3).   
4.4 
Early Withdrawal of Subjects 
4.4.1 When and How to Withdraw Subjects 
Permanently discontinue ixazomib in individual subjects if any of the following occur: 
• Death of any subject  
• Anaphylactic reaction in any subject 
• A life-threatening adverse event in any subject 
• Any Grade 3 AE (except if there is clear evidence that it is not study drug related – i.e. a 
plausible alternative reason).  
• Any Grade 4 AE 
• A decrease in CD4 count below 350 cells/mm3, confirmed on repeat testing within 2 
weeks, and associated with a relative drop in CD4 T cell percentage by >25% from 
baseline values, in the absence of an alternative explanation 
• Confirmed virologic rebound (HIV-1 RNA >500 copies/ml confirmed by a second assay 
done within 14 days of the initial one).  If the HIV-1 viral load is >20 copies/mL and 
<500 copies/mL, the HIV-1 viral load will be rechecked every 2 weeks until it becomes 
<20 copies/mL or >500 copies/mL. 
• Development of ≥ Grade 1 peripheral neuropathy with or without pain 
 
An independent Data Safety and Monitoring Board will review the case.  The committee 
and principal investigators will determine whether there is an association between the event and 
the ixazomib. The DSMB will make a recommendation accordingly as to whether the study 
should proceed, or if a modification in trial design is required, or if a replacement subject should 
be enrolled at the same dose as the withdrawn subject. 
 
Reasons for early withdrawal of individual subjects may include: 
• Subjects refuse to return for any remaining study visits. 
• Subjects discontinue the ixazomib at any time during the study. 
• Safety reasons, either at the discretion of the Investigator or at the subject’s request.  
• Protocol violations at the discretion of the principal investigators. 
• Initiation of concomitant therapy that could interfere with the results of the study or 
ixazomib 
• Subject’s decision to withdraw, at any time. 
 


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All subjects are free to withdraw from participating in this study at any time and for 
whatever reason, specified or unspecified, and without prejudice. 
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 
All premature discontinuations and their causes must be carefully documented by the 
investigators.  Subjects not completing the entire study should be fully evaluated when possible. 
If subjects choose to withdraw before completing the study, they should return to complete the 
following safety and limited efficacy assessments: 
• Patient tolerability and side effects questionnaire. 
• Vital signs. 
• A complete physical exam. 
• A review of recent clinical laboratory results, including a complete blood count, serum 
electrolytes, creatinine, fasting glucose, liver function tests, CD4 T cell count and HIV 
viral load. 
 
If subjects who prematurely withdraw from the study do not return for this limited 
evaluation visit, then the relevant and available clinical data will be abstracted from the patient’s 
medical record at the appropriate time based upon the study schedule. 
 
Patients who withdraw from the study for virologic failure or a decrease in CD4 T cell 
counts will be followed until these values return to their baseline values. 
 
 


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5 Study Drug  
5.1 
Description 
 
Background 
Ixazomib is a second-generation small molecule inhibitor of the 20S proteasome that is 
under development for the treatment of non-hematologic malignancies, lymphoma, and multiple 
myeloma.  Ixazomib (MLN2238) refers to the biologically active, boronic acid form of the drug 
substance, ixazomib citrate.  The transition to MLN2238 occurs in any aqueous system. 
 
Formulation 
The ixazomib capsule drug product formulation consists of drug substance, 
microcrystalline cellulose, talc, and magnesium stearate.  Ixazomib capsules are individually 
packaged in cold form foil-foil blisters with a paper backing for child resistance. 
 
The ixazomib drug product is provided in strengths of 4.0-, 3.0-, and  2.0-, and 0.5-  mg 
capsules as the active boronic acid. The different dose strengths are differentiated by both 
capsule size and color as described below: 
 
Dose Strength 
Capsule Size 
Capsule Color 
4.0 mg 
Size 4 
Ivory 
3.0 mg 
Size 3 
Light gray 
2.3 mg 
Size 2 
Light pink 
2.0 mg 
Size 2 
Swedish orange 
0.5 mg 
Size 3 
Dark green 
0.2 mg 
Size 4 
White opaque 
 
For additional details, please see the ixazomib IB. 
5.2 
Treatment Regimen 
Patients will be treated in sequential groups after the safety evaluation of each group by the 
DSMB. Visits 1 and 2 do not have a time window, and can occur without DSMB review as 
patients will not receive drug at those visits. The following groups will be studied in this 
protocol: 
 
Group 1: 3 patients with HIV infection, who meet inclusion criteria, will receive ixazomib 1 mg 
on days 1, 8 and 15 for 28 days. After 28 days, the DSMB will meet to decide if enrollment of 
the next cohort should proceed. After 84 days (28 days x 3 cycles), the DSMB will meet and 
decide if patients will receive either (i) ixazomib once weekly for 12 weeks or (ii) ixazomib 1 mg 
on days 1, 8 and 15 for an additional three 28 days cycles. Visits 3-15 will have a window of ± 3 
days. 
 
Group 2: 3 patients with HIV infection, who meet inclusion criteria, will receive ixazomib 2 mg 
on days 1, 8 and 15 for 28 days. After 28 days, the DSMB will meet to decide if enrollment of 
the next cohort should proceed. After 84 days (28 days x 3 cycles), the DSMB will meet and 
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on days 1, 8 and 15 for an additional three 28 days cycles. Visits 3-15 will have a window of ± 3 
days. 
 
Group 3: 3 patients with HIV infection, who meet inclusion criteria, will receive ixazomib 3 mg 
on days 1, 8 and 15 for 28 days. After 28 days, the DSMB will meet to decide if enrollment of 
the next cohort should proceed. After 84 days (28 days x 3 cycles), the DSMB will meet and 
decide if patients will receive either (i) ixazomib once weekly for 12 weeks or (ii) ixazomib 3 mg 
on days 1, 8 and 15 for an additional three 28 days cycles. Visits 3-15 will have a window of ± 3 
days. 
 
Group 4: 3 patients with HIV infection, who meet inclusion criteria, will receive ixazomib 4 mg 
on days 1, 8 and 15 for 28 days. After 28 days, the DSMB will meet to decide if enrollment of 
the next cohort should proceed. After 84 days (28 days x 3 cycles), the DSMB will meet and 
decide if patients will receive either (i) ixazomib once weekly for 12 weeks or (ii) ixazomib 4 mg 
on days 1, 8 and 15 for an additional three 28 days cycles. Visits 3-15 will have a window of ± 3 
days. 
 
Group 5: 5 patients with HIV infection, who meet inclusion criteria will receive the maximum 
tolerated dose of ixazomib as determined by DSMB recommendation. Visits 3-15 will have a 
window of ± 3 days. 
 
 
 
All participants will continue their current ART regimen. 
 
The DSMB will review each dose group and the following recommendations can be made after 
each review:  


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5.3 
Method for Assigning Subjects to Treatment Groups 
Each group (or dose level) will enroll and after 4 weeks of follow up of 3 patients treated with 
ixazomib at a particular dose will be reviewed for safety by DSMB before initiating enrollment 
into the next group (or dose level).  All patients in the current cohort must have completed 4 
weeks and the cohort’s data reviewed by the by the Safety Monitoring Committee before patients 
in the next cohort can receive drug. Patients from the next cohort may complete the first two 
visits prior to DSMB review of earlier cohorts. 
 
Dose limiting toxicities are defined in Section 6.17 Table 2.  After all the subjects in each dosing 
group has completed Cycle 1 of ixazomib, the DSMB may recommend: 
(i) 
Proceed to next dose level, or 
 
(ii) 
Terminate trial, or 
 
(iii) 
Enroll a replacement subject for a withdrawn subject, or 
(iv) 
Add 5 more patients at previous dose group (maximum tolerated dose). The maximum 
tolerated dose is that level where no more than 0 out of 3 patients experience dose 
limiting toxicity. 
 


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If no DLT are observed in 3 subjects, the next dosing level will be initiated. 
 
After DSMB review of the 4 mg dosing level, if no DLT are observed, the DSMB may 
recommend: 
(i) 
Terminating the trial 
(ii) 
Enroll 5 more patients at the maximum tolerated dose. 
 
The NNRTIs etravirine and efavirenz are moderate CYP3A enzyme inducers and have potential 
to decrease systemic exposure to ixazomib. That possibility will be explored by measuring 
trough ixazomib levels in all patients and comparing ixazomib concentrations in patients on 
different ART regimens.  The DSMB will have to consider the number of patients treated with 
NNRTIs in a particular group as the safety profile of a group may be underestimated if the 
majority of patients are treated with NNRTIs as part of their ARV regimen.  
 
Additional data review by the DSMB will occur after Day 84 for each dosing cohort. At this 
time, the DSMB may recommend: 
(i) 
Proceed to uninterrupted weekly dosing on days 1, 8, 15 and 22 of a 28 day cycle, or 
(ii) 
Continue previous dosing on days 1, 8, and 15 of a 28 day cycle, or 
(iii) 
Enroll a replacement subject for a withdrawn subject, or 
(iv) 
Terminate dosing. 
This determination will be made on an individual participant basis, and not a dosing 
cohort basis, and will be made based on the presence or absence of adverse drug 
effects that do not meet the definition of dose related toxicities.   
 
5.4 
Preparation and Administration of Study Drug 
Preparation and storage 
Ixazomib capsules, individually packaged in foil blister strips with child resistant paper backing 
(see below),  will be stored unopened at 2-8ºC (36-46ºF) Of note, Ixazomib can be stored up to 
40ºC (104ºF) for up to 30 days, and up to 50º (122ºF) for up to 3 days). Study medication will be 
stored and dispensed by the Research Pharmacy at Mayo Clinic. Ixazomib that is dispensed to 
the patient for take-home dosing should remain in the blister packaging until the point of use. 
The investigative site will be providing the medication to the patient in units that comprise the 
correct dose configurations, which are prepackaged by the manufacturer.  Capsules should 
remain in the blisters until the point of use. Comprehensive instructions will be provided to the 
patient in order to ensure compliance with dosing procedures.  Patients will be instructed to store 
the medication in the refrigerator until the time of use.   


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Administration 
All protocol-specific criteria for administration of ixazomib must be met and documented 
before drug administration.  Ixazomib will be administered or dispensed only to eligible patients 
under the supervision of the investigator or identified subinvestigator(s).  Patients should be 
monitored for toxicity, as necessary, and doses of ixazomib should be modified as needed to 
accommodate patient tolerance to treatment; this may include symptomatic treatment, dose 
interruptions, and adjustments of ixazomib dose. Capsules of ixazomib will also be referred to as 
study drug.  Study drug will be supplied by Millennium as capsules of  0.5mg, 2.0mg, and 
3.0mg, and 4.0mg ixazomib. Patients will be instructed to swallow ixazomib capsules whole, 
with water, and not to break, chew, or open the capsules.  Ixazomib should be taken on an empty 
stomach (no food or drink) at least 1 hour before or 2 hours after a meal.  Each capsule should be 
swallowed separately with a sip of water.  A total of approximately 8 ounces (240 mL) of water 
should be taken with the capsules.   
 
Missed doses can be taken as soon as the patient remembers if the next scheduled dose is 
72 hours or more away.  A double dose should not be taken to make up for a missed dose.  If the 
patient vomits after taking a dose, the patient should not repeat the dose but should resume 
dosing at the time of the next scheduled dose. 
 


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5.5 
Subject Compliance Monitoring 
Reconciliation will occur accordingly when the patient returns for their next cycle of 
therapy. Any extremes in temperature should be reported as an excursion and will be managed 
on a case by case basis. Returned unused capsules should be discarded in a proper biohazard 
container. 
 
5.6 
Prior and Concomitant Therapy 
See section 4.2 Exclusion criteria above. 
 
 


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5.7 
Packaging 
See section 5.4 above. 
5.8 
Receiving, Storage, Dispensing and Return 
5.8.1 Receipt of Drug Supplies 
Drug procurement:  Investigational product will be supplied free of charge to trial participants 
by Takeda Pharmaceuticals, Inc. 
5.8.2 Storage 
See section 5.4 above.   
 
5.8.3 Inventory/Dispensing/Labeling of Study Drug 
See section 5.4. regarding storage of drug  
 
 
A drug inventory/accountability log will be utilized to track study drug 
inventory/accountability. Upon shipment receipt, invoices will be verified and the inventory 
updated upon receipt. The inventory/accountability log will note name, dose, lot number, 
expiration, and quantity. A dispensing record will be tracked to note drug name, strength, 
quantity, lot number, drug expiration, patient name, date of dispensing.  
 
 
Upon dispensing, a designated Research Pharmacist will label the medication with a label 
containing the following (Patient name, drug name/dose, dispense date, PI’s name, address, 
telephone #, IRB #, directions for use, expiration date) and an auxiliary label noting “Caution-
new drug limited by law to investigational use.” The labeling and packaging will be double 
checked by an additional study team member prior to release to the patient. Counseling and drug 
information will be provided to the patient upon study drug dispensing.  
 
Drug specific patient education/counseling 
• 
Ixazomib capsules must be administered intact and should not be opened or manipulated 
in any way. Additionally, ixazomib capsules should remain in the blister packs until they 
are ready to be taken. 
 
• 
Ixazomib should be taken on an empty stomach, at least 1 hour before or no sooner than 
2 hours after a meal (as described in section 5.4 above). 
 
• 
Thrombocytopenia has been observed with ixazomib. Patients will be instructed to 
report any unusual bruising or bleeding to the study team. 
 
• 
GI side effects have been observed in patients treated with ixazomib (nausea, diarrhea, 
vomiting). If this should occur, patients should seek medical advice to ensure 
compliance with HIV ARV medications and effective management of the adverse event. 
 
• 
Patients will be instructed to report any rash to study team. 
 


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• 
Patients will be instructed not to start any new medications or supplements without 
checking with the study team first. Our investigator team will assess patient’s 
concomitant medications, including over the counter and supplements. Ixazomib is 
metabolized through both CYP enzymes and non-CYP proteins, and drug to drug 
interactions exist with strong inducers of CYP3A.  
 
• 
The NNRTIs etravirine and efavirenz are moderate CYP3A enzyme inducers and have 
potential to decrease systemic exposure to ixazomib. That possibility will be explored by 
measuring trough ixazomib levels in all patients and comparing ixazomib concentrations 
in patients on different ART regimens. 
5.8.3 Return or Destruction of Study Drug 
At the completion of the study, there will be a final reconciliation of drug shipped, drug 
dispensed, drug returns, and drug remaining.  This reconciliation will be logged on the drug 
reconciliation form, signed and dated.  Any discrepancies noted will be documented and 
investigated, prior to destruction of unused study drug.  Drug destroyed on site will be 
documented in the study files. 
6 
Study Procedures 
 
 
Study related procedures include leukapheresis performed at baseline and at treatment 
completion..  
 
Leukapheresis  
Fenwal Amicus (Fenwal Inc., Lake Zurich IL, USA) apheresis systems (Version 3.1) will be 
used for mononuclear cell (MNC) collections.  Access will be obtained using peripheral veins.  
Each subject will be evaluated after enrollment but prior to collection by the nursing staff of the 
Therapeutic Apheresis Treatment Unit to ensure that they have adequate venous access for the 
collection procedure.  Venous access will consist of a 16 gauge steel needle placed into a vein in 
an antecubital fossa of one arm as a draw.  The return will consist of an IV placed in the opposite 
arm.  All MNC collections will have an endpoint of four hours processing time.  Anticoagulant 
will be acid-citrate-dextrose solution-A (ACD-A) (Baxter Healthcare Corp., Deerfield, IL, USA) 
for subjects with a contraindication to heparin or a mixture of ACD-A, normal saline, and 
heparin for subjects without a contraindication to heparin. The heparin/ACD-A anticoagulant is 
preferred due to the lower incidence of citrate toxicity because of the lower citrate dose 
administered during the procedure.  The lower dose of heparin administered also minimizes the 
length of systemic anticoagulation.The MNC offset will be 1.5 ml.  The RBC offset start at 5.0 
ml and will be adjusted during the procedure.  The operator will observe the first cycle MNC 
transfer to determine if the RBCs pass completely through the right cassette.  If the RBCs did not 
reach the top of the cassette, the operator will increase the RBC offsets to the next level for each 
succeeding cycle until the RBCs reached the top of the right cassette.  Once the RBCs reach the 
top of the right cassette, no further adjustment will be made. If the subject’s pre-procedure PLT 
count is ≥300 x 109/L, then the RBC offset will started at 6.0 ml. Only PBMCs will be harvested 
by this procedure.  Nearly all blood is returned to the patient with only a small amount of blood 
lost in the process and in the tubing.  PBMCs harvested by apheresis will be stored and used in 


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associated laboratory assays in the Badley HIV Immunology Laboratory BSL-3 facility on 
Guggenheim 5th floor in accordance with all appropriate Biosafety requirements. 
 
 
 
 
Screening for peripheral neuropathy 
Screening for peripheral neuropathy will occur at baseline (for study exclusion), and at each 
study visit (for safety monitoring), by administration of the European Organization for Research 
and Treatment of Cancer QLQ-CIPN20 Questionnaire. The 20-item CIPN-specific questionnaire 
which includes three scales assessing sensory (9 items), motor (8 items), and autonomic (3 items) 
symptoms and functioning with each item formatted the same as items of the EORTC QLQ-C30.  
It was developed to be used in conjunction with the EORTC QLQ-C30 following the EORTC 
guidelines for module development (15911236).  The EORTC QLQ-CIPN20 has been tested in 
cancer patients receiving a variety of chemotherapies and has been shown to have internal 
consistency reliability based on Cronbach’s alpha coefficients of 0.82, 0.73, and 0.76 for the 
three scales, respectively (PMID 23543373).  The questions that comprise the QLQ-CIPN20 are 
listed in Appendix II.  Any affirmative answer to questions in the QLQ-CIPN20 will trigger a 
further clinical assessment, and grading of suspected peripheral neuropathy according to the 
(CTCAE) Version 4.0 scale (Appendix I). Further clinical assessment of suspected peripheral 
neuropathy will be referred to a qualified physician in the area of drug-induced 
 peripheral 
neuropathy. 
 
The following toxicities will be considered to be dose limiting toxicities: 
 
• Any ≥Grade 4 toxicity   
• Any Grade 3 AE (except if there is clear evidence that it is not ixazomib related – i.e. 
a plausible alternative reason). 
• Any ≥Grade 1 peripheral neuropathy (PN)  
 
Specific management of toxicities in individual participants is discussed below.  
  
Study related biology assays for the secondary outcomes include: 
• CD4 T cell count, CD8 T cell count and CD4/CD8 ratio – by routine clinical flow 
cytometry (Mayo Medical Labs). 
• Plasma HIV RNA viral load – by routine clinical RT-qPCR (Mayo Medical Labs). 
• HIV Proviral DNA – by clinical qPCR (Mayo Medical Labs) 
• Cell associated HIV DNA by digital droplet PCR – Peripheral blood CD4 T cells will 
be isolated from baseline and post-treatment leukapheresis samples by magnetic bead 
negative selection. Cell associated HIV DNA will be quantified by digital droplet 
PCR using RainDance™ ddPCR system (Badley HIV Laboratory). 
• Culturable HIV – replication competent latent viral reservoir will be estimated using a 
quantitative viral outgrowth assay, considered the gold standard measurement, on 
peripheral blood CD4 T cells from baseline and post-treatment leukapheresis samples 
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6.1 
Visit 1 (Screening and Enrollment) 
• Screen for study eligibility based on inclusion and exclusion criteria. 
• Obtained informed consent. 
• Obtain vital signs, including weight. 
• Document medical history. 
• Document concomitant medications and non-drug treatments (including prescription and 
over-the-counter medications, nutritional supplements and herbal remedies, taken within 
30 days of screening) 
• Perform complete physical exam. 
• Review and record results of routine clinical monitoring labs, including CBC, 
electrolytes, glucose, creatinine, LFTs, CD4 T cell count and percentage, CD8 T cell 
count and percentage and CD4/CD8 ratio, HIV RNA viral load, HIV proviral DNA, urine 
pregnancy test and ECG.  Obtain an additional four (4) heparin tubes of whole blood for 
stored PBMC and plasma. 
 
When screening clinical laboratory test results are received, they must be reviewed by the 
Principal Investigator or designee physician for clinically significant abnormalities. If no 
clinically significant abnormalities or exclusions are found, the subject should be scheduled to 
attend Visit 2. Visits 1 and 2 may occur on the same date, but not before visit 3. 
6.2 
Visit 2  
• Leukapheresis performed at Clinical Apheresis Unit, Charlton 8th floor.  
6.3 
Visit 3 (Study Entry, Time 0 weeks) 
• Obtain vital signs, including weight. 
• Perform brief physical exam. 
• Monitor for changes to concomitant drug and non-drug treatments  
• Obtain CBC and an additional four (4) heparin tubes of whole blood for stored PBMC 
and plasma.   
• QLQ-CIPN20 Questionnaire 
• Prescribe study medication.   
6.3.5 
Visit 3.5 (Optional post-dose blood draw) 
• Obtain four (4) heparin tubes of whole blood for stored PBMC and plasma.   
6.4 
Visit 4 (Treatment phase, Time 1 week ±3 days) 
• Obtain vital signs, including weight. 
• Perform brief physical exam. 
• Monitor for changes to concomitant drug and non-drug treatments  
• Obtain CBC and confirm lack of hematologic toxicity prior to administering day 8 dose.  
• QLQ-CIPN20 Questionnaire 
• Monitor and record adverse events  
• Prescribe study medication.   


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6.5 
Visit 5 (Treatment Phase, Time 2 weeks ±3 days) 
• Obtain vital signs, including weight. 
• Perform brief physical exam. 
• Monitor for changes to concomitant drug and non-drug treatments.  
• Obtain CBC and confirm lack of hematologic toxicity prior to administering day 15 dose.   
• Obtain electrolytes, glucose, creatinine, LFTs. 
• QLQ-CIPN20 Questionnaire 
• Monitor and record adverse events    
• Prescribe study medication  
6.6 
Visit 6 (Treatment phase, Time 3 weeks ±3 days) 
• Obtain vital signs, including weight. 
• Perform brief physical exam. 
• Monitor for changes to concomitant drug and non-drug treatments  
• Obtain CBC, CD4 and CD8 count, and confirm lack of hematologic toxicity.  
• QLQ-CIPN20 Questionnaire 
• Monitor and record adverse events   
• Prescribe study medication.   
6.7 
Visit 7 (Treatment Phase, Time 4 weeks ±3 days) 
• Obtain vital signs, including weight. 
• Monitor for changes to concomitant drug and non-drug treatments.  
• Monitor and record adverse events. 
• Perform brief physical exam.   
• Obtain CD4 and CD8 count, HIV RNA viral load, HIV proviral DNA, and an additional 
four (4) heparin tubes of whole blood for stored PBMC and plasma.   
• Obtain electrolytes, glucose, creatinine, LFTs  
• Obtain CBC and confirm lack of hematologic toxicity prior to starting next 28 day cycle. 
• QLQ-CIPN20 Questionnaire   
• Prescribe study medication.  
 
6.8 
Visit 8 (Treatment Phase, Time 6 weeks ±3 days) 
• Obtain vital signs, including weight. 
• Monitor for changes to concomitant drug and non-drug treatments.  
• Monitor and record adverse events. 
• Perform brief physical exam 
• 
Obtain CBC, electrolytes, glucose, creatinine, LFTs 
• 
Prescribe study medication.QLQ-CIPN20 Questionnaire 
• Draw ixazomib trough level (pre dose sample for drug level determination) and 1 hour 
post-dose level. 


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6.9 
Visit 9 (Treatment Phase, Time 8 weeks ±3 days) 
• Obtain vital signs, including weight. 
• Monitor for changes to concomitant drug and non-drug treatments.  
• Monitor and record adverse events. 
• Perform brief physical exam.   
• Obtain CBC, CD4 and CD8 count, HIV RNA viral load, HIV proviral DNA and an 
additional four (4) heparins tube of whole blood for stored PBMC and plasma.   
• 
Obtain electrolytes, glucose, creatinine, LFTs 
• QLQ-CIPN20 Questionnaire 
• Prescribe study medication. 
6.10 Visit 10 (Treatment Phase, Time 12 weeks ±3 days) 
• Obtain vital signs, including weight. 
• Monitor for changes to concomitant drug and non-drug treatments.  
• Monitor and record adverse events. 
• Perform brief physical exam.   
• Obtain CBC, CD4 and CD8 count, HIV RNA viral load, HIV proviral DNA and an 
additional four (4) heparin tubes of whole blood for stored PBMC and plasma.   
• Obtain electrolytes, glucose, creatinine, LFTs 
• QLQ-CIPN20 Questionnaire 
• Prescribe study medication. 
6.11 Visit 11 (Treatment Phase, Time 16 weeks ±3 days) 
• Obtain vital signs, including weight. 
• Monitor for changes to concomitant drug and non-drug treatments.  
• Monitor and record adverse events. 
• Perform brief physical exam.   
• Obtain CBC, CD4 and CD8 count, HIV RNA viral load, HIV proviral DNA and an 
additional four (4) heparin tubes of whole blood for stored PBMC and plasma.   
• Obtain electrolytes, glucose, creatinine, LFTs 
• 
QLQ-CIPN20 Questionnaire 
• Prescribe study medication. 
 
6.12 Visit 12 (Treatment Phase, Time 20 Weeks ±3 days) 
• Obtain vital signs, including weight. 
• Monitor for changes to concomitant drug and non-drug treatments.  
• Monitor and record adverse events. 
• Perform brief physical exam.   


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• Obtain CBC, CD4 and CD8 count, HIV RNA viral load, HIV proviral DNA and an 
additional four (4) heparin tubes of whole blood for stored PBMC and plasma.   
• Obtain electrolytes, glucose, creatinine, LFTs 
• 
QLQ-CIPN20 Questionnaire 
• Prescribe study medication. 
 
6.13 Visit 13 (Leukapheresis, Time 22 weeks ±3 days) 
• Leukapheresis performed at Clinical Apheresis Unit, Charlton 8th floor.  
• Monitor and record adverse events. 
• Obtain CBC. 
• Draw ixazomib trough level (pre dose sample for drug level determination) and 1 hour 
post-dose level.  
6.14 Visit 14 (Treatment Completion, Time 24 weeks ±3 days) 
• Obtain vital signs, including weight. 
• Monitor for changes to concomitant drug and non-drug treatments.  
• Monitor and record adverse events. 
• Perform brief physical exam.   
• Obtain CBC, CD4 count, HIV RNA viral load, HIV proviral DNA and an additional four 
(4) heparin tubes of whole blood for stored PBMC and plasma.   
• Obtain electrolytes, glucose, creatinine, LFTs 
• 
QLQ-CIPN20 Questionnaire 
• Obtain ECG. 
 
 
6.15 Visit 15 (Final Study Visit, Time 28 weeks ±3 days) 
• Obtain vital signs, including weight. 
• Monitor for changes to concomitant drug and non-drug treatments.  
• Monitor and record adverse events. 
• Perform brief physical exam.   
• Obtain CBC, CD4 count, HIV RNA viral load, HIV proviral DNA  
• Obtain electrolytes, glucose, creatinine, LFTsQLQ-CIPN20 Questionnaire 


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6.16 Table 1.  Table of Study Activities 
 
Study Activity 
V 1 
V 2 
V 
3c 
V 
3.5 
V 4 
V 5 
V 6 
V 7 
 
V 8 
V 9 
 
V 
10 
 
V 
11 
V 
12 
 
V 
13 
V 
14 
V 
15 
Eligibility Screening 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Peripheral Neuropathy 
self-reported questionnaire 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Concurrent meds 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Complete physical exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Brief physical exam 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Vital Signs 
X 
 
X 
 
X 
X 
X 
X 
 X 
X 
X 
X 
X 
 
X 
X 
CD4/CD8 T cell counts 
X 
 
 
 
 
 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
HIV RNA Viral load 
X 
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
HIV Proviral DNA 
X 
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
 
X 
X 
CBC w/diff, platelet a 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Heparin tubes for PBMC 
and plasma storage 
X 
 
X 
X 
 
 
 
X 
 
X 
X 
X 
X 
 
X 
 
Serum chemistry b 
X 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
X 
 
X 
X 
Pre- and post-dose 
ixazomib level 
 
 
 
 
 
 
 
 
X 
 
 
 
 
X 
 
 
Adverse event evaluation 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Urine Pregnancy test 
 X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Leukapheresis 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
a – Increased blood draw frequency may be required in the presence of hematologic toxicity. This will occur prior to 
each dose of ixazomib. In addition, increased blood draw frequency may be required if ixazomib is dosed weekly 
uninterrupted in Cycles 4-6. 
b - Potassium, Creatinine, Alkaline phosphatase, ALT, AST, Total bilirubin, Direct bilirubin, Sodium, BUN, 
glucose, Chloride, Bicarbonate. 
c- Visits 1 and 2 can occur on the same date, as long as they occur before visit 3. 
6.17 Table 2: Instructions for treatment delays and dose reductions prior to ixazomib 
administration 
 
CTCAE 
System/Organ/ 
Class (SOC) 
 
ADVERSE EVENT 
 
AGENT 
 
ACTION** 
BASED ON INTERVAL ADVERSE EVENT  


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CTCAE 
System/Organ/ 
Class (SOC) 
 
ADVERSE EVENT 
 
AGENT 
 
ACTION** 
Investigations 
Grade 2 
thrombocytopenia 
(PLT count <75,000 - 
50,000/mm3) and/or 
neutropenia <1500 - 
1000/mm3 
 
Ixazomib 
Ixazomib dose should be omitted until 
the PLT and/or ANC has recovered to 
baseline values (counts >lower limit 
of normal).  Ixazomib may be 
reinitiated at the same dose if the 
recovery has occurred in less than 
7days. If the abnormality lasts ≥7 days 
or recurs, then the dose of ixazomib 
should be reduced by one dose level.  
If a patient is already at the lowest 
dose level, ixazomib should be 
permanently discontinued.  Patients 
should have CBC checked weekly 
until resolution of abnormality. 
 
 
≥Grade 3 
thrombocytopenia 
and/or neutropenia  
 
Permanently discontinue ixazomib 
 
≥Grade 2 increase in 
AST, ALT or total 
bilirubin 
Ixazomib 
Ixazomib dose should be omitted until 
the abnormal LFT has recovered to 
baseline values.  Ixazomib may be 
reinitiated at the same dose if the 
recovery has occurred in less than 
7days. If the abnormality lasts ≥7 days 
or recurs, then the dose of ixazomib 
should be reduced by one dose level.  
If a patient is already at the lowest 
dose level, ixazomib should be 
permanently discontinued.  Patients 
should have LFTs checked weekly 
until resolution of abnormality. 
Skin and 
subcutaneous tissue 
disorders 
Any Grade ≤2 Rash 
Ixazomib 
Omit ixazomib until rash resolves.  
Restart at same dose. If the rash 
recurs, reduce dose by one dose level.  
If a patient is already at the lowest 
ixazomib dose, ixazomib should be 
permanently discontinued.  
 
Any rash, ≥Grade 3 
Ixazomib 
Ixazomib should be permanently 
discontinued. 


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CTCAE 
System/Organ/ 
Class (SOC) 
 
ADVERSE EVENT 
 
AGENT 
 
ACTION** 
Nervous System 
Disorders 
Grade 1 peripheral 
neuropathy  
Ixazomib 
Ixazomib should be permanently 
discontinued. 
Other 
Any Grade 3 AE 
(except if there is 
clear evidence that it 
is not ixazomib 
related – i.e. a 
plausible alternative 
reason). 
 
Ixazomib 
Permanently discontinue ixazomib. 
 
Grade 4 AE 
 
Ixazomib 
Permanently discontinue ixazomib  
 
Plasma HIV RNA  
>500copies/ml on 2 
consecutive 
occasions. 
(except if there is 
clear evidence that it 
is not ixazomib 
related – i.e. a 
plausible alternative 
reason – for example 
missed ART doses).    
Ixazomib 
Permanently discontinue ixazomib.  
Perform HIV genotypic resistance 
testing.   
Use the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0* unless 
otherwise specified for all Adverse Events. 
 
6.18 Excluded Concomitant Medications and Procedures 
The following medications and procedures are prohibited during the study. 
Systemic treatment with any of the following metabolizing enzyme inducers should be avoided, 
unless there is no appropriate alternative medication for the patient’s use:  
• Strong CYP3A inducers:  rifampin, rifapentine, rifabutin, carbamazepine, phenytoin, and 
phenobarbital 
• Excluded foods and dietary supplements include St. John’s wort.  


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6.19 Permitted Concomitant Medications and Procedures 
The following medications and procedures are permitted during the study: 
• Antiemetics, including 5-HT3 serotonin receptor antagonists, may be used at the 
discretion of the investigator. 
• Loperamide or other antidiarrheal should be used for symptomatic diarrhea at discretion 
of the investigator. The dose and regimen will be according to institutional guidelines. 
fluid replacement should be given to prevent volume depletion. 
• Antiviral therapy (in addition to the patient’s antiretroviral regimen) such as acyclovir 
may be administered if medically appropriate. 
• Supportive measures consistent with optimal patient care may be given throughout the 
study. 
6.20 Precautions and Restrictions  
Fluid deficit should be corrected before initiation of treatment and during ixazomib 
treatmentNonsteroidal anti-inflammatory drugs (NSAIDs) should be avoided with impaired renal 
function given reported NSAID-induced renal failure in patients with decreased renal function.  
The NNRTIs etravirine and efavirenz are moderate CYP3A4 enzyme inducers and have 
potential to decrease systemic exposure to ixazomib. That possibility will be explored by 
measuring ixazomib levels in all patients and comparing ixazomib concentrations in patients on 
different ART regimens.   
Pregnancy 
It is not known what effects ixazomib has on human pregnancy or development of the 
embryo or fetus.  Therefore, female patients participating in this study should avoid becoming 
pregnant, and male patients should avoid impregnating a female partner.  Male patients should 
use effective methods of contraception through defined periods during and after study treatment 
as specified below. 
Female patients must meet 1 of the following:  
Postmenopausal for at least 1 year before the screening visit, or surgically sterile.  
Regardless of above criteria, females must have a negative urine pregnancy test prior to 
receiving the 1st dose of ixazomib. 


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Male patients, even if surgically sterilized (ie, status postvasectomy) must agree to 1 of the 
following: 
• Practice effective barrier contraception AND a second method of contraception for 
female partners of childbearing potential during the entire study treatment period and 
through 90 days after the last dose of ixazomib, or 
• Agree to practice true abstinence, when this is in line with the preferred and usual 
lifestyle of the subject.  (Periodic abstinence [eg, calendar, ovulation, symptothermal, 
postovulation methods for the female partner] and withdrawal are not acceptable methods 
of contraception.) 
• AND 
• Agree to forego sperm donation for the same period as above. 
6.21 Management of Clinical Events  
Adverse drug reactions such as thrombocytopenia, diarrhea, fatigue, nausea, vomiting, 
and rash have been associated with ixazomib treatment.  Management guidelines regarding these 
events are outlined below.  Further details of management of ixazomib AEs are described in the 
ixazomib IB.   
 
Prophylaxis Against Risk of Reactivation of Herpes Infection 
Patients may be at an increased risk of infection including reactivation of herpes zoster 
and herpes simplex viruses.  Antiviral therapy such as acyclovir, valacyclovir, or other antivirals 
may be initiated as clinically indicated.   
Nausea and/or Vomiting 
Standard anti-emetics including 5-hydroxytryptamine 3 serotonin receptor antagonists are 
recommended for emesis if it occurs once treatment is initiated; prophylactic anti-emetics may 
also be considered at the physician’s discretion. Fluid deficit should be corrected before the 
patient takes ixazomib. 
Diarrhea 
Prophylactic antidiarrheals will not be used in this protocol. However, diarrhea should be 
managed according to clinical practice, including the administration of antidiarrheals once 
infectious causes are excluded. Fluid intake should be maintained to avoid dehydration. Fluid 
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Erythematous Rash With or Without Pruritus 
As with bortezomib, rash with or without pruritus has been reported with ixazomib, 
primarily at the higher doses tested and when given with agents where rash is an overlapping 
toxicity.  The rash may range from limited erythematous areas, macular and/or small papular 
bumps that may or may not be pruritic over a few areas of the body, to a more generalized 
eruption that is predominately on the trunk or extremities.  Rash has been most commonly 
characterized as maculopapular or macular.  To date, when it does occur, rash is most commonly 
reported within the first 3 cycles of therapy.  The rash is often transient, self-limiting, and is 
typically Grade 1 to 2 in severity.  
Symptomatic measures such as antihistamines or corticosteroids (oral or topical) have 
been successfully used to manage rash and have been used prophylactically in subsequent cycles.  
The use of a topical, IV, or oral steroid (eg, prednisone ≤ 10 mg per day or equivalent) is 
permitted.  Management of a Grade 3 rash may require intravenous antihistamines or 
corticosteroids.  Administration of ixazomib (and/or other causative agent if given in 
combination) should be modified per protocol and re-initiated at a reduced level from where rash 
was noted (also, per protocol).   
In line with clinical practice, dermatology consult and biopsy of Grade 3 or higher rash or 
any SAE involving rash is recommended.  Prophylactic measures should also be considered if a 
patient has previously developed a rash (eg, using a thick, alcohol-free emollient cream on dry 
areas of the body or oral or topical antihistamines).  The rare risks of Stevens-Johnson syndrome, 
TEN, DRESS syndrome and pemphigus vulgaris have been reported in oncology studies when 
ixazomib (or placebo) has been given in a multi-drug regimen, with concomitant medications 
known to cause rash, and/or in the setting of confounding treatment emergent adverse events.  
These severe, potentially life-threatening, or deadly conditions may involve rash with skin 
peeling and mouth sores and should be clinically managed according to standard medical 
practice.  Study medication should be discontinued in the event of severe, potentially life-
threatening rash.  Punch biopsies for histopathological analysis are encouraged at the discretion 
of the investigator. 
Thrombocytopenia and anemia 
Blood counts should be monitored regularly as outlined in the protocol with additional 
testing obtained according to standard clinical practice. Thrombocytopenia has been seen in 
patients with cancer and patients that were treated with combination anti-cancer therapy which 
included ixazomib, and may be severe but has been manageable with platelet transfusions 
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per dose modification recommendations in the protocol when thrombocytopenia occurs (See 
Page 39 above). Therapy can be reinitiated at a reduced level upon recovery of platelet counts. A 
rare risk is thrombotic thrombocytopenic purpura (TTP), a rare blood disorder where blood clots 
form in small blood vessels throughout the body characterized by thrombocytopenia, petechiae, 
fever, or possibly more serious signs and symptoms. TTP should be managed symptomatically 
according to standard medical practice. Red blood cell transfusions should be administered for 
the treatment of anemia according to standard clinical practice. 
Neutropenia 
Blood counts should be monitored regularly as outlined in the protocol with additional 
testing obtained according to standard clinical practice.  Neutropenia may be severe but has been 
manageable. Ixazomib administration should be modified as noted as per dose modification 
recommendations in the protocol when neutropenia occurs (see Page 39 above).  Therapy can be 
reinitiated at a reduced level upon recovery of ANCs. 
Fluid Deficit 
Dehydration should be avoided since ixazomib may cause vomiting, diarrhea, and 
dehydration.  Acute renal failure has been reported in patients treated with ixazomib, commonly 
in the setting of the previously noted gastrointestinal toxicities and dehydration. 
Fluid deficit should be corrected before initiation of ixazomib and as needed during 
treatment to avoid dehydration. 
Hypotension 
Symptomatic hypotension and orthostatic hypotension with or without syncope have been 
reported with ixazomib.  Blood pressure should be closely monitored while the patient is on 
study treatment and fluid deficit should be corrected as needed, especially in the setting of 
concomitant symptoms such as nausea, vomiting, diarrhea, or anorexia.  Patients taking 
medications and/or diuretics to manage their blood pressure (for either hypo- or hypertension) 
should be managed according to standard clinical practice, including considerations for dose 
adjustments of their concomitant medications during the course of the trial.  Fluid deficit should 
be corrected before initiation of ixazomib and as needed during treatment to avoid dehydration. 
Posterior Reversible Encephalopathy Syndrome 
One case of posterior reversible encephalopathy syndrome, which ultimately resolved, 
has been reported with ixazomib.  This condition is characterized by headache, seizures and 
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magnetic resonance imaging (MRI).  If the syndrome is diagnosed or suspected, symptom-
directed treatment should be maintained until the condition is reversed by control of 
hypertension or other instigating factors. 
Transverse Myelitis 
Transverse myelitis has also been reported with ixazomib. It is not known if ixazomib 
causes transverse myelitis; however, because it happened to a patient receiving ixazomib, the 
possibility that ixazomib may have contributed to transverse myelitis cannot be excluded. 
7 Statistical Plan 
7.1 
Sample Size Determination 
For this Phase I/II single-center study, the sample size is primarily determined by the 
estimated number of patients that can be successfully recruited during the study time period.   
7.2 
Statistical Methods 
Descriptive Statistics 
Univariate descriptive statistics and frequency distributions will be calculated, as 
appropriate for all variables.  Baseline values for demographic, clinical, and outcome variables 
will be tabulated for the treatment groups.  These analyses will help identify potential 
confounding variables to be used as covariates in sensitivity analyses.  Putative prognostic 
variables that will be investigated through these descriptive analyses include variables such as 
age, sex, race/ethnicity, and duration of HIV infection, baseline CD4 T cell count and HIV viral 
load, mode of transmission, duration of ART, and duration of viral suppression. 
 
Multiplicity 
No a priori correction to the type I error rate is warranted for this study. 
 
Primary Analysis 
Ixazomib will be safe and well tolerated in HIV positive persons on suppressive ART. 
 
Description statistics will be used to describe the rates of adverse effects by CTC grade of 
ixazomib among the dosing groups.   
 
 
 


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Secondary Analysis 1:  
Ixazomib will reduce HIV DNA and replication competent viral reservoir size and 
improve CD4 T cell counts in HIV positive patients on suppressive ART. 
 
To test this hypothesis, a mixed model will be estimated for each outcome separately. 
This model will consider linear trajectories and more flexible distribution of outcomes over time 
using each assessment point as a factor (i.e., a “repeated measures ANOVA formulation”). Given 
the limitations of the sample size, random effects for only subject (random intercept) models will 
be considered. These models will examine the treatment by time interaction and the linear 
trajectory model will be expected to have greater, but relatively poor, power to detect differences 
in outcomes such as viral load over time. An alpha of 0.1 or lower will be considered sufficiently 
significant to serve as preliminary evidence of a biologic effect to warrant further studies.   
 
In the event the models are not able to be estimated from the data available, a planned 
post-hoc analysis will compare changes in secondary endpoints between participants on integrase 
inhibitor-based ART and non-nucleoside reverse transcriptase-based ART.   
 
Interim Analysis 
There is no interim analysis in this protocol, each cohort/dose level will be reviewed by 
the DSMB and decide upon escalation to the next dose level.  This review will be conducted 
when all treated patients/cohort have received 4 weeks of treatment, or earlier if a DLT has 
occurred. Thereafter DSMB will review each cohort monthly. 
7.3 
Subject Population(s) for Analysis 
 
The per-protocol analysis set will be of participants followed according to the study 
schedule. This subset of the entire study sample will be of particular interest to the mechanistic 
studies since all data will be available. The results of this potentially non-representative subset 
will be compared to the results obtained using the Safety evaluable Analysis Set as a sensitivity 
analysis. Should the results disagree qualitatively, the ITT results will be considered the less-
biased results; however, a careful examination of putative causes for the differences will be fully 
investigated to inform a Phase II/III confirmatory study.  
 
The Safety Evaluable Analysis Set will consist of all participants that have at least one 
dose of study medication.  This analysis set will serve as the basis for the primary analysis noted 
above and adverse event reporting of treatment emergent and/or worsening events. 
 
8 Safety and Adverse Events 
8.1 
Definitions 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets the following three criteria:  
• Serious: Serious problems or events that results in significant harm, (which may be 
physical, psychological, financial, social, economic, or legal) or increased risk for the 
subject or others (including individuals who are not research subjects). These include: (1) 


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death; (2) life threatening adverse experience; (3) hospitalization - inpatient, new, or 
prolonged; (4) disability/incapacity - persistent or significant; (5) birth defect/anomaly; (6) 
breach of confidentiality and (7) other problems, events, or new information (i.e. 
publications, DSMB reports, interim findings, product labeling change) that in the opinion 
of the local investigator may adversely affect the rights, safety, or welfare of the subjects 
or others, or substantially compromise the research data, AND 
• Unanticipated: (i.e. unexpected) problems or events are those that are not already 
described as potential risks in the protocol, consent document, not listed in the 
Investigator’s Brochure, or not part of an underlying disease. A problem or event is 
"unanticipated" when it was unforeseeable at the time of its occurrence. A problem or 
event is "unanticipated" when it occurs at an increased frequency or at an increased 
severity than expected, AND 
• Related: A problem or event is "related" if it is possibly related to the research procedures. 
 
Adverse Event 
An untoward or undesirable experience associated with the use of a medical product (i.e. drug, 
device, biologic) in a patient or research subject. An AE can therefore be any unfavorable and 
unintended sign (including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of a medicinal (investigational) product whether or not it is related to the 
medicinal product.  This includes any newly occurring event, or a previous condition that has 
increased in severity or frequency since the administration of ixazomib. 
An abnormal laboratory value will not be assessed as an AE unless that value leads to 
discontinuation or delay in treatment, dose modification, therapeutic intervention, or is 
considered by the investigator to be a clinically significant change from baseline.   
 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  Serious problems/events can be well 
defined and include; 
• death 
• life threatening adverse experience 
• hospitalization 
• inpatient, new, or prolonged; disability/incapacity 
• persistent or significant birth defect/anomaly 
 
and/or per protocol may be problems/events that in the opinion of the sponsor-investigator 
may have adversely affected the rights, safety, or welfare of the subjects or others, or 
substantially compromised the research data. 
 
All adverse events that do not meet any of the criteria for serious, should be regarded as non-
serious adverse events.  
 
Adverse Event Reporting Period 


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For this study, the study treatment follow-up period is defined as 30 days following the last 
administration of study treatment.  
 
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A preexisting condition 
should be recorded as an adverse event if the frequency, intensity, or the character of the 
condition worsens during the study period. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting 
condition.  At the end of the study, any new clinically significant findings/abnormalities that 
meet the definition of an adverse event must also be recorded and documented as an adverse 
event.  
 
Post-study Adverse Event 
All unresolved adverse events should be followed by the sponsor-investigator until the events are 
resolved, the subject is lost to follow-up, or the adverse event is otherwise explained.  At the last 
scheduled visit, the sponsor-investigator should instruct each subject to report, to the sponsor-
investigator, any subsequent event(s) that the subject, or the subject’s personal physician, 
believes might reasonably be related to participation in this study.  
 
Table 3. Adverse Events to be Graded at Each Evaluation 
 
 
SYSTEM ORGAN 
CLASS 
 
Adverse event/Symptoms 
 
Baseline 
Each 
evaluation 
Investigations 
Creatinine increased 
X 
X 
Neutrophil count decreased 
X 
X 
Platelet count decreased 
X 
X 
General disorders and 
administration site 
conditions 
Edema limbs 
X 
X 
Fatigue  
X 
X 
Gastrointestinal 
Disorders 
Nausea 
X 
X 
Vomiting 
X 
X 
# of Stools 
X 
 
Diarrhea 
 
X 
Constipation 
 
X 
Infections and 
infestations 
Sepsis 
X 
X 
Blood and lymphatic 
system disorders 
Febrile neutropenia 
X 
X 
Skin and 
subcutaneous tissue 
disorders 
Rash, maculopapular 
X 
X 
Nervous system 
Peripheral sensory neuropathy  
X 
X 


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disorders 
 
Peripheral motor neuropathy  
X 
X 
Cognitive disturbance 
X 
X 
 
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be 
documented and reported as a serious adverse event unless specifically instructed otherwise in 
this protocol.  Any condition responsible for surgery should be documented as an adverse event 
if the condition meets the criteria for an adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an 
adverse event in the following circumstances:  
• Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures 
for a preexisting condition.  Surgery should not be reported as an outcome of an adverse 
event if the purpose of the surgery was elective or diagnostic and the outcome was 
uneventful. 
• Hospitalization or prolonged hospitalization required to allow efficacy measurement for 
the study. 
• Hospitalization or prolonged hospitalization for therapy of the target disease of the study, 
unless it is a worsening or increase in frequency of hospital admissions as judged by the 
clinical investigator. 
8.2 
Recording of Adverse Events 
At each contact with the subject, the study team must seek information on adverse events by 
specific questioning and, as appropriate, by examination.  AEs may be spontaneously reported by 
the patient and/or in response to an open question from study personnel or revealed by 
observation, physical examination, or other diagnostic procedures.  Any clinically relevant 
deterioration in laboratory assessments or other clinical finding is considered an AE.  When 
possible, signs and symptoms indicating a common underlying pathology should be noted as one 
comprehensive event.  For serious AEs, the investigator must determine both the intensity of the 
event and the relationship of the event to ixazomib administration.  
Information on all adverse events should be recorded immediately in the source document, and 
also in the appropriate adverse event section of the case report form (CRF).  All clearly related 
signs, symptoms, and abnormal diagnostic, laboratory or procedure results should recorded in the 
source document. 
 
All adverse events occurring during the study period must be recorded.  The clinical course of 
each event should be followed until resolution, stabilization, or until it has been ultimately 
determined that the study treatment or participation is not the probable cause.  Serious adverse 
events that are still ongoing at the end of the study period must be followed up, to determine the 
final outcome.  Any serious adverse event that occurs after the study period and is considered to 
be at least possibly related to the study treatment or study participation should be recorded and 
reported immediately. 


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8.3 
Reporting of Serious Adverse Events and Unanticipated Problems 
 
When an adverse event has been identified, the study team will take appropriated action 
necessary to protect the study participant and then complete the Study Adverse Event Worksheet 
and log.  The sponsor-investigator will evaluate the event and determine the necessary follow-up 
and reporting required. 
8.3.1 Sponsor-Investigator reporting: notifying the Mayo IRB 
 
The sponsor-investigator will report to the Mayo IRB any UPIRTSOs and Non-UPIRTSOs 
according to the Mayo IRB Policy and Procedures.  According to Mayo IRB Policy any serious 
adverse event (SAE) which the Principal Investigator has determined to be a UPIRTSO must be 
reported to the Mayo IRB as soon as possible but no later than 5 working days after the 
investigator first learns of the problem/event. 
 
Information collected on the adverse event worksheet (and entered in the research database) will 
include:  
• Subject’s name:  
• Medical record number:  
• Disease/histology (if applicable):  
• The date the adverse event occurred:  
• Description of the adverse event:  
• Relationship of the adverse event to the research (drug, procedure, or intervention):  
• If the adverse event was expected:  
• The severity of the adverse event:  
• If any intervention was necessary:  
• Resolution: (was the incident resolved spontaneously, or after discontinuing treatment) 
• Date of Resolution:  
 
The investigator will review all adverse event reports to determine if specific reports need to be 
made to the IRB and FDA.  The sponsor-investigator will sign and date the adverse event report 
when it is reviewed.  For this protocol, only directly related SAEs/UPIRTSOs will be reported to 
the IRB. 
 
 
 


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8.3.2 Sponsor-Investigator reporting: Notifying the FDA 
The sponsor-investigator will report to the FDA all unexpected, serious suspected adverse 
reactions according to the required IND Safety Reporting timelines, formats and requirements. 
 
Unexpected fatal or life threatening suspected adverse reactions where there is evidence to 
suggest a causal relationship between the ixazomib and the adverse event, will be reported as a 
serious suspected adverse reaction.  This will be reported to the FDA on FDA Form 3500A, no 
later than 7 calendar days after the sponsor-investigator’s initial receipt of the information about 
the event. 
 
Other unexpected serious suspected adverse reactions where there is evidence to suggest a causal 
relationship between the ixazomib and the adverse event, will be reported as a serious suspected 
adverse reaction.  This will be reported to the FDA on FDA Form 3500A, no later than 15 
calendar days after the sponsor-investigator’s initial receipt of the information about the event. 
 
Any clinically important increase in the rate of serious suspected adverse reactions over those 
listed in the protocol or product insert will be reported as a serious suspected adverse reaction.  
This will be reported to the FDA on FDA Form 3500A no later than 15 calendar days after the 
sponsor-investigator’s initial receipt of the information about the event. 
 
Findings from other studies in human or animals that suggest a significant risk in humans 
exposed to the drug will be reported.  This will be reported to the FDA on FDA Form 3500A, no 
later than 15 calendar days after the sponsor-investigators initial receipt of the information about 
the event. 
AEs which are serious must be reported to Millennium Pharmacovigilance (or designee) from 
the first dose of ixazomib through 30 days after administration of the last dose of ixazomib. Any 
SAE that occurs at any time after completion of ixazomib treatment or after the designated 
follow-up period that the sponsor-investigator and/or sub-investigator considers to be related to 
ixazomib must be reported to Millennium Pharmacovigilance (or designee).  In addition, new 
primary malignancies that occur during the follow-up periods must be reported, regardless of 
causality to ixazomib, for a minimum of three years after the last dose of ixazomib, starting from 
the first dose of ixazomib. All new cases of primary malignancy must be reported to Millennium 
Pharmacovigilance (or designee).    
Planned hospital admissions or surgical procedures for an illness or disease that existed before 
the patient was enrolled in the trial are not to be considered AEs unless the condition deteriorated 
in an unexpected manner during the trial (e.g., surgery was performed earlier or later than 
planned).  All SAEs should be monitored until they are resolved or are clearly determined to be 
due to a patient’s stable or chronic condition or intercurrent illness(es). 
Since this is an investigator-initiated study, the principal investigator is responsible for reporting 
serious adverse events (SAEs) to any regulatory agency and to the Mayo Clinic’s IRB.    
Regardless of expectedness or causality, all SAEs (including serious pretreatment events) must 
also be reported in English to Millennium Pharmacovigilance (or designee): 


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Fatal and Life Threatening SAEs within 24 hours of the sponsor-investigator’s 
observation or awareness of the event 
 
All other serious (non-fatal/non life threatening) events within 4 calendar days of the 
sponsor-investigator’s observation or awareness of the event 
 
See below for contact information for the reporting of SAEs to Millennium Pharmacovigilance.   
The sponsor-investigator must fax or email the SAE Form per the timelines above.  A sample of 
an SAE Form will be provided.   
The SAE report must include at minimum: 
• Event term(s) 
• Serious criteria 
• Intensity of the event(s): Sponsor-investigator’s or sub-investigator’s 
determination.  Intensity for each SAE, including any lab abnormalities, will be 
determined by using the NCI CTCAE version specified in the protocol, as a 
guideline, whenever possible.  The criteria are available online at 
http://ctep.cancer.gov/reporting/ctc.html. 
• Causality of the event(s): Sponsor-investigator’s or sub-investigator’s 
determination of the relationship of the event(s) to ixazomib administration.  
Follow-up information on the SAE may be requested by Millennium.   
Intensity for each SAE, including any lab abnormalities, will be determined by using the NCI 
CTCAE version used at your institution, as a guideline, whenever possible.  The criteria are 
available online at http://ctep.cancer.gov/reporting/ctc.html. 
In the event that this is a multisite study, the sponsor-investigator is responsible to ensure that the 
SAE reports are sent to Millennium Pharmacovigilance (or designee) from all sites participating 
in the study.  Sub-investigators must report all SAEs to the sponsor-investigator so that the 
sponsor-investigator can meet his/her foregoing reporting obligations to the required regulatory 
agencies and to Millennium Pharmacovigilance, unless otherwise agreed between the sponsor-
investigator and sub-investigator(s).   
Relationship to ixazomib for each SAE will be determined by the investigator or sub-investigator 
by responding yes or no to the question: Is there a reasonable possibility that the AE is associated 
with the ixazomib? 


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Sponsor-investigator must also provide Millennium Pharmacovigilance with a copy of all 
communications with applicable regulatory authorities related to ixazomib as soon as possible 
but no later than 4 calendar days of such communication. 
 
 
SAE and Pregnancy Reporting Contact Information 
 
US & Canada 
Fax Number: 1-800-963-6290 
Email: TakedaOncoCases@cognizant.com 
 
Rest of World 
Fax #: 1 202 315 3560 
E-mail: TakedaOncoCases@cognizant.comSuggested Reporting Form:   
• 
SAE Report Form (provided by Millennium) 
• 
US FDA MedWatch 3500A:  
http://www.fda.gov/Safety/MedWatch/HowToReport/DownloadForms/default.htm 
• 
Any other form deemed appropriate by the sponsor-investigator 
 
8.4 Procedures for Reporting Drug Exposure During Pregnancy and Birth Events 
If a woman becomes pregnant or suspects that she is pregnant while participating in this study or 
within 90 days after the last dose of ixazomib, she must inform the investigator immediately and 
permanently discontinue ixazomib .  The sponsor-investigator must immediately fax a completed 
Pregnancy Form to the Millennium Department of Pharmacovigilance or designee (see Section 
8.2).  The pregnancy must be followed for the final pregnancy outcome. 
If a female partner of a male patient becomes pregnant during the male patient’s participation in 
this study, the sponsor-investigator must also immediately fax a completed Pregnancy Form to 
the Millennium Department of Pharmacovigilance or designee (see Section 8.2).  Every effort 
should be made to follow the pregnancy for the final pregnancy outcome. 
Suggested Pregnancy Reporting Form: 
• Pregnancy Report Form (provided by Millennium)   


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9 ADMINISTRATIVE REQUIREMENTS  
9.1 
Product Complaints 
A product complaint is a verbal, written, or electronic expression that implies dissatisfaction 
regarding the identity, strength, purity, quality, or stability of a drug product.  Individuals who 
identify a potential product complaint situation should immediately contact Millennium (see 
below) and report the event.  Whenever possible, the associated product should be maintained in 
accordance with the label instructions pending further guidance from a Millennium Quality 
representative. 
For Product Complaints, 
• Phone: 1-844-N1-POINT (1-844-617-6468) 
• E-mail: GlobalOncologyMedinfo@takeda.com 
• FAX: 1-800-881-6092 
• Hours: Mon-Fri, 9 a.m. – 7 p.m. ET (US) 
 
Product complaints in and of themselves are not AEs.  If a product complaint results in an SAE, 
an SAE form should be completed and sent to Millennium Pharmacovigilance 
 
9.2 
Unmasking/Unblinding Procedures 
 
Not applicable as this is an open-label study. 
9.3 
Stopping Rules 
If any of the following occur: 
• Death of any subject 
• Anaphylactic reaction in any subject 
• A life-threatening adverse event in any subject 
• Any Grade 3 AE (except if there is clear evidence that it is not study drug related – i.e. a 
plausible alternative reason).  
• Any Grade 4 AE 
• A decrease in CD4 count below 350 cells/mm3 
• Confirmed virologic rebound (HIV-1 RNA >500 copies/ml  confirmed by a second assay 
done within 14 days of the initial one).  If the HIV-1 viral load is >20 copies/mL and 
<500 copies/mL, the HIV-1 viral load will be rechecked every 2 weeks until it becomes 
<20 copies/mL or >500 copies/mL. 
 
An independent Data Safety and Monitoring Committee will review the case.  The committee 
and principal investigators will determine whether there is an association between the event and 
the ixazomib and make a recommendation accordingly as to whether the study should proceed or 
if a modification in trial design is required.  If this should occur, the principal investigators will 
report this action to the IRB and the FDA within the timelines noted in Sections 8.3.1 and 8.3.2. 
 


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9.4 
Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study at all study 
sites.  This safety monitoring will include careful assessment and appropriate reporting of 
adverse events as noted above, as well as the construction and implementation of a site data and 
safety-monitoring plan (see section 10  “Study Monitoring, Auditing, and Inspecting”).  Medical 
monitoring will include a regular assessment of the number and type of serious adverse events. 
9.4.1 Internal Data and Safety Monitoring Board 
 
An Internal Data and Safety Monitoring Board (DSMB) will be established based on the FDA 
Guidance on Establishment and Operation of Clinical Trial Data Monitoring Committees, 
(http://www.fda.gov/RegulatoryInformation/Guidances/ucm127069.htm).  The DSMB will have 
a charter describing its function, standard operating procedures, responsibilities, and an analysis 
plan for pre-planned safety analyses.  The DSMB will consist of an HIV physician (Zelalem 
Temesgen, Division of Infectious Diseases, Rochester MN), a clinician with expertise in clinical 
use of ixazomib (Vincent Rajkumar, Division of Hematology, Rochester MN), and a general 
infectious disease physician (Rana Chakraborty, Division of Infectious Diseases, Rochester 
MN).  The DSMB will review the clinical and laboratory safety data after each group of patients 
have completed the first month of therapy and a decision will be made as to whether to proceed 
with enrolling the subsequent group.   Subsequent review of the safety data will be done on a 
regular basis, at a minimum every quarter. 
10 Data Handling and Record Keeping 
10.1 Confidentiality 
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 (long term survival status that 
the subject is alive) at the end of their scheduled study period. 
10.2 Source Documents 
Source data is 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, 


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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. 
 
10.3 Data Management 
 
The investigator is responsible to ensure the accuracy, completeness, legibility, and 
timeliness of the data reported.  All source documents should be completed in a neat, legible 
manner to ensure accurate interpretation of data.  Black ink is recommended to ensure clarity of 
reproduced copies.  When making changes or corrections, cross out the original entry with a 
single line, and initial and date the change.  DO NOT ERASE, OVERWRITE, OR USE 
CORRECTION FLUID OR TAPE ON THE ORIGINAL.  Data reported in the eCRF derived 
from source documents should be consistent with the source documents or the discrepancies 
should be explained.   
 
10.4 Data Processing 
 
Qualified study staff at each site will perform primary data collection from source-
document reviews.  This study will use eCRFs developed through a validated, Medidata RAVE, 
the information technology endorsed by Mayo Clinic’s Clinical Trial Management System 
(CTMS).   
 
10.5 Records Retention 
 
The sponsor-investigator will maintain records and essential documents related to the 
conduct of the study.  These will include subject case histories and regulatory documents.  
Subject-specific data and Case Report Forms will receive a study-specific unique code for each 
subject.  These materials, and the subject identification code list, will be stored in the principal 
investigator’s office in a locked cabinet so as to protect the subjects’ confidentiality.  Subject 
names or other directly identifiable information will not appear on any reports, publications, or 
other disclosures of clinical study outcomes. 
 
The sponsor-investigator will retain the specified records and reports for: 
1. Up to 2 years after the marketing application is approved for the drug; or, if a marketing 
application is not submitted or approved for the drug, until 2 years after shipment and 
delivery of the drug for investigational use is discontinued and the FDA has been so 
notified. OR 
2. As outlined in the Mayo Clinic Research Policy Manual –“Access to and Retention of 
Research Data Policy” http://mayocontent.mayo.edu/research-policy/MSS_669717, 
whichever is longer. 
 


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11 Study Monitoring, Auditing, and Inspecting 
11.1 Study Monitoring Plan 
 
This study will be monitored on a routine basis during the conduct of the trial.  The Mayo 
Clinic Office of Research Regulatory Support will provide clinical monitoring for the trial as a 
service for the sponsor-investigator.  Clinical trial monitoring requires review of the study data 
generated throughout the duration of the study to ensure the validity and integrity of the data 
along with the protection of human research subjects.  This will assist sponsor-investigators in 
complying with Food and Drug Administration regulations. 
 
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 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, and government regulatory agencies, 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 compliance offices. 
12 Ethical Considerations 
This study is to be conducted according to United States government regulations and 
Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted local 
Institutional Review Board (IRB), in agreement with local legal prescriptions, for formal 
approval of the study.  The decision of the IRB concerning the conduct of the study will be made 
in writing to the sponsor-investigator 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 Approved IRB 
consent form, must be obtained before that subject undergoes any study procedure.  The consent 
form must be signed by the subject or the subject’s legally authorized representative, and the 
individual obtaining the informed consent. 
 


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13 Study Finances 
13.1 Funding Source 
 
Funding for portions of this study and ixazomib will be provided from Takeda. This 
funding will be used for study personnel costs, patient care costs including leukapheresis, and 
use of the Clinical Research and Trials Unit, study drug storage and administration, and 
measurements of secondary outcomes (CD4 T cells counts, HIV RNA viral loads, HIV proviral 
DNA, QVOA, safety and monitoring labs). 
13.2 Conflict of Interest 
 
Any study team member who has a conflict of interest with this study (patent ownership, 
royalties, or financial gain greater than the minimum allowable by their institution, etc.) must 
have the conflict reviewed by a properly constituted Conflict of Interest Committee with a 
Committee-sanctioned conflict management plan that has been reviewed and approved by the 
study sponsor-investigator prior to participation in this study. 
13.3 Subject Stipends or Payments 
 
Participants will be remunerated for participation based on the following schedule: 
• Leukapheresis $150 each, for a possible total of $300. 
• Research Blood draws $25 each, for a possible total of $225. 
• $50 for each study visit (not leukapheresis) 
• Total potential compensation $1,175. 
• Patients referred from HCMC will be reimbursed for travel costs. 
• Participants will also receive parking passes for each study visit. 
14 Publication Plan 
 
The principal investigators hold the primary responsibility for publication of the results of 
the 
study. 
 
This 
study 
will 
be 
registered 
with 
ClinicalTrials.gov 
(https://register.clinicaltrials.gov/) prior to subject recruitment and enrollment.  In addition, 
results of the study will be posted to ClinicalTrials.gov within 12 months of final data collection 
for the primary outcome.   
 
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16 Appendix I 
 
Peripheral Neuropathy Scale 
Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0 
Published: May 28, 2009 (v4.03: June 14, 2010) 
U.S.DEPARTMENT OF HEALTH AND HUMAN SERVICES 
National Institutes of Health 
National Cancer Institute 
 
Adverse 
Event 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Grade 5 
Peripheral 
neuropathy 
Asymptomatic; 
clinical or 
diagnostic 
observations 
only; 
intervention 
not indicated 
Moderate 
symptoms; 
limiting 
instrumental 
ADL 
Severe 
symptoms; 
limiting self 
care ADL; 
assistive 
device 
indicated 
Life-
threatening 
consequences; 
urgent 
intervention 
indicated 
Death 
Questions 
Sample answers 
for each toxicity 
grade 
 
 
 
 
Do you have 
problems tying 
your 
shoe 
laces, 
buttoning your 
shirts, 
fastening 
buckles 
or 
pulling 
up 
zippers? 
“No, I might 
feel some 
tingling in my 
hands, but I 
have no 
problems tying 
laces, buttoning 
shirts, fastening 
buckles or 
pulling up 
zippers” 
“It is a bit 
harder than 
before, but I 
can still tie 
laces, button 
shirts, fasten 
buckles or pull 
up zippers” 
“I have severe 
difficulties 
tying shoe 
laces, 
buttoning 
shirts, 
fastening 
buckles or 
pulling up 
zippers” or “I 
cannot tie 
laces, button 
shirts, fasten 
buckles or pull 
up zippers 
anymore” 
“I haven’t been 
able to tie 
laces, button 
shirts, fasten 
buckles or pull 
up zippers for 
weeks” 
 
Do you have 
problems 
writing? 
“No, I might 
feel some 
tingling in my 
hands, but I 
have no 
problems 
writing” 
“It is a bit 
harder than 
before, but I 
can still write” 
“I have severe 
difficulties 
writing” or “I 
cannot write 
anymore” 
“I haven’t been 
able to write 
for weeks” 
 
Do you have 
problems 
“No, I might 
feel some 
“It is a bit 
harder than 
“I have severe 
difficulties 
“I haven’t been 
able to put on 
 


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putting on your 
jewelry or your 
watch? 
tingling in my 
hands, but I 
have no 
problems 
putting on my 
jewelry or my 
watch” 
before, but I 
can still put on 
my jewelry or 
my watch” 
putting on my 
jewelry or my 
watch” or “I 
cannot put on 
my jewelry or 
my watch 
anymore” 
my jewelry or 
my watch for 
weeks” 
Do your have 
problems 
walking? 
“No, I might 
feel some 
tingling in my 
feet, but I have 
no problems 
walking” 
“It is a bit 
harder than 
before, but I 
can still walk” 
“I have severe 
difficulties 
walking” or “I 
cannot walk 
anymore” 
“I haven’t been 
able to walk for 
weeks 
 
 
 
 


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Appendix II 
 
European Organization for Research and Treatment of Cancer QLQ-CIPN20 
Questionnaire. 
Lavoie Smith EM, Barton DL, Qin R, Steen PD, Aaronson NK, Loprinzi CL. Qual Life Res. 
2013 Dec;22(10):2787-99. doi: 10.1007/s11136-013-0379-8. Epub 2013 Mar 30. 
 
 
1.Do you have tingling fingers or hands? 
 
 
2.Do you have tingling toes or feet? 
 
 
3.Do you have numbness in your fingers or hands? 
 
 
4.Do you have numbness in your toes or feet? 
 
 
5.Do you have shooting or burning pain in your fingers or hands? 
 
 
6.Do you have shooting or burning pain in your toes or feet? 
 
 
7.Do you have cramps in your hands? 
 
 
8.Do you have cramps in your feet? 
 
 
9.Do you have problems standing or walking because of difficulty feeling the ground under your 
feet? 
 
 
10.Do you have difficulty distinguishing between hot and cold water? 
 
 
11.Do you have a problem holding a pen, which made writing difficult? 
 
 
12.Do you have difficulty manipulating small objects with your fingers (for example, fastening 
small buttons)? 
 
 


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13.Do you have difficulty opening a jar or bottle because of weakness in your hands? 
 
 
14.Do you have difficulty walking because your feet dropped downwards? 
 
 
15.Do you have difficulty climbing stairs or getting up out of a chair because of weakness in 
your legs? 
 
 
16.Are you dizzy when standing up from a sitting or lying position? 
 
 
17.Do you have blurred vision? 
 
 
18.Do you have difficulty hearing? 
 
Please answer the following question only if you drive a car 
 
 
19.Do you have difficulty using the pedals? 
 
Please answer the following question only if you are a man 
 
 
20.Do you have difficulty getting or maintaining an erection?