Clinical trial NCT02700841
Conditions: Plasma Cell Myeloma
Sponsor: University of Nebraska
Start date: 2020-01-09; Completion date: 2022-12-21
Enrollment: 8 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This pilot randomized Phase II trial (10 subjects per arm) will compare immune reconstitution following transplantation of an autologous mobilized graft product to reconstitution following transplantation of a mobilized graft product followed by an autologous lymphocyte infusion collected prior to G-CSF mobilization. All subjects will receive tetanus vaccines pre and post-transplant. The primary end point will be tetanus vaccine immune responses post-transplant.

Detailed description:
PRIMARY OBJECTIVES:

1. To compare the cellular and humoral vaccine response post-transplant between the two arms by performing Elisa, and T-cell enzyme-linked immunospot (ELISPOT) assays
2. To determine the feasibility and safety of this approach

SECONDARY OBJECTIVES:

1. To compare post-transplant recovery of innate and adaptive immune cells (CD8, CD4, CD19, NK, γδ T-cells), in addition to T-cell phenotype markers between the two arms.
2. To compare post-transplant recovery of T-regs and MDSCs between the two arms.
3. To compare progression free survival (PFS) at 2 years post-transplant

Arms: arm 1: ARM I: Patients receive 3 doses of tetanus before transplant and on days 15, and 60 post transplant. arm 2: Patients receive 3 doses of tetanus as in Arm I. Patients receive high-dose melphalan IV on day -2 and undergo AHSCT on day 0.
Interventions: intervention 1: Melphalan intervention 2: Peripheral Blood Stem Cell Transplantation--CD34 HSCT intervention 3: Peripheral Blood Stem Cell Transplantation--AHSCT intervention 4: T Cell-Depleted Hematopoietic Stem Cell Transplantation intervention 5: Tetanus Toxoid Vaccine
Intervention descriptions: intervention 1: Given IV intervention 2: Undergo autologous CD34 HSCT intervention 3: Undergo AHSCT intervention 4: Undergo autologous CD34 HSCT intervention 5: Given IM

Protocol documents:
Prot_SAP_000.pdf: 
IRB # 669-19 
Protocol Version 1.5,  
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Title: 
A Pilot Study of Enhancing Anti-Tetanus Vaccine 
Response After Autologous Stem Cell 
Transplantation 
  
Principal Investigator:  Christopher D’Angelo, MD 
 
986840 Nebraska Medical Center 
 
Omaha, Ne 68198-6840 
 
 
  Co-Principal Investigator: Muhamed Baljevic, MD 
 
986840 Nebraska Medical Center 
Omaha, Ne 68198-6840 
 
           Co-Principal Investigator: Zaid Al-Kadhimi, MD 
 
986840 Nebraska Medical Center  
Omaha, Ne 68198-6840 
 
 
UNMC Secondary Investigators: 
 
Sarah A. Holstein MD, PhD 
 Julie M. Vose, MD 
 Matthew A. Lunning, DO 
 James Talmadge, PhD 
 Alison G. Freifeld, MD, FIDSA 
  
 BMT Immunotherapy  
University of Nebraska Medical Center  
  
Biostatistician: Lynette Smith, PhD 
  
  
Study Drugs: Anti-Tetanus Vaccine 
Study Device: CliniMacs CD34 beads 
IND: 016450 
Clinical Trials.gov # NCT02700841 
NCI-CTRP #: NCI-2015-00743 
  
Protocol Version: Version 1.2, Dated 11-Oct-2019 
Version 1.3, dated 27-Jan-2020 


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Version 1.4, dated 05-Sep-2020 
Version 1.5, dated 10-May-2021 
 
 
 
Abstract 
 
3 
Schema 
 
4 
Section 1.0 
Objectives 
5 
Section 2.0 
Introduction, including Background Data and Future Objectives 
7 
Section 3.0 
Eligibility Criteria 
14 
Section 4.0 
Randomization / Registration Procedures 
14 
Section 5.0 
Treatment Plan or Research Design 
16 
Section 6.0 
Measurement of Effect 
20 
Section 7.0 
Study Parameters 
21 
Section 8.0 
Drug Formulation and Procurement 
24 
Section 9.0 
Toxicity and Adverse Event Reporting Guidelines 
25 
Section 10.0 
Biostatistical Considerations including Stopping Criteria 
29 
Section 11.0 
Records to be Kept 
30 
Section 12.0 
Patient Consent Form Statement 
31 
Section 13.0 
References 
31 
Section 14.0 
Data Collection Forms 
37 
Appendix A 
Eligibility Checklist 
38 
Appendix B 
Correlative Lab Specimen Collection schedule  
40 
 
 
 


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Abstract:  
Because curative therapy for multiple myeloma is not yet available, the treatment goal is 
to produce lengthy asymptomatic disease remissions. High dose chemotherapy with 
autologous hematopoietic stem cell transplant (AHSCT) provides optimal remission 
outcomes, and is the current standard of care for transplant eligible patients. However, 
some patients experience early relapse. One possible explanation for early relapse is poor 
immune recovery after high dose chemotherapy given before AHSCT. Harvesting an 
autologous transplant graft product from circulating blood is facilitated by administration 
of granulocyte colony stimulating factor (G-CSF) to mobilize large numbers of 
hematopoietic stem cells into the circulation. The cells are collected from the blood with 
apheresis and stored until the transplant. Our preliminary studies show that immune 
suppressor cells are also mobilized and collected in the graft product. Depletion of these 
suppressor cells in the graft before transplant provided enhanced T-cell reactivity to 
allogeneic antigens and lymphocyte homeostatic expansion. The hypothesis is that 
depleting majority of immune suppressive cells from G-PBSC graft in vitro in the setting of 
AHSCT will tip the balance towards accelerated lymphocyte expansion in-vivo, which will 
be reflected in high ALC numbers on day 15 (>500/μl). The goal is to achieve this depletion 
via selection and infusion of CD34+ cells, thereby eliminating MDSC’s and T-regs which 
have an immunosuppressing effect on immune reconstitution.  This pilot randomized 
Phase II trial (10 subjects per arm) will compare immune reconstitution following 
transplantation of an autologous mobilized graft product to reconstitution following 
transplantation of a mobilized graft product followed by an autologous lymphocyte infusion 
collected prior to G-CSF mobilization. All subjects will receive tetanus vaccines pre and 
post-transplant. The primary end point will be tetanus vaccine immune responses post-
transplant.  
 
 
 


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Schema:  
 
 
 
As shown in the schema above: 
1- Subjects in all groups will receive tetanus vaccine 7-10 days prior to mobilization. 
  
2- Subjects in group 1 will have mononuclear cells collected and frozen 7-10 days after tetanus vaccination pre-
transplant. These subjects will then receive a CD34+ selected graft on day 0 and autologous donor lymphocyte infusion 
(DLI) on day +7.       
 
3- Subjects in group 2 will receive a standard G-CSF mobilized graft on day 0 without any DLI post-transplant. 
 
4- Subjects in all groups will receive G-CSF as subcutaneous injections daily (10 µg/kg) x 5 days starting on day -11 
(following the DLI pheresis in the experimental group). Subjects will receive one dose of plerixafor, a stem cell mobilizer, 
followed by pheresis on the next day to collect G-PBSC graft, with the goal of > 6.0 × 106 CD34+ cells/kg.  
 
5- On day of G-PBSC pheresis, the graft in group 2 will be analyzed for CD34, lymphocyte content, and T-regs and 
MDSCs (cell differential and flow cytometry). Then it will be processed and frozen as per local institutional standards.  
 
6- The stem cell product will start to be washed while waiting for the CD34+ content to be known. CD34+ stem cells 
will then be selected out of the G-PBSC mobilized graft the same day using clinical grade CD34+ magnetic beads and 
Clinimacs device from Miltenyi as per manufacturer’s instructions and as described below. The CD34 content will be 
analyzed before freezing in liquid nitrogen. 
 
7- The subjects in all groups will receive high dose chemotherapy Melphalan at 200 mg/m2 on day -1. 
 


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8- On day 0, subjects in group 1 will receive the thawed CD34+ stem cell graft, while subjects in the group 2 will receive 
the standard thawed G-PBSC graft. Transplanted CD34+ stem cell dose both groups will be in the range of 2-10 X 
106/Kg body weight.  
 
9- On day +7 post-transplant, the stored autologous donor lymphocyte product in group 1 will be thawed and infused 
back to the subjects. 
 
10- No planned G-CSF post-transplant (unless medically deemed necessary due to slow engraftment by day 15)  
 
11- Subjects will be followed closely in the hospital until engraftment and resolution of all transplant related toxicities. 
 
12- Subjects will be closely evaluated for any trial related adverse events, specifically: delayed engraftment, auto-
immune side effects (autologous graft versus host disease), and infections. 
 
13- At day +15(+/-3) and day +60 (+/-3) post-transplant, all subjects will receive tetanus vaccines.  
 
14- At days + 30(+14), +70(+14), +100(+/-10), and 180(+/-10) post-transplant subjects will undergo immune evaluation 
for tetanus vaccine responses.   
 
 
 
 
 
 
 
Section 1.0  Objectives: 
Hypotheses 
1. The abundance of immune suppressive cells in G-PBSC grafts results in poor immune 
reconstitution post AHSCT, leading to higher risk for infection and myeloma relapse. 
2. 
Depletion of immune suppressor cells from G-PBSC graft will allow a robust in vivo 
expansion of the adoptively transferred lymphocytes as well as a robust recovery of 
antigen specific tetanus immune response post-transplant compared with the control 
group. 
3. 
It is safe and feasible to deplete all immune suppressive cells from G-PBSC grafts 
by positively selecting CD34+ cells from G-PBSC grafts and combining that with an 
autologous donor lymphocyte infusion product. 
  
1.1  
Primary:  
 
1- To compare the cellular and humoral vaccine response post-transplant between the 
two arms by performing Elisa, and T-cell enzyme-linked immunospot (ELISPOT) 
assays 
 
 
 
 
 
 
 
 
 
 
 
2- To determine the feasibility and safety of this approach 
 
 
 
1.2 
Secondary: 
1- To compare post-transplant recovery of innate and adaptive immune cells (CD8, CD4, 
CD19, NK, γδ T-cells), in addition to T-cell phenotype markers between the two arms. 
 
2- To compare post-transplant recovery of T-regs and MDSCs between the two arms. 
 
3- To compare progression free survival (PFS) at 2 years post-transplant  
 
1.3 
Exploratory objective: 


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To compare the depth of multiple myeloma (MM) response by international myeloma 
working group (IMWG) criteria, including analyzing minimal residual disease (MRD) 
status at 3 and 6 months post-transplant for those subjects who are close to achieving 
CR (defined as monoclonal serum protein of ≤ 0.1 g/dL, with negative urine protein 
electrophoresis) between the two arms.  
 
 
 
 


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Section 2.0  Introduction:  
 
2.1 Myeloma relapse is the main challenge post autologous hematopoietic Stem cell transplant 
(AHSCT) 
 
Multiple Myeloma (MM) is a hematologic malignancy with a rising incidence in the US (about 
30,000 cases newly diagnosed per year and 12,000 related myeloma deaths per year). 
Unfortunately, myeloma is considered incurable despite widening available standard therapies.  
New and innovative approaches are needed to improve the outcomes of this growing disease1. 
AHSCT has significantly improved disease free and overall survival (OS) for patients with MM.  
However, relapses continue to limit long term survival of these patients.2 The efficacy of AHSCT 
as a treatment modality has relied mainly on the hope that high dose chemotherapy will eradicate 
the resistant tumor clones that survived standard chemotherapy. This paradigm has not changed 
in more than 30 years. Unfortunately, the same intensive preparative transplant regimen that 
overcomes resistance to standard dose chemotherapy, depletes various immune cells as well as 
compromises their function, increasing the risk for serious infections from various agents as well 
as potentially increasing the risk of myeloma relapse post-transplant.3  Advances in modifying the 
chemotherapy preparative regimen and supportive care have resulted in less transplant related 
morbidity and mortality, but no improvement in relapse rates.  Attempts at improving AHSCT 
efficacy by tumor cell purging, CD34 selection or switching the preparative regimen have not been 
successful.4,5  Therefore, new interventions are needed to improve autologous transplant 
outcomes.  
 
 
2.2 High dose chemotherapy and autologous peripheral blood stem cell transplant can be a 
favorable vaccine platform for multiple myeloma. 
The state of minimal residual disease and abundance of homeostatic cytokines (IL-7 & IL-15)  can 
make the early phase post AHSCT a favorable platform for vaccine response.6 The recovery 
phase of lymphopenia enhances tumor vaccine responses in multiple animal models. 7-14 Mitchell 
et al. has demonstrated a favorable immune response to PP65 pulsed dendritic cell vaccine given 
during chemo induced lymphopenia in patients with glioblastoma.15  Sampson  et al. showed that 
greater chemotherapy-induced lymphopenia enhances tumor-specific immune responses that 
eliminate EGFRvIII-expressing tumor cells in patients with glioblastoma.15  However, studies that 
tested early vaccine responses post AHSCT showed poor immune responses, which reflects the 
immune suppression state in the first year post AHSCT.17,18      
 
2.3 Immune reconstitution after AHSCT.  
Today’s AHSCTs are almost all done with G-CSF mobilized PBSC (G-PBSC), rather than bone 
marrow grafts based on faster engraftment, decreased risk of infections and days of 
hospitalization.19  Early hematopoietic reconstitution of white blood cells, hemoglobin and platelets 
occur within the first two weeks post AHSCT. However, there is a substantial delay in 
reconstitution of the non-granulocytic immune components.  B-lymphocytes (B-cell) recover to 
normal numbers within 2-4 months.20  Natural killer cells (CD56+3- or CD16+3- NK) reached 
normal values within one month post-transplant.20  Total T-lymphocyte (T-cell) CD3+ 
reconstitution occurs within 2-4 months post AHSCT. All patients showed a low CD4/CD8 ratio 
during the first year post transplant, due both to low numbers of CD4+ cells and elevated numbers 
of CD8+ cells. The low number of CD4+ cells was due to a persistently low level of naive 
CD4+CD45RA+ cells. While a high proportion of the CD8+ cells displayed a phenotype 
compatible with activated T-cells (CD8+DR+) up to 10 months after AHSCT. This seems to be 
secondary to the intrinsic difference between CD4 versus CD8 regeneration pathways post 
intensive chemotherapy. CD4 regeneration post intensive chemotherapy is more dependent on 
thymic function, which starts to regress even in young adults.21-23 Therefore CD4 reconstitution 
post AHSCT is mainly dependent on expansion of mature T-cells in the graft.24 A clear global 


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suppression of T-cell proliferative, and cytokine release in response to mitogenic agents in vitro 
has been observed in the first 6-12 months post AHSCT.23-26  
 
2.4 Antigen specific immune reconstitution 
Riemer et al. evaluated the antigen specific humoral immune response post AHSCT in 38 
patients.  He found that IgG antibody titers against measles, mumps, rubella, and polio were 
present in almost all patients pre-transplant and during 12 months post-transplant, indicating 
persistent humoral immunity.27  Svane et. al. evaluated the antigen specific cellular immune 
responses post AHSCT in breast cancer patients.28    Peripheral blood was collected from five 
breast cancer patients at serial time points in connection with chemotherapy treatment and in a 
follow-up period of 1 year.  The frequencies of CD8 and CD4 T-cells responsive to 
cytomegalovirus (CMV), varicella zoster virus (VZV), and tetanus in antigen-activated whole blood 
were determined by flow cytometric analysis of CD69, (intra-cellular) TNFα, and IFNγ expression.  
Mononuclear cells were labeled with PKH26 dye and the CMV, VZV, and tetanus toxoid-specific 
proliferation of T-cell subpopulations was analyzed by flow cytometry.  In none of the patients did 
the treatment result in loss of overall T-cell reactivity for any of the antigens.  Prior to 
chemotherapy 5/5 patients possessed TNF expressing T-cells specific for CMV, 4/5 for VZV, and 
3/5 for tetanus.  One year after stem cell transplantation all patients possessed TNF expressing 
T-cells specific for CMV, VZV and tetanus.  The highest percentages of cytokine responding T-
cells were seen after stimulation with CMV antigen. In general, the lowest antigen specific 
immune reactivity (close to zero) was measured in G-CSF-mobilized blood at the time of 
leukapheresis.  Despite a continuously reduced CD4 to CD8 ratio after transplantation, recovery 
of antigen specific CD4 T-cells usually occurred prior to CD8 recovery and often to a higher level. 
This study demonstrated that natural as well as vaccine-induced specific immunity established 
prior to AHSCT can be regained after stem cell transplantation.  
 
2.5 The impact of post AHSCT immune suppression on risk of infections:   
The risk of viral infections is higher in the first year post AHSCT.  Varicella zoster virus VZV seems 
to be the main one, with a risk of 23-50%.29,30  The risk of VZV infections was higher when 
CD4<200/μl, and CD8<800/μl.  Patients who met both of these predictors had a VZV risk of about 
48% at one year.31  Other less common infections include CMV.  Wingard et. al. in 1988 reported 
a 45% rate of CMV infections in 165 patients post auto transplant (based on a rise in CMV 
antibody titer).  However, the rate of CMV pneumonia in that study was 2%.32 Kadakia et. al. 
reported on human herpes virus 6 (HHV6) viral infections in the first year post autologous 
transplant.  By using direct viral isolations, HHV6 DNA, and HHV6 antibody titer testing, they could 
detect active HHV6 infection in 10/11 autologous transplant patients.  Furthermore there was a 
55% association between HHV6 isolation and sinusitis (P=.002).33, 19  The risk of CMV, VZV, 
parainfluenza and bacterial infections were all increased in CD34 selected AHSCT compared to 
standard AHSCT, based on the excessive immune suppression post-transplant caused by T-cell 
depletion.34  
 
 
2.6 The impact of post AHSCT immune suppression on the risk of cancer relapse:   
There is no clear direct evidence to link the post AHSCT immune suppression, slow numerical 
and functional T-cell recovery to rates of relapse.  However, animal models and clinical data 
strongly suggest a link between immunosuppression and occurrence/progression of cancer. 
Furthermore, retrospective studies and one prospective study in AHSCT suggest that such link 
exists. 
   
Animal studies of gene-targeted and lymphocyte subset-depleted mice were used to establish the 
relative importance of NK and NK1.1+ T (natural killer T, NKT) cells in protecting against tumor 
initiation and metastasis. In these models, CD3+ NK cells were responsible for rejection and 


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protection from metastasis of chemically induced (methylcholanthrene MCA) tumor.35,36  Similarly 
Rag2-/- mice (no T-cells, and B cells) showed increased susceptibility to chemically MCA induced 
tumors.37 Studies of T-cell receptor deficient mice demonstrated a non-overlapping role of γδ T-
cells and αβ T-cells in blocking the formation and progression of chemically induced tumors.38,39  
These studies seem to suggest that γδ T-cells act to inhibit initial tumor formation, while αβ T-
cells directly inhibit tumor progression by using their cytotoxic mechanisms to kill tumor cells. 
 
Clinical studies have shown that the high-grade density of CD8+ T-cells in cancer cell nests 
correlated with prognosis. The presence of tumor infiltrating T-cells (TILs) was able to predict a 
better survival as an independent prognostic factor in various types of cancers including colon 
cancer,40,41 esophageal cancer,42 oral squamous cell carcinoma,43  breast cancer,44 ovarian 
cancer,45,31 and malignant melanoma.46Other studies have shown a similar positive correlation 
between NK cell infiltration and the survival for gastric cancer,47 colorectal cancer,48 and 
squamous cell lung cancer.49   
 
Increased relative risk for various types of cancers has been observed in immunosuppressed 
solid organ transplant recipients that have no apparent viral origin.  Information on 5692 Nordic 
recipients of renal transplants in 1964–82 was linked to the national cancer registries in 1964–86 
and to population registries.  Significant overall excess risks of two-to-five-fold were seen in both 
sexes for cancers of the colon, larynx, lung and bladder, and in men for cancers of the prostate 
and testis.  Notable high risks ranging from 10-30 fold above expectations, were associated with 
cancers of the lip, skin (non-melanoma), kidney, endocrine glands, non-Hodgkin’s lymphoma, and 
in women with cancers of the cervix and vulva–vagina.50  Additionally, there have been case 
reports of growth and flare up of epithelial cancers in chronic lymphocytic leukemia (CLL) patients 
after the use of nucleoside analogs fludarabine or 2-chlorodeoxy adenosine (2CdA).51,52  
Allogeneic bone marrow transplants have been associated with increased risk for squamous cell 
carcinoma of the buccal cavity and skin.53  
      
In AHSCT patients, the group from Mayo clinic has retrospectively evaluated the role of early 
lymphocyte count recovery post-transplant.  In multiple reports, they found that absolute 
lymphocyte count ALC of 500/μl on day 15 post AHSCT correlated with improved overall and 
disease free survival in patients with Hodgkin’s lymphoma, NHL, acute myeloid leukemia, multiple 
myeloma, amyloidosis, and breast cancer.54-58  Porrata et al reported one prospective study on 
50 NHL patients undergoing AHSCT, confirming the same above observation.59 
More recently peripheral blood absolute monocyte count (AMC) has been used as a surrogate 
marker of tumor microenvironment immune suppression.60 Porrata et al. showed a correlation 
between AMC excess at day 15 post-transplant with risk of relapse and low PFS.  He showed 
that day+ 15 ALC/AMC ratio> 1.0 correlated with better survival. Furthermore, he demonstrated 
that the recovery of ALC and AMC at day 15 post AHSCT correlated with the numbers of these 
cells in the autologous graft.   
 
The value of immune recovery was further highlighted in a recent report that looked at immune 
signatures associated with improved PFS and OS for myeloma patients treated with AHSCT.61 In 
this study, MM patients underwent comprehensive immune profiling (IP) in peripheral blood and 
had the marrow tested for MRD before and approximately 100 days after AHSCT. It was found 
that higher γδ T-cell counts post-AHSCT correlated with improved 2-year PFS and OS61. 
Furthermore, the higher CD4+ central memory (CM) cell counts post-AHSCT were associated 
with improved 2-year OS but not PFS. The higher γδ T-cell and CD4+ CM-cell count associations 
were primarily observed in MRD-negative patients post-AHSCT and in patients not receiving 
maintenance therapy.61  
 
 


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Collectively these studies suggest that high ALC and low AMC on day 15 post AHSCT are 
markers of accelerated immune recovery, and lower immune suppression, resulting in lower risk 
of relapse and better long-term survival. 
2.7 The autologous graft is enriched with immune suppressive cells:   
 
 
     
One of the immune suppressive cellular elements enriched in the G-PBSC grafts are regulatory 
T cells (T-reg).  These CD4+CD25+hi T-reg is a specialized subset of T-cells that act to suppress 
immune activation, and thereby maintain immune tolerance to self.  FOXP3 is thought to be a 
characteristic marker for T-regs (a master transcriptional regulator in the development and 
function of regulatory T-cells).62 Seddiki et al. showed that expression of CD4+CD25+CD127low 
phenotype gave an accurate estimation of T-reg cell numbers and correlated well with FOXP3 
expression. 63,47  The bone marrow is an important reservoir for functional CD4+CD25+hi T-regs.  
G-CSF mobilizes T regs from the bone marrow to the peripheral blood through down regulation 
of bone marrow CXCL12, a ligand for CXCR4 receptor expressed on the surface of T-reg.64     
Ruttella et al. showed that in vivo exposure to G-CSF induced suppressor T-cells with a peculiar 
cytokine profile (IL-10 high, TGF-β1 high, IL-2 low, IL-4 low), and suppression of antigen-driven 
proliferation.65  A clinical trial evaluating the role of AHSCT in juvenile idiopathic arthritis 
demonstrated an accelerated recovery of CD4+CD25+hi T-regs in peripheral blood post- 
transplant.66  This phenomenon correlated with the clinical quiescence of the autoimmune 
process.  In vitro depletion of these T-regs from peripheral blood samples post-transplant partially 
corrected the T-cell dysfunctional state.  Early after AHSCT, T-reg expansion occurs through 
homeostatic proliferation from the G-PBSC graft rather than through thymic differentiation. 65  
Another immune suppressive population abundant in the G-PBSC grafts is immature monocytes.  
Fraser et al. showed an increased proportion of CD14+ monocytes in G-PBSC grafts. Majority of 
these monocytes were immature, expressed low CD14, had surface bound IL-10, and induced 
significant T-cells suppression in vitro.67  Activation of STAT3 pathway and expression of FAS 
ligand are possible mechanisms of their immunosuppressive phenotype.68,69 G-CSF mobilized 
immature monocytes are recruited to the tumor microenvironment, where their phenotype is 
further modified to become myeloid suppressor cells (MDSC).70  These MDSCs are usually 
CD33+, CD11b+,HLA-DR low and CD15+/-.71  
 
As mentioned above Saven et al. noticed the lowest level of antigen specific T-cell immune 
responses in G-PBSC grafts.28 The abundance of immune suppressor cells in G-PBSC grafts 
raises concern about their in vivo effect on the immune recovery post-transplant.  
   
2.8 The effect of T-regs on T-cell homeostatic expansion in vivo 
In an autoimmune disease animal model, Shen et al. demonstrated that T-regs inhibited 
homeostatic proliferation of adoptively transferred T-cells, reduced their survival, and reduced 
their functional differentiation into cytokine producing effector/memory T-cells.  Eventually these 
T-regs prevented the development of clinical autoimmune disease.72  A similar observation of 
enhanced T-reg recovery was made by Alexander et al. in patients with refractory systemic lupus 
erythematosus (SLE) who underwent AHSCT.73 Collectively the above data offer a possible 
explanation for the lack of robust T-cell homeostatic expansion post AHSCT.  Therefore, in the 
absence of immune suppressor cells (no G-CSF), chemotherapy induced lymphopenia can 
stimulate significant in vivo expansion and activation of donor derived lymphocyte infusions both 
in animal models and human clinical trials. 74,75   
 
2.9 In-vivo depletion/modulation of immune suppressive cells improves anti-tumor immune 
response: 


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Kline et al. demonstrated that depletion of T-regs enhanced in vivo expansion of adoptively 
transferred tumor specific T-cells, increased their cytokine secretion and their ability to eradicate 
pre-injected subcutaneous tumors in RAG2-/- (lymphocytes deficient) B16 melanoma animal 
model. To simulate a transplant model, similar results were observed when C57BL/6 (immune 
intact) mice irradiated and received adoptive transfer of total or T-reg depleted splenocytes.76  
Collectively the above data showed that T-reg depletion not only enhanced T-cell homeostatic 
expansion, but also improved their ability to eradicate tumors. Jennifer Ko et al. demonstrated 
that sunitinib dropped the proportion of MDSC and T regs in the peripheral blood of renal cancer 
patients, as well as improved T-cell immune function.71 The use of monoclonal antibodies (CD33 
or CD15) for in vitro purging of myeloid leukemic cells from PBSC grafts have been attempted 
before in clinical trials.77,78  However, significant delays in neutrophil and platelet engraftment post-
transplant have prevented further development of this approach.  
2.10 Feasibility of CD34+ selection for clinical applications   
The feasibility of CD34+ hematopoietic stem cell selection in AHSCT was demonstrated in the 
late 90s as a strategy of purging the graft from cancer cells.79 – 83  This approach was tested in a 
large randomized phase III MM trial and shown to be safe, however there was no improvement in 
PFS or OS.4 CD34+ stem selection using the immune-magnetic bead approach is now FDA 
approved allogeneic stem cell transplant in acute myeloid leukemia. Previously completed phase 
II trial demonstrated feasibility of engraftment with as low as 2.0 million x 106/Kg body weight 
CD34+ selected stem cell graft for patient with hematologic malignancy93.    
Therefore, combining a pre G-CSF peripheral blood mononuclear cells (PBMC) pheresis 
product (autologous DLI) with CD34+ selected stem cells may produce a graft with minimum 
immune suppresser cells, enhanced immune reconstitution and vaccine immune response post 
AHSCT. 
 
2.11  Preliminary data: 
2.11.1 - G-CSF mobilized grafts are enriched with myeloid derived suppressor cells 
MDSCs.   
 
A- We have evaluated the proportion of MDSC in G-PBSC graft samples and compared 
them with healthy donor’s peripheral blood.  As shown below in Figure 1 (first two columns on 
the left), the proportion of CD33+/HLA DR low, CD14+/HLA DR low, CD14 low/side scatter high 
population (immature monocytes), were all higher in the G-PBSC graft compared with healthy 
donor blood samples (six patient samples).  While all the CD33+/HLADR low cells were positive 
for CD11b, a proportion of these MDSCs were positive for CD15 (data not shown). We 
demonstrated the in vitro feasibility of depleting MDSCs using CD33/15 Miltenyi’s magnetic beads 
(third column from the left showed the depleted graft), while the fourth shows the positively 
selected population. 
 
 
Figure 1 


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2.11.2- Depletion of T-regs and or MDSC led to improved T-cell proliferation, in response 
to an allogeniec mixed lymphocyte reaction.   
We performed a standard one way mixed lymphocyte reaction (MLR) with responder G-PBSC 
graft cells (1 x 105 cells) co-cultured with a similar number of irradiated (2500 rads) allogeneic 
stimulator PBMC for 6 days in a round bottomed 96-well microtiter plates in 37o humidified 5% 
CO2 incubator. The cells then were pulsed with 2 μCi of 3H-thymidine for 16 hours, then harvested 
and counted in a beta scintillation counter. G-PBSC graft samples (six patients) were divided to 
5 groups: (1) un-manipulated G-PBSC graft sample, (2) CD25 depleted, (3) CD33/CD15 depleted, 
(4) CD25 enriched, (5) CD33/CD15 enriched.  Controls included: (1) patients’ own pre G-CSF 
PBMC sample MLR (2) a healthy donor MLR, and (3) G-PBSC effecter cells only with no targets.  
We observed an increase in proliferation of CD25 or CD33/15 depleted G-PBSC graft sample 
over un-manipulated G-PBSC graft in 6 patients, while CD25 or CD33/15 enriched groups in all 


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the patients had very little reaction as shown in the Figure 2 below. Each bar shows the average 
counts per minute reflecting 3H-thymidine uptake for patients in that group.  
   
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
2.11.3- Summary data on two patients treated on the experimental arm of this protocol at 
Emory University.   
Two high risk MM patients were treated on the experimental arm of this protocol at Emory 
University. They received all pre- and post-transplant vaccines in addition to graft 
manipulation. Both patients started to show rise in WBC on days (15 and 16) with ANC 
engraftment on days (17 & 18). Platelets started to rise on day 2 13 & 14 with platelet 
engraftment on days 15 & 16.  Both patients experienced transient neutropenic fever with 
no positive cultures. Both patients were discharged from the hospital and had no further 
complications post-transplant. Both patients continue to be in remission >12 months post-
transplant. Day 15 post-transplant ALC/AMC ratio was >1 in both patients.     
 
 
 
  H3Thymidin uptake assay of graft PBMC MLR
Unmanipulated
Pre G-CSF
CD25-
CD33-/15-
CD25+
CD33+/15+
Healty allogeniec control
Effectors alone
0
5000
10000
15000
Avarege CPM
Figure 2 


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Section 3.0  Eligibility Criteria:  
 
3.1  Inclusion Criteria 
1. Age ≥ 19 years to 70 years old at time of study entry (consent)  
2. Diagnosis of Multiple Myeloma (MM) as per updated International Myeloma Working 
Group (IMWG) criteria84.  
3. Must have received bortezomib, lenalidomide and dexamethasone (VRd) as a form of 
induction therapy pre-AHSCT (use of cyclophosphamide, bortezomib and 
dexamethasone may be allowed for up to 2 weekly doses before initiation of VRd 
induction, if necessary clinically for cytoreduction)  
4. Able to understand and sign a consent form. 
5. Creatinine clearance equal or > 60 ml/min (calculated) 
6. Ejection fraction equal or > 50% before admission for transplant as per institutional 
standards. Patients with coronary heart disease (recent myocardial infarctions, angina, 
cardiac stent, or bypass surgery in the last 6 months or arrhythmia) need to be cleared by 
cardiology as per institutional BMT standards. 
7. Total bilirubin ≤2.5 times the upper limit of the institutional normal values, and 
total AST (SGOT) and ALT (SGPT) ≤2.5 times the upper limit of the institutional normal 
values.   
8. FVC, FEV1 or DLCO equal or >50% predicted before admission for transplant as per 
institutional standards. Patients on home oxygen are not allowed on the protocol. 
9. No more than 6 months of pre-transplant MM chemotherapy is allowed (from the date of 
the start of the induction therapy).  
10. KPS ≥ 70%or ECOG 0-2. 
11. Must be eligible to receive Melphalan dose of 200mg/m2 
12. A female of child-bearing potential, must have two negative urine pregnancy test results 
at time of eligibility testing and prior to GCS-F pre-transplant as a way of ensuring safe 
transplant planning.   
 
3.2  Exclusion Criteria 
1. Participation in another clinical study with an investigational product during the last 28 
days. 
2. Prior stem cell transplant (either autologous or allogeneic) 
3. Creatinine clearance < 60 ml/min (calculated) 
4. Subjects who failed to achieve ≥ PR to induction therapy (i.e. VRd) and required salvage 
induction prior to AHSCT. 
5. Documented central nervous system or extramedullary disease. 
6. Significant organ dysfunction deemed to carry inappropriate risk for AHSCT. 
7. Intention or plans for cyclophosphamide mobilization. 
8. Known allergic reactions after previous tetanus diphtheria vaccination or had a condition 
of Guillain Barre Syndrome (GBS) 
9. Known active hepatitis B, C or HIV infections on initial assessment. 
10. Enrollment on any other transplant related protocols. 
 
3.3   Minorities and Women 
Both genders and from all racial/ethnic groups will be recruited equally into this trial. 
 
Section 4.0  Registration Procedure:  
The date of enrollment is the date of consent. However, all eligibility criteria do not need to be 
met until the date of the first study related treatment. 


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All subjects need to sign the consent form in order to enroll on the study. They will receive a copy 
of the consent form. In case of any issues, the treating physician or PI should be called for 
clarification. The subject will be registered as describe below in section 4.2.  
 
4.1  
Recruitment 
Subjects, who are referred to the Nebraska Medical Center (NMC)/UNMC with a diagnosis of MM 
and considered eligible for autologous hematopoietic stem cell transplantation will be identified 
from the outpatient clinics or inpatient service. Potential subjects will be recruited for the study by 
the principal investigator (PI), co-investigators or participating clinicians. 
 
Screening eligibility is based on standard clinical care performed by Bone Marrow Transplant 
(BMT) physician. Pre-transplant work up performed for determining the eligibility of the subject for 
high dose chemotherapy and autologous transplant, prior to signing of consent form will be 
acceptable. All other study related interventions and evaluation can only be performed after 
signing the consent form.  
 
If the subject is screened as potentially eligible, he/she will then be offered the option to 
participate. An informed consent will be signed by the subject after thorough review of the study 
is completed with the physician and his/her designee.  
 
4.2 
Eligibility Verification/Registration  
 
Before potential subjects are registered into the study, an eligibility checklist (Appendix A) must 
be completed to confirm the subject meets the eligibility criteria. The eligibility checklist will be 
maintained in the study file as source documentation if it has been reviewed, signed, and dated 
prior to registration by the treating physician.  
 
Subjects will be registered through the sponsor site (UNMC) by contacting the UNMC Project 
Coordinator. Study personnel from UNMC will contact the UNMC Research Project Coordinator 
if a subject appears to meet the eligibility criteria. They will email the following information:   
• 
Registration request with Demographics cover sheet (located in the Study site Manual) 
• 
Signed, completed eligibility checklist (Appendix A)  
• 
Copy of the signed and dated consent form  
Once the UNMC Research Project Coordinator confirms that the subject meets criteria, and 
target accrual has not been met, approval for the subject will be given and study subject number 
assigned. An email confirmation of registration is sent by the UNMC Research Project 
Coordinator to the site. The project coordinator can assign a subject study number prior to final 
registration confirmation of a subject. 
 
Each subject consented to the protocol is loaded into the UNMC Clinical Trial Management 
System (CTMS) within 7 days of consent.   CTMS registration includes entering the required 
demographic information as stated in the SRC policies and procedures.  
 
4.3 Randomization 
The protocol will randomize subjects to two subsequent arms (10 subjects per arm): group 1 will 
get a CD34+ selected graft + autologous DLI on day+7, while group 2 will get standard G-CSF 
mobilized graft only.    
The process of a 1:1 randomization between the experimental and control arms. During the 
registration procedures, study personnel are provided the randomization assignment from the  the 


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IIT Office project coordinator.  The 1:1 randomization will be conducted using blocked 
randomization with blocks of size 2 or 4. 
 
 
4.4 Instructions for Subjects Who Do Not Start Assigned Protocol Treatment  
If a subject does not receive any assigned protocol treatment after consenting, baseline data will 
be collected and included in the electronic data capture system. 
 
Section 5.0  Treatment Plan of Research Design:  
 
5.1  
Outline of research plan 
 
The protocol will randomize subjects to two subsequent arms (10 subjects per arm): group 1 
(experimental) will get a CD34+ selected graft + autologous DLI on day+7, while group 2 (control) 
will get standard G-CSF mobilized graft only.   Nebraska Medicine Biological Production Facility 
(BPF) personnel will perform processes outlined in Sections 5.1.4 through 5.1.9 below. 
 
5.1.1 
Pre-transplant Tetanus Vaccine 
Vaccination can start whenever the subject is in reasonable physical condition to move 
forward to AHSCT (VRd 3-6 cycles) with a minimum of partial response by IMWG, and at least 
2 weeks since the last does of bortezomib, lenalidomide or dexamethasone.  Tetanus (without 
pertussis) is given pre-transplant as an intramuscular injection. Tetanus vaccination will be 
documented in the medical record per usual clinical practice.  Data needed for the study data 
set will be recorded from this information. 
 
5.1.2 
 Immune Correlative Studies  
In all subjects, 70 mls of peripheral blood will be obtained (four tablespoon full) will be 
collected from all subjects for baseline immune correlative studies . A similar amount of 
blood will be collected at pre-G-CSF, graft pheresis and on days +15 (+/- 3), +30 (+14), +70 
(7-14 days post last dose of tetanus vaccine), +100 (+/-10), and 180 (+/-10) post-transplant 
to analyze initial post-vaccine immune response.   More details in section 5.7.  
 
Test 
Time 
point 
Pre vaccine 
Pre - PBMC 
harvest/Pre 
G-CSF 
Graft 
Pheresis/PBSC 
graft day 
Day + 15 
(+/- 3) 
Day + 30 
(+14) 
Day + 70 
(+14) 
 
Day + 100 
(+/- 10) 
Day + 180 
(+/- 10) 
T-cell Elispot 
ELISA 
X 
X 
 
 
X 
X 
X 
X 
Flow 
X 
X 
X 
X 
X 
X 
X 
X 
ELISA to 
Tetanus 
X 
X 
 
 
X 
X 
X 
X 
 
5.1.3 
 Pre-G-CSF PBMC (DLI) Pheresis  
Seven to 10 days after vaccine, subjects in group 1 will undergo mononuclear leukapheresis 
to collect and freeze PBMC product for DLI.  Subjects will be apheresed for a target blood 
processed volume of 24-28 liters or as the patient tolerates and per standard UNMC BPF 
practice. Lymphocyte content of the apheresed product as well as subset analysis will be 
determined by a cell differential count and flow cytometry. 
 
5.1.4 
 Stem cell lab processing of the pheresis product   
Depending on the volume of the product and the total nucleated cell count (TNC) in it, 
additional processing may be required to minimize volume. The recommended storage cell 
concentration should not exceed 2x108/mL. This will require centrifugation and plasma 


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reduction at 1000X G for approximately 15 minutes. The excess plasma is discarded then. 
The total cell count and volume of the concentrated product is determined. The proportion of 
the following components in the final product should be approximately: PBMC 75%, 
plasma/serum 5%, Plasma-Lyte 9.5%, DNase 0.5% and DMSO 10% per unit volume. For 
example for a 100 ml final product volume the PBMC should be 75ml, plasma/serum 5 ml, 
plasma-Lyte 9.5 ml, DNase 0.5 ml, and DMSO 10 ml. The product is then taken to the 
controlled rate freezer before final transfer to liquid nitrogen.   
 
 
5.1.5  
Stem cell mobilization  
Subjects in all groups will receive G-CSF as subcutaneous injections daily (10 µg/kg/day) x 5 
days starting on day -11 (after 1st leukapheresis for groups 1). Subjects will receive one dose 
of plerixafor followed by pheresis on the next day to collect G-PBSC graft. No 
Cyclophophamide mobilization is allowed on this protocol. Follow standard of care for stem 
cell collection.  
 
5.1.6 
 Graft processing on the experimental arm  
The stem cell product may start to be washed while waiting for the CD34+ content to known.  
Subjects who fail to mobilize a minimum of 6 million CD34/kg on day 1 of leukapheresis will 
be removed from the experimental arm of the study (these subjects will be treated and 
followed like group 2 for a separate analysis at the end).  
• 
Subjects who mobilize 6-10 million CD34/kg on day 1: 
o  will proceed to CD34 stem cell selection on the Milteny device, provided that 
the TNC content does not exceed the column capacity.  They will proceed to 
day 2 collection to complete CD34 need for second stem cell transplant 2 
million CD34/kg + 2 million CD34/kg reserve for potential “boost” need in case 
of graft failure on protocol (hence 4 total).  
• 
Subjects who mobilize >10 million CD34/kg on day 1:  
o They can have approximately up to 10 million CD 34/kg processed on the 
Miltenyi column, if the TNC content does not exceed the column capacity 
(remainder difference for the second AHSCT and the boost will be collected on 
the following day 2 of collection).  
• 
Collecting at least 14 million CD34/kg on day 1 will complete stem cell collection 
process(10 for Miltenyi, 2 for second AHSCT and 2 for boost).  
 
5.1.7 
 Stem Cell lab processing for CD34+ selection  
The PBSC pheresed graft is washed initially with PBS containing 5mM EDTA and 0.05% 
human albumin as per manufacture’s standard operating procedure SOP.  After reducing the 
total volume to 95 mls, the anti CD34 antibody (directly conjugated to magnetic micro beads 
Miltenyi) will be added and incubated for 30 minutes. The cells then washed with Max buffer 
and attached to Clinimax tubing set. The enriched cells will be cryopresrved with DMSO 7.5%, 
PBS/HAS 4%/heparin and stored in the gas phase of liquid nitrogen.  CD34 content of the 
final product should be no less than 2 million/Kg body weight.  
 
All subjects in the control group will have the graft absolute lymphocytes quantified (differential 
count).  A sample of the graft will be sent to the stat hematology lab for white cell count and 
differential. In addition, another sample will be sent for flow cytometry for subset analysis.   
 


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5.1.8 
 Conditioning Regimen   
Transplant preparative regimens:  a single intravenous dose of Melphalan 200mg/m2 on day 
-1 is used as the standard preparative regimen in subjects with multiple myeloma.   
 
5.1.9 
Day zero  
The CD34 stem cells will be thawed and infused back to subjects in group 1. Subjects in group 
2 will receive their unmanipulated graft as per standard procedures. The CD34 graft content 
should be in the range of 2-10 million/kg body weight in all groups. Subjects will receive 
maximal standard of care premedication excluding glucocorticoids.  
 
Glucocorticoid Use 
Due to lymphodepleting effect on the immune system, no steroids will be allowed from day 
0 of ASCT until day+181, unless unavoidable for urgent clinical use (such as autoimmune 
diagnosis or adrenal insufficiency).  
 
5.1.10 
On day +7 post-transplant 
Subjects in groups 1 will receive the stored autologous donor lymphocyte product after 
thawing via intravenous infusion. Subjects will receive maximal standard of care  
premedication excluding glucocorticoid use before donor lymphocyte infusion. No 
dexamethasone should be given on or after the day of DLI infusion.  
 
 
G-CSF post AHSCT will not be given.  
Subjects who show no rise in WBC as a sign of engraftment by day +15 post-transplant are 
allowed to receive G-CSF at 5 μg/kg daily until absolute neutrophil count is equal or > 500/μl.  
 
5.1.11 
  Other Therapy:  
All standard of care therapy provided for multiple myeloma subjects undergoing autologous 
transplant will be allowed. 
 
5.1.12   
At day +15 (+/-3) and day +60 (+/-3) post-transplant 
All subjects will receive tetanus vaccines as intramuscular injection. Platelets will be 
transfused if platelet count is < 30k on day +15 to allow for the intramuscular injection.  
 
5.1.13 
 Immune evaluation assays (anti-tetanus & immunophenotyping)  
Assays will be performed on day +15 (+/- 3 days), +30 (+14days), day+70 (+14days), day+100 
(+/-10 days), and Day 180(+/-10) post-transplant on both groups.  
 
 
 
5.2 
Post transplant Myeloma Management:    
 
5.2.1 All post-transplant myeloma staging and therapy will be based on local standard of 
clinical practice for myeloma subjects. Subjects will undergo Euroflow-based85 MRD staging 
if they are deemed to be close to CR (serum protein electrophoresis < 0.1 gdL, but serum 
immunefixation positive) based on IMWG criteria.    
  
5.2.2 For all subjects, post-transplant myeloma specific therapy may not be started before the 
day 100 (+/-10) visit on this protocol. The only exception would be disease progression that 
occurs before day+100, in which case the subject will be taken off study. 
  
5.2.3 Subjects on this protocol may not enroll on other  treatment/interventional protocols for 
the first 100 (+/-10) days post-transplant. The only exception would be disease progression 
before day+100, in which case, the subject is off study. 


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5.3  
Possible Complications:  
Subjects in the experimental arm (group 1) will be closely monitored after day zero for any 
possible treatment related toxicity. These toxicities may include but are not limited to:  
 
1- Engraftment failure:  
Although quite unlikely based on previous studies with CD34+ selected grafts in AHSCT3, in 
the event of slow engraftment or failure, the subject can receive part or all of his back up 
CD34+ grafts. No engraftment failure was observed in the two subjects treated on the 
experimental arm at Emory University. 
 
2- 
Autoimmunity 
(including 
autologous 
graft 
versus 
host 
disease 
autoGVHD):  
Subjects will be closely monitored for any clinical evidence of auto-immunity secondary to 
excessive immune suppressor cells depletion. Subjects can be treated according to the 
severity and organ involved based on physician discretion after consulting with the study PI.    
 
3- Severe lymphopenia: 
Has been described in CD34+ selected AHSCT34,86. In this trial, the chance of lymphopenia 
is small, since subjects will receive a DLI product on day +7, and these lymphocytes have 
shown evidence of accelerated in vivo expansion in the past.74  Nevertheless, we will closely 
watch subjects for lymphopenia beyond the general expected range at 4 weeks post 
AHSCT. If three consecutive subject’s had ALC equal or <1000 cells/µl at 4 weeks post 
AHSCT, further accrual will be held until their ALC >1000 with no infectious complications 
(including: CMV, EBV, Adeno- virus, mold). These subjects will be closely monitored for 
infections (CMV, EBV, adeno virus screening on lymphopenic subjects is optional). 
 
5.4 
 Duration of the study.  
The study is expected to last approximately 24 months.  
 
5.5 
 Number of Subjects: 
In this pilot phase II study, 10 subjects per arm will be enrolled for a total of 20 subjects.  To 
account for screen failures up to 29 subjects may be screened for the whole trial.     
 
5.6  
Removal from the study   
Subjects who for whatever reason do not meet the cardiac ejection fraction, pulmonary function 
test requirement or develop significant organ dysfunction before the transplant admission will be 
removed from the study. 
 
The investigator will withdraw a subject from the study treatment whenever: 
 
1. Continued participation is no longer in the subject’s best interests. Reasons for 
discontinuing treatment may include the occurrence of a SAE or an inter-current illness. 
These subjects will be taken off treatment and followed according to the study calendar. 
2.  The subject requests to end treatment. 
3. Subjects who fail to collect a minimum of 6 million CD 34 cells/kg body weight at the end 
of 1st day of Leukapheresis will be either removed or transferred to arm 2 of the protocol 
after discussion with the PI.   
 
5.7 
Immuno-Correlative Laboratory Studies 
 


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5.7.1 
 Laboratory studies to determine the general immunologic benefit of depleting 
immune suppressive cells from G-PBSC grafts.  
 
The recovery of ALC and AMC will be recorded at days 15(+/-3) and 30 (+14) post-transplant, 
as surrogates for immune recovery and predictors of relapse and survival. These values will 
be obtained as part of the standard clinical care of these subjects post AHSCT. ALC/AMC 
ratio represents a surrogate for the balance between the host adaptive immune response 
versus tumor microenvironment immune suppression, as has been shown in the background 
section. The hypothesis is that depleting majority of immune suppressive cells from G-PBSC 
graft in the setting of AHSCT will tip the balance towards accelerated lymphocyte expansion 
in-vivo, which will be reflected in high ALC numbers on day 15 (>500/μl).  The above 
intervention is hypothesized to lead to minimum AMC expansion by day 15 post AHSCT, 
leading to a high ALC/AMC ratio (>1). Such effect may jeopardize self-immune tolerance and 
trigger an auto-immune reaction or auto-GVHD. The development of autoimmune side effects 
in the context of cancer immunotherapy has been associated with augmented anti-tumor 
immune response and survival advantage. This was demonstrated in the use of CTLA4 
(Cytotoxic T-Lymphocyte Antigen) blockade in renal cell carcinoma.90            
 
5.7.2 
 Laboratory studies to determine phenotypic recovery of immune cells post 
AHSCT. 
 
The phenotypic recovery can play a role in cancer immune surveillance and tolerance, 
specifically T-cells (CD8, CD4), B cells, NK cells, γδ T-cells, regulatory T-cells 
(CD4/CD25+/FOXP3+/CD26-) and MDSCs (CD33+/CD11b+/HLADR low/CD14 
low/CD15+/-).These recovery patterns of immune phenotype data collected between the 
experimental group and the control group in the trial will be compared.  PBMCs will be 
collected for these subjects on day of graft pheresis, +15 (+/-3), +30 (+14), +70(+14, 7-10 
days post last tetanus vaccine), +100 (+/-10), and day 180(+30/-7) post-transplant.  The 
number/proportion of MDSCs and T-regs are hypothesized to be much lower in the 
experimental group compared with the control group. 
 
Subject’s blood Samples (70 mls) will be collected on days: Pre-G-CSF, PBSC graft day, 
days +15 (+/- 3), +30 (+14), +70 (7-14 days post last tetanus vaccine), +100 (+/- 10), and 
day 180(+/-10) post-transplant.   
 
5.7.3  
Studies to determine antigen specific immune response post AHSCT. 
To determine whether depletion of immune suppressive cells from G-PBSC graft will improve 
post AHSCT antigen specific (Ag) immune reconstitution, tetanus vaccine immune response 
post-transplant in the graft manipulation group will be studied and compared with the control 
groups and pre-transplant levels.  The tetanus specific immune response will be determined 
by performing both ELISPOT assay to anti-IFNᵞ and ELISA to anti-tetanus IgG antibody. 
 
 
Section 6.0  Measurement of Effect  
Statistical analysis of both T-cell Elispot, ELISA and flow cytometric assays will be conducted 
among the subjects in the two groups of subjects, to determine if meaningful and/or statistically 
significant response has occurred after study intervention in experimental group compared to 
the control group.  


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 Section 7.0  Study Parameters 
Examinations and 
Assessments 
Baseline 
(Screening) 
Pre-
Tra
nspl
ant 
(SC
T) 
 
 
 
Trans
plant 
Post Transplant Period 
Follow 
up 
Period 
Pre vaccine 
Vac
cine 
(day 
-19) 
Day -
12  to 
-9 
Day 
-11 
to -
7 
Day 
-6  
to -
4 
AHSCT 
Hospita
lization 
Day + 
7 
Day 
+15 
Day
+30 
Day
+60 
Day 
+70 
Day + 
100 
(mo. 
3) 
Day 
+ 
130 
(mo
. 4) 
Day 
+ 
150 
(mo
. 5) 
Day + 
180 
(mo. 
6) 
Day 
+ 
365 
(1 
yr) 
Survival 
status 
(2 
years) 
Window Days for 
Study Visit4 
-30 days 
(+/-10 
days) 
 
 
 
 
Day 0  
 
+/-3 
+14 
+/-3 
+14 
+/-10 
+/-
7 
+/-7 
+/-10 
+/-
30 
+/-30 
days 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion 
Criteria  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy testing 
X5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Clinical 
Assessment 
 
X2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data collection to 
assess organ 
function (labs)6 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Disease response3 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
X 
X 
Data collection for 
Disease 
Assessment 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 


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Section 7.0  Study Parameters 
Examinations and 
Assessments 
Baseline 
(Screening) 
Pre-
Tra
nspl
ant 
(SC
T) 
 
 
 
Trans
plant 
Post Transplant Period 
Follow 
up 
Period 
Pre vaccine 
Vac
cine 
(day 
-19) 
Day -
12  to 
-9 
Day 
-11 
to -
7 
Day 
-6  
to -
4 
AHSCT 
Hospita
lization 
Day + 
7 
Day 
+15 
Day
+30 
Day
+60 
Day 
+70 
Day + 
100 
(mo. 
3) 
Day 
+ 
130 
(mo
. 4) 
Day 
+ 
150 
(mo
. 5) 
Day + 
180 
(mo. 
6) 
Day 
+ 
365 
(1 
yr) 
Survival 
status 
(2 
years) 
(imaging and bone 
marrow) 
Data collection for 
Disease 
assessment (blood 
and urine) 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
X 
X 
Adverse Events 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assessment for 
Subject/Graft 
Survival 
 
 
 
 
 
 
 
X 
X 
 
X 
X 
 
 
X 
 
X 
Hematology 
(complete blood 
count (CBC) with 
differential ALC 
platelets) 
X 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
X 
X 
Randomization 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1 Pre-PBMC harvest in group 1 7-10 day post vaccine only  
2Clinical Assessment is billed as physical exam with performance score.  Data will be collected for MM disease specifics.  Height and weight are also collected only at 
screening. 
3 Disease response to be done at discretion of physician in keeping with standard of care.   
4 Transplant day is day 0. 
Section 7.0  Study Parameters 
Examinations and 
Assessments 
Baseline 
(Screening) 
Pre-
Tra
nspl
ant 
(SC
T) 
 
 
 
Trans
plant 
Post Transplant Period 
Follow 
up 
Period 
Pre vaccine 
Vac
cine 
(day 
-19) 
Day -
12  to 
-9 
Day 
-11 
to -
7 
Day 
-6  
to -
4 
AHSCT 
Hospita
lization 
Day + 
7 
Day 
+15 
Day
+30 
Day
+60 
Day 
+70 
Day + 
100 
(mo. 
3) 
Day 
+ 
130 
(mo
. 4) 
Day 
+ 
150 
(mo
. 5) 
Day + 
180 
(mo. 
6) 
Day 
+ 
365 
(1 
yr) 
Survival 
status 
(2 
years) 
Tetanus Vaccine 
 
X 
 
 
 
 
 
X 
 
X 
 
 
 
 
 
 
 
Group 11/ Pre G-
CSF PBMC 
pheresis 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Group 1 – Post-
AHSCT PBMC 
infusion 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
G-CSF (both 
groups) 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pre  Stem Cell 
harvest / Graft 
Pheresis (both 
groups)7 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Immune correlative 
study blood draw  
 
X 
X1 
 
X 
 
 
X 
X 
 
X 
X 
 
 
X 
 
 


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5 Female of child-bearing potential must have two negative urine pregnancy test results within at time of eligibility testing and prior to GCS-F pre-transplant as a way of 
ensuring safe transplant planning. 
 
6Organ function measured by standard clinical care labs collected from CBC, complete metabolic panel to assess the kidney, liver function.  
 
7 Subjects will receive one dose of plerixafor, a stem cell mobilizer, followed by pheresis on the next day to collect G-PBSC graft 


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7.1 
SCREENING AND BASELINE EVALUATION 
A flow chart of study procedures and assessments is provided above.   
The transplant patients that meet the study eligibility criteria and are consented for the study will 
undergo baseline evaluations. Much of the information listed below will be obtained at the time of 
determining eligibility/screening prior to enrollment on the protocol. 
The following baseline evaluations should represent those assessments/labs performed prior to 
the start of the conditioning regimen according to institutional standards. 
• 
History, physical examination, height and weight. 
• 
CBC with differential, platelet count 
• 
Blood for immunocorrelative studies at pre-transplant pre-vaccine (day -30 to day -20) 
 
 
 
7.2  PRE-TRANSPLANT PERIOD 
The following procedures will be performed pre-transplantation: 
 
• 
Blood for immunocorrelative studies on days: 7-10 days post last vaccine (pre-PBMC 
harvest and Pre-G-CSF), stem cell harvest day 
 
7.3 STUDY PROCEDURES: POST-TRANSPLANT PERIOD 
The following procedures will be performed post-transplantation 
• 
History and physical examination to assess morbidity daily during the in-
patient phase, then after transplant at designated timepoint on study 
parameters table 
• 
Record specific adverse events and opportunistic infections. 
• 
CBC (hemoglobin, hematocrit, WBC with differential and platelet count). 
• 
Blood for immunocorrelative studies all time points noted on the study 
parameter table. 
 
Section 8.0  Investigational Agents  
8.1 CliniMacs CD34 beads and CliniMacs Devise by Miltenyi  
The CliMacs devise and accompanying beads is approved for CD34 stem cell selection in 
allogeneic stem cell transplant from sibling donors. CD34+ stem cell selection in autologous stem 
cell transplant has been shown to be feasible and safe by Imai et al. BMT 2005 & Stewart et al. 
JCO 2005. 
 
8.2 Tetanus Vaccine 
The Tetanus vaccine that will be administered to subjects for this study is Tetanus/Diphtheria 
Toxoid (Td).  Td will be provided as 5-2FLU/0.5mL ready to inject prefilled syringes. Active 
ingredients include Clostridium Tetani, Toxoid antigen (Formaldehyde inactivated) 5Lf/0.5mL, 
Corynebacterium Diphtheriae Toxoid antigen (Formaldehyde inactivated) 2Lf/0.5mL. Inactive 
ingredients present are Aluminum phosphate, Formaldehyde, Latex, Sodium Chloride and 
Sterile Water for injection.  
 
Td is commercially available and is manufactured by Sanofi under the name Tenivac. Td will be 
obtained from Cardinal Health by the Investigational Drug Service (IDS) Pharmacy.  
 


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If patients experience any grade ≥ 3 adverse event/vaccine related toxicity following the first 
vaccine administration, they will be excluded from subsequent vaccine administration and taken 
off trial. 
 
8.2.1 Contrainidications92[Tenivac-FDA Approved Sanofi Pasteur Limited. Full Prescribing 
Package Insert]: 
Hypersensitivity:  A severe allergic reaction (e.g., anaphylaxis) after a previous dose of 
TENIVAC vaccine or any other tetanus toxoid or diphtheria toxoid-containing vaccine or any 
other component of this vaccine is a contraindication to administration of TENIVAC vaccine.  
Because of uncertainty as to which component of the vaccine may be responsible, none of the 
components should be administered. 
 
8.2.2  Warnings/ Precautions92 [Tenivac-FDA Approved Sanofi Pasteur Limited. Full 
Prescribing Package Insert]:   
A. Acute allergic anaphylactic reactions could occur.  Epinephrine hydrochloride solution 
(1:1,000) and other appropriate agents and equipment must be available for immediate 
use in case an anaphylactic or acute hypersensitivity reaction occurs. 
B. Latex is present in this TD preparation.  The tip caps of the TENIVAC prefilled syringes 
may contain natural rubber latex, which may cause allergic reactions in latex sensitive 
individuals. 
C. Arthus reactions can occur. Persons who experienced an Arthus-type hypersensitivity 
reaction following a prior dose of a tetanus toxoid-containing vaccine usually have high 
serum tetanus antitoxin levels and should not receive TENIVAC vaccine more frequently 
than every 10 years, even for tetanus prophylaxis as part of wound management. 
8.2.3 Adverse Reactions92 [Tenivac-FDA Approved Sanofi Pasteur Limited. Full Prescribing 
Package Insert]:  
A. Injection site reactions could occur such as pain, redness and swelling. 
B. Systemic reactions consist of fever, headache, muscle weakness, general malaise, 
pain in joints, dizziness, syncope, vomiting and lymphadenopathy. 
 
Section 9.0  Toxicity and Adverse Event Reporting Guidelines:   
This protocol will comply with monitoring and adverse event reporting requirements of the 
UNMC Fred & Pamela Buffett Cancer Center Data Monitoring plan. The protocol will adhere to 
both institutional IRB and FDA guidelines for toxicity reporting.  
 
All subjects will be closely followed for toxicity from between the start of any protocol 
intervention and day 100 during the study and all grade 3 or 4 adverse events (or highly unusual 
grade 2 adverse events) will be recorded on the CRF. 
 
Per NCI guidelines, Serious Adverse Events (SAEs) and Adverse Events (AEs) will be assessed 
and graded by reports from subjects to their physician/Investigator and by physical examinations. 
SAEs and AEs will be graded and toxicity assessed using the revised NCI CTCAE version 5.0.  
AEs will be followed until resolution, return to baseline or ≤ grade 1 levels. The outcome of all 
SAEs will be graded as resolved; recovering; ongoing; resolved with sequela; fatal. 
 
SAEs will be followed until resolution, return to baseline or ≤ grade 1 levels, death, or until no 
further improvement is reasonably expected. Deaths occurring within 30 days of study treatment 
regardless of relationship will be reported to the UNMC DSMC.  
 
9.1 Definitions:  


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Adverse Event  
An AE is defined as any untoward medical occurrence in a clinical investigation subject 
administered a pharmaceutical product and which does not necessarily have a causal 
relationship with this treatment. 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 the event is considered causally 
related to the use of the product.  
 
Any worsening of a pre-existing condition or illness is considered an AE.  Laboratory 
abnormalities and changes in vital signs are considered to be AEs if they result in 
discontinuation from the study, necessitate therapeutic medical intervention, meet protocol 
specific criteria (see Section 5.0, Treatment Plan) and/or if the investigator considers them to be 
AEs. 
 
Expected Adverse Event 
Any event in which the severity or specificity is consistent with the risk information described in 
the protocol, or is anticipated based on the subject's medical history. 
 
Unexpected Adverse Event  
An AE in which the specificity, severity, or frequency is not consistent with (a) the IRB 
application and detailed protocol; (b) Risk information in the ICF; (c) and the event is not 
anticipated from the subject's disease history or status. 
 
Serious Adverse Event  
 
An SAE is one which results in any of the following outcomes:  
• 
Death  
• 
Is a serious injury, or otherwise seriously impacts the health, safety or welfare-fare of 
subject1  
• 
Inpatient hospitalization or prolongation of existing hospitalization 
• 
Required intervention to prevent permanent impairment or damage 
• 
Persistent or significant disability or incapacity2  
• 
Is a congenital anomaly or birth defect  
• 
Other serious important medical event3  
• 
Any event that requires treatment to prevent one of the outcomes listed above 
 
1 means that the subject was at immediate risk of death at the time of the SAE.  
2 “Persistent or significant disability or incapacity” means that there is a substantial disruption of 
a person’s ability to carry out normal life functions.  
3 Medical and scientific judgment will be exercised in deciding whether expedited reporting is 
appropriate in situations where none of the outcomes listed above occurred. Important 
medical events that may not be immediately life-threatening or result in death or 
hospitalization but may jeopardize the subject or may require intervention to prevent one of 
the other outcomes listed in the definition above should also usually be considered serious. 
Examples of such events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse. A new diagnosis of 
cancer during the course of a treatment should be considered as medically important. 
 


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9.2 Adverse Event Reporting Requirements Per University of Nebraska Medical Center, 
IRB and Fred & Pamela Buffett Cancer Center Data and Safety Monitoring Committee 
(DSMC)  
 
9.2.1 IRB Reporting  
 
All internal adverse events (AEs) will be reported to the local IRB promptly per institutional 
human research protection program policy. 
  
 
9.2.2 Fred & Pamela Buffett Cancer Center Data and Safety Monitoring Committee 
(DSMC) Reporting 
 
In its initial review, the DSMC will make a recommendation for the frequency of monitoring 
based on an assessment of risk associated with study-associated therapy, per the DSMC 
policy.  Reporting of the following will be done in accordance with DSMC guidelines: 
 
• 
All SAEs and toxicity reporting will be reported to the DSMC. 
 
• 
All AE grade 3 or higher (expected or unexpected, regardless of attribution) will be 
reported to the DSMC. In certain investigator-initiated institutional trials where virtually 
100% of subjects are guaranteed to experience very specific known grade 3 or higher 
toxicities (e.g., hematologic toxicity in stem cell transplant studies) , the requirement for 
reporting of those toxicities may be waived at the discretion of the DSMC based on initial 
protocol review and in conjunction with investigator input if required. 
 
Attribution of AE: The likelihood of relationship of the AE to the study drugs will be determined 
by the investigator based on the following definitions:  
 
Not related:  The subject was not exposed to the study treatment or another cause that is 
obvious.  
Probably not related: The AE is most likely explained by another cause, and the time of 
occurrence of the AE is not reasonably related to the study treatment.  
Possibly related: Study treatment administration and AE occurrence reasonably related in 
time, and the AE is explained equally well by causes other than study treatment, or 
treatment administration and AE occurrence are not reasonably related in time, but the AE 
is not obviously a result of other causes.  
Probably related: Study treatment administration and AE occurrence are reasonably related in 
time, and the AE is more likely explained by study treatment than by other mechanisms.  
Definitely related: The occurrence and timing of the AE are clearly attributable to the study 
treatment.  
 
AEs will be collected from the time the start of any protocol intervention and day 100 post 
transplant. All AEs will be followed until resolution or a cause is identified. AEs judged by the 
investigator as not related or probably not related to the treatment will NOT be followed beyond 
the 100 days post transplant. 
 
 As a part of this study subjects will receive an autologous hematopoietic stem cell transplant 
(AHSCT). As part of a routine AHSCT process there are many adverse events expected to 
potentially occur. Below are known or frequent grade 3 or above Adverse Events that occur post 
AHSCT. 
 


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Hematologic Toxicities  
Electrolyte Abnormalities  
Mucositis  
Fatigue  
Fevers, including Neutropenic Fevers  
 
Per DSMC approval, for this protocol these AEs will not be reported to the PRMS office during 
the trial. All other grade 3 and above AEs and any Serious Adverse Event that is unexpected 
will still be reported using the DSMC approved forms.  
  
All SAEs and AE reporting will be completed using DSMC approved forms.  Detailed policy and 
procedures for this section may be reviewed at: 
http://www.unmc.edu/cancercenter/clinical/prms.html  
 
 
9.3 Monitoring  
  
Various methods will be implemented to exchange information with study personnel:  
 
• Site Initiation/Orientation  
• Investigator meetings as feasible  
• Email distributions/reports as needed  
 
9.3.1 Data Monitoring 
 
For this study, data monitoring is the act of overseeing the progress of a clinical trial, and of 
ensuring that it is conducted, recorded, and reported in accordance with the protocol, standard 
operating procedures (SOPs), Good Clinical Practice (GCP), and applicable regulatory 
requirement(s).  Monitoring is a Quality Control, continuous process during the entire trial. 
 
Scheduled meetings will occur depending upon the activity of the protocol. These meetings will 
include the Principal Investigator, Co-PI, data managers, and anybody deemed important to 
attend by the PI.  
 
During these meetings the following points will be reviewed and discussed: 
 
1. Safety of protocol participants (AE reporting). 
2. Validity and integrity of the data. 
3. Enrollment rate relative to expectations and the characteristics of participants. 
4. Retention of participants and adherence to the protocol (potential or real protocol 
violations). 
5. Completeness of collected data. 
 
9.4 Auditing   
 
Auditing is a systematic and independent examination of trial-related activities and documents 
to determine: 
• 
whether the evaluated trial-related activities were conducted 
• 
 the data were recorded, analyzed, and accurately reported, according to the protocol, 
to the sponsor's SOPs, GCP, and applicable regulatory requirement(s).  
 
Auditing is a Quality Assurance, one point process during the trial. 


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The UNMC Fred & Pamela Buffett Cancer Center Scientific Review Committee (SRC) will 
review this protocol on at least an annual basis.  
 
This study will undergo audit on at least a semi-annual basis by the UNMC Fred & Pamela 
Buffett Cancer Center Audit Committee.  
 
Detailed policy and procedures for this section may be reviewed at: 
https://www.unmc.edu/cancercenter/clinical/prms.html . 
 
Section 10.0  Statistical Considerations:  
10.1 Primary Objectives 
Given the known lack of vaccine response in the first few months post autologous stem cell 
transplantation, we intend to look for evidence of vaccine response in the experimental arm via 
increase in tetanus specific T-cell Elispots and anti-tetanus antibody (Elisa) on days 30, 70, 100 
and 180 post-transplant. Given the pilot/explorative nature of this trial and the lack of good pre 
trial assumptions about vaccine response, we will evaluate the vaccine response by Elisa and 
Elispot on days 30, 70, 100 and 180 will be compared between groups using a Mann-Whitney 
test. 
10.2 Secondary Objectives 
For the secondary objectives, engraftment, development of any auto immune side effects, and 
infections will be treated as binary variables. Chi-square tests will be used to compare each of 
these 3 outcome variables between the two groups, respectively. A Wilcoxon signed-ranks test 
will be employed to test the change in absolute lymphocyte (ALC), absolute monocyte counts 
(AMC), immune cells (T cells CD8, CD4), natural killer cells, γδ T-cells, T-regs and MDSCs) at 
days 15 and 30 post AHSCT from the baseline among each group, respectively. Wilcoxon 
signed-ranks test will be further used to compare the change of change in ALC, AMC and 
immune cells between the two groups at days 15 and 30 post- AHSCT, respectively. 
Generalized linear mixed models will be employed to compare the change pattern of ALC, AMC 
and immune cells between the two groups over the entire follow up periods. Time to relapse and 
overall survival will plotted using the methods of Kaplan and Meier and the log rank test will be 
used to compare the time to event curves between the two treatment groups. 
 
Lastly, as an explorative objective will also be depth of anti-myeloma response as evaluated by 
the standard IMWG criteria, including the rate of sCR, and subsequent MRD status for the 
subjects in sCR. 
 
10.3 Study Design: 
This is a pilot phase II trial with 1:1 randomization between the experimental and control arms.  
10.4 Stopping Rules 
If more than three subjects on the experimental arm develop a treatment related grade 3-4  
toxicity, which is irreversible, protocol will be halted and the Institutional Review Board will be 
notified. 
 
10.5 Sample Size and Power Consideration 


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This is exploratory trial with a total sample size of 20 subjects total (10 subjects in each phase II 
arm). There is no preliminary data on the primary endpoint of the study (baseline tetanus 
vaccine response early after transplant). However, it is known that vaccine response in general 
is muted in the first three months post-transplant. We hypothesize to see a wide difference in 
Elispot values between the two arms. Two-sided two sample t-test is used to for power 
calculation. The sample size of 20 will achieve a power of at least 80% to detect an effect size 
of d=1.36 for a Mann-Whitney test comparing two independent groups at the significance level 
of 5% assuming that the actual distribution is Norma91. Because of the exploratory nature of the 
study, corrections for multiple comparisons will not be performed. Signiﬁcance for each test will 
be set at P< .05. 
 
10.6 Expected Accrual Rate, Accrual Duration, and Study Duration 
The estimated accrual rate is 20 subjects enrolled in 12 -18 months.   
 
Section 11.0  Records to be Kept: 
Information regarding the actual treatments, adverse effects, radiographic and laboratory 
information are to be recorded on appropriate forms. See attached Data form plan. Serious 
adverse events, when noted, will be recorded on site via the standard serious adverse effects 
form. 
 
11.1 Quality assurance 
Complete records must be maintained in a research chart on each subject treated on the 
protocol. Research chart can be maintained either as an electronic record, paper chart or a 
combination or both. These records should include primary documentation (e.g., lab. report 
slips, physician notes, etc.) which confirm that:  
- The subject met the eligibility criteria.  
- Signed informed consent was obtained prior to treatment.  
- Treatment was given according to protocol (dated notes about doses given & reasons 
for any dose modifications).  
- Toxicity was assessed according to protocol (laboratory report slips, etc.).  
- Response was assessed according to protocol (dated notes on clinical assessment, lab 
reports as appropriate).  
 
11.2 
Responsibilities of the Principal Investigator  
The PI is responsible for ensuring that the protocol and its appendices are scrupulously 
followed, particularly when other departments are involved in the trial.  The PI oversees the 
quality of the data collected in the case reports.  The data obtained during the trial are recorded 
directly, with all modifications of data signed, dated, and justified as stated above.  Modifications 
must conform to the procedures defined for paper and electronic records. 
 
For the entire group of persons involved in carrying out the trial (day, night and emergency 
personnel), the PI is responsible for the following: 
 
1. 
Ensuring that the personnel are informed of the protocol used and that they understand 
the part they are responsible for implementing. 
2. 
Training personnel if necessary. 
3. 
Designating individual(s) specifically responsible for the administrative management of the 
trial. 
4. 
Ensuring that other departments or services involved in this trial are informed of the trial 
and determining with them the specific operating procedures necessary to conduct the 
trial. 


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11.3   Records Retention   
The Principal Investigator will maintain all records related to the study as required by regulations 
and institutional policy. 
 
11.4 
Forte Electronic Data Capturing (EDC) System 
Data will be stored electronically for this study on the Forte secure server. Data forms will not 
differ from the paper versions with the exception of an electronic format containing the UNMC 
Fred & Pamela Buffett Cancer Center and Forte logo.  
 
Forte EDC provides for remote data collection that meets FDA 21 CFR Part 11 requirements as 
well as HIPAA and other regulatory requirements designed to enhance data security and protect 
subject confidentiality. Authorized users log into Forte through a secure connection and must 
provide a valid username, password, and database ID. This data may be made available to the 
public at large. 
 
Section 12.0  Patient Consent: 
In accordance with Title 21 CFR (Part 50), the PI commits to obtaining the informed consent of 
the subject by signature and clearly indicates the content of the information given to the subject 
and the means by which consent is obtained. 
 
The PI commits him/herself to conform to GCP, 21 CFR Parts 50, 54, 56 and 312 regulations, 
concerning his/her duties. 
 
12.1  Subject Competency 
Subjects will be eligible to participate in the study only if they are competent to give informed 
consent. A subject that the investigators judges to be incompetent will not be enrolled. 
 
12.2 Process of Informed Consent and subject comprehension 
If the patient chooses to be a participant in this study informed consent will be  obtained by the 
investigators. The study and procedures involved including the risks will be explained in detail to 
each subject and that participation is entirely on a voluntary basis. The participating 
investigators will be available to discuss the study with them. 
 
The subject will be asked to state in his/her own words the purpose of the study, the procedures 
that will be carried out, potential risk, potential benefits to the subject, the alternatives and the 
right to withdraw from the study. If there are any indications that a given subject's 
comprehension is anything less than accurate, the points of confusion will be discussed and 
clarified. 
 
 
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Attal M, Harousseau JL, Stoppa AM, et al. A prospective, randomized trial of autologous 
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Section 14.0  Data Collection Forms:  
A data collection plan for electronic data capture forms (eCRF) is submitted as part of this 
protocol for the scientific review committees review.  This plan includes data capture forms for 
the following:  
• 
Adverse Event Log 
• 
Clinical Laboratory Values 
• 
Death Record 
• 
Eligibility Checklist (including both inclusion and exclusion criteria to match the protocol)  
• 
Medical History  
• 
Physical Examination 
 
In addition, the study team has the following items provided, at the time of site activation, as an 
accompaniment to the protocol.  These items are provided electronically as a study site manual 
and Manual of Operations (MOP) to guide the study operations. Below are the included forms. 
• 
Manual of Operations document 
• 
Study site manual 
o Delegation log template 
o Training log template 
o Enrollment/Registration documents – registration request, eligibility exception 
request and ineligibility report and enrollment log 
o Td Vaccine Information sheet for subjects 
o Investigator’s brochure, package insert 
o DSMC/ Federal AE/SAE reporting documents 
o Specimen collection and prep worksheet – Lab Manual 
o eCRF completion guidelines 
o FDA 1572, FDA 3455 Financial disclosure, as appropriate 
 
 


IRB # 669-19 
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Appendix A: Eligibility checklist 
Date Completed: 
 
Institution: UNMC 
Subject ID#: 
 
IRB # 669-19 Title: A Pilot Study of Enhancing Anti-Tetanus 
Vaccine Response After Autologous Stem Cell Transplantation 
PI: Muhamed Baljevic, MD 
Waiver #: 
 
Inclusion criteria: 
  Yes     No    N/A 
1. Age ≥19 years to 70 years old at time of study entry (consent).  
 
[   ]   [   ]   [   ]  
2. Diagnosis of Multiple Myeloma as per updated International Myeloma Working 
Group (IMWG) criteria84. 
[   ]   [   ]   [   ] 
3. Must have received bortezomib, lenalidomide and dexamethasone (VRd) as a 
form of induction therapy pre-AHSCT (use of cyclophosphamide, bortezomib and 
dexamethasone may be allowed for up to 2 weekly doses before initiation of VRd 
induction, if necessary clinically for cytoreduction)  
 
[   ]   [   ]   [   ] 
4. Able to understand and sign a consent form. 
 
[   ]   [   ]   [   ] 
5. Creatinine clearance equal or > 60 ml/min (calculated) 
 
[   ]   [   ]   [   ] 
6. Ejection fraction equal or > 50% before admission for transplant as per institutional 
standards. Patients with coronary heart disease (recent myocardial infarctions, 
angina, cardiac stent, or bypass surgery in the last 6 months or arrhythmia) need 
to be cleared by cardiology as per institutional BMT standards. 
 
[   ]   [   ]   [   ] 
7. Total bilirubin ≤2.5 times the upper limit of the institutional normal values, and 
total AST (SGOT) and ALT (SGPT) ≤2.5 times the upper limit of the institutional 
normal values. 
[   ]   [   ]   [   ] 
8. FVC, FEV1 or DLCO equal to or >50% predicted before admission for transplant 
as per institutional standards. Patients on home oxygen are not allowed on the 
protocol. 
[   ]   [   ]   [   ] 
9. Is there less than 6 months of pre-transplant MM chemotherapy (from the date of 
the start of the induction therapy)?  
 
[   ]   [   ]   [   ] 
10. KPS ≥ 70%or ECOG 0-2. 
 
[   ]   [   ]   [   ] 
11. Must be eligible to receive Melphalan dose of 200mg/m2 
[   ]   [   ]   [   ] 
12. A female of child-bearing potential, must have two negative urine pregnancy test 
results at time of eligibility testing and prior to GCS-F pre-transplant 
[   ]   [   ]   [   ] 
All of the above must be yes to be eligible.   
 
Exclusion criteria    
  Yes     No    N/A 


IRB # 669-19 
Protocol Version 1.5,  
Page 40 of 41 
 
1. Participation in another clinical study with an investigational product during the last 
28 days. 
[   ]   [   ]   [   ] 
2. Prior stem cell transplant (both autologous and allogeneic) 
 
[   ]   [   ]   [   ] 
3. Creatinine clearance < 60 ml/min (calculated) 
 
[   ]   [   ]   [   ] 
4. Subjects who failed to achieve ≥ PR to induction therapy (i.e. VRd) and required 
salvage induction prior to AHSCT. 
 
[   ]   [   ]   [   ] 
5. Documented central nervous system or extramedullary disease. 
 
[   ]   [   ]   [   ] 
6. Significant organ dysfunction deemed to carry inappropriate risk for AHSCT. 
 
[   ]   [   ]   [   ] 
7. Intention or plans for cyclophosphamide mobilization. 
 
[   ]   [   ]   [   ] 
8. Known allergic reactions after previous tetanus diphtheria vaccination or had a 
condition of Guillain Barre Syndrome (GBS) 
 
[   ]   [   ]   [   ] 
9. Known active hepatitis B, C or HIV infections on initial assessment. 
 
[   ]   [   ]   [   ] 
10. Enrollment on any other transplant related protocols 
 
[   ]   [   ]   [   ] 
All of the above must be no to be eligible. 
 
Eligibility:      [    ]  Subject satisfies all criteria.  
                     [    ]  Subject not formally eligible, but admitted to study because (state reason) 
_______________________________________________________________________________________
__ 
             
 Subject Initials:  ______________MR #______________DOB ______________ 
   
ELIGIBILITY reviewed and confirmed  
                     
 Site Investigator Signature _______________________Date_____________        
 
 
 


IRB # 669-19 
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Page 41 of 41 
 
Appendix B – Correlative Lab Specimen Collection Schedule 
 
Visit 
Specimen 
Type 
Collection 
amount 
Tube 
Handling Instructions 
 
Pre vaccine 
 
Blood 
70 mls 
6 Sodium Heparin tubes 
– 10 mls each 
1 red top tube – 10 mls 
Sodium heparin tubes 
must be placed in 4°C 
temperature if not 
delivers to the lab 
within 30 minutes. 
 
Red top tube – deliver 
to Al-Kadhimi lab 
directly. 
Pre G-CSF PBMC 
pheresis 
 
 
Blood 
70 mls 
6 Sodium Heparin tubes 
– 10 mls each 
1 red top tube – 10 mls 
Graft 
Pheresis/PBSC 
graft day 
 
Blood 
10 mls 
1 Sodium Heparin tube 
 
Sodium heparin tubes 
must be placed in 4°C 
temperature if not 
delivers to the lab 
within 30 minutes. 
Day + 15 (+/- 3) 
 
Blood 
70 mls 
6 Sodium Heparin tubes 
– 10 mls each 
1 red top tube – 10 mls 
 
Sodium heparin tubes 
must be placed in 4°C 
temperature if not 
delivers to the lab 
within 30 minutes. 
Red top tube - deliver 
to Al-Kadhimi lab 
directly. 
Day + 30 (+14) 
 
Blood 
70 mls 
6 Sodium Heparin tubes 
– 10 mls each 
1 red top tube – 10 mls 
 
Day + 70 (+14) 
 
Blood 
70 mls 
6 Sodium Heparin tubes 
– 10 mls each 
1 red top tube – 10 mls 
 
Day + 100 (+/- 10) 
 
Blood 
70 mls 
6 Sodium Heparin tubes 
– 10 mls each 
1 red top tube – 10 mls 
 
Day + 180 (+/- 10) 
 
Blood 
70 mls 
6 Sodium Heparin tubes 
– 10 mls each 
1 red top tube – 10 mls