Enhanced Epidermal Antigen Specific Immunotherapy Trial -1 (EE-ASI-1): A Phase 1a Study of Gold Nanoparticles Administered Intradermally by Microneedles to Deliver Immunotherapy With a Proinsulin Derived Peptide in Type 1 Diabetes
Phase: PHASE1
Conditions: Type 1 Diabetes
Sponsor: Cardiff University

EE-ASI WP5 Clinical Trial Protocol Version 3.2, 06 July 2018 
EudraCT no: 2015-003934-28, IRAS 190330, Rec 16/LO/0020, Sponsor no: SPON1455-15 
                     
                             
 
 
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Protocol Title:  
Enhanced Epidermal Antigen Specific Immunotherapy trial -1 (EE-ASI-1): A 
Phase 1a study of gold nanoparticles administered intradermally by 
microneedles to deliver immunotherapy with a proinsulin derived peptide in 
Type 1 diabetes. 
 
 
 
 
Short title:  
EE-ASI – 1. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
 
1 
LIST OF ABBREVIATONS ...................................................................................................... 5 
2 
TRIAL MANAGEMENT ......................................................................................................... 6 
2.1 
Sponsor ........................................................................................................................ 6 
2.2 
Trial Centre/Trials Unit ................................................................................................ 6 
2.3 
Trial Investigators ........................................................................................................ 7 
3 
BACKGROUND INFORMATION ........................................................................................... 7 
3.1 
Overview of the intervention in this protocol ............................................................ 7 
3.2 
Background to approach to immunotherapy explored in the protocol. .................... 7 
3.3 
Route of administration, dosage, regimen, and treatment period .......................... 12 
3.4 
Sample size and population to be studied ................................................................ 12 
3.5 
Conduct of trial .......................................................................................................... 13 
4 
TRIAL OBJECTIVES AND DESIGN ....................................................................................... 13 
4.1 
Trial Objectives .......................................................................................................... 13 
4.2 
Trial Design ................................................................................................................ 13 
4.3 
Primary endpoint ...................................................................................................... 13 
4.4 
Secondary endpoints ................................................................................................. 13 
5 
TRIAL MEDICATION ........................................................................................................... 17 
5.1 
IMP ............................................................................................................................ 17 
5.2 
Dosing regimen ......................................................................................................... 17 
5.3 
Drug accountability ................................................................................................... 17 
5.4 
Subject compliance ................................................................................................... 17 
5.5 
Dosing strategy: ......................................................................................................... 18 
5.6 
Concomitant medication ........................................................................................... 18 
5.7 
Concomitant health issues ........................................................................................ 19 
5.8 
Measures to avoid bias ............................................................................................. 19 
5.9 
Timescales ................................................................................................................. 19 
6 
SELECTION AND WITHDRAWAL OF SUBJECTS .................................................................. 19 
6.1 
Study population ....................................................................................................... 19 
6.2 
Inclusion criteria ........................................................................................................ 19 
6.3 
Exclusion criteria ....................................................................................................... 20 
6.4 
Recruitment and informed consent .......................................................................... 21 
6.5 
General assessments ................................................................................................. 22 
6.6 
Follow up assessments .............................................................................................. 22 
6.7 
General laboratory tests ........................................................................................... 22 
6.8 
Study Drug Administration ........................................................................................ 23 
6.9 
Metabolic outcome assessments .............................................................................. 23 
6.10 Mechanistic immunological studies .......................................................................... 23 
6.11 General guidance on glycaemic control during the study ........................................ 24 

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6.12 Ultrasound guided Fine Needle Aspiration of axillary lymph node .......................... 24 
6.13 Sample management ................................................................................................ 25 
6.14 Specimen handling .................................................................................................... 26 
6.15 Laboratory procedures .............................................................................................. 28 
7 
Risk Mitigation Strategy .................................................................................................... 30 
8 
WITHDRAWAL/EXIT CRITERIA ........................................................................................... 31 
8.1 
At any time ................................................................................................................ 31 
8.2 
After receipt of first dose .......................................................................................... 31 
8.3 
Subject becomes pregnant........................................................................................ 31 
8.4 
Treatment of Withdrawn Subjects ............................................................................ 32 
9 
END OF STUDY .................................................................................................................. 32 
10 
PHARMACOVIGILANCE ................................................................................................. 32 
10.1 Specification, timing and recording of safety parameters ........................................ 32 
10.2 
10.3 Hypersensitivity ......................................................................................................... 33 
10.4 Disease exacerbation ................................................................................................ 34 
11 
ADVERSE EVENTS .......................................................................................................... 34 
11.1 Definitions ................................................................................................................. 34 
11.2 Eliciting Adverse Events ............................................................................................ 35 
11.3 Principal Investigator/Trial Centre Responsibilities .................................................. 35 
11.4 Assessing AEs ............................................................................................................. 36 
11.5 Reporting procedures ............................................................................................... 36 
11.6 Sponsor and Chief Investigator Responsibilities ....................................................... 37 
11.7 Urgent Safety measures ............................................................................................ 37 
11.8 Treatment Stopping Rules ............................................................................................. 
11.9 Safety Report/ Line Listing ........................................................................................ 38 
12 
ASSESSMENT OF EFFICACY............................................................................................ 38 
12.1 Efficacy Parameters ................................................................................................... 38 
12.2 Procedures for Assessing Efficacy Parameters ......................................................... 39 
13 
STATISTICS ..................................................................................................................... 39 
14 
SAMPLE SIZE .................................................................................................................. 39 
15 
PLANNED ANALYSIS ...................................................................................................... 40 
15.1 Final ........................................................................................................................... 40 
16 
QUALITY ASSURANCE, SITE MONITORING AND DIRECT ACCESS TO SOURCE DATA AND 
DOCUMENTS ............................................................................................................................ 40 
17 
ETHICS AND REGULATORY APPROVALS ........................................................................ 41 
18 
FINANCIAL AND INSURANCE ASPECTS .......................................................................... 41 
19 
PATIENT INVOLVEMENT ............................................................................................... 42 
20 
PUBLICATION POLICY .................................................................................................... 42 
21 
TRIAL MANAGEMENT ................................................................................................... 42 
21.1 Trial Steering Committee (TSC) ................................................................................. 42 
21.2 Trial Management Group (TMG) ............................................................................... 42 
21.3 Data safety Monitoring Board (DSMB) members: .................................................... 43 

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22 
SIGNATURE PAGE .......................................................................................................... 43 
23 
REFERENCES .................................................................................................................. 44 
 
LIST OF FIGURES 
Figure 1. Flow chart of ascertainment, recruitment and screening through to IMP 
administration. ......................................................................................................................... 16 
Figure 2. Chart indicating dosing schedule …………………………………………………………………..…….19 
Figure 3. Blood draw schedule ….……………………………………………..………………………………………..26 
Figure 4. Sample management flowchart ……………………………………........................................27 
 
 
LIST OF TABLES 
Table 1: Schedule of study visits ………………………………………………………………………………………..16 
 
 
 
 
 
 
 
 

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1 
LIST OF ABBREVIATONS 
 
ADDQoL: Audit of diabetes-dependent quality of life 
AE:  Adverse Event 
AGA: Antigen-induced activation and gene array assay 
AICD: Activation-induced cell death 
APC: Antigen presenting cell 
AR: Adverse reaction 
ASI: Antigen-specific immunotherapy  
CI: Chief Investigator 
CSRI: Client Service Receipt Inventory 
CRF: Case Report Form 
CU: Cardiff University 
DSMB: Data Safety Monitoring Board 
DTSQc: Diabetes treatment satisfaction questionnaire, change version 
DTSQs: Diabetes treatment satisfaction questionnaire 
ELISPOT: Cytokine enzyme-linked immunospot assay 
GAD-65: Glutamic acid decarboxylase-65 
GCP: Good Clinical Practice 
GNP: Gold nanoparticles 
HbA1c: Glycated haemoglobin 
HFS: Hypoglycaemia fear survey 
HLA class II: Human Leukocyte Antigen class II molecule 
IB: Investigator’s brochure 
i.d.: Intradermal 
IA-2:  Insulinoma-associated antigen-2  
IFN-γ: Interferon-γ 
IL: Interleukin 
LN: Lymph node 
IMP: Investigational Medicinal Product 
IMPD: Investigational Medicinal Product Dossier 
MAGE:  Mean amplitude of glucose excursion 
MHRA: Medicines & Healthcare Products Regulatory Agency 
MMTT: Mixed meal tolerance test 
MPA: Medical Products Agency 
NIHR: National Institute for Health Research 
NISCHR CRC: National Institute for Social Care & Health Research Clinical Research Centre 
PBMC: peripheral blood mononuclear cells 
PI: Principal Investigator 
PIS: Patient information sheet 
PIT: Peptide immunotherapy 
REC: Research Ethics Committee 
RERB: Regional Ethical Review Board 

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RIES: Research, Innovation and Enterprise Services 
SAE: Severe Adverse Event 
SAR: Severe Adverse Reaction 
s.c.: Subcutaneous 
SPC: Summary of product characteristics 
STU: Swansea Trials Unit 
SUSAR: Suspected Unexpected Serious Adverse Reaction 
TGF-β: transforming growth factor-β 
Th1, Th2: T helper 1, T helper 2 
TMG: Trial Management Group 
TNF-α: tumour necrosis factor-α 
TSC: Trial Steering Committee 
Treg: Regulatory T lymphocytes 
UAR: Unexpected adverse reaction 
UCPCR: Urine C-peptide creatinine ratio 
ZnT8: Zinc transporter 8 
2 
TRIAL MANAGEMENT 
2.1 
Sponsor  
Cardiff University 
Chris Shaw 
Research Governance Coordinator 
Research, Innovation and Enterprise Services (RIES) 
Cardiff University 
7th Floor, 30-36 Newport Road 
Cardiff 
CF24 0DE 
Tel: (+44) 0 29 208 ex79130 
Fax: (+44) 0 29 20874189 
Email: ShawC3@cardiff.ac.uk 
2.2 
Trial Centre/Trials Unit 
Swansea Trials Unit 
Institute of Life Science 2 
College of Medicine 
Swansea University 
Singleton Park 
Swansea 
SA2 8PP  
Tel: 01792 606545 
 

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2.3 
Trial Investigators 
2.3.1 Chief Investigator and Principal Investigator 
Colin M Dayan MA FRCP PhD 
Professor of Clinical Diabetes and Metabolism 
Director, Institute of Molecular and Experimental Medicine 
Cardiff University School of Medicine 
University Hospital of Wales, Heath Park, Cardiff CF14 4XN 
Tel:(+44)  029 20 742182 
Fax:(+44)  029 20 744671 
Email: DayanCM@cf.ac.uk 
2.3.2 Principal Investigator 
Professor Johnny Ludvigsson MD PhD 
Div of Pediatrics, Dept of Clinical and Experimental Medicine, 
Linköping university 
SE-58185 Linköping, Sweden 
Tel +46 (0)13 28 6854 
Fax +46 (0)13 148265 
Email:Johnny.Ludvigsson@liu.se 
 
 
3 
BACKGROUND INFORMATION 
3.1 
Overview of the intervention in this protocol 
This protocol describes the use of the investigational medicinal product (IMP) C19-A3 GNP in 
a first-in-man study of safety of administration in patients with Type 1 diabetes. C19-A3 GNP 
comprises gold nanoparticles (GNPs) of size less 5nm covalently coupled to an 18-amino acid 
human peptide the sequence of which is identical to the residues from position 19 in the C-
peptide of proinsulin through to position 3 on the A-chain of the same molecule 
(GSLQPLALEGSLQKRGIV or using 3-letter designation:  H-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-
Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-OH). The peptide is synthesised with a linker to 
facilitate binding to the GNPs: 3-Mercaptopropionyl-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Glu-
Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-OH) 2 acetate salt (Disulfide bond). GNP C 19-A3 will be 
delivered via CE marked microneedles made by NanoPass.  
 
3.2 Background to approach to immunotherapy explored in the protocol. 
The challenge of glycaemic control with insulin in Type 1 diabetes and the need for 
immunotherapy.  
Type 1 diabetes is an autoimmune disease in which the insulin-secreting β-cells of the 
endocrine pancreas are destroyed to a sufficient extent that the patient relies on injection of 
exogenous insulin to adequately control blood glucose levels for the remainder of his/her life. 

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Most patients with Type 1 diabetes are unable to achieve levels of long-term metabolic 
control sufficient to avoid long-term complications, including loss of sight, renal failure and 
premature cardiovascular disease. Even with the most sophisticated insulin regimes, such as 
sensor augmented pump therapy, used in the setting of clinical trials, less than 30% of 
patients achieve an HbA1c < 7.0% (53 mmol/mmol)l(1, 2) and in the “real world” rates are 
typically less than 16%(3). The personal effort required to achieve these glycaemic targets is 
so high (4) that compliance is poor especially amongst young people (5). Although advances 
in insulin delivery systems continue to be made, there is little indication that they will be able 
to match the level of metabolic control and flexibility that can be achieved by functioning 
pancreatic beta cells(6). This is well illustrated by data from islet transplantation, in which 
restoration of even a small amount of beta cell function, results in complete correction of 
hypoglycaemia and excellent levels of HbA1c (< 7.0%, 53mmol/mol)(7). At diagnosis of Type 
1 diabetes, 90% of adults and 80% of children have sufficient residual beta cell function (peak 
insulin c-peptide > 200pmol/L)(8) to achieve these goals. Prospective studies show that this 
level of beta cell function, even if it declines or is lost over the next few years, is nevertheless 
associated with a 50% reduction in hypoglycaemia and a 40% reduction in the development 
of long-term complications over 9 years(9). Furthermore, evidence suggests that the “legacy” 
of such a period of good glycaemic control will result in a 50% relative reduction in retinopathy 
even 30 years later(10). Clearly, if this level of beta cell function were maintained for longer, 
the health gains would be even greater. Hence slowing the autoimmune process and thereby 
preserving the residual beta cell function present at diagnosis of type 1 diabetes, even for a 
few years, has the potential to deliver very major quality of life improvements (hypoglycaemia 
reduction) and long-term health gains (complications reduction). Importantly, since 
metabolic control delivered by functioning beta cells does not require high levels of 
compliance from patients, it is likely that these benefits could be realised in almost all 
individuals, at lower personal cost and inconvenience to patients than insulin delivery 
systems. 
An effective immunological therapy which could preserve beta cell function by preventing 
immune-mediated damage in type 1 diabetes would therefore represent an important 
advance in achieving improved glycaemia control. Such a therapy might also have application 
in individuals with long-standing diabetes by protecting new or regenerating beta cells from 
destruction by the immune system. Furthermore, such a therapeutic could also be used in 
“pre-diabetes” to prevent the development of clinical diabetes in individuals identified by 
autoantibody screening to be at high risk(11).  
 
Immunosuppression. Studies in the 1980s demonstrated that immunosuppressive treatment 
with cyclosporine can slow the loss of endogenous insulin production in newly diagnosed 
patients (including children) with type 1 diabetes but the toxicity was unacceptable (12-14). 
Since that time, two approaches have been taken to reduce toxicity: “smarter” 
immunosuppression and targeted immunoregulation. Of the immunosuppressive 
approaches, phase 2 efficacy has recently been reported for B- cell depletion with anti-CD20 
(rituximab) monoclonal antibody therapy(15), T cell modulation with anti-CD3 (otelixizumab, 
teplizumab)(16, 17), blockade of co-stimulation using CTLA-4 Ig (abatacept)(18) , LFA-2 

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(alefacept)(19), as well as antityhymocyte globulin and G-CSF(20). Although phase 2 studies 
with limited follow-up reported that these treatments were well tolerated, they are 
expensive, are likely to have to be given repeatedly and have the potential long-term risks 
associated with immunosuppression including opportunistic infection and increased cancer 
incidence(21). It should be noted that not all immunosuppressive regimes have met with 
success: recently negative results have been reported with the combination of 
mycophenolate mofetil and anti-CD25 (daclizumab) (22). Induction of remission has also been 
reported for the use of autologous stem cell transplantation (23), but controlled studies using 
the conditioning agents alone and replication of this approach in different centres are 
awaited. 
Immunoregulation. In recent years we have demonstrated the presence of T cells in humans 
specific for islet-derived peptides that have a regulatory (protective) phenotype (Treg). These 
cells secrete IL-10 or a combination of IL-10 and interferon gamma, are more prevalent in 
healthy controls and first-degree relatives than in patients with type 1 diabetes (24, 25), and 
have been shown to mediate immunoregulation (26). Hence expansion of these T cells or 
subsets with similar function would be expected to result in a slowing of the autoimmune 
process and preservation of beta cells. Amongst approaches to achieve such an 
immunoregulatory outcome, beta cell preservation with non-depleting anti-CD3 monoclonal 
antibody therapy has been extensively studied. The exact mechanism of action is 
uncertain(27) but the induction of CD8 regulatory cells has been postulated(28). Despite very 
promising results from initial phase II studies and evidence of efficacy over 4 years (16, 29, 
30), more recent results attempting to lower the dose to reduce toxicity or use alternative 
regimes have been less encouraging (31, 32). In parallel with these studies, others have taken 
a cellular approach to trying to increase levels of Tregs that might slow the autoimmune 
process. Bluestone and colleagues are currently reinfusing purified and expanded 
CD4+CD127lo/-CD25+ polyclonal Tregs into patients with newly-diagnosed type 1 diabetes 
(NCT01210664). Trucco and colleagues are exploring the administration of autologous 
dendritic cells stabilised in a tolerogenic state by antisense oligonucleotides in patients with 
type 1 diabetes(33). However, these studies are at an early stage, and even if they proved 
effective would require very substantial investment in facilities for cellular manipulation to 
enable patients to be treated in this way. 
Antigen-specific immunotherapy (ASI). In animal models, there is extensive evidence that 
antigen-specific immunotherapy (ASI) is possible (34, 35). In this approach, beta cell antigens, 
or peptides comprising T cell epitopes derived from them, are administered to animals either 
intranasally, subcutaneously or intraperitoneally, with the aim of inducing a regulatory 
response targeted to the beta cells, without immunosuppression(35-40). Evidence for the 
induction of antigen specific Tregs has been obtained in several experimental systems (35, 
41-44). In the majority of cases one, or a short series of treatments, was sufficient to protect 
the animal from disease for the rest of its life and it was also possible to reverse established 
disease at the time of disease onset (35, 37). Advantages of ASI over immunosuppressive 

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treatments or non-antigen targeted immunoregulatory approaches include lower long-term 
risk of infection and malignancy, lower systemic toxicity and lower cost; hence several groups 
have attempted to develop this approach for use in humans (reviewed in(44)). Many different 
approaches have been used including whole antigen alone or in adjuvant(45) (46) (47-49), 
given by the mucosal route (orally or nasally),  (50, 51) (52, 53) , DNA vaccination (54, 55) 
(http://www.bayhilltx.com)(55), the use of peptide epitopes or altered peptide ligands (56) 
(57), modified peptide epitopes that bind directly to HLA molecules without modification 
(“apitopes”, www.apitope.com) or preformed MHC peptide complexes(58), although the 
latter two approaches have not yet been applied to human type 1 diabetes.  These 
approaches have generally proved safe and very well tolerated, but sufficient efficacy to 
reliably reverse established disease in humans has proved difficult to generate(48, 51, 52, 59). 
Analysis of the antigen specific trials to date, suggests that this is less likely to be due to the 
particular antigen used, the dose or the route of administration, and more related to the 
context in which the antigen is presented to the immune system. 
Enhancing the efficacy of antigen specific immunotherapy – recruiting the potential of 
tolerogenic dendritic cells. Studies in animal models suggest that to achieve and maintain 
remission in established autoimmune disease via antigen-specific therapy, it is important to 
efficiently generate regulatory T cells specific for the antigen target(35).  In recent years, much 
work has been directed to determining the optimal conditions for the generation of 
regulatory T cells (60). It has become apparent that the status of the antigen presenting cells 
(APCs) that activate T cells is pivotal in this process. If the APCs express increased levels of 
MHC class II and costimulatory molecules (e.g. CD80, CD86) as well as key immunostimulatory 
cytokines such as IL-12 and IL-23, they generate T cells with proinflammatory properties(61). 
By contrast, expression of immunoregulatory cytokines such as IL-10 by APCs, promotes the 
induction of regulatory T cells (62-65). This effect can be further enhanced by the expression 
of certain surface molecules, such as ILT3(66), by the upregulation of Indoleamine-Pyrrole 
2,3,-Dioxygenase (IDO)(67) and surface CD155 activation by the T cell ligand TIGIT(68), as well 
as by increased levels of TGFβ and retinoic acid signalling(62). As a result, some groups are 
currently generating tolerogenic APC ex-vivo (from blood derived dendritic cells) and re-
infusing these into patients as a tolerogenic therapy (44, 69, 70). While this may prove very 
effective, this therapy will inevitably be costly and require GMP cell handling facilities in each 
clinical setting to deliver.  
Targeting antigen presentation through the superficial layers of the skin improves efficiency 
of antigen delivery. The skin has repeatedly been shown to be not only the most convenient 
but also the most efficient site for delivery of antigen to the immune system due to the very 
high density and specialised nature of its antigen presenting cells(71, 72). Recent studies have 
shown that not only is there an interlinked network of relatively immobile Langerhans Cells 
at the dermo-epidermal junction, but this is supplemented by very motile dermal dendritic 

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cells present in the superficial layers of skin just below the epidermis(73). The density of APCs 
reduces rapidly below this level, and hence antigen presentation is more efficient, the more 
superficially it is delivered in the skin. Consistent with this, intradermal immunisation requires 
less antigen than subcutaneous administration to achieve the same level of 
immunisation(74). Traditionally, intradermal immunisation has been achieved using angled 
hypodermic needle injection. However, a wide range of ultrashort needles (typically 
650micrometres in length or less) have now been developed which can deliver antigen into 
the superficial layers of the skin reliably with direct (perpendicular) injection. This approach 
has the advantage that it ensures intradermal rather than subcutaneous delivery ensuring 
high efficiency -  recent trials have confirmed that 10 fold less antigen than subcutaneous 
administration is required to achieve similar levels of immunisation(75).  
The use of gold nanoparticles to enhance uptake of antigen by dendritic cells and reduce 
inflammation.  Dendritic cells are particularly active in taking up particulate material and 
hence there has been increasing interest in the use of microparticles or nano-scale particles 
to further target antigen to DCs (76). For the current application, it is important that any 
particles used are both safe in humans and inert, since the generation of inflammation is likely 
to antagonise the development of regulatory T cells (76). Gold is an inert metal which has an 
extensive safety record dating back to its use as a therapy for rheumatoid arthritis. However, 
safety can be further enhanced by the use of very small nanoparticles (< 5nm) as these can 
be excreted through the kidney reducing the potential for accumulation with repeated 
therapy. Gold nanoparticles of this size have also been reported to have low toxicity in cellular 
assays(77).  
 
Antigen delivery to be used in the current protocol. In the current protocol, we will explore 
the safety of antigen specific immunotherapy using very small (< 5nm) gold nanoparticles 
coupled to a peptide of proinsulin C19-A3 which we have identified as being a naturally 
processed and presented T cell epitope of proinsulin that is frequently targeted in patients 
with type 1 diabetes who carry the susceptibility molecule, HLADRB1*0401 (~35% of the 
patient population)(24, 78). We have completed a phase 1A study of intradermal delivery of 
this peptide (alone) in patients with type 1 diabetes (24) up to doses of 100ug and no evidence 
of toxicity was observed (79). At the lower dose tested (10mcg) potentially protective IL-10 
responses to the peptide were induced in 5/18 subjects (79). We have also recently 
completed a phase 1b study of this peptide in new-onset type 1 diabetes patients giving more 
doses (12 versus 3), more frequently (every 14 days rather than monthly) with no evidence of 
toxicity other than transient local red reactions seen previously. The small size and high 
solubility of this peptide allows it to be easily coupled to small nanoparticles. In addition, it 
does not have the bioactivity of insulin and hence is free from the risk of hypoglycaemia 
associated with the intact molecule. In a future study, we plan to study the additional effect 
of linking delivery of these particles to IL-10 to reduce inflammation and promote the 
generation of regulatory T cells. We have termed the overall approach, “Enhanced Epidermal 
– Antigen Specific Immunotherapy” (EE-ASI). 

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Preclinical data 
Gold nanoparticles (GNP) were formulated with a thiol three amino-acid linker to covalently 
attach to the peptide proinsulin C19-A3 (C19-A3 GNP). Addition of C19-A3 GNP to human 
monocyte-derived immature dendritic cells did not induce maturation of these cells. 
However, following exposure to a maturation signal (lipopolysaccharide, LPS), dendritic cells 
treated with C19-A3 GNP released less cytokines – notably less TNF, IL-12 as well as IL-10. 
Presentation of the peptide in C19-A3 GNP particles to peptide specific T cells by these 
dendritic cells was as efficient as non-conjugated peptide.  
Intradermal injection of fluorescent nanoparticles via NanoPass 600micron microneedles into 
ex-vivo human skin resulted in accumulation of fluorescence in the upper dermis. Injection of 
GNP resulted in a more widespread distribution, including “reflux” into the epidermis. Flow 
cytometry of cells extracted from the epidermis following injection of fluorescently 
conjugated GNP into human skin, showed that over 98% of dendritic cells took up GNP as 
compared to 38% of non-dendritic cells.  
In murine studies, intradermal injection of peptide coupled GNPs resulted in antigen 
presentation in draining and distant lymph nodes and spleen within 3 days, as compared to 
peptide alone which was only presented in the draining lymph node. Studies with CCR7 
deficient mice, in which dendritic cells do not migrate from the skin to the draining following 
local stimuli, suggested that this distant dissemination of GNPs at least in part requires 
migration of dendritic cells, and is not wholly a fluid phase process. 
Taken together, this data provides evidence that the coupling of peptide to GNPs results in 
efficient  uptake and antigen presentation by dendritic cells, increased diffusion into the 
surrounding skin including epidermis (where there is a high density of dendritic (Langerhans) 
cells with regulatory potential), an anti-inflammatory effect on local DCs and enhanced 
dissemination of the antigen to distant lymphoid tissue. All of these features may be expected 
to increase the capability of GNP coupled peptide to induce a regulatory T cell response not 
only in skin at the site of administration but at distance sites including the pancreas draining 
lymph nodes. In addition, C19-A3 GNP has the potential in future studies to carry a dual cargo, 
for example a combination of peptide antigen with a “tolerogenic adjuvant”.  
 
3.3 Route of administration, dosage, regimen, and treatment period 
C19-A3 GNP will be administered intradermally via CE marked Nanopass 600nm 
microneedles. 3 doses will be given at 4 weekly intervals. The dose given will be equivalent to 
10ug of C19-A3 peptide.  
 
3.4 Sample size and population to be studied 
8 subjects aged 16 to 40 years with a diagnosis of Type 1 diabetes and some residual 
endogenous insulin (c-peptide) production will be included in the study. The Cardiff site will 
recruit subjects from the age of 18 years only and the Swedish site will recruit subjects from 
the age of 16 years, as the PI is an experienced paediatrician. Patients will be required to 
possess the HLA DRB1*0401 genotype and residual c-peptide production as evidenced by a 
stimulated urine c-peptide: creatinine level > 0.2  nmol/mmol or random serum sample level 

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of > 60 pmol/l. Residual c-peptide production is required to be able to monitor for disease 
exacerbation (ie unexpectedly rapid loss of insulin c-peptide production).  
 
3.5 
Conduct of trial 
The trial will be conducted according to the protocol and in compliance with the principles of 
the Declaration of Helsinki (2013), the principles of Good Clinical Practice (GCP) and in 
accordance with Medicines for Human Use (Clinical Trials) Regulations 2004, as amended in 
2006, the Research Governance Framework for Health and Social Care, the Data Protection 
Act 1998 and other regulatory requirements as appropriate. The Protocol will be submitted 
for approval by an NHS Research Ethics Committee (REC), UK and Regional Ethical Review 
Board (RERB), Sweden.  
 
4 
TRIAL OBJECTIVES AND DESIGN 
4.1 Trial Objectives 
The primary objective of the study is to examine the risk of C19-A3 GNP administration in 
terms of general safety and induction of hypersensitivity.  
 
Secondary objectives are: 
• To study the feasibility of delivering C19-A3 GNP via microneedles to humans. 
• To study the size and nature of immune responses to C19-A3 GNP generated in blood 
and the draining (axillary) lymph node. 
 
4.2 Trial Design 
The study is a two centre, open-label, uncontrolled single group phase 1A study of C19-A3 
GNP  peptide (10 μg peptide equivalent content) administered via Nanopass microneedles 
every 28 days for 8 weeks (3 doses), with follow-up for 12 weeks (20 weeks in total from first 
dose). Treatment will be given into the arm at a volume of 50ul. 
 
4.3 Primary endpoint 
Assessment of the safety of C19-A3 GNP administration in terms of adverse events at the dose 
used. 
 
4.4 Secondary endpoints 
• T cell responses to C19-A3 GNP as determined by changes from baseline of interferon 
gamma and IL-10 ELISPOT responses to this peptide in blood following treatment at weeks 
0, 9 and 14. 
• T cell responses to C19-A3 GNP as determined by changes from baseline of interferon 
gamma and IL-10 ELISPOT responses to this peptide in draining axillary lymph node before 
treatment and following the last treatment administration. 
• Changes in additional immunological biomarkers (e.g. flow cytometry profiles, T reg 
assays, autoantibodies, beta cell and T cell free DNA markers) from baseline at week 0, 9 
and 14.  

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• Effects on residual insulin requirement and c-peptide secretion at week 14 as compared 
to baseline as assessed by a mixed meal tolerance test and a stimulated urine c-peptide 
test. 
• Effects on glycaemic control assessed by blood sugar profiles and HbA1c at week 14 as 
compared to baseline 
• Questionnaires on quality of life and diabetes self-management.  
 
Trial Flowchart 
The schedule of visits for the study is shown in Table 1. Figure 1 shows the steps from 
ascertainment to first injection.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table 1: Schedule of study visits  
 
Screening 
 
Treatment 
 
Follow up 
Optional 
Visit number 
-2 
-1 
Pre 
V1 
1 
1b 
1C 
2 
3 
3b 
3C 
4 
Pre V5 
5 
6 
7 
any     
             time   
              post  
             dosing
Weeks 
These visits can 
be 
combined. 
Treatment must 
start within 3 
months 
of 
screening 
commencing1 
A 
mini
mum 
of 72 
hour
s 
prior 
to 
visit 
1 
0 
1 day 
post 
visit1 
3 to 
5 
days 
post 
visit 
1 
4 
8 
1 day 
post 
visit3 
3 to 5 
days 
post 
visit 3 
9 
A 
minim
um of 
72 
hours 
prior to 
visit 5 
14 
20 
52 
Days 
0 
28 
56 
63 (or 
one 
week 
post 
final 
injecti
on) 
98 
140 
(85 
days 
post 
final 
dose
) 
366       
(311        
days      
post.   
final 
dose  
+/- 14 
days) 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study drug administration 
 
 
 
X 
 
 
X 
X 
 
 
 
 
 
 
 
Follow up telephone call 24 
hours post IMP 
 
 
 
X 
 
 
X 
X 
 
 
 
 
 
 
 
Physical Exam & ECG 
X 
 
 
X 
 
 
X 
X 
 
 
 
 
X 
 
X 
2Serum for AAbs and immune 
markers 
 
 
 
X 
 
 
 
 
 
 
X 
 
X 
 
X 
DRB1*0401 genotype 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MMTT  
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
X 
Photographs at injection site 
 
 
 
X 
 
 
X 
X 
 
 
X 
 
X 
X 
X 
Blood sample to accompany 
skin studies (optional) 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
Stitch removal from biopsy site 
(optional) 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
urine or random serum c-
peptide post meal 
 
X 
 
X 
 
 
 
 
 
 
 
 
X 
 
X 
72 hour continuous glucose 
monitoring 
 
 
Attach 
CGM 
X 
 
 
 
 
 
 
 
Attach 
CGM 
X 
 
X 
Serum for Cystatin C 
 
 
 
X 
 
 
 
 
 
 
X 
 
X 
X 
X 
Urinalysis for blood, protein, 
and beta-2 microglobulin 
 
X 
 
X 
 
 
X 
 
 
 
X 
 
X 
X 
X 
Serum and urine (morning) 
sample 
to 
measure 
gold 
concentrations 
 
 
 
X  
pr
e 
do
se 
X2 
 
 
 
X2 
 
X 
 
X 
X 
X 
T cell (immune biomarker) 
assays (blood) 
 
 
 
X 
 
 
 
- 
 
 
X 
 
X 
 
X 
3LN aspiration 0/3-5d 
 
 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
3Safety blood samples 
 
X 
 
X 
 
 
X 
 
 
 
X 
 
X 
X 
X 
HbA1c measurement 
 
X 
 
X 
 
 
X 
 
 
 
X 
 
X 
 
X 
Vital signs 
X 
X 
 
X 
 
 
X 
X 
 
 
X 
 
X 
X 
X 
Adverse Event assessment/con 
meds 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urine pregnancy test 
 
X 
 
X 
 
 
X 
X 
 
 
 
 
X 
X 
 
Record of insulin use 
X 
 
 
X 
 
 
X 
X 
 
 
 
 
X 
 
X 
3Blood glucose profile/insulin 
usage 
 
 
 
X 
 
 
X 
X 
 
 
 
 
X 
X 
X 

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Screening can be up to 3 months 
Treatment visits 1, 2 and 3 should be every 28 days +/- 3 days and follow up visit 5 should be at 14 weeks +/- 1 week 
1If dosing is delayed beyond 3 months, all test other than genotype should be repeated.  
2Samples to be taken post dose. 
3Details supplied in outcome assessment section 6 and safety assessment section 10. 
4 Contact hypersensitivity testing will be done once post dose.  It involves 3 visits (the 2nd visit can be done at home). 
5 These tests will be done in subjects with a persistent reaction (optionally, post-dose). 
 
 
Figure 1. Flow chart of ascertainment, recruitment and screening through to IMP administration. 
 
Recruiting clinical sites 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Assessment 
of 
glycaemic 
control/review 
blood 
sugar 
recordings  
 
 
 
X 
 
 
X 
X 
 
 
 
 
X 
 
X 
Skin biopsy (optional)5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                 X 
Contact hypersensitivity testing 
(optional)4,5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                 X 
Suction blister (optional)5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                 X 
XHFS survey 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
ADDQoL 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
X 
DTSQs 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
X 
DTSQc 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Cardiff 
Linköping 
Not DRB1*0401 and/or post 
meal UCPCR <0.2 nmol/mmol or 
random serum c-peptide  <60 
pmol/l. 
DRB1*0401  
and post meal UCPCR >0.2  
nmol/mmol or random serum 
c-peptide > 60 pmol/l. 
Exclude 
Confirm diagnosis of type 1 
diabetes for > than 3 months  
Proceed to 1st 
injection/visit 1 
Confirm that subject meets all 
inclusion/exclusion criteria  

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5 
TRIAL MEDICATION 
5.1 
IMP 
The IMP will be delivered as sterilised solution in small vials produced by Baccinex SA 
(Courroux, CH), and stored, QP released and shipped to the clinical sites by PharmaKorell 
GmbH (Lörrach, Germany). 
The vials should be stored refrigerated at 2-8 °C. 
The IMP will be 10ug of C19-A3 peptide Gold NPs injected by Nanopass microneedles, 
delivered intradermally into the upper arm, in 50ul volume.  
  
5.2 
Dosing regimen 
C19-A3 GNP will be administered intradermally via CE marked Nanopass 600nm 
microneedles. 3 doses will be given at 4 weekly intervals. The dose given will be equivalent to 
10ug of C19-A3 peptide. Thus the cumulative dose will be 30ug. 
 
5.3 
Drug accountability 
The will arrange transfer of IMP to local IMP Storage and Supply Sites.  The local Site Pharmacy 
will then take responsibility for IMP accountability by ensuring that: the IMP is stored in a 
secure location, segregated from other medicines; used and returned medication is kept 
separate from unused medication; storage conditions are monitored and recorded; IMP is 
dispensed to participants in accordance with the trial protocol; unused medication is returned 
to the study team or disposed of as authorised by the study team. Full accountability records 
will be kept for all aspects of IMP handling. IMP accountability records will be monitored by 
the Sponsor. 
 
5.4 
Subject compliance 
Patients will be allowed 28 days +/- 3 days between treatments, but every effort should be 
made to dose every 28 days. Where a visit is > 3 days late it should be documented as a 
protocol violation and scheduled as soon as possible, with subsequent dosing visits 
recommenced on a 28 day schedule after this. Where the interval between 2 doses is > 42 
days, the dose will be considered to have been missed and the subsequent dose should be 
given. Subject attendance and compliance will be recorded. If a treatment is missed the 
subject will continue in follow-up but an additional subject will be recruited, so that a total of 
8 subjects will receive all 3 doses.  
Patients will be allowed to attend for follow up visit 4 (week 9 or one week post final injection) 
+/- 1 day of schedule, visit 5 (week 14), +/- 1 week of visit schedule and visit 6 (week 20) must 
be a minimum of 85 days post final dose/+2 weeks.  There will also be an optional visit 7 (week 
52) at a minimum of 311 days post final dose +/- 2 weeks. There will be an optional skin blister, 
an optional skin biopsy and an optional contact hypersensitivity testing performed anytime 
post-dose (referring to post final dose).  Any visits outside of this guidance will be considered 
a protocol deviation.  

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Figure 2 Chart indicating dosing schedule  
 
 
 
 
 
                0                4                  8               20              52 weeks 
screening                treatment                      F/U 
 
 
5.5 
 Dosing strategy: 
This is an open label uncontrolled early phase safety study, so no blinding or 
randomisation will be performed. In keeping with standard phase 1 study designs, no 
placebo or control group is included as the primary aim is to establish whether there 
are any major unexpected safety issues in the use of this IMP for the first time in man. 
 
5.6 
Concomitant medication 
Patients will be supported to receive optimal care during the study, to include daily 
insulin injections. Routine vaccinations (flu and others) should be avoided for the 
duration of the study unless clinically essential (eg during periods of high risk flu 
exposure). Use of β-cell stimulants (e.g. sulphonylureas), glucagon-like peptide-1 
agonists, dipeptidyl peptidase-IV inhibitors, insulin sensitisers (e.g. metformin, 
thiazolidinediones) that 
may 
affect 
insulin 
production, 
and the use 
of 
immunosuppressive or immunomodulatory therapies (including systemic but not 
topical or inhaled steroids) which may modulate immune responses is not permitted. 
Use of other drugs with potential effects on the immune system, insulin sensitivity or 
β-cell function, where these are considered clinically essential for treatment of 
concomitant medical conditions, will be discussed with the Chief Investigator. See also 
6.3 
 
Key:  Dosing  

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5.7 
Concomitant health issues 
Study drug should not be administered if a subject has had a febrile illness within the 
last 3 days as this may activate T cells non-specifically. Under these circumstances, the 
missed study visit should be delayed to the earliest next feasible date (but no longer 
than 14 days) and the study continued. 
 
Females will be advised not to become pregnant within 85 days following the date of 
their final treatment of IMP and will be advised to continue using adequate 
contraception until this time point See section 6.2 
Males will be advised not to father a child within 85 days following the date of their final 
treatment of IMP. 
 
5.8 
Measures to avoid bias 
The study will be open label. Assessment of immune responses will be conducted 
blinded from metabolic parameters in the first instance. All samples will be labelled with 
a study ID only, and laboratory staff will know whether samples are from the same 
subject but not the identity of the subjects.  
 
5.9 
Timescales  
5.9.1 Proposed Trial Start: July 2016 
 
5.9.2 Projected Trial Completion (amended): January 2019 
 
5.9.3 Trial Duration: 26 months 
 
5.9.4 Duration of Each Patient’s Participation: 12 months (52weeks excluding 
screening), the last visit (visit 7) is optional and if the participants choose not to 
take it the duration would be 5 months (20 weeks excluding screening), there will 
be three optional tests performed post final dose 
 
6 
SELECTION AND WITHDRAWAL OF SUBJECTS 
6.1 
Study population 
8 subjects with a diagnosis of Type 1 diabetes will be included in the study. Autoimmune 
diabetes will be confirmed by measurement of islet cell autoantibodies. Patients will be 
required to possess the HLA-DR4 (B1*0401) genotype. 
 
6.2 
Inclusion criteria 
1. Clinical diagnosis of type 1 diabetes for > 3 months (dated from the first insulin 
injection). 
2. Commenced on insulin treatment within 1 month of diagnosis. 
3. Age 16 to 40 years  

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4. 2 hour post-meal UCPCR > 0.2   nmol/mmol or random serum sample > 60 pmol/l on 
at least one occasion (maximum 3 tests on different days) 
5. Possession of  0401 allele at the HLA-DRB1 gene locus 
6. The following birth control methods should be used (considered highly effective with 
a failure rate of less than 1% per year when used consistently and correctly]: 
• combined (estrogen and progestogen containing) hormonal contraception 
associated with inhibition of ovulation: 
➢ oral 
➢ intravaginal 
➢ transdermal 
• progestogen-only hormonal contraception associated with inhibition of ovulation: 
➢ oral 
➢ injectable 
➢ implantable  
• _ intrauterine device (IUD)  
• _ intrauterine hormone-releasing system ( IUS) 
• _ bilateral tubal occlusion 
• _ vasectomised partner (provided that the partner is the sole sexual partner of the 
trial participant and that medical assessment of azoospermia has been confirmed) 
• Sexual abstinence (defined as refraining from hetrosexual intercourse during the 
duration of the trial) 
7. Written and witnessed informed consent to participate. 
 
 
6.3 
Exclusion criteria 
1. HbA1c > 86mmol/L (10%). 
2. Females who are pregnant, breast-feeding or not using adequate forms of 
contraception. 
3. Previous diagnosis of renal disease including glomerulonephritis or nephropathy. 
4. Raised serum creatinine or abnormal urine albumin/creatinine ratio (ACR) (values 
above the laboratory reference range). If the initial ACR is raised, this should be 
repeated on two further occasions as first morning samples. The subject can be 
included if both of these samples are negative (within the reference range).  
5. Use of immunosuppressive or immunomodulatory therapies, including systemic 
steroids within 1 month prior to receiving the IMP and any monoclonal antibody 
therapy given for any indication.  Note that previous exposure to proinsulin peptide 
C19-A3 in a clinical trial is an exclusion criterion. 
6. Use of cannabis within one month prior to trial entry. 
7. Use of any hypoglycaemia agents other than insulin, for more than 6 weeks, at any 
time prior to trial entry. 
8. Use of inhaled insulin. 
9. 
Known alcohol abuse, drug abuse, HIV or hepatitis. 

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10. Allergies to drug components or any excipients. 
11. Any other medical condition which, in the opinion of investigators, could affect the 
safety of the subject’s participation or outcomes of the study, including 
immunocompromised states and autoimmune conditions. 
12. Subjects should not have had immunisations (flu and others) for 1 month prior to trial 
entry and should not receive any during their time in the trial – see section 5.6. 
13. Recent subject’s involvement in other research studies which, in the opinion of 
investigators, may adversely affect the safety of the subjects or the results of the 
study. 
14.   Abnormal ECG findings. 
 
6.4 
Recruitment and informed consent  
             Subjects will be recruited into the study according to the following steps: 
 
1. Patients with type 1 diabetes will be identified by their local diabetes teams (hospital 
doctors or diabetes nurses), from clinic records, during clinic visits or inpatient 
admissions. 
2. Some patients may self-refer and contact the trial team directly, from seeing internet 
information, news articles or posters regarding participating in diabetes research, or 
be identified by the ADDRESS-2 registry of recently diagnosed patients with type 1 
diabetes. Patients who self-refer will have their details passed to the local coordinator 
of the study or research nurse/doctor and will be contacted initially by phone, email 
or in person by the local centre research team who will outline and explain the aim of 
the study and provide copies of the Participant Information Sheet (PIS). 
3. For subjects identified by health records or clinical contacts, the study will be 
discussed with the identified patients by a member of their clinical care team and, 
with the subject’s agreement, details will be passed to the local coordinator of the 
study. 
4. Subjects who have consented to be on the ADDRESS2 registry and consented to be 
contacted regarding research that they may be eligible to take part in, will be 
contacted initially by the lead centre. 
5. Subjects will then be contacted by phone, email or in person by the local centre 
research team who will outline and explain the aim of the study and provide copies of 
the Participant Information Sheet (PIS). 
6. Subjects will have a minimum of 24 hours to consider this information and usually at 
least 5-7 days. Subjects will be invited to call the local research nurse or Local Principal 
Investigator for any queries about the PIS and to indicate if they wish to take part. 
7. An appointment will then be made for interested subjects, at which the Local Principal 
Investigator or their appointed deputy can check the subject’s understanding of the 
study and obtain written consent. Subjects may then take part in assessments 
required for the first screening visit (visit -2). 

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8. If the subject has the HLA DRB1*0401 genotype then the local research team will 
arrange an appointment with him/her for the second screening visit (visit -1) which 
will include a post meal UCPCR or random serum c-peptide, blood samples and urine 
pregnancy test (if female). 
9.  If appropriate, screening visits -2 & -1 can be combined.  
10. Subjects who fulfil all inclusion criteria without exclusion criteria will be informed of 
their screening results, and arrangements made for enrolment and the first study visit.  
11. A schedule of study visits and procedures carried out at each study visit are shown in 
table 1. 
12. Where the start of dosing is delayed more than three months beyond screening visit -
1, all the tests and physical assessment required at screening (excluding genotyping) 
should be repeated to confirm continues eligibility.  
 
6.5 
General assessments 
General assessments for this protocol are: 
• Inclusion/exclusion criteria 
• Medical history 
• Physical examination & ECG recording 
• Concomitant medications 
• Adverse event recording 
• Record of insulin usage 
• Assessment of glycaemic control 
• Quality of life questionnaires 
• Photographs at injection sites at each visit 
 
6.6 
Follow up assessments 
The primary outcome measure for the trial is the safety (adverse event) profile of this 
investigational agent.  
 
The key components of the follow-up visits are:  
1. Diabetes assessments; 
2. Adverse events; 
3. Any changes in medications; 
4. Review of clinical safety blood tests. 
 
6.7 
General laboratory tests 
A study specific laboratory manual will be provided and the following general laboratory 
assessments will be performed during the study: 
 

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• Full blood count; urea, electrolytes and creatinine; liver function tests (prothrombin 
time, total bilirubin, total protein, albumin, AST (SGOT), SGPT (ALT), alkaline 
phosphatase; thyroid stimulating hormone; immunoglobulins (G, A, M); calcium; 
magnesium, phosphate, lipid profile (total cholesterol, LDL, HDL, triglyceride). These 
will be tested in the local laboratories of the study sites.   
• Urine pregnancy test (females only, at all trial visits) 
• Urinalysis for pH, blood and protein by dipstick urinalysis and laboratory analysis for 
albumin/creatinine ratio, urine beta-2-microglobulin and cystatin-C. 
• Autoantibodies against insulin, GAD-65, IA-2 and ZnT8 (performed in the Diabetes 
Research Unit Cymru Laboratories, Swansea University) 
• Anti-drug (GNP-peptide C19-A3) antibodies 
6.8 
Study Drug Administration  
Study subjects will attend a clinical research facility or hospital ward (Linkoping) for receiving 
the IMP. Staff will be experienced in CTIMPs and facilities for managing a severe reaction will 
be to hand (see section 10.2). The drug will be prepared and injected intradermally in the 
upper arm of the patient in a 50ul volume, according to the schedule (see figure 2). 
 
All subjects will be observed 6 hours after their first dosing/injection, however the first subject 
will be dosed at least 24 hours before subsequent subjects as per sentinel dosing strategy 
 (see risk mitigation strategy section 7). If no SAEs are observed after this first dose, all 
subjects will be observed for a minimum of one hour after each injection and up to 2 hours if 
there are any signs of hypersensitivity. All local and systemic reactions will be documented.  
 
6.9 
Metabolic outcome assessments 
Metabolic assessments will consist of: 
• Glucose records and reports of hypoglycaemia (clinical care measurement) 
• Insulin dose (clinical care measurement) 
• HbA1c level (laboratory test) 
• Urine or random serum post-meal c-peptide/creatinine ratio (laboratory test) 
• Mixed meal tolerance test (MMTT) (laboratory test) 
Laboratory measurements for these will be performed in the laboratories of the Diabetes 
Research Unit Cymru laboratory, Swansea University.  
 
6.10 Mechanistic immunological studies 
Mechanistic assessments will consist of: 
• Ultra-sound guided Axillary LN aspiration and ELISPOT data before and 3-5days after 
administration of the first dose and the final dose of IMP (total of 4 aspirations for 
each patient)  

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• T cell assays to include enzyme-linked immunospot (ELISPOT) to measure T cell 
responses to PPI C19-A3 and other islet cell autoantigenic peptides using LN cells and 
fresh whole heparinised blood sent by courier to the assay laboratory 
• Storage of peripheral blood mononuclear cells (PBMCs) for subsequent analysis of T 
cell responses (measurement of Tregs and antigen-specific T cells by tetramer and 
Diab-Q-kit) 
• Storage of DNA for subsequent analysis of gene-phenotype interactions 
• Storage of serum for islet autoantibody  assessments and cell free DNA measurements  
• Additional mechanistic assays by the T1D UK Consortium Mechanistic Core 
 
6.11 General guidance on glycaemic control during the study 
Glycaemic control will be maintained according to clinical guidelines and conducted in 
collaboration with the patient’s diabetes health care team. 
• HbA1c will be measured every 1-3 months with a target value set by agreement with 
the patient and their clinical care team. Where this target is not met, advice will be 
given as clinically required. 
• Glycaemic control will be reviewed at every study visit. 
• Where insulin requirements fall to less than a total of 8 IU per day, continuation of 
insulin therapy should be discussed with the Chief Investigator: continuation of a low 
dose of insulin where possible is considered preferable.  
 
6.12 Ultrasound guided Fine Needle Aspiration of axillary lymph node 
Ultrasound guided Fine Needle Aspiration of axillary lymph node (US-guided FNA) will be 
performed by an experienced consultant radiologist, before and after Visits 1 and 3. 
 
The procedure is as follows: Under aseptic technique and ultrasound guidance and following 
local anaesthetic, a 21G needle is inserted into an axillary lymph node and few drops of fluid 
aspirated and sent for immunological studies (as in section 6.10). 
 
6.13 Optional Skin biopsy and blister sampling.  
To determine how long the gold nanoparticles stay in the skin and the immunological 
effects, subjects will be invited to undergo an optional skin biopsy and an optional suction 
blister. In order not to disturb the systemic effects of nanoparticle administration, skin 
sampling will not be performed until after visit 3 (post final dose). For the optional skin 
biopsy, a 4mm skin biopsy using standard dermatological techniques will be taken at the site 
of the first injection (ie 2 months after the final dose) to determine residual gold 
distribution.  For the optional suction blister, a standard suction blister sample (82,83) will 
be taken from the site of the second injection (ie 1 month after the final dose) to study any 
infiltrating lymphocytes (83).   

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Skin biopsies will be taken by a 4mm standard punch technique. The material obtained will 
snap frozen for immunohistochemistry and gold staining and part stored for RNA extraction 
and PCR/RNA seq analysis.  
Skin suction blisters will be performed by gradually applying negative pressure (up to 50 
kPa) from a suction pump machine VP25 (Eschmann, Lancing, UK) through a suction blister 
cup with a 15-mm hole in the base for 2–6 hr until a unilocular blister had formed within the 
cup. The blister fluid is then aspirated after blister formation and spun to obtain any 
infiltrating lymphocytes. These will be analysed for reactivity to the administered peptide by 
ELISPOT and for surface molecule expression by cytospins and fluorescence microscopy if 
sufficient sample in obtained.   
 
 
6.14 Photographs at injection sites 
After the injection it is possible, in some patients, to see the gold as a small grey mark under 
the skin less than 5mm across and this may still be there for 3 months or more after the 
injection. In some individuals there may be a painless red area around the grey mark which 
may also stay there for 3 months or more.  We intend to take photographs of the injection 
sites at each visit to compare the marks at subsequent visits.  
6.15 Contact hypersensitivity or skin patch testing 
Contact hypersensitivity testing or patch testing is to determine delayed Type 4 
hypersensitivity to allergens including gold for this study.  We would like to check for 
development of hypersensitivity to gold in patients who received trial medication. This is an 
optional test performed anytime post-dose (referring to post final dose).  The test itself can 
involve 3 visits.  For the participant’s convenience, visit 2 can done at home by the patient.  
These visits include day 0 (patches applied to back or arm of patient), day 2 (patches taken 
off (home or clinic) and first reading (if in clinic)) and final reading (day 4 or day 7).  We are 
looking for red, raised, itchy, vesicular reactions to detect contact allergy and these results 
are graded according to internationally agreed standards. The allergens applied have to be 
diluted to correct dilutions considered suitable by experts for patch testing and application 
to patients’ backs. The patient needs to keep the back (or other site of application for 
example the arm) dry during the whole test period and be prepared to attend all 3 visits to 
perform the test.  Participants will be asked for blood sample (5ml) to measure serum gold 
concentration before the test.  
 
6.16 Sample management 
Sample management will be carried out according to GCP and according to the approval of 
the Research Ethics Committee. 
 

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6.17 Specimen handling  
Samples for local laboratory assessment may be taken at a time convenient for same day 
processing by the local laboratory. Samples for immunologic or metabolic studies, HLA typing 
or autoantibodies will be drawn before 12.00 and sent by approved courier to the appropriate 
laboratory or stored locally for later analysis. At dosing visits blood draw will take place prior 
to administration of the IMP.  
 
 
 

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Figure 3 Blood draw schedule: 
  
Screening 
Treatment 
Follow-up 
 
 
Visit no: 
-2 
-1 
1 
1b 
2 
3 
3b 
4 
5 
6 
7 
post 
Weeks 
  
  
0 
  
4 
8 
  
9 
14 
20 
52 
final  
Days 
  
  
0 
1 
28 
56 
57 
63 
98 
140 
366 
dose 
Safety blood 
samples HbA1c & 
glucose 
  
11 
  
  
11 
  
  
11 
11 
11 
 
11 
 
DRB1*0401 and 
A*02 
2 
  
  
  
  
  
  
  
  
  
 
 
Serum for Aabs 
2 
  
  
  
  
  
  
  
  
  
 
 
Serum for insulin 
cell free DNA 
8 
  
  
  
  
  
  
  
  
  
 
 
Serum for: Aabs, 
cystatin-c, insulin 
cell free DNA, 
other 
  
  
10 
  
  
  
  
10 
10 
10 
 
10 
 
 
MMTT 
  
  
25 
  
  
  
  
  
25 
  
 
 
Blood to 
accompany skin 
biopsies 
 
 
 
 
 
 
 
 
 
 
 
 
20 
Serum for gold 
concentrations 
(morning urine 
also to be sent) 
  
  
1 
1 
  
  
1 
 1 
1  
1 
 
 1 
 
 
PBMC's T reg 
assay, T cells, CD4 
& CD8 tetramers, 
Elispots & biobank 
storage. 
  
  
64 
  
  
  
  
64 
64* 
  
 
 
64 
 
Flow cytometry 
  
  
2 
  
  
  
  
2 
2 
  
 
2 
 
Serum for gold 
concentration 
before contact 
hypersensitivity 
testing 
 
 
 
 
 
 
 
 
 
 
 
 
 
1 
Total 
12 
11 
102 
1 
11 
20 
1 
88 
113 
22 
113 
1 
*in subjects with immune responses of interest visit 4,  an additional blood sample up to 70mls (35mls for under 18s) may be requested 
between visit 4 and visit 6.  
 

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Volumes for the study within an 8-week period coincide with guidelines of the National Institute of Health (ie blood collection should be 
<550ml per 8-week period for adults, and no more than 5ml/kg in a single day and no more than 9.5ml/kg in any 8 week period for under 
18’s) and are those used by the Type 1 diabetes clinical trial consortium, Type 1 Diabetes Trial Net (www.diabetestrialnet.org). 
 
 
Figure 4 Sample management flowchart 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6.18 Laboratory procedures 
6.18.1 Stimulated secretion of C-peptide 
For screening, a post-meal random serum sample in the clinic or urine c-peptide/creatinine 
ratio will be measured. Patients are provided with a suitable container (boric acid 
preservative) and asked to void their bladder (and discard this urine) prior to their first meal 
of the day. They are then asked to substitute their first meal of the day with Ensure Plus and 
asked not to pass any urine until 2 hours after the Ensure Plus meal and take a sample of urine 
into the container at this time. These samples will be assayed in the laboratory in Diabetes 
Research Unit Cymru Laboratories in Swansea for urine c-peptide and creatinine.  
Li.U 
Blood, urine, skin & 
lymph node samples 
Cardiff 
Blood, urine, skin & 
lymph node samples 
Cardiff 
Cardiff samples 
returned to 
Cardiff for storage 
Li.U 
Li.U samples 
returned to Li.U 
for storage 
Local lab - Cardiff 
Local lab - 
Linkoping 
Diabetes Research Unit 
Cymru Laboratory (DRUC 
Swansea) 
Midatech Pharma 
Laboratory, Spain 
LUMC (Leiden 
University Medical 
Centre) 
King’s College 
London 
Bristol 
University 
Cardiff 
Queen 
Mary 
University 
London 

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During the study secretion of c-peptide will also be tested using the Mixed Meal Tolerance 
Test method (MMTT). Briefly, the test is performed in the morning (between 7 and 10 AM). 
The participant must be fasting and have had no food or drink (with the exception of water), 
and no smoking is permitted from 12 (midnight) or 8 hours prior to the start of the test. Tests 
should be conducted only if fasting value by capillary blood glucose meter is between 3.9-
11.1mmol/L. Participants should withhold taking long acting insulin on the morning of the 
test. They can take very short acting insulin (e.g. Humalog, Apidra, or Novorapid) up to 2 hours 
before the test. They can take regular insulin up to 6 hours before the test. If the subject is 
using an insulin pump, they should be advised to continue their basal regime but not have a 
bolus.(80) 
The procedures are as follows. Start IV line. Obtain baseline blood samples at 0 minutes, and 
any other test samples required at that visit (e.g. HbA1c, T cell studies, PBMCs islet cell 
autoantibodies). For each time point blood samples are 1.2 ml fluoride tube for glucose and 
1.2 ml serum tube for C-peptide. In addition, for each test the first sample (0 minutes) and 
the last sample (120 minute for MMTT) will be used to obtain a meter ketone reading, which 
is recorded. A first blood sample is drawn immediately before the participant starts drinking 
the liquid meal (formal “baseline samples”). The patient is given the standardized liquid meal: 
Ensure Plus 6 ml/kg (Maximum 360ml) to be ingested within 5 minutes. Blood samples are 
drawn at times: 15, 30, 60, 90 and 120 minutes, after the end of ingestion of Ensure Plus 
(note: time runs from the start of ingestion).  After the test is completed, the participant eats 
and receives insulin as appropriate and prescribed by the local investigator. A urine sample at 
the 120 minute time point will also be collected for c-peptide creatinine ratio as an alternative 
marker of insulin production. 
6.18.2 Mechanistic assays for the assessment of immune biomarkers 
6.16.2.1 Cytokine ELISPOT 
Cytokine ELISPOTS for the detection of IFN-γ and IL-10 producing CD4 T cells in response to 
PPI C19-A3 and other islet autoantigenic peptides will be performed as previously described 
(24, 79, 81). These assays provide a means to monitor potentially favourable changes in the 
immune response to PPI C19-A3 (eg a shift from IFN- to IL-10 secreting cells). These will be 
performed on both peripheral blood mononuclear cells and LN aspirate cells as well as on skin 
derived leukocytes where an optional skin blister aspiration sample is provided.  
6.18.2.2 Storage of peripheral blood mononuclear cells for additional T and B cells assays 
and immune gene profiling 
Blood samples will be taken at 0, 9 and 14 weeks and the peripheral blood mononuclear cells 
cryopreserved as well as samples stored for later DNA analysis. These will be performed by 
the T1D UK Mechanistic Core laboratories.  
6.18.2.3 Storage of serum for islet autoantibody and cell free DNA studies.  
Blood will be taken at 0, 9 and 14 weeks and spun down for serum which will be aliquoted 
and frozen at -80C. Sample will be stored for later islet autoantibody and cell free DNA 

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analysis. Islet autoantibodies to GAD, IA-2 and ZnT8 will be measured in the Diabetes 
Research Unit Cymru Laboratories, Swansea University and cell free DNA assays (for beta 
cell and T cell apoptosis) will be performed via the T1D UK Mechanistic Core laboratories.  
6.18.3 Analysis of skin biopsies.  
In individuals who agree to provide a skin biopsy from the first injection site anytime post-
dose, this material will divided for immunohistology/gold distribution analysis and RNA 
extraction for RNA seq/QPCR.  
7 
Risk Mitigation Strategy 
Communication between trial sites. The clinical research teams of the two sites involved 
will be in weekly telephone contact (teleconference) during the recruitment and dosing 
periods. All adverse reactions will be discussed and information shared. 
All serious adverse events will be shared within 24 hours with the other recruiting site. 
Sentinel dosing. The first patient dosed with GNP-peptide will be observed for 6 hours post 
dose. Temperature, blood pressure and heart rate will be recorded at the following intervals 
post injection: 15 mins, 30 mins, 45 mins, 1 hour, 1 hour 30mins, 2 hours, 2 hours 30 mins, 3 
hours, 4 hours, 5 hours and 6 hours. The injection site will be observed at the following 
intervals post injection: 5mins, 10 mins, 15 mins, 30 mins, 45 mins and 1 hour. A follow-up 
phone call will be made to the subject 24 hours after the first dose. No additional patients will 
be able to be recruited until this 24 hour period is completed (“lock-out period”).  
If no serious adverse events are observed, subsequent subjects will also be observed for 6 
hours after their first injection. If no serious adverse events are observed, then subjects will 
be observed for a minimum of 1 hour post dose, or 2 hours if there are any symptoms of signs 
of hypersensitivity. This time period has been chosen as the only anticipated severe reactions 
is one of hypersensitivity which, if systemic, would be apparent within 60 mins of intradermal 
dosing. See section 10.2 for further discussion of hypersensitivity.  
Each subject will be contacted by telephone 24 hours after each dose. 
The trial manager will communicate with clinical staff by email and telephone to keep both 
sites up to date with screening and recruitment activity. Weekly teleconferences between 
sites will take place during the screening and recruitment phase and the trial manager will 
inform clinical staff at both sites when the first patient is going to be dosed and report to 
them afterwards regarding whether any AE’s were experienced. This will negate the 
possibility of two subjects starting treatment on the same day. Close communication between 
sites will be maintained throughout the trial and details of any adverse reactions will be 
communicated to the other trial treatment site by email, in addition to the standard 
pharmacovigilance procedures. 

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Conditions for interruption of dosing to individuals.  
If there is evidence of hypersensitivity (i.e. wheal as well as flare), subjects can have 
paracetamol and antihistamines and continue in the trial as per the study schedule.  
Injections and treatment will be immediately discontinued (but follow up will continue) in 
any subject who: 
1. Experiences a worsening local wheal or flare reaction at the injection site exceeding 5 
cm. 
2. Experiences a serious adverse reaction or an allergic event suggestive of systemic 
hypersensitivity requiring more than local therapy (e.g. requiring steroids, bronchodilators, 
or adrenaline). 
3. Becomes pregnant (see section 8 withdrawal/exit criteria). 
Conditions for suspending further dosing to all subjects. If any individual in the study 
experiences a SUSAR further dosing of all subjects at all sites will be suspended pending 
review by the Data Safety Monitoring Board and discussion with the CI and Sponsor. Advice 
will be sought from the DSMB regarding re-starting the study. 
A substantial amendment application will be submitted to the Regulatory Agencies to 
resume the trial.  
8 
WITHDRAWAL/EXIT CRITERIA 
8.1 At any time 
• 
Patient withdrawal of consent 
 
8.2 After receipt of first dose 
• 
Potential Serious Adverse Event attributable to the IMP. 
 
8.3 Subject becomes pregnant 
• 
No further doses will be given and the subject will be invited to attend trial follow up 
visits, to obtain outcome data in accordance with the planned analysis. 
• 
The pregnancy will be recorded on a pregnancy notification form and the subject will 
be followed up until child birth. 

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8.4 
Treatment of Withdrawn Subjects 
8.4.1 Follow-up of Withdrawn Subjects 
Care for withdrawn subjects will return to their referring diabetologist, however they will also 
be invited to attend trial follow up visits, to obtain outcome data in accordance with the 
planned analysis. 
8.4.2 Management of Data from Withdrawn Subjects 
All withdrawn patients will be invited to attend follow up visits to provide outcome data for 
the purposes of the planned analysis.  
 
9 
END OF STUDY 
REC, the MHRA, MPA and the Regional Ethical Review Board in Linköping will be notified of 
end of study within 60 days of the last visit of the last participant taking part in the trial.  
 
10 PHARMACOVIGILANCE 
 
10.1 Specification, timing and recording of safety parameters 
The major associated safety parameters are evidence of induction of hypersensitivity and 
exacerbation of β-cell specific autoimmunity with accelerated β-cell loss. To address general 
safety concerns at screening and 0, 4, 8 and 14 weeks a physical examination will be 
conducted. A review of AEs will be performed at all visits and blood will be drawn at screening, 
weeks 4, 9, 14 & 20 to examine the full blood count; urea, electrolytes and creatinine; liver 
function tests; (prothrombin time, total bilirubin, total protein, albumin, AST (SGOT), SGPT 
(ALT), alkaline phosphatase; thyroid stimulating hormone; immunoglobulins (G, A, M); 
calcium; magnesium, phosphate, lipid profile (total cholesterol, LDL, HDL, triglyceride). 
Urinalysis for pH blood, protein, urine beta-2-microglobulin and albumin/creatinine ratio will 
be done at screening and visits 1, 2, 4, 5 and 6 and urine for cystatin-c will be collected at 
visits 1, 4, 5 & 6. A urine pregnancy test will be completed on females only, at all trial visits. 
At visits 1, 1b, 3b, 4, 5 and 6 blood and urine samples will be taken for gold concentrations to 
enable assessment of gold excretion. Laboratory measurements for these will be performed 
at the Midatech Pharma laboratory, Spain.  
 
 
The safety of the investigational medicinal product (IMP) C19-A3 GNP has been extensively 
studied in pre-clinical toxicity studies. No signs of toxicity and no significant adverse effects in 
the organs and the >40 tissues examined were seen, not even at doses that were up to 500-
fold higher than the intended dose in this clinical study, and given daily during 14 consecutive 
days. The studies revealed that the major organ of accumulation and excretion of the gold 
nanoparticles is the kidney, and to a much lesser extent the liver and spleen. Significant 
quantities of gold were only detectable in kidneys, which collected on average 30 % of the 
total dose administered (in rats) over the 14 day period, with a half-life of elimination of circa 

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17 days. Because the kidney was the most exposed vital organ in the animals used for the pre-
clinical safety studies, a number of markers of kidney function will be monitored in the 
patients. Electrolytes, urea, creatinine, calcium, phosphate, and magnesium levels will be 
tested in blood. Urinalysis for blood, protein (cystatin-c) and beta-2-microglobulin will be 
performed in addition. A final analysis will be performed 12 weeks after the last dosing.  There 
will also be an optional analysis at 12 months/ 52 weeks post last dose.  For safety reasons, 
there will be an optional contact hypersensitivity/ skin testing to be performed anytime post 
final dose.   
10.2 Hypersensitivity 
Induction of hypersensitivity to C19-A3 GNP will be assessed by a period of observation of 
subjects and during the immediate period after peptide injection. Local hypersensitivity may 
manifest as an increasing wheal and flare reaction at the site of peptide injection. The first 
patient dosed with GNP-peptide will be observed for 6 hours post dose – see section 7.3. Any 
adverse reactions will be communicated to the other trial treatment site by email, in addition 
to the standard pharmacovigilance procedures. Subsequent subjects will also be observed for 
6 hours. If no serious adverse events are observed post first dose, subsequent visits will have 
an observation period of a minimum of 1 hour post dose, or 2 hours if there are any symptoms 
of signs of hypersensitivity. This time period has been chosen as the only anticipated severe 
reactions is one of hypersensitivity which, if systemic, would be apparent within 60 mins of 
intradermal dosing. 
Observation will be by inspection of the injection site at 5 minute intervals for the first 15 
minutes and then 15 minute intervals for a further 45 minutes. If any reaction is present and 
not resolving over this period, observation will be extended to 15 minute intervals until the 
reaction is reducing and < 1cm in diameter. Maximum size of any redness or swelling at the 
injection site will be recorded at each visit at which study drug is given (visits 1-3). Systemic 
hypersensitivity may manifest as shortness of breath; skin rash; tissue swelling; collapse. 
Subjects will be inspected for such signs at 5-minute intervals for the first 15 minutes and 
then at 15 minute intervals for a further 45 minutes. Pulse and blood pressure will be recorded 
at 15 minute intervals over this time.  Any adverse events during this observation period will 
be recorded. To minimise any risk that might result from hypersensitivity: 
• IMP administration should ONLY performed in a clinical research environment 
appropriate for phase 1 studies in which resuscitation facilities are immediately 
available. 
• Facilities for treatment of a major hypersensitivity reaction, adrenaline, oxygen, 
intravenous infusion, head-down position, blood pressure and pulse monitoring 
should be available in the immediate environment of the subject, with access to 
hydrocortisone and antihistamines. 
• Subjects should not be treated by a lone-worker unless he/she has immediate access 
to clinically trained support. 
• Subjects should be directly observed for a minimum of 60 minutes after IMP 
administration and for up to an additional 2 hours or more if there are symptoms/signs 
of hypersensitivity. 

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There is an optional contact hypersensitivity testing or patch testing that can be performed 
anytime post final dose.  This test was amended in per suggestion of the Data Steering 
Monitoring Board as a safety measure.  This test determines delayed Type 4 hypersensitivity 
to allergens including gold for this study.  This test can involve 3 visits (visit 2 is flexible and 
can be done at home) for the patient including day 0 (patches applied to back or arm of 
patient), day 2 (patches taken off and first reading- this can be done at home by the patient) 
and final reading (day 4 or day 7). 
10.3 Disease exacerbation 
Laboratory tests for exacerbation of β-cell specific autoimmunity are (i) measurement of islet 
cell autoantibodies (against insulin, GAD-65, IA-2 and ZnT8) at 0 and 14 weeks; and (ii) 
measurement of pro-inflammatory β-cell specific T cell responses at 0, and 14 weeks.  
Clinical manifestation of disease exacerbation will be monitored by measurement of secreted 
C-peptide AUC after MMTT and stimulated urine c-peptide at 0 and 14 weeks ; HbA1c levels 
(0, 4, 8 and 14 weeks.); and the record of blood sugars and insulin use at each study visit. 
Disease specific quality of life questionnaires including hypoglycaemia fear (HFS), treatment 
satisfaction DTSQ and quality of life ADDQoL will be performed at 0 and 14 weeks.  
11 ADVERSE EVENTS 
AE collection will be recorded from the time the participant receives their first dose of IMP 
until one month after the patient’s last trial visit.   
Adverse events will be monitored, reported in the trial publications and to the Trial Sponsor, 
Research Ethics Committee (REC), Medicines and Healthcare products Regulatory Agency 
(MHRA), Medical Products Agency (MPA) and the Regional Ethical Review Board in Linköping 
if appropriate. 
 
Definitions used will be those under the Medicines for Human Use (Clinical Trials) Regulations 
2004 and Amended Regulations 2006 as follows. 
 
11.1 Definitions 
An Adverse Event (AE) is any untoward medical occurrence in a participant to whom an 
investigational medicinal product (IMP) has been administered, including occurrences which 
are not necessarily caused by or related to that product. 
 
An Adverse Reaction (AR) is any untoward and unintended response, in a participant, to an 
IMP which is related to any dose administered to that participant. An Adverse Reaction is an 
Adverse Event with a causal relationship to the IMP. 
 

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Serious Adverse Event (SAE), Serious Adverse Reaction (SAR) or Serious Unexpected 
Suspected Adverse Reaction (SUSAR) is defined as an Adverse Event, Adverse Reaction or 
unexpected Adverse Reaction that: 
 
• results in death; 
• is life-threatening; 
The term “life-threatening” in the definition of serious refers to an event in which the 
participant was at risk of death at the time of the event; it does not refer to an event 
which hypothetically might have caused death if it were more severe; 
• Requires hospitalisation or prolongation of existing hospitalisation.  Hospitalization is 
defined as an inpatient admission, regardless of the length of stay, even if the 
hospitalization is a precautionary measure, for continued observation. Pre-planned 
hospitalisation e.g. for pre-existing conditions which have not worsened or elective 
procedures does not constitute an adverse event; 
• results in persistent or significant disability or incapacity; 
• consists of a congenital anomaly or birth defect; or 
• is medically significant; 
Other events that may not result in death are not life-threatening, or do not require 
hospitalisation may be considered as a serious adverse event when, based upon 
appropriate medical judgement, the event may jeopardise the participant and may 
require medical or surgical intervention to prevent one of the outcomes listed above. 
 
 
Pregnancy in either a participant or the partner of a participant taking trial medication will be 
recorded on a pregnancy notification form and the subject will be followed up until child birth. 
 
A Suspected Unexpected Serious Adverse Reaction (SUSAR) is an adverse reaction that is both 
serious and unexpected.  
 
11.2 Eliciting Adverse Events 
All trial subjects will have an enquiry about co-morbidities, medications and new or 
unexpected symptoms at each follow-up visit, as well as diabetes assessment.  
 
11.3 Principal Investigator/Trial Centre Responsibilities 
The PI will be required to report all AEs/SAEs which occur during the Trial on an Adverse Event 
Form and keep a record in the case report form (CRF). AEs will be collected from the time the 
participant receives their first dose of IMP until one month after the last trial visit (visit 6, 
week 20). The AEs will be reported at agreed intervals to the DSMB and if there is any concern 
a DSMB meeting will be convened.  
 

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11.4 Assessing AEs 
The PI should assess the AE for seriousness and causality. The PI should exercise medical 
judgement in deciding whether an Adverse Event/Reaction is serious in other situations. All 
SAEs should be reported to STU by the PI immediately (within 24hrs) of becoming aware of 
the event. The PI will be asked to grade all AEs in relationship to the study treatment according 
to their clinical judgement as follows: 
 
Causality: 
• 
Not related – temporal relationship of the onset of the event, relative to 
administration of the product, is not reasonable or another cause can by itself explain the 
occurrence of the event. 
• 
Unlikely to be related – temporal relationship of the onset of the event, relative to 
administration of the product, is likely to have another cause which can by itself explain the 
occurrence of the event. 
• 
Possibly related – temporal relationship of the onset of the event, relative to 
administration of the product, is reasonable but the event could have been due to another, 
equally likely cause. 
• 
Probably related – temporal relationship of the onset of the event, relative to 
administration of the product, is reasonable and the event is more likely explained by the 
product than any other cause. 
• 
Very likely – temporal relationship of the onset of the event, relative to administration 
of the product, is reasonable and there is no other cause to explain the event, or a re-
challenge is positive. 
11.5 Reporting procedures 
All SAEs must be reported immediately (within 24 hrs of being made aware of the event) by 
the PI to STU within 24 hours of receipt. 
Initial reports should be submitted as soon as the following minimum criteria are met: 
• A suspected SAE is identified; 
• An identifiable participant (e.g. trial participant code number); 
• An AE assessed as serious and unexpected, and for which there is a reasonable 
suspected causal relationship; 
• An identifiable reporting source (e.g. clinical research fellow). 
 
Following the Initial Report, all SAEs should be followed to resolution. Following the initial 
report the PI may be requested to provide further information. The participant should only 
be identified by trial number, date of birth and initials. The participant’s name should not be 
used on any correspondence. The PI is also responsible for reporting AEs to their NHS Trust 
as per their local NHS Trust procedures. 
 

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11.6 Sponsor and Chief Investigator Responsibilities 
The Sponsor (or delegate – STU) is responsible for ensuring all SAEs, SARs and SUSARs 
(except those specified in this protocol as not requiring reporting) will be reported in the 
appropriate timescale to the MHRA, REC, MPA and the Regional Ethical Review Board 
(RERB) in Linköping. 
STU is responsible for ensuring all SUSARs from Cardiff and Linkoping (except those specified 
in this protocol as not requiring reporting) will be reported in the appropriate timescale to 
the MHRA, REC. MPA and RERB. 
Once an SAE is received by STU, the SAE will be sent to the CI (or appropriate delegate) for 
clinical review (assessment of causality and expectedness).  Fatal and life threatening SAEs 
should be assessed by the CI within 24 hours of receipt.  Non-fatal or non-life-threatening 
SAEs should be assessed by the CI within 4 days of receipt. 
Only SUSARs should be expedited  to MHRA, MPA, RERB and REC.   STU (UK) and Karolinska 
Trial Alliance (Sweden) will report according to the following timelines: 
Fatal and life threatening SUSARs not later than 7 days after receipt; Non-fatal or non-life-
threatening SUSARs not later than 15 days after receipt. Follow up information should be 
reported within 8 days of receipt of the follow up information. A copy of the SUSAR report 
should be provided to the Sponsor.  
In addition to reporting to the relevant REC, RERB, MHRA and MPA, SUSARs will also be 
reported to all members of the TMG.  The CI shall ensure that all co-investigators receive 
regular safety updates of SAE’s and SUSARs that occur in relation to the IMP in the trial. 
 
Dose interrupting and suspension of dosing will be managed as per the risk mitigation, see 
section 7. 
 
The CI is responsible for assessing expectedness 
No events other than those listed in section 10.2 are expected. 
 
11.7 Urgent Safety measures  
Urgent safety measures which are implemented immediately by the PI should be notified to 
the Trial Manager immediately, along with the reasons for the measure being taken. 
The CI (or delegate – STU) shall be responsible for reporting urgent safety measures to MHRA, 
MPA, RERB and REC.  This should include an initial contact with a medical assessor at the 
MHRA/MPA to discuss the urgent safety measure, followed by a detailed written report 
within 3 days.  The report should include details of the urgent safety notification, the 
measures taken and the reason for the measures and details of any discussion with the MHRA 
and MPA medical assessor.  
See section 8 for treatment stopping and withdrawal criteria. 
 

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11.8 Safety Report/ Line Listing 
One year following the granting of a CTA, and thereafter annually, the CI (or delegate – STU) 
should compile an annual safety report & DSUR for submission to the MHRA and REC, UK 
and the MPA and the Regional Ethical Review Board in Linköping. 
 
12 ASSESSMENT OF EFFICACY 
Although not primarily a study designed to assess efficacy, laboratory and clinical 
measurements will be made that may indicate beneficial therapeutic trends associated with 
C19-A3 GNP administration. These have been specified as secondary endpoints. 
 
12.1 Efficacy Parameters 
12.1.1 Metabolic outcomes 
Residual insulin production will be assessed by measurement of stimulated C-peptide 
production during the MMTT using the area under the curve (AUC) approach. The normalized 
AUC will be calculated for each subject at 14 weeks and compared versus baseline (visit 1). 
Stimulated urine c-peptide creatinine ratio will also be measured as an alternative 
measurement.  
Likewise, change in glycated haemoglobin (as measured by % HbA1c levels), daily insulin 
usage, and mean amplitude of glucose excursions will be measured/recorded as specified in 
the schedule of visits and compared with baseline.  
For objective assessment of hypoglycaemia continuous glucose monitoring will be conducted 
for 3 days at baseline and prior to the final follow up visit. Hypoglycaemia frequency and 
standard deviation and interquartile range of glucose variability will be calculated from this 
data. 
12.1.2 Immunological outcomes 
Change in level or quality of T lymphocyte biomarkers of β-cell specific immune response will 
be assessed at 14 weeks (8 weeks for lymph nodes sampling) versus baseline, Levels of T 
lymphocyte biomarkers are measured as the numbers of cytokine-producing T cells 
responding to PPI C19-A3 or other β-cell autoantigenic peptides and the numbers of cytokine 
and other immune gene transcripts produced in response to stimulation with PPI C19-A3 or 
other β-cell autoantigenic peptides in vitro. A potentially important biomarker of therapeutic 
efficacy would be an increase in the number of responder T cells synthesizing the immune 
regulatory cytokine IL-10, or a decrease in the number of responder T cells synthesizing the 
pro-inflammatory cytokines IFN-γ. 
Changes in the quality of the immune response can also be measured by assays that 
specifically detect the ability of post-treatment CD4 T cells to regulate pre-treatment pro-
inflammatory responses. 
Changes in the level or quality of islet cell autoantibody biomarkers of β-cell specific immune 
response will be measured at 0, 14 weeks and compared with levels at baseline and between 

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groups. A potentially important biomarker would be a reduction in levels of islet cell 
autoantibodies. Changes in serum levels of cell free DNA derived from beta cells and T cells 
will be measured to provide potentially important biomarkers of beta cell and T cell death. 
 
 
12.2 Procedures for Assessing Efficacy Parameters 
12.2.1 Insulin secretion 
C-peptide production in the MMTT assay will be assessed using the AUC approach. Levels of 
glucose, and C-peptide will be assessed from blood samples taken during the test. Stimulated 
C-peptide will be the area under the (C-peptide/time) curve from time 0 to 120 minutes, and 
this will be calculated by the trapezoidal rule. The AUC will be normalized for time interval by 
dividing it by the number of minutes over which AUC was determined; the number is expected 
to be 120 minutes but may differ in some cases. A urine sample after 120 minutes will be 
taken for urine C-peptide creatinine ratio which has been proposed as a surrogate marker of 
insulin secretion. 
12.2.2 Insulin usage 
Subjects will record the daily insulin usage in their capillary blood glucose testing meters 
supplied for the study. Insulin usage will be recorded daily from the screening stage through 
to the end of the study. In particular, insulin usage should be recorded accurately for at least 
7 consecutive days during the 2 weeks prior to visits at 0, 4, 8 weeks. The mean daily insulin 
usage over the 7 consecutive days (IU units/kg body weight/day) will thus be calculated for 
each subject.   
12.2.3 Blood glucose variability 
Mean amplitude of glucose excursion (MAGE) will be calculated at 0 and 14 weeks using 
records from 72 hour CGM recordings.  
12.2.4 Immunological outcomes 
See sections 6.15 and 12.1.  
 
  
13  STATISTICS 
The primary aim of this study is provide patient safety data.  
The study will be open label. 
 
14 SAMPLE SIZE 
The single group study provides 8 patients on active treatment from whom it will be possible 
to collect safety data. This is considered optimal to achieve sufficient data to inform future 
study designs and on which to base future sample size calculations.  
 
 

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15 PLANNED ANALYSIS 
 
15.1 Final 
Data cleaning and preparation process will be carried out prior to analysis.  
All patients enrolled will be followed up and included unless they withdrew from the study 
before the administration of the first dose. An intention to treat analysis will be carried out. 
Per protocol analyses may also be carried out alongside the intention to treat analysis if 
necessary. The analyses will mostly consist of tables of data such as mean values (or geometric 
means if the data is log normal) together with standard deviations and ranges.  For data such 
as scores, medians, and ranges may be used instead, and binary outcome variables will use 
percentages. Comparisons against baseline will be made using Student’s t-tests for paired and 
unpaired samples;  Mann-Whitney U test; Wilcoxon matched pairs test;  p values <0.05 will 
be considered significant.  
Since the primary aim of the study is to provide patient safety data and guide the 
development of an antigen specific immunotherapy programme, it may be a requirement for 
strategic planning of subsequent stages of clinical study development, that early analysis of 
trial data is conducted.  
 
 
16 QUALITY ASSURANCE, SITE MONITORING AND DIRECT ACCESS TO SOURCE DATA AND 
DOCUMENTS 
 
The Investigator(s) will permit trial-related monitoring, audits, REC review, and regulatory 
inspections (where appropriate) by providing direct access to source data and other 
documents (ie patients’ case sheets, blood test reports, X-ray reports, histology reports etc). 
Monitoring of this trial to ensure compliance with Good Clinical Practice and scientific 
integrity will be conducted by STU for the Cardiff site and Forum Östergötland at Linkoping 
who will have direct access to source data.  
 
Source documents produced for this trial will be kept in the patient’s hospital records and 
source data will be transcribed into trial-specific Case Report Forms (CRFs) at the end of each 
patient visit. These CRFs will be coded with the study number and will not include patients’ 
names and addresses.  
 
Selected anonymised data will also be stored in electronic format. Paper records will be kept 
in a locked cabinet in secure premises at all times when the record is not in use for a study 
visit. Access to the records will be restricted to researchers working on the study, Sponsor 
representatives and representatives of regulatory authorities required to audit the conduct 
of the research study.  
 
Relevant data will be transferred (with double data entry/data checking) from the paper 
record in the trial office to an electronic database which will be stored and regularly backed 
up on a Cardiff University server. Identifiable data including the link between the patients’ 

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names and the study number will be stored separately from other data. All files will be 
password protected. Electronic data containing personalised information will be saved on 
Cardiff University computers only in password protected files and backed up regularly to hard 
copy on secure remote Cardiff University servers. Participant data will be anonymised by the 
use of study numbers. A copy of the study number code identifying subjects will be kept in a 
secure cabinet at local study sites accessible to the investigators at all times. Analysis will be 
conducted by the study team. Analysis will only be conducted on anonymised data.  
 
The Chief Investigator, Professor Colin Dayan will act as custodian of the data, however for 
practical purposes this role will be delegated to STU. Personal data will be stored for a 
minimum of 15 years. Access will be controlled by Professor Dayan who will continue to act 
as custodian for all data held by the Sponsor and will permit trial related monitoring, audits, 
REC review, and regulatory inspections (where appropriate) by providing direct access to 
source data and all other documents (i.e. patients’ case sheets, blood test reports, etc). 
 
17 ETHICS AND REGULATORY APPROVALS 
The trial will be conducted in compliance with the principles of the Declaration of Helsinki 
(2013), the principles of GCP and in accordance with all applicable regulatory requirements 
including but not limited to the Research Governance Framework and the Medicines for 
Human Use (Clinical Trial) Regulations 2004, as amended in 2006 and any subsequent 
amendments. 
 
This protocol and related documents will be submitted via the Integrated Research 
Application System for review by a Research Ethics Committee (REC), Regional Ethical Review 
Board (RERB) and to the Medicines and Healthcare products Regulatory Agency (MHRA) for 
Clinical Trial Authorisation and to the Medical Products Agency (MPA) for approval.  
 
The Chief Investigator will submit annual progress and safety reports and a final report at 
conclusion of the trial to the REC and the MHRA within the timelines defined in the 
Regulations. 
 
18 FINANCIAL AND INSURANCE ASPECTS 
Support for this trial has been granted by the European Commission FP7 programme. 
 
Cardiff University has arranged clinical research insurance to cover the legal liability of the 
University for negligent harm. In addition the study doctors hold substantive or honorary NHS 
contracts, giving them the protection of the NHS clinical negligence arrangements. Swedish 
patients will be covered by Patientskadeförsäkringen. 
 

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19 PATIENT INVOLVEMENT 
Involving patients from the outset strengthens the application by showing that the research 
is patient-centred and that patient participation has been welcomed, encouraged and 
facilitated. Our patient representative(s) will be involved in: 
• Trial management meetings; 
• Trouble shooting to support recruitment and retention of participants; 
• Reporting the final results to the trial participants; 
• Preparing information for web site inclusion. 
 
The study will be registered on the NIHR INVOLVE and HCRW Involving People (Cynnwys Pobl) 
databases once ethical approval is granted. These are national advisory groups that support 
greater public involvement in NHS, public health and social care research. INVOLVE provides 
a public information pack, newsletters and public networking to facilitate public involvement. 
Locally, Involving People provides excellent training and support for public involvement via 
local conferences, newsletters and public network.  
 
Patient involvement, will also facilitate dissemination of the study findings to as wide an 
audience as possible and will help set the agenda for future research in this field. Training and 
support for our patient representatives will be flexible and tailored to their individual needs. 
It will include training on the study background, methods and outcomes and where indicated 
IT training will be provided. Patients will be reimbursed in line with HCRW’s AcoRD guidance 
(Attributing the costs of Health and Social Care Research & Development).  
 
20 PUBLICATION POLICY 
It is intended that the results of the study will be reported and disseminated at international 
conferences and in peer-reviewed scientific journals. Written feedback will also be provided 
to the study participants. 
 
 
21 TRIAL MANAGEMENT 
21.1 Trial Steering Committee (TSC) 
1. For a small phase 1 trial such as this, it is not considered that a TSC is required. 
 
21.2 Trial Management Group (TMG) 
The project will be run by a Trial Management Group comprising PI’s, trial manager, clinical 
research fellow, statistician (CTU staff), chief investigator and patient representatives and 
Sponsor representation. The TMG will oversee the day to day trial management and will meet 
in person or by teleconference at a minimum 2 monthly for the duration of the study. The 
TMG will overview and provide guidance on all aspects of regulatory approval, set-up, 
recruitment, protocol deviations, adverse events, data management, data analysis and 
dissemination. The TMG will report at agreed intervals to the DSMB and to the study sponsor. 

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21.3 Data safety Monitoring Board (DSMB) members: 
An independent Data Safety Monitoring Board (DSMB) will be convened for the duration of 
the trial comprising a consultant diabetes physician, a clinical immunologist and a consultant 
rheumatologist. The DSMB will meet (face-to-face or by teleconference) at agreed intervals 
throughout the trial and receive reports of all adverse events from the Chief Investigator 
and/or other members of the trial team, as well as assessing progress in recruitment. The 
chairman of the DSMB may also call additional meetings as necessary. The DSMB will have 
responsibility for suspending or terminating treatment of one or a group of subjects if subject 
safety is considered at risk. 
 
 
 
 
 
 
 
22 SIGNATURE PAGE 
 
Investigator’s Statement of Agreement: I have read, understand, and will conduct the 
study according to this protocol and Good Clinical Practice 
 
 
 
 
                
     
Chief Investigator  
 
 
 
 
Date:  18th  July 2018 
Professor Colin Dayan 
 
 
 
 
 
 
 
 20th July  2018 
 
_______________________________ 
 
 
___________________ 
Principal Investigator 
 
 
 
 
Date 
Professor Johnny Ludvigsson 
 
 

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