1604 diabetes care volume 44, july 2021 emerging therapies

Intralymphatic Glutamic AcidDecarboxylase With Vitamin DSupplementation in Recent-OnsetType 1 Diabetes: A Double-Blind,Randomized, Placebo-ControlledPhase IIb TrialDiabetes Care 2021;44:1604–1612 | https://doi.org/10.2337/dc21-0318

Johnny Ludvigsson,1 Zdenek Sumnik,2

Terezie Pelikanova,3 Lia Nattero Chavez,4

Elena Lundberg,5 Itxaso Rica,6

Maria A. Mart�ınez-Brocca,7

Marisol Ruiz de Adana,8

Jeanette Wahlberg,9

Anastasia Katsarou,10 Ragnar Hanas,11

Cristina Hernandez,12

Maria Clemente Le�on,13

Ana G�omez-Gila,14 Marcus Lind,15,16

Marta Ferrer Lozano,17 Theo Sas,18

Ulf Samuelsson,1 Stepanka Pruhova,2

Fabricia Dietrich,19 Sara Puente Marin,19

Anders Nordlund,20 Ulf Hannelius,21 and

Rosaura Casas19

OBJECTIVE

To evaluate the efficacy of aluminum-formulated intralymphatic glutamic acid de-carboxylase (GAD-alum) therapy combined with vitamin D supplementation inpreserving endogenous insulin secretion in all patients with type 1 diabetes (T1D)or in a genetically prespecified subgroup.

RESEARCH DESIGN AND METHODS

In a multicenter, randomized, placebo-controlled, double-blind trial, 109 patientsaged 12–24 years (mean ± SD 16.4 ± 4.1) with a diabetes duration of 7–193 days(88.8 ± 51.4), elevated serum GAD65 autoantibodies, and a fasting serum C-pep-tide>0.12 nmol/L were recruited. Participants were randomized to receive eitherthree intralymphatic injections (1 month apart) with 4 mg GAD-alum and oral vi-tamin D (2,000 IE daily for 120 days) or placebo. The primary outcome was thechange in stimulated serum C-peptide (mean area under the curve [AUC] after amixed-meal tolerance test) between baseline and 15 months.

RESULTS

Primary end point was not met in the full analysis set (treatment effect ratio1.091 [CI 0.845–1.408]; P 5 0.5009). However, GAD-alum–treated patients carry-ing HLA DR3-DQ2 (n 5 29; defined as DRB1*03, DQB1*02:01) showed greaterpreservation of C-peptide AUC (treatment effect ratio 1.557 [CI 1.126–2.153];P 5 0.0078) after 15 months compared with individuals receiving placebo withthe same genotype (n 5 17). Several secondary end points showed supportingtrends, and a positive effect was seen in partial remission (insulin dose–adjustedHbA1c #9; P5 0.0310). Minor transient injection site reactions were reported.

CONCLUSION

Intralymphatic administration of GAD-alum is a simple, well-tolerated treat-ment that together with vitamin D supplementation seems to preserveC-peptide in patients with recent-onset T1D carrying HLA DR3-DQ2. Thisconstitutes a disease-modifying treatment for T1D with a precision medicineapproach.

1Division of Pediatrics, Department of Biomedicaland Clinical Sciences, Faculty of Medicine andHealth Sciences and Crown Princess VictoriaChildren’s Hospital, Link€oping University, Link€oping,Sweden2Department of Pediatrics, 2nd Faculty ofMedicine, Charles University and Motol Uni-versity Hospital, Prague, Czech Republic3Diabetes Centre of the Institute of Clinical andExperimental Medicine, Prague, Czech Republic4Department of Endocrinology and Nutrition,Hospital Universitario Ram�on y Cajal, Madrid,Spain5Institution of Clinical Science, Department ofPediatrics, Umeå University, Norrland Uni-versity Hospital, Umeå, Sweden6Department of Pediatric Endocrinology, CrucesUniversity Hospital, CIBERDEM, Bilbao, Spain7Department of Endocrinology, VirgenMacarena Hospital, Sevilla, Spain8Department of Adult Endocrinology andDiabetology, General University Hospital,Instituto de Biomedicina de M�alaga,CIBERDEM, Malaga, Spain9Departments of Endocrinology Region€Osterg€otland and Health, Medicine and CaringSciences, Link€oping University, Link€oping,Sweden10Department of Endocrinology, Skåne Uni-versity Hospital, Malm€o, Sweden11Department of Pediatrics, NU Hospital Group,Uddevalla, Sweden12Department of Endocrinology and Nutrition,Vall d’Hebron Hospital, CIBERDEM, Barcelona,Spain13Department of Endocrinology and Nutrition,Vall d’Hebron Hospital, CIBERDEM, Barcelona,Spain14Pediatric Endocrinology Service, Virgen delRoc�ıo University Hospital, Sevilla, Spain15Department of Molecular and ClinicalMedicine, University of Gothenburg, Gothenburg,Sweden

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Many individuals with type 1 diabetes(T1D) have a long life, with reasonablygood quality of life, but even with mod-ern treatment, the disease still causesserious morbidity (1) and increasedmortality (2,3). Even a limited residualb-cell function facilitates glycemic con-trol, decreases the risk of both acuteand late complications, and reducesmortality (4). The most efficient immunetherapy evaluated to date for preserva-tion of b-cell function is treatment withanti-CD3 monoclonal antibodies (tepli-zumab) (5,6); tumor necrosis factor-ainhibitors (7), ATG (8), alefacept (9), andrituximab (10) have also displayed effi-cacy, which is encouraging, but some-times these therapies have adverseevents, risks, and heavy treatment bur-dens, and therefore, additional studiesare needed.An alternative approach is treatment

with autoantigens to modulate the im-mune system. Most autoantigen immu-notherapies have failed to meet theirtherapeutic end points or have showninconclusive results (11–14). One reasonfor this may be the heterogeneity ofT1D (15). Disease endotype should beconsidered in the design and evaluationof clinical trials (16). The appearance ofGAD65 autoantibodies (GADA) is linkedto the HLA DR3-DQ2 haplotype, and theemergence of insulin autoantibodies islinked to DR4-DQ8 (15,16). Importantly,a meta-analysis of data from clinical tri-als evaluating subcutaneous GAD-alumimmunotherapy (12–14) indicated thatpatients carrying the HLA DR3-DQ2 hap-lotype showed a significant and positivedose-dependent preservation of b-cellfunction following GAD-alum treatment(17).The immunological impact of anti-

gen can be improved by direct admin-istration into lymph nodes. Thisstrategy has been successful in allergyimmunotherapy (18) and was tested

in T1D in a pilot trial with GAD-aluminjected into an inguinal lymph node,in combination with oral vitamin D,with encouraging results (19).

Therefore, in this randomized, double-blind, placebo-controlled phase IIb trial,we aimed to determine the efficacy ofthree intralymphatic injections of 4 mgGAD-alum into inguinal lymph nodes inpreserving b-cell function in individualswith recent-onset T1D. Based on the re-sults from the meta-analysis suggestingefficacy of GAD-alum in individuals carry-ing the HLA DR3-DQ2 (defined asDRB1*03, DQB1*02:01) haplotype (17),the analysis of efficacy end points in thesubpopulation carrying this genotypewas introduced as an amendment to theoriginal clinical study protocol ahead oftreatment unblinding.

RESEARCH DESIGN AND METHODS

Study ConductThe coordinating investigator (J.L.) de-signed the study based on a previouspilot trial (DIAGNODE-1; EudraCT2014-001417-79) (19) together with thesponsor, Diamyd Medical (Stockholm,Sweden). The first and last authorsvouch for the data, analysis, and fideli-ty of the report to the study protocol.

The study was approved by therelevant regulatory authorities andresearch ethics boards of the partici-pating sites and countries. All pa-tients and their caregivers providedwritten informed consent. The studywas conducted in accordance withthe clinical study protocol and thestatistical analysis plan.

Study Design and PatientsThis study was a two-arm, multicenter,randomized, double-blind, placebo-controlled trial performed at 18 diabe-tes clinics in the Czech Republic, theNetherlands, Spain, and Sweden. Pa-tients were recruited by the clinicians,

and screening was performed be-tween 7 December 2017 and 16 April2019. The study intended to enroll106 patients aged between $12 and<25 years at screening with a diagno-sis of T1D within the previous 6months. Major inclusion criteria wereelevated serum GADA and fasting se-rum C-peptide >0.12 nmol/L (>0.36ng/mL). Major exclusion criteria wereprior or current treatment with immu-nosuppressant therapy, continuoustreatment with an anti-inflammatorydrug, treatment with any oral or in-jected antidiabetic medication (otherthan insulin), and concomitant treat-ment with vitamin D.

Randomization and MaskingPatients were randomized at a 1:1 ratioin blocks of four by an interactive webresponse system using a computer-gen-erated randomization list. Randomiza-tion was stratified by level of serumGADA and by country. The randomiza-tion list was kept strictly confidentialuntil database lock at 15 months. Theidentity of the treatments (GAD-alumand vitamin D) was masked usingmatching placebo with identical packag-ing, labeling, appearance, and scheduleof administration. Matching placeboused for GAD-alum was aluminum only.

Study Treatments and ProceduresPatients were randomized in a 1:1 ratioto receive one of the following:

• Three intralymphatic injections with4 mg Diamyd on days 30, 60, and 90and 2,000 IE oral vitamin D daily for4 months (from day 1 through 120)if the vitamin D serum level was<100 nmol/L (40 ng/mL) atscreening.

• Three intralymphatic injections ofplacebo for Diamyd on days 30, 60,and 90 and oral placebo for vitamin

16Department of Medicine, Uddevalla, Sweden17Department of Pediatric Endocrinology,Miguel Servet University Hospital, Zaragoza,Spain18Diabeter, National Treatment and ResearchCenter for Children, Adolescents and YoungAdults With Type 1 Diabetes, and Departmentof Pediatric Endocrinology, Erasmus UniversityMedical Center, Rotterdam, the Netherlands19Division of Pediatrics, Department ofBiomedical and Clinical Sciences, Faculty ofMedicine and Health Sciences, Link€opingUniversity, Link€oping, Sweden

20Trial Form Support, Lund, Sweden21Diamyd Medical AB, Stockholm, Sweden

Corresponding author: Johnny Ludvigsson,[email protected]

Received 6 February 2021 and accepted 15 April2021

Clinical trial reg. nos. NCT03345004, clinicaltrials.gov

This article contains supplementary material onlineat https://doi.org/10.2337/figshare.14427956.

© 2021 by the American Diabetes Association.Readers may use this article as long as thework is properly cited, the use is educationaland not for profit, and the work is not altered.More information is available at https://www.diabetesjournals.org/content/license.

care.diabetesjournals.org Ludvigsson and Associates 1605

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mailto:[email protected]

https://clinicaltrials.gov

https://clinicaltrials.gov

https://doi.org/10.2337/figshare.14427956

D daily for 4 months (from day 1through 120) if the vitamin D serumlevel was <100 nmol/L (40 ng/mL)at screening.

Each patient kit contained three vials,which were labeled with the sametreatment number.

Follow-up visits were performed after180 and 450 days. An extra follow-upvisit after 720 days will be performed ina subset of patients. Patients, investiga-tors, and study personnel will remainblinded to the treatment assignmentsuntil the follow-up visits are completed.

Medical examination was performedat all visits including baseline (day 1)and months 1, 2, 3, 6, and 15. Mixed-meal tolerance tests (MMTT) (20) wereperformed at baseline (day 1) andmonths 6 and 15. Investigator assess-ment of injection site reactions was col-lected after each injection, and adverseevents were collected throughout thestudy. Patients documented insulin doseinformation for 4 days before each visit.Patients wore blinded FreeStyle LibrePro continuous glucose monitoring devi-ces during 2 weeks postvisit for visit 1(screening), month 6, and month 15. In-jection site reactions after each injec-tion were reported by patients in adiary, and any severe episodes of hypo-glycemia were collected at each studyvisit postbaseline.

HLA genotyping (month-1 visit) aswell as clinical chemistry, hematology,urine, GADA, HbA1c, and C-peptideanalyses were performed centrally bySynlab Pharma Institute (Munich, Ger-many), using its standardized techni-ques. GADA levels were assessed bymeans of ELISA, with results measuredin IU/mL. C-peptide quantification wasperformed with the use of dual-sidedchemiluminescence immunoassay, us-ing two antibodies (Siemens C-peptideassay no. 03649928), with calibrationstandards based on the World HealthOrganization National Institute for Bio-logical Standards and Control Interna-tional Reference Reagent standards(product no. 84/510). HLA typing(HLA-DR-B1 and HLA-DQ-B1) was per-formed using a sequence-specific oli-gonucleotides kit (One Lambda).

For cytokine quantification, peripheralblood mononuclear cells (PBMC) were cul-tured for 7 days in the presence of 5 mg/mL recombinant human GAD65 (DiamydMedical) or in medium (AIM-V) alone at

37�C in 5% carbon dioxide, as previouslydescribed (21). Interleukin-10 (IL-10) andIL-13 were measured in cell culture super-natants using the Bio-Plex Pro CytokinePanel (Bio-Rad, Hercules, CA) according tothe manufacturer’s instructions. Data werecollected using the Luminex 200 (LuminexxMAP Corporation, Austin, TX). The anti-gen-induced cytokine secretion level wascalculated by subtracting the spontaneoussecretion (i.e., secretion from PBMC cul-tured in medium alone) from that follow-ing stimulation with GAD65.

To quantify PBMC proliferation,PBMC were resuspended in AIM-V me-dium and incubated in triplicate inround-bottom 96-well plates with 5mg/mL recombinant human GAD65(Diamyd Medical) or in AIM-V mediumalone. After 3 days, cells were pulsedfor 18 h with 0.2 mCi of [3H] thymidineper well (PerkinElmer) and thereafterharvested. Proliferation was recordedusing a cell counter and expressed asstimulation index, calculated as themedian of triplicates in presence ofstimulus divided by the median oftriplicates with medium alone.

OutcomesThe primary outcome was the change instimulated serum C-peptide (mean areaunder the curve [AUC] over the 2-h pe-riod after an MMTT between the base-line visit and the 15-month visit).

The key secondary outcomes werechanges between baseline and 15months in insulin dose–adjusted HbA1c(IDAA1c) (22), HbA1c, and daily exoge-nous insulin dose. Other secondary endpoints were:

• Change in glycemic variability/fluctu-ations (evaluated from data fromcontinuous glucose monitoring Free-Style LibrePro and flash glucosemonitoring) over a 14-day periodbetween screening and 15 months.

• Proportions of patients at 15months with IDAA1c $9, a stimulat-ed maximum C-peptide >0.2 nmol/L(0.6 ng/mL), and a stimulated 90-min C-peptide >0.2 nmol/L (0.6 ng/mL).

• Number of self-reported episodes ofsevere hypoglycemia (defined asneeding help from others and/orseizures and/or unconsciousness)between baseline and 15 months.

• Number of patients with at least 1severe hypoglycemic event betweenbaseline and 15 months

• Change in maximum C-peptide dur-ing MMTT between baseline and 15months

• Change in fasting C-peptide betweenbaseline and 15 monthsA number of secondary end points

were evaluated to assess safety, such asoccurrences of adverse events, reactionsat the injection site, physical and neuro-logical assessments, and clinical chemis-try and hematology measurements.

Analyses of primary and key sec-ondary end points were preplanned inthe statistical analysis plan and clinicaltrial protocol for the subgroup of pa-tients carrying the HLA DR3-DQ2 hap-lotype. This subgroup analysis was notpart of the initial protocol but ratherwas prespecified ahead of treatmentunblinding in the statistical analysisplan and in the clinical study protocolthrough a protocol amendment. Thescientific rationale for this subgroup isbased on the results of a meta-analy-sis (17) of previous GAD-alum studiesshowing efficacy mainly in patientscarrying HLA DR3-DQ2.

Statistical AnalysesSample size was calculated such thatthe study would have 90% power todetect a 50% difference in the pri-mary end point (mean C-peptideAUCmean 0–120 min) during an MMTTat 15 months between the activelytreated group and the placebo groupat a two-sided significance level of5%, including allowance for a 10%dropout rate. This was based on a ttest using ln(x 1 1) normalizingtransformation of the primary endpoint and assumed mean and SD es-timates, on the transformed scale,of 0.134 and 0.20, respectively (23).

Analyses of efficacy end points in-cluded all randomized patients who re-ceived at least one injection and had atleast one postbaseline assessment ofany efficacy end point.

For the primary efficacy and key sec-ondary end points, between-group com-parisons of mean changes from baselinewere analyzed using a restricted maxi-mum likelihood–based repeated meas-ures approach (mixed-model repeatedmeasures). The model for analysis in-cluded fixed categorical effects of

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treatment, randomization strata (GAD65antibody level), visit and treatment-by-visit interactions, and the continuousfixed covariate of log-transformed base-line C-peptide AUCmean 0–120 min duringan MMTT. The treatment effect withinthe impact of the HLA haplotype sub-groups on the primary efficacy analysiswas analyzed by adding a class variable(HLA haplotype as DR3-DQ2 vs. notDR3-DQ2) and the interaction term be-tween this variable and the treatmentvariable (i.e., treatment * Visit * HLA) inthe mixed-model repeated measuresanalysis. An unstructured (full sampleanalyses) and compound symmetry(HLA subgroup analyses) (co)vari-ance structure was used to modelthe within-patient errors. Becausethe model accounts for missingdata, no imputations were made.

Data and Resource AvailabilityThe data sets are available from thecorresponding author on reasonablerequest.

RESULTS

Patients (n 5 140) were screened be-tween 7 December 2017 and 16 April2019, of whom 109 were randomizedto active treatment (n 5 57) or placebo(n 5 52). The primary analysis was per-formed in randomized patients who

received at least one injection and hadbeen evaluated for at least one efficacyvariable at baseline and at one follow-up visit (full analysis set [FAS]). All infor-mation on eligibility, FAS, and subgroupswith HLA DR3-DQ2 is shown in Fig. 1.

The baseline characteristics of the pa-tients are shown in Table 1. Two pa-tients had vitamin D >100 nmol/L (40ng/mL) at screening and therefore re-ceived no vitamin D/placebo supple-mentation. Significant differences wereobserved between active and placebotreatment groups in the prespecifiedsubgroup defined by HLA DR3-DQ2 forHbA1c (P 5 0.0100) and IDAA1c (P 50.0101); a near-significant differencewas seen for C-peptide AUC (P 50.0523).

Primary end point was notreached (treatment effect ratio1.091 [CI 0.845–1.408]; P 5 0.5009).Therefore, there was no differencebetween the actively treated groupand the placebo group in change ofstimulated serum C-peptide (meanAUC over the 2-h period after anMMTT) between the baseline visitand the 15-month visit (Fig. 2A) inthe FAS. There was no significant dif-ference between the actively treatedgroup and the placebo group inmean daily insulin dose, HbA1c, orIDAA1c.

In the prespecified subgroup of pa-tients carrying the HLA DR3-DQ2 hap-lotype, there was a significantly betterpreservation of C-peptide AUC0–120 min

during an MMTT in the activelytreated group compared with the pla-cebo group (treatment effect ratio1.557 [CI 1.126–2.153]; P 5 0.0078)(Fig. 2B). This is also illustrated inSupplementary Fig. 1, where absoluteand change from baseline show thatthe C-peptide decline continued morerapidly up to 15 months in the place-bo group than in the actively treatedgroup. A higher proportion of activelytreated patients with HLA DR3-DQ2remained in partial remission (IDAA1c#9; 78.6% in active [CI 59.0–91.7] and40.0% in placebo [CI 16.3–67.7]; P 50.0310) and maintained a stimulatedmaximum C-peptide >0.2 nmol/L(96.6% in active [CI 82.2–99.9] and70.6% in placebo [CI 44.0–89.7]; P 50.0284) as well as stimulated 90-minC-peptide >0.2 nmol/L (96.6% inactive [CI 82.2–99.9] and 64.7% inplacebo [CI 38.3–85.8]; P 5 0.0086) at15 months (Fig. 2C; SupplementaryTable 1). There was a trend at month15 where actively treated patientswith HLA DR3-DQ2 were within targetrange (70–180 mg/dL) for a median of18.6 h per day compared with 14.8 hper day in the placebo group (P5 0.1488)

Figure 1—Trial profile. Between December 2017 and April 2019, a total of 140 patients were screened for a fasting C-peptide $0.12 nmol/L andpositivity for GAD65 antibodies (<50,000 IU/mL), and 109 underwent randomization. Of these study participants, 108 were included in the FAS,whose data were used in the analysis of clinical efficacy at 15 months. A total of 48 patients in the FAS were found to carry the HLA DR3-DQ2 hap-lotype. 1Information on HLA genotype was missing for one patient in the placebo group.


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Severe hypoglycemic events were only re-ported by one patient; therefore, analysisof this end point could not be performed.

Vitamin D levels increased for patientsreceiving active vitamin D throughout thesupplementation period (12.4 ng/mLmean increase at 3 months) and de-creased to starting levels by month 15,while these levels in placebo-treated pa-tients changed only slightly (4.2 ng/mLmaximum increase over trial period). Therewas no significant impact of vitamin D lev-el (average absolute levels at month 1, 2,and 3) on primary outcome across all

patients (P 5 0.1186), and Pearson corre-lation coefficient for patients with HLADR3-DQ2 was low (�0.0117). When pa-tients were categorized according to low,medium, and high levels, groups becamesmall, and influence of vitamin D on effica-cy was difficult to interpret.

A significant increase in GADA levelswas observed in individuals receivingGAD-alum treatment, both in individualspositive and in those negative for HLADR3-DQ2 haplotype, when comparedwith placebo (Fig. 3A). There was no sig-nificant difference in GADA levels

between actively treated patients posi-tive and negative for the DR3-DQ2 haplo-type, but patients with HLA DR3-DQ2had both significantly increased prolifera-tion index (Fig. 3A) and significantly in-creased GAD-stimulated IL-10 and IL-13compared with those negative for HLADR3-DQ2 or patients in the placebogroup (Fig. 3B).

The proportion of patients with anyadverse event was 49% in the activelytreated group and 58% in the placebogroup (Supplementary Table 2). Therewere three serious adverse events, all

Table 1—Baseline characteristics of study patients, according to treatment group

Safety set HLA DR3-DQ2 subgroup

CharacteristicGAD-alum(n = 57)

Placebo(n = 52)

GAD-alum(n = 29)

Placebo(n = 19)

Age, years 16.2 (3.8) 16.6 (4.3) 16.5 (4.0) 16.3 (3.9)

Time from diagnosis to first treatment, days 147.6 (54.7) 134.9 (48.1) 149.8 (52.0) 131.8 (58.0)

Sex, n (%)

Female 21 (36.8) 26 (50.0) 10 (33.3) 7 (36.8)Male 36 (63.2) 26 (50.0) 20 (66.7) 12 (63.2)

BMI category (children aged #18 years), n (%)*

<25th percentile 6 (10.5) 3 (5.8) 3 (10.0) 3 (15.8)25–75th percentile 23 (40.4) 25 (48.1) 10 (33.3) 9 (47.4)>75th percentile 14 (24.6) 9 (17.3) 8 (26.7) 3 (15.8)

BMI category (adolescents and young adults aged >18 years),kg/m2, n (%)*

$18.5 to <25.0 8 (14.0) 8 (15.4) 3 (10.0) 3 (15.8)$25.0 to <30.0 5 (8.8) 5 (9.6) 5 (16.7) 1 (5.3)>30.0 1 (1.8) 2 (3.8) 1 (3.3)

Tanner puberty stage, n (%)

1–3 13 (23.2) 7 (13.5) 6 (20.0) 2 (10.5)4–5 28 (50.0) 32 (61.5) 14 (46.7) 14 (73.7)Not applicable† 15 (26.8) 13 (25.0) 10 (33.3) 3 (15.8)

Median GADA, units/mL 76.2 77.9 57.9 80.3

HLA DR3-DQ2 haplotype, n (%)

No 27 (48.2) 32 (61.5)Yes 29 (51.8) 19 (36.5)

Fasting C-peptide, nmol/L 0.329 (0.165) 0.323 (0.168) 0.320 (0.132) 0.307 (0.130)

Stimulated C-peptide AUC 0.793 (0.381) 0.702 (0.314) 0.819 (0.341) 0.633 (0.263)

HbA1c, % 6.39 (0.96) 6.51 (1.13) 6.17 (0.71) 7.02 (1.23)

IDAA1c 7.96 (1.37) 8.16 (1.83) 7.46 (1.09) 8.68 (1.76)

Insulin dose, IU/kg body weight per day 0.393 (0.228) 0.411 (0.300) 0.324 (0.191) 0.415 (0.276)

Fasting plasma glucose, mmol/L 6.34 (1.70) 6.58 (2.07) 5.90 (1.14) 7.03 (2.45)

Data are presented as mean (SD) unless otherwise indicated. HLA DR3-DQ2 haplotype, fasting C-peptide, stimulated C-peptide AUC, HbA1c,IDAA1c, insulin dose, and fasting plasma glucose are based on FAS; others are based on safety set. Safety set includes 109 patients who re-ceived at least one injection (no exclusions were made). FAS includes 108 patients from the safety set. One patient was excluded for discon-tinuing study 1 month after first injection and therefore did not have enough efficacy data. One patient did not have HLA DR3-DQ2haplotype information. There were no missing data on other variables. In FAS, none of the differences between treatment groups were statis-tically significant. In HLA DR3-DQ2 haplotype subgroup, there were statistically significant differences between Diamyd and placebo groups inHbA1c (P = 0.0100) and IDAA1c (P = 0.0101) and near-significant difference in C-peptide AUC (P = 0.0523). Statistical testing of baseline differ-ences between treatment groups was performed using Fisher exact test for categorical variables with two categories, and Cochran-Mantel-Haenszel test (general association, nonstratified) was used for categorical variables with more than two categories. Wilcoxon test was usedfor the C-peptide AUC because of skewed distribution as well as for all tests in HLA DR3-DQ2 haplotype subgroup because of small numbers.Student t test was used in all other instances. *BMI categories according to percentiles from CDC growth charts for children aged #18 yearsand according to WHO obesity categories for adolescents and young adults aged >18 years. †Young adults aged >18 years.


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in individuals treated with placebo. Nopatient discontinued the treatment regi-men as a result of an adverse event.There were no physiological or neuro-logical concerns reported. Injection sitereactions were mild and similar in num-ber in the two groups, as were resultsof safety laboratory analyses.

CONCLUSIONS

GAD-alum administered in the current dos-ing regimen did not change the diseaseprogression of T1D across the entire trialpopulation, but it seemed to be beneficialin a genetically selected group of patients.

Immunotherapeutic interventions inT1D patients should combine efficacywith a low-risk profile and few adverseeffects to be widely accepted in clini-cal practice. In contrast to more bur-densome interventions that suppressthe immune system (5–10), GAD-alumtreatment has been shown to be easyfor patients and the health care sys-tem, and well-tolerated GAD-alumtherapy has to date shown strongsafety and patient convenience in tri-als involving >1,000 individuals, in-cluding children, adolescents, andadults.

Previous trials with GAD-alum re-vealed positive although inconsistentresults, warranting an alternative ap-proach to improve treatment efficacy(12–14,24). Animal studies have shownthat intralymphatic injections induce astrong and relevant T-cell response(25,26), and clinical studies in the aller-gy field have shown that presentationof the antigen/allergen directly into thelymph nodes seems to be more effec-tive than traditional administration(27). To test this hypothesis, a first-in-hu-man pilot trial (DIAGNODE-1) with GAD-alum administered intralymphatically

Figure 2—Primary and key secondary study outcomes. Relative change from baseline (back transformed from log scale model) in C-peptideAUCmean 0–120 min during an MMTT for the two treatment groups (GAD-alum and placebo) in the FAS (A) and the prespecified subgroup HLA DR3-DQ2(B). Error bars indicate SD. C: Forest plot depicting estimated treatment difference between GAD-alum and placebo groups for key secondary end points(IDAA1c and HbA1c in nmol/mol and daily exogenous insulin dose in units/kg/24 h) observed in the FAS and in the the prespecified subgroup HLA DR3-DQ2. Error bars indicate 95% CI. P values are indicated. Primary and key secondary efficacy end point variables were analyzed using a restricted maxi-mum likelihood–based repeated measures approach (mixed-model repeated measures), as described in RESEARCH DESIGN AND METHODS.


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commenced in a small group of adults.After showing promising results (28,29)and few adverse events, authorities al-lowed an expansion of the trial and theinclusion of children. The full pilot trialshowed encouraging results (19). To con-firm these results, a larger randomized,double-blind, placebo-controlled phaseII trial (DIAGNODE-2) was consequentlyinitiated.

While DIAGNODE-2 was ongoing, alarge meta-analysis showed clear posi-tive efficacy of GAD-alum in individualspresenting with the HLA DR3-DQ2 hap-lotype (17), whereupon this patientsubgroup was prespecified in anamendment to the study protocol. Con-firming the earlier findings described inthe meta-analysis, a significant preser-vation of b-cell function was found inthis prespecified subgroup. DIAGNODE-2 was not powered to show significancefor the prespecified subgroup, whichconstituted half of the full patient popu-lation. Even so, clear trends of improve-ment in several clinical parameterscould be observed in individuals positivefor HLA DR3-DQ2. In the prespecifiedpatient group, there were differences inbaseline HbA1c, IDAA1c, and C-peptide,but the statistical analysis adjusted forbaseline differences.

Vitamin D was supplemented for 120days in those patients with vitamin Dlevels <100 nmol/L, because adequatevitamin D concentrations may be

important to achieve an appropriate ef-fect on the immune system (30). VitaminD may have beneficial effects on thepreservation of b-cell function, but sev-eral studies have contradicted this (31).We were not able to see any significanteffect on C-peptide decline directly relat-ed to vitamin D, and because no positiveeffect was seen in the actively treatedHLA DR3-DQ2–negative patients, whoalso received supplementation with vita-min , it is unlikely that the observed effi-cacy was conferred by vitamin D.

Autoantigen treatment has been triedin many autoimmune diseases, withpoor results. There are several problemsto be solved, such as selection of auto-antigen, dose, timing, and route of ad-ministration (32). This study confirmedthe positive effects of intralymphaticautoantigen treatment, suggested in ourprevious first-in-human pilot trial (19),and this route of administration maytherefore be of interest for trials inother autoimmune diseases. Com-pared with subcutaneous administration,intralymphatic administration allows theuse of a smaller dose, reduces exposureto adjuvant, and potentially reduces im-munological interference by other vac-cines using traditional administrationroutes. It seems to be safe and easy forpatients, and the required ultrasoundcompetence needed for the injectionscan be found within the health caresystem.

There is an emerging consensus inthe field that T1D is a heterogeneousdisease (16). The importance of select-ing the right treatment for a certain en-dotype of disease has already beenshown in studies on the effect of immu-notherapy with proinsulin peptides (33)and with methyldopa (34). Individualscarrying the HLA DR3-DQ2 haplotypeare more prone to develop GADA (15).Our results show that patients with thisgenotype benefit from antigen immuno-therapy with GAD-alum. Notably, ourtrial points to the crucial importance ofchoosing the right therapeutic antigento treat specific endotypes of a disease(22). We observed significant differ-ences in immune response in patientspositive for HLA DR3-DQ2 comparedwith those negative for HLA DR3-DQ2,and further careful and extensive analy-sis of the immune responses in this trialwill be performed to gain a deeper un-derstanding of the mechanism of thetreatment.

The value of residual insulin secretionfor prevention of complications is wellknown, although it is generally difficultto show clinical efficacy in variablessuch as insulin dose or HbA1c in studieswith a follow-up of <24 months in pa-tients receiving modern intensive insulintreatment, because the study designaims for best possible metabolic controlin all patients, including those who re-ceive placebo treatment.

Figure 3—Pharmacological outcomes. Median changes from baseline of GADA and proliferation (Pr) of PMBC (stimulation index [SI]) (A) and GAD-stimulated secretion by PBMC of IL-10 and IL-13 levels (B) for GAD-alum–treated patients with and without the DR3-DQ2 haplotype as well as pla-cebo-treated patients. P values by Wilcoxon test are indicated.


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This study was not designed or pow-ered to specifically evaluate individuals car-rying HLA DR3-DQ2, nor was it stratifiedfor this variable. Therefore, the number ofpatients in the specified subgroup wassmaller, and there were some differencesin the baseline characteristics of the activeand placebo groups. Also, outside of theprimary and key secondary end pointsevaluated for the total patient population,there was no adjustment for multiple endpoints performed in the statistical analysisof efficacy. However, the statistical modelaccounts for baseline values, and the sub-group in focus was prespecified based ona comprehensive meta-analysis (17) ofdata from previous studies and was basedon a scientific rationale associated withthe genetic predisposition to autoimmuni-ty against GAD65. Therefore, the positiveresult in the primary end point in this sub-group supports that GAD-alum treatmentis efficacious. This needs to be confirmedin future placebo-controlled studies ofGAD-alum treatment in this subgroup.Thereafter, it will be natural to studywhether booster doses of GAD-alum willfurther prolong the preservation of b-cellfunction.In summary, intralymphatic adminis-

tration of GAD-alum in individuals withrecent-onset T1D is a relatively easy andtolerable treatment. The study did notmeet its primary end point in the fullpatient cohort. However, in the prespe-cified subgroup of patients with HLAhaplotype DR3-DQ2, who may consti-tute up to half of all individuals withGADA-positive T1D, those who receivedactive GAD-alum treatment had preser-vation of b-cell function significantlybetter than patients who received pla-cebo, and the clinical course of the dis-ease tended to be improved. Becausemodern medicine is about matching theappropriate drugs or treatments to thepatient populations most likely to bene-fit from them (35), this finding is prom-ising, and if confirmed in a larger clinicaltrial, it could pave the way for such aprecision medicine approach in T1D.

Acknowledgments. The authors thank all theparticipants who agreed to participate in thestudy. The authors are grateful to Dr. JoachimDavidsson (Department of Radiology, Link€opingUniversity Hospital, Link€oping, Sweden) for skillfulperformance of the needle-guided lymph nodeinjections and instructions to other centers. Theauthors also thank Isabel Leiva (Malaga, Spain)

and all research nurses and physicians at thesites, as well as Ingela Johansson and GosiaSmolinska (Division of Pediatrics, Department ofBiomedical and Clinical Sciences, Faculty of Medi-cine and Health Sciences, Link€oping University,Link€oping, Sweden) for skillful laboratory work.Funding. This study was supported by Barn-diabetesfonden (Swedish Child Diabetes Foun-dation) and Diabetesfonden. Diamyd Medicalprovided GAD-alum, vitamin D, and matchingplacebos.

Neither Barndiabetesfonden nor Diabetesfon-den was involved in the management of the tri-al, data collection, or analysis or in the decisionto submit the manuscript for publication.Duality of Interest. This study was fundedby an unrestricted grant from Diamyd Medi-cal. U.H. is employed by Diamyd Medical. Noother potential conflicts of interest relevantto this article were reported.

Diamyd Medical was involved in the man-agement of the trial, data collection, andanalysis, both directly and through contractresearch organizations.Author Contributions. J.L. was responsiblefor conceptualization, data curation, analyses,funding acquisition, investigation, methodology,project administration, resources, supervision,validation, and visualization; wrote the originaldraft; and has verified the underlying data. A.N.was responsible for statistical analyses and vali-dation. U.H. contributed to conceptualizationand was responsible for data curation, analyses,funding acquisition, project administration, re-sources, supervision, and validation. R.C. wasresponsible for data curation, analyses, method-ology, project administration, and validation andhas verified the underlying data. All otherauthors contributed to investigation and acquisi-tion of patients and data. All authors reviewedand edited the manuscript and approved the fi-nal version and submission. J.L. is the guarantorof this work and, as such, had full access to allthe data in the study and takes responsibility forthe integrity of the data and the accuracy of thedata analysis.

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1604 diabetes care volume 44, july 2021 emerging therapies

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