Formulation and Characterization of Conjugate Vaccines to Reduce

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Formulation and characterization of conjugate vaccines to reduce opioid use disorders suitable for pharmaceutical manufacturing and clinical evaluation. Federico Baruffaldi, M. D. Raleigh, Samantha King, Michaela Roslawski, Angela Birnbaum, Carla Hassler, F. Ivy Carroll, Scott P Runyon, Scott Winston, Paul R. Pentel, and Marco Pravetoni Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01296 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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Molecular Pharmaceutics

1 1

Formulation and characterization of conjugate vaccines to reduce opioid use disorders

2

suitable for pharmaceutical manufacturing and clinical evaluation.

3

1Baruffaldi

4

3Runyon

F, 1Raleigh MD, 1King SJ, 2Roslawski MJ, 2Birnbaum AK, 3Hassler C, 3Carroll FI,

SP, 4Winston S, 1,5Pentel PR, and 1,5,6,7Pravetoni M.

5 6

1Hennepin

7

Pharmacy, Minneapolis, MN;

8

Biopharmaceutical Consulting, CO; University of Minnesota

9

6Department

Healthcare Research Institute, Minneapolis, MN; 2University of Minnesota College of 3RTI

International, Research Triangle Park, NC; 5Department

4Winston

of Medicine,

of Pharmacology, and 7Center for Immunology, Minneapolis, MN.

10 11

Keywords: opioid use disorder, oxycodone, heroin, vaccine, antibody, conjugate, GMP, GLP,

12

FDA.

13 14

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2 16

ABSTRACT

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This study focused on formulating conjugate vaccines targeting oxycodone and heroin for

18

technology transfer, Good Manufacturing Process (GMP), and clinical evaluation. Lead

19

vaccines used the highly immunogenic carrier protein keyhole limpet hemocyanin (KLH),

20

which poses formulation problems because of its size. To address this barrier to translation,

21

an oxycodone-based hapten conjugated to GMP-grade subunit KLH (OXY-sKLH) and

22

adsorbed on alum adjuvant was studied with regard to carbodiimide coupling reaction time,

23

buffer composition, purification methods for conjugates, conjugate size, state of aggregation,

24

and protein:alum ratio. Vaccine formulations were screened for post-immunization antibody

25

levels and efficacy in reducing oxycodone distribution to the brain in rats.

26

conjugates were more immunogenic, their size prevented characterization of haptenation

27

ratio by standard analytical methods and sterilization by filtration. To address this issue,

28

conjugation chemistry and vaccine formulation were optimized for maximal efficacy, and

29

conjugate size was measured by dynamic light scattering prior to adsorption to alum. An

30

analogous heroin vaccine (M-sKLH) was also optimized for conjugation chemistry,

31

formulated in alum, and characterized for potency against heroin in rats. Finally, this study

32

found that the efficacy of OXY-sKLH was preserved when co-administered with M-sKLH,

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supporting the proof of concept for a bivalent vaccine formulation targeting both heroin and

34

oxycodone. This study suggests methods for addressing the unique formulation and

35

characterization challenges posed by conjugating small molecules to sKLH while preserving

36

vaccine efficacy.

37 38


While larger

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3 40

INTRODUCTION

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The United States is facing a public health crisis resulting from widespread opioid use disorders

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(OUD) and increased incidence of opioid-related overdoses. The incidence of opioid-related

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fatal overdoses has quadrupled since 1999 1 and over 42,249 people died from opioid overdose

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in 2016 alone

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Medication Assisted Treatment (MAT)

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molecule-based pharmacotherapies, vaccines may offer a safe and cost-effective treatment for

47

OUD. Vaccines stimulate the patient’s own immune system to produce opioid-specific polyclonal

48

antibodies that selectively bind the target opioid and prevent its distribution across the blood

49

brain barrier, subsequently reducing opioid-induced behavior and other opioid-induced

50

pharmacological undesired effects, such as respiratory depression 5. Although several studies

51

have shown pre-clinical efficacy of opioid vaccines in mouse

7-8,

52

primates

13.

53

based conjugate vaccine showed safety, but only limited information is available

54

the need for further clinical evaluation of this approach. Ultimately, human studies will answer

55

the question of whether opioid vaccines have the potential to become a viable treatment option

56

for OUD.

11-12,

2-4.

Only a limited subset of the population diagnosed with OUD is receiving 5-6.

As a complementary alternative to approved small

only one clinical trial has been conducted to date

rat

9-10,

and non-human

Evaluation of a morphine13,

suggesting

57

Some of the challenges for transitioning vaccine candidates into late-stage pre-clinical

58

development are: 1) generating a well-characterized hapten-protein conjugate (drug substance)

59

and hapten-protein conjugate adsorbed on adjuvant (drug product) that can be manufactured at

60

the scale required for clinical testing, 2) access to vaccine components produced under Good

61

Manufacturing Practices (GMP), and 3) establish or meet release criteria for vaccine products

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compliant to the guidelines of the Food and Drug Administration (FDA), European Medical

63

Agency (EMA) or other regulatory agencies. Although several candidate vaccines for OUD



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showed excellent pre-clinical efficacy, very few were made using GMP components, and their

65

manufacturing processes optimized, and fully characterized 11, 14-15.

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A first-generation vaccine consisting of an oxycodone-based hapten attached to the

67

native keyhole limpet hemocyanin (KLH, decamer or didecamer) carrier protein adsorbed to

68

alum adjuvant (OXY-KLH) has shown extensive pre-clinical safety and efficacy against

69

oxycodone in mice and rats

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therefore cannot be used in human trials. Because of its large molecular weight, native KLH is

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also difficult to characterize using standard analytical methods complicating translation of KLH-

72

based vaccines. A second-generation oxycodone vaccine containing a GMP-grade subunit

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dimer KLH (OXY-dKLH) has shown equivalent efficacy compared to OXY-KLH and OXY

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conjugated to tetanus toxoid (OXY-TT) 9, but has not been fully optimized for scale-up and

75

technology transfer. Finally, the lead OXY hapten has proven effective when conjugated to other

76

carrier proteins including a GMP-grade subunit monomer KLH (sKLH), CRM197, and TT as well

77

as E. coli-expressed CRM- (EcoCRM) and TT-based carriers

78

accessible GMP-grade carrier, our group has been seeking FDA-approval for clinical evaluation

79

of the OXY-sKLH, and the primary focus of this study was to facilitate manufacturing of OXY-

80

sKLH adsorbed on alum adjuvant. Because we have previously shown efficacy of the M-KLH,

81

M-sKLH, and M-EcoCRM vaccines targeting heroin and its metabolites in mice and rats 20-22, the

82

secondary goal of this study was to optimize formulation of M-sKLH for further development.

83

Finally, the tertiary goal of this study was to test whether both OXY-sKLH and M-sKLH could be

84

co-administered in a bivalent vaccine formulation targeting both heroin and oxycodone. First,

85

OXY-sKLH was conjugated under a range of conditions to optimize haptenation ratio, conjugate

86

appearance and stability, and subsequently tested for immunogenicity and potency in rats. To

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allow for sterile filtration, chelants and reducing agents were evaluated for controlling conjugate

88

size. Reducing conjugate size effectively decreased the degree of precipitation and loss on

16-19,

but native KLH is not available as GMP-grade material and


20.

Since the sKLH was the most

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5 89

filtration but also negatively impacted the immunogenicity of the vaccine. Larger OXY-sKLH

90

conjugates were characterized by dynamic light scattering (DLS) for size and an ELISA was

91

developed to measure relative haptenation of conjugates. To optimize M-sKLH, carbodiimide

92

and maleimide coupling chemistries were compared for generation of effective vaccine

93

formulations. In this study, addition of 10% DMSO during carbodiimide conjugation yielded the

94

most effective M-sKLH formulation. A previous study supported the rationale for developing a

95

bivalent formulation that combines OXY-sKLH and M-sKLH for use as a single vaccine in

96

humans, yet the study focused on a first-generation bivalent formulation containing native KLH

97

and injected intraperitoneally in Freund’s Complete and Incomplete Adjuvants

98

advance this concept, in the current study OXY-sKLH adsorbed on alum was administered IM in

99

combination with M-sKLH to determine whether a bivalent immunization regimen would interfere

23.

To further

100

with its efficacy against oxycodone.

101

effective as doubling the dose of OXY-sKLH and more effective than the single dose of

102

immunogen against oxycodone.

103

evaluation.

Co-administration of OXY-sKLH and M-sKLH was as

These studies will advance the OXY-sKLH toward clinical

104



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2. Material and methods

107

2.1 Synthesis of oxycodone (OXY) hapten and conjugation to sKLH and bovine serum

108

albumin (BSA). The oxycodone-based hapten containing a tetraglycine linker at the C6 position

109

was synthesized as previously described to generate or Li++ salt to scale for manufacturing

110

As part of a technology transfer effort to support synthesis of GMP-grade haptens, the

111

lyophilized form of the OXY(gly)4 hapten was synthesized at 30 g scale at Cambrex, NC.

112

Because the native KLH used in previous studies is not available as GMP source, the OXY(gly)4

113

hapten was conjugated to either the GMP-grade monomer KLH (Biosyn, Carlsbad, CA) or the

114

GMP-grade dimer KLH (Stellar, Port Hueneme, CA). Because these KLH subunit formulations

115

were equally immunogenic (Fig. S1), and for simplicity, both were labeled as sKLH throughout.

116

In addition, no differences in immunogenicity and efficacy were found when OXY-sKLH

117

containing lyophilized OXY(gly)4 hapten synthesized at 30 g scale was compared with OXY-

118

sKLH containing powder OXY(gly)4 hapten synthesized at 1.54 g scale (Fig. S1). For use as

119

coating antigen in ELISA assays, haptens were conjugated to BSA (Sigma Aldrich, St. Louis,

120

MO). All conjugation conditions for the OXY hapten to sKLH are detailed in Table 1. The OXY

121

hapten (range 5.2-52 mM) and ethyl-N′-(3 dimethylaminopropyl)carbodiimide hydrochloride

122

(EDAC, 5.2-208 mM) (Sigma-Aldrich, St. Louis, MO) were dissolved in 0.1M MES buffer (range

123

pH 4.5-6.0) and reacted for 10 min at room temperature (RT). sKLH or BSA were added and

124

reactions stirred for 3 hr at RT followed by ultrafiltration using Amicon filters with 50 or 100 kDa

125

molecular weight cutoff (Merk Millipore, Burlington MA). MES buffer was exchanged with PBS

126

0.1M pH 7.2 and the conjugate was stored at + 4°C.

127

2.2 Synthesis of morphine (M) hapten and conjugation to subunit KLH and BSA. The

128

intermediate morphine hapten structure without the tetraglycine linker was synthesize as

129

previously described

130

terminal thiol group (M(gly)4SH) as previously described for an analogous OXY(gly)4SH

23.

20.

A modified version of the M(gly)4OH hapten was generated by adding a


20.

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7 131

M(gly)4OH and M(gly)4SH Li++ haptens were conjugated to either sKLH or BSA as previously

132

described

133

avoid precipitation as detailed below. One condition involved conjugation of the M(gly)4OH

134

hapten to sKLH by dissolving 5.2 mM of hapten and 208 mM of EDAC in MES pH 4.5 in

135

presence of 10% of DMSO (conjugate M1, Fig. 5). A second condition involved activation of the

136

M(gly)4OH hapten with 52 mM EDAC in presence of 128 mM sulfo-NHS (N-hydroxysuccinimide,

137

Thermo Fisher Scientific, Waltham, MA) and 10% DMSO in MES buffer pH 5.0. Upon

138

conversion to the NHS ester the pH was increased to 7.0 using sodium hydroxide. Conjugations

139

proceeded for 2 hr at RT. After 2 hr, the conjugate was purified as described in the section

140

above (conjugate M2, Fig. 5). A third condition consisted of conjugating M(gly)4SH to maleimide

141

activated sKLH, which was activated with sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-

142

1-carboxylate (80:1 ratio of maleimide:sKLH moles) (sulfo-SMCC, Thermo Scientific, Waltham,

143

MA) for 2 hr at RT as described

144

ultrafiltration. The hapten was dissolved in 715 µL of PBS pH 7.2 containing 1 mM

145

Ethylenediaminetetraacetic acid (EDTA) and 50 mM tris(2-carboxyethyl)phosphine (TCEP)

146

(Sigma Aldrich, St. Louis, MO), added to the sKLH solution and the conjugation proceeded for 4

147

hr at RT. After 4 hr, the conjugate was purified by ultrafiltration and stored in PBS pH 7.2 at 4°C

148

(conjugate M3, Fig. 5).

149

2.3 Characterization of conjugates

150

2.3.1 Dynamic light scattering (DLS). Analysis was performed using a Zetasizer S90 (Malvern

151

Instruments Inc., Westborough, MA) equipped with a 633-nm laser and an output power in the

152

range of 10–50 mW. The size and diameter of the conjugates were measured under a 173-

153

degree backscatter. To analyze the aggregation of the conjugate over time, an autopiloted

154

measurement was applied, after manually mixing the sample. Measurements were performed

155

with 75 µL OXY-sKLH solution in 0.01M PBS buffer pH 7.2 at a constant temperature of 25oC

20, 23,

, using either carbodiimide or maleimide chemistry with minor modifications to

20.

The maleimide activated protein was desalted by



8 156

using a 40 µL cuvette (Malvern Instruments Inc., Westborough, MA). Data were analyzed using

157

Zetasizer software 7.12 and the raw data exported to Excel and analyzed with Prism v.7 (Graph

158

pad Software, La Jolla, CA). Analysis of three different OXY-sKLH batches by DLS validated

159

this method (Fig. S2). The size distribution of OXY-sKLH was consistent across the three

160

different batches, confirming the reproducibility of the OXY-sKLH conjugation reaction and the

161

DLS as a method to measure the size distribution of these conjugates.

162

2.3.2 Size exclusion chromatography-high performance liquid chromatography (SEC-

163

HPLC). Samples were analyzed on a Shimadzu Prominence LC-20AD dual pumps system with

164

a SPD-20A UV/VIS Spectrophotometer detector (Shimadzu Scientific Instruments Inc.,

165

Columbia, MD) using a GE Healthcare Superose 6 Increase column (GE Healthcare Bio-

166

Sciences, Marlborough, MA). The analysis was performed at 280 and 230 nm wavelength using

167

0.5 mL/min flow rate. A PBS solution pH 7.0 mobile phase was selected to allow good

168

separation, resolution and for compatibility with solutions contained in samples of interest

169

without hindering resolution and elution of the size standards. Several injections of mobile

170

phase alone were run throughout the batch to ensure there was no peak carry-over and/or

171

peaks from mobile phase contributing to the baseline. Run time was determined based on the

172

length of time it took for the size standards to elute (40 min). In instances where an aggregate

173

peak eluted into the next sample injection, run time was extended to include all sample peaks

174

and size standards were run again to verify column efficiency.

175

2.3.3 Measurement of hapten density by ELISA. Because sKLH is too large to measure

176

haptenation ratio (number of haptens per protein) using MALDI-TOF, semi-quantitative hapten

177

density was measured using a modified ELISA. Mouse sera was obtained from mice immunized

178

with OXY-CRM197, purified with Protein G (Thermo Fisher Scientific, Waltham, MA) and

179

oxycodone-specific antibodies were coated on a 96 well plate (costar 9018 EIA/RIA 96, Jackson

180

Research Laboratories, Inc., West Grove,PA) at 5 ng/well concentration using 0.5M carbonate


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9 181

buffer at pH 9.6 and stored overnight at 4oC. On the following day, the solution was discarded

182

and the plate washed 5 times with 0.05M phosphate buffered saline tween-20 pH 7.2-7.4

183

(PBST). The plate was blocked with 1% gelatin blocking buffer in PBST. After 1 hr the gelatin

184

was discarded and the plate was washed and stored overnight at 4°C. On day 3, a standard

185

curve was made using OXY-BSA conjugates with a range of haptenation ratios measured by

186

MALDI-TOF and diluted to 5 µg/mL in PBST (Fig. S3). BSA and sKLH controls, as well as OXY-

187

sKLH conjugates with unknown haptenation ratios, were also diluted to 5 µg/mL. All samples

188

were added to the plate in quadruplicate at 100 µL and incubated for 2 hr at room temperature,

189

slowly mixing at 60 rpm. After 2 hr, the samples were discarded and the plate was washed. Rat

190

sera which contained oxycodone-specific polyclonal antibodies derived from rats immunized

191

with OXY-TT was diluted 5400x and 100 µL added to three of the four wells per sample and 100

192

µL of 1:200 anti-sKLH rat serum was added to the remaining sample well to test for the

193

presence of sKLH. The plate was gently mixed at 60 rpm for 1.5 hr room temperature and

194

incubated without mixing for another 0.5 hr before discarding contents and washing with PBST.

195

Secondary antibody Fc-specific goat anti-rat coupled to horseradish peroxidase (Jackson

196

ImmunoResearch Laboratories, Inc., West Grove, PA) was diluted 1:50,000 and 100 µL was

197

added to the plate and stored at 4oC overnight. The following day, the contents were discarded

198

and the plate was washed with PBST. Enzyme substrate o-phenylenediamine (OPD) was

199

added to all sample wells (SIGMAFASTTM tablet set, Sigma Life Sciences, St Louis, MO). After

200

30 min of incubation, 2% oxalic acid was added to stop the enzymatic reaction. Plate was read

201

at 492 nm on a BioTek PowerWave XS (BioTek Instruments Inc., Winooski, VT). OXY-sKLH

202

haptenation ratio was measured based on the OXY-BSA standard curve (function plotted as

203

absorbance at 492 nm versus MALDI-TOF measured haptenation ratio), and the presence of

204

sKLH was observed based on the comparison of the OD values from the OXY-BSA vs. OXY-

205

sKLH conjugates. To further qualify this hapten density ELISA assay, results reproducibility was

206

determined within plates on the same day and across different days using representative OXY-



10 207

sKLH conjugates ranging in haptenation ratio. The results are reported in Table S1

208

(supplemental material).


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11 210

2.3.4 SDS-PAGE

211

Samples were diluted to 1 µg/ml need concentration here in sterile water (dH2O) and combined

212

with 4x laemmli sample buffer containing 2-mercaptoethanol according to manufacturer’s

213

instructions (BioRad, Hercules,CA). Samples incubated for 10 min at 25oC and 5 min at 95oC

214

before loading into a 3-8% tris-acetate protein gel submerged in XT-tricine running buffer

215

(BioRad, Hercules, CA). A large molecular weight (MW) protein ladder (30 to 60 KDa, Thermo

216

Fisher Scientific, Waltham, MA) was used to guide MW analysis. The gel was run at a constant

217

voltage of 50V for 30 min followed by 200V for 2 hr. After several dH2O washes, the gel was

218

stained in biosafe Coomassie G250 stain (BioRad, Hercules, CA) for 1 hr. The gel was then

219

washed in dH2O for 30 min before imaging.

220

2.4 Experimental design and immunization

221

2.4.1 Ethics statement. These studies were performed following the recommendations of the

222

Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal

223

protocols were approved by the Hennepin Healthcare Research Institute Animal Care and Use

224

Committee. Animals were euthanized by CO2 inhalation using AAALAC approved chambers,

225

and all efforts were made to minimize suffering.

226

2.4.2 Animals. Male Holtzman and Sprague Dawley rats (Envigo, Madison, WI) weighing 200-

227

225 g at day of arrival were double housed with 12/12 hr standard light/dark cycle and free-fed.

228

Testing occurred during the light phase.

229

2.4.3 Vaccine immunogenicity: antibody analysis. Oxycodone or heroin/morphine-specific

230

serum IgG antibody titers were measured using ELISA as previously described

231

ELISA plates (Costar 9018 EIA/RIA, Jackson Immunoresearch Laboratories Inc., West Grove,

232

PA) were coated with 5 ng/well of BSA conjugates or unconjugated protein control in carbonate

233

buffer at pH 9.6 and blocked with 1% gelatin. Primary antibodies were incubated with goat anti-


9, 23.

Briefly,


Page 12 of 38

12 234

rat IgG antibodies conjugated to horseradish peroxidase or rabbit anti-mouse IgG antibodies

235

(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) to measure immunized rat and

236

mouse sera.

237

2.4.4 Vaccine potency: hotplate nociception test and analysis of oxycodone and heroin

238

distribution to serum and to the brain. The effect of immunization in reducing opioid

239

nociception and opioid distribution to the brain was used to measure vaccine potency in rats

240

challenged with single doses of either oxycodone or heroin (NIDA Drug Supply Program). Rats

241

were habituated to the testing environment for 1 hr, and then tested on a hotplate (Columbus

242

Instruments, Columbus, OH) set to 54°C to obtain baseline latencies, a nociceptive response of

243

hindpaw lick or jumping. A maximum cutoff of 60 sec was used to avoid injury. 30 min later, rats

244

were injected subcutaneously (SC) with either oxycodone or heroin, and their post-drug latency

245

was obtained 30 min post-challenge. Serum and brain samples were collected immediately

246

afterward and oxycodone concentrations were measured by gas chromatography-mass

247

spectrophotometry

248

determined by liquid chromatography-mass spectrophotometry (LC-MS)

249

blocking opioid-induced antinociception was calculated as the percent maximum possible effect

250

(%MPE), calculated as (post-drug latency – pre-drug latency / maximum latency – pre-drug

251

latency) x 100 21.

252

2.4.5 Effect of pH and EDAC concentration on efficacy of OXY-sKLH conjugates. Rats

253

were vaccinated on days 0, 21, 42, and 63 IM with 60 μg OXY-sKLH conjugated under a range

254

of pH and EDAC concentrations as defined above (see section 2.1, and summary Table 1)

255

adsorbed to 90 μg of alum adjuvant (Alhydrogel, Brenntag Biosector, Denmark) and 0.01%

256

polysorbate 80 (PS80, Avantor, Center Valley, PA) in a final volume of 0.15 mL. Control rats

257

(n=10/group) were vaccinated IM with 60 μg unconjugated sKLH in 90 μg alum adjuvant in a

17.

Heroin, 6-AM, and morphine in serum and brain samples were


22.

Vaccine efficacy in

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13 258

final volume of 0.15 mL in PBS pH 7.2. On day 70, blood was collected via tail vein for antibody

259

characterization.

260

2.4.6 Effect of OXY-sKLH immunogen and alum dose on efficacy. Rats were vaccinated on

261

days 0, 21, 42, and 63 IM with 0.4 mg/mL OXY-sKLH in 0.6 mg/mL alum adjuvant and 0.01%

262

polysorbate 80 in a final volume of 0.15 mL (30 μg, 60 μg OXY-sKLH and 45 μg, 90 μg alum

263

adjuvant), or 0.3 mL (120 μg OXY-sKLH and 180 μg alum adjuvant). A group of 12 rats was

264

also vaccinated IM with 0.4 mg/mL unconjugated sKLH in 0.6 mg/mL alum adjuvant in a final

265

volume of 0.3 mL (120 μg sKLH and 180 μg alum adjuvant). Blood was collected via tail vein on

266

day 70 for serum antibody characterization. On day 77 all rats received 2.25 mg/kg oxycodone

267

SC and were tested for hot plate nociception 30 min later. Immediately following hotplate

268

testing, blood and brain were collected to measure oxycodone concentrations.

269

2.4.7 Effect of sterile filtration on immunogenicity of OXY-sKLH.

270

Rats were vaccinated on day 0 IM with 60 μg OXY-sKLH in 90 μg of alum adjuvant and 0.01%

271

polysorbate 80 in a final volume of 0.15 mL. Control rats were vaccinated IM with 60 μg

272

unconjugated sKLH in 90 μg alum adjuvant in a final volume of 0.15 mL in PBS pH 7.2. On day

273

7, blood was collected via tail vein for antibody characterization.

274

2.4.8 Effect of TCEP and EDAC on size and subsequent immunogenicity of OXY-sKLH.

275

Rats were vaccinated on day 0 IM with 60 μg OXY-sKLH in 90 μg of alum adjuvant and 0.01%

276

polysorbate 80 in a final volume of 0.15 mL. Control rats were vaccinated IM with 60 μg

277

unconjugated sKLH in 90 μg aluminum hydroxide in a final volume of 0.15 mL in PBS pH 7.2.

278

On day 7, blood was collected via tail vein for antibody characterization.

279

2.4.9 Effect of conjugation chemistry on M-sKLH immunogenicity and size. Rats were

280

vaccinated on days 0, 21, 42, and 63 IM with 60 μg M-sKLH in 90 μg of alum adjuvant and

281

0.01% polysorbate 80 in a final volume of 0.15 mL. This experiment compared M-sKLH



14 282

conjugates M1, M2 and M3, generated by carbodiimide, carbodiimide in presence of NHS ester,

283

or maleimide. Control rats were vaccinated IM with 60 μg unconjugated sKLH in 90 μg of alum

284

adjuvant in a final volume of 0.15 mL in PBS pH 7.2. On day 70, blood was collected via tail vein

285

for antibody characterization. On day 77 all groups of rats received 1 mg/kg heroin SC and were

286

tested for hot plate nociception 30 min later. Immediately following testing blood and brain were

287

collected to measure the concentration of heroin and its metabolites by LC-MS.

288

2.4.10 Effect of co-administration of M-sKLH and OXY-sKLH on oxycodone distribution.

289

Rats were vaccinated on days 0, 21, 42, and 63 IM with 0.4 mg/mL OXY-sKLH, M-sKLH or

290

OXY-sKLH plus M-sKLH in 0.6 mg/mL alum adjuvant and 0.01% polysorbate 80 in a final

291

volume of 0.15 mL or 0.3 mL (60 μg OXY-sKLH, 60 μg M-sKLH, 60 μg OXY-sKLH plus 60 μg

292

M-sKLH, and 120 μg OXY-sKLH in 90 μg and 180 μg alum adjuvant, respectively). Control rats

293

were vaccinated IM with 0.4 mg/mL unconjugated sKLH in 0.6 mg/mL alum adjuvant in a final

294

volume of 0.15 mL (60 μg sKLH and 90 μg alum adjuvant). Blood was collected via tail vein on

295

day 70 for serum antibody characterization. On day 77 all groups of rats received 2.25 mg/kg

296

oxycodone SC and were tested for hot plate nociception 30 min later. Immediately following

297

testing blood and brain were collected to measure oxycodone concentrations.

298

2.5 Statistical analysis.

299

Data were analyzed using Prism version 7.0 (GraphPad Software, San Diego, CA). The mean

300

antibody titer, serum and brain concentrations, percentage (%) MPE across groups were

301

analyzed by one-way ANOVA test paired with Tukey’s multiple comparisons post-hoc test.

302

Group that didn’t pass the D’Agostino & Pearson normality test were analyzed using Kruskal-

303

Wallis test.


Page 14 of 38

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15 305

RESULTS

306

EXPERIMENT 1. Characterizing haptenation ratio of OXY-BSA conjugates

307

Conjugation of oxycodone haptens, OXY(gly)4 Li++ salt and OXY(gly)4 TFA base, to BSA yielded

308

haptenation ratios of 22 and 20, respectively. OXY(gly)4 Li++ salt was subsequently synthesized

309

as a powder or lyophilized, conjugated to BSA, and yielded haptenation ratios of 27 and 28,

310

respectively. Because the lyophilized hapten is easier to reconstitute compared to powder and

311

because it showed the highest haptenation ratio, it was used in subsequent experiments.

312

EXPERIMENT 2. Effect of pH and EDAC concentration on haptenation ratio of OXY-BSA

313

and immunogenicity of OXY-sKLH

314

The OXY(gly)4 Li++ hapten conjugated to BSA using MES buffer (4.5 – 7 pH) yielded higher

315

haptenation ratios at lower pHs (Fig. 1A). The OXY(gly)4 Li++ lyophilized hapten was

316

subsequently conjugated to sKLH using MES pH 4.5 and 52 mM EDAC and its immunogenicity

317

was tested in rats. While this vaccine formulation elicited oxycodone-specific serum IgG

318

antibody titers of 68 ± 24 x 103 (mean ± SD, Fig. 1B), the conjugate itself precipitated in solution

319

after 2 hr from the beginning of the conjugation. To prevent precipitation, conjugation conditions

320

were optimized using OXY-BSA as a model immunogen over a range of EDAC concentrations

321

(52-208 mM) at pH 6 in MES buffer. Increasing the concentration of EDAC during the

322

conjugation of OXY-BSA led to higher haptenation ratios (Fig. 1A). OXY-sKLH was then

323

conjugated using 208 mM EDAC in the pH 4.5-6.0 range and showed relative hapten densities

324

of 33-38 as calculated by hapten density ELISA. OXY-sKLH conjugated at lower pH conditions

325

elicited higher oxycodone-specific IgG titers (Fig. 1B). The OXY-sKLH conjugated at pH 4.5

326

showed slight precipitation in solution, whereas the OXY-sKLH conjugated using pH 5.0 and

327

208 mM EDAC showed higher hapten density and elicited high oxycodone-specific IgG titers.

328

Hence, OXY-sKLH conjugated using pH 5.0 and 208 mM EDAC was chosen as the lead

329

vaccine for all subsequent studies.



16 330

331 332

Figure 1. Effect of pH and EDAC concentration on haptenation ratio and immunogenicity.

333

(A) MALDI-TOF was used to calculate haptenation ratios of OXY-BSA conjugated at pH 4.5 – 7

334

using 5.2 mM of hapten, 52 or 208 mM EDAC concentrations and (A, inset) at pH 6.0 from 52 –

335

208 mM EDAC added to BSA or sKLH for a final concentration of 2.3 or 2.8 mg/mL respectively.

336

(B) Male Holtzman rats (n= 10/group) were immunized by IM injection with 60 µg of OXY-sKLH

337

and 90 µg of alum on day 0, 21, 42 and 63. Serum was collected on day 70 and oxycodone-

338

specific serum IgG antibody titers were determined by ELISA. * p < 0.05 brackets indicate group

339

differences.


Page 16 of 38

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17 340

EXPERIMENT 3. Effect of dose on immunogenicity of OXY-sKLH

341

Rats were first vaccinated with 1, 3, 10 and 30 µg of OXY-sKLH to determine the lowest

342

effective dose of vaccine. Only rats that received 10 and 30 µg of OXY-sKLH developed high

343

titers, which were significantly different from titers elicited by 1 and 3 µg of OXY-sKLH

344

respectively (Fig. S4 Panel A). In addition, brain oxycodone concentration was reduced by 38

345

and 35% in rats that were immunized with 10 and 30 µg of OXY-sKLH respectively (Fig. S4

346

Panel B). In a subsequent experiment, vaccination with 30, 60, or 120 µg OXY-sKLH elicited

347

oxycodone-specific IgG antibody titers in all vaccinated rats (Fig. 2A). Rats that received 60 µg,

348

but not 30 or 120 µg, of OXY-sKLH showed reduced %MPE compared to controls (p < 0.01,

349

Fig. 2B). Serum oxycodone concentrations were significantly increased in all groups compared

350

to controls (p < 0.0001, Fig. 2C). Brain oxycodone concentrations were significantly reduced in

351

all groups compared to controls (p < 0.01, 0.001, Fig. 2D). 60 µg OXY-sKLH formulated with 90

352

µg aluminum was chosen as the lead formulation for subsequent experiments because it was

353

the lowest dose of vaccine that showed the greatest efficacy in the thermal nociception test.



18

354 355

Figure 2. Oxycodone-specific antibody titers, oxycodone distribution and antinociception

356

in rats. Male Holtzman rats (n= 12/group) were immunized by IM injection with 30 , 60 and

357

120 µg of OXY-sKLH and 45, 90 and 180 µg of alum, respectively, on day 0, 21, 42 and 63. (A)

358

Serum was collected on day 70 and oxycodone-specific antibody titers were measured. (B) On

359

day 77 animals received a SC injection of 2.25 mg/kg of oxycodone and 30 min later were

360

tested on a hotplate set to 54C for nociception. (C) Serum and (D) brain samples were

361

collected immediately following hotplate testing. Numbers above bars represent the percentage

362

of difference from controls. Distribution and behavioral data are the mean ± SD.** p < 0.01, *** p

363

< 0.001, **** p < 0.0001 compared to control.

364 365 366


Page 18 of 38

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19 367

EXPERIMENT 4. Effect of sterile filtration on immunogenicity of OXY-sKLH

368

Rats were immunized with OXY-sKLH conjugates that were filtered with either regenerated

369

cellulose (RC) 0.2 µm or RC 0.45 µm filters (Sartorius AG, Germany) and compared to non-

370

filtered conjugates. Vaccine batches sterile-filtered with 0.2 µm, but not 0.45 µm, filters elicited

371

significantly lower oxycodone-specific antibody titers compared to non-sterile filtered OXY-sKLH

372

(p < 0.05, Fig. 3A). Whereas 0.45 µm filtration did not affect the size of the drug substance, the

373

conjugate filtered using the 0.2 µm showed a reduction in size when analyzed by DLS (Fig. 3B),

374

suggesting that OXY-sKLH is too large to be sterile-filtered and 0.2 µm filtration might impact

375

the identity of the drug substance. Sterile filtration through 0.45 µm-size filters resulted in a 94%

376

recovery, whereas 0.2 µm-size filters resulted in a 40% recovery, suggesting that these

377

conjugates may be too large for standard sterile filtration methods. A subsequent experiment

378

compared the effect of purifying OXY-sKLH by either PES (polyethersulfone) or RC

379

(regenerated cellulose) membranes on the vaccine’s immunogenicity. Although both PES- and

380

RC-purified conjugates elicited effective antibodies, use of PES membranes resulted in

381

precipitation of the OXY-sKLH suggesting that RC membranes are more suitable for further

382

development (Fig. S5).



Page 20 of 38

20

383 384

Figure 3. Effect of filter size on OXY-sKLH immunogenicity and size.

385

(A) Male Holtzman rats (n = 3-6/group) were immunized by IM injection with 60 µg filtered or

386

unfiltered OXY-sKLH with 90 µg of alum on day 0. Serum was collected on day 7 and

387

oxycodone-specific antibody titers were measured. (B)

388

filtered and non-filtered OXY-sKLH. * p < 0.05 compared to the non-filtered OXY-sKLH.

Percent intensity of light scatter of

389 390

EXPERIMENT 5. Effect of TCEP and EDAC on size and immunogenicity of OXY-sKLH.

391

Varying concentrations of TCEP and EDTA were added to the conjugation reactions containing

392

various hapten:EDAC molar ratios (Table 1) to reduce the size of OXY-sKLH and allow

393

subsequent sterile filtration. To further improve conjugate formulation, the vaccine was stored

394

either in PBS 0.1M pH 7.2 or 1 mM EDTA in water pH 7.2 (Table 1). Batch 11 (Table 1) filtered


Page 21 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21 395

through a 0.22 µm-size filter showed a leftward shift in the DLS curve, indicating a reduction in

396

size compared to the batch 1 (Fig. 4A). However, batch 11 elicited titers of 8.7 ± 2.7 x 103 (mean

397

± SD), which were significantly lower than those elicited by batch 1 (p < 0.05), our lead

398

candidate vaccine (Fig. 4B). Reduction in size of batch 11 was also confirmed by SDS-PAGE,

399

with a band around 460 kDa (lane 8, Fig. S6) compared to the batch 1 which did not migrate

400

into the gel (lane 7, Fig. S6). Batches 5, 17 and 19, which were conjugated as described in table

401

1, had a band visible near the gel loading well, but the band further down was indistinct. Batch

402

12 and 18 showed a band around 460 kDa, which was comparable to batch 11. Size exclusion

403

chromatography (SEC) was used to analyze sKLH, the lead conjugate (batch 1), and the batch

404

11. Conjugate 11 had two major peaks. The first peak was similar in size to sKLH (Fig. 4C)

405

while the other was approximately 400 - 450 kDa (Fig. 4D). Batch 1 had a single peak close to

406

the MW limits for the column, suggesting that it was too large to be analyzed by SEC (Fig 4E).

407

To circumvent the limitation of sterile-filtering larger conjugates, individual reagents could be

408

sterile filtered prior to conjugation and manufacturing conducted under aspetic conditions.

409 410



22 411

Figure 4. Oxycodone-specific antibody titers and dynamic light scattering.

412

(A) Dynamic light scattering of sKLH and OXY-sKLH batch 1 and 11. (B) Male Holtzman rats

413

(n= 3-17/group) were immunized by IM injection with 60µg of OXY-sKLH with 90 µg of alum on

414

day 0. Serum was collected on day 7 and oxycodone-specific antibody titers were measured.

415

Data are mean ± SD (C-E) Size exclusion chromatography analysis performed on sKLH and

416

OXY-sKLH (batch 1 and 11). * p < 0.05 compared to batch 1.

417 418

EXPERIMENT 6. Effect of conjugation chemistry on heroin/morphine vaccine efficacy.

419

Conditions identified during optimization of the oxycodone vaccine were applied to the

420

optimization of the M-sKLH vaccine (Fig 5A). Conjugation of M-sKLH was performed using

421

EDAC, EDAC in presence of NHS ester, and maleimide and the relative M-sKLH conjugates

422

were characterized by DLS and then subsequently tested for efficacy in vivo. Conjugation of the

423

lead M(gly)4OH hapten to BSA using EDAC resulted in a haptenation ratio of 29, and the

424

resulting M-sKLH was effective (conjugate M1, Fig. 5B). The EDAC coupling reaction was not

425

improved by the presence of sulfo-NHS ester (conjugate M2, Fig 5B, haptenation ratio of 8).

426

Similarly, use of maleimide chemistry to conjugate the novel M(gly)4SH hapten to BSA

427

(conjugate M3, Fig 5B) showed a haptenation ratio of 7. The DLS panel for M1, 2 and 3 is

428

shown in Fig. 5F. Conjugate M2 and M3 are slightly smaller than conjugate M1, maybe due

429

lower haptenation or reduced crosslinking in solution during the conjugation reaction. Conjugate

430

M1 elicited titers of 147 ± 48 x 103 (mean ± SD), which were significantly higher than those

431

elicited by M2 and M3 (p < 0.001), which elicited titers of 18196 ± 7525 and 19025 ± 7356,

432

respectively (Fig. 5B). In contrast to M2 and M3, M1 showed a significant increase in serum

433

heroin, 6-AM, and morphine compared to controls (p < 0.001, Fig. 5C) following SC

434

administration of 1 mg/kg heroin. In fact, M1 reduced heroin-induced antinociception by 80%


Page 22 of 38

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23 435

and reduced drug distribution to the brain by 66% compared to M2, M3 and control (p < 0.001

436

for all comparisons, Fig. 5C, D, E). These data suggest that EDAC-conjugated M-sKLH (M1) is

437

the most effective conjugate vaccine for further development.

438 439

Figure 5. Characterization, immunogenicity and efficacy of the M-sKLH vaccine in rats.

440

(A) Morphine-based haptens. (B) Male Holtzman rats (n = 8/group) were immunized by IM

441

injection with 60 µg of M-sKLH and 90 µg of alum on day 0, 21, 42 and 63. Serum was collected

442

on day 70 and morphine-specific antibody titers were measured. On day 77 animals received a

443

SC injection of 1 mg/kg of heroin and 30 min later were tested on a hotplate set to 54C for

444

nociception. (C) Serum and (D) brain were collected following (E) hotplate testing. (F) Percent

445

intensity of light scattering of different batches of M-sKLH. Distribution and behavioral data are

446

the mean ± SD. *** p < 0.001 compared to control.

447 448 449



24 450

EXPERIMENT 7. Effect of co-administration of morphine and oxycodone vaccine on

451

immunogenicity and efficacy.

452

OXY-sKLH and M-sKLH were co-administered to test whether a bivalent vaccine formulation

453

would potentially affect the immunogenicity and efficacy of OXY-sKLH. Vaccination with the

454

monovalent OXY-sKLH at either 60 or 120 µg doses and the bivalent vaccine formulation (60 µg

455

OXY-sKLH and 60 µg M-sKLH) elicited high titers of oxycodone-specific antibodies. The

456

presence of M-sKLH in the bivalent formulation did not interfere with the development of

457

oxycodone-specific IgG antibodies (Table 2). Oxycodone-specific antibody titers elicited by the

458

monovalent OXY-sKLH vaccine (60 and 120 µg) cross-reacted with the morphine hapten by

459

15% and 17%, respectively. Both doses of monovalent OXY-sKLH (60 and 120 µg) and the

460

bivalent vaccine significantly increased the retention of oxycodone in serum compared to the

461

sKLH control group (p < 0.001, Fig. 6A). The increase in serum oxycodone elicited by 120 µg of

462

OXY-sKLH was significantly greater than the 60 µg dose of OXY-sKLH, suggesting an effect of

463

immunogen dose (p < 0.0001, Fig. 6A). Distribution of oxycodone to the brain was significantly

464

reduced by all monovalent and bivalent vaccine formulations compared to the sKLH control

465

group (p < 0.0001, Fig. 6B). The monovalent OXY-sKLH (120 µg) vaccine and bivalent vaccine

466

reduced oxycodone to the brain respectively by 61% and 62% compared to the sKLH group.

467

OXY-sKLH (120 µg) and the bivalent vaccine reduced oxycodone antinociception by 83% and

468

71%, respectively (p < 0.0001, 0.001, Fig. 6C). Vaccination with M-sKLH also produced a

469

significant decrease in distribution of oxycodone to the brain (Fig. 6B), consistent with ELISA

470

titers (Table 2) showing 33% cross-reactivity of anti-morphine antibodies with the oxycodone

471

hapten. M-sKLH also reduced oxycodone antinociception by 52% compared to the KLH control

472

group (p < 0.01, Fig 6C).


Page 24 of 38

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25

473 474

Figure 6. Immunogenicity and efficacy of OXY-sKLH co-administered in a bivalent

475

formulation in rats.

476

Male Holtzman rats (n= 12/group) were immunized on day 0, 21, 42 and 63 by IM injection with

477

60 μg of OXY-sKLH, 60 μg of M-sKLH, 60 μg of OXY-sKLH plus 60 μg of M-sKLH, and 120 μg

478

of OXY-sKLH adsorbed on either 90 μg or 180 μg of alum adjuvant, respectively. On day 77

479

animals received a SC injection of 2.25 mg/kg of oxycodone and 30 min later were tested on a

480

hotplate set to 54C for nociception. (A) Serum and (B) brain samples were collected

481

immediately following (C) hotplate testing. Numbers above bars represent the percentage of

482

difference from controls. Distribution and behavioral data are the mean ± SD. ** p < 0.01. *** p

483

< 0.001, **** p < 0.0001 compared to control. #### p < 0.0001 brackets indicate group

484

differences. (A) **** OXY-sKLH (120 µg) vs. M-sKLH. (B) **** sKLH (60 µg) vs. M-sKLH (60 µg),

485

*** OXY-sKLH (120 µg) vs. M-sKLH (60 µg), *** M-sKLH (60 µg) vs OXY-sKLH (60 µg) + M-

486

sKLH (60 µg).

487 488



26


Page 26 of 38

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27 490

Discussion

491 492

Vaccines offer a promising approach to treat OUD and potentially reduce incidence of

493

fatal overdoses. To date, several vaccine candidates have shown pre-clinical proof of selectivity

494

and efficacy in reducing the behavioral effects of heroin, oxycodone, hydrocodone, fentanyl and

495

fentanyl-like compounds

496

lethality

497

drug products that can be characterized and manufactured at scale under GMP. Hence, this

498

study focused on further advancing the lead OXY-sKLH and M-sKLH vaccines toward clinical

499

evaluation.

8-9, 24.

9, 19, 22, 24-25

as well as opioid-induced respiratory depression and

One of the biggest challenges to pre-clinical vaccine development is to generate

500

The major findings from this study were: 1) synthesis of OXY(gly)4 hapten as a lithium

501

salt led to a higher haptenation ratio than as TFA salt. A lyophilized version of the OXY(gly)4

502

hapten synthesized at our CMO site was easier to conjugate to sKLH than the same hapten

503

formulated as powder, 2) increasing the EDAC concentration in the conjugation reaction

504

increased the haptenation ratio and decreased precipitation, 3) when comparing vaccine doses,

505

60 µg of immunogen was the most effective dose, 4) larger OXY-sKLH conjugates were more

506

immunogenic than smaller conjugates, but filtration through a 0.22 µm filter resulted in

507

significant loss of vaccine and efficacy. As a proposed solution, individual vaccine components

508

could be sterile-filtered prior to conjugation and the conjugation could be conducted under

509

sterile conditions, 5) optimal vaccine efficacy against heroin was achieved by a morphine-based

510

hapten conjugated to the carrier protein using EDAC coupling chemistry in presence of DMSO,

511

and 6) co-administration of 60 µg of OXY-sKLH and 60 µg of M-sKLH yielded a bivalent vaccine

512

that was as effective as doubling the dose of OXY-sKLH (120 µg) and more effective than a

513

single dose of OXY-sKLH (60 µg).

514 515

Hapten and linker chemistry can greatly affect vaccine efficacy

11, 19, 26.

Previous studies

have shown that opioid vaccines containing haptens conjugated to carrier proteins via



Page 28 of 38

28 516

tetraglycine linkers using carbodiimide coupling were more effective that structurally-similar

517

haptens containing terminal thiol groups for maleimide chemistry 11, 17, 19. This study showed that

518

the OXY(gly)4 synthesized as lithium salt yielded a higher haptenation ratio after conjugation

519

compared to a TFA salt, perhaps due to an interference of TFA during the coupling reaction. It is

520

known that TFA provides carboxyl groups that may interfere with EDAC chemistry. In addition,

521

a lyophilized version of the OXY(gly)4 hapten synthesized at CMO site yielded a higher

522

haptenation ratio compared to the previously established powder form. In our experience,

523

lyophilized haptens offer the advantage of simplifying manufacturing, greater stability, and

524

greater solubility compared to the same haptens in powder form.

525

The OXY-sKLH vaccine precipitated when conjugated in MES buffer at pH 4.5 using 52

526

mM of EDAC, despite a trend for higher haptenation ratios at lower conjugation pH. Adding

527

higher concentrations of EDAC (208 mM) decreased precipitation and increased the

528

haptenation ratio, perhaps creating cross-linking, carrier protein-carrier protein interactions, and

529

stabilizing the conjugate in solution, causing random polymerization of polyproteins 27.

530

In exploring vaccine doses, OXY-sKLH showed to be effective at doses as low as 10 µg, 28-30.

531

which is within the range of doses previously used in clinical trials

532

immunogen was chosen because it was the most effective dose in reducing oxycodone

533

antinociception, even though no differences were found in titers, oxycodone serum, or brain

534

levels when compared to doses of 30 and 120 µg. These results suggest that a possible plateau

535

effect in rats was reached when 120 µg of conjugate and 180 µg of alum adjuvant were used.

536

Considering that opioid users self-reported orally abused oxycodone doses of 2 mg/kg

537

OXY-sKLH vaccine showed pre-clinical efficacy in reducing antinociception and brain

538

oxycodone levels when immunized rats or mice were challenged with SC doses of oxycodone

539

higher than commonly abused oral doses in humans.


The 60 µg dose of

31-32,

the

Page 29 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


29 540

In this study, OXY-sKLH could not be sterilized by filtration without affecting its potency.

541

Sterility is one of the requirements to obtain regulatory approval for human testing. Therefore,

542

sterile filtration of the drug substance was evaluated using 0.2 µm and 0.45 µm size filters as

543

advised by the FDA

544

these heroin and oxycodone vaccines because sterilization by heat would induce aggregation

545

and degradation of the carrier protein. In order to reduce the size and aggregation of OXY-sKLH

546

conjugate, a chelant and a reducing agent were tested during conjugation as well as quenching

547

strategies. TCEP is typically used as a reducing agent to selectively break disulfide bonds inside

548

or between proteins. In molecular models of KLH, the functional units of the KLH subunits are

549

stabilized by disulfide bonds which seem to be responsible to maintain the integrity of the

550

tertiary structure of KLH

551

conjugate size, but also lowered its immunogenicity and efficacy against oxycodone. There is a

552

correlation between size and immunogenicity, but the immunological and biochemical

553

mechanisms are not clear

554

antigens

555

of aggregation showed that the extent of aggregation correlated with individual vaccine efficacy

556

against nicotine in both mice and non-human primates

557

immunogenicity due to presence of aggregates is that T cell-dependent B cell activation is

558

promoted by repetitive epitopes in carrier proteins or aggregates, which may enhance B cell

559

activation

560

proteins, but it is not effective when conjugated to peptides

561

dextran 20, and its efficacy depends upon CD4+ T cell activation 7, 19. Since size of OXY-sKLH is

562

an essential requirement for retaining vaccine efficacy, filtration of individual components prior

563

to conjugation under sterile conditions could be a viable strategy to achieve sterility without

564

compromising efficacy.

37.

33.

Filtration is the only sterilization procedure that could be performed with

34-35.

36.

Use of TCEP during conjugation, reduced the OXY-sKLH

Protein aggregation is known to increase the immune response to

Immunization with several batches of nicotine vaccines exhibiting different degrees

41-42.

38-40.

Another hypothesis for increased

In fact, the OXY(gly)4 hapten is effective when conjugated to a variety of carrier


14

or polymers such as ficoll and


Page 30 of 38

30 565

Here, morphine-based haptens were conjugated to sKLH (M-sKLH) using a similar

566

strategy as the lead OXY-sKLH vaccine. We have previously shown that OXY haptens

567

equipped with a tetraglycine linker with a C-term carboxyl group were more effective against

568

oxycodone than OXY haptens containing the same linker with a C-term thiol (-SH) group or a

569

polyethylene glycol linker (PEG)

570

effective heroin vaccine using a PEGylated hapten equipped with a thiol group for maleimide

571

chemistry

572

or drug target. In this study, M-sKLH conjugated using EDAC had the highest haptenation ratio,

573

which was consistent with another heroin vaccine

574

effects of 1 mg/kg of heroin in rodents. Instead, M-sKLH conjugated using either NHS ester or

575

maleimide chemistry was not effective and therefore discarded from further development. As the

576

M(gly)4 hapten is less soluble in MES buffer than the analogous oxycodone hapten, optimal

577

conjugation required 10% DMSO. DMSO is a non-toxic polar aprotic solvent that dissolves both

578

polar and nonpolar compounds and it was added to the conjugation reaction to increase the

579

solubility of the morphine hapten and consequently stabilize the O-acylisourea intermediate that

580

is formed when EDAC reacts with carboxylic acid groups present on the morphine hapten 27 .

15, 43,

20.

Another group has shown the feasibility of developing an

suggesting that linker chemistry should be optimized for each individual hapten

11,

and effectively reduced the behavioral

581

Co-administration of OXY-sKLH and M-sKLH (60 ug of each) in a bivalent vaccine

582

formulation was as effective as doubling the OXY-sKLH dose (120 µg) and better than the

583

single OXY-sKLH dose (60 µg). Because of the cross-reactivity showed by the morphine-

584

specific antibodies with the oxycodone hapten (Table 2), it is possible that a B cell population

585

subset may recognize both the OXY and M haptens resulting in an augmented activation of

586

each respective hapten-specific B cell population. These data are consistent with a previous

587

study focusing on combination of OXY-KLH and M-KLH, which showed that M-KLH partially

588

reduced distribution of oxycodone to the brain in rats

589

demonstrated that the addition of M-sKLH did not interfere with the efficacy of OXY-sKLH. The

590

bivalent vaccine increased serum antibody titers compared to the monovalent vaccine, which is

23.


The current experiment further

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31 591

consistent with what has been shown previously

592

single formulation would appear to be a better option for increasing the efficacy of an oxycodone

593

vaccine than simply increasing the dose, due to limitations in the amount of alum adjuvant and

594

immunogen that can be administered clinically. This is an important implication considering that

595

previous addiction vaccines failed in clinical trials because of relatively low and variable

596

antibody titers

597

advantage to treat subjects who abuse a range of opioids.

598 599

28-30.

23.

Combining OXY-sKLH and M-sKLH in a

In addition, a bivalent or multivalent vaccine formulation could have an

In conclusion, this study identified optimal conditions for further advancement of the OXY-sKLH and M-sKLH candidate vaccines toward manufacturing and IND-enabling studies.

600 601



Page 32 of 38

32 602

TABLES AND FIGURE LEGENDS

603 604 605

TABLE 1 – Summary of conjugation conditions.

606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640

OXY-sKLH Batch #

641 642 643 644

Coating Immunogen

645 646 647 648 649 650

OXY-BSA 213 ± 30 * * compared to sKLH control group,

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Hapten mM 5.2 5.2 5.2 5.2 52 52 26 10.4 5.2 26 52 52 52 52 52 5.2 5.2 5.2 5.2 5.2 5.2 5.2 5.2 5.2 5.2 5.2

EDAC mM 208 208 52 52 52 52 26 10.4 5.2 104 52 52 52 52 52 208 208 208 208 208 208 208 52 208 208 208

EDTA mM

1 1

TCEP mM

NaCl Sucrose Quenching mM mM condition

15 15 15 15

1 1 1

15 15 15 30 8 30 0.5 40 pH 2.8 1% AcOH

Storage buffer PBS pH 7.2 H2O 1mMEDTA PBS pH 7.2 H2O 1mM EDTA PBS pH 7.2 H2O 1mM EDTA H2O 1mM EDTA H2O 1mM EDTA H2O 1mM EDTA H2O 1mM EDTA H2O 1mM EDTA PBS pH 7.2 H2O 1mM EDTA PBS pH 7.2 H2O 1mM EDTA H2O 1mM EDTA H2O 1mM EDTA H2O 1Mm EDTA PBS pH 7.2 H2O 1mM EDTA H2O 1Mm EDTA PBS pH 7.2 PBS pH 7.2 PBS pH 7.2 PBS pH 7.2 PBS pH 7.2

TABLE 2 – Serum antibodies titers (x103) and cross-reactivity (%) #.

M-BSA

OXY-sKLH 60 µg 31 ± 12 (15%)

OXY-sKLH 120 µg

M-sKLH 60 µg

56 ± 18 (17%)

243 ± 75

334 ± 92 *

M-sKLH + OXY-sKLH 60 µg + 60 µg

80 ± 26 (33%)

164 ± 63 226 ± 71 *

# Cross-reactivity of serum antibodies for the non-targeted hapten was calculated by dividing the antibody titers measured using the non-targeted coating antigen by the antibody titers measured using the targeted coating antigen and multiplying by 100 to obtain a percentage.

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33

653 654 655

Acknowledgment: this study was funded by the National Institute of Health DA038876 to MP and PRP. The authors thank Theresa Harmon and Jenny Vigliaturo for technical support. The authors thank Cambrex (previously Avista), Durham, NC for synthesis of the OXY(gly)4 hapten as part of the technology transfer process to generate the GMP-grade OXY(gly)4-sKLH vaccine. Supporting Information Available: [Supplementary figures and methodology.] This material is available free of charge via the Internet at http://pubs.acs.org.

656 657

REFERENCES

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The manuscript addresses some of the challenges of developing conjugate vaccines for opioid use disorders and overdose suitable for pharmaceutical manufacturing and FDA approval. Vaccination elicits antibodies that selectively bind opioids and reduce opioid distribution to the brain.


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Formulation and Characterization of Conjugate Vaccines to Reduce

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