Enhancing durability of CIS43 monoclonal antibody by Fc mutation or AAV delivery for prevention

Neville Kielau Kisalu, … , Joseph R. Francica, Robert A. Seder

JCI Insight. 2020. https://doi.org/10.1172/jci.insight.143958.

Research In-Press Preview Infectious disease

CIS43 is a potent neutralizing human monoclonal antibody (mAb) that targets a highly conserved ‘junctional’ epitope in the (Pf) circumsporozoite protein (PfCSP). Enhancing the durability of CIS43 in vivo will be important for clinical translation. Here, two approaches were used to improve the durability of CIS43 in vivo while maintaining potent neutralization. First, the Fc domain was modified with the “LS” mutations (CIS43LS) to increase CIS43 binding affinity for the neonatal Fc receptor (FcRn). CIS43LS and CIS43 showed comparable in vivo protective efficacy. CIS43LS had nine to thirteen-fold increased binding affinity for human (6.2 nM vs. 54.2 nM) and rhesus (25.1 nM vs. 325.8 nM) FcRn at endosomal pH 6.0 compared to CIS43. Importantly, the half-life of CIS43LS in macaques increased from 22 days to 39 days compared to CIS43. The second approach for sustaining antibody levels of CIS43 in vivo is through adeno- associated virus (AAV) expression. Mice administered once with AAV expressing CIS43 had sustained antibody levels of ~ 300 μg/mL and mediated protection against sequential malaria challenges up to 36 weeks. Based on these data, CIS43LS has advanced to Phase I clinical trials and AAV delivery provides a potential next generation approach.

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1 Enhancing durability of CIS43 monoclonal antibody by Fc mutation or AAV delivery for

2 malaria prevention

3

4

5 Neville K. Kisalu1, Lais D. Pereira1, Keenan Ernste1, Yevel Flores-Garcia2, Azza H. Idris1,

6 Mangaiarkarasi Asokan1, Marlon Dillon1, Scott MacDonald3, Wei Shi1, Xuejun Chen1,

7 Amarendra Pegu1, Arne Schön4, Fidel Zavala2, Alejandro B. Balazs3, Joseph R. Francica1,

8 Robert A. Seder1*

9

10

11

12 1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National

13 Institutes of Health, Bethesda, MD, USA

14 2Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD,

15 USA

16 3Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and

17 Harvard University, Cambridge, MA, USA

18 4Department of Biology, Johns Hopkins University, Baltimore, MD, USA

19 *Corresponding author: Correspondence and requests for materials should be addressed to Robert

20 A. Seder ([email protected])

21

22 Keywords: CIS43, monoclonal antibody, malaria, PfCSP, junctional epitope, protectkion, half-

23 life, LS mutation, FcRn, AAV, Phase 1 clinical trial.

24 25 Abstract

26 CIS43 is a potent neutralizing human monoclonal antibody (mAb) that targets a highly

27 conserved ‘junctional’ epitope in the Plasmodium falciparum (Pf) circumsporozoite protein

28 (PfCSP). Enhancing the durability of CIS43 in vivo will be important for clinical translation.

29 Here, two approaches were used to improve the durability of CIS43 in vivo while maintaining

30 potent neutralization. First, the Fc domain was modified with the “LS” mutations (CIS43LS) to

31 increase CIS43 binding affinity for the neonatal Fc receptor (FcRn). CIS43LS and CIS43 showed

32 comparable in vivo protective efficacy. CIS43LS had nine to thirteen-fold increased binding

33 affinity for human (6.2 nM vs. 54.2 nM) and rhesus (25.1 nM vs. 325.8 nM) FcRn at endosomal

34 pH 6.0 compared to CIS43. Importantly, the half-life of CIS43LS in rhesus macaques

35 increased from 22 days to 39 days compared to CIS43. The second approach for sustaining

36 antibody levels of CIS43 in vivo is through adeno-associated virus (AAV) expression. Mice

37 administered once with AAV expressing CIS43 had sustained antibody levels of ~ 300 μg/mL

38 and mediated protection against sequential malaria challenges up to 36 weeks. Based on these

39 data, CIS43LS has advanced to Phase I clinical trials and AAV delivery provides a potential

40 next generation approach for malaria prevention.

41

42

43

44

45

2 46 Introduction

47 Malaria is a mosquito-borne parasitic disease causing high morbidity and mortality

48 primarily in infants and young children in sub-Saharan Africa. Several interventions have

49 significantly contributed to the decrease of malaria case incidence and mortality, including

50 vector control, insecticide-treated bednets, and seasonal malaria chemoprevention. However,

51 reductions in malaria cases have plateaued globally since 2015 and are actually increasing in

52 some countries (1,2). Thus, there is an urgent need to develop new approaches for controlling

53 and eventually eradicating malaria.

54 The most transformative modality to control malaria would be a vaccine that provides

55 high-level and durable protection. RTS,S, a truncated version of PfCSP containing NANP

56 repeats and the C- terminal region administered with the AS01 adjuvant, induces ~ 50%

57 protection at 1 year and ~30% protection after 4 years primarily due to the high level of

58 antibodies required for protection (3,4). While some vaccine strategies focused on T cell

59 mediated protection such as whole sporozoite-based vaccines conferred high-level, durable

60 protection of ~ 1 year in malaria naïve individuals following controlled human malaria infection

61 (CHMI) (5-7), there is more limited immunity and protection in malaria endemic regions against

62 CHMI (8) or in areas of intense natural transmission (9). Factors that may influence vaccine

63 efficacy include prior malaria exposure (9,10), parasite diversity (11,12), and age (13,14). Thus,

64 an alternative immune approach independent of the potential factors that limit immunity by

65 vaccination, that would induce high level protection for defined periods of time is through

66 passive immunization with a highly potent monoclonal antibody (mAb).

67

3 68 We recently reported on the discovery of CIS43, a human mAb isolated from a subject

69 immunized with an attenuated Pf whole-sporozoite (SPZ) vaccine (® PfSPZ Vaccine)

70 (15) that was protected against CHMI (16). CIS43 preferentially recognizes the ‘junctional’

71 epitope positioned between the N-terminus and the central repeat domain of PfCSP which is

72 highly conserved across 99% of Pf strains. Passive transfer of CIS43 provided high-level, sterile

73 protection in two different mouse models of malaria infection (17). In addition to potency, the

74 clinical utility of mAbs such as CIS43 will be strongly influenced by improving the antibody

75 half-life (T½).

76 Here, two distinct strategies are used to extend the durability of CIS43 in vivo. First, we

77 introduced the LS mutations in the Fc (constant) region of CIS43 to enhance its binding affinity

78 for the neonatal Fc receptor (FcRn), to limit degradation of the antibody and increase recycling

79 (18,19). Second, vectored immunoprophylaxis (VIP), an adeno-associated virus (AAV) gene

80 based delivery strategy, which has been used for expressing antibodies in vivo against a number

81 of viral antigens or PfCSP was used to express CIS43 (20-24). Together, the data presented

82 provide evidence for optimizing the ability of a malaria mAb to mediate durable protection in

83 vivo.

4 84 Results

85 Generation of CIS43LS mAb

86 To improve the durability of CIS43, this mAb was modified by site-directed mutagenesis to

87 introduce the LS point mutations, which replaced methionine with leucine at amino acid position

88 451 and asparagine with serine at amino acid position 457 in the CH3 domain of the Fc region

89 (M451L/N457S) (Supplemental Figure 1). The LS mutations increase the binding affinity of the

90 Fc region for FcRn, preventing its degradation and thereby increasing the serum T½ (18,19).

91 To establish that CIS43LS retained the biophysical properties of CIS43, the binding

92 specificity, affinity and stoichiometry were characterized. CIS43LS showed dose-dependent

93 binding to rPfCSP by ELISA similar to CIS43 with a maximal effective concentration (EC50) of

94 0.039 and 0.037 g/ml, respectively (Figure 1A). mAb 317, a human antibody specific for the

95 NANP-repeat region of PfCSP used as a control antibody, bound to rPfCSP with an EC50 of

96 0.011 g/ml (Figure 1A). Epitope mapping of CIS43LS and CIS43 confirmed comparable

97 specificity with potent binding to peptide 21, the preferred junctional epitope of CIS43, with an

98 EC50 of 0.06 g/ml and binding to a NANP-containing peptide (peptide 29) with an EC50 of 5.1

99 g/ml. mAb 317 did not bind to peptide 21 and had an EC50 of 0.010 for binding to peptide 29

100 (Figure 1A). Similar to CIS43, the stoichiometry and thermodynamic parameters of CIS43LS

101 measured by isothermal titration calorimetry (ITC) showed two sequential binding events to

102 rPfCSP (Figure 1B). The first binding event to the junctional epitope involved a single binding

103 site per antibody with an apparent affinity of 11.0 nM (CIS43LS) and 7.9 nM (CIS43), whereas

104 the second binding event encompassed five to six additional sites within the NANP repeat region

105 with an apparent affinity of 35.5 nM for CIS43LS and 42.0 nM for CIS43 (Figure 1B). These

5 106 data show that the LS mutations did not disrupt the binding of CIS43 to its cognate antigen and

107 confirmed that the junctional epitope is the preferred epitope for this mAb.

108

109 CIS43LS mediates protection in vivo in mice

110 To determine whether CIS43LS can protect against malaria challenge in vivo and to

111 compare its potency with CIS43, mice were challenged with a transgenic parasite strain of

112 chimeric Plasmodium berghei (Pb) expressing PfCSP and luciferase (Pb-PfCSP-LUC) (25)

113 following passive transfer of the mAb. First, in two independent experiments, we assessed the

114 ability of CIS43 and CIS43LS to mediate reduction of liver-stage parasite burden as measured by

115 bioluminescence in vivo in C57BL/6 mice following challenge with 2000 Pb-PfCSP-LUC

116 sporozoites (Pb-PfCSP-LUC SPZ) by intravenous (IV) injection (17). Passive administration of

117 CIS43 or CIS43LS led to similar and signficant reduction (~2.5 logs) of the liver-stage parasite

118 burden in a dose-dependent manner (p = 0.0019 for CIS43 and p = 0.0099 for CIS43-LS; Figure

119 1C and D) compared to untreated mice or to mice that received VRC01, a human anti-HIV-1

120 isotype control mAb. The protective capacity of CIS43LS was next assessed following

121 challenge with infectious mosquito bites, the natural route of transmission for malaria. In two

122 independent experiments, all mice that received 300 μg CIS43 or CIS43LS had no detectible

123 parasites in blood (parasitemia) up to 12 days following infection (p = 0.0001, log-rank test)

124 while the untreated or isotype control-treated mice were all parasitemic by day 4 (Figure 1E).

125 Together, these data show that CIS43LS had comparable protective efficacy as CIS43 in vivo.

126

127 CIS43LS shows pH-dependent increased affinity for FcRn

6 128 FcRn binding increases antibody T½ by preventing its degradation in lysosomes,

129 allowing it to recycle back into circulation (18,19). This association is pH-dependent, whereby

130 high binding occurs at low pH, but not at physiological pH. To confirm that CIS43LS had this

131 pH dependency, we measured the capacity of CIS43LS to bind to human and rhesus FcRn at

132 endosomal or physiological pH (6.0 or 7.4, respectively) using biolayer interferometry. Indeed,

133 at pH 6.0 CIS43LS had a higher apparent binding affinity to human (6.2 nM) and rhesus (25.1)

134 FcRn, compared to CIS43 binding to human (54.2 nM) and rhesus (325.8 nM) FcRn (Fig. 1F and

135 Supplemental Table 1). At pH 7.4, the affinity of CIS43LS for both human (234.1 nM) and

136 rhesus (262.1 nM) FcRn was significantly reduced, while there was limited to no detectable

137 binding to either human or rhesus FcRn for CIS43 (Figure 1F and Supplemental Table 1).

138 To extend the analysis, the binding affinities of CIS43 and CIS43LS for all human Fc

139 gamma receptors (Supplemental Table 2) was assessed at physiologic pH 7.4. There were no

140 significant differences in the binding between these two antibodies for any of the Fc gamma

141 receptors. Thus, the LS mutation primarily affected pH dependent FcRn-binding, the primary

142 mechanism by which antibody T½ is increased (18,19,26).

143

144 Pharmacokinetics of CIS43LS in non-human primates (NHP)

145 The next studies focused on whether CIS43LS had enhanced T½ compared to CIS43 in vivo.

146 Accordingly, NHP can be a useful pre-clinical model for pharmacokinetic (PK) analysis of

147 human mAbs based on high FcRn conservation compared to humans (27). Rhesus macaques

148 (Macaca mulatta) were infused intravenously (IV) with 10 mg/kg of CIS43LS or CIS43 and the

149 concentrations of both mAbs were assessed in serum and skin up to 140 days post-infusion. The

150 mean maximum serum antibody concentration (CMAX) was comparable between both mAbs

7 151 (287.3 μg/ml for CIS43LS and 280.4 μg/ml for CIS43) and was attained within 1h post mAb

152 administration (Supplemental Table 3). Although mAb titers were similar between CIS43 and

153 CIS43LS up to 14 days post infusion (Figure 2A), CIS43LS exhibited a longer T½ after this

154 timepoint, resulting in a titer of ~15 μg/ml at 98 days compared to ~1 μg/ml for CIS43.

155 Remarkably, CIS43LS remained detectable in serum for more than 140 days and persisted at

156 significantly higher levels (~3.7 μg/ml) than CIS43, which was no detectible after 126 days

157 following mAb administration (Figure 2A, p = 0.0003). We next analyzed the T½ for each mAb

158 using a two-compartment model (Supplemental Table 3) and CIS43LS had ~ 2.0-fold increase in

159 T½ (CIS43LS, 38.7 ± 7.8 days vs. CIS43, 22.3 ± 2.2). The clearance of antibody, a

160 measurement of plasma volume from which the antibody is entirely removed per day per Kg

161 body weight, was decreased ~2.5-fold for CIS43LS compared to the parental antibody (CIS43,

162 5.8 ± 0.5 ml/day/kg; CIS43LS, 2.1 ± 0.3 ml/day/kg) (Supplemental Table 3). Consequently, the

163 area under the curve (AUC), which measures the exposure of all the body to the drug, for

164 CIS43LS was ~3 fold larger than for CIS43. These data are consistent with previous reports

165 demonstrating prolonged mAb T½ for FcRn binding mutants(18,28,29).

166

167 As the skin has a critical role in the initation of malaria infection, we also assessed

168 whether the LS mutations would alter the distribution of CIS43 in this tissue. Skin biopsy

169 samples were collected at various time points from the macaques that received CIS43 or

170 CIS43LS and antibody concentrations were measured in skin homogenates up to 140 days post-

171 infusion. Following antibody administration, each mAb showed a consistent titer pattern to that

172 observed in the blood samples, with CIS43LS persisting longer in the skin (Figure 2B, p =

173 0.0148). Both mAb titers were similar for up to seven days, but by day 14, CIS43 and CIS43LS

8 174 titers were approximately 29.4 and 56.9 µg/ml, respectively. CIS43LS was still detectible in skin

175 tissue for up to 84 days. By contrast, CIS43 titers became undetectable by 56 days post infusion.

176 These data show that, as in blood, the LS mutant significantly increased the T½ of CIS43 in the

177 skin.

178 CIS43LS does not induce anti-idiotypic responses in NHP

179 Anti-drug antibodies (ADA) may develop in response to passively administered mAbs, which

180 may influence their neutralization capacity and durability (30,31). To assess whether the LS

181 mutant induced immune responses in passively immunized NHP, ADA was assessed up to 20

182 weeks after infusion. Two assays were used for ADA. First, a competition assay was performed

183 in which CIS43LS or CIS43 were pre-incubated with serum before and after mAb

184 administration; the mixture was then added to PfCSP-coated plates to determine any change of

185 the mAb binding to PfCSP. As a positive control, a CIS43 anti-idiotype mAb was used to block

186 CIS43LS or CIS43 binding to rPfCSP (Figure 2C). Both pre- and post- mAb infusion sera

187 showed similar binding to PfCSP for all timepoints tested (Figure 2C), showing that there was no

188 detectable ADA. A second direct approach for measuring ADA was used, in which plates were

189 pre-coated with CIS43LS or CIS43, sera were added and then detected with an anti-monkey IgG-

190 specific secondary antibody. In this assay, no ADA could be detected at any time point (Figure

191 2D). Together these data suggest that CIS43 and CIS43LS do not develop ADA in NHP up to 20

192 weeks after a single infusion.

193

194 CIS43LS is not polyreactive

195 A final consideration for clinical development of an antibody is to demonstrate the absence of

196 polyreactivity or autoreactivity. CIS43LS was tested for binding to a panel of mammalian

9 197 autoantigens and showed no evidence of autoreactivity for CIS43LS or CIS43 (Supplemental

198 Figure 2A, B) .

199

200 Vectored immunoprophylaxis (VIP) for CIS43 using an adeno-associated virus (AAV)

201 vector

202 To provide an orthogonal approach for increasing antibody durability in vivo, VIP was used as a

203 gene based delivery approach (20-24). A previous study using an AAV vector encoding 2A10, a

204 mouse mAb against PfCSP, demonstrated high expression levels (> 1000 µg/ml), but only

205 partial protection following malaria challenge in mice after 8 weeks (23). Based on the enhanced

206 potentcy of CIS43 compared to 2A10, we hypothesized that VIP with CIS43 would elicit higher

207 protection than 2A10 for longer periods of time.

208 C57BL/6 mice were given a single intramuscular (IM) administration of 1 × 1011

209 genomic copies (GC) of the AAV expressing CIS43, 2A10, or VRC01 (negative control) mAb.

210 Three weeks post administration, the protective efficacy of AAV-encoded PfCSP mAbs against

211 malaria infection was assessed in C57BL/6 mice challenged with infectious mosquito bites.

212 providing evidence that the antibodies generated in vivo to PfCSP can inhibit sporozoites in the

213 skin (32,33). Nine out of 10 mice injected with CIS43-AAV and 6 of 10 mice that passively

214 received 300 μg of CIS43 mAb as a positive control had no detectable parasitemia up to 12 days

215 after infection (p = 0.0001, log-rank test). In contrast, all mice that received 2A10-AAV,

216 VRC01-AAV, or untreated mice exhibited parasitemia by day 4 (Figure 3A). To extend these

217 findings and assess durability of protection, C57BL/6 albino mice were challenged by the IV

218 route 8 weeks after AAV administration with Pb-PfCSP-LUC SPZ to demonstrate the in vivo

219 expressed antibodies can neutralize sporozoites in the blood. CIS43-AAV (p = 0.0001) treated

10 220 mice and mice that passively received CIS43 (p = 0.0159) had a significant reduction (~2 logs)

221 of the liver-stage parasite burden compared to VRC01-AAV administered or untreated mice

222 when assessed two days post challenge (Figure 3B). 2A10-AAV treated mice had a smaller

223 reduction (~1 log) in parasite burden (Figure 3B). Remarkably, when these mice were further

224 assessed for blood stage infection 6 days post challenge, 9 out of 10 CIS43-AAV (p = 0.0005)

225 mice and 4 out of 5 mice that passively received CIS43 (p = 0.0102) had no detectable

226 parasitemia, whereas all mice treated with 2A10-AAV, VRC01-AAV or untreated mice had high

227 levels of parasitemia (Figure 3B). Eight weeks post AAV administration, serum concentrations

228 of total human IgG were signficantly higher for 2A10 compared to the CIS43 (p = 0.0248) or

229 VRC01 (p = 0.0058) group (averages, 1114.3, 372.0, and 333.2 µg/ml, and ranges, 858.8-1302.5,

230 157.2-458.3 and 65.1-582.2 µg/ml, respectively (Figure 3C). rPfCSP-specific Ab concentrations

231 were significantly higher for 2A10 than CIS43 (averages, 954.4 vs. 354.1 µg/ml and ranges,

232 608.5-1399.3 μg/ml vs. 186.9-470.5 μg/ml (p = 0.0409, Figure 3D). Similarly, 2A10 had higher

233 average skin concentrations of total human IgG (162.0 µg/g tissue weight) than CIS43 (61.1 µg/g

234 tissue weight, p = 0.0169) or VRC01 (56.0 µg/g tissue weight, p = 0.0051); ranges were 54.4-

235 235.8 for 2A10, 38.1-110.5 for CIS43, and 6.1-94.3 µg/g tissue weight for VRC01 (Figure 3E).

236 Skin homogenate samples from CIS43-AAV and 2A10-AAV–but not VRC01-AAV

237 administered mice, bound to rPfCSP (average, 59.0 µg/g tissue weight and range, 40.0-115.0

238 µg/g tissue weight for CIS43 vs. average, 175.9 µg/g tissue weight and range, 58.0-299.0 μg/g

239 tissue weight for 2A10), but the difference in Ab concentrations was not significant between

240 both groups (Figure 3F). In assessing the kinetics of CIS43 in sera from a separate group of mice

241 following CIS43 AAV administration over time, the levels peaked ~2 weeks after administration

242 at a concentration of ~300 µg/ml, and remained stable up to 16 weeks (Supplemental Figure 3A).

11 243 CIS43 was also sustained in the skin up to 16 weeks (Supplemental Figure 3B). To show that the

244 antibody expressed in vivo following AAV administration was functional, serum from mice

245 administered 2A10- and CIS43-AAV showed comparable binding to rPfCSP as recombinant

246 2A10 or CIS43 antibody by biolayer inferometry (Supplemental Figure 3C). Taken together

247 these data demonstrate that CIS43 expressed in vivo by AAV is highly protective and more

248 potent than 2A10 (17), despite 2A10 having higher serum antibody titers.

249

250 CIS43 AAV mediates long-term protection following malaria challenge

251 A potential advantage of using AAV is the ability to sustain antibody expression in vivo leading

252 to durable protection. To assess the long-term protective efficacy of CIS43-AAV, mice that

253 received CIS43-AAV, 2A10-AAV, or VRC01-AAV were challenged by the IV route 36 weeks

254 post-AAV administration. Mice that received CIS43-AAV or 300 µg of CIS43 given at the time

255 of challenge showed a significant reduction of both liver-stage parasite burden (~2 logs, p =

256 <0.0001) and blood-stage infection (p = 0.0013 for CIS43-AAV and p = 0.0235 for CIS43)

257 respectively compared to untreated mice (Figure 4A). In contrast, mice that received 2A10-AAV

258 had a modest reduction in liver-stage parasite burden (~0.5-1 log, Figure 4A) and had

259 parasitemia similar to that seen in untreated mice (Figure 4A). Thirty two weeks post AAV-

260 administration, consistent with the findings in Figure 3, serum concentration of total human IgG

261 in these mice were signficantly higher for 2A10 (701.8 µg/ml) compared to CIS43 (186.9 µg/ml,

262 p = 0.0199) or VRC01 (184.5 µg/ml, p = 0.0279); ranges were 500.9-1000.1 µg/ml for 2A10;

263 99.8-348.4 µg/ml for CIS43; and 60.0-283.2 µg/ml for VRC01 (Figure 4B).

264

12 265 A key feature of Ab-mediated protection would be the ability to protect against repeated

266 infection over prolonged periods of time. Thus, CIS43-AAV-administered mice initially

267 challenged by mosquito bites and protected against malaria infection at week 3 (Figure 3A) were

268 rechallenged by mosquito bites 11 weeks following AAV administration. Eight out of 9 mice

269 that received CIS43-AAV had no detectable parasitemia up to 12 days after rechallenge (p =

270 0.0001, log-rank test). In contrast, untreated mice exhibited parasitemia by day 4 (Figure 4C).

271 Moreover, CIS43-AAV-administered mice initially challenged by the IV route and protected

272 against malaria infection at week 8 (Figure 3B) were rechallenged by the same route at week 36

273 following AAV administration. Following rechallenge, mice that received CIS43-AAV (p =

274 0.0005) or 300 µg of CIS43 (p = 0.0004) at the time of challenge had a significant reduction of

275 both liver-stage parasite burden (~2.5 logs) and blood-stage infection (p = 0.0002 for CIS43-

276 AAV and p = 0.0111 for CIS43) (Figure 4D). Because antibodies induced after the primary

277 challenge at week 8 may potentially play a role in the protection observed following rechallenge,

278 we determined the level of mouse antibodies against PfCSP 32 weeks after CIS43-AAV

279 administration, just prior to rechallenge at 36 weeks. Mouse anti-PfCSP antibodies were

280 significantly lower than the AAV-expressed CIS43 (OD 0.5 vs. OD 3.0) (Supplemental Figure

281 4). Together, these data demonstrate that CIS43-AAV mediates sustained antibody production in

282 vivo and durable protection against primary and repeated malaria challenge.

283

284

13 285 Discussion

286 Potent neutralizing mAbs are a promising approach for conferring high level protection

287 against malaria. While mAbs could be used to prevent malaria infection in travelers, health care

288 workers and military personnel, the greatest public health impact of this intervention would be

289 for seasonal control or elimination campaigns in Africa. Both of these indications would require

290 mechanisms to improve antibody durability. Here we provide two orthogonal approaches for

291 increasing the durability of mAb CIS43 in vivo. The present study shows that CIS43LS has a

292 longer serum T½ (38.7 ± 7.8 days) compared to CIS43 (22.3 ± 2.2 days) in NHP, with a titer of

293 ~10 g/ml at 15 weeks post-infusion. The serum T½ of CIS43LS reported here is higher

294 compared to a prior study with the HIV mAb VRC01LS (11.8 days) in NHP (18). Remarkably,

295 the T½ of VRC01LS was ~ 71 days in humans (34), suggesting that the durability of CIS43LS in

296 humans may be greater than predicted by NHP. The lack of detection of ADA anti-CIS43LS

297 responses in NHP is consistent with previous reports showing that the FcRn binding site

298 mutations, including the LS mutations, are well tolerated and have limited ADA in humans, even

299 after multiple infusions (34,35).

300

301 Although altering the pharmacokinetic profile of antibodies will favorably impact their utility for

302 various clinical use cases, repeated administration might still be required for elimination

303 campaigns of malaria. An alternative solution to sustain Ab titers for longer periods of time with

304 a single adminstrtion is the VIP strategy reported here using AAV. AAV vectors have been used

305 safely in humans for gene therapy (36-40) and VIP (41). The demonstration here that persistent

306 VIP expression of CIS43 confers protection up to 36 wpa against a single or sequential malaria

307 challenges provides clear evidence for the promise of this approach for sustaining high levels of

14 308 antibody. In terms of the protection against repeated infection in this model, immune responses

309 generated after the initial challenge are potential confounding variable to the protection by

310 rechallenge. However, we observed only very low mouse antibody responses following the

311 primary challenge which likely had limited contribution to the protection we observed after

312 rechallenge.

313

314 For clinical translation, a possible limitation for the VIP approach is whether ADA are induced

315 that would limit durability of the antibody. The data presented here show no indication of NHP

316 ADA and a recent report demonstrated AAV induction of long-lived SIV antibodies in an NHP

317 over six years with limited ADA responses (42).

318

319 A notable finding was that AAV can result in differential mAb expression in vivo. While it is

320 unclear why the 2A10-AAV expression was significantly higher than that of CIS43 or VRC01,

321 these data are consistent with a previous study, showing very high antibody levels by AAV

322 encoding 2A10 (23). Importantly, despite having higher antibody titers, 2A10 had more limited

323 protection than CIS43. These data highlight the primary importance of antibody potency for

324 mediating protection, which will be complemented by increased durability.

325

326 In summary, this report provides two approaches that could be used individually or potentially

327 together to enhance the durability of CIS43 in vivo. Based on the data reported here, a Phase I

328 clinical trial evaluating different doses of CIS43LS was initated this year to assess safety,

329 pharmacokinetics and protection against CHMI. As monoclonal antibody concentrations required

330 for protection against malaria infection in humans remains unknown, the results of the ongoing

15 331 clinical trial with CIS43LS will provide important information for the future use of monoclonal

332 antibodies and potentially AAV as new modalities to prevent malaria.

333

334

16 335 Methods

336 Site-directed mutagenesis and rPfCSP probes generation

337 CIS43LS was generated through site-directed mutagenesis (GenScript). For rPfCSP production,

338 the codon-optimized synthetic gene corresponding to the full-length circumsporozoite (CS)

339 protein (NCBI gene ID: PF3D7_0304600) of 3D7, a clone of the NF54 strain of P. falciparum,

340 was used (GenScript). The 15-mer linear PfCSP peptides that overlapped by 11 residues

341 spanning the full length of PfCSP were described previously (17) and were synthesized at

342 GenScript.

343

344 Production of CIS43 and CIS43LS

345 DNA constructs encoding the heavy and light chain of CIS43 or CIS43LS were expressed in

346 Expi293 (GenScript) or CHO cells to produce both mAbs following purification using a

347 recombinant protein-A column (GE Healthcare). Both mAbs bound equally well to rPfCSP and

348 protected against malaria challenge in vivo. Control mAb 317 (human antibody specific for the

349 NANP-repeat region of PfCSP) was produced in-house using the published sequence of this

350 antibody (43).

351

352 Generation of anti-idiotypic antibody against CIS43 Fab

353 Female Balb/c (Jackson Laboratory), 6-8 weeks old, were immunized with 20 μg of CIS43 Fab

354 in PBS mixed with an equal volume of Ribi (Sigma) adjuvant in a total volume of 100 µl by

355 intramuscular injection, followed by a booster immunization four weeks later with the same

356 dose. Ten days following the boost, serum reactivity against CIS43 Fab was tested by ELISA. A

357 germline-reverted CIS43 mAb was generated by cloning the unmutated heavy and light chain

17 358 genes of CIS43. Mice that strongly reacted to CIS43 Fab but not to germline-reverted CIS43 Fab

359 were selected for cell fusion. Three days before cell fusion, mice with desired reactivity were

360 boosted with 20 μg of CIS43 Fab in a total volume of 100 µl PBS by IV injection via the tail

361 vein. Spleens were excised and purified splenocytes were fused with myeloma cells Sp2/0

362 (ATCC, Manassas, VA) in 2:1 ratio according to established fusion protocols (44). Ten days post

363 cell fusion, hybridoma supernatants were screened for binding to the CIS43 Fab but not to the

364 germline-reverted CIS43 Fab. Following three rounds of screening, positive clones were adapted

365 to serum-free hybridoma culture medium (Invitrogen) to facilitate monoclonal antibody

366 purification. Ultimately, to increase the yield, the DNA constructs encoding the heavy and light

367 chain of selected hybridomas were cloned into the mouse IgG2a expression vectors (GenScript)

368 and expressed in Expi293 cells. Antibodies were purified in-house using a recombinant protein-

369 A column (GE Healthcare). Anti-idiotypic mAb 1-1 was selected for use in the downstream

370 experiments because of its potent binding to CIS43 Fab and lack of reactivity to germline-

371 reverted CIS43 Fab.

372

373 Processing of skin punches to quantitate mAb

374 Macaque or mouse skin punches were processed as previously described (18). Briefly, skin

375 punches were weighed and ground for 1 min in a 1.5-ml tubes containing 400 ml PBS with

376 EDTA-free protease inhibitor (Roche). After grinding, skin homogenates were centrifuged at

377 12,000 r.p.m for 15 min at 4 °C. Supernatants were collected and stored at -80 °C until testing.

378 Concentrations of passively infused CIS43 and CIS43LS or CIS43-, 2A10-, VRC01-AAV

379 induced mAb were quantified by ELISA. The amount of each monoclonal antibody in the

380 mouse skin biopsy homogenate was normalized to the skin tissue weight.

18 381

382 ELISA

383 Binding of CIS43 and CIS43LS to rPfCSP or PfCSP peptides was carried out as described

384 previously (17). Briefly, serially diluted (0.00006–5.0 µg/ml) CIS43, CIS43LS, or control

385 antibodies including the negative control VRC01 (human anti-HIV-1 IgG1) (45) or mAb 317

386 (43) were added to MaxiSorp ELISA plates (Thermo Scientific Nunc) coated with 100 µl of

387 rPfCSP (1 µg/ml) or PfCSP peptides (5 µg/ml) per well for 1 h at room temperature. After 1 h

388 incubation, plates were washed and incubated with 100 µl/well of 0.2 µg/ml peroxidase-labeled

389 goat anti–human IgG antibody (KPL, cat. no. 04-10-06) before adding the ABTS peroxidase

390 (KPL, cat. no. 50-62-10). The optical density was read at 405nm following addition of stopping

391 solution (100 µl/well). Amino acid sequences: peptide 21, NPDPNANPNVDPNAN; peptide

392 29, NANPNANPNANPNAN.

393 To quantitate CIS43, 2A10 and VRC01 induced by AAV administration in serum or

394 skin, ELISA plates were coated for 1h with 100μl of 1 μg/ml of goat anti-human IgG-Fc

395 antibody (Bethyl, cat. no. A80-104) for measuring total human IgG or plates coated with

396 100μl of 1 μg/ml PfCSP to assess PfCSP-specific mAbs. Serum or skin homogenate samples

397 serially diluted (1:100 to 1:312,500 for serum or 1:20 to 1: 62,500 for skin homogenate

398 respectively in 5-fold increments) in blocking solution were added to plate in duplicates and

399 the ELISA proceeded as described above. A horseradish peroxidase (HRP)-conjugated goat

400 anti- human kappa light chain antibody (Bethyl, cat. no. A80-115P) was used as a secondary

401 antibody. A standard curve was generated using each mAb. Antibody quantitation was made

402 from sample dillutions that fell within the linear range of the standard curve. For each

403 specimen, 6 five-fold serial dilutions starting at 1:100 (serum) or 1:20 (skin homogenate)

19 404 were run in duplicate.

405 To assess the murine endogenous PfCSP antibody responses after the first malaria

406 challenge, serum samples collected 32 weeks following AAV administration, prior to the

407 second challenge, were diluted 1:100 in blocking solution and tested against rPfCSP by

408 ELISA as as described above and mouse PfCSP antibodies were detected with a peroxidase-

409 labeled goat anti-mouse IgG (Invitrogen, cat. no. 62-6520).

410 Anti-CIS43 antibody (ADA) responses were evaluated in pre-immune macaque serum

411 samples collected 7-14 days prior to antibody infusion and in sera collected 4, 8, 12, 16, and 20

412 weeks after CIS43LS or CIS43 infusion. For the competition assay, 1 µg/ml CIS43LS or CIS43

413 was pre-incubated with pre- and post-infusion serum diluted 1:100 in blocking solution or, as

414 positive control, with serum spiked with an anti-idiotypic mAb recognizing the CIS43 Fab (0.01-

415 1000 ug/mL). The mixture was then added to PfCSP-coated plates. After 1 h incubation, plates

416 were washed and incubated with peroxidase-labeled goat anti–human IgG antibody (KPL, cat.

417 no. 04-10-06) before detection as outlined above. For the direct assay, plates were appropriately

418 pre-coated with CIS43LS or CIS43 and macaque or mouse sera diluted 1:100 in blocking

419 solution were added to the plates. ELISA was performed as described above and macaque ADA

420 were detected with a peroxidase-labeled anti-monkey IgG (Alpha Diagnostic International, cat.

421 no. 7054).

422

423 Isothermal titration calorimetry

424 Isothermal titration calorimetry (ITC) was carried out as described previously (17). Briefly,

425 CIS43 and CIS43LS were diluted to a concentration of ~100 µM (expressed per antigen-binding

426 fragment site) in PBS (pH 7.4) (Sigma-Aldrich) before injection in 10 µl aliquots into the

20 427 calorimetric cell containing ~1 µM rPfCSP. All titrations were performed at 25 °C.

428 Concentrations reactants in each experiment were calculated from the protein absorbance at 280

429 nm. Heat deriving from individual injection of antibody was determined by the integral of the

430 calorimetric indicator. Heat related to the binding of the antibody to rPfCSP within the cell was

431 determined by deducting the heat of dilution from the heat of reaction. The enthalpy variation

432 (∆H), the association (Ka) and dissociation (Kd = 1 / Ka) constant, and the stoichiometry (N)

433 were determined using a nonlinear regression of the individual heats as a function of the

434 concentrations to fit a model that takes into account the binding to two sets of sites with different

435 binding energetics for rPfCSP. The variation in Gibbs energy (∆G), was determined by the

436 equation ∆G = –RTlnKa, where R is the is the universal gas constant [1.987 cal/(K × mol)] and T

437 is the absolute temperature in kelvin. The entropy contribution to Gibbs energy, –T × ∆S, was

438 computed from the known relation ∆G = ∆H – (T × ∆S).

439

440 Kinetic binding assay using biolayer interferometry

441 mAbs were purified on a Superdex200 Hi Load 16/600 column to remove aggregates. BLI

442 analysis was performed on a Octet RED384 system using receptors (Acro Biosystems, #FCM-

443 H5286, FCM-C5284 and FCM-M82W5) or rPfCSP and PfCSP peptides loaded on HIS1K or

444 SAX biosensors (ForteBio #18-5122 and 18-5117) and probed into mAbs in a 2-fold dilution

445 series. Assay conditions were individually optimized for each receptor-ligand pair. Data were

446 fit using a 1:1 model on Octet Data Analyis Software version 12.0.2.3 (Molecular Devices

447 LLC). Serial antibody concentrations used are 20, 10, 5, 2.5, and 1.25 µg/ml for pH 6.0 and

448 2000, 100, 50, 25, and 12.5 µg/ml for pH 7.4 for binding to the receptors, whereas antibody

449 concentrations used for binding to rPfCSP are 2, 1, 0.5, 0.25, and 0.125 µg/ml.

21 450

451 Mouse challenge studies with chimeric Pb-PfCSP-LUC SPZ

452 Malaria challenges to assess the antibody protective efficacy were done as previously described

453 (17). Briefly, stephensi (Nijmegan, Netherlands) mosquitoes were obtained and

454 reared from a colony maintained at the Laboratory of Malaria and Vector Research, NIAID,

455 NIH. Female mosquitoes were allowed to feed on 6-8 weeks female Balb/c mice (Jackson

456 Laboratory) infected with blood-stage Pb-PfCSP-LUC parasites. Eighteen to 21 days following

457 mosquito infections, mosquito salivary glands were dissected and ground in 400 ul of L-15

458 medium (Sigma Aldrich), and viable sporozoites were counted in a Neubauer chamber.

459 Female C57BL/6 mice (6 to 8 weeks old) from Jackson Laboratory were IV injected with

460 VRC01 or varying concentrations of CIS43 or CIS43LS diluted in PBS (pH 7.4) for a total

461 volume of 200 µl per mouse and challenged 2h later with sporozoites IV (25).

462 For the VIP studies, female C57BL/6 albino mice (Jackson Laboratory) were challenged by the

463 IV route 8 or 36 weeks after AAV administration. Protected mice were rechallenged 36 weeks

464 post AAV administration. All IV challenges were made by the tail vein with 2,000 Pb chimeric

465 Pb-PfCSP-LUC SPZ diluted in L-15 medium (100uL /mouse). For infection assessment, mice

466 were injected IP with 150μl D-luciferine (30 mg/ml) (PerkinElmer) substrate prior imaging with

467 the IVIS® Spectrum in vivo imaging system (PerkinElmer). Parasite liver load was assessed 40-

468 42 h after challenge, whereas parasitemia was measured 6 days following challenge. Parasite

469 load was quantified by analyzing a region of interest (ROI) in the upper abdominal region and by

470 determining the total flux or bioluminescent radiance (photons/sec). For assessment of liver stage

471 infection, a defined region of interest around the liver is assessed for all mice, while for

22 472 parasitemia IVIS is performed on the whole animal. This more comprehensive analysis of the

473 whole animal can result in a shift in the background signal.

474 The mosquito bite challenge was done as previously described (17). Briefly, C57BL/6 mice were

475 administered with the AAV constructs or were IV injected with 300 μg of CIS43, CIS43LS, or

476 VRC01 as detailed above. Mice were anesthetized with 2% avertin (Alfa Aesar) and 10 minutes

477 later mice were exposed to 6 infected mosquitoes/mouse for ∼10 min, after which engorged

478 mosquito abdomens were visually inspected for blood to determine whether the mosquito has

479 bitten. Mouse parasitemia was monitored daily by Giemsa staining of blood smears from day 4

480 through day 12 post-infection. The mosquito bite challenge was done 3 weeks (primary

481 challenge) and 11 weeks (rechallenge) post AAV administration.

482

483 Pharmacokinetic (PK) studies in rhesus macaques

484 Male Indian rhesus macaques (5.6 to 8.2 kg body weight, 2 macaques per group) from Covance

485 (Texas, USA) were randomly selected and assigned into groups, and passively infused IV with 10

486 mg/kg body weight of CIS43LS or CIS43. Blood samples were collected two weeks before mAb

487 infusion and post infusion at 5 min, 30 min, 6 h, and 1, 2, 5, 7, 11, 14 d, and weekly thereafter up

488 to 140 days. Similarly, skin punches were collected two weeks prior infusion and after mAb

489 infusion at 30 min, 2, 7, 11, 14 days and bi-weekly thereafter up to 140 days. Ab concentrations

490 were quantified by a PfCSP-based ELISA using the KPL kit. Antibodies were quantitated in 6

491 five-fold serial dilutions run in duplicate starting at 1:20 (serum) or 1:10 (skin homogenate).

492 Blood pharmacokinetic parameters including T½, clearance, and area under the curve were

493 determined with a two-compartment model using the WinNonlin software (Pharsight) (18) to

23 494 estimate the area under the curve (AUC), clearance and T½.

495

496 HEp-2 Cell Staining

497 Autoreactivity staining assays were performed on HEp-2 cells (Zeus Scientific, Branchburg, NJ)

498 per the manufacturer recommendations. MAbs were diluted to 50 and 25 μg/mL using SAVe

499 diluent. Twenty microliters of the appropriate dilution was coated onto cells fixed on the slide

500 and incubated for 30 min at room temperature in a humidified chamber. Slides were rinsed in 1X

501 PBS, washed twice in 1X PBS in Coplin jars for 3–5 min and then stained with 20 μl of FITC-

502 conjugated secondary antibody for 30 min in a humidified chamber. Slides were rinsed in 1X

503 PBS, washed twice in 1X PBS in Coplin jars for 3–5 min and mounted with 15 μl of mounting

504 media per well and a cover glass (Thermo Scientific). Slides were imaged on a Nikon Eclipse

505 E800 microscope at 20X in the RGB mode for 2 s using SPOT 5.0 software (SPOT Imaging,

506 Sterling Heights, MI). VRC01, 4E10, VRC07-523 and VRC07-G54W were used as control

507 mAbs for staining and given a score of 0, 1, 2, and 3 respectively based on their staining

508 intensity. Test mAbs were assigned scores based on visual comparisons of staining intensity to

509 the control antibodies.

510

511 Cardiolipin ELISA

512 mAb binding to cardiolipin was tested by ELISA per the manufacturer’s protocol (Inova

513 Diagnostics, San Diego, CA). Starting at 100μg/ml, mAbs were tested in a 3-fold series. Assays

514 were validated using positive and negative controls and standards provided in the kit. OD values

515 were converted to IgG anti-phospholipid (GPL) units by linear regression. 4E10 and VRC01

516 were used as additional positive and negative controls. Cardiolipin binding was scored as

24 517 follows: no binding for <15 GPL, indeterminate for 15–20 GPL, low positive for 20–80 GPL and

518 high positive for >80 GPL.

519

520 Construction and production of VIP vectors

521 DNA segments corresponding to the heavy and light chain variable regions of VRC01 (45),

522 2A10 (46) or CIS43 (17) mAbs were synthesized (Integrated DNA Technologies) and cloned

523 into the human IgG1 heavy chain and kappa light chain constant region framework in VIP

524 expression vectors as previously described (21). Briefly, the resulting vector consists of the

525 heavy and light chain antibody coding regions separated by a picornavirus F2A self-processing

526 peptide sequence that enables expression of both proteins from a single transcript. Transcription

527 is driven by a CASI promoter, and includes a Woodchuck hepatitis virus post transcriptional

528 regulatory element (WPRE) and SV40 polyadenylation signal. The entire transgene is flanked

529 by AAV2 ITR elements in which the antibody coding region is driven by VIP. Vector

530 production, quantitation, and storage were done as previously described (21,22). Briefly, 293T

531 cells were cotransfected with the AAV backbone vector, helper plasmid pHELP (Applied

532 Viromics), and plasmid pAAV2/8 SEED (University of Pennsylvania Vector Core) at a ratio

533 of 0.25:1:2. Culture supernatants were collected at 36, 48, 72, 96, and 120 h after transfection

534 and pooled. Virus was precipitated by a 40% poly- ethylene glycol solution. Precipitated virus

535 was banded to equilibrium in a 1.36 g mL−1 cesium chloride solution in a Beckman 70.1 Ti rotor

536 at 60,000 rpm for 24 h. Collected fractions exhibiting a refractive index between 1.3755 and

537 1.3655 were combined and diluted in test formulation buffer 2 (TFB2; 100 mM sodium citrate,

538 10 mM Tris, pH 8). Virus was concentrated and washed with TFB2 by centrifugation at 500 g at

25 539 4 °C using 100 kDa molecular weight cutoff centrifugal filters (Millipore). Virus was aliquoted

540 and stored at −80 °C. Viral genomes were quantified by qPCR.

541

542 VIP vector administration

543 After thawing, each AAV construct was diluted with PBS (pH 7.4) to 1 × 1011 GC in a 50 μL

544 volume and injected into mice in a single injection (50 μL) into the cranial thigh muscle. Mice

545 were bled by the tail vein or skin punches were sampled bi-weekly following AAV

546 administration to assess the Ab expression.

547

548 Statistical analyses

549 All data were plotted and graphed using GraphPad Prism, version 8.4.3, unless otherwise stated.

550 For stoichiometry, errors with 95% confidence were analyzed from the fits of the data. To

551 measure CIS43, CIS43LS, 2A10 and VRC01 in macaque or mouse serum and skin, standard

552 curves were fitted with a hyperbolic parameter curve, and concentration values were

553 interpolated. For mAb in macaques, differences in serum concentrations between the CIS43LS

554 and CIS43 group over days 0-140 were calculated using the no sphericity 2way ANOVA

555 analysis. Differences in Ab concentrations between groups and liver parasite burden were

556 determined for significance using the non-parametric One-way ANOVA Kruskal–Wallis test

557 (with Dunn’s correction for multiple comparisons). Parasitemias for the mosquito bite challenge

558 were analyzed by Kaplan–Meier curves using the log-rank test. A statistically significant

559 difference is indicated by P < 0.05.

560

561 Study Approval

26 562 All non-human primate and mouse research studies complied with the Institutional Animal Care

563 and Use Committee (IACUC) of the NIH Vaccine Research Center (protocols VRC-18-755.1

564 and VRC-19-0824) and Johns Hopkins University (Approved protocol permit no. MO18H419).

565

27 566 Author contributions

567 NKK., JF and RAS conceived, designed, and supervised the project studies. NKK, LDP, KE,

568 YF-G, MA, WS, XJ, AP, MD and AS performed experiments. NKK and WS generated mAb 1-

569 1, the CIS43 anti-idiotypic antibody. ABB and SM produced and provided the AAV constructs.

570 NKK, LDP, KE, YF-G, MA, AHI, WS, XC, AP, MD, AS, FZ, JF, RAS analyzed results from

571 the studies. NKK wrote the first draft of the manuscript. NKK, JF, AHI, and RAS wrote the final

572 version of the paper. All authors reviewed, edited and approved the manuscript.

573

574 Competing interests: NKK, AHI, and RAS have submitted a patent application (E-038-2017)

575 describing CIS43 and related mutants. All other authors declare no competing financial interests.

576

577 Data and materials availability: All data associated with this study are present in the paper or

578 the Supplementary Materials.

579

580 The findings and conclusions in this report are those of the authors and do not necessarily reflect

581 the views of the funding agency or collaborators. The views expressed in this publication are

582 those of the authors and do not necessarily reflect the official policy or position of the

583 Department of Health and Human Services or the United States Government.

584

28 585 Acknowledgments

586 We thank T. Zhou and S. Narpala [Vaccine Research Center (VRC), National Institutes of

587 Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH)] for providing

588 monoclonal antibody VRC01. We thank Sung-Youl Ko (VRC, NIAID, NIH) for running the

589 two-compartment analysis for the half-life calculation. We thank John-Paul Todd, Elizabeth

590 McCarthy, the Translational Research Program, and the Vaccine Production Plant (VRC,

591 NIAID, NIH) for technical support. We thank Lawrence Wang (VRC, NIAID, NIH) for his

592 comments on this manuscript. Funding: This work was supported by the following grants:

593 Award (P2DA040254) to ABB from the National Institutes for Drug Abuse (NIDA) Avenir New

594 Innovator; grant (INV-001763) to FZ from the Bill and Melinda Gates Foundation; Contract

595 (HHSN261200800001E) to AS from the National Cancer Institute (NCI), NIH. YFG and FZ

596 thank the Bloomberg Philanthropies for continued support.

597

598

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721

722

35 723 Figures and Tables

724

725 Figure 1. Characterization of CIS43LS

726 (A) Binding of CIS43LS to rPfCSP and PfCSP peptides by ELISA. CIS43, VRC01 (human anti-

727 HIV-1 IgG1) and 317 (human antibody specific for the NANP-repeat region of PfCSP) were

728 used as control antibodies. (B) Thermodynamic parameters and stoichiometry of binding of

729 CIS43LS to rPfCSP by isothermal calorimetry. The dissociation constant (Kd), change in Gibbs

36 730 energy (ΔG) of binding, enthalpy (ΔH), entropy contribution to Gibbs energy (−TΔS) and

731 stoichiometry (N) are shown. Data displayed are representative of two independent experiments.

732 (C) Protective effect of CIS43LS on liver burden. Following passive transfer of CIS43LS or

733 CIS43, C57BL/6 mice were challenged intravenously (IV) with Pb-PfCSP-LUC SPZ before

734 imaging by IVIS. Differences in liver-stage parasite burden reduction between the VRC01,

735 CIS43, or CIS43LS group compared to the untreated group were determined using the non-

736 parametric Kruskal-Wallis test for multiple comparisons with Dunn’s correction. P values are

737 displayed on the panels; ns = not significant. (D) Protective effect on liver burden by CIS43LS at

738 varying concentrations (30–300 μg/ml). Data represent geometric mean with 95% CI (C and D).

739 (E) Sterile protection by CIS43LS following infection by mosquito bites. C57BL/6 mice were

740 challenged with infected mosquitoes following passive transfer of CIS43LS or CIS43 (300 µg

741 per mouse). The presence of parasites were determined through Giemsa staining of blood up to

742 12 d following challenge. Kaplan Meier curves, analyzed by the log rank test, show frequencies

743 of mice free of parasites as determined by Giemsa staining of blood. Differences between

744 CIS43LS and CIS43 as compared to untreated mice are shown (P = 0.0001). n = 5 per group (C-

745 E). (F) Apparent affinity of CIS43LS to human and rhesus FcRn at acidic pH 6.0 and

746 physiological pH 7.4. Antibody binding curves are shown in red (raw data) and black (fitted

747 data). The apparent affinity is displayed as KD (equilibrium dissociation constant) in nM. A

748 representative of 2 independent experiments is shown. ND, no fit could be determined.

749

750

37 751

752

753 Figure 2. Pharmacokinetic study in Indian Rhesus macaques

754 (A) Serum concentrations of CIS43LS were measured by ELISA following IV injection of 10

755 mg per kg body weight of CIS43 or CIS43LS in rhesus macaques. ****P = 0.0003. (B) The

756 amount of mAb in skin punch homogenates from each group of CIS43- or CIS43LS-treated

757 macaques displayed in (A) was quantitated over time and is shown. *P = 0.0148. Data represent

758 the mean with SD and differences in mAb concentrations between both groups were determined

759 using the 2-way ANOVA analysis (A and B). (C and D) ELISA to assess the NHP serum

760 antibodies elicited against CIS43 or CIS43LS (anti-drug antibodies, ADA). Competitive ADA

761 ELISA (C) showing CIS43 or CIS43LS binding to rPfCSP in the presence of NHP sera before

762 (Pre) or at the indicated timepoints after mAb infusion. mAb groups and animals infused with

38 763 CIS43LS (12M069 and 14D017) or CIS43 (13D084 and 15C049) are indicated. Varying

764 concentrations of mouse mAb 1-1, a CIS43 mouse anti-idiotype antibody, were used as a

765 positive control for the competitive binding of CIS43LS or CIS43 to rPfCSP. Pre, pre-immune

766 serum collected at baseline prior to antibody infusion. OD405nm, optical density at 405 nm. Data

767 collected at different timepoints from the animals that received CIS43LS (red) or CIS43 (blue)

768 are shown; black, mAb 1-1. Direct ADA ELISA (D). CIS43LS or CIS43 was coated onto ELISA

769 plates and NHP sera were analyzed. Color codes are as in (C). Data represent the mean ± SEM

770 (C and D). For all panels, n = 2 per group.

771

772

773

774

39 775

776 Figure 3. Protective efficacy and expression of PfCSP-AAV-induced mAbs in C57BL/6

777 mice.

778 (A) Sterile protection by mosquito bite 3 weeks following IM administration of 1011 genome

779 copies (GC) AAV encoding CIS43, 2A10 or VRC01. C57BL/6 mice were challenged by

780 mosquito bites 3 weeks following AAV administration of the indicated mAb. As a control 300

781 μg-rCIS43 was administered 2h prior to challenge. Kaplan–Meier curves, analyzed using the log-

782 rank test, show the frequencies of mice free of parasites. Differences between CIS43-AAV-

783 administered or 300 μg rCIS43-treated mice and untreated mice are shown (P = 0.0001). wpa,

784 weeks post-AAV administration. n = 10 per group; 2A10-AAV, n = 8. 300 μg-rCIS43, mice that

40 785 passively received the protective dose of 300 μg of CIS43 at the time of challenge. (B) Protective

786 effect of PfCSP-AAV expressed mAbs on liver burden 8 weeks following AAV administration.

787 AAV-treated C57BL/6 albino mice were challenged IV with Pb-PfCSP-LUC SPZ and imaged by

788 IVIS. Data represent the geometric mean with 95% CI. For liver burden (2 dpc), *P = 0.0159 and

789 ***P = 0.0001; for parasitemia (6 dpc), *P = 0.0102 and ***P = 0.0005; ns = not significant.

790 Max burden, naïve infected mice; 8 wpa, 8 weeks post AAV administration; dpc, days post-

791 challenge. CIS43 and VRC01, n = 10 per group; 2A10, n = 8. hIgG; human IgG1. Serum hIgG

792 (C) and PfCSP-specific mAb (D) levels, and skin hIgG (E) and PfCSP-specific mAb (F) levels

793 from mice shown in (B) were measured by ELISA 8 wpa. Differences in antibody concentration,

794 parasite liver burden or parasitemia between each group and the untreated group were

795 determined using the Kruskal-Wallis test for multiple comparisons with Dunn’s correction. P

796 values are shown on panels. Means with SD are displayed (C-F).

797

798

41 799

800 Figure 4. Long-term expression and protective capacity of AAV-induced mAbs.

801 (A) C57BL/6 albino mice were challenged IV with Pb-PfCSP-LUC SPZ 36 weeks after a single

802 administration of AAV encoding CIS43, 2A10 or VRC01. As a control, rCIS43 (300ug)

803 administered 2h prior to challenge. For all groups, n = 10; 2A10-AAV, n = 8. *P = 0.0235; **P

804 = 0.0013; ****P = <0.0001; ns, not significant. (B) Serum hIgG levels of AAV-injected mice

805 shown in (A) were measured by ELISA 32 weeks post AAV administration prior rechallenge (36

806 weeks). P values are shown on the panel. Data represent the mean ± SEM. (C) AAV-treated

42 807 C57BL/6 mice previously protected at 3 weeks post CIS43-AAV administration (Figure 3A)

808 were rechallenged by mosquito bites 11 weeks following the AAV administration. Kaplan–Meier

809 curves, analyzed using the log-rank test, show the frequencies of mice free of parasites as

810 determined through Giemsa staining of blood up to 12 d following challenge. Differences

811 between CIS43-AAV-administered mice and untreated mice are shown (P = 0.0001). Untreated

812 mice, n = 7; CIS43-AAV, n = 9. (D) C57BL/6 albino mice previously protected when challenged

813 8 weeks after AAV administration (Figure 3B) were rechallenged IV with Pb-PfCSP-LUC SPZ

814 at 36 weeks. Liver burden (left) and parasitemia (right) are shown. P values are displayed on the

815 panels. CIS43-AAV, n = 8; 300 μg-rCIS43; n = 10. Differences in parasite liver burden,

816 parasitemia, or serum antibody concentration between groups were determined using the

817 Kruskal-Wallis test for multiple comparisons with Dunn’s correction. Data represent the

818 geometric mean with 95%CI (A and D).

819

820

43 821

822 Figure S1. Amino acid sequence alignment resulting from the DNA sequencing of CIS43

823 and CIS43LS heavy chain.

824 The LS point mutations are boxed in red.

825

826

827

44 A No mAb VRC01LS 4E10 VRC07-523LS VRC07-G54W CIS43 CIS43LS

- -

l

m

/

g

µ

0 5

No mAb VRC01LS 4E10 VRC07-523LS VRC07-G54W CIS43 CIS43LS - - -

0 0 1 2 3 0 0

l

m

/

g

µ

5 2 A

B OD at 450 nm GPL units mAb Interpretation 100 µg/ml 33.3 µg/ml 100 µg/ml 33.3 µg/ml

VRC01LS 0.03 0.03 -0.52 -0.52 Negative 4E10 1.58 1.53 162.34 157.57 High positive <15 Negative VRC07-523LS 0.24 0.11 20.83 7.02 Negative 15-20 Indeterminate VRC07-G54W 0.43 0.18 41.36 14.44 Negative CIS43 0.04 0.03 -0.46 -0.65 Negative 20-80 Low to medium positive 828 CIS43LS 0.04 0.04 -0.19 -0.25 Negative >80 High positive

829 Figure S2. Autoreactivity of CIS43LS.

830 (A), Representative confocal fluorescence microscopy images of human HEp-2 epithelial cell

831 staining following reactivity with CIS43LS or CIS43. Test antibodies and concentrations (25 and

832 50 µg) used are indicated. Anti-HIV1 antibodies were used as negative (VRC01LS) and positive

833 (4E10, VRC07-523LS and VRC07-G54W) controls. Images were acquired with 20x objective

834 N.A. = 0.7. Reactivity was assigned scores of 0, 1, 2, and 3 in the 25 μg/mL samples, using the

835 no-mAb sample as baseline. (B) Binding of varying concentrations (100 and 33.3 μg/ml) of

836 mAbs to cardiolipin determined by ELISA. Test and control antibodies are as in (A). Values are

837 expressed as optical density at 450 nm (OD450nm) and IgG phospholipid signal (GPL) units. One

838 unit represents 1 μg control IgG antibody.

839

840

45 841

842 Figure S3. Binding kinetics of CIS43-expressed AAV mAb in mouse serum to PfCSP.

843 CIS43 mAb levels in serum (A) and skin (B) at indicated times after IM administration with 1011

844 GC of CIS43-AAV in C57BL/6 albino mice (n = 5). For (B), differences in CIS43 concentration

845 at indicated timepoints were determined using the Kruskal-Wallis test for multiple comparisons

846 with Dunn’s correction. ns, not significant. Data represent the mean with SD (A and B). (C)

847 Apparent binding affinity of CIS43-, 2A10-, VRC01-AAV in mouse sera to PfCSP, 8 weeks

848 following AAV administration. CIS43 and 2A10 IgG have been used as controls. Antibody

849 binding curves are shown in red (raw data) and black (fitted data). The apparent affinity is

850 displayed as KD (equilibrium dissociation constant) in nM on each panel. ND, no fit could be

851 determined. mAb serial concentrations (2, 1, 0.5, 0.25, and 0.125 µg/ml ) used are displayed.

852

46 3.0

2.0

m

n

5

0

4

D O 1.0

0.0 e e n iv s a a u m N o u -m h ti ti- n n 853 A A

854 Figure S4. Mouse PfCSP-specific antibody responses in CIS43-AAV-administered mice

855 following the first malaria challenge.

856 Previously challenged and protected mice that received CIS43-AAV (Figure 3B) were assessed

857 for mouse PfCSP-specific antibodies (anti-mouse) measured by ELISA at 32 weeks just prior to

858 rechallenge. Human IgG induced by CIS43-AAV (anti-human) are shown for comparison. Naïve

859 indicates serum samples collected prior to AAV administration and prior to any challenge.

860 OD405nm, optical density at 405 nm. Data points represent the mean ± SEM. Naïve and anti-

861 human, n = 4 per group; anti-mouse, n = 8.

862

47 863 Supplemental Table 1. Summary of binding of CIS43 or CIS43LS to rhesus or human

864 neonatal Fc receptor (FcRn).

Fc Receptor Sample ID Kd (nM) Kd (M) Kd Error Kon (1/Ms) Kon Error Koff (1/s) Koff Error Fold Kd *

Human FcRn CIS43 54.2 5.4E-08 1.5E-09 4.8E+05 1.2E+04 2.6E-02 3.5E-04

pH 6.0 CIS43LS 6.2 6.2E-09 6.7E-11 2.7E+05 2.8E+03 1.7E-03 3.6E-06 9.0

Human FcRn CIS43 ND No Fits Obtained

pH 7.4 CIS43LS 234.1 2.3E-07 5.8E-09 4.8E+05 1.1E+04 1.1E-01 8.9E-04

Rhesus FcRn CIS43 325.8 3.3E-07 5.4E-09 5.9E+04 9.0E+02 1.9E-02 1.2E-04

pH 6.0 CIS43LS 25.1 3.1E-08 3.4E-10 3.7E+04 2.2E+02 1.1E-03 1.1E-05 13.0

Rhesus FcRn CIS43 ND No Fits Obtained

pH 7.4 CIS43LS 262.1 2.6E-07 3.8E-09 2.35E+05 2.9E+03 6.15E-02 4.4E-04 865

866 * Fold change in KD versus unmodified CIS43.

867 KD, equilibrium dissociation constant; Kon, association rate constant; Koff, dissociation rate

868 constant.

869

48 870 Supplemental Table 2. Binding affinities of CIS43 and CIS43LS for human Fc gamma

871 receptors.

Human Sample ID KD (nM) KD (M) KD Error Kon (1/Ms) Kon Error K0ff (1/s) Koff Error Fold KD * Fc Receptor (pH 7.4)

CIS43 14.84 1.48E-08 2.15E-10 1.87E+05 2.17E+03 2.78E-03 2.41E-05 FcγRI CIS43LS 15.84 1.58E-08 1.96E-10 1.94E+05 1.99E+03 3.08E-03 2.14E-05 1.0

CIS43 3835 3.84E-06 6.85E-07 1.76E+05 3.11E+04 6.76E-01 1.91E-02 FcγRIIA H167 CIS43LS 2489 2.49E-06 2.76E-07 2.40E+05 2.60E+04 5.98E-01 1.35E-02 1.5

CIS43 1479 1.48E-06 1.16E-07 3.48E+05 2.64E+04 5.14E-01 9.79E-03 FcγRIIA R167 CIS43LS 763.5 7.64E-07 6.45E-08 4.57E+05 3.68E+04 3.49E-01 9.00E-03 1.9

CIS43 3132 3.13E-06 8.28E-07 2.59E+05 6.74E+04 8.12E-01 3.81E-02 FcγRIIB CIS43LS 3803 3.80E-06 1.15E-06 2.30E+05 6.84E+04 8.74E-01 4.11E-02 0.8

CIS43 652 6.52E-07 1.55E-08 1.61E+05 3.44E+03 1.05E-01 1.09E-03 FcγRIIIA V176 CIS43LS 550 5.50E-07 2.22E-08 7.87E+04 2.50E+03 4.33E-02 1.08E-03 1.2

CIS43 1736 1.74E-06 1.21E-07 1.57E+05 1.06E+04 2.73E-01 FcγRIIIA 5.11E-03 F176 CIS43LS 2919 2.92E-06 2.54E-07 4.40+05 3.73E+04 1.29E-01 2.56E-03 0.6

CIS43 1027 1.03E-06 1.20E-07 4.30E+05 4.80E+04 4.42E-01 1.56E-02 FcγRIIIB NA1 Protein CIS43LS 1187 1.19E-06 7.75E-08 3.00E+05 1.88E+04 3.56E-01 6.68E-03 0.9

CIS43 1283 1.28E-06 1.01E-07 3.27E+05 2.46E+04 4.19E-01 9.08E-03 FcγRIIIB NA2 Protein CIS43LS 853.6 8.54E-07 5.20E-08 2.93E+05 1.70E+04 2.50E-01 4.61E-03 1.5 872

873 * Fold change in KD versus unmodified CIS43; KD, equilibrium dissociation constant; Kon,

874 association rate constant; Koff, dissociation rate constant. Human polymorphisms: FcγRIIA

875 H167, FcγRIIA H167; FcγRIIA R167; FcγRIIIA V176; FcγRIIIA F176; FcγRIIIB NA1;

876 FcγRIIIB NA2.

877

49 878 Supplemental Table 3: Pharmacokinetic parameters in sera from rhesus macaques

879 administered with CIS43LS or CIS43 (10 mg/kg) by IV route.

Observed Observed AUC Clearance Serum Concentration (µg/mL) Animal ID mAb Route T (Day) 1/2 Tmax (Day) Cmax (µg/ml) (Day x µg/ mL) (mL/Day/Kg) Day 49 Day 98

12M069 CIS43LS IV 46.5 0.01 261.5 5,467 1.83 37.3 18.8

14D017 CIS43LS IV 31.0 0.01 313.2 4,107 2.43 28.3 11.5

AVG 38.7 0.01 287.3 4,787 2.13 32.8 15.1

SE 7.8 0.0 25.9 680 0.30 4.5 3.7

13C084 CIS43 IV 24.5 0.01 289.2 1,887 5.30 4.1 1.1

15C049 CIS43 IV 20.1 0.01 271.7 1,592 6.28 1.9 0.5

AVG 22.3 0.01 280.4 1,739 5.79 3.0 0.8

SE 2.2 0.0 8.8 148 0.49 1.1 0.3 880

881 T½ (elimination half-life), time taken for the plasma concentration of a drug to fall by half its

882 original value; Tmax, time taken to attain Cmax; Cmax, maximum drug concentration in plasma;

883 AUC (Area Under the Curve), measure of exposure of all the body to the drug; Clearance,

884 measurement of plasma volume from which a drug is entirely removed per day per Kg body

885 weight.

886

50