VIRAL HEPATITIS

Prevention of Hepatitis C Virus Infection and Spread in Human Liver Chimeric Mice by an Anti-CD81 Monoclonal Antibody Changhua Ji,1 Yang Liu,1 Chandra Pamulapati,1 Sandhya Bohini,1 Georg Fertig,2 Michael Schraeml,2 Werner Rubas,3 Michael Brandt,2 Stefan Ries,2 Han Ma,1 and Klaus Klumpp1 CD81 is a required receptor for hepatitis C virus (HCV) infection of human hepatocytes in vitro. We generated several high-affinity anti-human CD81 monoclonal antibodies (mAbs) that demonstrated potent, specific, and cross-genotype inhibition of HCV entry. One of these mAbs, K04, was administered to human liver chimeric mice before or after HCV infection to determine its ability to prevent HCV infection or spread of HCV infection, respectively. All vehicle control mice established HCV infection, reaching steady-state levels of serum HCV RNA by day 21. Pretreatment of mice with K04 prevented HCV infection in all mice (n 5 5). Treatment of mice with mAb K04 every 3 days for 21 days, starting at 6 hours postinfection, resulted in effective inhibition of virus spread. In 3 mice that were sacrificed on day 24, serum HCV levels remained detectable, below the limit of quantification (LOQ), indicating that infection was established, but virus spread was blocked, by the anti-CD81 mAb. In 5 additional mice that were followed for a longer time, virus remained detectable, below LOQ, until days 24 and 30 in 4 of 5 mice. In the fifth mouse, viral load was quantifiable, but reduced to 64-fold below the mean viral load in vehicle control at day 24. In addition, 2 of 5 mice cleared the infection by day 30 and 1 mouse had undetectable virus load from day 6 onward. Conclusion: These results demonstrate that CD81 is required for HCV infection and virus spread in vivo, and that antiCD81 antibodies such as K04 may have potential as broad-spectrum antiviral agents for prevention and treatment of HCV infection. (HEPATOLOGY 2015;61:1136-1144)

H

epatitis C virus (HCV) infection is a major cause of severe liver disease, cirrhosis, and liver cancer. It is estimated that, among the approximately 160 million people who are living with chronic HCV infection globally, 10%-20% will develop complications of chronic liver disease and 1%5% will develop liver cancer.1 Treatment options for chronic HCV infection have been significantly improved by the recent introduction of potent directacting antivirals (DAAs), such as the antiviral nucleoside analog, sofosbuvir (SOF), in 2013.2,3 SOF in combination with ribavirin (RBV) has become the first interferon (IFN)-free treatment option for HCV geno-

type 2 and 3 infection, whereas SOF in combination with pegylated IFN alpha and RBV has shortened treatment duration and increased efficacy of treatment for patients infected with genotype 1. Research and development activities are currently focusing on morepotent DAA combinations that will enable more efficacious IFN-free regimens against all genotypes. Another important area of research is the study of prophylactic treatment options to prevent HCV infection after liver transplantation (LT). Reinfection of the liver graft is common and the recurrence of HCV infection is usually more progressive, compared to the natural course of the disease in nontransplanted patients. It has been

Abbreviations: Ab, antibody; DAAs, direct-acting antivirals; ECL2, extracellular loop 2 of CD81; HCV, hepatitis C virus; HCVcc, cell-culture–derived HCV; HCVpp, pseudotyped HCV; HSA, human serum albumin; IC50, half-maximal inhibitory concentration; IC90, 90% inhibitory concentration; IFN, interferon; LOD, limit of detection; LOQ, limit of quantification; LT, liver transplantation; mAb, monoclonal antibody; NMRI, Naval Medical Research Institute; PHHs, primary human hepatocytes; PK, pharmacokinetics; RBV, ribavirin; RI, replacement index; SC, subcutaneous; SCID, severe combined immune deficiency; SOF, sofosbuvir; SR-BI, scavenger receptor class B type I; uPA, urokinase-type plasminogen activator; VSV, vesicular stomatitis virus; VSVpp, pseudotyped VSV. From the 1Hoffmann-La Roche Inc., Nutley, NJ; 2Roche Penzberg, Penzberg, Germany and 3Roche Palo Alto, Palo Alto, CA Changhua Ji is currently affiliated with Roche Innovation Center (China), Shanghai, China. Received June 19, 2014; accepted November 13, 2014. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27603/suppinfo. 1136

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reported that 30% of liver graft recipients developed graft cirrhosis within 5 years after LT.4-6 Reinfection of the liver graft begins with the entry of virus into hepatocytes, and thus the prevention of HCV entry is an attractive approach for preventing HCV infection and HCV recurrence after transplantation. HCV enters hepatocytes by fusion of the viral envelope with the cellular endosomal membrane. Current data suggest an entry mechanism mediated by the interaction between the viral envelope proteins, E1 and E2, and four essential cellular receptors: CD81; scavenger receptor class B type I (SR-BI); claudin I; and occludin.7,8 CD81 was the first identified essential HCV receptor.9-13 An essential role of CD81 in HCV entry was suggested from the infection of primary human hepatocytes (PHHs) with laboratory-generated virus (cell-culture–derived HCV; HCVcc), pseudotyped HCV viruses (HCVpp), and clinical HCV isolates obtained from HCV-infected donors using a variety of methods, including small interfering RNA– mediated suppression of CD81 expression and treatment with anti-CD81 antibody (Ab).7,14 It was also shown that an anti-CD81 Ab could protect human liver chimeric mice from infection with HCV.15 Association of CD81 with claudin I may be necessary for their interaction with HCV E2 envelope protein and subsequent HCV entry.16,17 It has also been suggested that HCV may infect cells in contact with infected cells by a cell-to-cell transmission pathway. The role of CD81 in this pathway and the ability of anti-CD81 monoclonal Ab (mAb) to control HCV infection dissemination are still debated.18-21 The current study describes the generation of a new set of anti-CD81 mAb molecules with potent inhibitory activity on HCV entry in vitro. We show here that the anti-CD81 mAb, K04, could completely block infection of humanized mice with HCV, when it was administered before viral challenge (prophylactic setting), consistent with previous results with a different Ab.15 In addition, we report here, for the first time, that an anti-CD81 mAb can also effectively suppress virus spread in the infected liver when treatment is started after infection. There was effective suppression of virus dissemination and no viral rebound dur-

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ing a 3-week treatment of HCV infection with the anti-CD81 mAb, K04, and viral clearance was observed in 2 of 5 treated mice.

Materials and Methods Additional information on the materials and methods used in this study are available in the Supporting Information. CD81 mAb Generation. Anti-CD81 mAbs were generated from immunization of Balb/c and Naval Medical Research Institute (NMRI) mice with CD81 extracellular loop 2 (ECL2) protein or with CHOCD81 cells. HCVpp and HCV Entry Inhibition Assay. HCVpp or pseudotyped VSV (VSVpp) were generated using a luciferase encoding human immunodeficiency virus genome with an env gene deletion and HCV or vesicular stomatitis virus (VSV) envelope gene expression plasmids.10,22 Compounds or test Abs were serially diluted in 96-well plates, mixed with HCVpp or VSVpp containing culture supernatant, and then added to Huh-7 cells. Luciferase activity was measured 3 days after infection. Generation of HCVcc and HCVcc Replication Inhibition Assay. Full-length genomic RNA of HCV strain H77S (genotype 1a)23 was transfected into Huh-7-derived Rof0c-S3 cells24 to produce HCVcc viral stock. Rof0c-S3 cells were infected with HCVcc in the presence of serially diluted Abs. After 4 days, HCV RNA was extracted and quantified by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Surface Plasmon Resonance. Abs were captured onto chips coated with polyclonal rabbit anti-mouse (RAMIgG) Ab. The analyte CD81, ECL2, was injected for a 3-minute association time in a concentration series. The dissociation was monitored for 10 minutes. The sensorgrams were evaluated using a BIAcore T100 instrument and software (GE Healthcare Life Sciences, Pittsburgh, PA). Determination of Anti-CD81 mAb-Binding Specificity and Affinity by Fluorescence-Activated Cell Sorting. Cells were incubated with anti-CD81 Abs for 45 minutes, then with a labeled secondary Ab

Address reprint requests to: Changhua Ji, M.D., Ph.D., Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110. E-mail: [email protected]; fax: 186-21-50790293 or Klaus Klumpp, Ph.D., Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110. E-mail: [email protected]; fax: 1-973-235-3518. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27603 Potential conflict of interest: Dr. Klumpp owns stock in Riboscience. Dr. Ma is employed by and owns stock in Hoffman-La Roche.

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Table 1. Antiviral Activities of Anti-CD81 mAbs in Huh-7 Cells* IC50 Mean 6 SD (ng/mL)

CD81 mAb

JS81 K02 K04 K07 K13 K21

HCVpp GT1b (Con1)

HCVpp GT1a (H77)

HCVcc GT1a (H77)

53 6 13 49 6 11 50 6 1 46 6 15 38 6 10 12 6 6

55 6 12 50 6 18 45 6 23 63 6 21 30 6 5 20 6 6

167 6 27 78 6 12 136 6 12 128 6 3 77 6 4 44 6 6

*From three or more independent experiments. Abbreviations: SD, standard deviation; GT, genotype.

for 30 minutes. Stained cells were analyzed with an LSRFortessa cell analyzer (BD Biosciences, San Jose, CA). In Vivo Pharmacokinetics and Efficacy Studies. These studies were conducted at PhoenixBio Co., Ltd. (Hiroshima, Japan).25,26 A single-dose pharmacokinetics (PK) study was conducted using subcutaneous (SC) administration of K04 Ab at 20 or 60 mg/kg to severe combined immune deficiency (SCID) mice (n 5 3/ group) and human chimeric liver mice (PXB mice; n 5 4/group). In the prophylactic study, mice were dosed SC with 60 mg/kg of K04 or vehicle. Twelve hours later, these mice were inoculated with HCV (time 0) and two other doses of 60 mg/kg of K04 or vehicle were administered at 24 and 60 hours. All mice were sacrificed on day 28. In the postexposure treatment study, K04 Ab was administered to 8 mice at 60-mg/kg doses for 3 weeks, every 3 days, starting 6 hours after HCV infection. Three mice (group A) were sacrificed on day 24, and the remaining 5 (group B) were sacrificed on day 48.

Results Anti-CD81 mAbs Inhibit HCV Entry. AntiCD81 mAbs were generated from immunization of Balb/c and NMRI mice with recombinant CD81 ECL2 protein or CHO cells expressing human CD81 (CHOCD81 cells). Abs were selected based on their ability to bind to CHO or Huh-7 cells expressing human or monkey CD81, but not to parental cells and not to CHO cells expressing human CD9. Five Abs (K02, K04, K07, K13, and K21) were characterized further and compared to anti-CD81 mAb JS81. These Abs were different from one another, as determined by variable region sequencing (Supporting Fig. 1A). This was consistent with epitope differences, as determined by profiling of binding to cells expressing different singlepoint mutants of human CD81 (Supporting Fig. 1C).

All Abs showed potent inhibition of HCVpp genotype 1a (H77) and 1b (Con1) entry with mean halfmaximal inhibitory concentration (IC50) values ranging from 12 to 63 ng/mL (77-405 pM; Table 1). All Abs similarly inhibited HCV entry into PHHs (IC50 range: 56-166 ng/mL; Supporting Table 1; Fig. 1), and infection by HCVcc (IC50 range: 44-167 ng/mL; Table 1) was similarly within 2-fold across human hepatocytes from up to 5 different donors for most Abs, suggesting robust HCV entry inhibition with little donor-to-donor variability (Fig. 1A). All mAbs displayed steep inhibition curves and small 90% inhibitory concentration (IC90)/IC50 ratios ranging from 1.6 to 2.3 (Supporting Table 1), consistent with a threshold effect of CD81 blockage required for entry inhibition. HCVpp were generated from a total of 28 clinical samples (HCV patient sera), including genotypes 1a (n 5 11), 1b (n 5 11), 2 (n 5 3), and 4 (n 5 3). The sequence analysis of the clinical isolate panel indicates significant sequence diversity beyond the common laboratory sequences of Con1 and H77 (Supporting Fig. 2). The anti-CD81 mAb, K21, showed potent antiviral activity against all 28 HCVpp variants, with all IC50 values of similar magnitude, ranging from 10 to 81 ng/mL. Data obtained with the other anti-CD81 mAbs were also similar, with IC50 values ranging from 12 to 143 ng/mL (data not shown). These results are consistent with CD81 being an essential receptor for HCV entry across genotypes and suggest a broad antiviral spectrum potential of anti-CD81 mAbs. The anti-CD81 Abs bound specifically to cells expressing human CD81 (Huh-7), but not to BxPC3 or HepG2 cells, that do not express CD81, as determined by fluorescence-activated cell sorting analyses (Supporting Fig. 3). In comparison, an anti-CD9 Ab did not bind to CD81-expressing Huh-7 cells, but did bind to CD9-expressing HepG2-CD9 and BxPC3 cells. The anti-CD81 Abs, K02, K04, and K21, showed high binding affinity to ECL2 at both 25  C and 37  C, with Kd values ranging from 0.35 to 4.9 nM as determined by surface plasmon resonance (SPR; Table 2). Prophylactic Treatment With Anti-CD81 mAb K04 Prevents HCV Infection. To determine whether an anti-CD81 mAb can protect against HCV infection in vivo, we tested K04 in the urokinase-type plasminogen activator (uPA)11/SCID human liver chimeric mouse model (PhoenixBio, Hiroshima, Japan). Mice with high human serum albumin (HSA) levels (7.2 mg/mL), indicative of high human hepatocyte repopulation in the chimeric liver (70%) and thus of

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Fig. 2. Evaluation of anti-CD81 mAb K04 PK in SCID and uPA11/ SCID human liver chimeric mice (PXB mice). Single 20- or 60-mg/kg doses of K04 Ab were administered SC, and serum concentrations of K04 were determined at different times. (A) PK parameters and (B) concentration-time profiles are shown. Abbreviations: Cmax, maximum plasma concentration; Conc., concentration; Tmax, time to maximum plasma concentration.

Fig. 1. CD81 mAbs showed broad-spectrum and potent antiviral activity. (A and B) PHHs from 5 different donors (except K21, with 3 donors) were infected with HCVpp con1 and treated with serially diluted CD81 mAbs K02, K04, K07, K13, K21, and JS81. Three days later, luciferase activity was measured and IC50 values were determined by using the sigmoidal dose-response model with one binding site in Microsoft XLfit. Individual IC50 values for each mAb and donor are shown in (B), and pooled multidonor hepatocyte HCVpp inhibition curves are shown in (A). (C) Huh-7 cells were infected with HCVpp viral particles carrying HCV envelope proteins derived from various clinical isolates belonging to GT1a, GT1b, GT2, and GT4 subtypes and treated with serially diluted CD81 mAb K21. Three days later, luciferase activity was measured and IC50 values were determined. Individual IC50 values for each HCVpp within each subtype were graphed. Abbreviation: GT, genotype.

high susceptibility for HCV infection, were used (PXB mice). The PK obtained after SC administration of single SC 20- and 60-mg/kg K04 doses were assessed first (Fig. 2). Serum exposure to K04 in PXB mice was significantly lower, as compared to SCID mice, at the same dose. The increase in exposure comparing the 20- with the 60-mg/kg dose was approximately dose proportional in SCID mice, whereas it was more than dose proportional in PXB mice. These results are consistent with the lack of binding of K04 Ab to mouse cells (SCID mice) and effective binding of K04 Ab to human hepatocytes in PXB mice. Therefore, human CD81 in liver of PXB mice serves as a sink for Ab binding and Ab extraction from circulation. We estimated that 3 SC doses of 60 mg/kg given at 36hour intervals would maintain a K04 minimal serum exposure (Cmin) significantly above its IC90 value to inhibit HCVpp entry into PHHs (Supporting Table 1), at least for 5 days, which we expected to be sufficient for mice to clear all of the free virions from the inoculum. Five PXB mice were injected with a 60-mg/kg SC dose of mAb K04. Twelve hours later, mice were infected with 1 3 104 copies of genotype 1b HCV virus. Two additional SC doses of 60 mg/kg of K04 were administered at 36 and 72 hours after the first injection. A control group of 5 mice were treated in the same way with vehicle formulation. HCV-RNA

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Table 2. Binding Affinity of Anti-CD81 Abs to CD81 ECL2 Determined by SPR Measured at 25 C or 37 C CD81 mAb

JS81 K02 K04 K21

ka (1/Ms) 

25 C 37 C 25 C 37 C 25 C 37 C 25 C 37 C

3.63 7.41 3.83 7.98 3.43 6.87 5.75 8.52

E105 E105 E105 E105 E105 E105 E105 E105

Estimated Error

6 6 6 6 6 6 6 6

5.18 3.91 3.68 5.22 1.95 1.42 6.56 7.67

E101 E102 E101 E101 E101 E102 E101 E101

levels in mouse serum at days 7, 14, 21, and 28 after infection were quantified by RT-PCR, as indicated in the experimental layout scheme in Fig. 3A. All 5 vehicle control mice were infected with HCV, as indicated by the presence of HCV RNA in serum, detectable as early as day 7 after infection. Serum viremia reached maximal steady levels by day 21 at approximately 107 copies/mL in all control mice (Fig. 3B). In contrast, none of the K04-treated mice developed viremia. Serum concentrations of K04 were 44, 40, and 22 ug/ mL at 12, 72, and 168 hours after the first dose, respectively, and became undetectable by day 28 (Fig. 3C). Both vehicle- and Ab-treated mice showed reductions of HSA levels in the blood. K04-treated mice showed larger reductions of HSA levels, which reached nadir by day 14 (31% decrease from baseline) and then rebounded, whereas vehicle-treated mice showed a reduction of 5.4% from baseline over this time frame (Fig. 3D; Supporting Table 2). The lowest human albumin level among any of the mice at any time point of the study was 6.2 mg/mL, indicating that all mice had high levels of HSA and human hepatocyte chimerism sufficient for robust HCV infection throughout the study. A transient slight body-weight reduction was also observed during the study. The maximal mean body-weight reduction of the vehicle control group was 2.5% on day 1, whereas for K04treated group it was 6.7% on day 10. Both groups reached a similar mean body weight by the end of the study (Fig. 3E). These results demonstrate that prophylactic dosing of an anti-CD81 mAb to serum concentrations of approximately 40 mg/mL could completely protect from HCV infection in the humanized mouse model. Postexposure Treatment With Anti-CD81 mAb K04 Blocked Viral Spread In Vivo. We next tested the ability of anti-CD81 mAb K04 to block HCV infection dissemination (spread) in the chimeric mouse liver. For this purpose, treatment with the Ab at a dose of 60 mg/kg was initiated at 6 hours after virus inoculation and continued for 3 weeks (Fig. 4A). Serum viral load was monitored up to 48 days. The

kd (1/s)

1.24 3.37 5.96 6.24 1.76 7.49 2.90 4.53

E-03 E-03 E-04 E-04 E-04 E-04 E-04 E-04

Estimated Error

6 6 6 6 6 6 6 6

1.80 4.56 4.18 4.05 7.76 4.05 7.80 2.77

E-06 E-06 E-06 E-06 E-06 E-06 E-06 E-05

Kd (M)

3.41 4.55 1.56 7.83 5.12 1.09 5.05 5.32

E-09 E-09 E-09 E-10 E-10 E-09 E-10 E-10

Estimated Error

6 6 6 6 6 6 6 6

5.43 8.55 1.11 5.43 2.27 6.12 1.36 5.43

E-12 E-12 E-11 E-12 E-11 E-12 E-11 E-12

vehicle-treated control group of 5 mice showed first detectable levels of serum HCV RNA between days 6 and 12 after inoculation and reached maximal levels of serum HCV RNA between days 24 and 42 (2.6-13 million copies/mL; Supporting Table 3; Fig. 4B). These viral load kinetics were similar to those obtained in the prophylactic study. Of 8 K04-treated mice, 3 were sacrificed on day 24, shortly after the end of treatment (group A, animal ID #201, #202, and #203). All three group A mice showed detectable HCV RNA in the blood, which remained below the limit of quantification (LOQ) for the whole treatment period. These results demonstrate that all group A mice were infected, and that the presence of K04 Ab established an effective block of virus spread in the liver. Five K04-treated mice (group B, animal ID #204-#208) had serum HCV RNA monitored up to day 48 postinfection (Supporting Table 3). Mouse #205 maintained HCV levels below the limit of detection (LOD) at all time points. Mice #206 and #207 showed detectable serum levels of HCV RNA by day 6, which became undetectable by days 24 and 30, respectively, and remained undetectable for the residual time points, consistent with clearance of the infection. Mouse #204 showed detectable serum HCV RNA below LOQ by day 6 after infection and until day 30. On day 30, viral load became quantifiable (0.37 million copies/mL) and then increased with time until reaching 5.3 million copies/mL by day 42. Mouse #208 had low levels of serum HCV RNA slightly above the LOQ by day 6, which remained suppressed at similar low levels until the end of treatment with K04. Viral load rebounded after the end of treatment to >1 million copies/mL by day 36. The HCV-RNA data for the individual mice are plotted and shown in Fig. 4B and Supporting Table 3. These results indicate an effective block of virus spread in liver of HCVinfected mice by the presence of K04 Ab and a functional clearance of the Ab within 15-21 days after the end of treatment. Serum levels of K04 remained above 100 times the antiviral 90% effective concentration in PHHs (i.e.,

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Fig. 3. Anti-CD81 mAb K04 prevented HCV infection of PXB mice. Mice received 3 SC doses of 60 mg/kg of K04 Ab or vehicle control at 12 hours before HCV inoculation and 24 and 60 hours after HCV inoculation. (A) In the outline of the study design, day 0 is defined as day of virus inoculation. (B) Serum HCV-RNA levels of treated mice. Limit of Quantitation (LOQ 5 4 3 104 copies/mL) and Limit of Detection (LOD 5 1 3 104 copies/mL) are shown as dotted lines. Serum HCV RNA of the 5 K04-treated mice were below LOD (1 3 104 copies/mL) at any time point. Data points that were below LOQ were arbitrarily set at 20,000 copies/mL and data points that were below LOD were arbitrarily set at 12,000 copies/mL to visualize them on the time course graph. (C) Population PK of K04. Serum concentrations of K04 mAb were obtained from pooled blood from all treated mice, except for the first time point (12 hours) and the last time point (day 28) right before sacrifice. Serum concentration of K04 on day 28 was below LOQ (250 ng/mL), shown as 1 ng/mL on the graph. (D) Mean serum concentrations of HSA. (E) Mean mouse body weights.

above 21 mg/mL) for the duration of treatment and until 3 days after the end of treatment (day 24 after HCV infection; Fig. 4C). Mean K04 serum concentration decreased to 3.4 mg/mL by day 30 and to an undetectable level by day 36. The decline of K04 serum concentration after day 24 correlated with serum HCV levels increasing only after day 30 for mice #204 and #208. Similar to the prophylaxis study result, a decrease of HSA levels was observed in all K04-treated mice (maximal mean reduction observed in group B mice was 54% on day 42). HSA levels remained above 3 mg/mL at all time points (Support-

ing Table 4; Fig. 4D). It is noteworthy that mice in the vehicle control group also experienced gradual HSA reduction, with maximal mean reduction of 32% on day 42. A transient and slight decrease in body weight was also observed for K04-treated mice in this study (maximal mean reduction of 6.7% on day 9; Fig. 4E). A body-weight decrease was not observed in animals dosed with vehicle. These data demonstrate that postexposure treatment of HCV-infected human chimeric liver mice with an anti-CD81 mAb could effectively block HCV dissemination in the liver in all treated mice. In addition, treatment of 5 HCV-

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Fig. 4. Anti-CD81 mAb K04 prevented HCV spread in PXB mice. Mice received 9 SC doses of 60 mg/kg of K04 Ab starting at 6 hours after HCV infection. (A) In the study design, day 0 is defined as day of virus inoculation. (B) Serum viral load kinetics of vehicle-treated animals (5 of 5 animals) is shown in red and of K04-treated animals are shown in blue (group A in Supporting Table 2) or green (group B in Supporting Table 2). Note that only 2 of 8 K04-treated mice showed quantifiable HCV RNA at any time point during the study. HCV RNA data points below LOQ (4 3 104 copies/mL) were arbitrarily set at 20,000 copies/mL and data points below LOD (1 3 104 copies/mL) were set at 2,000 copies/mL to visualize them on the graph. (C) Population PK of K04. Serum concentration of K04 mAb was determined from pooled blood from all treated mice, except for the last time point before sacrifice (day 24) for the 3 sacrificed mice (#201, #202, and #203) and day 48 for the other 5 treated mice #204-#208). Starting from day 36, K04 serum concentrations were below LOQ (1 ng/mL). (D) mean serum concentrations of HSA. (E) Mouse mean body weights.

infected PXB mice for 3 weeks, starting 6 hours after infection, may have resulted in prevention of infection in 1 mouse and cure of infection in 2 mice, monitored up to 48 days postinfection.

Discussion With an aging HCV-infected population, the number of patients with end-stage liver disease and need of transplantation is estimated to increase worldwide over the coming years. Reinfection of grafted liver in LT recipients occurs with high frequency and results in

poor clinical outcomes.5,6,27,28 Selective and efficacious prophylactic treatment options could improve treatment options in this population, but the discovery of neutralizing Abs targeting the viral envelope proteins has been hampered by the difficulty to achieve broad-spectrum protection against highly heterogeneous HCV quasi-species.29-31 Abs targeting host HCV receptors, such as CD81, have potential for intrinsic broad-spectrum antiviral activity. We have demonstrated here that several selective anti-CD81 mAbs were nearly equally potent in inhibiting HCV entry across HCV genotypes 1a, 1b, 2, and 4.

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The anti-CD81 mAb, JS81, has previously been shown to enable protection of humanized mice from HCV infection.15 However, only a small number of mice (n 5 3) were used in that study at the effective dose of 400 mg (approximately 20 mg/kg). In this article, we report that a different anti-CD81 mAb, K04, could completely block HCV infection in 100% (5 of 5) of mice. The HCV dose used for inoculation was sufficient to achieve infection and high serum viral load in 100% of untreated mice. Full protection from infection was achieved with 3 administered doses of K04 Ab that maintained Ab levels at >100-fold mean IC90 of HCV entry inhibition in PHHs for 7 days. These results demonstrate that CD81 is essential for HCV infection in vivo. The experiment was performed at high exposure of protective antibody. Further studies will be needed to determine the minimal exposure of Ab needed to provide full protection from infection. At the high dose of Ab used in this experiment, treatmentassociated transient reductions in body weight and HSA levels were observed. Separate toxicology studies are necessary to evaluate the safety of the anti-CD81 mAbs before further development. It has been reported, from in vitro studies, that HCV may also be able to infect neighboring hepatocytes by means of cell-to-cell transmission, albeit in a lessefficient manner. It is still unclear whether cell-to-cell transmission is biologically relevant in vivo. The role of CD81 in this process also remains unclear. It was initially suggested that CD81 may not be required for HCV cell-to-cell transmission in vitro, based on the inability of neutralizing Abs targeting the HCV E2CD81 interaction and an anti-CD81 mAb, M38, to inhibit this process.18,19 In contrast, it was recently shown that HCV cell-to-cell transmission was resistant to neutralizing Abs, but sensitive to inhibition by antiCD81 mAbs.20,21 Meuleman et al. reported that antiCD81 mAb JS81 failed to block HCV spread in the chimeric human liver mouse model when dosing started after infection.15,32 In the current study, we demonstrate that when anti-CD81 mAb K04 was administered 6 hours after HCV inoculation, followed by repeated dosing up to 21 days, all 8 treated mice showed either undetectable (1 mouse), unquantifiable (6 mice), or low (1 mouse) serum HCV RNA during the 3-week treatment period. Among 5 mice that were followed until day 48 (27 days after end of treatment), 2 increased viral load to untreated levels by 15-21 days after the end of treatment, consistent with the time required for clearance of the K04 Ab from serum. The other 3 mice in this group did not experience viral load rebound. These results demonstrate that an anti-CD81 Ab can

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be highly efficacious in a postexposure treatment setting and that CD81 is essential for HCV spread in vivo. The outcome of our treatment study is different to observations reported with a different anti-CD81 Ab,15,32 which may be explained by differences in Abbinding epitopes, binding affinity, PK, or dosing frequency and duration. K04 Ab showed 5- to 7-fold improved intrinsic binding affinity to CD81 ECL2, as compared to JS81 Ab, as determined by SPR, which correlated with a reduced dissociation rate. In addition, K04 was dosed at 60 mg/kg, whereas JS81 had been dosed at approximately 3-fold lower dose. K04 and JS81 also showed differences in binding to cells expressing CD81 with different point mutations in the ECL2, suggesting differences in binding epitope (Supporting Fig. 1C). We dosed the K04 Ab at a concentration and frequency to maintain serum exposure above 100-fold of the concentration needed to inhibit HCVpp entry into PHHs in vitro by 90% (IC90) for 24 days. The assessment of lower doses will be informative as part of the preclinical evaluation of this Ab. It is noteworthy that of the 5 anti-CD81 mAb-treated mice that were followed up to 48 days, 2 cleared the virus after day 24 or 30, and 1 never developed detectable viremia, suggesting that postexposure treatment of HCV infection with an anti-CD81 Ab could lead to HCV clearance. In summary, we have generated high-affinity antiCD81 mAbs with different variable region sequences and CD81-binding epitopes that show potent, broadspectrum antiviral activity in various in vitro assays. The anti-CD81 mAb, K04, could completely block HCV infection and spread in vivo, indicating that CD81 is essential for infection and spread of HCV in the liver. Based on the ability of K04 to clear HCV infection in a postexposure treatment setting, antiCD81 mAbs may become useful therapeutic agents for the treatment and prophylaxis of HCV infection. Acknowledgment: The authors thank PhoenixBio for conducting mouse studies and their Roche colleagues, Johannes Auer, Marianna Dioszegi, Ford Kirschenbaum, Junjun Gao, Priya Sriraman, Sophie Le Pogam, Alan Kosaka, Jun Zhang, Lena Liang, Charles Belunis, and Daniel Chin, for scientific discussions and technical assistance. The authors thank Prof. Stanley Lemon for providing the H77S sequence variant of HCV for use in the HCVcc cell-culture system.

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Author names in bold designate shared co-first authorship.

Supporting Information Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27603/suppinfo.