key: cord-0257293-z3zr2meq authors: Loo, Y.-M.; McTamney, P. M.; Arends, R. H.; Gasser, R. A.; Abram, M. E.; Aksyuk, A.; Diallo, S.; Flores, D. J.; Kelly, E. J.; Ren, K.; Roque, R.; Rosenthal, K.; Streicher, K.; Tuffy, K. M.; Bond, N. J.; Cornwell, O.; Bouquet, J.; Cheng, L. I.; Dunyak, J.; Huang, Y.; Rosenbaum, A. I.; Andersen, H.; Carnahan, R. H.; Crowe, J. E.; Kuehne, A. I.; Herbert, A. S.; Dye, J. M.; Bright, H.; Kallewaard, N. L.; Pangalos, M. N.; Esser, M. T. title: AZD7442 demonstrates prophylactic and therapeutic efficacy in non-human primates and extended half-life in humans date: 2021-08-31 journal: nan DOI: 10.1101/2021.08.30.21262666 sha: 1e33fe44901b65a13683662467f8ee50d432b27c doc_id: 257293 cord_uid: z3zr2meq Despite the success of SARS-CoV-2 vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of COVID-19. Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19, and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct non-overlapping epitopes on the spike protein receptor binding domain to potently neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and abrogate effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry. Together, these two mAbs create a higher barrier to viral escape and a wider breadth of coverage, neutralizing all known SARS-CoV-2 variants of concern. In a non-human primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, while therapeutic administration accelerated virus clearance from lung. In an ongoing Phase I study in healthy participants (NCT04507256), 300 mg intramuscular AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers >10-fold above those of convalescent sera for >=3 months, which remained 3-fold above those of convalescent sera 9 months post-AZD7442 administration. Approximately 1-2% of serum AZD7442 levels were detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentrations suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19. The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to cause substantial morbidity and mortality world-wide. While roll out of effective COVID-19 vaccines has reduced hospitalizations and death in several countries (1, 2), SARS-CoV-2 infection continues to spread globally, as variants with increased transmissability and immune evasion continue to emerge. The need for new therapies to protect individuals who remain at risk of COVID-19 persists, which includes unvaccinated individuals, individuals who are unable to mount an adequate immune response following vaccination (3) (4) (5) (6) (7) , and individuals with breakthrough infections despite full vaccination (8). SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) represent an approach for both the prevention and/or treatment of 10) . The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 mediates attachment to human angiotensinconverting enzyme 2 (ACE2) receptor resulting in viral entry into host cells (10). Many individuals with SARS-CoV-2 infection develop neutralizing antibodies to the spike protein (11, 12) , which correlate with protection against symptomatic infection (13). Antibodies targeting the spike protein have also been shown to limit the progression of SARS-CoV-2 infection and the development of COVID-19 (14) (15) (16) . (19) . Their progenitor mAbs were shown to potently and synergistically neutralize SARS-CoV-2 in vitro and confer protection in animal models of SARS-CoV-2 infection when co-administered (19) , supporting further development of this combination. The variable regions of the progenitor mAbs were reformatted as immunoglobulin 1 kappa (IgG1) with amino acid substitutions in the fragment crystallizable (Fc) regions to extend their serum half-lives (20, 21) and reduce Fc gamma receptor (FcγR) and complement binding, to create AZD8895 and AZD0161 (22) . In this study we describe the preclinical and translational characteristics of AZD8895, AZD1061, and AZD7442, and evaluate the potential of AZD7442 to both prevent and treat SARS-CoV-2 infection in non-human primate (NHP) models. Additionally, we characterize AZD7442 pharmacokinetics and transudation to the nasal mucosae in healthy adult participants enrolled in a Phase 1 clinical study . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 .08.30.21262666 doi: medRxiv preprint 26 August 2021 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. rhesus macaques given 4 and 40 mg/kg AZD7442, respectively (Fig. S5A-B) ; median SARS-CoV-2 plaque reduction neutralization test (PRNT50) titers of 3.4 and 4.6 log10 were measured for cynomolgus macaques administered 4 and 40 mg/kg AZD7442, respectively (Fig. S5C-D) . Low neutralizing titers were detected in the isotype controltreated rhesus macaques beginning Day 10 post-infection consistent with the development of an adaptive humoral immune response ( Fig. S5A-B) . Rhesus macaques were challenged on Day 0 with 10 5 plaque forming units (PFU) of SARS-CoV-2 strain USA-WA1/2020 (Fig. 3A) . SARS-CoV-2 viral subgenomic messenger RNA (sgmRNA) was measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in bronchoalveolar lavage (BAL) and nasal swab samples up to 14 days after virus challenge. In rhesus macaques treated prophylactically with isotype control, peak levels of viral sgmRNA were detected on Day 2 post-infection, with means of 4.67 log10 copies/mL (BAL) and 4.73 log10 copies/swab (nasal swab) of sgmRNA detected (Fig. 3B-C) . In contrast, sgmRNA was undetectable in the BAL samples of rhesus macaques receiving AZD7442 or AZD7442-YTE ( Figure 3B ); low levels of sgmRNA were detected transiently (Day 2 only) in nasal swab samples from two of four rhesus macaques receiving 4 mg/kg AZD7442 (3.32-3.60 log10 copies/swab) and from one of four that received 4 mg/kg AZD7442-YTE (4.95 log10 copies/swab) (Fig. 3C) . The undetectable levels of sgmRNA in the BAL samples from rhesus macaques that received as little as 4 mg/kg AZD7442 (comparable to the (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 .08.30.21262666 doi: medRxiv preprint 26 August 2021 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. human 300 mg dose) indicates that prophylactic administration of AZD7442 can protect against SARS-CoV-2 lower respiratory tract infection in rhesus macaques. The lack of Fc effector function and complement binding did not affect efficacy in this model as the non-TM (AZ7442-YTE) and the TM-modified AZD7442 showed similar in vivo efficacies ( Fig.3B and 3C) . Rhesus macaques treated with AZD7442 24 hours after inoculation showed a reduction in viral sgmRNA 24 hours later (day 2) in BAL ( Fig. 3D) but not in nasal swabs as compared to isotype control-treated NHPs (Fig. 3E) . AZD7442 administration resulted in accelerated viral clearance as detected in both BAL and nasal swabs within 4 and 7 days post-infection, respectively. In comparison, NHPs that received the isotype mAb did not fully clear the virus until Day 10 post-infection (Fig. In the second study, cynomolgus macaques were challenged on Day 0 with 10 5 tissue culture infection dose (TCID50) of SARS-CoV-2 strain USA-WA1/2020 (Fig. 4A) . SARS-CoV-2 burden was measured in BAL and nasal swab samples up to 5 days after virus challenge. Prophylactic AZD7442 administration demonstrated dose-dependent reduction of infectious virus titers in BAL samples (Fig. 4B) , and reduction in viral sgmRNA concentrations in BAL and nasal swab samples ( Fig. S4A and S4B) compared with isotype control antibody. Importantly, the 4 mg/kg dose (comparable to the human 300 mg dose) was fully protective in both NHP studies. Also consistent with observations from the rhesus macaque study, faster virus clearance was observed in T h i s d o c u m e n t h a s n o t b e e n p e e r r e v i e w e d All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 .08.30.21262666 doi: medRxiv preprint 26 August 2021 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. cynomolgus macaques administered 40 mg/kg AZD7442 therapeutically compared with isotype control ( Figure 4C ). AZD7442 treatment reduced viral titer similarly to AZD7442-YTE (no TM modification) at an equivalent dose, suggesting that Fc effector function and/or complement binding are not required for treating SARS-CoV-2 infection. Lung sections from control cynomolgus macaques showed histologic changes consistent with SARS-CoV-2 infection (Fig. 4D) . In sections of lung parenchyma, there was mild to marked perivascular cuffing of medium-caliber blood vessels by lymphocytes, alveolar wall thickening, and mixed inflammatory cell infiltrates comprising lymphocytes, neutrophils, and macrophages within alveolar spaces. There was mild to marked bronchial/bronchiolar inflammation with loss and/or blunting of bronchial/bronchiolar epithelium and infiltrates of lymphocytes and neutrophils within the lamina propria and submucosa. Although there were some variability between sections consistent with focal SARS-CoV-2 infection, lung sections from animals administered AZD7442 either prophylactically or therapeutically showed an overall reduction in inflammation compared with those treated with isotype control. The 4 mg/kg prophylaxis group demonstrated minimal to mild histological changes in the lung parenchyma and bronchial epithelium compared with all other AZD7442 doses tested in both prophylaxis and treatment settings in NHPs. Pathology scores were similarly reduced for animals that received 40 mg/kg AZD7442 or AZD7442-YTE as treatment (Fig. 4E) (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 .08.30.21262666 doi: medRxiv preprint 26 August 2021 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. that AZD7442 with TM modification provides equivalent protection against SARS-CoV-2-induced lung injury to AZD7442 without TM modification. Taken together, these two NHP studies indicate that AZD7442 administration prophylactically protects NHPs against SARS-CoV-2 infection, and therapeutically accelerates viral clearance. Importantly, AZD7442 reduced pulmonary inflammation and protected animals against alveolar damage associated with SARS-CoV-2 infection, suggesting AZD7442 can provide clinical benefit in both prevention and treatment settings. The pharmacokinetic characteristics and transudation of AZD7442 to the nasal mucosae were evaluated in a randomized, double-blind, placebo-controlled, Phase I study of 60 healthy adults (NCT04507256). This study is ongoing and the data presented herein are preliminary. Full results will be published in due course. Participants in the active arm received 300 mg of IM AZD7442 (150 mg of each mAb administered sequentially), IV AZD7442 at doses of 300 mg, 1,000 mg or 3,000 mg (150 mg, 500 mg or 1,500 mg, respectively of each mAb administered sequentially), or 3,000 mg AZD7442 co-administered by IV (1,500 mg AZD8895 plus 1,500 mg AZD1061); all groups n=10. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021 Serum concentrations of AZD8895 and AZD1061 were measured up to 9 months postdose and confirmed the extended t1/2 of approximately 90 days for both antibodies in each dose cohort following either IV or IM administration (Fig. 5A) . After a single 300 mg IM dose, the geometric mean Cmax of AZD8895 (16.5 g/mL) and AZD1061 (15.3 g/mL) were similar and reached at a median Tmax of 14 days. SARS-CoV-2 neutralizing antibody titers in sera conferred by AZD7442 were considerably higher than titers associated with convalescent plasma (Fig. 5B) . Geometric mean neutralization titers (GMT) afforded by AZD7442 300 mg IM and IV were ~22 and 41-fold higher, respectively, 7 days after dosing than those of convalescent sera. At Day 270 post dose, GMT following AZD7442 administration remained 3-fold higher than the GMT in convalescent plasma samples (n=28). For the 300 mg IM dose, median AZD8895 and AZD10161 nasal lining fluid (NLF) concentrations were ~200 ng/mL through 30 days post-dose ( Figure 5C ). This correlates to 1-2% of AZD8895 and AZD1061 levels in the sera, and importantly, suggest high levels of neutralizing antibodies at a key site of SARS-CoV-2 infection. Extrapolating serum and nasal AZD7442 concentrations and anti-SARS-CoV-2 neutralizing antibody titers to 15 months post dose using an estimated t1/2 of ~90 days suggest that a 300 mg intramuscular dose of AZD7442 could provide protection against COVID-19 for up to 12 months ( Figure 5A ). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 .08.30.21262666 doi: medRxiv preprint 26 August 2021 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. Antibody combinations comprising mAbs targeting distinct epitopes on the SARS-CoV-2 spike protein provide a higher threshold against virus escape from neutralization than single antibodies (24) , as the virus would have to acquire mutations at both epitopes to escape neutralization. Structural analysis of the mAbs comprising AZD7442 (AZD8895 and AZD1061) showed that they simultaneously bind the RBD at distinct, nonoverlapping epitopes at the RBD-ACE2 interface (24) . These data reinforce prior analyses of the AZD8895 and AZD1061 progenitor antibodies, COV-2196 and COV-2130, which demonstrated that they neutralize SARS-CoV-2 by blocking binding to ACE2 (19, 25) . Furthermore, we show that a different Fc domain containing TM and YTE modifications did not affect binding to the RBD. Mutations in the spike protein have been shown to increase viral infectivity and transmissibility (26, 27) and/or threaten the effectiveness of vaccines and therapeutics by providing escape from neutralizing antibodies (28) (29) (30) (31) . Monoclonal antibody combinations that target distinct SARS-CoV-2 spike protein epitopes have demonstrated a higher threshold against virus escape than the individual mAbs (28, 32) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021 mutations that confer reduced susceptibility to AZD8895 or AZD1061 individually. Taken together, the data presented here and in other studies confirm that AZD7442 retains activity against all known VOCs, including the Alpha (24, (28) (29) (30) 33) , Beta (24, (29) (30) (31) 33) , Gamma (28, 30, 34) and importantly, Delta (24, 35) . In some virus infections, antibody-enhanced disease mediated by Fc engagement of immune cells and immune complex formation leading to enhanced immune activation has been proposed as a mechanism of exacerbation of immunopathology and inflammation (36). The TM modification in the Fc region was therefore introduced to reduce this potential risk by preventing FcγR and complement binding. This study confirmed that the introduction of the TM modification to AZD7442 abrogated Fc effector function without affecting neutralization activity. While Fc effector function has been shown to enhance viral clearance by some mAbs in mouse models of SARS-CoV-2 infection (37, 38), SARS-CoV-2 clearance was not affected by the TM substitution in the NHP studies of AZD7442 reported here. These findings support the continued clinical evaluation of this TM-modified mAb combination for the prevention and treatment of AZD7442 was also modified to include the half-life extending YTE substitutions (20, 21) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021 AZD1061 have higher affinities for human FcRn at low endosomal pH than antibodies without the YTE modification, which translated into extended half-lives in NHPs and humans, consistent with other studies of YTE-enhanced mAbs (20, 40, 41) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021 Pharmacokinetic predictions support that AZD7442 could provide up to 12 months of protection from COVID-19. AZD7442 therefore has the potential to provide almost immediate protection in unvaccinated individuals or bolster the immune response of individuals who respond sub-optimally to vaccination, such as those with hematologic malignancies (7) or solid organ transplant recipients (6) . Accelerated viral clearance and reduced immunopathology in NHPs support potential use of AZD7442 as treatment for (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Additional materials and methods are provided in the Supplementary Materials describing: hydrogen-deuterium exchange mass spectrometry; protein expression and purification; binding assays; virus neutralization assays; and Fc effector function analyses. The pharmacokinetic analyses were part of an 8-week GLP toxicity study conducted at Charles River Laboratories. Cynomolgus monkeys were received from Orient BioResource (Alice, Tx, USA) and were between 2.2 and 5.1 years-old; males weighing between 1.9 and 2.6 kg and females weighing between 2.2 and 2.5 kg at the time of dosing. Animals were randomized and assigned to groups using a computer-based procedure prior to transfer to study; males and females were randomized separately. AZD7442 was administered to cynomolgus monkeys by a IV infusion at 600 mg/kg (n = 10) or by a IM injection at 300 mg/kg (n = 4); control animals were administered vehicle alone at volumes equivalent to AZD7442 by a single IV infusion (n = 12) or a single IM injection (n = 4). For the IV administered animals, all toxicology/safety assessment endpoints were evaluated at Day 15 (n = 9 control animals and n = 6 AZD7442 animals) and Day 57 (n = 4 for control and AZD7442 animals) necropsy timepoints. The IM (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. animals had only general in-life procedures, observations and measurements collected. IV infusion was via a suitable peripheral vein using an infusion pump connected to a temporary indwelling catheter once for 15 minutes. AZD8895 and AZD1061 were administered via separate 15 minute infusions, for a total infusion time of 30 minutes. A 1 mL saline flush was administered following each 15 minute infusion. IM injections to the anterior thigh was performed on temporarily restrained but not sedated animals. AZD8895 and AZD1061 were administered separately via 2 injections, 1 injection per thigh. In-life measurements included post-dose and detailed clinical observations, injection site dermal scoring, body weights, food consumption, veterinary physical examinations, ophthalmic examinations, electrocardiography exams, neurologic examination, blood pressure and heart rate, respiratory rate and body temperature. Blood was collected by venipuncture at various times before AZD7442 administration and up to 8 weeks post dose to measure AZD8895 and AZD1061 serum concentations and ex vivo pharmacodynamic evaluations. At study termination, animals were euthanized by IV injection of a commercially available veterinary euthanasia solution, followed by exsanguination. Animal studies (IACUC protocol no. 20-035 and 21-018P) were approved by the (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. For the first NHP study, 14 rhesus macaques (Macaca mulatta) of Indian origin, aged 5-6 years and weighing between 3.6 and 7.3 kg, received prophylactic 10 mL IV infusions of the following mAbs 3 days prior to SARS-CoV-2 challenge: 40 mg/kg isotype control mAb R347-TM-YTE (Group 1; 2 male, 1 female); 40 mg/kg AZD7442 (Group 2; 2 male, 1 female); 4 mg/kg AZD7442 (Group 3; 2 male, 2 female); or 4 mg/kg AZD7442-YTE (Group 4; 3 male, 1 female). Four additional rhesus macaques weighing between 4.6 and 6.6 kg received a therapeutic 10 mL IV infusion of 40 mg/kg AZD7442 one day after SARS-CoV-2 challenge (Group 5; 2 male, 2 female). In the second NHP study, 18 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. measurements. BAL samples and nasal swabs were collected for virologic analyses. One rhesus macaque from each of Groups 3-5 was euthanized on Day 2 post-infection for histopathology analyses. Cynomolgus macaques from all groups were euthanized on Day 5 post-infection for histopathology analyses. The design for each study is summarized in Fig. 3A and Fig. 4A . All NHPs were challenged on Day 0 with 10 5 PFU (rhesus macaques) or 10 5 TCID50 (cynomolgus macaques) SARS-CoV-2 variant USA-WA1/2020. Rhesus macaques were challenged with an in-house generated stock, lot no. 022320-1100, expanded in Vero E6 cells from BEI Resources (Manassas, VA, USA), cat. no. NR-52281, lot no. 70033175 (45) . Cynomolgus macaques were challenged with a stock, obtained from BEI Resources, cat. no. NR-53872, lot 70040665, 6.9 × 10 4 TCID50 per mL in Vero E6 cells. Virus inoculum was prepared on the day of administration at BIOQUAL, Inc. by research associates. SARS-CoV-2 was administered to NHPs under ketamine sedation, with the virus inoculum split between intratracheal and intranasal routes. Blood was collected under anesthesia by femoral venipuncture into BD Vacutainer SST gel tubes. BAL samples were collected under anesthesia via rubber feeding tubes (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. flocked swabs (COPAN Diagnostics Inc., Carlsbad, CA, USA). RNA was extracted from BAL and nasal swab samples using a QIAcube HT (Qiagen, Germantown, MD, USA) and the Cador pathogen HT kit and eluted in nuclease-free water. RNA was reverse transcribed using superscript VILO (Thermo Fisher Scientific, Waltham, MA, USA) and tested in duplicate with QuantStudio 6 and 7 Flex RTPCR System (Thermo Fisher Scientific). Viral titers were calculated to give copies/mL using a viral RNA standard curve, using the primers and probes that target the SARS-CoV-2 E gene sub-genomic mRNA (sgmRNA). To generate a standard curve for the SARS-CoV-2 E gene sgmRNA assay, the SARS-CoV-2 E gene sgmRNA was cloned into a pcDNA3.1 expression plasmid. This insert was transcribed using the AmpliCap-Max-T7 High Yield Message Marker Kit (Cellscript, Madison, WI, USA) to obtain RNA (LOD 50 copies/mL). The concentrations of AZ7442, AZD7442-YTE, and control R347-TM-YTE in NHP serum samples were determined by ELISA for human IgG against a known standard curve. The absorbance at 450 nm was recorded using a VersaMax or Omega (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. to a non-linear, sigmoidal (4PL) curve using GraphPad Prism, and the ensuing curve used to calculate concentrations of human IgG in each serum sample. A pseudovirus neutralization assay (46) was used to quantify neutralizing antibody titers in rhesus macaque serum samples. Lungs were removed immediately after euthanasia. NHP lung tissues were fixed in 10% neutral-buffered formalin and submitted in 70% ethanol to the histology laboratory at AstraZeneca (Gaithersburg, MD, USA). Paraffin-embedded lung samples were sectioned at 4 μM and stained with hematoxylin and eosin for evaluation by a boardcertified veterinary pathologist. This is an ongoing Phase I, randomized, double-blind placebo-controlled, doseescalation study of the safety, tolerability, and pharmacokinetics of AZD7442 in healthy adult participants with no prior history of COVID-19 and no prior receipt of a COVID-19 vaccine (NCT04507256). Participants, investigators, clinical staff, and the study monitor were all blinded from the assigned intervention. The study was conducted in compliance (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. with the ethical principles originating in or derived from the Declaration of Helsinki and in compliance with the International Confederation on Harmonization Good Clinical Practice Guidelines. All participants provided written informed consent before entering the study. The study protocol and informed consent documentation were reviewed and approved by the study site institutional review board. Sixty healthy participants aged 18-55 years at a single site in the United Kingdom were randomized 5:1 to one of five dose cohorts to receive one of four AZD7442 doses (300 mg IM, 300 mg IV, 1,000 mg IV or 3,000 mg IV, each mAb administered sequentially at 50% of the total AZD7442 dose; 3,000 mg IV co-administration of AZD8895 and AZD1061 as a single mixed infusion) or placebo. To determine the pharmacokinetics of AZD7442, serum samples were collected and analyzed pre-dose (all cohorts), during infusion and after infusion (IV cohorts only), at 8 hours post-dose and 1, 3, 5, 7, 14, 30, 60, 90, 150, and (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 interim results of serum and NLF antibody concentrations, and neutralizing antibody titers are reported to confirm preclinical information of AZD7442. The overall results of the Phase 1 study will be reported separately. Qualified assays were used to measure the concentration of AZD7442 and urea in NLF eluent. NLF samples were collected by Nasosorption™ FX·i. To extract NLF, 300 µL of elution buffer (1.0 mg/mL BSA, 1% NP-40 in 1x PBS, pH = 7.4) was added to each synthetic absorptive matrix. Immediately upon elution, up to 60 μL of NLF eluant was used to measure the urea concentration with an enzymatic-colorimetric assay. Next, 180 μL of NLF eluant was subjected to immunocapture and enzymatic digestion treatments identical to the method described for serum sample analysis. The urea concentrations in NLF eluant and serum were used for normalization of AZD7442 concentrations in the NLF to correct for different extraction volumes. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 incubation with a monoclonal antibody targeting the viral nucleocapsid protein, followed by a secondary anti-human IgG peroxidase conjugate and KPL TrueBlue™ substrate. Images of all wells were acquired by a CTL ImmunoSpot analyzer, equipped with software to quantitate the nucleocapsid-positive cells (virus signal). The 80% neutralization titer (PRNT80) was calculated as described previously (47) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 .08.30.21262666 doi: medRxiv preprint 26 August 2021 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. reduction, alkylation, and trypsin digestion. After digestion, the extract was fortified with stable isotope labelled peptide internal standard working solution. The final extract was analyzed via ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) with positive electrospray. A linear, 1/concentration² weighted, least-squares regression algorithm was used to quantify unknown samples. Descriptive statistics were used to present data. GraphPad Prism software (versions 8.4.3 or higher) was used for data analysis and graph production. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 .08.30.21262666 doi: medRxiv preprint 26 August 2021 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. Figure S1 . Hydrogen-deuterium exchange confirms that AZD8895 and AZD1061 bind distinct, non-overlapping epitopes on the SARS-CoV-2 spike protein RBD Figure S2 . In vitro assays confirm that AZD8895, AZD1061 and AZD7442 demonstrate reduced Fc effector function consistent with mAbs with TM substitutions (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2021. ; Data shown in solid circles have been published previously (24, 28, 29, 31, (33) (34) (35) . Data T h i s A. AZD8895 and AZD1061 binding affinities for human FcRn, measured by SPR with mAbs immobilized and titrated binding of huFcRn at pH 6.0. B. AZD8895 and AZD1061 exhibited extended in vivo serum half-lives in NHPs. Data shown are mean ± SD C. AZD8895 and AZD1061 exhibited reduced binding to FcγR and C1q at physiological serum mAb concentrations. Isotype control = R347-WT, an antibody to HIV glycoprotein gp120 with no TM or YTE modifications in the Fc. Binding response = mAb binding response / ligand binding to probe (background subtraction). A. Timeline of the in vivo SARS-CoV-2 challenge study. Six-year-old rhesus macaques weighing ~3.6-7.3 kg in prophylaxis groups 1, 2, 3, and 4 (n=3 for Groups 1 and 2; n=4 for Groups 3 and 4) received IV infusions of 40 mg/kg isotype control mAb R347-TM-YTE, 40 mg/kg AZD7442, 4 mg/kg AZD7442, or 4 mg/kg AZD7442-YTE, respectively, 3 days prior to challenge. Rhesus macaques in treatment group 5 (n=4) received an IV infusion of 40 mg/kg AZD7442 1 day after challenge. Rhesus macaques were challenged with 10 5 PFU of SARS-CoV-2, split between IT and IN delivery on Day 0. BAL and nasal swab samples were collected at days 0, 1, 2, 4, 7, 10, and 14. One rhesus macaque from each of Groups 3, 4, and 5 was euthanized on Day 2 for histopathology analyses (data not shown as minimal inflammation was observed on Day 2 rendering results inconclusive). B. Geometric mean ± SD viral burden in BAL samples from rhesus macaques receiving isotype control mAb, AZD7442 or AZD7442-YTE as prophylaxis 3 days prior to SARS CoV-2 challenge C. Geometric mean ± SD viral burden in nasal swab samples from rhesus macaques receiving isotype control mAb, AZD7442 or AZD7442-YTE as prophylaxis 3 days prior to SARS CoV-2 challenge D. Geometric mean ± SD viral burden in BAL samples from rhesus macaques receiving isotype control mAb, challenge. Cynomolgus macaques in treatment Group 5 (n=3) received an IV infusion of 40 mg/kg AZD7442 1 day after challenge. Cynomolgus macaques were challenged with 10 5 TCID50 of SARS-CoV-2, split between intratracheal and intranasal delivery on Day 0. BAL and nasal swab samples were collected at days 0, 1, 2, 4, 7, 10, and 14. Serum was collected on days -3, 0, 1, 2, 4, 7, 10 and 14. One cynomolgus macaque from each of Groups 3, 4 , and 5 was euthanized on Day 2 for histopathology analyses. B. Mean ± SD viral burden in BAL samples from cynomolgus macaques receiving isotype control mAb or AZD7442 as prophylaxis 3 days prior to SARS CoV-2 challenge. C. Mean ± SD viral burden in BAL samples from control cynomolgus macaques receiving isotype control mAb, or cynomolgus macaques receiving AZD7442 or AZD7442-YTE as treatment 1 day after SARS CoV-2 infection. A. Symbols are observed mean ± SD serum concentrations of AZD7442 over 9 months following single IM or IV administration of AZD7442 in healthy participants, and lines represent the predicted mean with shaded area representing the 90% prediction interval up to 15 months. Geometric mean neutralizing antibody titers against SARS-CoV-2 over 9 months following single IM or IV doses in healthy adult participants; data represent geometric mean PRNT80 titer ± SD for placebo or AZD7442 and GMT ± 95% CI for convalescent plasma samples. Concentrations of AZD8895, AZD1061 and AZD7442 (AZD8895 + AZD1061) in the NLF following 300 mg IM or 3000 mg IV doses of AZD7442. Graph shows individual and median concentration ± 95% confidence interval. Effectiveness of the Pfizer-BioNTech and Oxford-AstraZeneca vaccines on covid-19 related symptoms, hospital admissions, and mortality in older adults in England: test negative case-control study Impact and effectiveness of mRNA BNT162b2 vaccine against SARS-CoV-2 infections and COVID-19 cases, hospitalisations, and deaths following a nationwide vaccination campaign in Israel: an observational study using national surveillance data Humoral response to the Pfizer BNT162b2 vaccine in patients undergoing maintenance hemodialysis vaccine BNT162b2 for patients with cancer: interim analysis of a prospective observational study Immunogenicity of a single dose of SARS-CoV-2 messenger RNA vaccine in solid organ transplant recipients Suboptimal response to COVID-19 mRNA vaccines in hematologic malignancies patients Effect of bamlanivimab as monotherapy or in combination with etesevimab on viral load in patients with mild to moderate COVID-19: a randomized clinical trial Early Covid-19 treatment with SARS-CoV-2 neutralizing antibody sotrovimab NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice REGN-COV2, a neutralizing antibody cocktail, in outpatients with Covid-19 Phase III double-blind, placebo-controlled study of AZD7442 for post-exposure prophylaxis of COVID-19 in adults Accessed 2021 Phase III double-blind, placebo-controlled study of AZD7442 for pre-exposure prophylaxis of COVID-19 in adult (PROVENT). Accessed Potently neutralizing and protective human antibodies against SARS-CoV-2 A novel investigational Fc-modified humanized monoclonal antibody, motavizumab-YTE, has an extended half-life in healthy adults Safety, tolerability, and pharmacokinetics of MEDI4893, an investigational, extended-half-life, antistaphylococcus aureus alpha-toxin human monoclonal antibody, in healthy adults Structural characterization of a human Fc fragment engineered for lack of effector functions Antibody pharmacokinetics and pharmacodynamics Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serumderived polyclonal antibodies Antibody evasion by the P.1 strain of SARS-CoV-2 Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera Antibody cocktail to SARS-CoV-2 spike protein Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 Increased resistance of SARS-CoV-2 variant P.1 to antibody neutralization The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2 SARS-CoV-2 infection protects against rechallenge in rhesus macaques Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques Development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Comparing hydrogen deuterium exchange and fast photochemical oxidation of proteins: a structural characterisation of wild-type and DeltaN6 beta2-microglobulin Safety, tolerability and pharmacokinetics of MEDI8897, an extended half-life single-dose respiratory syncytial virus prefusion F-targeting monoclonal antibody administered as a single dose to healthy preterm infants Safety, tolerability, and pharmacokinetics of MEDI8897, the respiratory syncytial virus prefusion F-targeting monoclonal antibody with an extended half-life, in healthy adults COMBACTE Consortium, the SAATELLITE Study Group, Efficacy and safety of suvratoxumab for prevention of Staphylococcus aureus ventilator-associated pneumonia (SAATELLITE): a multicentre, randomised, double-blind, placebo-controlled, parallel-group, phase 2 pilot trial Overall survival with durvalumab after chemoradiotherapy in stage III NSCLC Type I interferon inhibitor anifrolumab in active systemic lupus erythematosus (TULIP-1): a randomised Anifrolumab in systemic lupus erythematosus Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis Neutralization of SARS-CoV-2 by destruction of the prefusion spike AIR Writing -review & editing: All Competing interests YML NLK were employees of AstraZeneca at the time of this study. NLK is currently an employee of and holds stock options of Eli Lilly The authors would like to thank all investigators and researchers involved in this study, including the investigators on the Phase 1 clinical study Dr Muna Albayaty This study was supported by AstraZeneca.