The SARS-CoV-2 monoclonal antibody combination, AZD7442, is protective in nonhuman primates and has an extended half-life in humans

Abstract

Despite the success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of coronavirus disease 2019 (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, nonoverlapping epitopes on the spike protein receptor binding domain to 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 reduce effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry, and neutralize all tested SARS-CoV-2 variants of concern. In a nonhuman primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, whereas therapeutic administration accelerated virus clearance from the lung. In an ongoing phase 1 study in healthy participants (NCT04507256), a 300-mg intramuscular injection of AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers greater than 10-fold above those of convalescent serum for at least 3 months, which remained threefold above those of convalescent serum at 9 months after AZD7442 administration. About 1 to 2% of serum AZD7442 was detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentration suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19.

Fig. 1.. Simultaneous binding of AZD1061 and AZD8895 (AD7442) to the SARS-CoV-2 RBD blocks binding to ACE2 and potently neutralizes SARS-CoV-2 variants of concern.

(A) AZD8895 and AZD1061 simultaneously bind to distinct, nonoverlapping epitopes on the spike protein RBD and sterically block RBD binding to ACE2. A side-view depiction shows cartoon representations of AZD8895 (blue) and AZD1061 (red) on top of the RBD (white) in surface representation based on co-crystal structures of AZD8895 and AZD1061 with RBD (24). (B) AZD8895, AZD1061, and AZD7442 binding kinetics on the SARS-CoV-2 S trimer (S2P ectodomain protein) are shown. Data shown are from a representative experiment from at least two independent assays (58). (C) AZD8895, AZD1061, and AZD7442 block binding of the RBD to ACE2. Measurements were taken across a series of mAb concentrations and the resulting nonlinear regression curves were used to calculate IC₅₀ values. Data shown were performed in duplicate and represent at least two independent assays. (D) AZD8895, AZD1061, and AZD7442 neutralize the USA-WA1/2020 strain of SARS-CoV-2 in vitro. Nonlinear regression dose-response curves from a representative experiment are shown, with mean and SD error from two or more technical replicates. Mean IC₅₀ values were calculated from three independent experiments. (E) AZD8895, AZD1061, and AZD7442 neutralize SARS-CoV-2 VOC in vitro. Data shown represent fold-change in neutralization potencies (IC₅₀) of AZD8895, AZD1061 and AZD7442 against the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) VOC as compared with the USA-WA1/2020 or AUS/VIC01/2020 reference strains. Data shown in solid circles have been published previously (, , , , –35). Data shown in open circles are from this study, performed at Public Health England (Alpha, Beta, Gamma, and Delta), United States Army Medical Research Institute of Infectious Diseases (Alpha and Beta), and Integrated Research Facility, National Institute of Allergy and Infectious Diseases (Alpha and Beta). hACE2, human angiotensin-converting enzyme 2; IC₅₀, half maximal inhibitory concentration; k_a, association rate constant; k_d, dissociation rate constant; K_D, equilibrium dissociation constant; mAb, monoclonal antibody; RBD, receptor binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SD, standard deviation; VOC, variant of concern.

Fig. 2.. AZD7442 shows extended half-life and reduced Fc effector function in vivo.

(A) AZD8895 and AZD1061 binding affinities for human FcRn are shown, 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). (D) AZD8895, AZD1061, and AZD7442 exhibited reduced Fc effector functions as compared with WT mAbs. Ratings were based on normalized AUC values with –, +, ++, and +++ assigned to values <25%, ≥25-50%, ≥50-75%, and ≥75% AUC, respectively. Negative control is an antibody to Ebola virus glycoprotein with no TM or YTE modifications in the Fc. Positive controls are two antibodies to SARS-CoV-2 spike protein RBD with no TM or YTE modifications in the Fc (Pos. Ctrl 1 and 2) and their combination (Pos. Ctrl 3). Ab, antibody; ADCC, antibody-dependent cellular cytotoxicity; ADCD, antibody-dependent complement deposition; ADCP, antibody-dependent cellular phagocytosis; ADEI, antibody-dependent enhancement of infection; ADNKA, antibody-dependent natural killer cell activation; AUC, area under the concentration curve; Fc, fragment crystallizable; FcγR, Fc gamma receptor; huFcRn, human neonatal Fc receptor; IM, intramuscular; IV, intravenous; K_D, equilibrium dissociation constant; mAb, monoclonal antibody; Neg Ctrl, negative control; Pos Ctrl, positive control; SD, standard deviation; SPR, surface plasmon resonance; TM, substitution L234F/L235E/P331S in the antibody Fc region; WT, wild-type antibody (no substitution in Fc region).

Fig. 3.. AZD7442 administration protects rhesus macaques against SARS-CoV-2 infection in prophylaxis or treatment settings.

(A) A timeline of the in vivo SARS-CoV-2 challenge study is shown. Six-year-old rhesus macaques weighing 3.6 to 7.3 kg were used in this study. Rhesus macaques in prophylaxis groups received IV infusions of 40 mg/kg isotype control mAb R347-TM-YTE (n = 3), 40 mg/kg AZD7442 (n = 3), 4 mg/kg AZD7442 (n = 4), or 4 mg/kg AZD7442-YTE (n = 4), respectively, 3 days prior to challenge. Rhesus macaques in treatment group (n = 4) received an IV infusion of 40 mg/kg AZD7442 1 day after challenge. Rhesus macaques were challenged with 10⁵ 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. (B) Geometric mean ± SD viral burden are shown 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 are shown 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 are shown in BAL samples from rhesus macaques receiving isotype control mAb, AZD7442 or AZD7442-YTE as treatment 1 day after SARS-CoV-2 infection. (E) Geometric mean ± SD viral burden are shown in nasal swab samples from rhesus macaques receiving isotype control mAb, AZD7442 or AZD7442-YTE as treatment 1 day after SARS-CoV-2 infection. In (D) and (E), the arrow indicates the day of dosing relative to challenge on day 0. BAL, bronchoalveolar lavage; D, day; IN, intranasal; IT, intratracheal; IV, intravenous; LOD, limit of detection; mAb, monoclonal antibody; PFU, plaque forming unit; RNA, ribonucleic acid; sgmRNA, subgenomic messenger RNA; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SD, standard deviation.

Fig. 4.. AZD7442 administration protects cynomolgus macaques against SARS-CoV-2 infection and associated lung immune pathology in prophylaxis and treatment.

(A) A timeline of the in vivo SARS-CoV-2 challenge study is shown. 6-year-old cynomolgus macaques weighing 3.2 to 6.3 kg were used in this study. Cynomolgus macaques in prophylaxis groups (all groups n = 3), received IV infusions of 40 mg/kg isotope 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. Cynomolgus macaques in treatment group (n = 3) received an IV infusion of 40 mg/kg AZD7442 1 day after challenge. Cynomolgus macaques were challenged with 10⁵ TCID₅₀ 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, and 5. Serum was collected on days -3, 0, 1, 2, 4, and 5. All animals were euthanized on day 5 post-infection for histopathology analyses. (B) Geometric mean ± SD viral burden are shown in BAL samples from cynomolgus macaques receiving isotype control mAb or AZD7442 as prophylaxis 3 days prior to SARS-CoV-2 challenge. (C) Geometric mean ± SD viral burden are shown 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. Arrow (↓) indicates day of dosing relative to challenge on day 0. (D) Lung histology following AZD7442 administration is shown for cynomolgus macaques receiving the indicated treatments. Hematoxylin/eosin-stained lung parenchyma and bronchus are shown from a representative animal (10x magnification). Inset shows 20x magnified image of bronchus. (E) A pathology score was assigned by board-certified veterinary pathologist based on histologic findings on eight lung sections per animal. Data are presented as mean + SD. The scores for one animal from the AZD7442 4 mg/kg prophylaxis dose group was excluded from analysis due to evidence of foreign material (plant matter) in multiple sections and observed inflammation inconsistent with SARS-CoV-2 infection. BAL, bronchoalveolar lavage; D, day; IN, intranasal; IT, intratracheal; IV, intravenous; LOD, limit of detection; mAb, monoclonal antibody; proph, prophylaxis; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SD, standard deviation; TCID₅₀, tissue culture infection dose; treat, treatment.

Fig. 5.. AZD7442 exhibits extended half-life, confers high anti-SARS-CoV-2 neutralizing antibody concentrations, and transudates to the mucosal epithelium in healthy adult participants.

(A) Serum concentrations of AZD7442 were measured over 9 months following single IM or IV administration of AZD7442 in healthy participants. Symbols are observed mean ± SD and lines represent the predicted mean with shaded area representing the 90% prediction interval up to 15 months. (B) Geometric mean neutralizing antibody titers against SARS-CoV-2 are shown 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 (n = 28). (C) Fold-difference or ratio of SARS-CoV-2 neutralizing antibody titers to convalescent antibody titers for AZD7442 and vaccinated individuals is shown; vaccine range and median based on published data for seven different vaccines (13). (D) Concentrations of AZD8895, AZD1061 and AZD7442 (AZD8895 + AZD1061) were measured in the nasal lining fluid following 300 mg IM or 3000 mg IV doses of AZD7442. Graph shows individual and median concentration ± 95% CI. CI, confidence interval; IM, intramuscular; IV, intravenous; PRNT₅₀, median plaque reduction neutralization titer; GMT, geometric mean neutralizing antibody titer; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SD, standard deviation.