Engineered anti-spike protein IgG3 antibodies

Abstract

The present invention relates generally to immunology and antibody engineering, in particular to engineered antibodies with improved properties and hinge engineering methods for improving properties, e.g., IgG1 antibodies. More specifically, the present invention provides engineered antibodies comprising an IgG3 subclass specific domain and a variable domain having a viral spike protein binding moiety.

Description

TECHNICAL FIELD

[0001] The present invention relates to the fields of immunology, vaccines, and pharmaceuticals. In particular, the present invention relates to antibodies, pharmaceutical compositions, antibody engineering, viral vaccines, and methods of making and using the same. BACKGROUND

[0002] SARS-CoV-2 has caused millions of deaths worldwide since its first appearance in 2019 1 . Monoclonal antibodies (mAbs) were successfully used as therapeutic drugs early in the pandemic 2 . These antibodies disrupted the ACE2 receptor-spike protein receptor binding domain (RBD) interaction, thereby neutralizing the virus and protecting the host 3 . Neutralizing antibodies against the SARS-CoV-2 spike protein constitute only a part of the antibody immune response 4 . It has been previously demonstrated that non-neutralizing opsonic mAbs are as good as potent neutralizing mAbs in protecting animals from SARS-CoV-2 infection 5 . These findings are consistent with other studies indicating that Fc effector functions are critical for viral control 6,7 . In fact, the protection provided by vaccines against mutated variants such as Omicron can be partially explained by the intact antibody Fc-effector functions against non-RBD sites or RBD sites that did not mutate 8 .

[0003] Antibody class and subclass play an important role in determining functional outcomes, especially in Fc and complement receptor-mediated phagocytosis. There are four subclasses of the human IgG class, defined by differences in the heavy chain constant region 9 . The constant region has historically been considered independent of the variable region and responsible only for antibody effector functions. However, increasing evidence of subclass-switched antibodies suggests that the constant region can influence affinity for target antigens 10 . Nonetheless, antibody subclass constant region studies have remained largely focused on affinity for Fc receptors and complement activation. IgG1 and IgG3 are the most potent activators of Fcy receptors on immune cells, likely because of their high affinity for these receptors 11 . IgG3 is also the most potent activator of the classical complement pathway, followed by IgG1, 2, and 4 12 ,13 . In the case of HIV-1, IgG3 has a stronger opsonic capacity and mediates stronger Fc-mediated phagocytosis of bead-bound gp140 antigen than IgG1, despite similar affinities of IgG3 and IgG1 anti-HIV antibodies for their target 14The functional differences are attributed to the longer hinge region on IgG3, which gives the Fc tail greater spatial flexibility. In a related study, replacing the IgG1 hinge with the IgG3 hinge enhanced intracellular immunity against adenovirus infection, supporting the flexibility hypothesis 15 . Thus, IgG subclass is a determinant in optimizing the immunological function of monoclonal antibodies against viral pathogens. It was recently shown that subclass switching to IgG3 can enhance neutralization of SARS-CoV-2 16 , and that the IgG3 backbone can facilitate ACE2-Fc protein effector functions 17 . Some aspects remain unclear, such as the effect of IgG subclass on antibody affinity for the spike protein, its effect on complement activation, its relationship to subsequent Fc-mediated immune functions, and how combinations of various anti-spike protein antibodies can influence Fc-mediated antibody functions.

[0004] mAb therapy against SARS-CoV-2 is based on the ability of neutralizing mAbs to bind the RBD, thereby disrupting the interaction of the spike protein with the ACE2 receptor. However, as a large number of mutations were found in the RBD of emerging variants of concern (VOC), most of these antibodies lost their binding, leading to loss of function and therapeutic failure 20,21 . This prompted the adoption of alternative strategies, either using combinations of neutralizing antibodies (nAbs) to treat these new variants 22 or searching for universal neutralizing antibodies 23 .

[0005] It was previously shown that ACE2-Fc constructs utilizing the IgG3 backbone have higher potency in vitro than their IgG1 counterparts 17 . In the non-SARS-CoV-2 context, IgG3 mAbs have been shown to efficiently mediate Fc-mediated HIV virus phagocytosis 14 . Similar benefits were observed for enhanced intracellular activity against adenovirus through the TRIM21 receptor 15 .

[0006] WO2020 / 254591 discloses an immunoreceptor comprising one or more IgGS intermediate hinge repeat domain motifs, wherein the immunoreceptor does not comprise IgGS CH2 and / or CH3 domains.

[0007] There are some biological limitations to the use of IgG3 as a drug in vivo. Among the four subclasses, IgG3 has the shortest half-life 9 , due to its lower affinity for the neonatal Fc receptor 9 . Through Fc engineering, its affinity for the neonatal Fc receptor can be improved without weakening Fc effector functions30, 31This change has been shown to be essential for in vivo protective effects in mouse models 30 .

[0008] There is a need for improved monoclonal antibodies for the treatment and prevention of viral infections, such as SARS-CoV-2 infections. There is in particular a need for more potent and capable of defending against more viral variants monoclonal antibodies and compositions containing such antibodies. SUMMARY

[0009] It is an object of the present invention to overcome certain deficiencies of antibodies and their use in the treatment or prevention of viral infections. The present inventors have designed novel antibodies (monoclonal antibodies, mAbs) which are engineered antibody formats prepared by immunization using viral spike proteins as antigens.

[0010] The present inventors have surprisingly and unexpectedly found that the conversion of the IgGl antibody subclass to IgG3 antibodies can enhance Fc- and complement receptor (CR)-mediated phagocytosis.

[0011] The present inventors have further surprisingly found that the engineering of IgGl antibodies by replacing the IgGl hinge with an IgG3 hinge can facilitate the generation of novel antibodies with improved properties. Thus, the alteration of the constant region of the antibody can significantly enhance the immune response against, for example, SARS-Cov-2 virus. Furthermore, it has been found that the repetition of a specific number of exons (e.g. 1, 2 or 3) after the core part of the IgG3 hinge influences the properties of the prepared IgG3 antibodies.

[0012] Furthermore, it has been surprisingly found that the combination of multiple antibodies in a mixture comprising several different antibodies can enhance the immune function of the antibodies. It has been found that oligoclonal mixtures of IgG3 subclass mAbs are even more effective in Fc- and CR-mediated phagocytosis than the best performing monoclonal antibodies.

[0013] Thus, antibody subclass engineering and mixture construction can modulate the efficacy of antibodies as antiviral infection therapeutics.

[0014] Based on the above findings, the present invention provides in a first aspect an engineered antibody comprising an IgG3 subclass specific domain and a variable domain with a viral spike protein binding moiety.

[0015] The present invention further provides a composition comprising at least two different engineered antibodies of the present invention.

[0016] The present invention further provides the use of an engineered antibody of the present invention for the prevention, treatment or alleviation of a viral infection caused by a virus comprising said spike protein.

[0017] The application also provides the use of the engineered antibody of the application for Fc-mediated phagocytosis of a virus comprising said Spike protein.

[0018] The application also provides the use of the composition of the application for the activation of the complement pathway.

[0019] The application also provides the use of the composition of the application for Fc-mediated phagocytosis of a virus comprising said Spike protein.

[0020] The application also provides a method of preparing an engineered antibody of the application comprising the steps of:

[0021] - immunizing a host organism with said viral Spike protein or a protein comprising said viral Spike protein,

[0022] - isolating at least one viral Spike protein-reactive B cell to isolate an IgGl antibody that binds to said viral Spike protein,

[0023] - preparing at least one recombinant DNA molecule encoding a modified version of said IgGl antibody comprising the replacement of the IgGl subclass-specific domain of said isolated IgGl antibody with an IgG3 subclass-specific domain,

[0024] - expressing said at least one recombinant DNA molecule in a suitable host cell,

[0025] to obtain said engineered antibody.

[0026] BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A-F. Conversion of IgGl constant region to IgG3 can change avidity to Spike protein. A: Schematic of heavy and light chain plasmids containing variable and constant regions. Generation of IgG3 mAbs requires conversion of the constant region of the heavy chain from IgGl (blue) to IgG3 (orange). B: Spike protein-coated microspheres were used as a model for SARS-CoV-2 virions. Antibody binding assays were done by opsonizing Spike protein beads with mAbs and adding an Alexa 488-conjugated secondary antibody that shows binding of IgG to Spike protein beads. C: Binding curves show the percentage of IgG-positive Spike protein beads as a function of IgG concentration. Each clone shows the presence of two subclasses (IgGl and IgG3). Three independent experiments were performed, the mean is shown in the graph, error bars represent SEM. D: Table summarizes the subclass of each clone K DValues and original subgroups. Affinity was calculated using a non-linear regression model in GraphPad Prism. E: Binding kinetics of intact spike protein (trimer) based on surface plasmon resonance. Binding of 40 nM spike protein (thin line) to immobilized IgG for clones 11, 36, 57 and their respective subgroups compared to 40 nM RBD (thick line) and PBS (lower curve). F: K D values and K A determinations. Statistical analysis was performed using one-way ANOVA and multiple comparisons with correction using Tukey. p values higher than 0.05 are indicated as ns, p values lower than 0.05 as *, p values lower than 0.01 as **, p values lower than 0.001 as *** and p values lower than 0.0001 as ****. G: ELISA data with spike protein coated wells and bound IgG for clones 11 and 57 and their respective subgroups are shown. Three independent experiments were performed using titration curves to plot binding curves. One representative experiment with 4 technical replicates is shown, the mean and error lines represent SEM.

[0028] Figure 2A-E. IgG3 antibodies outperform IgG1 antibodies in inducing Fc-mediated phagocytosis and are further enhanced when used as oligoclonal mixtures. A: The graph shows the three different phagocytic states a cell can be in and how they appear in flow cytometry gating: the first represents unbound cells, the second bound but not internalized cells, and the third cells with internalized beads. The gating on the right shows the double positive cell population representing cells with internalized beads. The far right graph shows the IgG isotype control. The Y axis shows the percentage of THP-1 cells with internalized beads. The experimental conditions used were the same as in B-C. B: Both IgG1 and IgG3 variants of each antibody clone mediate phagocytosis to different extents. The bar graph shows the percentage of cells with internalized beads. Beads were opsonized with 10 pg / ml of the respective mAb (3 million beads per 100,000 phagocytes, at a predator to prey ratio of 30 (MOP 30). C: Comparison of the average bead signal across the THP-1 cell population between IgG1 and IgG3 for each clone. D: This graph shows the comparison of the number of beads phagocytosed by the whole cell population by the monoclonal antibodies versus the mix. The table on the right shows the average of the median bead signal (fluorescence intensity) for each treatment. E: This graph shows the comparison of the percentage of THP-1 cells with internalized beads by the IgG1 and IgG3 monoclonal antibodies versus the mix. The table on the right summarizes the data from the average of the three experiments. In D-E, 1.5 million beads per 100,000 phagocytes (MOP 15) were opsonized with 5 pg / ml of the monoclonal antibodies or IgG mix. In B-C, statistical analysis was performed using the two-tailed Welch t-test. For D-E, statistical analysis was performed using one-way ANOVA and Tukey’s test for multiple comparisons correction. P values lower than 0.05 are represented by *, P values lower than 0.01 by **, and higher than 0.05 by ns. Three independent experiments were performed. The horizontal line represents the mean, and the error bars represent the SEM. All experiments were performed in triplicate.

[0029] Figure 3 A-F. IgG subclass and oligoclonality influence complement activation and phagocytosis of spike protein beads by neutrophils. A: Individual gating of C1q deposition for Ab81, Ab94, and OctomAb IgG3 at 10 pg / mL. The far right graph shows all three graphs combined. B: Graph showing C1q deposition data for the indicated monoclonal antibodies and oligoclonal mixtures. C: Graph showing the percentage of C1q deposition as a function of antibody concentration, including EC 50 values. D: Graph showing the fluorescence signal of C1q deposition as a function of antibody concentration, including EC 50Values. In C-D, IgG3 isotype control data is shown as a small panel in each corresponding graph. E: Comparison of different treatment methods and heat-inactivated serum (HI) versus complement-active serum (C+) with internalized spike protein beads on neutrophils. F: Bead signal of neutrophils associated with beads. E-F: Isotype controls are shown on the far right. Three independent experiments were performed for A-E. Statistical analysis was performed using one-way ANOVA for A-E, and multiple comparison correction using Tukey's test. * indicates p-value less than 0.05, ** indicates p-value less than 0.01, *** indicates p-value less than 0.001, ns indicates p-value greater than 0.05.

[0030] Figure 4 A-J. Real-time imaging of Fc-mediated protection in hACE2-K18 mice and phagocytosis by human neutrophils. A: Super-resolution structured illumination microscopy images showing phagocytosis of spike protein beads opsonized by the indicated treatments by human neutrophils. Microbeads (dye), cell surface (Alexa 594-WGA), and DNA (Hoechst) are visible in the images. PHrodo green was used as an internalization marker and is not shown in the images. Different antibody treatments resulted in different numbers of internalized beads and percentages of cells with internalized beads. SIM images are shown as maximum intensity projections in the Z-axis. Example images are from one of four experiments. Scale bar in the lower right corner is 5 pm. B: Mean bead number of neutrophils with internalized beads over time. Bar graphs in C show results after 60 minutes. D: Time course of phagocytosis is monitored and shown as neutrophils with internalized spike protein beads. Table below the graph shows ET 50Values, with 95% confidence intervals in parentheses. E: Humanized ACE2 mice were prophylactically injected intraperitoneally with the therapeutic antibody or vector. Eight hours after treatment, mice were intranasally infected with SARS-CoV-2 (original strain). Body weight was measured for ten days. F: Mean relative body weight of each group on days 0-7, with red dashed lines highlighting the time of death for all mice in the control group. G: Status (survival / euthanasia) of each mouse in each treatment group on day 6. H: Relative body weight of each mouse in each treatment group on day 6, with euthanized mice set to 0. I: Survival curves for each treatment group during the 10-day experiment. J: Terminal viral load titer in BAL fluid of euthanized mice was analyzed, regardless of the date of death. qPCR was performed on BAL fluid, and the cycle thresholds were recorded and plotted. Median values ​​are shown. Figures BD and FJ show the mean, with error bars at SEM. Statistical analysis was performed using one-way ANOVA and corrected for multiple comparisons using Tukey's test. * indicates a p-value less than 0.05, ** indicates less than 0.01, *** indicates less than 0.001, **** indicates less than 0.0001, and ns indicates a p-value greater than 0.05 that is not displayed. In BD, the data comes from four independent experiments by four different donors.

[0031] Figure 5 .Transformation from IgG1 to IgGh 47 The subclass of SARS-CoV-2 mAb exhibited strong opsonization capabilities. A: The figure shows SARS-CoV-2 virions carrying the spike protein trimer antigen, and IgGh formed from the original IgG1. 47 Three different clones, Ab11, Ab36, and Ab77. B: MOP curves for different mAbs (MOP in parentheses). 50 Statistically significant differences between the two mAbs (with 95% CI and no overlap) are highlighted in green (*). IgGh 47 The above shows the data points and curves. C: Phagocytosis score for different MOPs. All figures show Ab11 IgG1 (left) and Ab11 IgGh. 47 (Middle) and control IgG1 (left). DE shows binding and phagocytosis scores at MOP 30. Statistical comparisons were performed using the Mann-Whitney U two-tailed test to compare IgG1 and IgGh. 47 Version. The figure shows the average value, and the error bar is SEM. *** indicates p-value < 0.001, ** indicates p-value < 0.01, * indicates p < 0.05, and p-value > 0.05 is in ns. The positive control was DuomAb IgG3, and the negative control was Xolair IgG1. Three independent experiments were conducted in group BC (N=3), while four independent experiments were conducted in group D (N=4). Invention Details

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of immunology, biochemistry, genetics, and microbiology.

[0034] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0035] The practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology and recombinant DNA, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Harries & S.J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory).Kuby Immunology (2018, JPunt & S Stranford). Therapeutic Antibody Engineering (2012, 1st Ed), W Strohl & L Strohl.

[0036] The present application provides an engineered antibody comprising an IgG3 hinge and a variable domain having a viral spike protein binding portion.

[0037] The present application provides the following items, and is further characterized as the following items:

[0038] 1. An engineered antibody comprising an IgG3 hinge and a variable domain having a viral spike protein binding portion.

[0039] 2. The engineered antibody of item 1, wherein the Fc region is an IgG3 region.

[0040] 3. The engineered antibody of item 2, wherein the Fc region comprises an IgG3 heavy chain constant domain SEQ ID NO: 1 or a variant thereof having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 1.

[0041] 4. The engineered antibody of any one of items 1-2, comprising an IgG3 Fc region comprising one or more substitutions selected from R435H, N392K, and M397V.

[0042] 5. The engineered antibody of item 4, wherein the IgG3 region comprises the three substitutions R435H, N392K, and M397V.

[0043] 6. The engineered antibody of any one of items 3-5, wherein the IgG3 region comprises a polypeptide having at least 95% identity or at least 98% identity to the modified IgG3 heavy chain constant domain of SEQ ID NO: 2.

[0044] 7. The engineered antibody of any one of items 3-6, wherein the IgG3 region comprises the modified IgG3 heavy chain constant domain of SEQ ID NO: 2.

[0045] 8. The engineered antibody of item 1, wherein the Fc region is an IgG1 region.

[0046] 9. The engineered antibody of item 8, wherein the engineered antibody comprises an IgG1 constant domain SEQ ID NO: 3 engineered with an IgG3 hinge.

[0047] 10. The engineered antibody of any one of items 1-9, wherein the viral spike protein is from a coronavirus.

[0048] 11. The engineered antibody of any one of items 1-10, wherein the viral spike protein is a SARS-Cov-2 spike protein.

[0049] 12. The engineered antibody of any one of items 1-11, wherein the viral spike protein is selected from the group consisting of a wild-type spike protein of SARS-Cov-2 (SEQ ID NO: 4), a Delta (B1.617.2) spike protein of SARS-Cov-2 (SEQ ID NO: 5), and an Omicron (B1.1529) spike protein of SARS-Cov-2 (SEQ ID NO: 6).

[0050] 13. The engineered antibody of any one of items 1-12, wherein the viral spike protein is a wild-type spike protein of SARS-Cov-2 (SEQ ID NO: 4).

[0051] 14. The engineered antibody of any one of items 1-12, wherein the viral spike protein is a Delta (B1.617.2) spike protein of SARS-Cov-2 (SEQ ID NO: 5).

[0052] 15. The engineered antibody of any one of items 1-12, wherein the viral spike protein is an Omicron (B1.1529) spike protein of SARS-Cov-2 (SEQ ID NO: 6).

[0053] 16. The engineered antibody of any one of items 1-9, wherein the viral spike protein is from an orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, arenavirus, or retrovirus, such as from HIV, herpesvirus, rubella virus, or filovirus.

[0054] 17. The engineered antibody of any one of items 1-16, wherein the IgG3 hinge comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence ELKTPLGDTTHTCPRCP EPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSC DTPPPCPRCP (SEQ ID NO: 7).

[0055] 18. The engineered antibody of any of items 1-17, wherein the IgG3 hinge comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence X-(Y)n, wherein

[0056] X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8),

[0057] Y is EPSKCDTPPPCPRCP (SEQ ID NO: 9), and

[0058] n is an integer in the range of 2 to 8, preferably in the range of 2 to 5, more preferably in the range of 2 to 4.

[0059] 19. The engineered antibody of any of items 1-18, wherein the IgG3 hinge comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence X-Y, wherein

[0060] X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8),

[0061] Y is EPSKCDTPPPCPRCP (SEQ ID NO: 9).

[0062] 20. The engineered antibody of any of items 1-19, wherein the IgG3 hinge has the amino acid sequence X-(Y)m, wherein

[0063] X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8),

[0064] Y is EPSKCDTPPPCPRCP (SEQ ID NO: 9), and

[0065] m is an integer in the range of 1 to 8, preferably in the range of 1 to 5, more preferably in the range of 1 to 3.

[0066] 21. The engineered antibody of any of items 1-17, wherein the IgG3 hinge comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence X-(Y)n, wherein

[0067] X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8),

[0068] Y is EPKSCDTPPPCPRCP (SEQ ID NO: 26), and

[0069] n is an integer in the range of 2 to 8, preferably an integer in the range of 2 to 5, more preferably an integer in the range of 2 to 4.

[0070] 22. The engineered antibody of any one of items 1-17, wherein the IgG3 hinge comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence X-Y, wherein

[0071] X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8),

[0072] Y is EPKSCDTPPPCPRCP (SEQ ID NO: 26).

[0073] 23. The engineered antibody of any one of items 1-17, wherein the IgG3 hinge has the amino acid sequence X-(Y)m, wherein

[0074] X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8),

[0075] Y is EPKSCDTPPPCPRCP (SEQ ID NO: 26), and

[0076] m is an integer in the range of 1 to 8, preferably an integer in the range of 1 to 5, more preferably an integer in the range of 1 to 3.

[0077] 24. The engineered antibody of item 20 or 23, wherein m is 1.

[0078] 25. The engineered antibody of item 20 or 23, wherein m is 2.

[0079] 26. The engineered antibody of item 20 or 23, wherein m is 3.

[0080] 27. The engineered antibody of item 18 or 21, wherein n is 2.

[0081] 28. The engineered antibody of item 18 or 21, wherein n is 3.

[0082] 29. The engineered antibody of item 18 or 21, wherein n is 4.

[0083] 30. The engineered antibody of any one of items 1-29, which is an isolated antibody.

[0084] 31. The engineered antibody of any one of items 1-30, which promotes opsonization.

[0085] 32. The engineered antibody of any one of items 1-30, which does not bind to a neutralizing epitope.

[0086] 33. The engineered antibody of any one of items 1-30, wherein the engineered antibody is selected from the group consisting of Abll, Ab36, Ab57, Ab59, Ab66, Ab77, Ab81, and Ab94.

[0087] 34. The engineered antibody of any one of items 1-33, wherein the engineered antibody is selected from the group consisting of Abll, Ab36, Ab57, Ab59, Ab66, and Ab77.

[0088] 35. The engineered antibody of any one of items 1-34, wherein the heavy chain variable region is SEQ ID NO: 10 and the light chain variable region is SEQ ID NO: 11.

[0089] 36. The engineered antibody of any one of items 33-35, which is Abll.

[0090] 37. The engineered antibody of any one of items 1-34, wherein the heavy chain variable region is SEQ ID NO: 12 and the light chain variable region is SEQ ID NO: 13.

[0091] 38. The engineered antibody of any one of items 33, 34, or 37, which is Ab36.

[0092] 39. The engineered antibody of any one of items 1-34, wherein the heavy chain variable region is SEQ ID NO: 14 and the light chain variable region is SEQ ID NO: 15.

[0093] 40. The engineered antibody of item 33, 34, or 39, which is Ab57.

[0094] 41. The engineered antibody of any one of items 1-34, wherein the heavy chain variable region is SEQ ID NO: 16 and the light chain variable region is SEQ ID NO: 17.

[0095] 42. The engineered antibody of item 33, 34, or 41, which is Ab59.

[0096] 43. The engineered antibody of any one of items 1-34, wherein the heavy chain variable region is SEQ ID NO: 18 and the light chain variable region is SEQ ID NO: 19.

[0097] 44. The engineered antibody of item 33, 34, or 41, which is Ab66.

[0098] 45. The engineered antibody of any one of items 1-34, wherein the heavy chain variable region is SEQ ID NO: 20 and the light chain variable region is SEQ ID NO: 21.

[0099] 46. The engineered antibody of item 33 or 45, which is Ab77.

[0100] 47. The engineered antibody of any one of items 1-33, wherein the heavy chain variable region is SEQ ID NO: 22 and the light chain variable region is SEQ ID NO: 23.

[0101] 48. The engineered antibody of item 33 or 47, which is Ab81.

[0102] 49. The engineered antibody of any one of items 1-33, wherein the heavy chain variable region is SEQ ID NO: 24 and the light chain variable region is SEQ ID NO: 25.

[0103] 50. The engineered antibody of item 33 or 49, which is Ab94.

[0104] 51. A composition comprising at least two different engineered antibodies as defined in any one of items 1-50.

[0105] 52. The composition of item 51, comprising at least three, at least four, at least five, at least eight, or at least ten different engineered antibodies as defined in any one of items 1-50.

[0106] 53. The composition of any one of items 51-52, comprising 3 to 15, 3 to 12, 3 to 9, 5 to 15, 5 to 10, or 6 to 10 different engineered antibodies as defined in any one of items 1-50.

[0107] 54. The composition of any one of items 51-52, wherein the at least two, at least three, at least four, at least five, at least eight, or at least ten different engineered antibodies have different viral spike protein binding moieties.

[0108] 55. The composition of item 53, wherein the 3 to 15, 3 to 12, 3 to 9, 5 to 15, 5 to 10, or 6 to 10 different engineered antibodies have different viral spike protein binding moieties.

[0109] 56. The composition of any one of items 51-55, which is a pharmaceutical composition.

[0110] 57. The composition of any one of items 51-56, further comprising a pharmaceutically acceptable excipient.

[0111] 58. The composition according to any one of items 51-57, which is sterile.

[0112] 59. A nucleic acid molecule encoding a protein comprising an engineered antibody as defined in any one of items 1-50.

[0113] 60. The nucleic acid molecule according to item 59, encoding an engineered antibody as defined in any one of items 1-50.

[0114] 61. A cell comprising a nucleic acid as defined in item 59 or 60.

[0115] 62. Use of an engineered antibody as defined in any one of items 1-50 for preventing, treating or alleviating a viral infection caused by a virus comprising said spike protein.

[0116] 63. Use of an engineered antibody as defined in any one of items 1-50 for Fc-mediated phagocytosis of a virus comprising said spike protein.

[0117] 64. Use of an engineered antibody as defined in any one of items 1-50 for enhancing opsonization.

[0118] 65. Use of a composition as defined in any one of items 51-58 for preventing, treating or alleviating a viral infection caused by a virus comprising said spike protein.

[0119] 66. Use of a composition as defined in any one of items 51-58 for activating the complement pathway.

[0120] 67. Use of a composition as defined in any one of items 34-41 for Fc-mediated phagocytosis of a virus comprising said spike protein.

[0121] 68. Use according to any one of items 54-58, wherein said spike protein is from a coronavirus.

[0122] 69. Use according to any one of items 54-59, wherein said viral spike protein is a SARS-Cov-2 spike protein.

[0123] 70. Use according to any one of items 62-69, wherein said viral spike protein is selected from the group consisting of a wild type spike protein of SARS-Cov-2 (SEQ ID NO: 4), a Delta (B1.617.2) spike protein of SARS-Cov-2 (SEQ ID NO: 5) and an Omicron (B1.1529) spike protein of SARS-Cov-2 (SEQ ID NO: 6).

[0124] 71. The use according to any one of items 62-70, wherein the viral spike protein is the original spike protein of SARS-Cov-2 (SEQ ID NO: 4).

[0125] 72. The use according to any one of items 62-70, wherein the viral spike protein is the Delta (B1.617.2) spike protein of SARS-Cov-2 (SEQ ID NO: 5).

[0126] 73. The use according to any one of items 62-70, wherein the viral spike protein is the Omicron (B1.1529) spike protein of SARS-Cov-2 (SEQ ID NO: 6).

[0127] 74. The use according to any one of items 62-67, wherein the viral spike protein is from an orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, arenavirus or retrovirus, such as from HIV, herpesvirus, rubella virus or filovirus.

[0128] 75. A method of making an engineered antibody as defined in any one of items 1-50, comprising the steps of:

[0129] - immunizing a host organism with the viral spike protein or a protein comprising the viral spike protein,

[0130] - isolating at least one viral spike protein reactive B cell to isolate an IgGl antibody that binds to the viral spike protein,

[0131] - making at least one recombinant DNA molecule encoding a modified version of the IgGl antibody comprising replacing the IgGl subclass specific domain of the isolated IgGl antibody with an IgG3 subclass specific domain,

[0132] - expressing the at least one recombinant DNA molecule in a suitable host cell,

[0133] to obtain the engineered antibody.

[0134] 76. A method of making an engineered antibody as defined in any one of items 1-50, comprising the steps of:

[0135] - immunizing a host organism with the viral spike protein or a protein comprising the viral spike protein,

[0136] - isolating at least one viral spike protein reactive B cell to isolate an IgGl antibody that binds to the viral spike protein,

[0137] - preparing at least one recombinant DNA molecule encoding a modified version of said IgGl antibody, said modification comprising replacing the IgGl hinge of said isolated IgGl antibody with an IgG3 hinge,

[0138] - expressing said at least one recombinant DNA molecule in a suitable host cell,

[0139] to obtain said engineered antibody.

[0140] 77. The method according to item 76, wherein said IgG3 hinge comprises an amino acid sequence which is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 7).

[0141] 78. The method according to item 76, wherein said IgG3 hinge comprises an amino acid sequence which is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence X-(Y)n, wherein

[0142] X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8),

[0143] Y is EPSKCDTPPPCPRCP (SEQ ID NO: 9), and

[0144] n is an integer in the range of 2 to 8, preferably in the range of 2 to 5, more preferably in the range of 2 to 4.

[0145] 79. The method according to item 76, wherein said IgG3 hinge comprises an amino acid sequence which is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence X-(Y)n, wherein

[0146] X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8),

[0147] Y is EPKSCDTPPPCPRCP (SEQ ID NO: 26), and

[0148] n is an integer in the range of 2 to 8, preferably in the range of 2 to 5, more preferably in the range of 2 to 4.

[0149] 80. The method according to item 78 or 79, wherein n is 2, 3, or 4.

[0150] 81. The method of any of items 75-80, wherein the Spike protein is from a coronavirus.

[0151] 82. The method of any of items 75-81, wherein the viral Spike protein is a SARS-Cov-2 Spike protein.

[0152] 83. The method of any of items 75-82, wherein the viral Spike protein is selected from the group consisting of a Spike protein of SARS-Cov-2 (SEQ ID NO: 4), a Spike protein of SARS-Cov-2 Delta (B1.617.2) (SEQ ID NO: 5), and a Spike protein of SARS-Cov-2 Omicron (B1.1529) (SEQ ID NO: 6).

[0153] The compositions and formulations of the present application are prepared according to known standards for the preparation of pharmaceutical compositions and formulations. For example, the compositions and formulations are prepared in such a way that they are easy to store and to administer, for example by using pharmaceutically acceptable components, such as carriers, excipients or stabilizers. The amount of these pharmaceutically acceptable components should be non-toxic when the pharmaceutical composition or formulation is administered to a patient. The pharmaceutically acceptable components added to the pharmaceutical composition or formulation can depend on the chemical nature of the inhibitor and the targeting agent present in the composition or formulation (depending on whether the targeting agent is, for example, an antibody or a fragment thereof or a cell expressing a chimeric antigen receptor), the specific intended use of the pharmaceutical composition and the route of administration.

[0154] In a preferred embodiment of the present application, the composition or formulation is suitable for administration to a human, preferably the formulation is sterile and / or non-pyrogenic.

[0155] Definitions of some terms

[0156] Unless defined otherwise herein, the terms used in the present application shall be interpreted according to the general meaning known to a person skilled in the art.

[0157] The term "antibody" (e.g. monoclonal antibody) as described herein shall be interpreted according to the general meaning known to a person skilled in the art. The antibody can be a derivative antibody, or linked to a different molecule. For example, molecules that can be linked to the antibody are other proteins (e.g. other antibodies), molecular labels (e.g. fluorescent molecules, luminescent molecules, colored molecules or radioactive molecules), drugs and / or toxic agents. The antibody or antigen binding portion can be linked directly (e.g. in the form of a fusion of the two proteins) or via a linker molecule (e.g. any suitable type of chemical linker known in the art).

[0158] The term “isolated antibody” described herein is understood to mean an antibody that is recovered or purified to some extent. In one embodiment, the extent means that it is free from a substantial amount of antibodies of different structure, e.g., more than 25 antibodies. In another embodiment, the extent means that it is free from different antibodies that do not bind to the spike protein.

[0159] The term “engineered antibody” described herein is understood in accordance with the usual meaning known to the person skilled in the art. Engineered antibodies refer to novel recombinant antibody molecules that are produced, typically, by recombinant DNA and protein engineering techniques, with improved antigen specificity and effector functions.

[0160] The term “IgG3 hinge” used herein is understood to mean a hinge region from an antibody of the IgG3 class, including variants thereof. A non-limiting example of an IgG3 hinge is a peptide having an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 7). Further examples of an IgG3 hinge have an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to the amino acid sequence X-(Y)n, wherein X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8), Y is EPKSCDTPPPCPRCP (SEQ ID NO: 26), and n is an integer in the range of 2 to 8, preferably in the range of 2 to 5, more preferably in the range of 2 to 4.

[0161] The terms “IgGh 17 ”, “IgGh 32 ”, “IgGh 47 ”, “IgGh 62 ” used herein are understood to mean the core hinge (SEQ ID NO: 8) as an IgG3 hinge variant, and the variant has the core hinge (SEQ ID NO: 8) with one, two, or three extracellular repeats (all of SEQ ID NO: 9). Thus, the suffix indicates the number of amino acid residues in each hinge.

[0162] The term “viral spike protein” used herein refers to a spike-like protein located on the exterior of many viruses. Unlike membrane proteins and envelope proteins that are primarily involved in viral assembly, spike proteins play a crucial role in penetrating host cells and initiating the infection process.

[0163] The term "binds" in relation to an antibody as used herein refers to the formation of a complex with the molecule to be bound, e.g. the spike protein of SARS-CoV-2. Binding usually occurs in a non-covalent manner by intermolecular forces such as ionic, hydrogen, and van der Waals bonds and is usually reversible. A variety of methods and assays to determine binding capacity are known in the art. Binding is usually high affinity binding, wherein the affinity is determined with a KD value of preferably less than 1 mM, more preferably less than 100 nM, even more preferably less than 10 nM, even more preferably less than 1 nM, even more preferably less than 100 pM, even more preferably less than 10 pM, even more preferably less than 1 pM.

[0164] As used herein, each occurrence of the term such as "includes" or "comprising" can be optionally replaced with "consisting essentially of.

[0165] The terms "treatment", "prevention" or "relief" in the present invention refer to therapeutic treatment. Whether a therapeutic treatment is effective can be assessed, for example, by assessing whether the treatment is able to prevent or inhibit viral infection in a treated patient. Preferably, the prevention or inhibition is statistically significant, which can be assessed by appropriate statistical tests known in the art. Inhibition or prevention of viral infection can be assessed by comparing the degree of viral infection in a group of subjects infected or exposed to the virus but treated with the engineered antibody of the present invention to a control group of subjects infected or exposed to the virus but treated with a placebo. Alternatively, it can also be assessed by comparing a group of infected patients receiving an existing standard anti-viral treatment plus the treatment of the present invention to a control group of patients receiving only the existing standard anti-viral treatment. Such studies to assess the effect of inhibition or prevention of viral infection are designed according to accepted clinical study standards, e.g. double-blind randomized studies with sufficient statistical power.

[0166] The term "pharmaceutically acceptable" as used herein refers to approved by a federal or state governmental regulatory agency, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.

[0167] The sequences mentioned in the specification are as follows:

[0168] SEQ ID NO: 1 (IgG3 heavy chain constant domain)

[0169] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0170] SEQ ID NO: 2 (modified IgG3 heavy chain constant domain)

[0171] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHFTQKSLSLSPGK

[0172] SEQ ID NO: 3 (IgG1 constant domain, engineered with IgG3 hinge)

[0173] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0174] SEQ ID NO: 4 (original Sars-CoV-2 spike protein)

[0175]

[0176] SEQ ID NO: 5 (Delta (B1.617.2) spike protein Sars-CoV-2 variant)

[0177]

[0178] SEQ ID NO: 6 (Omicron (B1.1529) spike protein Sars-CoV-2 variant)

[0179]

[0180] SEQ ID NO: 7 (IgG3 hinge region)

[0181] ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

[0182] SEQ ID NO: 8 (IgG3 hinge portion)

[0183] ELKTPLGDTTHTCPRCP

[0184] SEQ ID NO: 9 (IgG3 hinge portion)

[0185] EPSKCDTPPPCPRCP

[0186] SEQ ID NO: 10 (clone 11 heavy chain variable domain)

[0187] QVTLKESGPVLVKPTETLTLTCTVSGFSLSNAKMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSSLTISKDTSKSQVVLTMTNMDPVDTATYYCARLLWFGGNYFDYWGQGTLVTVSS

[0188] SEQ ID NO: 11 (clone 11 light chain variable domain)

[0189] SYELTQPLSVSVALGQTARITCGGNNIGSKMHWYQQKPGQAPVLVIYRDSNRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSTVVFGGGTKLTVL

[0190] SEQ ID NO: 12 (clone 36 heavy chain variable domain)

[0191] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHAVHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDPVLRYFDWTTPYYFDYWGQGTLVTVSS

[0192] SEQ ID NO: 13 (clone 36 light chain variable domain)

[0193] EIVLTQSPGTLSLSPGERATLSCRASQSFSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPLTFGGGTKVEIK

[0194] SEQ ID NO: 14 (clone 57 heavy chain variable domain)

[0195] QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLARIDWDDDKYYSTSLRTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARMTVTTAFDIWGQGTMVTVSS

[0196] SEQ ID NO: 15 (clone 57 light chain variable domain)

[0197] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTKVEIK

[0198] SEQ ID NO: 16 (clone 59 heavy chain variable domain)

[0199] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTAYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARGGRYCSSTSCYSGVPNDYWGQGTLVTVSS

[0200] SEQ ID NO: 17 (clone 59 light chain variable domain)

[0201] DIQLTQSPSFLSASVGDRVTITCRASQGVSSYLAWYQQKPGKAPKLLIYPASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK

[0202] SEQ ID NO: 18 (clone 66 heavy chain variable domain)

[0203] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRSFVSSSTDDYYYYYD MDVWGQGTTVTVSS

[0204] SEQ ID NO: 19 (clone 66 light chain variable domain)

[0205] SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVLVIYKDSERPSGI PERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSGTYVVFAGGTKLTVL

[0206] SEQ ID NO: 20 (clone 77 heavy chain variable domain)

[0207] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVALISYDGS NTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVFYCAKTIYSYALKPNYFDY WGQGTLVTVSS

[0208] SEQ ID NO: 21 (clone 77 light chain variable domain)

[0209] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPPTFGGGTKVGIK

[0210] SEQ ID NO: 22 (clone 81 heavy chain variable domain)

[0211] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYN GNTNYAQKVQGRVTMTRDTSTSTAYMELRSLRSDDTAVYYCARDGIAVAEDWFDPW GQGTLVTVSS

[0212] SEQ ID NO: 23 (clone 81 light chain variable domain)

[0213] SYELTQPPSVSVAPGKTARITCGENNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHVVFGGGTKLTVL

[0214] SEQ ID NO: 24 (clone 94 heavy chain variable domain)

[0215] QVQLQESGPRLVKPSGTLSLTCAVSGGSISSSNWWTWVRQPPGKGLEWIGEIYHSGSTNYTPSLKSRVTISVDKSKNQFSLRLNSVTAADTAVYYCARGWSSSWYGLDYWGQGTLVTVSS

[0216] SEQ ID NO: 25 (clone 94 light chain variable domain)

[0217] QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLVIYEATKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCYSYAGSSTWVFGGGTKLTVL

[0218] SEQ ID NO: 26 (IgG3 hinge portion)

[0219] EPKSCDTPPPCPRCP Examples

[0220] Materials and Methods

[0221] Generation, production, and sequencing of IgG3 plasmids

[0222] The anti-spike protein monoclonal antibodies used in this study were made from spike protein reactive B cells from a convalescent patient infected with SARS-CoV-2 six weeks prior (who has been clinically validated). Briefly, V(D)J coding sequences of spike protein reactive B cells were generated using 10X Genomics. The variable regions were cloned into a pTwist IgGl vector (Twist Biosciences) containing an IgGl constant heavy chain domain. The plasmid containing the IgG3 heavy chain constant domain gene was previously generated (Izadi et al 2024, Nature Comm. accepted). The sequence of the recombinant vector containing the light and heavy chain plasmids with constant domains is found in Supplementary Material file 1. Primers were designed such that the insert (IgG3 heavy chain constant domain) contained overhangs complementary to the vector, such that homologous recombination could occur. This allowed the IgGl constant domain to be replaced with the IgG3 constant domain, while the variable region remained unchanged. The swapping of the heavy chain constant domain was achieved by performing PCR on the IgGl vector backbone and the IgG3 constant region, which would replace the IgGl specific domains. Subsequent HIFI-DNA assembly (NEB, biolAbs) would mediate the process of homologous recombination. PCR products were loaded onto an agarose gel, and the correct amplification was assessed by size. Bands were excised and purified by QIAGEN PCR purification kit according to the manufacturer’s instructions. The provided competent E. coli was transformed with the newly assembled IgG3 plasmid and plated on agar plates containing ampicillin. The IgG3 plasmid purified from the transformed colonies was sequenced to verify.

[0223] Cell culture and antibody production

[0224] Expi293F suspension cells were purchased from Gibco (ThermoFisher) and routinely cultured in 125 ml conical flasks (Nalgene) with 30 ml Expi293 medium (Gibco) in an Eppendorfs 41 i shaker incubator at 37°C, 8% CO2, 120 rpm. Cells were routinely passaged and split every 3 to 4 days to a density of 0.5 x 10 6 cells / ml. One day prior to transfection, cells were seeded at a density of 2 x 10 6 cells / ml. The following day, cells were transfected at a density of 7.5 x 10 7One cell was seeded in 25.5 ml Expi293 medium. Transfection of heavy and light chain plasmids was performed using the Expifectamine 293 kit (Gibco) according to the manufacturer’s instructions. Briefly, 20 pg of heavy and light chain plasmids were mixed with 2.8 ml OptiMEM (Gibco) and 100 mI Expifectamine, respectively, and incubated for 15 minutes at room temperature. The transfection mix was then added to the Expi293F cells. The next day, 1.5 ml Enhancer 1 and 0.15 ml Enhancer 2 (both from the Expifectamine 293 kit) were added and the cells were incubated for 72 hours.

[0225] Cells were removed from the cell culture medium by centrifugation (400 x g, 5 minutes) and the supernatant was transferred to a new tube. To capture IgG from the culture medium, Protein G Sepharose 4 Fast Flow (Cytiva) was added to the culture medium and incubated for 2 hours at room temperature under inversion. The beads were collected by gravity flow chromatography column (Biorad) and washed twice with 50 ml PBS. Antibodies were eluted using 5 ml Glycine-HCI (0.1 M, pH 2.7). The pH was neutralized using Tris (1 M, pH 8, 1 ml). The buffer was exchanged to PBS using Amicon Ultra-15 centrifugal filters (Sigma) with a molecular cut-off of 30,000 Da. The concentration and quality of the purified antibodies were spectrophotometrically measured using the IgG settings of a Nanodrop (Denovix), while the antibodies were subjected to SDS-PAGE and the band intensity was compared to serial dilutions of Xolair (Omalizumab).

[0226] Generation of Spike protein coated beads

[0227] Spike protein was generated by transfection of Expi293F cells with 40 pg of plasmid containing the Spike protein gene (using a CS / PP Spike protein encoding a secreted form of the protein for purification from cell culture supernatant), which was previously a kind gift from the lab of Dr. Florian Krammer 5 Biotinylation of 200 pg of Spike protein was performed according to the instructions of the EZ-Link™ Micro Sulfo-NHS-LC Biotinylation Kit (ThermoFisher). Then, 500 mI (5 x 10 7 Fluorescent (APC) Streptavidin Microspheres (1 pm, Bangs Laboratories) were conjugated to the biotinylated Spike protein (200 pg) according to the manufacturer’s instructions.

[0228] Affinity measurements based on flow cytometry

[0229] Binding assays were performed in 96-well plates that had been pre-coated with 200 μΐ of 2% BSA (in PBS) overnight at 4°C. 100,000 spike protein coated beads were used in all wells, with antibody concentrations variable between 0.02-0.2-2 μg / ml. 100 μΐ volumes of beads were incubated in IX PBS for 30 minutes at 37°C on a heated shaking module. Wells were washed twice with PBS. To assess antibody binding to spike protein beads, 50 μΐ (1 :500 dilution) of Fab-specific fluorescent secondary antibody (#109-546-097, Jackson ImmunoResearch) was used to generate a fluorescent signal. The secondary antibody was incubated with the spike protein-bead antibody complex for 30 minutes at 37°C on a heated shaking module. 100 μΐ of PBS was added to the wells prior to flow cytometry analysis. Beads were analyzed using a Beckman Coulter Cytoflex flow cytometer, with 20,000 beads acquired per sample. Data was processed using Flowjo. Gating for spike protein beads was based on forward scatter and side scatter settings. Gating for antibody positive spike protein beads was based on reactivity with non-reactive IgG control mAb. Data was subsequently analyzed using Graphpad Prism, with a non-linear regression model: Hill slope unconstrained, maximum (Bmax) set to 100%, Kd D constrained to be between 0-10000. The best fit values are displayed in the graph along with the model fit.

[0230] SPR kinetic assay

[0231] To study the binding kinetics to the Spike protein trimer, RBD or NTD, we immobilized high capacity amine sensor chips (Bruker) with 25 pg / ml anti-human IgG (Fc) antibody (Cytiva BR-1008-39) in 10 mM sodium acetate buffer pH 5 at a flow rate of 10 pl / min for 300 seconds. The operation was performed on a MASS-16 biosensor instrument (Bruker). The running buffer was PBS + 0.05% Tween20. The antibody was diluted in PBS and then injected to the surface at 10 pL / min for 90 seconds. The running buffer was PBS with 0.01% Tween 20. The RBD antigen used was produced in HEK293 cells obtained from Sinobiological (Beijing, China: RBD WT 40592-V08H). The Spike protein trimer was prepared as described above. The NTD antigen was obtained from Sinobiological (Beijing, China: NTD 40591-V9H and NTD-Omicron 40591-V08H42). The RBD antigen was serially diluted by two-fold, from 60 nM to 3.75 nM. The NTD antigen was serially diluted by two-fold, from 80 nM to 2.5 nM. The Spike protein trimer was added at 40 nM. The antigens were injected at these concentrations to allow them to interact with the sensor for 2 minutes at a flow rate of 30 pl / min, followed by a dissociation of 6 minutes. After each cycle, the surface was regenerated with 3 M MgCl. All RBD and NTD experiments were performed in triplicate, and the Spike protein trimer once. The data were analyzed using Sierra Analyser software version 3.4.3 (Bruker) program to determine the apparent dissociation constant (K D ).

[0232] ELISA affinity measurements

[0233] For ELISA, 1 pg / ml of Spike protein in PBS was immobilized on ELISA high binding plates (Sarstedt) overnight at 4°C. Wells were washed 3 times with PBST (1 x PBS with 0.05% Tween 20) and then blocked with 2% BSA / PBS for 1 hour at room temperature. After washing 3 times, a gradient dilution of primary antibody (anti-Spike protein mAb) was added (100 mI mAb at a concentration of 0.08 to 20 pg / ml) for 1 hour at room temperature. Wells were washed 3 times. Rabbit anti-human light chain-HRP secondary antibody (clone 57 is anti-kappa and clones 11 and 66 are anti-lambda) (Abeam ab202549 and ab200966) diluted 1 :5000 in PBS was added for 1 hour at room temperature. Wells were washed again 3 times with PBST. Finally, 100 mI of color reagent (20 ml sodium citrate pH 4.5 + 1 ml ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 0.2 g in 10 ml water, Sigma) + 0.4 ml 0.6% H2O2) was added. OD450 was recorded after 15-30 minutes. Data were plotted using GraphPad Prism.

[0234] C1q deposition assay

[0235] To investigate complement activation, 500,000 Spike protein beads were opsonized with 0.1-1-10 pg / ml of monoclonal antibodies and 1% antibody-depleted serum as a source of complement in 96-well plates previously coated with 2% BSA. After opsonization for 30 minutes at 37°C in a heating shaker module (300 RPM), wells were washed twice with PBS and then FITC fluorescently labeled anti-C1q antibody (ab4223, Abeam) was added (diluted 1 :250 from stock, 50 mI). Anti-C1q antibody was left with the beads for 30 minutes at 37°C in a heating shaker module (300 RPM). PBS was added to suspend the beads in a final volume of 150 mI. Gating of Spike protein beads was set according to forward and side scatter and gating of C1q deposition was set according to results with control IgG1 (supplemental Figure 5 A). Collected data were analyzed using Flowjo and plotted in GraphPad Prism. EC 50 Analysis was performed on GraphPad Prism using a non-linear regression model with bottom constraint of 0 and top value constraint of 100% for % beads-C1q deposition analysis. For MFI EC 50 analysis, top was set to shared and greater than 0.

[0236] Flow cytometry-based phagocytosis assay

[0237] Phagocytosis experiments were performed using the same batch of spike protein beads as in the binding assay. To assess internalization, red fluorescent beads were stained with the pH sensitive dye pHrodo green (P35369, ThermoFischer) for 30 minutes at 37°C with 5 pl of 10 mM pHrodo green (ThermoFischer) dissolved in 95 pl Na2CO3(pH 9). Excess dye was washed away with PBS. THP-1 cells (Sigma-Aldrich) were cultured as previously described 5 . All experiments used 1 x 10 5 THP-1 cells. In one set of experiments, the ratio of cells to beads was 1 :30 (predator- to-organism- ratio, MOP, 30) and in another set of experiments 1 :15 (MOP 15). For MOP 30 experiments, 3 x 10 6 THP-1 cells were opsonized with 10 pg / ml antibody in a volume of 100 pl of sodium medium (pH adjusted to 7.3 with NaOH; 5.6 mM glucose, 10.8 mM, 127 mM NaCl KCl, 2.4 mM KH2PO4, 1.6 mM MgSO4, 10 mM HEPES, 1.8 mM CaCl2). For MOP 15 experiments, 1.5 x 10 6 THP-1 cells were opsonized with 5 pg / ml mAb. Both opsonizations were performed for 30 minutes at 37°C on a heating shaker module (300 RPM) in a volume of 100 pl. During this incubation period, THP-1 cells were counted (using a Bürker chamber) and the medium was exchanged from RPMI to sodium medium. THP-1 cells (50 pl, 2 x 10 6 THP-1 cells were added to each well and allowed to phagocytose the beads for 30 minutes at 37°C on a heating shaker module (300 RPM). The 96-well plate was then incubated on ice for 15 minutes and analyzed directly in a Beckman Coulter Cytoflex flow cytometer. The gating of THP-1 cells was set according to forward and side scatter (Supplemental Figure 4 A). The gating of internalization and binding was set only on the negative control of cells Figure 3 A). The analysis was stopped after 5000 events were captured in the THP-1 gate. The data was analyzed in the Flowjo program by setting similar gates as described above. The Flowjo processed data was further analyzed in GraphPad Prism and the internalization and bead signal (APC-A) of the THP-1 gate were plotted to compare the different antibodies.

[0238] Mononuclear cells were isolated from blood by first obtaining the PBMC layer using polymorphoprep (Abbot). Mononuclear cells were purified from the PBMC layer by positive selection using CD14 microbeads (Cat# 130-050-201, Miltenyi Biotec) according to the manufacturer’s instructions. After isolation, mononuclear cells were counted using a XN-350 hematology analyzer (Sysmex). Neutrophils were isolated using polymorphoprep gradient according to the manufacturer’s instructions and counted using a Bürker chamber. Isolated cells were suspended in sodium medium and adjusted to 2x10 6 cells / ml and then placed on ice for 1 hour. Donors had given written and oral consent to participate in the study and were orally informed of the purpose of donation, which was only for isolation and use of mononuclear cells and neutrophils. The study used an ethical approval previously approved by the Swedish Ethical Review Authority (2020 / 01747).

[0239] In the mononuclear phagocytosis experiment, spike protein beads were opsonized with 10 pg / ml of monoclonal antibody (IgG control monoclonal antibody or OctomAb IgG3) but no serum was added. Mononuclear cells (1 x 10 5 cells) were added in a volume of 50 mI. Cells were placed on a heated shaking module (300 RPM) at 37°C for 30 minutes of phagocytosis. After this, cells were placed on ice and stained with a volume of 20 mI of CD14 antibody (Brilliant Violet 421 TM anti-human CD14 antibody, Biolegend) diluted 1 :50 to 3 pg / ml for 20 minutes. Wells were analyzed using a Beckman Coulter Cytoflex flow cytometer. Mononuclear cells were gated on size and granularity (Supplemental Figure 2 C). Further selection was performed by gating on CD14 positive cells. Analysis was stopped after 3000 events were recorded in the CD14+ gate. In Prism Graphpad, the percentage of cells binding spike protein beads (APC+) and the percentage of cells internalizing beads (APC+, FITC+) were plotted against MOP (Supplemental Figure 2 C).

[0240] For neutrophil experiments, Spike protein beads were opsonized with 10 pg / ml of monoclonal antibody and 1% antibody-depleted serum (Pel-Freeze, Cat# 34014-10) for 30 minutes at 37°C in a heat-shaking module (300 RPM) (final volume of 100 mΐ). To study only the effect of Fc-mediated phagocytosis, we heat-inactivated the serum at 56°C for 1 hour. Heat-inactivated serum and normal human serum were added to the respective wells. Neutrophils (1 x 10 5 6 / ml) were added to each well in a volume of 50 mΐ. Cells were phagocytosed for 30 minutes at 37°C in a heat-shaking module (300 RPM). The plate was placed on ice to stop the phagocytosis process. On ice, cells were stained with anti-CD18 antibody (BV421 mouse anti-human CD18, BD Biosciences) diluted 1 : 100 to 2 pg / ml, 20 mΐ of diluted antibody was added to the cells. Cells were stained on ice for 20 minutes and then analyzed in a Beckman Coulter Cytoflex flow cytometer. Gating was set for FSC-A and SSC-A to mark neutrophils (Supplemental Figure 2 D). This cell population was further selected by CD18 labeling (Supplemental Figure 6B). Analysis was stopped after 3000 events were captured in the CD18+ gate and data was processed in the Flowjo program using similar gating as described above. The percentage of cells with internalized beads and bead signal (APC) in the CD18 gate was used as an indicator to compare mAbs.

[0241] IgG mAb aggregation quality control experiments

[0242] After the production and purification of the mAbs were complete, we performed quality control on the mAbs to test whether aggregation would affect the results. We used clone 81 IgGl and IgG3 as sample organisms. We centrifuged the samples at 16,000 RCF for 10 minutes at room temperature. After centrifugation, we removed the supernatant and sterile filtered through a 0.2-micron syringe filter. We then compared the treated Ab81 IgGl and IgG3 with their untreated variants to see if there was a difference in function. We performed phagocytosis experiments on THP-1 cells as described above in MOP 30.

[0243] In vivo imaging phagocytosis assay

[0244] Cell preparation and spike protein bead opsonization

[0245] Neutrophils were isolated from 4 healthy donors using Polymorphs isolation solution as described above. Cells were then opsonized with 10 pg / ml of monoclonal antibody and 1% antibody-depleted serum (Pel-Freeze, Cat# 34014-10) for 30 minutes at 37°C in a heat-shaking module (300 RPM) (final volume of 100 mΐ). Heat-inactivated serum and normal human serum were added to the respective wells. Neutrophils (1 x 10 6 ​1 x 105cells / ml were placed on ice for 1 hour. Cells were seeded at a density of 1 x 105cells / well on 8-well Ibidi plates (cat. no. 80827, Ibidi GmbH, Germany) for 1 hour at 37°C in a 5% CO2environment. All Ibidi plates were pre-coated with 3.3 pg / ml human fibronectin (F0895-1MG, Sigma) diluted in PBS. After incubation, cells were stained with Hoechst 1 :20,000 (ThermoFisher, Germany) and wheat germ agglutinin (WGA) conjugated with Alexa Fluor 594 diluted 1 :333 in sodium medium for a final volume of 300 mΐ / well. Excess stain was washed away with 300 mΐ / well of sodium medium for 3 times before adding 250 mΐ / well of sodium medium. 5 6 5

[0246]

[0247]

[0248] ​​​​​Time-lapse images of phagocytosis were recorded every 10 minutes for 60 minutes using a Nikon N-SIM microscope. Images were acquired through a fluorescence microscope equipped with a 20X Plan Apo lambda objective (NA = 0.40) and a Perfect Focus System (PFS). All images were taken using an ORCA-Flash 4.0c CMOS camera (Hamamatsu Photonics K.K) with 8 positions per well. The filters used were DAPI (Ex. 340-380 nm, Em. 435-485 nm), TxRed (Ex. 540-580 nm, Em. 600-660 nm), FITC (Ex. 465-495 nm, Em. 515-555 nm) and Cy5 (Ex. 625-650 nm, Em. peak 670 nm). Samples were imaged live in an incubator (Okolab) at 37°C, 5% CO2. All image analysis was performed through the Nikon software General Analysis 3. Graphs and statistical data were prepared in Microsoft Excel and Prism 9. ET50 analysis was performed on Prism Graphpad using a nonlinear regression model with slope not constrained, bottom value limited to 0% and top value limited to 100%.

[0249] SIM image acquisition

[0250] After 60 minutes of phagocytosis, samples were fixed with 4% paraformaldehyde and PBS for 15 minutes at 37°C. Wells were washed 3 times with sodium medium before SIM imaging. All SIM images were acquired using a Nikon N-SIM microscope equipped with LU-NV laser units (488 / 561 / 640 nm), CFI SR HP Apochromat TIRF 100X oil objective (NA = 1.49) and ORCA-Flash 4.0c CMOS camera (Hamamatsu Photonics KK). The used filter cubes were N-SIM 488 (Ex. 484-496 nm, Em. 500-545 nm), N-SIM 561 (Ex. 557-567 nm, Em. 579-640 nm) and N-SIM 640 (Ex. 629-645 nm, Em. 663-738 nm). Images in the DAPI channel of the SIM system were acquired using a standard fluorescence microscope. For widefield excitation, a SPECTRA X light engine (Lumecore inc) was used. 15 steps of Z-axis were acquired for each image in all channels. SIM images were reconstructed using Nikon SIM software through NIS Elements Ar (NIS-A 6D and N-SIM Analysis). Widefield images were processed using NIS Element Clarify.ai and Denoise.ai to minimize blurring caused by the fluorescence microscope and cropped from a 3x5 grid. All Z-axis images were compiled into maximum intensity projection images and then SIM images and fluorescence images were merged in the NIS Elements software.

[0251] Animal experiments

[0252] Thirty 7- to 10-week-old female K18 hACE2 (B6.Cg-Tg(K18-ACE2)2Prlmn / J) mice were randomized into 5 groups of 6 mice each and the antibodies were administered intraperitoneally as a single dose. Eight hours after antibody administration, the animals were inoculated intranasally with 10 5 PFU of SARS-CoV-2 virus (original strain, isolate SARS-CoV-2 / 01 / human / 2020 / SWE, derived from the Swedish health authorities). Body weight and health status of the mice were recorded daily and if the mice lost more than 20% of their body weight or had a severe deterioration of their health status, they were euthanized. After 10 days of infection, the mice were then euthanized. Blood, tissues and bronchoalveolar lavage fluid were collected and stored accordingly. All animal experiments were approved by the Regional Animal Ethics Committee in Stockholm (2020-2021). Virus titers in BAL fluid were analyzed using qPCR 5 .

[0253] Statistical analysis

[0254] Statistical analysis was performed in Graphpad Prism software. For comparisons between different subclasses in a particular clone or between different mixes, a two-tailed Welch’s t-test was used for statistical analysis. When more than 2 treatments were analyzed, a one-way ANOVA with multiple comparison test was used, with the multiple comparison correction using Tukey’s test.

[0255] Example 1: Switching IgGl constant region to IgG3 to change avidity to Spike protein

[0256] Previously we generated 96 antibodies against SARS-CoV-2 original Spike protein by isolating Spike protein reactive B cells and performing single cell sequencing 5 . Out of the 96 clones, there were 11 that were strongly reactive to Spike protein and all 11 clones had opsonic activity in vitro. These mAbs were produced in the IgGl subclass, which was not the original subclass of the patient’s B cells. Using PCR and homologous recombination, we swapped the subclass specific domain of the heavy chain in the heavy chain expression plasmid. We successfully generated 8 new heavy chain plasmids that contain the original variable heavy chain domain and the IgG3 constant domain gene. There are a large number of allotypes for human IgG3 constant domain 9 . The allotype used in this study is IGHG3*11, which contains a long 62 amino acid hinge among other differences 9 . The heavy chain plasmids were transfected into Expi293F cells along with the original light chain plasmids to produce the IgG3 variants of these antibodies Figure 1 A).

[0257] To measure the binding avidity of our newly produced mAbs, we conjugated biotinylated Spike protein to fluorescent streptavidin microbeads (1 pm) as a model for SARS-CoV-2 virions 5 . We incubated our mAbs with the Spike protein beads and then added a fluorescent anti-Fab secondary antibody so that IgG binding on the beads could be detected using flow cytometry as previously described 5 ( Figure 1 B). Avidity was calculated by fitting a non-linear regression model to the dose binding data. We observed that out of our 8 IgG3 antibodies, 6 had equivalent avidity to their IgGl counterparts Figure 1 C-D). However, Ab57 had a significant change where the IgG3 version exhibited higher avidity compared to the IgGl parent Figure 1C-D). On the other hand, Ab11 as an IgG3 antibody had severely reduced binding to the spike protein, while the IgG1 version maintained high reactivity. It is important to note that Ab57 and Ab11 were derived from different clones. Ab57 and Ab11 evolved in vivo to IgG1 and IgG3. Unfortunately, the information obtained from sequencing the IgG3 constant domain of clone 11 was incomplete. Therefore, it was not possible to determine the isotype present in the donor patient. In both cases, the original subclass in the donor B cell (IgG3 for clone 11 and IgG1 for clone 57) had lower binding affinity to the spike protein than the subclass switched mAb variants IgG1 and IgG3 Figure 1 D).

[0258] To complement the flow cytometry-based binding data, we performed surface plasmon resonance (SPR) to investigate the affinity of the spike protein to the mAbs in the context of the respective monovalent domains. For clone 11-81, we used the receptor binding domain (RBD) of the spike protein as analyte, while for clone 94 we used the N-terminal domain (NTD). These clones were derived from previous studies where they were known to bind to these respective domains 5 . For both subclasses, Ab11, 36, and 57 were not able to bind to the RBD in this experimental setup. For Ab59, Ab66, Ab77, Ab81, and Ab94, we observed no big differences in affinity (in k A and K D aspect) (supplemental Figure 2 A), which confirmed the previous affinity data using spike protein bead flow cytometry. To measure the binding of Ab11, 36, and 57, we performed a similar experiment but replaced the RBD with the complete spike protein expressed in trimeric form as ligand. In line with our previous findings, Ab11 was less reactive to the spike protein when expressed as IgG3, more than 25-fold lower Figure 1 E-F). However, we observed no difference between IgG1 and IgG3 for clone 36. Clone 57 showed a 2-fold difference in apparent K D values in this experimental setup Figure 1 E-F). The results of the SPR validated our data that both lab-generated Ab11 (IgG1) and Ab57 (IgG3) have higher affinity to the spike protein than their respective native subclass.

[0259] We further validated our findings on Ab11 and Ab57 by coating ELISA plates with the spike protein and testing mAb reactivity. The results of the ELISA experiment showed that Ab57 was more strongly bound to the spike protein when expressed as IgG3, several-fold stronger signal than IgG1 Figure 1G, Supplement Figure 3 A). Furthermore, as expected, Ab11 IgG3 was less reactive to Spike protein than its IgG1 counterpart Figure 1 G, Supplement Figure 3 A), while the IgG1 was more reactive. Taken together, three different approaches indicated that for clones 36, 59, 66, 77, 81, and 94, replacing the constant region from IgG1 to IgG3 did not affect the avidity to Spike protein antigen. However, for clones 11 and 57, there was a large change in avidity after changing the constant region. In both cases, the best binding subclass was not the original one from the donor B cell, as the artificially created subclass had higher binding avidity Figure 1 C-D, Figure 1 E-F and Figure 1 G).

[0260] Example 2: IgG3 is more effective than IgG1 in inducing Fc-mediated phagocytosis

[0261] In our previous work, we utilized the THP-1 cell line to investigate how IgG1 monoclonal antibodies promote Fc-mediated phagocytosis of Spike protein beads 5 . These findings were correlated with protective effects in animal models. Using a similar approach, we incubated opsonized Spike protein-coated microsphere beads with THP-1 cells Figure 2 A) to measure Fc-mediated phagocytosis. Since all of our monoclonal antibodies were discovered by using the original Wuhan-1 Spike protein decoy B cells, we chose to use this antigen for the follow-up analysis. We stained the beads with a pH-sensitive dye (pHrodo green) to distinguish the cell population with internalized beads from the one that only bound to the beads Figure 2 A).

[0262] On average, IgG3 mAbs increased the percentage of cells with internalized beads by more than 2-fold compared to their IgG1 counterparts. Except for Ab11 IgG3, all mAb clones presented a consistent trend Figure 2 B), which reflected their reduced affinity. The fold change was the largest for Ab36 IgG3 (3-fold) when switching from IgG1 to IgG3 Figure 2 B). In terms of potency measured by the percentage of internalization, Ab94 (anti-NTD) IgG3 (47%) was the most potent, followed by Ab59 (38%) and Ab81 IgG3 (35%) Figure 2 B, Supplement Figure 4A). Combining these values with the context, the most potent IgGl antibody, Ab94 IgGl, was similar in efficacy to IgG3 Ab66 (17% vs 20%), which was the second weakest IgG3 antibody. In addition to leading to a greater proportion of phagocytes with internalized beads, IgG3 led to an increase in the number of opsonized beads bound to phagocytes compared to IgGl ( Figure 2 C, supplemental Figure 4 A). Ab94 IgG3 was 7-fold more potent than Ab94 IgGl. Ab81 IgG3 was 6-fold more potent than its IgGl counterpart, while no difference was observed for Ab11 and Ab57 IgG3. The differences between IgG3 clones were much greater in this metric than the percentage of phagocytes with internalized beads. Ab94 IgG3 led to more beads bound to THP-1 cells (73,000 MFI) than any other antibody, followed by Ab81 IgG3 (44,000). Likewise, the most potent IgGl antibody, Ab59 (10,500), was comparable to the weakest IgG3.

[0263] Our results suggest that the proportion of cells with internalized beads is not entirely correlated with the number of beads bound to cells, and that each clone exhibits a unique pattern in these metrics. Ab59 IgG3 and Ab81 IgG3 were comparable in the percentage of cells with internalized beads (38% vs 35%), but Ab81 IgG3 induced much more bead uptake, resulting in a higher bead signal (44,000 vs 28,000). Likewise, while Ab36 IgG3 was similar to Ab77 IgG3 in the percentage of internalized cells (31% vs 30%), there was a large difference in bead signal (29,000 vs 17,000). Furthermore, Ab94 and Ab81 IgG3 performed best in both metrics, being the most potent monoclonal antibodies ( Figure 2 B-C). Overall, replacing the constant region with IgG3 enhanced Fc-mediated phagocytosis for most of the mAb clones tested. Finally, it is worth noting that in the case of clones 36, 57, 66, 77, and 81, the switched IgG3 forms were superior in function to the form originally encoded by the donor B cells.

[0264] Example 3: IgG3 oligoclonal mixtures can enhance Fc-mediated phagocytosis compared to individual monoclonal antibodies

[0265] While the monomeric IgG3 exhibited strong opsonic capacity as a single mAb, the mAb-based experimental setup does not represent the in vivo environment. In serum, multiple antibody clones influence the overall phagocytic performance. To mimic this situation, we constructed a mixture of all 8 mAbs, called Octomab. To observe the contribution of the most potent clones (Ab94 and Ab81 IgG3), we constructed a mixture composed of these two mAbs, called DuomAb. The remaining Abs 11-77 were combined into a third mixture, called HexamAb. To observe the effect of the mixtures, we reduced the predator multiplicity (ratio of beads to phagocytes) from 30 to 15 and halved the mAb concentration (from 10 to 5 pg / ml) to avoid saturation of THP-1 cells with too many prey and mAbs.

[0266] The IgG3 mixtures significantly enhanced the phagocytic potency of the IgG3 mAbs. The proportion of cells with internalized beads was increased 3-fold in all three mixtures compared to the respective IgG1 mixtures Figure 2 D). HexamAb, which did not contain the potent Abs 94 and 81 IgG3, reached similar phagocytosis as the monomeric Ab94 IgG3 (32% vs. 27%). The difference was even more pronounced when observing the bead signal (40000 and 29000, respectively) Figure 2 E). In terms of bound beads per cell, the potency of the IgG3 mixtures was much higher than their IgG1 counterparts. The potency of the IgG3 DuomAb was 8-fold higher than IgG1, 9-fold higher for HexamAb, and 12-fold higher for Octomab Figure 2 E). Most importantly, all three antibody mixtures outperformed the most potent IgG3 mAb (Ab94), with HexamAb increasing by 38%, Octomab by 58%, and DuomAb by 79% Figure 2 E). Taken together, these results indicate that even a mixture of less potent mAbs promotes strong phagocytosis, even exceeding the best-performing mAb at the same concentration.

[0267] Example 4: IgG3 monoclonal antibodies efficiently activate complement, while their IgG1 counterparts do not

[0268] We wanted to measure the effect of our IgG3 monoclonal antibodies and cocktails to activate the classical complement pathway. We measured complement activation by adding 1% serum to opsonized spike protein beads. Using an anti-C1q secondary antibody (FITC), we could measure C1q deposition on spike protein beads by flow cytometry. Since clones 81 and 94 performed best in previous assays, we used them as representatives of monoclonal antibodies. The DuomAb and Octomab cocktails were the most potent of all cocktails, so we used them to assess the benefit of cocktails. To avoid donor differences in the presence of spike protein reactive antibodies, we used pooled human serum depleted of antibodies as a source of complement.

[0269] The only mAb that was able to induce C1q deposition by itself was Ab94 IgG3 Figure 3 A-B). When Ab81 IgG3 and Ab94 IgG3 were together as DuomAb, they deposited C1q on spike protein beads 2.5 times more than Ab94 by itself (38% vs 15%, Figure 3 A-B). This potent complement activation was not observed with IgG1 DuomAb, suggesting a subclass-dependent phenomenon. Octomab, which contains all antibodies, induced a similar degree of C1q deposition as DuomAb, but only in its IgG3 form. Subsequently, we analyzed the relationship between C1q deposition and mAb concentration. We calculated the EC 50 of DuomAb IgG3 and compared it to Ab94 IgG3. The analysis revealed that the addition of Ab81 IgG3 to Ab94 IgG3 increased the percentage of beads with C1q deposition more than 3-fold (EC 50 from 37 to 11 pg / ml, Figure 3 C). When looking at the C1q deposition signal, the efficacy increased 20-fold (EC 50 from 230 to 15 pg / ml) Figure 3 D). This means that the addition of DuomAb to beads with C1q deposited on them resulted in a stronger deposition compared to using Ab94 alone. In summary, anti-spike protein IgG3 mAbs have a clear increase in potency to activate the classical complement pathway compared to IgG1, which is enhanced when IgG3 mAbs are used as cocktails.

[0270] Example 5: Neutrophil phagocytosis of spike protein coated beads is predominantly Fc-mediated and is enhanced by IgG3 monoclonal antibodies and their cocktails

[0271] We decided to validate our findings on phagocytosis with primary cells. We first used monocytes and determined how changing the predator- prey ratio (MOP) would affect internalization, as this is an important experimental factor to consider in phagocytosis 18 At low MOP (<1), even with the potent OctomAb IgG3, monocytes hardly internalized the spike protein beads. However, at higher MOP (1-10), the opsonic efficacy of the mAbs increased. To study the effect of IgGl and IgG3 mAbs on primary cells, we used a MOP of 5. We chose to use neutrophils instead of monocytes in the subsequent experiments, as they are more efficient in mediating phagocytosis and are the most abundant phagocytic cells in the blood 19 .

[0272] The results show only minor differences between heat-inactivated (HI) serum and complement-active (C+) treatment. This indicates that Fc-mediated phagocytosis is the main functional mode of both subclasses of anti-spike protein monoclonal antibodies, while complement-mediated phagocytosis plays a minor role Figure 3 E). When comparing subclasses, all IgG3 treatments outperformed their IgGl counterparts in the proportion of neutrophils with internalized beads Figure 3 E). As mentioned before, the IgG3 mixture was equal or slightly better than Ab94 IgG3 alone (C+: OctomAb: 15.0 ± 1.6%, DuomAb: 15.5 ± 1.1%, Ab94: 13.1 ± 2.9%) Figure 3 E). Octomab IgGl (C+, 3.4 ± 0.3%) was slightly better than Ab81 (C+, 0.4 ± 0.1%) and Ab94 IgGl (C+, 0.6 ± 0.1%), which was not seen before with THP-1 cells Figure 2 D-E). However, a clear difference between Octomab IgGl and OctomAb IgG3 appeared again (3.4% vs 15.0%).

[0273] We also investigated the number of beads phagocytosed by the cells. Due to the lower MOP, more cells were non-reactive compared to THP-1 cells (MOP 30 vs 5). We analyzed the bead signal of bead-positive neutrophils (APC+). This analysis showed that the effect of all IgG3 treatments remained superior to their IgGl counterparts Figure 3 F), which is in line with the situation with THP-1 cells. Interestingly, IgGl Octomab showed a stronger phagocytosis compared to the single IgGl monoclonal antibodies, which was not observed with THP-1 cells Figure 2 B, Figure 3F). This indicates that even IgG1 monoclonal antibodies, when used in mixtures, are beneficial in enhancing phagocytic efficiency. Taken together, the results show that IgG3 monoclonal antibodies and their mixtures are superior to their IgG1 counterparts in promoting phagocytosis (mediated by Fc receptors and complement receptors). Both subclasses benefit from using a mixture-based oligoclonal approach to enhance phagocytic performance.

[0274] To investigate the temporal dynamics of monoclonal antibody-induced phagocytosis, we used in vivo fluorescence microscopy. We focused on comparing the optimal monoclonal antibody Ab94 IgG3 and the IgG1 and IgG3 forms of the oligoclonal OctomAb. To include CR-mediated phagocytosis, we did not heat-inactivate the serum. To compare flow cytometry data, we used neutrophils from the same donor. Figure 3 EF) and the same experimental conditions (MOP 5). We observed neutrophils with internalized beads and quantified the percentage of these cells and the number of their internalized beads (EF). Figure 4 A). OctomAb IgG3 produced more cells with internalized beads during the 60-minute experimental period, thus standing out among all treatments. We calculated the time required for 50% of neutrophils to have internalized beads (ET). 50 The efficiency of IgG mAbs was analyzed. This analysis showed that OctomAb IgG3 promoted spike protein bead internalization at twice the rate of Ab94 IgG3 and Octomab IgG1 (35 min, 66 min, and 62 min, respectively). Figure 4 D). All mAb treatments were significantly better than the untreated negative control (165 minutes). Figure 4 D) is more effective. Nevertheless, only OctomAb IgG3 resulted in a statistically significant difference. Interestingly, Octomab IgG1 and Ab94 IgG3 showed similar performance over time ( Figure 4 D). This confirms once again. Figure 3 The results in EF show that the IgG1 mixture enhanced the weaker-performing IgG1 mAb. While we previously used the median bead signal to quantify the number of phagocytosed beads, in vivo imaging allows us to do so more precisely. Figure 4 A). This analysis indicates that, compared to IgG1 Octomab, cells with internalized beads, on average, had more beads internalized by neutrophils when the opsonins were IgG3 Octomab and IgG3 Ab94. Figure 4 BC). However, considering that IgG3 Octomab activates more cells than IgG3 Ab94 ( BC). Figure 4D) (72% and 47% respectively after 60 minutes), thus the IgG3 octomab overall promoted stronger bead-cell interaction. Overall, these results highlight the advantage of using an oligoclonal mixture of monoclonal antibodies to improve monoclonal antibody efficacy for both IgGl and IgG3.

[0275] Example 6: Opsonic anti-Spike protein mAbs mediating Fc-mediated functions can protect from SARS-CoV-2 infection

[0276] Data from isolated primary human neutrophils from multiple donors complemented our THP-1 monocyte in vitro experiments. These experiments were performed ex vivo using microsphere beads. Next, we wanted to validate the biological potential of these Fc-mediated functions in a clinically relevant setting, in protecting from real SARS-CoV-2 infection. We tested the protective function of our mAbs in a hACE2-K18 mouse infection model Figure 4 E). We prophylactically injected each mouse intraperitoneally with a single dose (200 ug / mouse) of our mAbs (Ab94 IgGl, Ab94 IgG3, Ab81 IgG3, DuomAb IgG3 or vehicle control). Eight hours after mAb administration, we inoculated mice intranasally with 1 x 10 5 PFU (SARS-CoV-2; original strain from Swedish isolate) virus. Remarkably, these clones were all validated by three independent methods 5 , none of them had neutralizing activity and were considered strictly opsonic.

[0277] We speculate that all our antibodies have a protective effect through their Fc-mediated effector functions compared to the vehicle control. Ab94 IgGl and Ab81 IgG3 had less weight loss than the vehicle control from day 0-7, measured daily during the experiment. More importantly, all monoclonal antibodies prevented mice from dying from euthanasia (mice were euthanized when they were in poor clinical condition or had lost more than 20% of their body weight) Figure 4 E). On day 7, all control mice had died, while none of the monoclonal antibody treated groups had died Figure 4 , shown in red dashed line in Fig. F, showing mice survival after day 6). This data also reflects the mice survival and body weight on day 6 Figure 4 G). On day 6, four of the six control mice had died and one mouse from the Ab94 IgG3 group was euthanized Figure 4 G). At this time, none of the other antibody treated groups had died and maintained healthy average body weight Figure 4H). Notably, only the Ab94 IgGl and Ab81 IgG3 groups had mice surviving to day 10 Figure 4 I). Bronchoalveolar lavage fluid (BAL) was collected from all mice in each control group, regardless of the day they died, and analyzed by qPCR for viral titers calculated as cycle threshold (CT). Higher values indicate lower viral loads. This analysis showed that all mAb-treated animals, except DuomAb, had higher CT thresholds and thus lower viral loads at the time of euthanasia Figure 4 J). The Ab94 IgGl had the highest CT value (24.5) compared to the control group (17.8), followed by Ab94 IgG3 (19) and then Ab81 IgG3 (18.1) Figure 5 I). Our data show that mAb-treated mice were overall healthier in terms of average body weight, increased survival, and lower viral loads in BAL fluid. Taken together, our results suggest that opsonizing anti-Spike protein antibodies, regardless of isotype, can exert protective effects in a preclinical animal model of authentic SARS-CoV-2 infection. It is difficult to draw conclusions about differences between antibody treatments beyond Ab94 IgGl, which performed the best in this experiment. Our data serve as a proof of concept that highlights the biological relevance of studying Fc-mediated effector functions in the context of SARS-CoV-2 adaptive immunity.

[0278] Example 7: Discussion

[0279] This study focused on how to enhance Fc-mediated immune functions of anti-Spike protein antibodies. We found that changing the antibody subclass can enhance immunity. Our data show that the IgG3 versions of a range of Spike protein-specific antibodies outperformed their IgGl counterparts - even though they originally derived from IgGl B cells. Moreover, our data also show that Fc-mediated functions are significantly enhanced when antibodies are combined into a cocktail.

[0280] At any given time, an astonishing number of IgG antibody clones circulate in serum. Serum is thus a multifunctional polyclonal antibody cocktail that, upon infection, is tuned towards antigen-specific antibodies. In our current work, we show that a single IgG3 antibody (e.g., Ab94) can very effectively mediate Fc-mediated immune functions in the hierarchy of efficacy. However, when multiple less potent antibodies are combined together (Hexamab), they are equally effective in mediating immune functions. This can be due to the enhanced Fc receptor activation caused by multiple Fc clustering on antigen trimers, which is theoretically important for efficient phagocytosis 25–27 . The finding about the importance of cocktails also translated into an enhancement of the efficacy of complement deposition.

[0281] C1q requires multiple Fc to activate to form hexamers 28 Our results suggest that the IgG3 mixtures are more effective at forming hexameric clusters than the IgG1 mixtures. Another important thing to note is that the benefit of the mixtures is more pronounced with neutrophils than THP-1 cells when used to opsonize spike protein beads. This can be attributed to neutrophils being professional phagocytes with a more extensive repertoire of phagocytic receptors 29 We have shown how immune function can be enhanced by combining multiple opsonic mAbs. We believe that it would be a promising approach to consider an IgG3 platform for future mAb therapies against emerging variants of concern and other pathogens, as IgG3 is more functionally active than IgG1 in vitro, especially in mixtures.

[0282] The lower bioactivity of IgG3 in vivo can be reflected in clone 94. We believe that the performance of IgG3 Ab94 compared to its IgG1 counterpart can be due to a shorter half-life or less efficient Fc interaction. Human IgG has a shorter half-life in mice - on the order of days rather than weeks - compared to humans, and a greater proportion of the IgG3 subclass can be eliminated compared to IgG1 after 8 hours of virus administration, and this can also vary from mAb to mAb 32 It is notable that Ab81 IgG3 is close to Ab94 IgG1 in protective effect. We attribute this to it having a higher affinity than the two subclasses of clone 94, and thus likely to be more functionally active in vivo. We believe that the hACE2-K18 model is a good model to demonstrate as a proof of concept the biological relevance of Fc effector function in combating SARS-CoV-2 infection. However, the biological transferability of studying human subclasses in this model and predicting the outcome in humans is more limited and deserves caution. Among other notable differences, the Fc receptors expressed by mouse phagocytes are different from those expressed by human phagocytes 33 These Fc receptors, while having very similar affinity to human Fc receptors 34 are less well studied, especially in the context of IgG3 and its many isotypes. Those studies that have looked at human subclass affinity to mouse Fc-gamma receptors have observed that human IgG3 has as high a binding efficiency as IgG1. However, despite this, human IgG3 has been shown to be less functional in vitro than human IgG1 by mouse phagocytes 35,36 This is in contrast to in vitro studies with human phagocytes, where human IgG3 has been shown to be more functional than IgG1 12-14,30We believe that more work is needed to better link in vitro assays, in vivo animal models, and potential clinical benefits when studying human IgG subclasses as therapeutic agents. Progress has been made in generating mice with humanized immune systems to address these differences across species 37,38 but the currently available models are not without limitations 39 including model availability, relative complexity, and the impact of human donor variability required for implantation of human tissues or cells. Nonetheless, our in vivo data support the notion that Fc effector functions of non-neutralizing mAbs can strongly protect against SARS-CoV-2 infection from the perspective of understanding the role of Fc-mediated functions in SARS-CoV-2 immunity. In this regard, generating human IgG3 is a promising alternative to IgG1, especially if its shorter half-life pharmacokinetic properties are addressed by appropriate engineering.

[0283] In addition, we present evidence that swapping constant domains affects the ability of the variable domains to bind to the spike protein. Ab11 IgG3 has a reduced affinity compared to Ab11 IgG1. On the other hand, Ab57 IgG3 outperforms IgG1. This is interesting because clone 57 was originally IgG1, while clone 11 was originally IgG3 (according to sequencing data) 5 . IgG1 antibodies are formed as a result of the IgG3 constant domain gene being deleted during class switch recombination 9,40 . Thus, IgG1 can evolve from IgG3, but IgG3 cannot evolve from a clone that produces IgG1 antibodies. While IgM can directly switch to IgG1 antibodies without an IgG3 intermediate, B cells that produce IgG3 have not evolved to produce IgG1. Subsequently, clone 11 is derived from a B cell that produces IgG3, meaning that Ab11 IgG1 has not previously undergone subclass switching in this B cell. We find it interesting that artificial Ab11 IgG1 has a much higher affinity for the spike protein than the original IgG3. Furthermore, Ab57 IgG3 suggests that a theoretically less evolved mAb has a higher reactivity to the antigen than the naturally evolved Ab57 IgG1. These examples raise the question of whether the most reactive mAb expressed by B cells is the one that is circulating in the B cell at any given time point within the 6-8 week window after infection (in Bahnan et al 2022, patient B cells were obtained 6-8 weeks after the first SARS-CoV-2 infection 5). Our results suggest that the change in avidity does not always follow the expected subclass switching hierarchy of the IGHV loci. However, our data are based on only 8 clones, but raise some new questions worth thinking about. Our results further confirm the evidence of the reciprocal influence of antibody constant and variable domains 10,4142 . We further show that Fc effector functions are most effective in the IgG3 subclass, regardless of their original subclass. Thus, it is necessary to carry out future studies of subclass-switched mAbs to broaden our understanding of the reciprocal interaction of variable and constant domains and how they affect immune functions in specific diseases.

[0284] Overall, this study highlights the benefit of subclass switching of monoclonal antibodies to IgG3 and using them in oligoclonal mixtures to enhance opsonic functions in vitro. These results are crucial for the future development of therapeutic monoclonal antibodies that require Fc- and CR-mediated phagocytosis. In the context of SARS-CoV-2 and its VOCs, the use of opsonic monoclonal therapeutics could be a promising path for further investigation, especially since they are less dependent on binding to the easily mutable epitope in the RBD. Indeed, our study shows the prospect of using non-neutralizing but opsonic monoclonal antibodies in combating SARS-CoV-2 infection and the use of IgG3 monoclonal antibodies instead of IgG1 due to the powerful Fc effector functions of IgG3 monoclonal antibodies. We highlight the importance of future optimization of preclinical models for the therapeutic development of monoclonal antibodies using our in vitro data with human phagocytes and the differences between animal results. Moreover, the study presented here reminds of the complexity involved in B cell subclass switching and highlights the importance of different subclasses in the immune response.

[0285] Example 8: From IgG1 to IgG3 47 Subclass engineered SARS-CoV-2 mAbs exhibit strong opsonic functions.

[0286] Finally, we wanted to test whether hinge engineering and increasing antibody flexibility could enhance mAb functions in other biological settings. We focused on SARS-CoV-2 because Fc-mediated effector functions of antibodies (non-neutralizing and neutralizing) are highly relevant for immune protection against SARS-CoV-2 and its mutant variants 43-48 . We have previously generated anti-SARS-CoV-2 IgG1 mAbs against the spike antigen 48 , and showed that these mAbs have opsonic functions 47-48 . We aimed to elucidate whether increasing the Fc flexibility of some of these opsonic anti-spike protein IgG1 mAbs would also improve Fc-mediated functions and possibly play a role in the SARS-CoV-2 context.

[0287] We generated three IgGh molecules: mAb Ab11, Ab36, and Ab77. 47 These constructs, like IgG1, bind to the spike protein with nanomolar affinity. 47 ( Figure 5 A) Same as before. 38 We used biotinylated spike proteins conjugated with streptavidin microspheres as a virion model. These mAbs all elicited Fc-mediated effector functions. 47-48 We plotted the MOP curve and calculated the MOP for each corresponding mAb. 50 Values. We used Xolair (anti-IgE) as a negative control and a mixture of two antibodies, IgG3 mAb with emphasizing physiological function. 47 As a positive control, interestingly, we observed a significant enhancement in the function of all three clones. For Ab11, IgGh... 47 The version enhanced bead-phagocytic interactions by 1.4 times (MOP). 50 27 vs. 37, P<0.05), while for Ab77, a 1.7-fold functional enhancement was observed (MOP). 50 26 vs. 43, P>0.05)( Figure 5 B). Ab36IgGh 47 It exhibits significantly enhanced functionality, with the MOP50 value increasing by 4.3 times (9 vs 39, P < 0.05). Figure 5 B). Similar to Ab25, we calculated the phagocytic score (PS) to examine engineered IgGh. 47 Is there a difference in overall bead uptake between IgG1 mAb and IgG1 mAb? The differences between different IgG versions become more pronounced with increasing MOP: for Ab11, it increased fourfold at MOP 30 (3000 vs. 800). Figure 5 C). For Ab77, we observed a 6-fold increase in PS (3300 vs. 550), while for Ab36, the improvement relative to IgG1 was most significant, with a 13-fold increase in PS (8400 vs. 600). Figure 5 C). We conducted additional experiments at MOP 30 to assess whether these changes were statistically significant using the corresponding mAbs and investigated binding and PS. Here, we observed similar results for all three clones, namely IgGh 47 Both functions were significantly enhanced compared to IgG1 ( List of references DE), which was statistically significant for both clones 36 and 77. More interestingly, Ab36IgGh 47was shown to be comparable to our most potent opsonin (37) DuomAb IgG3. In summary, increasing the flexibility of antibodies by hinge engineering showed a significant enhanced efficacy in two different biologically relevant systems. In this regard, Ab36 IgGh 47 Due to its strong Fc-mediated effector functions, it is a promising candidate for further in vivo testing in the SARS-CoV-2 environment.

[0288] Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Definitions of some terms SEQ ID NO: 1 (IgG3 heavy chain constant domain) SEQ ID NO: 2 (modified IgG3 heavy chain constant domain) SEQ ID NO: 3 (IgG1 constant domain, engineered with IgG3 hinge) SEQ ID NO: 4 (original Sars-CoV-2 spike protein) SEQ ID NO: 5 (Delta (B1.617.2) spike protein Sars-CoV-2 variant) SEQ ID NO: 6 (Omicron (B1.1529) spike protein Sars-CoV-2 variant) SEQ ID NO: 7 (IgG3 hinge region) SEQ ID NO: 8 (IgG3 hinge portion) SEQ ID NO: 9 (IgG3 hinge portion) SEQ ID NO: 10 (clone 11 heavy chain variable domain) SEQ ID NO: 11 (clone 11 light chain variable domain) SEQ ID NO: 12 (clone 36 heavy chain variable domain) SEQ ID NO: 13 (clone 36 light chain variable domain) SEQ ID NO: 14 (clone 57 heavy chain variable domain) SEQ ID NO: 15 (clone 57 light chain variable domain) SEQ ID NO: 16 (clone 59 heavy chain variable domain) SEQ ID NO: 17 (clone 59 light chain variable domain) SEQ ID NO: 18 (clone 66 heavy chain variable domain) SEQ ID NO: 19 (clone 66 light chain variable domain) SEQ ID NO: 20 (clone 77 heavy chain variable domain) SEQ ID NO: 21 (clone 77 light chain variable domain) SEQ ID NO: 22 (clone 81 heavy chain variable domain) SEQ ID NO: 23 (clone 81 light chain variable domain) SEQ ID NO: 24 (clone 94 heavy chain variable domain) SEQ ID NO: 25 (clone 94 light chain variable domain) Figure 5 Figure 4 Figure 3 Figure 2 Figure 2 Figure 2 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 2 Figure 1 Figure 1 Figure 1 Figure 3 Figure 1 Figure 3 Figure 1 Figure 1 Figure 1 Figure 2 Figure 2 Figure 2 Figure 2 Figure 2 Figure 4 Figure 2 Figure 4 Figure 2 Figure 2 Figure 2 Figure 2 Figure 2 Figure 3 Figure 3 Figure 3 Figure 3 Figure 3 Figure 3 Figure 3 Figure 2 Figure 3 Figure 2 Figure 3 Figure 3 Figure 4 Figure 4 Figure 4 Figure 4 Figure 3 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 List of references

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Claims

1. An engineered antibody comprising an IgG3 hinge and a variable domain having a viral spike protein binding portion.

2. The engineered antibody of claim 1, wherein the Fc region is an IgG3 region.

3. The engineered antibody of claim 2, wherein the Fc region comprises an IgG3 heavy chain constant domain of SEQ ID NO: 1 or a variant thereof having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:

1.

4. The engineered antibody of claim 2, wherein the IgG3 region comprises three substitutions R435H, N392K, and M397V.

5. The engineered antibody of any one of claims 3-4, wherein the IgG3 region comprises a polypeptide having at least 95% identity or at least 98% identity to the modified IgG3 heavy chain constant domain of SEQ ID NO:

2.

6. The engineered antibody of any one of claims 3-5, wherein the IgG3 region comprises the modified IgG3 heavy chain constant domain of SEQ ID NO:

2.

7. The engineered antibody of claim 1, wherein the Fc region is an IgG1 region.

8. The engineered antibody of claim 7, wherein the engineered antibody comprises an IgG1 constant domain of SEQ ID NO: 3 engineered with an IgG3 hinge.

9. The engineered antibody of any one of claims 1-8, wherein the viral spike protein is from a coronavirus.

10. The engineered antibody of any one of claims 1-9, wherein the viral spike protein is a SARS-Cov-2 spike protein.

11. The engineered antibody of any one of claims 1-10, wherein the IgG3 hinge comprises an amino acid sequence having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 7).

12. The engineered antibody of any one of claims 1-10, wherein the IgG3 hinge comprises an amino acid sequence having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the amino acid sequence X-(Y)n, wherein X is ELKTPLGDTTHTCPRCP (SEQ ID NO: 8), Y is EPKSCDTPPPCPRCP (SEQ ID NO: 26), and n is an integer ranging from 2 to 8, preferably an integer ranging from 2 to 5, more preferably an integer ranging from 2 to 4.

13. The engineered antibody of any one of claims 1-12, which promotes opsonization.

14. The engineered antibody of any one of claims 1-12, which does not bind to a neutralization epitope. ​ ​ ​ 15. The engineered antibody according to any one of claims 1-14, wherein the engineered antibody is selected from the group consisting of: Abll, Ab36, Ab57, Ab59, Ab66 and Ab77.

16. A composition comprising at least two different engineered antibodies as defined in any one of claims 1-15.

17. The composition according to claim 16, comprising 3 to 15, 3 to 12, 3 to 9, 5 to 15, 5 to 10 or 6 to 10 different engineered antibodies as defined in any one of claims 1-15.

18. A nucleic acid molecule encoding a protein comprising an engineered antibody as defined in any one of claims 1-15.

19. A cell comprising a nucleic acid as defined in claim 18.

20. Use of an engineered antibody as defined in any one of claims 1-15 for preventing, treating or alleviating a viral infection caused by a virus comprising said spike protein.

21. Use of an engineered antibody as defined in any one of claims 1-15 for Fc-mediated phagocytosis of a virus comprising said spike protein.

22. Use of a composition as defined in any one of claims 16-17 for activating the complement pathway.

23. A method of making an engineered antibody as defined in any one of claims 1-15, comprising the steps of: - immunizing a host organism with said viral spike protein or a protein comprising said viral spike protein, - isolating at least one viral spike protein reactive B cell to isolate an IgGl antibody that binds to said viral spike protein, - making at least one recombinant DNA molecule encoding a modified version of said IgGl antibody, comprising replacing the IgGl subclass specific domain of said isolated IgGl antibody with an IgG3 subclass specific domain, - expressing said at least one recombinant DNA molecule in a suitable host cell, to obtain said engineered antibody.

24. A method of making an engineered antibody as defined in any one of claims 1-15, comprising the steps of: - immunizing a host organism with said viral spike protein or a protein comprising said viral spike protein, - isolating at least one viral spike protein reactive B cell to isolate an IgGl antibody that binds to said viral spike protein, - making at least one recombinant DNA molecule encoding a modified version of said IgGl antibody, said modification comprising replacing the IgGl hinge of said isolated IgGl antibody with an IgG3 hinge, - expressing said at least one recombinant DNA molecule in a suitable host cell, to obtain said engineered antibody.

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