Modified anti-spike IgG3 antibody

Abstract

The present invention relates in general to immunology and antibody engineering, and more specifically to modified antibodies having improved properties, and to hinge engineering methods for improving the properties of, for example, IgG1 antibodies. More specifically, the present invention provides a modified antibody comprising an IgG3 subclass-specific domain and a variable domain having a viral spike protein binding site.

Description

Technical Field

[0001] The present invention relates to the fields of immunology, vaccines, and pharmaceuticals. Specifically, the present invention relates to antibodies, pharmaceutical compositions, antibody engineering, viral vaccines, and methods for their preparation and use.

Background Art

[0002] Since its first appearance in 2019, SARS-CoV-2 has caused millions of deaths worldwide. 1 In the early days of the pandemic, monoclonal antibodies (mAbs) were successfully used as therapeutic agents. 2 These antibodies interfere with the ACE2 receptor-spike receptor-binding domain (RBD) interaction, thus 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 Non-neutral but opsonizing mAbs have already been shown to be equivalent to potent neutralizing mAbs in protecting SARS-CoV-2-infected animals. 5 These findings are consistent with other studies showing that Fc effector functions are essential for virus control. 6,7 Indeed, the protection provided by vaccines against variants such as Omicron can be partially explained by the Fc effector functions of intact antibodies against non-RBD sites or RBD sites that have not undergone mutations. 8

[0003] Antibody classes and subclasses play an important role in determining the functional outcome, especially in phagocytosis via Fc and complement receptors. The human IgG class has four subclasses defined by differences in the constant domains of the heavy chains 9 The constant domains have historically been considered independent of the variable domains and are responsible only for the effector functions of the antibody. However, there is increasing evidence for subclass-switching antibodies, and the constant domains target the antigen 10It has been shown that this affects affinity for the receptor. Nevertheless, research on the constant domains of antibody subclasses has mainly focused on affinity for Fc receptors and complement activation. IgG1 and IgG3 are the most potent activators of Fc gamma receptors on immune cells, and this is thought to be due to their high affinity for these receptors. 11 IgG3 is also the most potent activator of the classical complement pathway, along with IgG1, 2, and 4. 12,13 This continues. In the case of HIV-1, IgG3 has stronger opsonizing activity compared to IgG1, mediating a stronger Fc-mediated phagocytosis of beads bound to the gp140 antigen. This is despite the fact that IgG3 and IgG1 anti-HIV antibodies have similar affinities to their respective targets. 14 This functional difference stems from the more extended hinge region of IgG3, which gives it spatial flexibility due to its Fc tail. Related studies have shown that replacing the hinge of IgG1 with that of IgG3 enhances intracellular immunity against adenovirus infection, supporting the flexibility hypothesis. 15 Therefore, the IgG subclass is a determinant in optimizing the immune function of monoclonal antibodies against viral pathogens. Recently, it has been shown that subclass switching to IgG3 may enhance the neutralization of SARS-CoV-2. 16 Furthermore, it has been shown that the IgG3 backbone can enhance ACE2-Fc protein effector function. 17 Several aspects remain unclear, such as the effect of IgG subclasses on antibody affinity for spike proteins, their impact on complement activation, their relationship to subsequent Fc-mediated immune function, and how various anti-spike antibody combinations may affect Fc-mediated antibody function.

[0004] The basis of mAb therapy against SARS-CoV-2 relies on the ability of neutralizing mAbs to bind to RBD, thereby interfering with the spike protein's ability to interact with the ACE2 receptor. However, the increasing number of large mutations observed in RBD in novel variants of concern (VOCs) has led most of these antibodies to lose binding, resulting in loss of function and treatment failure. 20,21 This is a new variant of 22 To treat or universal neutralizing antibodies 23 To find a solution, alternative strategies have emerged that utilize combinations of neutralizing antibodies (nAbs).

[0005] It has been previously shown that ACE2-Fc constructs utilizing the IgG3 backbone exhibit higher efficacy in vitro than their IgG1 counterparts. 17 In a non-SARS-CoV-2 context, IgG3 mAb has been shown to be efficient in mediating Fc-mediated phagocytosis of HIV virions. 14 Similar benefits are found in the TRIM21 receptor. 15 This has been observed in enhanced intracellular activity against adenoviruses via this pathway.

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

[0007] The usefulness of IgG3 as a drug in vivo has several biological limitations. Of the four subclasses, IgG3 is the neonatal Fc receptor 9 Due to its low affinity for, half-life 9 This is the shortest. Through Fc modification, affinity for the Fc neonatal receptor can be increased without reducing Fc effector function. 30,31 This modification affects the mouse model. 30 It has been shown to be essential for in vivo protection in [the field].

[0008] Improved monoclonal antibodies are needed for the treatment and prevention of viral infections, such as those caused by SARS-CoV-2. In particular, there is a need for monoclonal antibodies and compositions containing such antibodies that are more efficient and provide protection against a wider range of viral variants. [Overview of the project]

[0009] The objective of the present invention is to overcome certain drawbacks in antibodies and their use in the treatment or prevention of viral infections. This is achieved by the inventors, who have designed novel antibodies (monoclonal antibodies, mAbs), which are modified forms of antibodies prepared by immunization using viral spike protein as an antigen.

[0010] To our surprise and delight, we discovered that subclass switching of an IgG1 antibody to an IgG3 antibody enhances both Fc-mediated and complement receptor (CR)-mediated phagocytosis.

[0011] The inventors have further discovered, surprisingly, that modifying an IgG1 antibody by replacing the IgG1 hinge with an IgG3 hinge makes it possible to easily obtain novel antibodies with improved properties. Therefore, by modifying the constant domain of an antibody, it is possible to potently enhance, for example, the immune response to the SARS-CoV-2 virus. It has also been found that a specific number of exon repeats following the core portion of the IgG3 hinge, e.g., 1, 2, or 3, also affects the properties of the prepared IgG3 antibody.

[0012] Even more surprisingly, it was found that combining antibodies in a cocktail containing several different antibodies enhanced the immune function of those antibodies. An oligoclonal cocktail of IgG3 subclass mAbs was found to be more potent than the best-performing monoclonal antibodies in both Fc-mediated and CR-mediated phagocytosis.

[0013] Therefore, modification of antibody subclasses and the formation of cocktails modulate the effectiveness of antibodies as therapeutic agents against viral infections.

[0014] Based on the aforementioned findings, the present invention provides, in a first embodiment, a modified antibody comprising an IgG3 subclass-specific domain and a variable domain having a viral spike protein binding site.

[0015] The present invention further provides a composition comprising at least two different modified antibodies according to the present invention.

[0016] The present invention further provides the use of a modified antibody according to the present invention for preventing, treating, or mitigating viral infections caused by viruses containing the spike protein.

[0017] The present invention further provides the use of a modified antibody according to the present invention for Fc-mediated phagocytosis of a virus containing the spike protein.

[0018] The present invention further provides the use of compositions according to the present invention for activating the complement pathway.

[0019] The present invention further provides the use of compositions according to the present invention for Fc-mediated phagocytosis of viruses containing the spike protein.

[0020] The present invention further provides a method for preparing a modified antibody according to the present invention, comprising the following steps for obtaining the modified antibody: -Immunization of a host organism by the viral spike protein or a protein containing the viral spike protein, - Isolation of at least one viral spike protein-reactive B cell to isolate an IgG1 antibody that binds to the viral spike protein, - Preparation of at least one recombinant DNA molecule encoding a modified form of the isolated IgG1 antibody, comprising replacing the IgG1 subclass-specific domain of the isolated IgG1 antibody with an IgG3 subclass-specific domain. - To express at least one recombinant DNA molecule in a suitable host cell. [Brief explanation of the drawing]

[0021] [Figure 1]Switching the constant domain of IgG1 to IgG3 can alter the avidity to the spike protein. A Schematic diagram of the heavy and light chain plasmids containing the variable and constant domains. The generation of IgG3 mAbs involves switching the constant domain of the heavy chain from IgG1 (blue) to IgG3 (orange). B Spike-coated microspheres are used as a model of the SARS-CoV-2 virion. Antibody binding assays were performed by opsonizing spike beads with mAbs and adding an Alexa 488-conjugated secondary antibody that reports IgG binding to the spike beads. C Binding curves showing the percentage of IgG-positive spike beads as a function of IgG concentration. Each clone is shown with both subclasses (IgG1 and IgG3) present. Three independent experiments were performed using the mean values ​​and error bars representing SEM shown in the graph. D A table summarizing the subclass KD value and original subclass for each clone. Avidity was calculated using a nonlinear regression model in GraphPad Prism. E. Surface plasmon resonance-based binding rates using the whole spike protein (trimer). Binding of 40 nM spike protein (thin line) to immobilized IgG compared to 40 nM RBD (dark line) and PBS (lower curve) for clones 11, 36, 57 and their respective subclasses. F. Table showing KD and KA values ​​for different subclasses of all clones (11-94), and the analytes (spike, RBD, or NTD) used in the SPR-based assays. Statistical analysis was performed using one-way ANOVA with multiple comparisons and Tukey for correction. P values ​​greater than 0.05 are indicated as ns, p values ​​less than 0.05 as *, p values ​​less than 0.01 as **, p values ​​less than 0.001 as ***, and p values ​​less than 0.0001 as ****. G. ELISA data using spike-coated wells and bound IgG for clones 11 and 57 and their respective subclasses. Three independent experiments were performed using titration curves to plot the coupling curve. Here, representative experiments with four technical replicates are shown, along with the mean and error bars representing the SEM. [Figure 2]IgG3 antibodies are superior to IgG1 antibodies in that they induce Fc-mediated phagocytosis, which is further enhanced when used as an oligoclonal cocktail. Figure A shows three different phagocytic states that cells can be in and how they appear in flow cytometry gates: the first shows unassociated cells, the second shows associated but non-internalized cells, and the third shows cells with internalized beads. The gate on the right shows a double-positive population showing internalized beads. The far right is a graph showing IgG isotype controls. The Y axis shows the percentage of THP-1 cells with internalized beads. The same experimental conditions as B and C were used. B IgG1 and IgG3 variants of each antibody clone mediate phagocytosis to varying degrees. The bar graph shows the percentage of cells with internalized beads. Beads were opsonized using the corresponding mAb at 10 μg / ml (3 million beads per 100,000 phagocytic cells, phagocytic multiplicity 30 (MOP 30)). Statistical analysis in B was performed using a two-sided Welch's t-test. C. Comparison of IgG1 and IgG3 from each clone regarding the mean bead signal of the entire THP-1 population. Statistical analysis in C was performed using a two-tailed Welch's t-test. D. Graph showing how monoclonal antibodies compare to cocktails in terms of the amount of beads phagocytosed by the entire cell population. The table on the right shows the mean median bead signal (fluorescence intensity) for each treatment. In D, 1.5 million beads (MOP 15) per 100,000 phagocytic cells were opsonized with either 5 μg / ml monoclonal antibody or IgG cocktail. Statistical analysis in D was performed using one-way ANOVA with multiple comparisons corrected for Tukey's test. p-values ​​less than 0.05 are indicated by *, p-values ​​less than 0.01 are indicated by **, and values ​​greater than 0.05 are indicated by ns. Three independent experiments were performed. Bars represent mean values, and error bars represent SEM. Three independent experiments were performed for all experiments. E. Comparison of the proportion of THP-1 cells containing internalized beads with IgG1 and IgG3 monoclonal antibodies and cocktails. The table on the right summarizes the graph data, including the mean values ​​obtained from three experiments.In experiment E, 1.5 million beads (MOP 15) per 100,000 phagocytic cells were opsonized with either a 5 μg / ml monoclonal antibody or an IgG cocktail. Statistical analysis of experiment E was performed using one-way ANOVA with multiple comparisons corrected for Tukey's test. p-values ​​less than 0.05 are indicated by *, p-values ​​less than 0.01 are indicated by **, and values ​​greater than 0.05 are indicated by ns. Three independent experiments were performed. Bars represent the mean, and error bars represent SEM. Three independent experiments were performed for all experiments. [Figure 3]IgG subclass and oligoclonality affect complement activation by neutrophils and phagocytosis of spike beads. A. Individual gates for C1q deposition against 10 μg / mL Ab81, Ab94, and OctomAb IgG3. The far right panel shows all three plots superimposed. For A, three independent experiments were performed. For A, statistical analysis was performed using one-way ANOVA, with Tukey's test for multiple comparisons. * indicates a p-value less than 0.05, ** indicates a p-value less than 0.01, *** indicates a p-value less than 0.001, and ns indicates a p-value greater than 0.05. B. Data on C1q deposition for the shown monoclonal antibodies and oligoclonal cocktails are shown. For B, three independent experiments were performed. For B, statistical analysis was performed using one-way ANOVA, with Tukey's test for multiple comparisons. * indicates a p-value less than 0.05, ** indicates a p-value less than 0.01, *** indicates a p-value less than 0.001, and ns indicates a p-value greater than 0.05. C: Percentage of C1q deposition as a function of antibody concentration, using EC50 values ​​in the graph. In C, IgG3 isotype control data is shown as a small graph within each graph. Three independent experiments were performed for C. Statistical analysis was performed using one-way ANOVA, and Tukey's test was used for correction for multiple comparisons. * indicates a p-value less than 0.05, ** indicates a p-value less than 0.01, *** indicates a p-value less than 0.001, and ns indicates a p-value greater than 0.05. D: Fluorescence signal of deposited C1q as a function of antibody concentration, using EC50 values ​​present in the graph. In D, IgG3 isotype control data is shown as a small graph within each graph. Three independent experiments were performed for D. Statistical analysis was performed using one-way ANOVA, and Tukey's test was used for correction for multiple comparisons. * indicates a p-value less than 0.05, ** indicates a p-value less than 0.01, *** indicates a p-value less than 0.001, and ns indicates a p-value greater than 0.05. E Neutrophils with internalized spike beads compared to complement-activated serum (C+) with different treatments and thermally inactivated serum (HI). Isotype controls are shown on the far right. Three independent experiments were performed for E.For E, statistical analysis was performed using one-way ANOVA, and Tukey's test was used for correction for multiple comparisons. * indicates a p-value less than 0.05, ** indicates a p-value less than 0.01, *** indicates a p-value less than 0.001, and ns indicates a p-value greater than 0.05. F: Bead signal of neutrophils associated with beads. Isotype control is shown on the far right. [Figure 4]Live imaging of Fc-mediated protection and human neutrophil phagocytosis in vivo using hACE2-K18 mice. A. Super-resolution structured illumination microscopy images showing human neutrophil phagocytosis of opsonized spike beads with the indicated treatment. Beads are visible in the image (stained), on the cell surface (Alexa 594-WGA), and in DNA (Hoechst). pHrodo green was used as an internalization marker not visible in the image. Differences in antibody treatment result in differences in the number of internalized beads and the percentage of cells internalizing beads. SIM images are seen in the maximum intensity projection of the Z stack. An example image from one of four experiments is shown. The scale bar in the lower right corner is 5 μm. B. Time course of the mean number of beads in neutrophils with internalized beads. Figure B shows the mean value, and the error bars represent SEM. In B, the data are from four independent experiments using four different donors. The bar graph in C shows the results after 60 minutes. Figure C shows the mean value, and the error bars represent SEM. In C, the data are from four independent experiments using four different donors. D Monitors and displays the temporal changes in phagocytosis for neutrophils with internalized spike beads. Below the graph, the ET50 values ​​are shown in the table with the 95% confidence interval (in parentheses). Figure D shows the mean values, and the error bars represent SEM. In D, the data are from four independent experiments using four different donors. E Humanized ACE2 mice were prophylactically injected with antibody treatment or vehicle. Eight hours after treatment, the mice were intranasally infected with SARS-CoV-2 (Wuhan strain). Body weight was measured over 10 days. F Average relative body weight for each group from day 0 to day 7, with the red dashed line highlighting the point at which all mice in the control group died. Figure F shows the mean values, and the error bars represent SEM. G Status of each mouse in each treatment group on day 6 (survival / euthanasia). Figure G shows the mean values, and the error bars represent SEM. Figure H shows the relative body weight of each mouse in each treatment group on day 6 (euthanized mice are set to 0). The mean values ​​are shown in Figure H, and the error bars represent SEM. Figure I shows the survival curves for each treatment group over the 10-day experiment.Figure I shows the mean values, and the error bars represent SEM. J The final titer of viral load was analyzed from BAL fluid of euthanized mice, regardless of the date of death. qPCR was performed on the BAL fluid, the cycle threshold was recorded, and the data was graphed. The median is shown. Figure J shows the mean values, and the error bars represent SEM. Statistical analysis was performed using one-way ANOVA, and Tukey's test was used for correction for multiple comparisons. * indicates a p-value less than 0.05, ** indicates a p-value less than 0.01, *** indicates a p-value less than 0.001, **** indicates a p-value less than 0.0001, and ns indicates a p-value greater than 0.05, but is not shown (in the figure). In B-D, the data are from four independent experiments using four different donors. [Figure 5]SARS-CoV-2 mAbs modified from IgG1 to the IgGh47 subclass exhibit potent opsonizing function. A. Figure shows the spike protein trimer antigen and SARS-CoV-2 virions with three different clones Ab11, Ab36, and Ab77 modified from the original IgG1 to IgGh47. B. MOP curves for different mAbs, with MOP50 and 95% CI in parentheses, where statistically significant differences (non-overlapping 95% CI) between two mAbs are highlighted in green (*). IgGh47 is the data point and curve at the top. Three independent experiments were performed in B (N=3). C. Phagocytosis scores at different MOPs. Ab11 IgG1 (left), Ab11 IgGh47 (center), and control IgG1 (left) of the entire panel. Three independent experiments were performed in C (N=3). D. Shows association and phagocytosis scores at MOP 30. Statistical comparisons were performed by comparing the IgG1 and IgGh47 versions using a two-tailed Mann-Whitney U test. Mean values ​​are shown throughout the figure, and error bars are SEM. *** indicates a P value < 0.001, ** indicates a P value < 0.01, * indicates P < 0.05, and P > 0.05 is ns. The positive control was DuomAb IgG3, and the negative control was Xolair IgG1. Four independent experiments were performed in D (N=4). E shows the association and phagocytosis scores at MOP 30. Statistical comparisons were performed by comparing the IgG1 and IgGh47 versions using a two-tailed Mann-Whitney U test. Mean values ​​are shown throughout the figure, and error bars are SEM. *** indicates a P value < 0.001, ** indicates a P value < 0.01, * indicates P < 0.05, and P > 0.05 is ns. The positive control was DuomAb IgG3, and the negative control was Xolair IgG1. [Modes for carrying out the invention]

[0022] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the fields of immunology, biochemistry, genetics, and microbiology.

[0023] All methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, and preferred methods and materials are described herein. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, including definitions, this specification shall prevail. Furthermore, materials, methods, and examples are merely illustrative and are not intended to limit unless otherwise specified.

[0024] In carrying out the present invention, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, and recombinant DNA, which are within the scope of the art of the art, will be used. Such techniques are well described in the literature. 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 (MJ Gait ed., 1984); Mullis et al. al.USPat.No.4,683,195;Nucleic Acid Hybridization(BDHarries & SJHiggins eds.1984);Transcription And Translation(BDHames & SJHiggins eds.1984);Culture Of Animal Cells(RIFreshney,Alan R.Liss,Inc.,1987);Immobilized Cells And Enzymes(IRL Press,1986);B.Perbal,A Practical Guide To Molecular Gene Transfer Vectors For Mammalian Cells(JHMiller and MPSee Calos eds., 1987, Cold Spring Harbor Laboratory; Kuby Immunology (2018, J. Punt & S. Stranford); and Therapeutic Antibody Engineering (2012, 1st Ed.), W. Strohl & L. Strohl.

[0025] The present invention provides a modified antibody comprising an IgG3 hinge and a variable domain having a viral spike protein binding site.

[0026] The present invention provides, and is further characterized by, the following items. 1. A modified antibody containing an IgG3 hinge and a variable domain having a viral spike protein binding site. 2. The modified antibody described in item 1, wherein the Fc region is the IgG3 region. 3. The modified antibody according to item 2, wherein the Fc region comprises a variant thereof having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to the IgG triple heavy chain constant domain (SEQ ID NO: 1) or SEQ ID NO: 1. 4. A modified antibody according to any one of items 1 to 2, comprising an IgG3 Fc region and including one or more substitutions selected from the group consisting of R435H, N392K, and M397V. 5. The modified antibody according to item 4, wherein the IgG3 region comprises three substitutions: R435H, N392K, and M397V. 6. A modified antibody according to any one of items 3 to 5, wherein the IgG3 region comprises a polypeptide having at least 95% identity or at least 98% identity with respect to the modified IgG3 heavy chain constant domain of SEQ ID NO: 2. 7. A modified antibody according to any one of items 3 to 6, wherein the IgG3 region includes the modified IgG3 heavy chain constant domain of SEQ ID NO: 2. 8. The modified antibody described in item 1, wherein the Fc region is the IgG1 region. 9. The modified antibody according to item 8, wherein the modified antibody comprises an IgG1 constant domain (SEQ ID NO: 3) modified with an IgG3 hinge. 10. A modified antibody according to any of items 1 to 9, wherein the viral spike protein is derived from coronavirus. 11. A modified antibody according to any of items 1 to 10, wherein the viral spike protein is the SARS-CoV-2 spike protein. 12. A modified antibody according to any of items 1 to 11, wherein the viral spike protein is selected from the group consisting of SARS-CoV-2 Wuhan spike protein (SEQ ID NO: 4), SARS-CoV-2 delta (B1.617.2) spike protein (SEQ ID NO: 5), and SARS-CoV-2 omicron (B1.1529) spike protein (SEQ ID NO: 6). 13. A modified antibody according to any of items 1 to 12, wherein the viral spike protein is the SARS-CoV-2 Wuhan spike protein (SEQ ID NO: 4). 14. A modified antibody according to any of items 1 to 12, wherein the viral spike protein is the SARS-CoV-2 delta (B1.617.2) spike protein (SEQ ID NO: 5). 15. A modified antibody according to any of items 1 to 12, wherein the viral spike protein is the omicron (B1.1529) spike protein (SEQ ID NO: 6) of SARS-CoV-2. 16. A modified antibody according to any of items 1 to 9, wherein the viral spike protein is derived from orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, arenavirus, or retrovirus, for example, HIV, herpesvirus, rubella, or filovirus. 17. A modified antibody according to any one of items 1 to 16, wherein the IgG3 hinge contains an amino acid sequence that has at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 7). 18. The IgG3 hinge includes an amino acid sequence having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to the amino acid sequence X-(Y)n, where, X is ELKTPLGDTTHTCPRCP (sequence code 8), Y is EPSKCDTPPPCPRCP (Sequence ID 9), A modified antibody according to any of items 1 to 17, wherein n is an integer in the range of 2 to 8, preferably in the range of 2 to 5, and more preferably in the range of 2 to 4. 19. The IgG3 hinge includes an amino acid sequence having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to amino acid sequence XY, where, X is ELKTPLGDTTHTCPRCP (sequence code 8), Y is the modified antibody described in any of items 1-18, which is EPSKCDTPPPCPRCP (Sequence ID 9). 20. The IgG3 hinge has the amino acid sequence X-(Y)m, where, X is ELKTPLGDTTHTCPRCP (sequence code 8), Y is EPSKCDTPPPCPRCP (Sequence ID 9), A modified antibody according to any of items 1 to 19, wherein m is an integer in the range of 1 to 8, preferably in the range of 1 to 5, and more preferably in the range of 1 to 3. 21. The IgG3 hinge includes an amino acid sequence having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to the amino acid sequence X-(Y)n, where, X is ELKTPLGDTTHTCPRCP (sequence code 8), Y is EPKSCDTPPPCPRCP (Sequence ID 26), A modified antibody according to any of items 1 to 17, wherein n is an integer in the range of 2 to 8, preferably in the range of 2 to 5, and more preferably in the range of 2 to 4. 22. The IgG3 hinge includes an amino acid sequence having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to amino acid sequence XY, where, X is ELKTPLGDTTHTCPRCP (sequence code 8), Y is the modified antibody described in any of items 1-17, which is EPKSCDTPPPCPRCP (SEQ ID NO: 26). 23. The IgG3 hinge has the amino acid sequence X-(Y)m, where, X is ELKTPLGDTTHTCPRCP (sequence code 8), Y is EPKSCDTPPPCPRCP (Sequence ID 26), A modified antibody according to any of items 1 to 17, wherein m is an integer in the range of 1 to 8, preferably in the range of 1 to 5, and more preferably in the range of 1 to 3. 24. A modified antibody as described in item 20 or 23, where m is 1. 25. A modified antibody as described in item 20 or 23, wherein m is 2. 26. A modified antibody as described in item 20 or 23, wherein m is 3. 27. A modified antibody as described in item 18 or 21, where n is 2. 28. A modified antibody as described in item 18 or 21, wherein n is 3. 29. A modified antibody as described in item 18 or 21, wherein n is 4. 30. An isolated antibody, a modified antibody as described in any of items 1-29. 31. A modified antibody described in any of items 1-30 that promotes opsonization. 32. A modified antibody described in any of items 1-30 that does not bind to the neutralizing epitope. 33. The modified antibody is selected from the group consisting of Ab11, Ab36, Ab57, Ab59, Ab66, Ab77, Ab81, and Ab94, and is one of the modified antibodies described in any of items 1 to 30. 34. The modified antibody is one of the modified antibodies described in any of items 1 to 33, selected from the group consisting of Ab11, Ab36, Ab57, Ab59, Ab66, and Ab77. 35. A modified antibody according to any one of items 1 to 34, wherein the heavy chain variable domain is SEQ ID NO: 10 and the light chain variable domain is SEQ ID NO: 11. 36. Ab11, a modified antibody as described in any of items 33-35. 37. A modified antibody according to any one of items 1 to 34, wherein the heavy chain variable domain is SEQ ID NO: 12 and the light chain variable domain is SEQ ID NO: 13. 38. The modified antibody described in any of items 33, 34, or 37, which is Ab36. 39. A modified antibody according to any one of items 1 to 34, wherein the heavy chain variable domain is SEQ ID NO: 14 and the light chain variable domain is SEQ ID NO: 15. 40. The modified antibody described in item 33, 34, or 39, which is Ab57. 41. A modified antibody according to any one of items 1 to 34, wherein the heavy chain variable domain is SEQ ID NO: 16 and the light chain variable domain is SEQ ID NO: 17. 42. The modified antibody described in item 33, 34, or 41, which is Ab59. 43. A modified antibody according to any one of items 1 to 34, wherein the heavy chain variable domain is SEQ ID NO: 18 and the light chain variable domain is SEQ ID NO: 19. 44. The modified antibody described in item 33, 34, or 41, which is Ab66. 45. A modified antibody according to any one of items 1 to 34, wherein the heavy chain variable domain is Sequence ID No. 20 and the light chain variable domain is Sequence ID No. 21. 46. ​​The modified antibody described in item 33 or 45, which is Ab77. 47. A modified antibody according to any one of items 1 to 33, wherein the heavy chain variable domain is SEQ ID NO: 22 and the light chain variable domain is SEQ ID NO: 23. 48. The modified antibody described in item 33 or 47, which is Ab81. 49. A modified antibody according to any one of items 1 to 33, wherein the heavy chain variable domain is SEQ ID NO: 24 and the light chain variable domain is SEQ ID NO: 25. 50. The modified antibody described in item 33 or 49, which is Ab94. 51. A composition comprising at least two different modified antibodies as defined in any of items 1 to 50. 52. The composition according to item 51, comprising at least three, at least four, at least five, at least eight, or at least ten different modified antibodies as defined in any of items 1 to 50. 53. A composition according to any of items 51 to 52, comprising 3 to 15, 3 to 12, 3 to 9, 5 to 15, 5 to 10, or 6 to 10 different modified antibodies as defined in any of items 1 to 50. 54. The composition according to any one of items 51 to 52, wherein at least two, at least three, at least four, at least five, at least eight, or at least ten of the different modified antibodies have different viral spike protein binding moieties. The composition according to item 53, wherein the different modified antibodies of 55.3-15, 3-12, 3-9, 5-15, 5-10, or 6-10 have different viral spike protein binding sites. 56. A pharmaceutical composition, as described in any of items 51 to 55. 57. A composition according to any one of items 51 to 56, further comprising pharmaceutically acceptable excipients. 58. A composition described in any of items 51 to 57, which is sterile. 59. A nucleic acid molecule encoding a protein containing a modified antibody as defined in any of items 1-50. 60. A nucleic acid molecule described in item 59 that encodes a modified antibody as defined in any of items 1 to 50. 61. Cells containing nucleic acids as described in item 59 or 60. 62. Use of a modified antibody as defined in any of items 1 to 50 to prevent, treat, or mitigate a viral infection caused by a virus containing the spike protein. 63. Use of a modified antibody as defined in any of items 1 to 50 for Fc-mediated phagocytosis of a virus containing the spike protein. 64. Use of a modified antibody, as defined in any of items 1-50, to enhance opsonization. 65. Use of any composition defined in items 51 to 58 to prevent, treat, or mitigate a viral infection caused by a virus containing the spike protein. 66. Use of any composition defined in items 51-58 for activating the complement pathway. 67. Use of a composition defined in any of items 34 to 41 for the Fc-mediated phagocytosis of a virus containing the spike protein. 68. Use as described in any of items 54-58, wherein the spike protein is derived from coronavirus. 69. Uses described in any of items 54-59, wherein the viral spike protein is the SARS-CoV-2 spike protein. 70. Use as described in any of items 62 to 69, wherein the viral spike protein is selected from the group consisting of SARS-CoV-2 Wuhan spike protein (SEQ ID NO: 4), SARS-CoV-2 delta (B1.617.2) spike protein (SEQ ID NO: 5), and SARS-CoV-2 omicron (B1.1529) spike protein (SEQ ID NO: 6). 71. Use as described in any of items 62-70, wherein the viral spike protein is the SARS-CoV-2 Wuhan spike protein (SEQ ID NO: 4). 72. Use as described in any of items 62-70, wherein the viral spike protein is the SARS-CoV-2 delta(B1.617.2) spike protein (SEQ ID NO: 5). 73. Use as described in any of items 62-70, wherein the viral spike protein is the omicron (B1.1529) spike protein of SARS-CoV-2 (SEQ ID NO: 6). 74. Uses of any of items 62-67, wherein the viral spike protein is derived from orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, arenavirus or retrovirus, for example, HIV, herpesvirus, rubella or filovirus. 75. A method for preparing a modified antibody as defined in any of items 1 to 50, comprising the following steps for obtaining the modified antibody: -Immunization of a host organism by the viral spike protein or a protein containing the viral spike protein, - Isolation of at least one viral spike protein-reactive B cell to isolate an IgG1 antibody that binds to the viral spike protein, - Preparation of at least one recombinant DNA molecule encoding a modified form of the isolated IgG1 antibody, comprising replacing the IgG1 subclass-specific domain of the isolated IgG1 antibody with an IgG3 subclass-specific domain. - To express at least one recombinant DNA molecule in a suitable host cell. 76. A method for preparing a modified antibody as defined in any of items 1 to 50, comprising the following steps for obtaining the modified antibody: -Immunization of a host organism by the viral spike protein or a protein containing the viral spike protein, - Isolation of at least one viral spike protein-reactive B cell to isolate an IgG1 antibody that binds to the viral spike protein, - Preparing at least one recombinant DNA molecule encoding a modified form of the IgG1 antibody, wherein the modification includes replacing the IgG1 hinge of the isolated IgG1 antibody with an IgG3 hinge. - To express at least one recombinant DNA molecule in a suitable host cell. 77. The method according to item 76, 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 with respect to the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 7). 78. The IgG3 hinge includes an amino acid sequence having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to the amino acid sequence X-(Y)n, where, X is ELKTPLGDTTHTCPRCP (sequence code 8), Y is EPSKCDTPPPCPRCP (Sequence ID 9), The method according to item 76, wherein n is an integer in the range of 2 to 8, preferably in the range of 2 to 5, and more preferably in the range of 2 to 4. 79. The IgG3 hinge includes an amino acid sequence having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to the amino acid sequence X-(Y)n, where, X is ELKTPLGDTTHTCPRCP (sequence code 8), Y is EPKSCDTPPPCPRCP (Sequence ID 26), The method according to item 76, wherein n is an integer in the range of 2 to 8, preferably in the range of 2 to 5, and more preferably in the range of 2 to 4. 80. The method described in item 78 or 79, wherein n is 2, 3 or 4. 81. The method according to any one of items 75-80, wherein the spike protein is derived from a coronavirus. 82. The method according to any one of items 75 to 81, wherein the viral spike protein is the SARS-CoV-2 spike protein. 83. The method according to any one of items 75 to 82, wherein the viral spike protein is selected from the group consisting of SARS-CoV-2 Wuhan spike protein (SEQ ID NO: 4), SARS-CoV-2 delta (B1.617.2) spike protein (SEQ ID NO: 5), and SARS-CoV-2 omicron (B1.1529) spike protein (SEQ ID NO: 6).

[0027] The compositions and formulations according to the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions and formulations. For example, the compositions and formulations are prepared using pharmaceutically acceptable components, such as carriers, excipients, or stabilizers, in a manner that allows them to be appropriately stored and administered. Such pharmaceutically acceptable components are non-toxic in the amounts used when the pharmaceutical composition or formulation is administered to a patient. The pharmaceutically acceptable components added to the pharmaceutical composition or formulation may depend on the chemical properties of the inhibitors and targeting agents present in the composition or formulation (for example, whether the targeting agent is 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.

[0028] In preferred embodiments of the present invention, the composition or formulation is suitable for administration to humans, and preferably, the formulation is sterile and / or non-pyrogenic.

[0029] Definitions of some terms. Unless otherwise defined below, terms used in this invention shall be understood in accordance with the general meanings known to those skilled in the art.

[0030] The term “antibody” (e.g., monoclonal antibody) as used herein shall be understood in accordance with the general meaning known to those skilled in the art. Antibodies may be derivatized antibodies or may be linked to different molecules. For example, molecules that may be linked to an antibody include other proteins (e.g., other antibodies), molecular labels (e.g., fluorescent, luminescent, colored, or radioactive molecules), pharmaceuticals, and / or toxic agents. Antibodies or antigen-binding sites may be linked directly (e.g., in the form of fusion between two proteins) or via linker molecules (e.g., any suitable type of chemical linker known in the art).

[0031] As used herein, the term “isolated antibody” should be understood as an antibody that has been recovered or purified to some extent. In some embodiments, this means that it does not include a large number of antibodies with different structures, such as more than 25 antibodies. In other embodiments, this means that it does not include different antibodies that do not bind to the spike protein.

[0032] The term “modified antibody” as used herein shall be understood in accordance with the general meaning known to those skilled in the art. Modified antibodies are novel recombinant antibody molecules having improved antigen specificity and effector function, and are typically produced by recombinant DNA technology and protein engineering technology.

[0033] As used herein, the term “IgG3 hinge” is understood to mean a hinge region (including its variants) derived from an antibody of the IgG3 class. A non-limiting example of an IgG3 hinge is a peptide having an amino acid sequence that has at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to the amino acid sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 7). Another example of an IgG3 hinge is having an amino acid sequence that has at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to the amino acid sequence X-(Y)n, where 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 2 to 5, more preferably 2 to 4.

[0034] The term "IgGh" used herein 17 "IgGh" 32 "IgGh" 47 "IgGh" 62The term "<T>" is understood to mean the IgG3 hinge variant with a core hinge (SEQ ID NO: 8), and variants having a core hinge (SEQ ID NO: 8) and one, two, or three exon repeats (each SEQ ID NO: 9). Therefore, the suffix indicates the number of amino acid residues in each hinge.

[0035] As used herein, the term “viral spike protein” refers to the spike-like proteins on the outer surface of many viruses. In contrast to membrane proteins and envelope proteins, which are primarily involved in viral assembly, spike proteins play a crucial role in invading host cells and initiating infection.

[0036] As used herein, the terms “binding” or “conjugation” in relation to antibodies refer to the formation of a complex with the molecule to be bound, for example, the spike protein of SARS-CoV-2. Binding is usually non-covalent, occurring through intermolecular forces such as ionic bonds, hydrogen bonds, and van der Waals forces, and is typically reversible. Various methods and assays for determining binding ability are known in the art. Typically, binding is high-affinity binding, with affinity measured by KD value 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, and even more preferably less than 1 pM.

[0037] As used herein, each occurrence of terms such as “comprising” or “comprises” may be optionally replaced with “consisting of” or “consists of.”

[0038] In the present invention, terms such as “treating,” “preventing,” or “mitigating” refer to therapeutic treatments. The effectiveness of a therapeutic treatment can be evaluated, for example, by assessing whether the treatment prevents or suppresses viral infection in the patient(s) being treated. Preferably, prevention or inhibition is statistically significant, as can be evaluated by appropriate statistical tests known in the art. Inhibition or prevention of viral infection can be evaluated by comparing the degree of viral infection in a group of infected or exposed subjects administered the modified antibody according to the present invention with that of a control group of infected or exposed subjects who receive a placebo. Alternatively, it can be evaluated by comparing a group of infected patients receiving standard antiviral treatment in the art and treatment according to the present invention with a control group of patients receiving only standard antiviral treatment in the art. Such tests for evaluating inhibition or prevention of viral infection are designed according to criteria recognized as clinical trials, for example, according to double-blind, randomized trials with sufficient statistical power.

[0039] As used herein, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory authority, or that it is listed in the United States Pharmacopeia, the European Pharmacopoeia, or any other generally accepted pharmacopoeia for use in mammals, more specifically in humans. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffers, and combinations thereof.

[0040] The sequences referred to herein are as follows: Sequence ID 1 (IgG triple-chain constant domain) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK Sequence ID 2 (Modified IgG triple-stranded constant domain) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHFTQKSLSLSPGK Sequence ID 3 (IgG1 constant domain modified with IgG3 hinge) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 4 (Wuhan Sars-CoV-2 spike protein) Sequence ID 5 (Delta(B1.617.2) spike Sars-CoV-2 mutant) Sequence ID 6 (Omicron (B1.1529) spike Sars-CoV-2 mutant) Sequence ID 7 (IgG3 hinge region) ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP Sequence ID 8 (IgG3 hinge portion) ELKTPLGDTTHTCPRCP Sequence ID 9 (IgG3 hinge portion) EPSKCDTPPPCPRCP Sequence ID 10 (heavy chain variable domain of clone 11) QVTLKESGPVLVKPTETLTLTCTVSGFSLSNAKMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSSLTISKDTSKSQVVLTMTNMMDPVDTATYYCARLLWFGGNYFDYWGQGTLVTVSS Sequence ID 11 (Light chain variable domain of clone 11) SYELTQPLSVSVALGQTARITCGGNNIGSKMHWYQQKPGQAPVLVIYRDSNRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSTVVFGGGTKLTVL Sequence ID 12 (heavy chain variable domain of clone 36) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHAVHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDPVLRYFDWTTPYYFDYWGQGTLVTVSS Sequence ID 13 (Light chain variable domain of clone 36) EIVLTQSPGTLSLSPGERATLSCRASQSFSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPLTFGGGTKVEIK Sequence ID 14 (heavy chain variable domain of clone 57) QVTLRESGPALVRPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLARIDWDDDKYYSTSLRTRLTISKDTSKNQVVLTMTNMMDPVDTATYYCARMTVTTAFDIWGQGTMVTVSS Sequence ID 15 (Light chain variable domain of clone 57) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTKVEIK Sequence ID 16 (heavy chain variable domain of clone 59) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTAYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARGGRYCSSTSCYSGVPNDYWGQGTLVTVSS Sequence ID 17 (Light chain variable domain of clone 59) DIQLTQSPSFLSASVGDRVTITCRASQGVSSYLAWYQQKPGKAPKLLIYPASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK Sequence ID 18 (heavy chain variable domain of clone 66) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRSFVSSSTDDYYYYYDMDVWGQGTTVTVSS Sequence ID 19 (Light chain variable domain of clone 66) SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSGTYVVFAGGTKLTVL Sequence ID 20 (heavy chain variable domain of clone 77) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPG KGLEWVALISYDGSNTYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVFYCAKTIYSYALKPNYFDYWGQGTLVTVSS Sequence ID 21 (Light chain variable domain of clone 77) DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGK APKLLIYDASNLETGVPSRFSGSGSGTFTFTISSLQPEDIA TYYCQQYDNLPPTFGGGTKVGIK Sequence ID 22 (heavy chain variable domain of clone 81) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPG QGLEWWMGWISAYNGNTNYAQKVQGRVTMTRDTSTSTAYMELR SLRSDDTAVYYCARDGIAVAEDWFDPWGQGTLVTVSS Sequence ID 23 (Light chain variable domain of clone 81) SYELTQPPSVSVAPGKTARITCGENNIGSKSVHWYQQKPGQA PVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAD YYCQVWDGSSDHVVFGGGTKLTVL Sequence ID 24 (heavy chain variable domain of clone 94) QVQLQESGPRLVKPSGTLSLTCAVSGGSISSSNWWTWVRQPP GKGLEWIGEIYHSGSTNYTPSLKSRVTISVDKSKNQFSLRLN SVTAADTAVYYCARGWSSSWYGLDYWGQGTLVTVSS Sequence ID 25 (Light chain variable domain of clone 94) QSALTQPASVGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLVIYEATKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCYSYAGSSTWVFGGGTKLTVL Sequence number 26 (IgG3 hinge portion) EPKSCDTPPPCPRCP [Examples]

[0041] Materials and methods IgG3 plasmid generation, production, and sequencing The anti-spike monoclonal antibody used in this study was generated from spike-reactive B cells derived from convalescent patients infected with SARS-CoV-2 6 weeks prior (clinically validated). Briefly, the V(D)J coding sequence derived from spike-reactive B cells was generated using 10X Genomics. The variable region was cloned into a pTwist IgG1 vector (Twist Biosciences) containing the constant heavy chain domain of IgG1. A plasmid containing the constant heavy chain domain gene of IgG3 was previously generated (Izadi et al 2024, accepted by Nature Comm). The sequences of the recombinant vectors for the light and heavy chain plasmids containing the constant domains are shown in Supplementary Document 1. The primers were designed so that the insert (IgG3 heavy chain constant domain) contained a complementary overhang to the vector, allowing homologous recombination to occur. This made it possible to replace the constant domain of IgG1 with the constant domain of IgG3 while keeping the variable region intact. Heavy chain constant domain replacement was performed by PCR of the IgG1 vector backbone and by substituted the IgG1-specific domain in the IgG3 constant region. Subsequent HIFI-DNA assemblies (NEB, biolAb) mediate the homologous recombination process. PCR products were loaded onto agarose gels to assess correct amplification based on size. Bands were excised and purified using the QIAGEN PCR cleanup kit according to the manufacturer's instructions. Competent E. coli supplied with the kit were transformed with the newly assembled IgG3 plasmid and seeded on agar plates containing ampicillin. The purified IgG3 plasmid from the transformed colonies was validated by sequencing.

[0042] Cell culture and antibody production Expi293F suspension cells were purchased from Gibco (ThermoFisher) and cultured as prescribed in 30 ml of Expi293 medium (Gibco) at 37°C, 8% CO2, in a 125 ml Erlenmeyer flask (Nalgene) in an Eppendorf S41i shaker incubator at 120 rpm. The cells were subcultured as prescribed and 0.5 x 10⁶ cells were passed every 3-4 days.6 The cells were divided into 2x10⁶ cells / ml densities. The day before transfection, the cells were divided into 2x10⁶ cells. 6 The cells were seeded at a density of cells / ml. The next day, the cells were divided into 7.5 x 10 7 Cells were seeded in 25.5 ml of Expi293 medium. Transfection with heavy and light chain plasmids was performed using the Expifectamine293 kit (Gibco) according to the manufacturer's instructions. Briefly, 20 μg each of heavy and light chain plasmids were mixed with 2.8 ml of OptiMEM (Gibco) and 100 μl of Expifectamine, and incubated at room temperature for 15 minutes. The transfection mix was then added to Expi293F cells. The following day, 1.5 ml of enhancer 1 and 0.15 ml of enhancer 2 (both from the Expifectamine293 kit) were added, and the cells were cultured for a further 72 hours.

[0043] Cells were removed from the cell culture medium by centrifugation (400x g, 5 min), and the supernatant was transferred to a new tube. To capture IgG from the medium, Protein G sepharose 4 Fast Flow (Cytiva) was added to the medium and rotated incubated at room temperature for 2 hours. The beads were collected by passing the medium bead mix through a gravity flow chromatography column (Biorad) and washed twice with 50 ml of PBS. The antibody was eluted using 5 ml of HCl-glycine (0.1 M, pH 2.7). The pH was neutralized using Tris (1 M, pH 8, 1 ml). The buffer was replaced with PBS using an Amicon Ultra-15 centrifuge filter (Sigma) with a molecular cutoff of 30,000 Da. The concentration and quality of the purified antibody were measured by spectrophotometric analysis using the IgG setting of NanoDrop (Denovix), and the antibody was further subjected to SDS-PAGE, with band intensity compared to serial dilutions of Xolair (omalizumab).

[0044] Generation of spike-coated beads The spike protein was generated by transfecting Expi293F cells with a 40 μg plasmid containing the spike protein gene (a CS / PP spike encoding a secretible version of the protein was used to allow purification from the cell culture supernatant), and this plasmid was previously provided by Dr. Florian Krammer's laboratory. 5 200 μg of spike protein was biotinylated according to the instructions of the EZ-Link® Micro Sulfo-NHS-LC biotinylation kit (ThermoFisher). Then, 500 μl (5 x 10) was used. 7 One million fluorescent (APC) streptavidin microsphere beads (1 μm, Bangs Laboratories) were conjugated with biotinylated spike protein (200 μg) according to the manufacturer's instructions.

[0045] Flow cytometry-based avidity measurement Binding assays were performed in 96-well plates pre-coated overnight at 4°C with 200 μl of 2% BSA (in PBS). 100,000 spike-coated beads were used in all wells, with antibody concentrations ranging from 0.02 to 0.2 to 2 μg per ml. The beads were opsonized in 100 μl of 1X PBS at 37°C for 30 minutes on a shaking heat block. The wells were washed twice with PBS. To evaluate antibody binding to the spike beads, fluorescence signals were generated using 50 μl (1:500 dilution) of Fab-specific fluorescent secondary antibody (#109-546-097, Jackson ImmunoResearch). The secondary antibody was incubated with the spike-bead antibody conjugate at 37°C for 30 minutes on a shaking heat block. 100 μl of PBS was added to the wells before flow cytometry analysis. Beads were analyzed using a Beckman Coulter Cytoflex flow cytometer, acquiring 20,000 beads per sample. Data were processed using Flowjo. Gating was set for spike beads based on forward and side scattering. Gating of spike beads positive for the antibody was set based on reactivity to an unreactive IgG control mAb. The data were then analyzed using a nonlinear regression model in GraphPad Prism: Hill slope was unconstrained, and the maximum value (Bmax) was set to 100%. D The value was constrained to any value between 0 and 10000. The best fit is presented in a graph showing the model's goodness of fit. SPR kinetic assay

[0046] To test the binding reaction rate to spike trimers, RBD, or NTD, high-volume amine sensor tips (Bruker) were immobilized with anti-human IgG(Fc) antibody (Cytiva BR-1008-39) at 25 μg / ml in 10 mM sodium acetate buffer at pH 5 at a flow rate of 10 μl / min and kept in contact for 300 seconds. This was performed using a MASS-16 biosensor instrument (Bruker). Running buffer was PBS + 0.05% Tween 20. The antibody was diluted in PBS and injected onto the surface at 10 μL / min for 90 seconds. The running buffer was PBS containing 0.01% Tween 20. The RBD antigen used was produced using HEK293 obtained from Sinobiological (Beijing, China: RBD WT 40592-V08H). Spike trimers were generated as described above. NTD antigens were obtained from Sinobiological (Beijing, China: NTD 40591-V9H and NTD-Omicron 40591-V08H42). RBD antigens were diluted from 60 nM to 3.75 nM by sequential half dilutions. Similarly, NTD antigens were diluted from 80 nM to 2.5 nM by sequential half dilutions. A spike trimer was added at 40 nM. Antigens were injected at these concentrations and interacted with the sensor at a flow rate of 30 μl / min for 2 minutes, followed by dissociation for 6 minutes. After each cycle, the surface was regenerated with 3 M MgCl. All experiments using RBD and NTD were performed three times, and the experiment using the spike trimer was performed once. Data were analyzed using the Sierra Analyzer software version 3.4.3 (Bruker) program, and the apparent dissociation constant (K) was determined. D ) was decided.

[0047] ELISA avidity measurement For ELISA, spike protein at 1 μg / ml in PBS was immobilized overnight at 4°C on an ELISA high-binding plate (Sarstedt). The wells were washed three times with PBST (1x PBS containing 0.05% Tween 20) and then blocked with 2% BSA / PBS at room temperature for 1 hour. After the three washes, serial dilutions of the primary antibody (anti-spike mAb) were added (100 μl of mAbs at concentrations ranging from 0.08 to 20 μg / ml) and incubated at room temperature for 1 hour. The wells were washed three times with PBS. Rabbit anti-human light chain-HRP secondary antibody at a 1:5000 dilution in PBS (anti-kappa for clone 57, anti-lambda for clones 11 and 66) (Abcam ab202549 and ab200966) was added and incubated at room temperature for 1 hour. The wells were washed three more times with PBST. Finally, 100 μl of chromogenic reagent (20 ml of sodium citrate pH 4.5 + 1 ml of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 0.2 g in 10 ml of water, Sigma) + 0.4 ml of 0.6% H2O2) was added. OD450 was recorded after 15-30 minutes. The data were plotted using GraphPad Prism.

[0048] C1q deposition assay To test complement activation, 500,000 spike beads were opsonized with 0.1–1–10 μg / ml mAbs in a 96-well plate pre-coated with 2% BSA, and 1% antibody-depleted serum was used as the complement source. After opsonization for 30 minutes on a shaking heat block (300 RPM) at 37°C, the wells were washed twice with PBS, and FITC fluorescently labeled anti-C1q Ab (ab4223, Abcam) was added (diluted 1:250 from stock, 50 μl). The anti-C1q Ab was left with the beads on a shaking heat block (300 RPM) at 37°C for 30 minutes. PBS was added to resuspend the beads in a final volume of 150 μl. Gating for spike beads was set based on forward and side scattering, and gate for C1q deposition was set based on the results for control IgG1 (Supplementary Figure 5A). The acquired data was analyzed using Flowjo and plotted using GraphPad Prism. 50 The analysis was performed using a nonlinear regression model in GraphPad Prism. For the analysis of bead-C1q deposition %, the lower limit was constrained to 0 and the upper limit to 100%. MFI EC 50 For the analysis, an upper limit was shared and set to be greater than 0.

[0049] Flow cytometry-based phagocytic assay Phagocytosis experiments were performed using the same batch of spike beads as those used in the binding assay. To evaluate internalization, red fluorescent beads were stained with pH-sensitive dye pHrodo green (P35369, ThermoFischer) in 95 μl of Na2CO3 (pH9) using 5 μl of 10 mM pHrodo green (ThermoFischer) at 37°C for 30 minutes. Excess staining was washed off with PBS. THP-1 cells (Sigma-Aldrich) were cultured as previously described. 5 In all experiments, 1 x 10 5 THP-1 cells were used. In one experimental setup, the cell-to-bead ratio was 1:30 (predation multiplicity, MOP, 30), and in the other, it was 1:15 (MOP, 15). In the MOP 30 experiment, 3 x 10 6Each spike bead was opsonized with 10 μg / ml antibody in 100 μl 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). In the MOP 15 experiment, 1.5 x 10 6 Each spike bead was opsonized with 5 μg / ml mAb. Both opsonizations were performed in 100 μl volumes on a shaking heat block (300 RPM) at 37°C for 30 minutes. During this incubation period, THP-1 cells were counted (using a Burker chamber), and the medium was changed from RPMI to sodium medium. THP-1 cells (50 μl, 2 x 10⁶) 6 A solution ( / ml) was added to each well, and the cells were allowed to phagocytose the beads on a shaking heat block (300 RPM) at 37°C for 30 minutes. The 96-well plate was then incubated on ice for 15 minutes and analyzed directly using a Beckman Coulter Cytoflex flow cytometer. THP-1 cell gating was set based on their forward and side scattering (Supplementary Figure 4A). Internalization and association gating was set with cell-only negative controls (Figure 3A). Analysis was stopped after 5000 events had been taken up by the THP-1 gate. The data were analyzed using the Flowjo program by setting the same gates as above. The Flowjo-processed data was further analyzed using GraphPad Prism, and THP-1 gate internalization and bead signals (APC-A) were plotted to compare different antibodies.

[0050] Monocytes were isolated from blood by first obtaining the PBMC layer using polymorphoprep (Abbot). The monocytes were purified from the PBMC layer by positive selection using CD14 microbeads (catalog number 130-050-201, Miltenyi Biotec) according to the manufacturer's instructions. After isolation, monocytes were counted using an XN-350 blood analyzer (Sysmex). Neutrophils were then isolated using a polymorphprep gradient according to the manufacturer's instructions and counted in a Burker chamber. After resuspending the isolated cells in sodium medium, 2 x 10⁻⁶ cells were subjected to a 2x10⁻⁶ filtration rate. 6 The cells were adjusted to cell / ml and maintained on ice for 1 hour. Donors gave written and verbal consent to participate in this study and provided verbal information regarding the purpose of donation, which was to isolate and use monocytes and neutrophils only. This study utilized the previously approved ethical approval (2020 / 01747) from the Swedish Ethics Review Authority.

[0051] In the monocyte phagocytosis experiment, spike beads were opsonized with 10 μg / ml mAb (IgG control mAb or OctomAb IgG3), but serum was not included. Monocytes (1 x 10) 5 Cells were added in a volume of 50 μl. The cells were phagocytosed on a shaking heat block (300 RPM) at 37°C for 30 minutes. Subsequently, CD14 antibody (Brilliant Violet 421® anti-human CD14 antibody, Biolegend) was diluted 1:50 to 3 μg / ml and added in a volume of 20 μl, and the cells were stained on ice for 20 minutes. The wells were analyzed using a Beckman Coulter Cytoflex flow cytometer. Monocytes were gated by size and particle size (Supplementary Figure 2C). Further selection was performed by gating CD14-positive cells. Analysis was stopped after 3000 events were recorded with the CD14+ gate. The percentage of cells associated with spike beads (APC+) and cells internalized with beads (APC+, FITC+) (Supplementary Figure 2C) was plotted against MOP in Prism Graphpad.

[0052] In the neutrophil experiment, spike beads were opsonized with 10 μg / ml mAb containing 1% antibody-depleted serum (Pel-Freeze, catalog number 34014-10) on a shaking heat block (300 RPM) at 37°C for 30 minutes (final volume 100 μl). To study only the effect of Fc-mediated phagocytosis, the serum was heated and inactivated at 56°C for 1 hour. Both heat-inactivated serum and normal human serum were added to the respective wells. Neutrophils (1 x 10⁶) 5 50 μl (2 x 10 cells) 6 The solution was added to each well at a volume of ( / ml). Cells were phagocytosed on a shaking heat block (300 RPM) at 37°C for 30 minutes. The phagocytic process was stopped by placing the plate on ice. On ice, anti-CD18 antibody (BV421 mouse anti-human CD18, BD Biosciences) was diluted 1:100 to 2 μg / ml, and 20 μl of this diluted antibody was added to the cells to stain them. After staining the cells on ice for 20 minutes, they were analyzed using a Beckman Coulter Cytoflex flow cytometer. To mark neutrophils, gates were set for FSC-A and SSC-A (Supplementary Figure 2D). This population was further selected using the CD18 marker (Supplementary Figure 6B). After 3000 events were taken up by the CD18+ gate, the analysis was stopped, and the data were processed using a program for Flowjo with the same gates as above. The percentage of cells with internalized beads and bead signals (APCs) in the CD18 gate was used as an indicator for comparing mAbs.

[0053] Quality control experiment for IgG mAb aggregation After mAb generation and purification, quality control of the mAbs was performed, and testing was conducted to determine if aggregation could affect the results. IgG1 and IgG3 from clone 81 were used as sample subjects. The samples were centrifuged at 16000 RCF at room temperature for 10 minutes. After centrifugation, the supernatant was removed and sterile filtered through a 0.2 micron syringe filter. Next, the treated Ab81 IgG1 and IgG3 were compared with their untreated variants to determine if there were any differences in function. Phagocytic experiments were performed using THP-1 cells in MOP 30 as described above.

[0054] Live imaging phagocytic assay Cell preparation and opsonization of spike beads Neutrophils were isolated from four healthy donors using Polymorphprep as described above. Then, cells were divided into 2x10 cells per donor. 6 Cells were placed in aliquots of 1 / ml on ice for 1 hour. Cells were then placed in 8-well Ibidi plates (catalog number 80827, Ibidi GmbH, Germany) in a 1x10⁶ arrangement. 5 Cells were seeded at a cell / well density in 5% CO2 at 37°C for 1 hour. All Ibidi plates were pre-coated with 3.3 μg / ml human fibronectin (F0895-1MG, Sigma) diluted in PBS. After incubation, cells were stained with Alexa Fluor 594-conjugated wheat germ agglutinin (WGA), diluted 1:333 in Hoechst 1:20000 (ThermoFisher, Germany) and sodium medium to a final volume of 300 μl / well. Excess stain was washed three times with 300 μl / well of sodium medium, and then 250 μl / well of sodium medium was added.

[0055] Spike biotinylated beads were pre-stained with pHrodo Green and then opsonized with 10 μg / ml antibodies (IgG3 OctomAb, IgG1 OctomAb, Ab94 IgG3, control IgG1) and 1% antibody-depleted serum (Pel-Freeze, catalog number 34014-10). The beads were then mixed in 100 μl volumes (1 x 10⁻¹⁶). 6 The beads were opsonized on a shaking heat block at 37°C for 30 minutes. After opsonization, 5x10 5 Beads / well (50 μl) were added to pre-seed neutrophils immediately before the start of live imaging.

[0056] Live fluorescence imaging and data acquisition Time-lapse images of phagocytosis were recorded every 10 minutes for 60 minutes using a Nikon N-SIM microscope. Images were acquired using a fluorescence microscope with a 20x Plan Apo λ objective lens (NA=0.40) and a perfect focus system (PFS). All images were acquired at 8 positions per well using an ORCA-Flash 4.0 cCMOS camera (Hamamatsu Photonics KK). The filter cubes used were DAPI (Ex. 340~380nm, Em. 435~485nm), TxRED (Ex. 540~580nm, Em. 600~660nm), FITC (Ex. 465~495nm, Em. 515~555nm), and Cy5 (Ex. 625~650nm, Em. peak 670nm). Samples were live-imaged in an environmental chamber (Okolab) at 5% CO2 and 37°C. All image analysis was performed using Nikon software General Analysis 3. Graphs and statistics were compiled in Microsoft Excel and Prism 9. ET50 analysis was performed using Prism Graphpad with a nonlinear regression model, without constraints on the hill slope, and constrained to a minimum value of 0% and a maximum value of 100%.

[0057] SIM image acquisition After 60 minutes of phagocytosis, the samples were fixed in 4% paraformaldehyde containing PBS at 37°C for 15 minutes. After washing the wells three times with sodium culture medium, SIM imaging was performed. All SIM images were acquired using a Nikon N-SIM microscope equipped with a LU-NV laser unit (488 / 561 / 640nm), a CFI SR HP Apochromat TIRF 100X oil objective lens (NA=1.49), and an ORCA-Flash 4.0 cCMOS camera (Hamamatsu Photonics KK). The filter cubes used were N-SIM 488 (Ex. 484~496nm, Em. 500~545nm), N-SIM 561 (Ex. 557~567nm, Em. 579~640nm), and N-SIM 640 (Ex. 629~645nm, Em. 663~738nm). Images were acquired in the DAPI channel of the SIM system using a standard fluorescence microscope. A SPECTRA X light engine (Lumecore Inc.) was used for wide-field excitation. A 15-step Z-stack was collected for each image across all channels. The SIM images were reconstructed using Nikon SIM software with NIS Elements Ar (NIS-A 6D and N-SIM analysis). To minimize blurring from the fluorescence microscope, wide-field images were processed with NIS Element Clarify.ai and Denoise.ai and cropped from a 3x5 grid. After compiling all Z-stack images into maximum intensity projection images, the SIM and fluorescence images were merged into the NIS Element software.

[0058] Animal experiments Female K18 hACE2 (B6.Cg-Tg(K18-ACE2)2Prlmn / J) mice aged 37-10 weeks were divided into five groups of 6 mice each, and the antibody was administered intraperitoneally as a single dose. Eight hours after antibody administration, the animals were 10 5PFU's SARS-CoV-2 (Wuhan strain, isolated strain SARS-CoV-2 / 01 / human / 2020 / SWE, supplied by the Swedish Health Authority) was administered intranasally. Mice's weight and health status were recorded daily, and animals were euthanized if their weight decreased by more than 20% or if their health deteriorated severely. After 10 days of infection, the animals were euthanized. Blood, tissue, and bronchoalveolar lavage fluid were collected and stored as appropriate. All animal experiments were conducted under the approval of the Stockholm Regional Animal Experiment Ethics Committee (2020-2021). Viral titers were analyzed using BAL fluid analysis with qPCR. 5 .

[0059] statistical analysis Statistical analysis was performed using GraphPad Prism. A two-tailed Welch's t-test was used for statistical analysis to compare subclasses or mixes of specific clones. When analyzing two or more treatments, a one-way ANOVA with multiple comparison tests was used, and Tukey's test was applied to correct for multiple comparisons.

[0060] Example 1: Changes in avidity to spike proteins by switching the constant domain of IgG1 to IgG3. To date, we have generated 96 antibodies against the SARS-CoV-2 Wuhan spike protein through the isolation of spike-reactive B cells and subsequent single-cell sequencing. 5 Of these 96 clones, 11 were strongly reactive to the spike protein, and all 11 were opsonizable in vitro. These mAbs were produced in the IgG1 subclass, regardless of the original subclass of the patient B cells. Subclass-specific domains of the heavy chain in the heavy chain expression plasmid were replaced using PCR and homologous recombination. Eight new heavy chain plasmids were successfully generated, containing the original variable heavy chain domain and the IgG3 constant domain gene. Numerous allotypes exist for the human IgG3 constant domain. 9 The allotype used in this study was IHG3*11, which, among other differences, contains a long hinge of 62 amino acids.9 The heavy chain plasmid was transfected into Expi293F cells along with the original light chain plasmid to generate IgG3 variants of these antibodies (Figure 1A).

[0061] To measure the binding avidity of newly generated mAbs, biotinylated spike proteins were conjugated to fluorescent streptavidin microsphere beads (1 μm) as a model for SARS-CoV-2 virions. 5 After incubating the mAbs with spike beads, a secondary fluorescent anti-Fab antibody was added, and IgG binding on the beads was detected using flow cytometry as previously performed. 5 (Figure 1B). Avidity is calculated by fitting a nonlinear regression model to dose-binding data. The inventors observed that six of the eight IgG3 antibodies had comparable avidity to their IgG1 counterparts (Figures 1C-D). However, there was a significant change in Ab57, where the IgG3 version showed increased avidity compared to the IgG1 parent (Figures 1C-D). On the other hand, Ab11 showed a significant decrease in binding to the spike as an IgG3 antibody, while the IgG1 version maintained high reactivity. It is important to note the different clonal origins of Ab57 and Ab11. Ab57 and Ab11 evolved as IgG1 and IgG3 in vivo. Unfortunately, the information obtained from sequencing the IgG3 constant domain of clone 11 was incomplete. Therefore, the allotype present in vivo in the donor patient could not be determined. In both of these cases, the original subclass (donor B cells), IgG3 from clone 11, and IgG1 from clone 57 bound to the spike protein with lower avidity than the subclass-switched mAb mutants, IgG1, and IgG3, respectively (Figure 1D).

[0062] To complement flow cytometry-based binding data, surface plasmon resonance (SPR) was performed to investigate spike-mAb affinity in each monovalent domain. For clones 11–81, the receptor-binding domain (RBD) of the spike protein was used as the analyte, while for clone 94, the N-terminal domain (NTD) was used. These clones originated from previous studies where binding to their respective domains was known. 5 In this experimental setup, none of the Ab11, 36, or 57 subclasses were able to bind to RBD. For Ab59, Ab66, Ab77, Ab81, and Ab94, affinity (k A and K D No significant differences were observed (Supplementary Figure 2A), which supported the aforementioned avidity data obtained by spike bead flow cytometry. Similar experiments were performed to measure the binding of Ab11, 36, and 57, but RBD as the ligand was replaced with intact spike protein expressed as a trimer. Consistent with our previous findings, Ab11 showed low reactivity to the spike protein when expressed as IgG3, more than 25-fold lower (Figures 1E-F). However, no difference was observed between IgG1 and IgG3 in clone 36. In this experimental setup, clone 57 showed apparent K D This showed a twofold difference (Figures 1E-F). Results using SPR confirmed our data that laboratory-generated forms of Ab11 (as IgG1) and Ab57 (as IgG3) had higher avidity to the spike protein than their respective original native subclasses.

[0063] Furthermore, findings regarding Ab11 and Ab57 were demonstrated by coating ELISA plates with spikes and testing mAb reactivity. ELISA results showed that when expressed as IgG3, Ab57 bound more strongly to spikes, exhibiting a signal several times stronger than IgG1 (Figure 1G, Supplementary Figure 3A). Unsurprisingly, Ab11 IgG3 showed decreased reactivity to spikes (Figure 1G, Supplementary Figure 3A), while IgG1 showed strong reactivity. In summary, the three different methods demonstrate that for clones 36, 59, 66, 77, 81, and 94, replacing the constant domain from IgG1 to IgG3 does not affect avidity to the spike antigen. However, for clones 11 and 57, modification of the constant domain resulted in significant changes in avidity. In these two cases, the binding avidity of the artificially created subclass was superior, meaning the best-binding subclass was not the original subclass supplied from the donor B cells (Figures 1C-D, 1E-F, and 1G).

[0064] Example 2: IgG3 is a more potent inducer of Fc-mediated phagocytosis than IgG1. In our previous study, we used the THP-1 cell line to investigate how IgG1 monoclonal cells promote Fc-mediated phagocytosis of spike beads. 5 These findings correlated with defense in animal models. Using a similar approach, opsonized spike-coated microsphere beads were incubated with THP-1 cells (Figure 2A), and Fc-mediated phagocytosis was measured. Since all mAbs were discovered by inducing B cells with the original Wuhan-1 spike, we chose to use their antigens for subsequent analysis. The beads were stained with a pH-sensitive dye (pHrodo green) to distinguish between cell populations with internalized beads and cell populations that associated only with the beads (Figure 2A).

[0065] On average, IgG3 mAbs more than doubled the percentage of cells with internalized beads compared to their IgG1 counterparts. This was a consistent trend across all mAb clones except Ab11 IgG3 (Figure 2B), reflecting a decrease in its avidity. When switching from IgG1 to IgG3, the largest increase in magnification (3x) was observed with Ab36 IgG3 (Figure 2B). In terms of potency measured as internalization %, Ab94 (anti-NTD) IgG3 (47%) was the most potent, followed by Ab59 (38%) and Ab81 IgG3 (35%) (Figure 2B, Supplementary Figure 4A). Translating these values ​​into context, Ab94 IgG1, the most potent IgG1 antibody, was similar in potency to IgG3 Ab66, the second weakest IgG3 antibody (17% vs. 20%). Compared to IgG1, IgG3 increased the proportion of phagocytic cells with internalized beads, as well as the amount of opsonized beads associated with phagocytic cells (Figure 2C, Supplementary Figure 4A). Ab94 IgG3 showed a 7-fold increase compared to Ab94 IgG1. Ab81 IgG3 showed a 6-fold increase compared to its IgG1 counterpart, while no difference was observed in Ab11 and Ab57 IgG3. The differences between IgG3 clones in this metric were far greater than the proportion of phagocytic cells with internalized beads. Ab94 IgG3 associated far more beads with THP-1 cells compared to the other antibodies (73,000 MFI), followed by Ab81 IgG3 (44,000). Here again, the most potent IgG1 antibody, Ab59 (10,500), was comparable to the weakest IgG3.

[0066] Our results indicate that the proportion of cells with internalized beads and the amount of beads associated with the cells did not fully correlate, suggesting that each clone exhibited a unique pattern in these metrics. Ab59 IgG3 and Ab81 IgG3 were similar in terms of the proportion of cells with internalized beads (38% vs. 35%), but Ab81 IgG3 induced significantly more bead uptake and had a higher bead signal (44,000 vs. 28,000). Similarly, Ab36 IgG3 was similar to Ab77 IgG3 in terms of the proportion of internalized cells (31% vs. 30%), but there was a significant difference in bead signal (29,000 vs. 17,000). Furthermore, Ab94 and Ab81 IgG3 stood out as the most potent mAbs in both metrics (Figures 2B-C). Overall, swapping the constant domain with IgG3 enhanced Fc-mediated phagocytosis in most of the mAb clones tested. Finally, it is noteworthy that in the case of clones 36, 57, 66, 77, and 81, which were originally IgG1 in their supply form, the switched IgG3 form was functionally superior to that originally encoded by the donor B cells.

[0067] Example 3: The IgG3 oligoclonal cocktail enhances Fc-mediated phagocytosis compared to a single monoclonal. While monoclonal IgG3 exhibits potent opsonizing activity as a single mAb, mAb-based experimental setups do not represent the in vivo environment. In serum, multiple antibody clones contribute to overall phagocytosis. To mimic this, a cocktail of all eight mAbs, called OctomAb, was created. To observe the contribution of the most potent clones (Ab94 and Ab81 IgG3), a cocktail consisting of these two mAbs, named DuomAb, was created. The remaining Ab11–Ab77 were combined in a third cocktail called HexamAb. To observe the effect of the cocktails, the phagocytic multiplicity (ratio of beads to phagocytic cells) was reduced from 30 to 15, and the mAb concentration was halved (from 10 to 5 μg / ml) to prevent THP-1 cells from becoming saturated with excessive predators and mAbs.

[0068] The IgG3 cocktails clearly enhanced the phagocytic capacity of IgG3 mAbs. In all three cocktails, the percentage of cells with internalized beads increased threefold compared to their respective IgG1 cocktails (Figure 2D). HexamAbs without potent Ab94 and Ab81 IgG3 achieved similar phagocytosis to monoclonal Ab94 IgG3 (32% vs. 27%). The observed differences were more pronounced when looking at bead signaling (40,000 vs. 29,000, respectively) (Figure 2E). The IgG3 cocktails were far more potent than their IgG1 counterparts in terms of the amount of associated beads per cell. IgG3 DuomAb was 8 times potent, HexamAb was 9 times, and OctomAb was 12 times better than IgG1 (Figure 2E). Most importantly, all three cocktails outperformed the most potent IgG3 mAb (Ab94), with a 38% increase for HexamAb, a 58% increase for OctomAb, and a 79% increase for DuomAb (Figure 2E). Taken together, the results demonstrate that even lower-performing mAbs can promote potent phagocytosis in oligoclonal antibody cocktails at the same concentration, surpassing even the most powerful mAb.

[0069] Example 4: IgG3 monoclonal activates complement with higher potency than IgG1 counterparts. We aimed to measure the extent to which IgG3 monoclonals and cocktails activate the classical complement pathway. Complement activation was measured by adding 1% serum to opsonized spike beads. C1q deposition on the spike beads was measured by flow cytometry using a secondary anti-C1q antibody (FITC). Clones 81 and 94 were used as representative monoclonals because they showed the best results in previous assays. The advantages of using DuomAb and OctomAb cocktails were evaluated because they were the most potent among the cocktails. To avoid donor variability in the presence of spike-reactive antibodies, antibody-depleted human pooled serum was used as the complement source.

[0070] The only mAb capable of inducing C1q deposition on its own was Ab94 IgG3 (Figure 3A-B). When both Ab81 IgG3 and Ab94 IgG3 were present as a DuomAb, they deposited 2.5 times more C1q onto the spike beads than Ab94 alone (38% vs. 15%, Figure 3A-B). This potent complement activation was not observed with the IgG1 DuomAb, indicating that this activation is a subclass-dependent phenomenon. OctomAbs containing all antibodies caused C1q deposition to a similar degree as DiomAbs, but only in their IgG3 form. Next, C1q deposition analysis was performed as a function of mAb concentration. EC of DuomAb IgG3 50 The result was calculated and compared with Ab94 IgG3. The analysis showed that adding Ab81 IgG3 to Ab94 IgG3 increased the proportion of beads with C1q deposition by more than three times (EC 50 The concentration decreased from 37 μg / ml to 11 μg / ml (Figure 3C). Looking instead at the C1q deposition signal, the efficacy increased 20-fold (EC). 50 The concentration decreased from 230 μg / ml to 15 μg / ml (Figure 3D). This means that beads containing Daum mAb with C1q deposition resulted in much stronger deposition compared to Ab94 alone. Taken together, the potency of anti-spike IgG3 mAbs is clearly increased compared to IgG1 in activating the classical complement pathway, and this finding is enhanced when IgG3 mAbs are used as a cocktail.

[0071] Example 5: The neutrophil phagocytosis of spike-coated beads is governed by Fc-mediated phagocytosis enhanced by IgG3 monoclonal and its cocktail. We decided to investigate phagocytic findings using primary cells. First, we used monocytes to determine how changes in phagocytic multiplicity (MOP) affect internalization, as this is an essential experimental factor to consider in phagocytosis. 18At low MOP (<1), monocytes hardly internalize spike beads, even with potent OctomAb IgG3. However, at higher MOP (1-10), the opsonization efficiency of mAbs increases. Therefore, MOP 5 was used to investigate the effects of IgG1 and IgG3 mAbs on primary cells. In subsequent experiments, neutrophils were chosen instead of monocytes because they are more efficient at mediating phagocytosis and are the most abundant phagocytic cells in the blood. 19 .

[0072] The results show only slight differences between heat-inactivated (HI) serum and complement-activated (C+) treatment. This indicates that Fc-mediated phagocytosis is the primary mode of function for anti-spike monoclonals in both subclasses, with complement-mediated phagocytosis playing only a minor role (Figure 3E). Comparing the subclasses, all IgG3 treatments were superior to their IgG1 counterparts in terms of the proportion of neutrophils with endogenized beads (Figure 3E). The IgG3 cocktail was equal to or slightly superior to Ab94 IgG3 alone, as previously observed (C+: OctomAb: 15.0±1.6%; DuomAb: 15.5±1.1% and Ab94: 13.1±2.9%) (Figure 3E). OctomAb IgG1 (C+, 3.4±0.3%) was slightly superior to both Ab81 (C+, 0.4±0.1%) and Ab94 IgG1 (C+, 0.6±0.1%), which was not observed in previous THP-1 cells (Figures 2D-E). However, a clear difference was again observed between OctomAb IgG1 and OctomAb IgG3 (3.4% vs. 15.0%).

[0073] We also examined the amount of beads phagocytosed by cells. Due to the lower MOP, more cells were unresponsive compared to THP-1 cells (MOP 30 vs 5). We analyzed the bead signaling in bead-positive neutrophils (APC+). This analysis revealed that all IgG3 treatments still outperformed IgG1 counterparts, as seen in THP-1 cells (Figure 3F). Interestingly, IgG1 OctomAbs showed enhanced phagocytosis compared to single IgG1 monoclonals, which was not observed in THP-1 cells (Figure 2B, Figure 3F). This suggests that even IgG1 mAbs may benefit when used as a cocktail to increase phagocytic efficiency. Taken together, the results indicate that IgG3 monoclonals and their cocktails are superior to IgG1 counterparts in promoting phagocytosis (Fc and complement receptor-mediated). Both subclasses benefit from the use of a cocktail-based oligoclonal approach to enhance phagocytosis.

[0074] Live fluorescence microscopy was used to study the temporal dynamics of mAb-induced phagocytic processes. The focus was on comparing the best monoclonal Ab94 IgG3 with oligoclonal OctmAb in both IgG1 and IgG3 forms. Serum was not heat-inactivated to include CR-mediated phagocytosis. Neutrophils from the same donor (Figure 3E-F) and under the same experimental conditions (MOP 5) were used to allow comparison of flow cytometry data. Neutrophils with internalized beads were examined, and the percentage of these cells and the amount of beads they internalized were quantified (Figure 4A). OctomAb IgG3 stood out in the treatment by producing more cells with internalized beads over the course of a 60-minute experiment. The time required for 50% of total neutrophils to have internalized beads (ET) was measured. 50The efficiency of IgG mAbs was analyzed by calculating the median bead signal. This analysis showed that OctomAb IgG3 promoted spike bead internalization twice as fast as Ab94 IgG3 and OctomAb IgG1 (at 35, 66, and 62 minutes, respectively) (Figure 4D). All mAb treatments were more efficient than the untreated negative control (165 minutes, Figure 4D). Nevertheless, only OctomAb IgG3 yielded a statistically significant difference. Interestingly, OctomAb IgG1 and Ab94 IgG3 performed similarly over time (Figure 4D). This reaffirms the results in Figures 3E-F, where the IgG1 cocktail enhanced the less effective IgG1 mAbs. Previously, the median bead signal was used to quantify the number of phagocytosed beads, but live imaging allowed this to be done with greater accuracy (Figure 4A). The results of this analysis show that cells with internalized beads internalize more beads on average in each neutrophil when the opsonin is IgG3 OctomAb and IgG3 Ab94 compared to IgG1 OctomAb (Figures 4B-C). However, considering that IgG3 OctomAb activates more cells than IgG3 Ab94 (Figure 4D) (72% vs. 47% at 60 minutes), it promotes a stronger bead-cell interaction overall. In summary, these results strongly suggest the benefit of using an oligoclonal cocktail of mAbs to increase the efficacy of mAbs against both IgG1 and IgG3.

[0075] Example 6: Opsonized antispike mAbs that mediate Fc-mediated function prevent SArsCoV-2 infection. THP-1 monocyte in vitro experiments were supplemented with data from primary human neutrophils isolated from multiple donors. These experiments were performed ex vivo using microsphere beads. Next, we investigated the biological and potential clinical relevance of these Fc-mediated functions in the prevention of true SARS-CoV-2 infection. We tested the protective function of mAbs in an infection model using hACE2-K18 mice (Figure 4E). mAbs (Ab94 IgG1, Ab94 IgG3, Ab81 IgG3, DuomAb IgG3, or vehicle control) were prophylactically administered intraperitoneally to each mouse at a single dose (200 ug / mouse). Eight hours after mAb administration, 1 x 10⁶ 5 The PFU (SARS-CoV-2; Wuhan strain derived from a Swedish isolate) virus was inoculated into the nasal cavity of mice. It was verified by three independent methods that none of these clones possessed neutralizing activity. 5 It is important to note that, strictly speaking, it should be considered as being opsonized.

[0076] The inventors theorized that all antibodies were prophylactic compared to the vehicle control through Fc-mediated effector function. Daily measurements throughout the experimental period showed that Ab94 IgG1 and Ab81 IgG3 exhibited lower weight loss compared to the vehicle control from day 0 to day 7. More importantly, all mAbs prevented death by euthanasia (performed when mice experience non-human clinically healthy conditions or weight loss exceeding 20%) (Figure 4E). On day 7, all mice in the control group died, in contrast to the mAb-treated groups (highlighted by a red dashed line in Figure 4F as survival rate beyond day 6). This data also reflects the survival status and weight of mice on day 6 (Figure 4G). On day 6, four of the six mice in the control group died, and one mouse in the Ab94 IgG3 group was euthanized (Figure 4G). At this time, no mice died in the other antibody treatment groups and maintained a healthy average body weight (Figure 4H). Notably, only the Ab94 IgG1 and Ab81 IgG3 groups survived until day 10 (Figure 4I). After euthanasia, regardless of the day of death, bronchoalveolar lavage (BAL) fluid was collected from all mice in each control group, and the viral titer, measured as the cycle threshold (CT) by qPCR, was analyzed. A higher value indicates a lower viral load. This analysis showed that animals treated with all mAbs except DuomAb had higher CT thresholds and therefore lower viral loads at euthanasia (Figure 4J). Compared to the control (17.8), Ab94 IgG1 had the highest CT value (24.5), followed by Ab94 IgG3 (19), and then Ab81 IgG3 (18.1) (Figure 4I). Our data show that mAb-treated mice generally had a healthy average body weight, increased survival rates, and reduced viral loads in BAL fluid. Taken together, our results suggest that opsonized anti-spike antibodies, regardless of subclass, may be prophylactic in preclinical animal models of genuine SARS-CoV-2 infection. Except for Ab94 IgG1, which appeared to show the best results in this experiment, it is difficult to draw conclusions about differences between antibody treatments.Our data provide a proof-of-concept highlighting the biological relevance of studying Fc-mediated effector function in SARS-CoV-2 adaptive immunity.

[0077] Example 7: Discussion This study focuses on how the Fc-mediated immune function of antibodies against spike proteins can be enhanced. We found that modifying subclasses leads to improved immune efficacy. Our data demonstrate that the IgG3 version of our spike-specific antibody array functions better than its IgG1 counterpart, even when originally derived from IgG1 B cells. Furthermore, our data show that when antibodies are combined in a cocktail, they significantly enhance Fc-mediated function.

[0078] A very large number of IgG antibody clones are constantly circulating in the serum. Therefore, serum is a mixture of diverse polyclonal antibodies that specialize into antigen-specific antibodies after infection. In current research, we have shown that a single IgG3 antibody (e.g., Ab94) in the efficacy hierarchy can be highly efficient in mediating Fc-mediated immune function. However, pooling multiple less effective antibodies (Hexamab) can be equally potent in mediating immune function. This may be due to enhanced activation of Fc receptors by clustering multiple Fc molecules on the antigen trimer, which is theorized to be important for efficient phagocytosis. 25~27 The insights into the importance of the cocktail also led to increased effectiveness in activating complement deposition.

[0079] C1q requires multiple Fc cells formed into a hexamer to be activated. 28Our results suggest that the IgG3 cocktail is more efficient than the IgG1 cocktail in forming hexameric clusters. Another important point to note is that the cocktail's advantages were more pronounced compared to THP-1 cells when spike beads were used to opsonize neutrophils. This may be due to neutrophils being specialized phagocytes with a broader repertoire of phagocyte receptors. 29 The inventors demonstrated how immune function can be enhanced by using a combination of multiple opsonic mAbs. The inventors believe that because IgG3 is functionally more active in vitro than IgG1, particularly in cocktail form, exploring an IgG3 platform for future mAb therapies against novel variants and other pathogens of concern could be a promising approach.

[0080] The problem of reduced in vivo biological activity of IgG3 may have been reflected in clone 94. We believe that the performance of IgG3 Ab94 compared to its IgG1 counterpart can be explained by a shorter half-life or potentially lower Fc interaction effect. Compared to humans, the half-life of human IgG in mice is shorter (on the order of days rather than weeks), and a larger proportion of the IgG3 subclass compared to IgG1 may be eliminated 8 hours after viral administration, which may also vary by mAb. 32It is noteworthy that Ab81 IgG3 was close to Ab94 IgG1 in terms of protective effect. We believe this is due to its higher affinity than both subclasses of clone 94 and therefore potentially more functionally active in vivo. We believe the hACE2-K18 model is a good model for demonstrating the biological relevance of Fc effector function against SARS-CoV-2 infection, as a proof of concept. However, caution is advised as the biological applicability of testing human subclasses in this model and predicting results in humans is greatly limited. Among other notable differences, mice express different Fc receptors in their phagocytic cells compared to human phagocytic cells. 33 These Fc receptors have very similar affinity to those found in human Fc receptors. 34 In particular, research on IgG3 and many of its allotypes is insufficient. These studies investigating the affinity of human subclasses to the mouse Fc-γ receptor have observed that human IgG3 binds with the same efficiency as IgG1. Nevertheless, despite this, human IgG3 has been shown to exhibit reduced in vitro function in mouse phagocytic cells compared to human IgG1. 35,36 This contrasts with in vitro studies using human phagocytic cells, which have shown that human IgG3 has a more potent function compared to IgG1. 12~14,30 The inventors believe that more research is needed to better correlate in vitro assays, in vivo animal models, and potential clinical benefits when studying human IgG subclasses as therapeutic agents. To address these discrepancies across species, progress has been made in generating mice with humanized immune systems. 37,38 However, currently available models are not without their limitations, including their availability, relative complexity, and the impact of variability in human donors required for engraftment of human tissue or cells. 39Nevertheless, from the perspective of understanding the role of Fc-mediated function in SARS-CoV-2 immunity, our in vivo data support the Fc effector function of non-neutralizing mAbs, which can potently prevent SARS-CoV-2 infection. In this regard, the production of human IgG3 is a promising alternative to IgG1, especially if its pharmacokinetic properties, particularly its shorter half-life, are addressed by appropriate techniques.

[0081] Furthermore, the inventors present evidence that replacing the constant domain affects the ability of the variable domain to bind to the spike protein. Ab11 IgG3 shows lower avidity compared to Ab11 IgG1. On the other hand, Ab57 IgG3 binds better than IgG1. This is because clone 57 was originally IgG1 and clone 11 was originally IgG3 (according to sequencing data). 5 This is interesting. IgG1 antibodies are formed when the IgG3 constant domain gene is deleted during the class switch recombination process. 9,40 Therefore, IgG1 can evolve from IgG3, but IgG3 cannot arise from clones that produce IgG1 antibodies. IgM can directly switch to IgG1 antibodies without having an IgG3 intermediate, but B cells that produce IgG3 have not evolved to produce IgG1. Subsequently, clone 11 was supplied from IgG3-producing B cells, meaning that Ab11 IgG1 had not been previously subclass-switched in those B cells. Next, we find it interesting that the artificial Ab11 IgG1 has a much higher affinity for the spike than the original IgG3. Furthermore, Ab57 IgG3 shows that the theoretically less evolved mAb is more reactive 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 circulating within the B cell at any given time during the 6-8 week period after infection (Bahnan et al 2022, patient B cells were acquired 6-8 weeks after the initial SARS-CoV-2 infection). 5Our results indicate that avidity changes do not necessarily follow the expected subclass switching hierarchy of the IGHV locus. However, our data, although based on only eight clones, raise new questions worth considering. Our results further add to the growing evidence that the constant and variable domains of antibodies can influence each other. 10,41 42 Furthermore, the inventors demonstrated that the Fc effector function was most potent in the IgG3 subclass, regardless of the original subclass. Therefore, future research using subclass-switched mAbs is needed to broaden the inventors' understanding of the interaction between the variable and constant domains, and how this affects immune function in specific diseases.

[0082] Overall, this study highlights the advantages of subclass switching mAbs to IgG3 and using them in oligoclonal cocktails to enhance opsonization function in vitro. These results are important for the future development of therapeutic mAbs where Fc and CR-mediated phagocytosis is desirable. Regarding SARS-CoV-2 and its VOCs, the use of opsonic monoclonal therapeutics may be a promising tool for further research, particularly because it relies less on binding to mutagenic epitopes in RBDs. Indeed, our trials demonstrate the potential of using non-neutralizing but opsonic mAbs against SARS-CoV-2 infection, and the use of IgG3 mAbs instead of IgG1 due to their potent Fc effector function. The discrepancies between in vitro data using human phagocytic cells and results in animals highlight the importance of future research on optimizing preclinical models for mAb therapeutic development. Furthermore, the studies presented here are reminiscent of the complexities of B-cell subclass switching and highlight the importance of different subclasses in the immune response.

[0083] Example 8: From IgG1 to IgGh 47SARS-CoV-2 mAbs modified into subclasses exhibit potent opsonin function. Finally, the inventors performed hinge engineering to increase antibody flexibility and tested whether the function of mAbs in other biological contexts could be improved. The non-neutral and neutral Fc-mediated effector functions of antibodies are highly relevant to immune protection against SARS-CoV-2 and its mutants 43~48 so the focus was on SARS-CoV-2. Previously, anti-SARS-CoV-2 IgG1 mAbs against the spike antigen 48 were generated and it was shown that these mAbs are opsononic 47~48 . By increasing the flexibility of the Fc of some of these opsononic anti-spike IgG1 mAbs, it was our aim to clarify whether Fc-mediated functions could also be improved and become useful in the SARS-CoV-2 situation.

[0084] Three IgGh 47 constructs of mAbs Ab11, Ab36, and Ab77 were generated, and all of these bind to the spike protein with nanomolar affinity, similar to IgG1 47 (Figure 5A). As before 38 , biotinylated spike protein conjugated to streptavidin microsphere beads was used as a model of virions. All of these mAbs induce Fc-mediated effector functions 47~48 . MOP curves were created and MOP 50 values were calculated for each mAb. Xolair (anti-IgE) was used as a negative control and two antibody cocktails of IgG3 mAbs with potent opsonin function 47 were used as positive controls. Interestingly, the inventors observed a significant enhancement of function in all three clones. In the case of Ab11, the IgGh 47 version enhanced bead-phagocyte interaction 1.4-fold (MOP 50 27 vs 37, P<0.05), and in Ab77, a 1.7-fold enhancement of function was observed (MOP 5026 vs 43, P>0.05) (Figure 5B). Ab36 IgGh 47 A significant enhancement of function was observed, accompanied by a 4.3-fold improvement in MOP50 values ​​(9 vs. 39, P<0.05) (Figure 5B). Similar to the case of Ab25, the phagocytic score (PS) was calculated to determine the overall bead uptake of the modified IgGh mAb. 47 We checked whether there were differences between IgGh and IgG1 mAbs. As MOP increased, the differences between IgG versions became clearer: Ab11 showed improvement with a 4-fold increase at MOP 30 (3000 vs 800) (Figure 5C). Ab77 showed a 6-fold increase (3300 vs 550), and Ab36 showed the most substantial improvement with a 13-fold increase in PS compared to IgG1 (8400 vs 600) (Figure 5C). Additional experiments were conducted at MOP 30 to assess whether these changes were statistically significant for each mAb, and association and PS were examined. Here, for all three clones, there was a significant enhancement of IgGh compared to IgG1. 47 Similar findings were observed regarding (Figures 5D-E), which were statistically significant in both clones 36 and 77. Even more interestingly, Ab36 IgGh 47 It has been shown to be comparable to DuomAb IgG3, the most potent opsonin (37). Taken together, increasing the flexibility of the antibody through hinge engineering demonstrates a significant enhancement of efficacy in two different biologically related systems. In this respect, Ab36 IgGh 47 Due to its powerful Fc-mediated effector function, it is a promising candidate for further in vivo testing regarding SARS-CoV-2.

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Claims

1. A modified antibody containing an IgG3 hinge and a variable domain having a viral spike protein binding site.

2. The modified antibody according to claim 1, wherein the Fc region is an IgG3 region.

3. The modified antibody according to claim 2, wherein the Fc region includes a variant thereof having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to the IgG3 heavy chain constant domain (SEQ ID NO: 1) or SEQ ID NO:

1.

4. The modified antibody according to claim 2, wherein the IgG3 region includes three substitutions: R435H, N392K, and M397V.

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

2.

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

2.

7. The modified antibody according to claim 1, wherein the Fc region is an IgG1 region.

8. The modified antibody according to claim 7, wherein the modified antibody comprises an IgG1 constant domain (SEQ ID NO: 3) modified with an IgG3 hinge.

9. The modified antibody according to any one of claims 1 to 8, wherein the viral spike protein is derived from coronavirus.

10. The modified antibody according to any one of claims 1 to 9, wherein the viral spike protein is the SARS-CoV-2 spike protein.

11. The modified antibody according to any one of claims 1 to 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 with respect to the amino acid sequence ELKTPLGDTTHTCPRRCPEPKKSCDTPPPPCPRRCPEPKKSCDTPPPPCPRCP (SEQ ID NO: 7).

12. The IgG3 hinge includes an amino acid sequence having at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity with respect to the amino acid sequence X-(Y)n, where, X is ELKTPLGDTTHTCPRP (Sequence ID 8), Y is EPKSCDTPPPCPRCP (Sequence ID 26), The modified antibody according to any one of claims 1 to 10, wherein n is an integer in the range of 2 to 8, preferably in the range of 2 to 5, and more preferably in the range of 2 to 4.

13. A modified antibody according to any one of claims 1 to 12, which promotes opsonization.

14. A modified antibody according to any one of claims 1 to 12, which does not bind to a neutralizing epitope.

15. The modified antibody according to any one of claims 1 to 14, wherein the modified antibody is selected from the group consisting of Ab11, Ab36, Ab57, Ab59, Ab66, and Ab77.

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

18. A nucleic acid molecule encoding a protein containing a modified antibody as defined in any one of claims 1 to 15.

19. A cell containing nucleic acid according to claim 18.

20. Use of a modified antibody as defined in any one of claims 1 to 15 for the prevention, treatment, or mitigation of a viral infection caused by a virus containing the spike protein.

21. Use of a modified antibody as defined in any one of claims 1 to 15 for Fc-mediated phagocytosis of a virus containing the spike protein.

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

23. A method for preparing a modified antibody as defined in any one of claims 1 to 15, the method comprising the following steps for obtaining the modified antibody: - Immunization of a host organism by the viral spike protein or a protein containing the viral spike protein, - Isolation of at least one viral spike protein-reactive B cell for isolating an IgG1 antibody that binds to the viral spike protein, - Preparation of at least one recombinant DNA molecule encoding a modified version of the IgG1 antibody, comprising replacing the IgG1 subclass-specific domain of the isolated IgG1 antibody with an IgG3 subclass-specific domain. - Expression of at least one recombinant DNA molecule in a suitable host cell.

24. A method for preparing a modified antibody as defined in any one of claims 1 to 15, the method comprising the following steps for obtaining the modified antibody: - Immunization of a host organism by the viral spike protein or a protein containing the viral spike protein, - Isolation of at least one viral spike protein-reactive B cell for isolating an IgG1 antibody that binds to the viral spike protein, - Preparing at least one recombinant DNA molecule encoding a modified form of the IgG1 antibody, wherein the modification includes replacing the IgG1 hinge of the isolated IgG1 antibody with an IgG3 hinge. - Expression of at least one recombinant DNA molecule in a suitable host cell.

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