Compositions and methods for preventing and treating orthopoxvirus infections

Abstract

Provided herein are antibodies and functional fragments thereof for treating or preventing viral diseases, such as poxvirus diseases, such as smallpox and monkeypox. Any antibody or functional fragment thereof described herein can be an engineered antibody or an engineered antibody fragment. Also provided herein are compositions and pharmaceutical compositions containing one or more antibodies or functional fragments thereof. Further provided herein are kits containing one or more antibodies or functional fragments thereof, as well as methods, dosing schedules, dosages, and routes of administration of antibodies and functional fragments thereof. Finally, provided are methods for producing antibodies and functional fragments thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 402,900, filed August 31, 2022, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Contract No. 75A50119C00054 awarded by the U.S. Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response; Biomedical Advanced Research and Development Authority. The government has certain rights in this invention. Summary of the Invention [Means for solving the problem]

[0003] Summary of the Invention Disclosed herein are compositions for the prevention and treatment of orthopoxvirus infections, such as vaccinia virus, monkeypox virus, and variola virus, the causative agents of smallpox disease in humans, including compositions containing antibodies that bind to epitopes found on intracellular mature virions or mature virion forms of orthopoxviruses and / or antibodies that bind to epitopes found on extracellular enveloped virions or enveloped virion forms of orthopoxviruses. Also disclosed are compositions such as humanized antibodies and antibodies thereof modified to reduce immunogenicity and / or extend serum half-life, and methods for their use to confer passive immunity against orthopoxvirus infection in individuals at risk of or exhibiting vaccinia infection, including adverse events caused by certain smallpox vaccines, such as progressive vaccinia and eczema vaccinatum, monkeypox infection, or smallpox.

[0004] Provided herein are antibodies and fragments thereof for treating or preventing viral diseases, such as poxvirus diseases, for example, orthopoxvirus diseases, such as smallpox and monkeypox. Also provided herein are compositions and pharmaceutical compositions containing one or more antibodies or fragments thereof. Further provided herein are kits containing one or more antibodies or fragments thereof, as well as methods, dosing schedules, dosages, and routes of administration of antibodies and fragments thereof that may be contained in compositions. Finally, provided are methods for producing antibodies and fragments thereof.

[0005] Further provided is a composition comprising: (A) a first antibody that binds to an epitope found on the mature virion (MV) form of a virus of the Orthopoxvirus genus; and (B) a second antibody that binds to an epitope found on the enveloped virion (EV) form of a virus of the Orthopoxvirus genus.

[0006] Additionally, a composition is provided comprising: (A) a first antibody that binds to an epitope found on the MV form of a virus of the Orthopoxvirus genus; and (B) a second antibody that binds to a first epitope found on the EV form of a virus of the Orthopoxvirus genus; and (C) a third antibody that binds to a second epitope found on the EV form of a virus of the Orthopoxvirus genus, wherein the first epitope is different from the second epitope.

[0007] Additionally, a method for conferring passive immunity to smallpox in a subject is provided, comprising administering to the subject an effective amount of a composition comprising: (A) a first antibody that binds to an epitope found on the MV form of variola virus; and (B) a second antibody that binds to an epitope found on the EV form of variola virus.

[0008] Further provided herein is a method of conferring passive immunity in a subject against infection by monkeypox virus, the method comprising administering to the subject an effective amount of a composition comprising: (A) a first antibody that binds to an epitope found on the MV form of the monkeypox virus; and (B) a second non-blood derived antibody that binds to an epitope found on the EV form of the monkeypox virus.

[0009] Also provided herein is a method for conferring passive immunity to smallpox in a subject, the method comprising administering to the subject an effective amount of a composition comprising: (A) a first antibody that binds to an epitope found on the MV form of variola virus; and (B) a second antibody that binds to a first epitope found on the EV form of the smallpox virus; and (C) a third antibody that binds to a second epitope found on the EV form of the smallpox virus, wherein the second epitope is different from the first epitope.

[0010] Also disclosed herein is a method of conferring passive immunity to infection by monkeypox virus in a subject, the method comprising administering to the subject an effective amount of a composition comprising: (A) a first antibody that binds to an epitope found on an MV form of the monkeypox virus; and (B) a second antibody that binds to a first epitope found on an EV form of the monkeypox virus; and (C) a third antibody that binds to a second epitope found on the EV form of the monkeypox virus, wherein the second epitope is different from the first epitope.

[0011] The Summary provides exemplary embodiments and is not meant to limit in any way what is provided herein. Incorporation by Reference

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0013] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized. The accompanying drawings, in which: [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows an amino acid sequence illustrating an exemplary fully humanized heavy chain h7D11 variant VH2 amino acid sequence aligned with the c7D11 mouse-human chimeric sequence, with the amino acid changes made in the humanized variant VH2 marked in dark grey highlighting. Light grey highlighting indicates framework sequences, with no highlighting representing the signal peptide, CDR sequences 1, 2, and 3, and human IgG1 constant region.

[0015] [Figure 2] 2 shows an amino acid sequence illustrating an exemplary fully humanized light chain h7D11 variant VK3 amino acid sequence aligned with the c7D11 mouse-human chimeric sequence, with the amino acid changes made in the humanized variant VK3 highlighted in dark gray. Light gray highlighting indicates framework sequences, with no highlighting representing the signal peptide, CDR sequences 1, 2, and 3, and the human kappa constant region.

[0016] [Figure 3] 3 shows an amino acid sequence illustrating an exemplary fully humanized heavy chain h8A variant VH3 amino acid sequence aligned with the c8A chimeric sequence, with the amino acid changes made in the humanized variant VH3 highlighted in dark grey. Light grey highlighting indicates framework sequences, with no highlighting representing the signal peptide, CDR sequences 1, 2, and 3, and human IgG1 constant region.

[0017] [Figure 4]4 shows an amino acid sequence illustrating an exemplary fully humanized light chain h8A variant VK2 amino acid sequence aligned with the c8A chimeric sequence, with the amino acid changes made in the humanized variant VK2 highlighted in dark gray. Light gray highlighting indicates framework sequences, with no highlighting representing the signal peptide, CDR sequences 1, 2, and 3, and the human kappa constant region.

[0018] [Figure 5] 5 shows an amino acid sequence illustrating an exemplary fully humanized heavy chain h8A variant VH1 amino acid sequence aligned with the c8A chimeric sequence, with the amino acid changes made in the humanized variant VH1 highlighted in dark grey. Light grey highlighting indicates framework sequences, with no highlighting representing the signal peptide, CDR sequences 1, 2, and 3, and human IgG1 constant region.

[0019] [Figure 6] 6 shows an amino acid sequence illustrating an exemplary fully humanized light chain h8A variant VK3 amino acid sequence aligned with the c8A chimeric sequence, with the amino acid changes made in the humanized variant VK3 highlighted in dark gray. Light gray highlighting indicates framework sequences, and no highlighting represents the signal peptide, CDR sequences 1, 2, and 3, and the human kappa constant region.

[0020] [Figure 7] 7 shows an amino acid sequence illustrating an exemplary fully humanized heavy chain h6C variant 2 amino acid sequence aligned with the c6C chimeric sequence, with dark grey highlighting indicating the amino acid changes made in humanized variant 2. Light grey highlighting indicates framework sequences, with no highlighting representing the signal peptide, CDR sequences 1, 2, and 3, and the human IgG1 constant region.

[0021] [Figure 8]Figure 8 shows an amino acid sequence illustrating an exemplary fully humanized light chain h6C variant VK2 amino acid sequence aligned with the c6C chimeric sequence, with the amino acid changes made in the humanized variant VK2 highlighted in dark gray. Light gray highlighting indicates framework sequences, while no highlighting represents the signal peptide, CDR sequences 1, 2, and 3, and the human kappa constant region. Also highlighted in dark gray is an asparagine residue (N) in CDR3, indicating a potential N-linked glycosylation site.

[0022] [Figure 9] 9A-9D show amino acid sequences illustrating several exemplary FcRn affinity-enhancing variants aligned with the unmodified Fc region of h7D11 HC. No highlighting indicates the CH2 domain of the Fc region. Light gray highlighting indicates the CH3 domain of the Fc region. Dark gray highlighting indicates modified amino acids. Unmodified h7D11 HC Fc region (FIG. 9A). h7D11 Fc with amino acid substitutions M280Y, S282T, and T284E (FIG. 9B). h7D11 Fc with amino acid substitutions M456L and N462S (FIG. 9C). h7D11 Fc with amino acid substitutions M280Y, S282T, T284E, M456L, and N462S (FIG. 9D).

[0023] [Figure 10] 10A-10D show amino acid sequences illustrating several exemplary FcRn affinity-enhancing variants aligned with the unmodified Fc region of h8A HC. No highlighting indicates the CH2 domain of the Fc region. Light gray highlighting indicates the CH3 domain of the Fc region. Dark gray highlighting indicates modified amino acids. Unmodified h8A HC Fc region (FIG. 10A). h8A Fc with amino acid substitutions M282Y, S284T, and T286E (FIG. 10B). h8A Fc with amino acid substitutions M458L and N464S (FIG. 10C). h8A Fc with amino acid substitutions M282Y, S284T, T286E, M458L, and N464S (FIG. 10D).

[0024] [Figure 11]11 is a dot plot illustrating the mean group weight by study day for negative control groups 1 and 2. All groups had 10 male and 10 female BALBc mice (n=20). Group 1 was treated with vehicle and no ectromelia virus (ECTV) challenge, and group 2 was treated with vehicle and challenged with 200 PFU of ECTV.

[0025] [Figure 12] Figure 12 is a dot plot illustrating the mean body weight of Test Material 1 group by study day for groups 3, 4, 5, and 6. All groups had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Groups 3, 4, 5, and 6 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 3 was treated on day 3 post-challenge, Group 4 was treated on day 4 post-challenge, Group 5 was treated on day 5 post-challenge, and Group 6 was treated on day 6 post-challenge.

[0026] [Figure 13] Figure 13 is a dot plot illustrating the mean body weight of Test Material 2 Group by study day for Groups 7, 8, 9, and 10. All groups had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Groups 7, 8, 9, and 10 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 7 was treated on day 3 post-challenge, Group 8 was treated on day 4 post-challenge, Group 9 was treated on day 5 post-challenge, and Group 10 was treated on day 6 post-challenge.

[0027] [Figure 14]Figure 14 is a dot plot illustrating the mean body weight of the three test material groups by study day for groups 11, 12, 13, and 14. All groups had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Groups 11, 12, 13, and 14 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 11 was treated on day 3 post-challenge, Group 12 on day 4 post-challenge, Group 13 on day 5 post-challenge, and Group 14 on day 6 post-challenge.

[0028] [Figure 15] Figure 15 is a dot plot illustrating the mean body weight of the four test materials by study day for groups 15, 16, 17, and 18. All groups had 10 male and 10 female BALBc mice (n = 20). All mice were challenged with 200 PFU of ECTV on day 0. Groups 15, 16, 17, and 18 were treated with a cocktail of the original chimeric versions of the mAbs as controls for the humanized versions used in all other treatment groups: c7D11 (anti-L1 mAb) at 5 mg / kg, c8A (anti-B5 mAb) at 5 mg / kg, and c6C (anti-A33 mAb) at 5 mg / kg, at a total mAb cocktail dose of 15 mg / kg. Group 15 was treated on day 3 post-challenge, group 16 on day 4 post-challenge, group 17 on day 5 post-challenge, and group 18 on day 6 post-challenge.

[0029] [Figure 16]Figure 16 is a dot plot illustrating the mean body weights of the Test Material 5 Low Dose Group by study day for Groups 19, 20, 21, and 22. All groups had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Groups 19, 20, 21, and 22 were treated with a cocktail of 3.33 mg / kg h7D11 (anti-L1 mAb), 3.33 mg / kg h8A (anti-B5 mAb), and 3.33 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 19 was treated on day 3 post-challenge, Group 20 on day 4 post-challenge, Group 21 on day 5 post-challenge, and Group 22 on day 6 post-challenge.

[0030] [Figure 17] Figure 17 is a Kaplan-Meier time-to-death plot illustrating the results for Set 1. Every group had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle alone on day 3 post-challenge. Groups 3, 4, 5, and 6 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb) for a total mAb cocktail dose of 15 mg / kg. Group 3 was treated on day 3 post-challenge, Group 4 on day 4 post-challenge, Group 5 on day 5 post-challenge, and Group 6 on day 6 post-challenge.

[0031] [Figure 18]Figure 18 is a Kaplan-Meier time-to-death plot illustrating the results for Set 2. Every group had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle alone on day 3 post-challenge. Groups 7, 8, 9, and 10 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 7 was treated on day 3 post-challenge, Group 8 on day 4 post-challenge, Group 9 on day 5 post-challenge, and Group 10 on day 6 post-challenge.

[0032] [Figure 19] Figure 19 is a Kaplan-Meier time-to-death plot illustrating the results for Set 3. Every group had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle alone on day 3 post-challenge. Groups 11, 12, 13, and 14 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 11 was treated on day 3 post-challenge, Group 12 on day 4 post-challenge, Group 13 on day 5 post-challenge, and Group 14 on day 6 post-challenge.

[0033] [Figure 20]Figure 20 is a Kaplan-Meier time-to-death plot illustrating the results for Set 4. Every group had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle only on day 3 post-challenge. Groups 15, 16, 17, and 18 were treated with a cocktail of the original chimeric versions of the mAbs as controls for the humanized versions used in all other treatment groups: c7D11 (anti-L1 mAb) at 5 mg / kg, c8A (anti-B5 mAb) at 5 mg / kg, and c6C (anti-A33 mAb) at 5 mg / kg, at a total mAb cocktail dose of 15 mg / kg. Group 15 was treated on day 3 after loading, Group 16 was treated on day 4 after loading, Group 17 was treated on day 5 after loading, and Group 18 was treated on day 6 after loading.

[0034] [Figure 21] Figure 21 is a Kaplan-Meier time-to-death plot illustrating the results for Set 5. Every group had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle alone on day 3 post-challenge. Groups 19, 20, 21, and 22 were treated with a cocktail of 3.33 mg / kg h7D11 (anti-L1 mAb), 3.33 mg / kg h8A (anti-B5 mAb), and 3.33 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 19 was treated on day 3 post-challenge, Group 20 on day 4 post-challenge, Group 21 on day 5 post-challenge, and Group 22 on day 6 post-challenge.

[0035] [Figure 22]Figure 22 is a Kaplan-Meier time-to-death plot illustrating the results for day 3 of intervention. Every group had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle only. All groups in this plot were treated on day 3 after challenge. Group 3 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 7 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 11 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 15 was treated with a cocktail of the original chimeric versions of the mAbs as a control for the humanized versions used in all other treatment groups: 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 19 was treated with a cocktail of 3.33 mg / kg h7D11 (anti-L1 mAb), 3.33 mg / kg h8A (anti-B5 mAb), and 3.33 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg.

[0036] [Figure 23]Figure 23 is a Kaplan-Meier time-to-death plot illustrating the results for day 4 of intervention. Every group had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle alone on day 3 post-challenge. All groups in this plot except Group 2 were treated on day 4 post-challenge. Group 4 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 8 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 12 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 16 was treated with a cocktail of the original chimeric versions of the mAbs as a control for the humanized versions used in all other treatment groups: 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 20 was treated with a cocktail of 3.33 mg / kg h7D11 (anti-L1 mAb), 3.33 mg / kg h8A (anti-B5 mAb), and 3.33 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg.

[0037] [Figure 24]Figure 24 is a Kaplan-Meier time-to-death plot illustrating the results for day 5 of intervention. Every group had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle alone on day 3 post-challenge. All groups in this plot except Group 2 were treated on day 5 post-challenge. Group 5 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 9 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 13 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 17 was treated with a cocktail of the original chimeric versions of the mAbs as a control for the humanized versions used in all other treatment groups: 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 21 was treated with a cocktail of 3.33 mg / kg h7D11 (anti-L1 mAb), 3.33 mg / kg h8A (anti-B5 mAb), and 3.33 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg.

[0038] [Figure 25]Figure 25 is a Kaplan-Meier time-to-death plot illustrating the results for day 6 of intervention. Every group had 10 male and 10 female BALBc mice (n=20). All mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle alone on day 3 post-challenge. All groups in this plot except Group 2 were treated on day 6 post-challenge. Group 6 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 10 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 14 was treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 18 was treated with a cocktail of the original chimeric versions of the mAbs as a control for the humanized versions used in all other treatment groups: 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 22 was treated with a cocktail of 3.33 mg / kg h7D11 (anti-L1 mAb), 3.33 mg / kg h8A (anti-B5 mAb), and 3.33 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0039] array SEQ ID NO: 1 (Vaccinia virus L1 ectodomain with 6xHis tag) [ka]

[0040] SEQ ID NO: 2 (chimeric 7D11 [c7D11] heavy chain) [ka]

[0041] SEQ ID NO: 3 (humanized 7D11 [h7D11] variant VH2 heavy chain) [ka]

[0042] SEQ ID NO: 4 (chimeric 7D11 [c7D11] light chain) [ka]

[0043] SEQ ID NO: 5 (humanized 7D11 [h7D11] variant VK3 light chain) [ka]

[0044] SEQ ID NO: 6 (Vaccinia virus B5 ectodomain with 6xHis tag) [ka] [ka]

[0045] SEQ ID NO: 7 (chimeric 8A [c8A] heavy chain) [ka]

[0046] SEQ ID NO: 8 (humanized 8A [h8A] variant VH3 heavy chain) [ka]

[0047] SEQ ID NO: 9 (chimeric 8A [c8A] light chain) [ka]

[0048] SEQ ID NO: 10 (humanized 8A [h8A] variant VK2 light chain) [ka]

[0049] SEQ ID NO: 11 (chimeric 8A [c8A] heavy chain) [ka]

[0050] SEQ ID NO: 12 (humanized 8A [h8A] variant VH1 heavy chain) [ka] [ka]

[0051] SEQ ID NO: 13 (chimeric 8A [c8A] light chain) [ka]

[0052] SEQ ID NO: 14 (humanized 8A [h8A] variant VK3 light chain) [ka]

[0053] SEQ ID NO: 15 (Vaccinia virus A33 ectodomain with 6xHis tag) [ka]

[0054] SEQ ID NO: 16 (chimeric 6C [c6C] heavy chain) [ka]

[0055] SEQ ID NO: 17 (humanized 6C [h6C] variant VH2 heavy chain) [ka]

[0056] SEQ ID NO: 18 (chimeric 6C [c6C] light chain) [ka]

[0057] SEQ ID NO: 19 (humanized 6C [h6C] variant VK2 light chain) [ka] [ka]

[0058] SEQ ID NO: 20 (h7D11 Fc unmodified) [ka]

[0059] SEQ ID NO: 21 (h7D11 Fc YTE swap) [ka]

[0060] SEQ ID NO: 22 (h7D11 Fc LS swap) [ka]

[0061] SEQ ID NO: 23 (h7D11 Fc YTELS swap) [ka]

[0062] SEQ ID NO: 24 (h8A Fc unmodified) [ka]

[0063] SEQ ID NO: 25 (h8A Fc YTE swap) [ka]

[0064] SEQ ID NO: 26 (h8A Fc LS swap) [ka]

[0065] Sequence number 27 (h8A Fc YTELS swap) [ka] definition

[0066] Unless otherwise defined, all terms used herein have the same meaning as that commonly understood by those skilled in the art to which this disclosure pertains. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the relevant technical field and in the context of this disclosure, and will not be interpreted in an idealized and very formal sense unless expressly defined herein. In the event of a conflict, the present document, including definitions, will control.

[0067] In describing the present disclosure, it should be understood that several techniques and steps are disclosed. Each of these has its own advantages, and each can also be used in combination with one or more, or in some cases all, of the other disclosed techniques. Methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. Therefore, for clarity, the present description refrains from repeating every possible combination of individual steps. Nevertheless, the specification and claims should be read with the understanding that such combinations are within the scope of the present disclosure and claims as a whole.

[0068] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure. As used herein, the term "and / or" may include any and all combinations of one or more of the associated listed items. As used herein, the singular forms "a," "an," and "the" may include the plural and the singular unless the context clearly dictates otherwise. It will be further understood that the terms "comprises" and / or "comprising," as used herein, specify the presence of stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof, as used herein, may be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. All definitions contained herein are understood to include plural forms as well, unless the context clearly dictates otherwise.

[0069] For the recitation of numerical ranges herein, each intervening number therebetween with the same precision may be expressly contemplated. For example, for the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0070] As used herein, the term "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which may depend, in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean plus or minus 10%, according to practice in the art. Alternatively, "about" can mean a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within five-fold, or within two-fold of a value. When particular values ​​are described in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value. Also, when ranges and / or subranges of values ​​are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0071] As used herein, the term "nucleic acid construct" may refer to a linear polymer of nucleic acids. The polymer may include a promoter sequence that drives expression of one or more genes of interest. As used herein, the term "nucleic acid construct" may include, but is not limited to, oligonucleotides, RNA, linear DNA, closed-end linear DNA, ministrings, transposons, dogbone DNA, GenWand DNA, and minimalistic, immunologically defined gene expression (MIDGE) DNA. As used herein, a nucleic acid construct may be DNA or RNA, or a mixture of the two, and may include naturally occurring or artificial (non-natural) nucleotides. As used herein, "naturally occurring nucleotides" may include adenine, guanine, cytosine, thymine, uracil, inosine, 2,6-diaminopurine, 5-hydroxymethylcytosine, N4-methylated cytosine, N6-methylated adenine, archaeotin, and other nucleotide modifications that occur in normal cellular (eukaryotic, prokaryotic, or archaeal, e.g., virus-induced or phage-induced) metabolism. As used herein, "artificial nucleotides" or "non-natural nucleotides" are nucleotide analogs or linkages that do not occur in nature, including, but not limited to, peptide nucleic acids, morpholinos, phosphorothioate linkages, locked nucleic acids, glycol nucleic acids, addition of functional groups such as amino (-NH2), fluoro (-F), and O-methyl (-OCH3) at the 2' position of the ribose sugar, threose nucleic acids, hexitol nucleic acids, sugar modifications at the 2' position, pyrimidine modifications at the 5' position, purine modifications at the 8' position, modifications with exocyclic amines, 4'-substituted nucleotides, and the like. -thiouridine substitutions, 5-((3-indolyl)propionamido-N-allyl)-20-deoxyuridine, 5-bromo- or 5-iodouracil substitutions, as well as backbone modifications, anti-reverse cap analogs, pseudouridine, 5-methylcytidine and / or N1-methyluridine substitutions, methylation, and unusual base pair combinations including, but not limited to, the isobases isocytidine and isoguanidine and (7-(2-thienyl)imidazo[4,5-b]pyridine, Ds).

[0072] As used herein, the term "plasmid" may refer to a circular DNA molecule that is physically separated from chromosomal DNA. As used herein, the term "plasmid" includes, but is not limited to, bacterial plasmids, minicircles, episomal DNA, covalently closed circular DNA (cccDNA), extrachromosomal circular DNA (eccDNA), chromids, chloroplast DNA, baculovirus-derived circular DNA, bacmids, nanoplasmids, and mitochondrial DNA. The term "plasmid" includes, but is not limited to, small circular double-stranded DNA molecules that have a promoter sequence that drives transcription of one or more genes of interest. Optionally, a plasmid may also contain an origin of replication (ori) site, a marker gene (e.g., an antibiotic resistance gene) for selection and / or screening, an enhancer element, and a restriction endonuclease (RE) site that allows for cloning of an insert at a specific site. As used herein, a plasmid may contain naturally occurring and / or artificial nucleotides.

[0073] As used herein, "antibody" may mean a protein that acts like or is an immunoglobulin, or is designed to replace an immunoglobulin, and includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, single-chain variable fragments (scFv), Fab fragments, camelid antibodies, nanobodies, engineered ankyrin repeat proteins, monobodies, anticalins, knottins, affimers, avimers, affinity clamps, or affibodies that specifically bind to a substance.

[0074] As used herein, "selectively binds," "selectively bound," "specifically binds," "specifically bound," "specifically recognizes," or "specifically recognized" are used interchangeably. "Recognized" can usually mean a substance that binds to another substance to the exclusion of others, optionally in aqueous solution, optionally under prescribed or defined or stringent conditions, e.g., in a solution having a pH in the range of about 5.5 to about 8, e.g., 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, at a temperature in the range of about 23 to about 37 degrees Celsius, optionally at a defined salt (e.g., sodium chloride) concentration in the range of about 0.0001 to about 10 molar, optionally in a buffer such as a phosphate buffer, with a dissociation constant (Kd) of less than or equal to 1 micromolar.

[0075] As used herein, the term "subject" or "patient" may refer to any organism to which a composition or formulation according to the present disclosure may be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mice, rats, rabbits, non-human primates, and humans). In some embodiments, the subject is a human. The human may be greater than about 1, 2, 5, 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or about 120 years old. The human may be a pediatric, child, or adult subject.

[0076] A "therapeutically effective amount" may refer to an amount of a composition or pharmaceutical composition disclosed herein that is effective, with or without additional agents, to achieve its intended purpose, e.g., to treat a disease. The needs of an individual patient may vary. Generally, the dosage required to provide an effective amount of the composition will vary depending on the recipient's age, health, physical condition, sex, weight, extent of the disease, frequency of treatment, and the nature and extent of the disease or condition. For example, a therapeutically effective amount of a composition or pharmaceutical composition herein may range from about 0.0001 mg / kg to about 10,000 mg / kg, e.g., 0.001, 0.01, 0.1, 1.0, 10.0, or 100.0 mg / kg, where mg is mg of the composition or pharmaceutical composition and kg is kg of the subject's or patient's body weight.

[0077] As used herein, the term "treatment" or "treating" may refer to a pharmaceutical or other intervention regimen to obtain a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to the eradication or amelioration of one or more symptoms of the underlying disorder being treated. Therapeutic benefit may also be achieved with the eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that improvement may be observed in a subject even though the subject may still be suffering from the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, subjects at risk of developing a particular disease or reporting one or more physiological symptoms of a disease may receive the treatments disclosed herein, even if a diagnosis of the disease may not have been made.

[0078] As used herein, "transfection" can refer to the introduction of a nucleic acid construct or a plasmid or an engineered DNA (e.g., DNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0079] As used herein, " adjuvant " can refer to any substance that promotes a stronger immune response in the subject receiving the vaccine. Examples of adjuvants include but are not limited to monophosphoryl lipid A (MPL), oligodeoxynucleotide (ODN) containing unmethylated CpG motif (CpG ODN), AS01, AS02, AS03, AS04, MF59, QS-21, Matrix-M, α-mannosylceramide, D-(+)-trehalose 6,6'-dibehenate, trehalose 6,6-dibehenate, dimethyldioctadecylammonium, glucopyranosyl lipid and R848.

[0080] As used herein, the term "medication package" may refer to a box, packet, bag, or plastic used to package a patient package insert (PPI), medication guide (MG), or instructions for use (IFU).

[0081] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Detailed Description Overview

[0082] Disclosed herein are compositions for the prevention and treatment of orthopoxvirus, as well as compositions containing antibodies that bind (with mono- or polyepitope specificity) to epitopes found on intracellular mature virions or mature virion forms of orthopoxvirus and / or antibodies that bind to epitopes found on extracellular enveloped virions or enveloped virion forms of orthopoxvirus. Also disclosed herein are compositions, including non-blood-derived antibody compositions, such as humanized antibodies and compositions such as antibodies modified to reduce immunogenicity and / or extend serum half-life, and methods for their use (e.g., treatment or prevention of orthopoxvirus infection) to confer passive immunity against orthopoxvirus infection in individuals at risk of or exhibiting vaccinia infection, including adverse events caused by certain smallpox vaccines, such as progressive vaccinia and eczema vaccinatum, monkeypox infection, or smallpox infection.

[0083] Also described herein are compositions or pharmaceutical compositions, either administered separately (simultaneously or sequentially), of two or more modified and / or humanized monoclonal antibodies (mAbs) that can target viral proteins in the two main forms of viruses: mature virions (MVs), which are non-enveloped particles responsible for transmission, and enveloped virions (EVs), which are MV particles that acquire an envelope, bud from infected host cells, and are responsible for cell-to-cell spread within the infected host. When MV particles are still within infected host cells, they may be referred to as intracellular mature virions, or IMVs. In some instances, the terms MV and IMV may be used interchangeably. When EV particles are released from infected host cells, they may be referred to as extracellular enveloped virions, or EEVs. When EV particles initially bud from infected host cells but have not yet been released from the cells, they may be referred to as cell-associated enveloped virions, or CEVs. Antibodies and functional fragments thereof

[0084] Provided herein are antibodies and functional fragments thereof that bind to epitopes of orthopoxviruses. In some embodiments, the epitopes are found on the mature virion (MV) form of the orthopoxvirus. In some cases, this includes the IMV form of the orthopoxvirus. In some embodiments, the epitopes are found on the enveloped virion (EV) form of the orthopoxvirus. In some cases, this includes the EEV and CEV forms of the orthopoxvirus. In some embodiments, the antibodies and functional fragments thereof are engineered. In some embodiments, an initial antibody or functional fragment thereof (e.g., mammalian, murine, rat, or rabbit) is selected, or a composite engineered antibody is designed. The composite engineered antibody is constructed using one, two, three, four, five, or more sequence segments from the variable regions of unrelated antibodies as building blocks. In some embodiments, the initial antibody or composite engineered antibody is a chimeric antibody. In some embodiments, the initial antibody (e.g., 7d11) is an antibody against a poxvirus (e.g., vaccinia virus) L1 protein. In some embodiments, the initial antibody (e.g., 8A) is an antibody against a poxvirus (e.g., vaccinia virus) ectodomain of the B5 protein. In some embodiments, the initial antibody (e.g., 6C) is an antibody against a poxvirus (e.g., vaccinia virus) ectodomain of the A33 protein. In some embodiments, the engineered antibody or functional fragment thereof comprises a constant region (Fc region) having one or both HC2 and HC3 construct domains.

[0085] In some embodiments, the antibody or functional fragment thereof is altered, which may confer one or more desired characteristics, such as increasing the half-life of the antibody or functional fragment in a subject. In some embodiments, the antibody or functional fragment is humanized, which may confer one or more desired characteristics, such as reducing the immunogenicity of the antibody or functional fragment in a human subject. In some embodiments, one or more sequences of the engineered composite antibody or functional fragment thereof are humanized separately or individually. In some instances, the non-human framework sequence may be in the Fc region of the antibody. In some embodiments, the antibody or functional fragment thereof comprises one or more framework regions (constant regions), including light and / or heavy chain framework regions (constant regions), for example, one or more IgG1, IgG2, IgG3, and / or IgG4 framework regions (constant regions).

[0086] Non-limiting examples of antibody fragments include orthopoxvirus binding and / or effector regions of antibodies (e.g., Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, dual variable region antibodies, single variable region antibodies, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, diabodies, di-diabodies, disulfide-linked Fvs (dsFv), single domain antibodies (e.g., nanobodies), or other functional fragments. In general terms, the variable (V) region domains can be any suitable arrangement of heavy chain (VH) and / or light chain (VL) variable domains). In some instances, each antibody may independently be an IgG, IgA, IgD, IgE, or IgM antibody, or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a biologically active fragment of any of these, e.g., a Fab fragment, or a light or heavy chain of any of these.

[0087] In some embodiments, the antibody or functional fragment thereof comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NOs: 1-27. Methods for expressing and purifying antibodies

[0088] The antibodies disclosed herein can be produced using a variety of methods. The antibodies of the present invention and their functional fragments can be produced from host cells. A host cell refers to a vehicle containing the essential cellular components (e.g., organelles) necessary to express the polypeptides and constructs described herein from their corresponding nucleic acids. The nucleic acid may be contained in a nucleic acid vector, which can be introduced into a host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection). The choice of nucleic acid vector depends, in part, on the host cell used. Generally, preferred host cells are of either prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0089] Nucleic acid sequences encoding the amino acid sequence of the antibody of the present invention or functional fragments thereof can be prepared by various methods. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the antibody of the present invention or functional fragments thereof may be obtained using standard techniques (e.g., gene synthesis). Alternatively, nucleic acid molecules encoding wild-type antibodies or functional fragments thereof may be modified to contain defined amino acid substitutions using standard techniques in the art (e.g., mutagenesis). Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.

[0090] The nucleic acid sequence encoding the antibody of the present invention or its functional fragment can be inserted into a vector capable of replicating and expressing the nucleic acid molecule in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector may contain various components that can be adjusted and optimized for compatibility with a specific host cell. For example, vector components may include, but are not limited to, a replication origin, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding a protein of interest, and a transcription termination sequence. Vectors that exhibit improved transfection efficiency can be linear or supercoiled.

[0091] In some embodiments, mammalian cells are used as host cells for the present invention. In some embodiments, glutamine auxotrophy and cholesterol auxotrophy phenotypes are induced by genetic manipulation of non-glutamine auxotrophic and non-cholesterol auxotrophic cells, for example, by mutation or deletion of a gene essential for endogenous glutamine biosynthesis, such as glutamine synthetase. Common methods for genetic manipulation, such as site-directed mutagenesis, zinc finger nucleases, shRNA, and transposons, are well known to those skilled in the art; see, for example, Cytotechnology. 2007 Apr; 53(1-3): 65-73. For example, mouse myeloma cells designated NS0 are known glutamine auxotrophic and cholesterol auxotrophic cells (see, for example, Barnes et al. Cytotechnology. 2000. Advances in animal cell recombinant protein production: GS-NS0 expression system Feb; 32(2): 109-23; and US20100028940).

[0092] Additional examples of mammalian cell types that can be engineered for use as host cells include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D), CRL7030, and HsS78Bst cells. In other embodiments, E. coli cells are used as host cells for the present invention. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ 1776 (ATCC® 31,537), E. coli BL21(DE3) (ATCC® BAA-1025), and E. coli 294 (ATCC® 31,446). RV308 (ATCC® 31,608). Different host cells have characteristics and specific mechanisms for post-translational processing and modification of protein products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed antibody or functional fragment thereof. The expression vectors described above can be introduced into suitable host cells using conventional techniques in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vector is introduced into the host cells for protein production, the host cells are cultured in conventional nutrient media modified, as appropriate, to induce promoters, select transformants, or amplify the genes encoding the desired sequences.Methods for expressing antibodies or functional fragments thereof are known in the art; see, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 (July 20, 2004) and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2012 (June 28, 2012).

[0093] Host cells used to produce the antibodies or functional fragments thereof of the present invention can be grown in media known in the art that are suitable for culturing the selected host cells. Examples of suitable media for mammalian host cells include Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria Broth (LB) plus necessary supplements, such as a selective agent, such as ampicillin. Host cells are cultured at a suitable temperature, for example, about 20°C to about 39°C, for example, 25°C to about 37°C, preferably 37°C, and at a CO2 level, for example, 5% to 10% (preferably, 8%). The pH of the medium is generally about 6.8 to 7.4, for example, 7.0, depending primarily on the host organism. When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for promoter activation. Conventional cell culture conditions for producing antibodies or functional fragments thereof are known in the art; see, for example, Butler, Cell Culture and Upstream Processing, Taylor & Francis; 1st edition (May 25, 2007).

[0094] Protein recovery typically involves disrupting host cells, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris can be removed by centrifugation or filtration. The protein may be further purified. The antibodies of the present invention can be purified by any method known in the art of protein purification, such as protein A affinity, other chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification (see Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley & Sons, Inc., 2009). In some instances, the antibody or functional fragment thereof can be conjugated to a marker sequence, such as a peptide to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His tag), which binds to a nickel-functionalized agarose affinity column with micromolar affinity. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein.

[0095] In some embodiments, antibodies are empirically designed (i.e., improvements are made by trial and error). In some embodiments, antibodies are designed in silico using one or more algorithms to optimize one or more characteristics. For example, a database can be created that has antibody segments previously screened using immunogenetic assays (e.g., ex vivo T cell immunogenetic assays), has half-life information for each antibody segment, or has MHC class II binding information. One or more algorithms can then be used to evaluate the database and predict amino acid changes that will improve one or more desired characteristics, such as increasing the half-life of the antibody in a subject, reducing immunogenetic responses in a subject (e.g., by avoiding sequences homologous to T cell epitopes), or both. In some embodiments, the designed antibody is then codon-optimized to minimize the use of rare codons in the coding sequence and improve protein production yield. In some embodiments, the antibody is codon-optimized for expression in mammalian cells (e.g., murine cells). Treatment method

[0096] Disclosed herein are methods of treatment using the compositions or pharmaceutical compositions disclosed herein. In some embodiments, the methods described herein can be used in the development of antibodies or functional fragments thereof that can be used to produce pharmaceuticals. In some cases, the cells can be used in the manufacture of vaccines and biological products that include antibodies or functional fragments thereof.

[0097] In some embodiments, the methods described herein can be used for in vivo delivery of an antibody or functional fragment thereof. In some cases, the methods can be used herein to deliver an antibody or functional fragment thereof to a subject in need thereof, for example, a subject in need of treatment or prevention of a disease (e.g., a viral disease, orthopoxvirus infection). For example, orthopoxviruses including abacinomacapoxvirus, aquametavirus, alaskapoxvirus, camelpoxvirus, cowpoxvirus, ectromeliavirus, monkeypoxvirus, raccoonpoxvirus, skunkpoxvirus, taterapoxvirus, vaccinia virus, variolavirus, and volepoxvirus can be treated. In some embodiments, a vaccine comprising a composition or pharmaceutical composition disclosed herein can be administered to a subject to increase immunogenicity in the subject. In some embodiments, the composition or pharmaceutical composition can be used to prevent, ameliorate, and / or reduce the severity of an orthopoxvirus infection.

[0098] In some embodiments, the methods, systems, and compositions can be used in animal models, such as mice or any mammal, generated by methods according to the present disclosure specifically designed for the study of orthopoxviruses.

[0099] In some embodiments, a method of treatment may include a therapeutically effective dose (e.g., a unit dose) of an antibody or functional fragment thereof. In some cases, a composition disclosed herein can be administered to a subject in need of treatment to effect treatment. In some cases, the treatment may be prophylactic and / or therapeutic, and may target any viral disease (e.g., orthopoxvirus). In some cases, the treatment scheme may be determined by a physician in each case depending on factors such as the orthopoxvirus being treated, the patient's age, and weight. In some embodiments, the antibody or functional fragment thereof is administered to a patient by a route selected from the group consisting of topical, sublingual, buccal, intravenous, subcutaneous, enteral, intraarterial, intramuscular, intraperitoneal, epidural, intrathecal, intraventricular, intraarticular, intraosseous injection, intracardiac, intravitreal, parenteral, vaginal, intracavernous, intravesical, rectal, transdermal, and perivascular.

[0100] In some embodiments, a method of treating a patient in need with an antibody or functional fragment thereof may include a first antibody that binds to an epitope found on the mature virion (MV) form of a virus of the Orthopoxvirus genus; and a second antibody that binds to an epitope found on the enveloped virion (EV) form of a virus of the Orthopoxvirus genus.

[0101] In some embodiments, the present disclosure may include a method of treating a patient in need thereof with an antibody or functional fragment thereof, comprising: a first antibody that binds to an epitope found on an MV form of an Orthopoxvirus virus; and a second antibody that binds to a first epitope found on an EV form of an Orthopoxvirus virus; and a third antibody that binds to a second epitope found on the EV form of an Orthopoxvirus virus, wherein the first epitope is different from the second epitope. Vaccine application

[0102] In some embodiments, the antibody or functional fragment thereof can be delivered as a vaccine. In some embodiments, the present disclosure may include a method of conferring passive immunity to a subject against smallpox, comprising administering to the subject an effective amount of a composition comprising a first antibody that binds to an epitope found on the mature virion (MV) form of variola virus and a second antibody that binds to an epitope found on the enveloped virion (EV) form of variola virus.

[0103] In some embodiments, the present disclosure may include a method of conferring passive immunity to infection by monkeypox virus in a subject, comprising administering to the subject an effective amount of a composition comprising: a first antibody that binds to an epitope found on the mature virion (MV) form of the monkeypox virus; and a second antibody that binds to an epitope found on the enveloped virion (EV) form of the monkeypox virus.

[0104] In some embodiments, the present disclosure may include a method of conferring passive immunity to smallpox in a subject, the method comprising administering to the subject an effective amount of a composition comprising: a first antibody that binds to an epitope found on the mature virion (MV) form of variola virus; and a second antibody that binds to a first epitope found on the enveloped virion (EV) form of the variola virus; and a third antibody that binds to a second epitope found on the EV form of the variola virus, wherein the second epitope is different from the first epitope.

[0105] In some embodiments, the present disclosure may include a method of conferring passive immunity to infection by monkeypox virus in a subject, comprising administering to the subject an effective amount of a composition comprising: a first antibody that binds to an epitope found on the mature virion (MV) form of the monkeypox virus; and a second antibody that binds to a first epitope found on the enveloped virion (EV) form of the monkeypox virus; and a third antibody that binds to a second epitope found on the EV form of the monkeypox virus, wherein the second epitope is different from the first epitope. Formulations, and excipients, carriers and diluents

[0106] In some embodiments, the compositions disclosed herein can comprise pharmaceutical preparations containing one or more antibodies or functional fragments thereof described herein.In some cases, the compositions herein can comprise pharmaceutical compositions.In some cases, pharmaceutical compositions can be in unit dosage form.In some cases, the formulations containing compounds according to the present disclosure can be in liquid, solid, semi-solid form, such as solution, suspension, emulsion, or lyophilized powder form, preferably in unit dosage form suitable for easy administration of accurate dosage.

[0107] In some embodiments, pharmaceutical compositions can include a conventional pharmaceutical carrier or excipient, and can additionally include other medicinal agents, carriers, adjuvants, additives, etc. In some cases, the composition can be about 0.1% to about 85%, or about 0.5% to about 75%, by weight, of an antibody or functional fragment thereof of the present disclosure, with the remainder consisting essentially of suitable pharmaceutical excipients. In some embodiments, the amount of active ingredient, e.g., one or more antibodies or functional fragments thereof of the present invention, included in the pharmaceutical preparation is such that a suitable dosage within a designed range is provided (e.g., a dosage within the range of 0.01 to 500 mg / kg body weight).

[0108] Acceptable carriers and excipients in pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers, antioxidants, preservatives, polymers, amino acids, and carbohydrates. The pharmaceutical compositions of the present invention can be administered parenterally in the form of an injectable formulation. Pharmaceutical compositions for injection (i.e., intravenous injection) can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco's Modified Eagle's Medium (DMEM), alpha-Modified Eagle's Medium (α-MEM), F-12 medium). Formulation methods are known in the art; see, for example, Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Francis Group, CRC Press (2006). In some embodiments, the compositions herein may be formulated with or without the following excipients: acacia, acesulfame potassium, glacial acetic acid, acetone, acetyl tributyl citrate, acetyl triethyl citrate, adipic acid, agar, albumin, alcohol, alginic acid, aliphatic polyesters, alitame, allantoin, almond oil, alpha hydroxy acids, alpha tocopherol, aluminum hydroxide adjuvant, aluminum monostearate, aluminum oxide, aluminum phosphate adjuvant, ammonia solution, ammonium alginate, ammonium chloride, argan. Oil, ascorbic acid, ascorbyl glucoside, ascorbyl palmitate, aspartame, attapulgite, azelaic acid, azulene, bakuchiol, beta-glucan, beta-hydroxy acid, bentonite, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, boric acid, bronopol, butylated glycol, butylated hydroxyanisole, butylated hydroxytoluene, butylene glycol, butylparaben, calcium acetate, calcium alginate, calcium carbonate, calcium chloride, calcium hydroxide,Calcium lactate, dibasic calcium phosphate anhydrous, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, calcium silicate, calcium stearate, calcium sulfate, canola oil, glycol caprate, capric triglyceride, carbomer, carbon dioxide, calcium carboxymethylcellulose, sodium carboxymethylcellulose, carrageenan, castor oil, hydrogenated castor oil, microcrystalline cellulose, microcrystalline cellulose and sodium carboxymethylcellulose, cellulose powder, silicified microcrystalline cellulose, cellulose acetate, Cellulose acetate phthalate, ceramide, ceresin, cetostearyl alcohol, cetrimide, cetearyl alcohol, cetyl alcohol, cetylpyridinium chloride, chitosan, chlorhexidine, chlorobutanol, chlorocresol, chlorodifluoroethane (HCFC), chlorofluorocarbons (CFC), chloroxylenol, cholesterol, citric acid monohydrate, coconut oil, collagen, colloidal silicon dioxide, colorant, copper peptide, copovidone, corn oil, cornstarch and pregelatinized starch, cottonseed oil, cresol, croscarmellose Lumellose sodium, crospovidone, cyclodextrin, cyclomethicone, denatonium benzoate, desitin, dextrates, dextrin, dextrose, dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (hfc), dimethicone, dimethyl ether, dimethyl phthalate, dimethyl sulfoxide, dimethylacetamide, edetate disodium, docusate sodium, edetic acid, erythorbic acid, erythritol, ethyl acetate, ethyl lactate, ethyl maltol, ethyl oleate, Ethyl vanillin, ethyl cellulose, ethylene glycol stearate, ethylene vinyl acetate, ethylparaben, fatty acids, ferulic acid, fructose, fumaric acid, gelatin, liquid glucose, glycerin, glycerol, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate, glycine, glycofurol, glycolic acid, glycol stearate, guar gum, hectorite, heptafluoropropane (hfc), hexetidine, hydrocarbon (hc), hyaluronic acid, hydrochloric acid, hydrocortisone,Hydrophobic colloidal silica, mesoporous silica, hydroquinone, hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl betadex, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl starch, hypromellose, hypromellose acetate succinate, hypromellose phthalate, imidourea, inulin, iron oxide, isomalt, isoparaffin, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, jojoba oil, kaolin, koji Diacid, lactic acid, lactitol, lactose anhydrous, lactose inhalation, lactose monohydrate, lactose monohydrate and corn starch, lactose monohydrate and microcrystalline cellulose, lactose monohydrate and povidone, lactose monohydrate and powdered cellulose, spray dried lactose, lanolin, lanolin hydrate, lanolin alcohol, lauric acid, lecithin, leucine, linoleic acid, macrogol 15 hydroxystearate, magnesium aluminum silicate, magnesium carbonate, magnesium oxide, magnesium silicate, magnesium stearate, Magnesium trisilicate, maleic acid, malic acid, maltitol, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium chain triglycerides, meglumine, menthol, methionine, methylcellulose, methylparaben, mineral oil, light mineral oil, mineral oil and lanolin alcohol, monoethanolamine, monosodium glutamate, monothioglycerol, myristic acid, myristyl alcohol, neohesperidin dihydrochalcone, neotame, niacinamide, nitrogen, nitrous oxide, octyldodecanol, oleic acid, Oleyl alcohol, olive oil, palmitic acid, paraffin, peanut oil, pectin, PEG-8 stearate, pentetic acid, petrolatum, petrolatum and lanolin alcohols, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, phospholipids, phosphoric acid, phytic acid, phytosphingosine, polacrilin potassium, poloxamer, polycarbophil, polydextrose, poly(dl-lactic acid), polyethylene glycol, polyethylene oxide, polymethacrylate,Poly(methyl vinyl ether / maleic anhydride), polyoxyethylene alkyl ether, polyoxyethylene castor oil derivative, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, polyoxyglyceride, polyparaben, polysorbate 60, polysorbate 80, polyvinyl acetate phthalate, polyvinyl alcohol, potassium alginate, potassium alum, potassium benzoate, potassium bicarbonate, potassium chloride, potassium citrate, potassium hydroxide, potassium metabisulfite, potassium sorbate, povidone, pro Pionic Acid, Propyl Gallate, Propylene Carbonate, Propylene Glycol, Propylene Glycol Alginate, Propylparaben, Propylparaben Sodium, Pyrrolidone, Raffinose, Retin A, Retinol and Retinoic Acid Derivatives, Saccharin, Sodium Saccharin, Safflower Oil, Salicylic Acid, Saponite, Sesame Oil, Shellac, Simethicone, Sodium Acetate, Sodium Alginate, Sodium Ascorbate, Sodium Benzoate, Sodium Bicarbonate, Sodium Borate, Sodium Carbonate, Sodium Chloride, Sodium Citrate Dihydrate, Sodium Sodium chloramate, sodium formaldehyde sulfoxylate, sodium hyaluronate, sodium hydroxide, sodium lactate, sodium lauryl sulfate, sodium metabisulfite, dibasic sodium phosphate, monobasic sodium phosphate, sodium propionate, sodium starch glycolate, sodium stearyl fumarate, sodium ascorbyl phosphate, sodium deoxycholate, sodium hydroxide, sodium lauroyl lactylate, sodium lauryl sulfate, sodium palmitate, sorbitan stearate, sodium sulfite (E221), spinolactone, sodium sulfite, sodium thiosulfate, sorbic acid, sorbitan esters (sorbitan fatty acid esters), sorbitan monostearate, sorbitol, soybean oil, sphingomyelin, starch, pregelatinized starch, disinfected corn starch, stearic acid, stearyl alcohol, squalene, sucralose, sucrose, sucrose octaacetate, compressible sugar, confectioners' sugar, spherical sugar, sulfobutyl ether b-cyclodextrin, sulfur dioxide, sulfuric acid, sunflower oil, hard fat suppository base, tagatose, talc,The composition may contain one or more of the following: tartaric acid, tetrafluoroethane (HFC), thaumatin, thimerosal, thymol, titanium dioxide, tragacanth, trehalose, tretinoin, triacetin, tributyl citrate, tricaprylin, triethanolamine, triethyl citrate, triethanolamine, triolein, undecylenic acid, vanillin, hydrogenated vegetable oil, vitamins, vitamin E, polyethylene glycol succinate, water, anionic emulsifying wax, carnauba wax, cetyl ester wax, microcrystalline wax, nonionic emulsifying wax, white wax, yellow wax, xanthan gum, xylitol, zein, zinc acetate, and / or zinc stearate.

[0109] In some cases, compositions, e.g., pharmaceutical compositions, may include carriers or diluents. In some instances, carriers or diluents may include water, alcohol, salt solutions (e.g., physiological saline), or mixtures thereof. In some instances, carriers may include carbohydrates, buffers, salts, pH adjusters, or any combination thereof. In some cases, compositions herein may include buffering agents, polymers, antioxidants, preservatives, chelating agents, viscosity adjusters, isotonicity agents, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, or any combination thereof.

[0110] In some embodiments, injectable compositions for parenteral administration (e.g., intravenous, intramuscular, or intrathecal) can contain the compound in a suitable intravenous solution, such as sterile physiological salt solution. The composition can also be formulated as a suspension in an aqueous emulsion. Administration

[0111] In some cases, the pharmaceutical composition can be administered by a method selected from the group consisting of topical, oral, sublingual, buccal, mucosal, nasal, intravenous, subcutaneous, enteral, intraarterial, intramuscular, intraperitoneal, epidural, intrathecal, intraventricular, intraarticular, intraosseous injection, intracardiac, intravitreal, parenteral, vaginal, intracavernosal, intravesical, rectal, local, transdermal, inhalation, perivascular, ocular, ear canal, and any combination thereof.

[0112] In some cases, administering can include delivering the composition. In some cases, delivering can include injection, intravenous administration, subcutaneous administration, intramuscular administration, or a combination thereof. The compositions provided herein can be administered by any method. In some instances, the subject can administer the composition without supervision. In some instances, the subject can administer the composition under the supervision of a medical professional (e.g., a doctor, a nurse, a physician's assistant, a janitor, a hospice worker). In some cases, a medical professional can administer the composition. In some cases, the subject can administer the composition.

[0113] In some embodiments, administration of the compositions herein can occur at least about once a day, twice a day, three times a day, or more than four times a day. In some cases, administration can occur daily, weekly, monthly, or as needed. In some cases, administration or application of the compositions herein can occur over a treatment period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some cases, the treatment period can be about 1 to about 30 days, about 1 to about 60 days, about 1 to about 90 days, about 30 to about 90 days, about 60 to about 90 days, about 30 to about 180 days, about 90 to about 180 days, or about 180 to about 360 days. In some embodiments, administration of the compositions disclosed herein can be carried out over a treatment period of at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 3 months, at least about 6 months, at least about 12 months, at least about 1 year, or until death. Administration can be repeated over the course of the subject's life, such as once a month or once a year over the subject's life.

[0114] In some embodiments, the composition to be administered can contain a pharmaceutically effective amount of the selected compound for therapeutic use in a biological system, including a patient or subject according to the present disclosure.

[0115] In some embodiments, a method for treating a patient or subject for a particular disease state or infection can include administering an effective amount of a pharmaceutical composition comprising an antibody or functional fragment thereof according to the present disclosure and / or at least one additional biologically active (e.g., antiviral) agent. In some cases, a therapeutically effective amount of a composition herein ranges from about 0.000001 mg / kg to about 1000 mg / kg, where mg can be mg of the composition and kg can be kg of the subject's body weight. For example, about 0.000001 mg / kg, 0.00001 mg / kg, 0.0001 mg / kg, 0.001 mg / kg, 0.1 mg / kg, 1.0 mg / kg, 10 mg / kg, 100 mg / kg, or 1000 mg / kg of a composition can be administered to a subject in need thereof. kit

[0116] In some embodiments, disclosed herein are kits and containers thereof suitable for carrying out any one of the methods disclosed herein.

[0117] In some embodiments, such a kit may contain a composition comprising an antibody or functional fragment thereof, the composition may include, in a container for shipment, a first antibody that binds to an epitope found on the mature virion (MV) form of a virus of the Orthopoxvirus genus; and a second antibody that binds to an epitope found on the enveloped virion (EV) form of a virus of the Orthopoxvirus genus.

[0118] In some embodiments, such a kit may contain, in a container for shipment, a composition comprising an antibody or functional fragment thereof, comprising: a first antibody that binds to an epitope found on the MV form of a virus of the Orthopoxvirus genus; and a second antibody that binds to a first epitope found on the EV form of a virus of the Orthopoxvirus genus; and a third antibody that binds to a second epitope found on the EV form of a virus of the Orthopoxvirus genus, wherein the first epitope is different from the second epitope.

[0119] In some embodiments, such kits can contain antibodies or functional fragments thereof for delivery as a vaccine. In some embodiments, the kits include, in a container for transport, an effective amount of a composition comprising a first antibody that binds to an epitope found on the mature virion (MV) form of the smallpox virus and a second antibody that binds to an epitope found on the enveloped virion (EV) form of the smallpox virus.

[0120] In some embodiments, the kit comprises, in a container for shipment, an effective amount of a composition comprising: a first antibody that binds to an epitope found on the mature virion (MV) form of monkeypox virus; and a second antibody that binds to an epitope found on the enveloped virion (EV) form of said monkeypox virus.

[0121] In some embodiments, the kit comprises, in a container for shipment, an effective amount of a composition comprising: a first antibody that binds to an epitope found on the mature virion (MV) form of the smallpox virus; and a second antibody that binds to a first epitope found on the enveloped virion (EV) form of the smallpox virus; and a third antibody that binds to a second epitope found on the EV form of the smallpox virus, wherein the second epitope is different from the first epitope.

[0122] In some embodiments, the kit comprises, in a container for shipment, an effective amount of a composition comprising: a first antibody that binds to an epitope found on the mature virion (MV) form of monkeypox virus; and a second antibody that binds to a first epitope found on the enveloped virion (EV) form of the monkeypox virus; and a third antibody that binds to a second epitope found on the EV form of the monkeypox virus, wherein the second epitope is different from the first epitope.

[0123] In some cases, the kit can further comprise a suitable excipient, carrier, diluent, or any combination thereof.In some cases, the excipient, carrier, diluent, or any combination thereof can be any known excipient, carrier, diluent, or any combination thereof for storing antibody or its functional fragment.The storage device can be any device known in the art for storing antibody or its functional fragment, including but not limited to glass vial, plastic vial, pre-filled syringe, and ampoule. [Example]

[0124] Example 1 Humanization of mouse-human mAb c7D11 A set of nine humanized heavy and light chain V region sequences (three heavy and three light chains) was designed entirely from segments of human V region sequences with the goal of avoiding T cell epitopes. The variants were designed using a database containing antibody segments previously screened using an ex vivo T cell immunogenicity assay, an in silico tool for assessing MHC class II binding. After design, the three heavy and three light chain variable domain sequences were codon-optimized for expression in mouse cells, synthesized, and cloned into a cloning vector with flanking restrictions for subsequent cloning into a vector suitable for expression.

[0125] The mouse-human c7D11 mAb (SEQ ID NO: 1) was cloned and expressed. Figure 1 shows the fully humanized heavy chain h7D11 variant VH2 amino acid sequence (SEQ ID NO: 3) aligned with the original c7D11 heavy chain chimeric sequence (SEQ ID NO: 2), and notes the amino acid changes made in the humanized variant VH2. Figure 2 shows a representative fully humanized light chain h7D11 variant VK3 amino acid sequence (SEQ ID NO: 5) aligned with the c7D11 light chain chimeric sequence (SEQ ID NO: 4), and notes the amino acid changes made in the humanized variant VK3. Example 2 Variant c7D11 expression

[0126] A stable NS0 clonal cell line was generated that expresses a properly folded, fully functional IgG consisting of two mature heavy chains with the sequence VH2 and two mature light chains with the sequence VK3, each without a signal peptide that is cleaved during the translocation process in the cell. Example 3 chimp - Humanization of human chimeric mAb c8A

[0127] A full-sized chimp-human mAb was generated by fusing the heavy chain variable domain to a human IgG1 constant sequence and the light chain variable domain to a human lambda constant sequence. This chimeric mAb demonstrated in vitro antiviral activity against both VACV and VARV in a comet reduction assay. The mAb also demonstrated protective efficacy in a BALB / c mouse pneumonia model using a lethal challenge with VACV Western Reserve (WR).

[0128] Chimp-human mAb c8A was cloned and expressed (SEQ ID NO: 6). Figure 3 shows the fully humanized heavy chain h8A variant VH3 amino acid sequence (SEQ ID NO: 8) aligned with the original c8A heavy chain chimeric sequence (SEQ ID NO: 7), and the amino acid changes made in the humanized variant VH3 are noted. Figure 4 shows the fully humanized light chain h8A variant VK2 amino acid sequence (SEQ ID NO: 10) aligned with the original c8A light chain chimeric sequence (SEQ ID NO: 9), and the amino acid changes made in the humanized variant VK2 are noted. Example 4 Variant c8A expression

[0129] A stable NS0 clonal cell line, designated AD2, was generated that expresses a properly folded, fully functional IgG with two mature heavy chains with the sequence VH3 and two mature light chains with the sequence VK2, without the signal peptide cleaved during the translocation process in the cell. Example 5 chimp - Humanization of human chimeric mAb c8A

[0130] A full-sized chimp-human mAb was generated by fusing the heavy chain variable domain to a human IgG1 constant sequence and the light chain variable domain to a human lambda constant sequence. Chimp-human mAb c8A was cloned and expressed (SEQ ID NO: 6). Figure 5 shows the fully humanized heavy chain h8A variant VH1 amino acid sequence (SEQ ID NO: 12) aligned with the original c8A heavy chain chimeric sequence (SEQ ID NO: 11), and the amino acid changes made in the humanized variant VH1 are noted. Figure 6 shows the fully humanized light chain h8A variant VK3 amino acid sequence (SEQ ID NO: 14) aligned with the original c8A light chain chimeric sequence (SEQ ID NO: 13), and the amino acid changes made in the humanized variant VK3 are noted. Example 6 Variant c8A expression

[0131] A stable NS0 clonal cell line, designated BC9, was generated that expresses a properly folded, fully functional IgG consisting of two mature heavy chains with the sequence VH1 and two mature light chains with the sequence VK3, without the signal peptide, which is cleaved during the translocation process in the cell. Example 7 chimp - Humanization of human chimeric mAb c6C

[0132] The variable domains were used to generate a full-sized chimp-human mAb by fusing the heavy chain variable domain to a human IgG1 constant sequence and the light chain variable domain to a human lambda constant sequence. This chimeric mAb demonstrated in vitro antiviral activity against both VACV and VARV in a comet reduction assay. The mAb also demonstrated protective efficacy in a BALB / c mouse pneumonia model using a lethal challenge with VACV Western Reserve (WR).

[0133] Chimp-human mAb c6C was cloned and expressed (SEQ ID NO: 15). Figure 7 shows the fully humanized heavy chain h6C variant VH2 amino acid sequence (SEQ ID NO: 17) aligned with the original c6C heavy chain chimeric sequence (SEQ ID NO: 16), and the amino acid changes made in humanized variant VH2 are noted. Figure 8 shows the fully humanized light chain h6C variant VK2 amino acid sequence (SEQ ID NO: 19) aligned with the original c6C light chain chimeric sequence (SEQ ID NO: 18), and the amino acid changes made in humanized variant 2 are noted. Example 8 Engineering enhanced half-life in humanized mAbs

[0134] The neonatal Fc receptor (FcRn) belongs to the broad major histocompatibility complex (MHC) class of molecules whose general function involves antigen presentation. However, FcRn itself has a narrower range of functions, as it cannot present antigens; instead, it plays a role in serum half-life through high-affinity binding of IgG at low pH. This interaction may be driven by association of FcRn at the interface of the CH2 and CH3 domains in the Fc region of IgG. Engineering the unmodified humanized 7D11 and 8A mAbs to increase serum half-life may result in a reduction in the effective dose and / or number of doses, resulting in more sustained antiviral efficacy and protection from orthopoxvirus infection. Therefore, the modifications herein can generate Fc variants with increased affinity for FcRn.

[0135] Modifications were made to enhance the affinity of FcRn in the h7D11 heavy chain Fc region (SEQ ID NO: 20). Figures 9A-9D show variants of the amino acid sequence in the Fc region of the h7D11 heavy chain (SEQ ID NOs: 21-23) aligned with the unmodified repeats. Amino acid changes are noted in the variants. Example 9 Modifications in the h8A heavy chain Fc region to enhance FcRn affinity

[0136] Modifications were made to enhance the affinity of FcRn in the h8A heavy chain Fc region (SEQ ID NO: 24). Figures 10A-10D show variants of the amino acid sequence in the Fc region of the h8A heavy chain (SEQ ID NOs: 25-27) aligned with the unmodified repeats. The amino acid changes are noted in the variants. It is also important to note that while the Fc region is consistent between h7D11 and h8A, the amino acid changes are identical but located at different positions due to differences of two amino acids in the variable regions of each heavy chain. Example 10 Drug Product Composition

[0137] The combination of at least one anti-MV and one anti-EV can form an effective and efficacious drug product. Disclosed are pharmaceutical compositions that can be a combination of mAb h7D11 and h8A, mAb h7D11 and h6C, or a triple combination of mAb h7D11, mAb h8A, and mAb h6C. Variants of each of the mAb components can be selected from those disclosed herein, including variants of mAb h7D11 and mAb h8A with modifications that confer enhanced serum half-life, as described herein. The pharmaceutical composition can be in liquid, frozen, or lyophilized form. The amount of any antibody or fragment thereof can be independently present in a composition or pharmaceutical composition in an amount ranging from about 0.0001 mg to about 10,000 mg, e.g., about 0.001 mg, 0.01 mg, 0.1 mg, 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 500 mg, 1,000 mg, 5,000 mg, or 10,000 mg. The antibody or functional fragment thereof can be administered to a subject in an amount ranging from about 0.001 mg per kg to about 200 mg per kg, where mg per kg is mg of antibody or functional fragment thereof and kg is kg of the subject's body weight. The antibody can be administered intravenously, subcutaneously, intramuscularly, e.g., as separate formulations administered sequentially or simultaneously, or as part of a pharmaceutical composition containing a pharmaceutically acceptable excipient, diluent, carrier, or any combination thereof. Administration can be 1, 2, 3, 4, or 5 times daily; for example, administration can be about once daily, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, or as needed. In some embodiments, two or more antibodies, which can be monoclonal antibodies (mAbs), are to be administered to a subject, and each antibody or mAb can be mixed at the time of administration or given as separate injections, either intramuscularly, subcutaneously, or intravenously, simultaneously or sequentially. Any pharmaceutical or antibody composition or pharmaceutical or antibody formulation can be in unit dose form.The subject may be a mammal, which may be a human. The subject may be a subject in need thereof. The compositions, pharmaceutical compositions, antibodies, and functional fragments thereof may be contained in a container such as a bag, e.g., an intravenous bag, or a syringe, thereby forming a kit. Example 11 High-concentration drug product formulations for intramuscular administration

[0138] For example, an intramuscular dose volume of 5 ml per injection, with a concentration of >100 mg total IgG / ml, may be effective for delivery, with an estimated dose of 10-200 mg total IgG per kg of subject body weight. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Histidine, sucrose, and polysorbate-80 can be used individually or in any combination as components for monoclonal antibody formulations. The average pH for monoclonal antibody formulations may be 6.0±0.4. A stable formulation at pH 6.0 has been developed that may contain histidine (buffering agent), sucrose (stabilizer and excipient), and polysorbate-80 (stabilizer). In addition to liquid formulations, to minimize injection volume, the composition can be filled at the target dose and lyophilized. Prior to administration, the lyophilized drug product can be reconstituted to half the original fill volume. Similar formulations may be used for subcutaneously or intravenously delivered antibodies, functional fragments thereof, compositions comprising same, or pharmaceutical compositions comprising same. Example 12 Use of the composition

[0139] Indications for the antibodies and compositions herein may include: i) post-exposure prophylaxis against monkeypox in adult and pediatric individuals at high risk of progression to symptomatic monkeypox; ii) treatment of monkeypox in adult and pediatric patients who have had a positive monkeypox virus test result and are at high risk of progression to symptomatic monkeypox; and iii) pre-exposure prophylaxis for certain adult and pediatric individuals without known monkeypox virus exposure who have a moderate to severely compromised immune system or for whom vaccination with any available monkeypox vaccine is not recommended due to a history of severe adverse reactions to monkeypox vaccine and / or monkeypox vaccine components.

[0140] Medical conditions or treatments that may result in moderate to severe immunocompromise and an inadequate immune response to monkeypox vaccination include, but are not limited to, i) moderate or severe primary immunodeficiency (e.g., DiGeorge syndrome, Wiskott-Aldrich syndrome); ii) active treatment for solid tumors and hematologic malignancies receiving chemotherapy agents classified as severely immunosuppressive; iii) undergoing solid organ transplantation and taking immunosuppressants associated with the transplant; iv) undergoing chimeric antigen receptor (CAR)-T cell or hematopoietic stem cell transplantation (within 2 years of transplantation or taking immunosuppressive therapy); v) active or untreated HIV infection; or vi) active treatment with high-dose corticosteroids (e.g., ≧20 mg prednisone, or the equivalent per day if administered for ≧2 weeks), alkylating agents, tumor necrosis factor (TNF) blockers, and other biologic agents that are immunosuppressive or immunomodulatory (e.g., B-cell depleting agents). Example 13 Dosage and Administration

[0141] Dosages for emergency use of pre-exposure prophylaxis and post-exposure prophylaxis in the treatment of monkeypox in adult and pediatric patients will be determined in non-human animal studies and validated in Phase 1 human trials in healthy adult volunteers. Doses can be administered as two separate, sequential intramuscular injections into each gluteal muscle, as a single intramuscular injection containing two antibodies into one gluteal muscle, or via intravenous infusion.

[0142] The injection may include a pharmaceutical composition comprising the active pharmaceutical ingredient (API) humanized mAb h7D11, with or without engineered half-life modifications, in a single-dose vial as a liquid formulation or reconstituted from a lyophilized state. The injection may also include a pharmaceutical composition comprising the active pharmaceutical ingredient (API) humanized mAb h8A, with or without engineered half-life modifications, in a single-dose vial as a liquid formulation or reconstituted from a lyophilized state. Two or more antibodies or functional fragments thereof may be combined to be reconstituted in a single-dose vial as a liquid formulation or reconstituted from a lyophilized state.

[0143] The antibodies and compositions herein may be contraindicated in individuals with a previous severe hypersensitivity reaction, including anaphylaxis, to the antibody or any component of the composition.

[0144] Other indications may include pre-exposure prophylaxis of monkeypox in adult and pediatric individuals (12 years of age and older weighing at least 40 kg) who: i) are not currently infected with monkeypox and have no known recent exposure to a monkeypox-infected individual; ii) have moderate to severe immunocompromise due to a medical condition or are receiving immunosuppressive medications or treatments that may not mount an adequate immune response to monkeypox vaccination; or iii) for whom vaccination with any available approved or licensed monkeypox vaccine is not recommended due to a history of severe adverse reactions (e.g., severe allergic reactions) to monkeypox vaccine and / or monkeypox vaccine components.

[0145] Other indications may include post-exposure prophylaxis in adult and pediatric individuals (12 years of age and older weighing at least 40 kg) at high risk of progression to severe monkeypox disease, including hospitalization or death.

[0146] Other indications may include the treatment of active monkeypox disease in adult and pediatric individuals (12 years of age and older weighing at least 40 kg) who have laboratory-confirmed monkeypox infection, have a positive monkeypox virus test result, are symptomatic or asymptomatic patients who are at high risk of rapidly progressing to symptomatic monkeypox, and for whom other forms of treatment are contraindicated or deemed suboptimal.

[0147] This randomized, double-blind, placebo-controlled trial in adults will determine the pre-exposure prophylaxis dose. The primary endpoint will be a significant reduction in the incidence of monkeypox-positive symptomatic disease in participants receiving a single IM or IV dose compared with placebo over a median follow-up period determined relative to the half-life of the drug product.

[0148] Repeat dosing is necessary for approval of pre-exposure prophylaxis to ensure preventative efficacy for individuals determined to be suitable for long-term protection (e.g., immunocompromised or with comorbid conditions). Maintenance dosing to maintain drug exposure similar to or slightly higher than that observed near the time efficacy was determined for a single dose is extrapolated from clinical trial data. Safety may vary with repeat dosing based on hypersensitivity reactions and the development of anti-drug antibodies. Example 14 Dosage and administration in mice

[0149] Mice in groups 1 and 2 (10 male and 10 female BALBc mice; n=20) were used as control groups. Group 1 was treated with vehicle and no ectromelia virus (ECTV) challenge, and Group 2 was treated with vehicle and challenged with 200 PFU of ECTV. Figure 11 shows that mice in Group 1 had no change in mean body weight upon challenge, and Group 2 had a moderate change in mean body weight, indicating that animal health is affected by viral challenge but not by vehicle treatment.

[0150] Groups 3, 4, 5, and 6 were challenged and dosed. Mice were challenged with 200 PFU of ECTV on day 0. Groups 3, 4, 5, and 6 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 15 mg / kg. Group 3 was treated on day 3 after challenge, Group 4 on day 4 after challenge, Group 5 on day 5 after challenge, and Group 6 on day 6 after challenge. Figure 12 shows that Group 3 experienced the least weight loss, followed by Groups 4, 5, and 6. This indicates that earlier treatment during exposure to ECTV had a more pronounced benefit of treatment. FIG. 17 shows that survival probability was greater than 90% for all treatment time points compared to a 30% survival probability for Group 2 controls.

[0151] Groups 7, 8, 9, and 10 were challenged and dosed. Mice were challenged with 200 PFU of ECTV on day 0. Groups 7, 8, 9, and 10 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 7 was treated on day 3 after challenge, Group 8 on day 4 after challenge, Group 9 on day 5 after challenge, and Group 10 on day 6 after challenge. Figure 13 shows that Group 7 experienced the least weight loss, followed by Groups 10, 8, and 9. This generally indicates that earlier treatment during exposure to ECTV had a more pronounced benefit of treatment. Figure 18 shows that survival probability was greater than 90% at the time of treatment for groups 7 and 8, followed by 90% for group 9 and 70% for group 10, compared to a 30% survival probability for the control in group 2.

[0152] Groups 11, 12, 13, and 14 were challenged and dosed. Mice were challenged with 200 PFU of ECTV on day 0. Groups 11, 12, 13, and 14 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 11 was treated on day 3 post-challenge, Group 12 on day 4 post-challenge, Group 13 on day 5 post-challenge, and Group 14 on day 6 post-challenge. Figure 14 shows that Group 13 experienced the least weight loss, followed by Groups 11, 12, and 14. This generally indicates that earlier treatment during exposure to ECTV had a more pronounced benefit of treatment. FIG. 19 shows that survival probability was 100% for all treatment time points compared to a 30% survival probability for Group 2 controls.

[0153] Groups 15, 16, 17, and 18 were challenged and dosed. Mice were challenged with 200 PFU of ECTV on day 0. Groups 15, 16, 17, and 18 were treated with a cocktail of the original chimeric versions of the mAbs as controls for the humanized versions used in all other treatment groups: c7D11 (anti-L1 mAb) at 5 mg / kg, c8A (anti-B5 mAb) at 5 mg / kg, and c6C (anti-A33 mAb) at 5 mg / kg, at a total mAb cocktail dose of 15 mg / kg. Group 15 was treated on day 3 post-challenge, Group 16 on day 4 post-challenge, Group 17 on day 5 post-challenge, and Group 18 on day 6 post-challenge. Figure 15 shows that Group 15 experienced the least weight loss, followed by Groups 16, 18, and 17. This generally indicates that the earlier treatment upon exposure to ECTV, the more pronounced the benefit of treatment. Figure 20 shows that survival probability was greater than 90% for all treatment time points, compared to a 30% survival probability for Group 2 controls.

[0154] Groups 19, 20, 21, and 22 were challenged and dosed. Mice were challenged with 200 PFU of ECTV on day 0. Groups 19, 20, 21, and 22 were treated with a cocktail of 3.33 mg / kg h7D11 (anti-L1 mAb), 3.33 mg / kg h8A (anti-B5 mAb), and 3.33 mg / kg h6C (anti-A33 mAb) at a total mAb cocktail dose of 10 mg / kg. Group 19 was treated on day 3 post-challenge, Group 20 on day 4 post-challenge, Group 21 on day 5 post-challenge, and Group 22 on day 6 post-challenge. Figure 16 shows that Groups 19 and 20 experienced the least weight loss, followed by Groups 21 and 22. This generally indicates that earlier treatment during exposure to ECTV had a more pronounced benefit of treatment. FIG. 21 shows that survival probability was greater than 90% for all treatment time points compared to a 30% survival probability for Group 2 controls.

[0155] Groups 2, 3, 4, 5, and 6 were challenged and dosed. Mice were challenged with 200 PFU of ECTV on day 0. Group 2 (negative control) was treated with vehicle alone on day 3 postchallenge. Groups 3, 4, 5, and 6 were treated with a cocktail of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb) for a total mAb cocktail dose of 15 mg / kg. Group 3 was treated on day 3 postchallenge, Group 4 on day 4 postchallenge, Group 5 on day 5 postchallenge, and Group 6 on day 6 postchallenge.

[0156] Figure 22 shows a comparison of the intervention on day 3 for the composition compared to the control in Group 2. The survival probability for Groups 3, 7, 11, 15, and 19, which received treatment on day 3, was greater than 90% for all treatment groups, compared to a survival probability of 30% for the control in Group 2.

[0157] Figure 23 shows a comparison of the intervention on day 4 for the composition compared to the control in Group 2. The survival probability for Groups 4, 8, 12, 16, and 20, which received treatment on day 4, was 100% for all treatment groups, compared to a survival probability of 30% for the control in Group 2.

[0158] Figure 24 shows a comparison of the intervention on day 5 for the composition compared to the control in Group 2. The survival probability for Groups 5, 9, 13, 17, and 21, which received treatment on day 5, was equal to or greater than 90% for all treatment groups, compared to a survival probability of 30% for the control in Group 2.

[0159] Figure 25 shows a comparison of the intervention on day 6 for the composition compared to the control in Group 2. The survival probability for Groups 6, 10, 14, 18, and 22, which received treatment on day 6, was greater than 90% for all treatment groups except for Group 10, which had a survival probability of 70% compared to 30% for the control in Group 2.

[0160] While embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. (A) a first antibody that binds to an epitope found on the mature virion (MV) form of a virus of the Orthopoxvirus genus; and (B) A second antibody that binds to an epitope found on the enveloped virion (EV) form of a virus of the Orthopoxvirus genus. A composition comprising:

2. 2. The composition of claim 1, wherein the epitope found on the MV form of the virus is an epitope of the vaccinia virus L1 protein.

3. The composition of claim 2 , wherein the epitope of the L1 protein is located within a domain of the L1 protein having the amino acid sequence of SEQ ID NO:

1.

4. The composition of claim 3 , wherein the first antibody is a humanized 7D11 antibody.

5. 4. The composition of claim 3, wherein the first antibody is a humanized 7D11 antibody variant comprising the light chain amino acid sequence of SEQ ID NO:5 and the heavy chain amino acid sequence of SEQ ID NO:

3.

6. 6. The composition of claim 5, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

21.

7. 6. The composition of claim 5, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

22.

8. 6. The composition of claim 5, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

23.

9. 2. The composition of claim 1, wherein the epitope found on the EV form of the virus is an epitope of the vaccinia virus B5 protein.

10. The composition of claim 9, wherein the epitope of the B5 protein is located within a domain of the B5 protein having the amino acid sequence of SEQ ID NO:

6.

11. The composition of claim 10 , wherein the second antibody is a humanized 8A antibody.

12. The composition of claim 10, wherein the second antibody is a humanized 8A antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 10 and the heavy chain amino acid sequence of SEQ ID NO:

8.

13. 13. The composition of claim 12, wherein the humanized 8A antibody variant comprises an h8A Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

25.

14. 13. The composition of claim 12, wherein the antibody (B) is a humanized 8A antibody variant comprising an h8A Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

26.

15. 13. The composition of claim 12, wherein the antibody (B) is a humanized 8A antibody variant comprising an h8A Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

27.

16. The composition of claim 9, wherein the antibody (B) is a humanized 8A antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 14 and the heavy chain amino acid sequence of SEQ ID NO:

12.

17. 2. The composition of claim 1, wherein the epitope found on the EV form of the virus is an epitope of the vaccinia virus A33 protein.

18. The composition of claim 17, wherein the epitope of the A33 protein is located within a domain of the A33 protein having the amino acid sequence of SEQ ID NO:

15.

19. 19. The composition of claim 18, wherein the second antibody is a humanized 6C antibody.

20. 19. The composition of claim 18, wherein the second antibody is a humanized 6C antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 19 and the heavy chain amino acid sequence of SEQ ID NO:

17.

21. (A) a first antibody that binds to an epitope found on the MV form of a virus of the Orthopoxvirus genus; and (B) a second antibody that binds to a first epitope found on the EV form of a virus of the Orthopoxvirus genus; and (C) a third antibody that binds to a second epitope found on the EV form of a virus of the Orthopoxvirus genus, wherein the first epitope is different from the second epitope. A composition comprising:

22. 22. The composition of claim 21, wherein the epitope of the MV form of the virus is an epitope of the vaccinia virus L1 protein.

23. 23. The composition of claim 22, wherein the epitope of the L1 protein is located within a domain of the L1 protein having the amino acid sequence of SEQ ID NO:

1.

24. 24. The composition of claim 23, wherein the first antibody is a humanized 7D11 antibody.

25. 24. The composition of claim 23, wherein the first antibody is a humanized 7D11 antibody variant comprising the light chain amino acid sequence of SEQ ID NO:5 and the heavy chain amino acid sequence of SEQ ID NO:

3.

26. 26. The composition of claim 25, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

21.

27. 26. The composition of claim 25, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

22.

28. 26. The composition of claim 25, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

23.

29. 22. The composition of claim 21, wherein the first epitope found on the EV form of the virus is an epitope of the vaccinia virus B5 protein.

30. 30. The composition of claim 29, wherein the B5 protein is located within a domain of the B5 protein having the amino acid sequence of SEQ ID NO:

6.

31. 31. The composition of claim 30, wherein the second antibody is a humanized 8A antibody.

32. 31. The composition of claim 30, wherein the second antibody is a humanized 8A antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 10 and the heavy chain amino acid sequence of SEQ ID NO:

8.

33. 33. The composition of claim 32, wherein the humanized 8A antibody variant comprises an h8A Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

25.

34. 33. The composition of claim 32, wherein the humanized 8A antibody variant comprises an h8A Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

26.

35. 33. The composition of claim 32, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

27.

36. 22. The composition of claim 21, wherein the second epitope found on the EV form of the virus is an epitope of the vaccinia virus A33 protein.

37. 37. The composition of claim 36, wherein the epitope of the A33 protein is located within a domain of the A33 protein having the amino acid sequence of SEQ ID NO:

15.

38. 38. The composition of claim 37, wherein the third antibody is a humanized 6C antibody.

39. 38. The composition of claim 37, wherein the third antibody is a humanized 6C antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 19 and the heavy chain amino acid sequence of SEQ ID NO:

17.

40. 1. A method of conferring passive immunity to smallpox in a subject, comprising: (A) a first antibody that binds to an epitope found on the MV form of smallpox virus; and (B) A second antibody that binds to an epitope found on the EV form of smallpox virus. administering to said subject an effective amount of a composition comprising:

41. 41. The method of claim 40, wherein the subject is not infected with variola virus that causes smallpox, and the effective amount confers pre-exposure prophylactic passive immunity against smallpox.

42. 41. The method of claim 40, wherein the subject has been exposed to variola virus, which causes smallpox, and the effective amount confers post-exposure prophylactic passive immunity against smallpox.

43. 41. The method of claim 40, wherein the subject exhibits symptomatic smallpox and the effective amount confers therapeutic passive immunity against smallpox.

44. 44. The method of any one of claims 41 to 43, wherein the epitope found on the MV form of the virus is an epitope of the vaccinia virus L1 protein.

45. 45. The method of claim 44, wherein the epitope of the L1 protein is located within a domain of the L1 protein having the amino acid sequence of SEQ ID NO:

1.

46. 46. ​​The method of claim 45, wherein the first antibody is a humanized 7D11 antibody.

47. 46. ​​The method of claim 45, wherein the first antibody is a humanized 7D11 antibody variant comprising the light chain amino acid sequence of SEQ ID NO:5 and the heavy chain amino acid sequence of SEQ ID NO:

3.

48. 48. The method of claim 47, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

21.

49. 48. The method of claim 47, wherein the antibody (A) is a humanized 7D11 antibody variant comprising a h7D11 Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

22.

50. 48. The method of claim 47, wherein the antibody (A) is a humanized 7D11 antibody variant comprising a h7D11 Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

23.

51. 44. The method of any one of claims 41 to 43, wherein the epitope found on the EV form of the virus is an epitope of the vaccinia virus B5 protein.

52. 52. The method of claim 51, wherein the epitope of the B5 protein is located within a domain of the B5 protein having the amino acid sequence of SEQ ID NO:

6.

53. 53. The method of claim 52, wherein the second antibody is a humanized 8A antibody.

54. 53. The method of claim 52, wherein the second antibody is a humanized 8A antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 10 and the heavy chain amino acid sequence of SEQ ID NO:

8.

55. 55. The method of claim 54, wherein the humanized 8A antibody variant comprises an h8A Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

25.

56. 55. The method of claim 54, wherein the humanized 8A antibody variant comprises an h8A Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

26.

57. 55. The method of claim 54, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

27.

58. 53. The method of claim 52, wherein the humanized 8A antibody variant comprises the light chain amino acid sequence of SEQ ID NO: 14 and the heavy chain amino acid sequence of SEQ ID NO:

12.

59. 44. The method of any one of claims 41 to 43, wherein the epitope found on the EV form of the virus is an epitope of the vaccinia virus A33 protein.

60. 60. The method of claim 59, wherein the epitope of the A33 protein is located within a domain of the A33 protein having the amino acid sequence of SEQ ID NO:

15.

61. 61. The method of claim 60, wherein the second antibody is a humanized 6C antibody.

62. 61. The method of claim 60, wherein the second antibody is a humanized 6C antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 19 and the heavy chain amino acid sequence of SEQ ID NO:

17.

63. 1. A method of conferring passive immunity to infection by monkeypox virus in a subject, comprising: (A) a first antibody that binds to an epitope found on the MV form of the monkeypox virus; and (B) a second antibody that binds to an epitope found on the EV form of the monkeypox virus. administering to said subject an effective amount of a composition comprising:

64. 64. The method of claim 63, wherein the subject is not infected with the monkeypox virus and the effective amount confers pre-exposure prophylactic passive immunity against the monkeypox infection.

65. 64. The method of claim 63, wherein the subject has been exposed to the monkeypox virus and the effective amount confers post-exposure prophylactic passive immunity against the monkeypox infection.

66. 64. The method of claim 63, wherein the subject exhibits a symptomatic monkeypox infection and the effective amount confers therapeutic passive immunity against the monkeypox infection.

67. 67. The method of any one of claims 64 to 66, wherein the epitope found on the MV form of the virus is an epitope of the vaccinia virus L1 protein.

68. 68. The method of claim 67, wherein the epitope of the L1 protein is located within a domain of the L1 protein having the amino acid sequence of SEQ ID NO:

1.

69. 69. The method of claim 68, wherein the first antibody is a humanized 7D11 antibody.

70. 69. The method of claim 68, wherein the first antibody is a humanized 7D11 antibody variant comprising the light chain amino acid sequence of SEQ ID NO:5 and the heavy chain amino acid sequence of SEQ ID NO:

3.

71. 71. The method of claim 70, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

21.

72. 71. The method of claim 70, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

22.

73. 71. The method of claim 70, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

23.

74. 67. The method of any one of claims 64 to 66, wherein the epitope found on the EV form of the virus is an epitope of the vaccinia virus B5 protein.

75. 75. The method of claim 74, wherein the epitope of the B5 protein is located within a domain of the B5 protein having the amino acid sequence of SEQ ID NO:

6.

76. 76. The method of claim 75, wherein the second antibody is a humanized 8A antibody.

77. 76. The method of claim 75, wherein the second antibody is a humanized 8A antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 10 and the heavy chain amino acid sequence of SEQ ID NO:

8.

78. 78. The method of claim 77, wherein the humanized 8A antibody variant comprises an h8A Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

25.

79. 78. The method of claim 77, wherein the humanized 8A antibody variant comprises an h8A Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

26.

80. 78. The method of claim 77, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

27.

81. 76. The method of claim 75, wherein the humanized 8A antibody variant comprises the light chain amino acid sequence of SEQ ID NO: 14 and the heavy chain amino acid sequence of SEQ ID NO:

12.

82. 67. The method of any one of claims 64 to 66, wherein the epitope found on the EV form of the virus is an epitope of the vaccinia virus A33 protein.

83. 83. The method of claim 82, wherein the epitope of the A33 protein is located within a domain of the A33 protein having the amino acid sequence of SEQ ID NO:

15.

84. 84. The method of claim 83, wherein the second antibody is a humanized 6C antibody.

85. 84. The method of claim 83, wherein the second antibody is a humanized 6C antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 19 and the heavy chain amino acid sequence of SEQ ID NO:

17.

86. 1. A method of conferring passive immunity to smallpox in a subject, comprising: (A) a first antibody that binds to an epitope found on the MV form of smallpox virus; and (B) a second antibody that binds to a first epitope found on the EV form of the smallpox virus; and (C) a third antibody that binds to a second epitope found on the EV form of the smallpox virus, wherein the second epitope is different from the first epitope. administering to said subject an effective amount of a composition comprising:

87. 87. The method of claim 86, wherein the subject is not infected with the smallpox virus and the effective amount confers pre-exposure prophylactic passive immunity against smallpox.

88. 87. The method of claim 86, wherein the subject has been exposed to the smallpox virus and the effective amount confers post-exposure prophylactic passive immunity against smallpox.

89. 87. The method of claim 86, wherein the subject exhibits symptomatic smallpox and the effective amount confers therapeutic passive immunity against smallpox.

90. 90. The method of any one of claims 87 to 89, wherein the epitope found on the MV form of the virus is an epitope of the vaccinia virus L1 protein.

91. 91. The method of claim 90, wherein the epitope of the L1 protein is located within a domain of the L1 protein having the amino acid sequence of SEQ ID NO:

1.

92. 92. The method of claim 91, wherein the first antibody is a humanized 7D11 antibody.

93. 92. The method of claim 91, wherein the first antibody is a humanized 7D11 antibody variant comprising the light chain amino acid sequence of SEQ ID NO:5 and the heavy chain amino acid sequence of SEQ ID NO:

3.

94. 94. The method of claim 93, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

21.

95. 94. The method of claim 93, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

22.

96. 94. The method of claim 93, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

23.

97. 90. The method of any one of claims 87 to 89, wherein the first epitope found on the EV form of the virus is an epitope of the vaccinia virus B5 protein.

98. 98. The method of claim 97, wherein the epitope of the B5 protein is located within a domain of the B5 protein having the amino acid sequence of SEQ ID NO:

6.

99. 99. The method of claim 98, wherein the second antibody is a humanized 8A antibody.

100. 99. The method of claim 98, wherein the second antibody is a humanized 8A antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 10 and the heavy chain amino acid sequence of SEQ ID NO:

8.

101. 101. The method of claim 100, wherein the humanized 8A antibody variant comprises an h8A Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

25.

102. The method of claim 100, wherein the humanized 8A antibody variant comprises an h8A Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

26.

103. 101. The method of claim 100, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

27.

104. 90. The method of any one of claims 87 to 89, wherein the second epitope found on the EV form of the virus is an epitope of the vaccinia virus A33 protein.

105. 105. The method of claim 104, wherein the epitope of the A33 protein is located within a domain of the A33 protein having the amino acid sequence of SEQ ID NO:

15.

106. The method of claim 105, wherein the third antibody is a humanized 6C antibody.

107. 106. The method of claim 105, wherein the third antibody is a humanized 6C antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 19 and the heavy chain amino acid sequence of SEQ ID NO:

17.

108. 1. A method of conferring passive immunity to infection by monkeypox virus in a subject, comprising: (A) a first antibody that binds to an epitope found on the MV form of the monkeypox virus; and (B) a second antibody that binds to a first epitope found on the EV form of the monkeypox virus; and (C) a third antibody that binds to a second epitope found on the EV form of the monkeypox virus, wherein the second epitope is different from the first epitope. administering to said subject an effective amount of a composition comprising:

109. 109. The method of claim 108, wherein the subject is not infected with the monkeypox virus and the effective amount confers pre-exposure prophylactic passive immunity against monkeypox infection.

110. 109. The method of claim 108, wherein the subject has been exposed to monkeypox virus and the effective amount confers post-exposure prophylactic passive immunity against monkeypox infection.

111. 109. The method of claim 108, wherein the subject exhibits symptomatic monkeypox infection and the effective amount confers therapeutic passive immunity against monkeypox infection.

112. 112. The method of any one of claims 109 to 111, wherein the epitope found on the MV form of the virus is an epitope of the vaccinia virus L1 protein.

113. 113. The method of claim 112, wherein the epitope of the L1 protein is located within a domain of the L1 protein having the amino acid sequence of SEQ ID NO:

1.

114. 114. The method of claim 113, wherein the first antibody is a humanized 7D11 antibody.

115. 114. The method of claim 113, wherein the first antibody is a humanized 7D11 antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 5 and the heavy chain amino acid sequence of SEQ ID NO:

3.

116. 116. The method of claim 115, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

21.

117. 116. The method of claim 115, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

22.

118. 116. The method of claim 115, wherein the humanized 7D11 antibody variant comprises a h7D11 Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

23.

119. 112. The method of any one of claims 109 to 111, wherein the first epitope found on the EV form of the virus is an epitope of the vaccinia virus B5 protein.

120. 120. The method of claim 119, wherein the epitope of the B5 protein is located within a domain of the B5 protein having the amino acid sequence of SEQ ID NO:

6.

121. 121. The method of claim 120, wherein the second antibody is a humanized 8A antibody.

122. 121. The method of claim 120, wherein the second antibody is a humanized 8A antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 10 and the heavy chain amino acid sequence of SEQ ID NO:

8.

123. 123. The method of claim 122, wherein the humanized 8A antibody variant comprises an h8A Fc YTE swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

25.

124. 123. The method of claim 122, wherein the humanized 8A antibody variant comprises an h8A Fc LS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

26.

125. 123. The method of claim 122, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS swap modification comprising Fc HC2 and HC3 domains having the amino acid sequence of SEQ ID NO:

27.

126. 112. The method of any one of claims 109 to 111, wherein the second epitope found on the EV form of the virus is an epitope of the vaccinia virus A33 protein.

127. 127. The method of claim 126, wherein the epitope of the A33 protein is located within a domain of the A33 protein having the amino acid sequence of SEQ ID NO:

15.

128. 128. The method of claim 127, wherein the third antibody is a humanized 6C antibody.

129. 128. The method of claim 127, wherein the third antibody is a humanized 6C antibody variant comprising the light chain amino acid sequence of SEQ ID NO: 19 and the heavy chain amino acid sequence of SEQ ID NO: 17.

Images