Compositions and methods for preventing and treating orthopoxvirus infections

Abstract

Provided herein are antibodies and functional fragments thereof for the treatment or prophylaxis of viral diseases, such as poxvirus diseases, such as Chenpox and monkey pox. Any antibody or functional fragment thereof described herein may be an engineered antibody or an engineered antibody fragment. In addition, provided herein are compositions and pharmaceutical compositions containing one or more antibodies or functional fragments thereof. Also provided herein are kits containing one or more antibodies or functional fragments thereof, as well as methods, dosing regimens, dosage amounts, and pathways for administering one or more antibodies and one or more functional fragments thereof. Finally, methods of making the antibodies and functional fragments thereof are provided.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 402,900, filed Aug. 31, 2022, the entire content of which is incorporated herein by reference.

[0003] Statement Regarding Federally Sponsored Research or Development

[0004] This invention was made with government support under Contract No. 75A50119C00054 awarded by the Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response; Biomedical Advanced Research and Development Authority. The U.S. government has certain rights in this invention. SUMMARY OF THE INVENTION

[0006] Compositions for preventing and treating orthopoxvirus infections such as vaccinia virus, monkeypox virus, and variola virus (the causative agent of the human smallpox disease) are disclosed herein, including compositions comprising antibodies that bind to epitopes found on the intracellular mature virions or mature virion forms of orthopoxviruses and / or antibodies that bind to epitopes found on the extracellular enveloped virions or enveloped virion forms of orthopoxviruses. Compositions such as humanized antibodies and those that have been modified to reduce immunogenicity and / or extend serum half - life are also disclosed, as well as methods of using them in conferring passive immunity against orthopoxvirus infection to individuals at risk of orthopoxvirus infection or individuals presenting with vaccinia infection, including adverse events caused by certain smallpox vaccines such as progressive vaccinia and eczema vaccinatum, monkeypox infection, or smallpox.

[0007] Antibodies and fragments thereof for treating or preventing viral diseases, such as poxvirus diseases, such as orthopoxvirus diseases, such as smallpox and monkeypox, are provided herein. Compositions and pharmaceutical compositions comprising one or more antibodies or fragments thereof are also provided herein. Kits comprising one or more antibodies or fragments thereof are also provided, as well as methods, dosing regimens, dose amounts, and routes of administering one or more antibodies and one or more fragments thereof that may be comprised in the compositions. Finally, methods of preparing the antibodies and fragments thereof are provided.

[0008] A composition is also provided that comprises: (A) a first antibody that binds to an epitope found on the mature virion (MV) form of a virus of the genus Orthopoxvirus; and (B) a second antibody that binds to an epitope found on the enveloped virion (EV) form of a virus of the genus Orthopoxvirus.

[0009] Additionally provided is a composition comprising: (A) a first antibody that binds to an epitope found on the MV form of a virus of the genus Orthopoxvirus; and (B) a second antibody that binds to a first epitope found on the EV form of a virus of the genus Orthopoxvirus; and (C) a third antibody that binds to a second epitope found on the EV form of a virus of the genus Orthopoxvirus, wherein the first epitope is different from the second epitope.

[0010] Additionally provided is a method of conferring passive immunity against smallpox to 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 an epitope found on the EV form of variola virus.

[0011] Also provided herein is a method of conferring passive immunity against monkeypox virus infection to 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 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.

[0012] Also provided herein is a method of conferring passive immunity against smallpox to 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 variola virus; and (C) 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.

[0013] Also disclosed herein is a method of conferring passive immunity against monkeypox virus infection to 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 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.

[0014] The invention summary provides exemplary embodiments and is not meant to limit in any way what is provided herein.

[0015] Incorporation by reference

[0016] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Brief Description of the Drawings

[0018] The features disclosed herein are specifically set forth 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 of illustrative embodiments that utilize the principles of the present disclosure, along with the accompanying drawings, in which:

[0019] Figure 1 An amino acid sequence showing 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 occurring in the humanized variant VH2 highlighted in dark gray. The light gray highlighting indicates the framework sequences, and no highlighting indicates the signal peptide, CDR sequences 1, 2, and 3, and the human IgG 1 constant region.

[0020] Figure 2 An amino acid sequence showing 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 occurring in the humanized variant VK3 highlighted in dark gray. The light gray highlighting indicates the framework sequences, and no highlighting indicates the signal peptide, CDR sequences 1, 2, and 3, and the human κ constant region.

[0021] Figure 3 An amino acid sequence showing an exemplary fully humanized heavy chain h8A variant VH3 amino acid sequence aligned with the c8A chimeric sequence, with the amino acid changes occurring in the humanized variant VH3 highlighted in dark gray. The light gray highlighting indicates the framework sequences, and no highlighting indicates the signal peptide, CDR sequences 1, 2, and 3, and the human IgG 1 constant region.

[0022] Figure 4 An amino acid sequence showing an exemplary fully humanized light chain h8A variant VK2 amino acid sequence aligned with the c8A chimeric sequence, with the amino acid changes occurring in the humanized variant VK2 highlighted in dark gray. The light gray highlighting indicates the framework sequences, and no highlighting indicates the signal peptide, CDR sequences 1, 2, and 3, and the human κ constant region.

[0023] Figure 5An amino acid sequence showing an exemplary fully humanized heavy chain h8A variant VH1 amino acid sequence aligned with the c8A chimeric sequence, with amino acid changes occurring in the humanized variant VH1 highlighted in dark gray. The light gray highlighting indicates the framework sequence, and no highlighting indicates the signal peptide, CDR sequences 1, 2, and 3, and the human IgG 1 constant region.

[0024] Figure 6 An amino acid sequence showing an exemplary fully humanized light chain h8A variant VK3 amino acid sequence aligned with the c8A chimeric sequence, with amino acid changes occurring in the humanized variant VK3 highlighted in dark gray. The light gray highlighting indicates the framework sequence, and no highlighting indicates the signal peptide, CDR sequences 1, 2, and 3, and the human κ constant region.

[0025] Figure 7 An amino acid sequence showing an exemplary fully humanized heavy chain h6C variant VH2 amino acid sequence aligned with the c6C chimeric sequence, with amino acid changes occurring in the humanized variant VH2 highlighted in dark gray. The light gray highlighting indicates the framework sequence, and no highlighting indicates the signal peptide, CDR sequences 1, 2, and 3, and the human IgG 1 constant region.

[0026] Figure 8 An amino acid sequence showing an exemplary fully humanized light chain h6C variant VK2 amino acid sequence aligned with the c6C chimeric sequence, with amino acid changes occurring in the humanized variant VK2 highlighted in dark gray. The light gray highlighting indicates the framework sequence, and no highlighting indicates the signal peptide, CDR sequences 1, 2, and 3, and the human κ constant region. Also highlighted in dark gray is the asparagine residue (N) in CDR3, indicating a potential N-linked glycosylation site.

[0027] Figure 9 A- Figure 9 D shows the amino acid sequences of several exemplary FcRn affinity-enhanced variants aligned with the unmodified Fc region of h7D11 HC. No highlighting indicates the CH2 domain of the Fc region. The light gray highlighting indicates the CH3 domain of the Fc region. The dark gray highlighting indicates the modified amino acids. The unmodified h7D11 HC Fc region ( Figure 9 A). h7D11 Fc with amino acid substitutions M280Y, S282T, and T284E ( Figure 9 B). h7D11 Fc with amino acid substitutions M456L and N462S ( Figure 9 C). h7D11 Fc with amino acid substitutions M280Y, S282T, T284E, M456L, and N462S ( Figure 9 D).

[0028] Figure 10 A- Figure 10 Panel D shows the amino acid sequences of several exemplary FcRn affinity-enhanced variants compared to 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. The unmodified h8A HC Fc region ( Figure 10 A). h8A Fc with amino acid substitutions M282Y, S284T, and T286E ( Figure 10 B). h8A Fc with amino acid substitutions M458L and N464S ( Figure 10 C). h8A Fc with amino acid substitutions M282Y, S284T, T286E, M458L, and N464S ( Figure 10 D).

[0029] Figure 11 is a dot plot showing the group mean body weights 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 not challenged with ectromelia virus (ECTV), and group 2 was treated with vehicle and challenged with 200 PFU of ECTV.

[0030] Figure 12 is a dot plot showing the group mean body weights by study day for test material 1 in 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 mixture of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture 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 13 is a dot plot showing the group mean body weights by study day for test material 2 in 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 mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb), with a total mAb mixture 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 14 A dot plot showing the group mean weights of Test Material 3 for Groups 11, 12, 13, and 14 by study day. 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 mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture 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 15 A dot plot showing the group mean weights of Test Material 4 for Groups 15, 16, 17, and 18 by study day. 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 mixture of the original chimeric form of the mAbs as a comparator to the humanized form used in all other treatment groups at 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb), with a total mAb mixture 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.

[0034] Figure 16 A dot plot showing the low-dose group mean weights of Test Material 5 for Groups 19, 20, 21, and 22 by study day. 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 mixture 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), with a total mAb mixture 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 17It is a Kaplan-Meier survival curve showing the results of Set 1. All groups 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 3, 4, 5, and 6 were treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture 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.

[0036] Figure 18 It is a Kaplan-Meier survival curve showing the results of Set 2. All groups 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 7, 8, 9, and 10 were treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb), with a total mAb mixture 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.

[0037] Figure 19 It is a Kaplan-Meier survival curve showing the results of Set 3. All groups 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 11, 12, 13, and 14 were treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture 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.

[0038] Figure 20is a Kaplan-Meier survival time plot depicting the results of cohort 4. All groups consisted of 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 mixture of the original chimeric forms of the mAbs as a comparator to the humanized forms used in all other treatment groups at 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb), for a total mAb mixture 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.

[0039] Figure 21 is a Kaplan-Meier survival time plot depicting the results of cohort 5. All groups consisted of 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 19, 20, 21, and 22 were treated with a mixture 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), for a total mAb mixture 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.

[0040] Figure 22This is a Kaplan-Meier survival time plot depicting the results on day 3 of the intervention. All groups consisted of 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 on the plot were treated on day 3 post-challenge. Group 3 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture dose of 15 mg / kg. Group 7 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb), with a total mAb mixture dose of 10 mg / kg. Group 11 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture dose of 10 mg / kg. Group 15 was treated with a mixture of the original chimeric forms of the mAbs as a comparator to the humanized forms used in all other treatment groups at 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb), with a total mAb mixture dose of 15 mg / kg. Group 19 was treated with a mixture 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), with a total mAb mixture dose of 10 mg / kg.

[0041] Figure 23It is a Kaplan-Meier survival time plot showing the results on day 4 of the intervention. All groups 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 after challenge. All groups except Group 2 in the plot were treated on day 4 after challenge. Group 4 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture dose of 15 mg / kg. Group 8 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb), with a total mAb mixture dose of 10 mg / kg. Group 12 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture dose of 10 mg / kg. Group 16 was treated with a mixture of the original chimeric forms of the mAbs as a comparator to the humanized forms used in all other treatment groups at 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb), with a total mAb mixture dose of 15 mg / kg. Group 20 was treated with a mixture 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), with a total mAb mixture dose of 10 mg / kg.

[0042] Figure 24This is a Kaplan-Meier survival time plot depicting the results on day 5 of the intervention. All groups consisted of 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. All groups except Group 2 in the plot were treated on day 5 post-challenge. Group 5 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture dose of 15 mg / kg. Group 9 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb), with a total mAb mixture dose of 10 mg / kg. Group 13 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture dose of 10 mg / kg. Group 17 was treated with a mixture of the original chimeric form of the mAbs as a comparator to the humanized forms used in all other treatment groups at 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb), with a total mAb mixture dose of 15 mg / kg. Group 21 was treated with a mixture 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), with a total mAb mixture dose of 10 mg / kg.

[0043] Figure 25This is a Kaplan-Meier survival curve depicting the results on day 6 of the intervention. All groups consisted of 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. All groups except Group 2 were treated on day 6 post-challenge. Group 6 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture dose of 15 mg / kg. Group 10 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb), with a total mAb mixture dose of 10 mg / kg. Group 14 was treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture dose of 10 mg / kg. Group 18 was treated with a mixture of the original chimeric forms of the mAbs as a comparator to the humanized forms used in all other treatment groups at 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb), with a total mAb mixture dose of 15 mg / kg. Group 22 was treated with a mixture 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), with a total mAb mixture dose of 10 mg / kg.

[0044] Sequence

[0045] SEQ ID NO:1 (Vaccinia virus L1 extracellular domain with 6×His tag)

[0046] GAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNLTVKNMCSADA

[0047] DAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSA

[0048] VVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPKQVAG

[0049] TGVQHHHHHH

[0050] SEQ ID NO:2 (Chimeric 7D11 [c7D11] heavy chain)

[0051] MKCSWVIFFLMAVVTGVNSEVQLEQSGAELAKPGASVKMSCKASGYTFTRYWMHWVK

[0052] QRPGQGLEWIGYINPSTGYTEYNQKFKDKATLTADKSSSTVYMQLSSLTSEDSAVYYCA

[0053] RTTVDGYDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT

[0054] VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0055] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

[0056] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

[0057] KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

[0058] TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0059] SEQ ID NO:3 (Humanized 7D11 [h7D11] variant VH2 heavy chain)

[0060] MKCSWVIFFLMAVVTGVNSEVQLVQSGAEVKKPGSSVKVSCKASGYTFTRYWMHWVR

[0061] QPPGKGLEWIGYINPSTGYTEYNQKFKDRATLTADKSTSTVYMELSSLRSEDTAVYYCA

[0062] RTTVDGYDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT

[0063] VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0064] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

[0065] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

[0066] KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

[0067] TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0068] SEQ ID NO:4 (Chimeric 7D11 [c7D11] light chain)

[0069] MKLPVRLLVLMFWIPASSSDIVMSQSPSSLAVSAGEKVSMSCKSSQTLLNSRTRKNYLAWY

[0070] QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLWTF

[0071] GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS

[0072] QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0073] SEQ ID NO:5 (Humanized 7D11 [h7D11] variant VK3 light chain)

[0074] MKLPVRLLVLMFWIPASSSDIVMTQSPLSLPVTPGEPASISCRSSQTLLNSRTRKNYLAWY

[0075] QQKPGQAPRLLIYWASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVAVYYCKQSYNLW

[0076] TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

[0077] GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0078] SEQ ID NO:6(Vaccinia virus B5 extracellular domain with 6×His tag)

[0079] TVPTMNNAKLTSTETSFNDKQKVTFTCDQGYHSLDPNAVCETDKWKYENPCKKMCTVS

[0080] DYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEH

[0081] GSCQPVKEKYSFGEYITINCDVGYEVIGASYISCTANSWNVIPSCQQKCDMPSLSNGLISG

[0082] STFSIGGVIHLSCKSGFILTGSPSSTCIDGKWNPILPTCVRSNEKFDPVDDGPDDETDLSKLS

[0083] KDVVQYEQEIESLEATYHHHHHHH

[0084] SEQ ID NO:7(Chimeric 8A [c8A] heavy chain)

[0085] MKCSWVIFFLMAVVTGVNSEVQLLESGGGLIKPGGSLRLSCAASGFIFRDYNINWVRQAPGK

[0086] GLEWLGFIRTRASGRSTEYSASVKGRFTISRDDSKNIAYLHINSLKMEDTAVYYCAKKGDSY

[0087] YYMDFWGKGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

[0088] LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT

[0089] CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

[0090] KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

[0091] YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0092] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0093] SEQ ID NO:8 (Humanized 8A [h8A] Variant VH3 Heavy Chain)

[0094] MKCSWVIFFLMAVVTGVNSEVQLLESGGGLVQPGGSLRLSCAASGFIFRDYNINWVRQA

[0095] PGKGLEWLSFIRTRASGRSTEYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCA

[0096] KKGDSYYYMDFWGRGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

[0097] TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

[0098] VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0099] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI

[0100] EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

[0101] TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0102] SEQ ID NO:9 (Chimeric 8A [c8A] light chain)

[0103] MKLPVRLLVLMFWIPASSSDIVLTQPASVSGSPGQSITISCTGGRSDLGDSNFVSWYQQYPGK

[0104] APKLLIYQVNKRPSGVPDRFSASKSANTASLTISGLQTEDEADYFCSSYTTTSTYVFGIGTKVV

[0105] VLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTTPSK

[0106] QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0107] SEQ ID NO:10 (Humanized 8A [h8A] variant VK2 light chain)

[0108] MKLPVRLLVLMFWIPASSSQSALTQPASVSGSPGQSITISCTGGRSDLGDSNFVSWYQQLP

[0109] GTAPKLLIYQVNKRPSGVPDRFSASKSANTASLTISGLQAEDEADYFCSSYTTTSTYVFGT

[0110] GTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG

[0111] VETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0112] SEQ ID NO:11 (Chimeric 8A [c8A] heavy chain)

[0113] MKCSWVIFFLMAVVTGVNSEVQLLESGGGLIKPGGSLRLSCAASGFIFRDYNINWVRQAPGK

[0114] GLEWLGFIRTRASGRSTEYSASVKGRFTISRDDSKNIAYLHINSLKMEDTAVYYCAKKGDSY

[0115] YYMDFWGKGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

[0116] LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT

[0117] CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

[0118] KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

[0119] YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0120] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0121] SEQ ID NO:12 (Humanized 8A [h8A] variant VH1 heavy chain)

[0122] MKCSWVIFFLMAVVTGVNSEVQLLESGGGLVQPGGSLRLSCAASGFIFRDYNINWVRQA

[0123] PGKGLEWLGFIRTRASGRSTEYSASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCA

[0124] KKGDSYYYMDFWGRGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

[0125] TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR

[0126] VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0127] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI

[0128] EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK

[0129] TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0130] SEQ ID NO:13 (Chimeric 8A [c8A] light chain)

[0131] MKLPVRLLVLMFWIPASSSDIVLTQPASVSGSPGQSITISCTGGRSDLGDSNFVSWYQQYPGK

[0132] APKLLIYQVNKRPSGVPDRFSASKSANTASLTISGLQTEDEADYFCSSYTTTSTYVFGIGTKVV

[0133] VLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTTPSK

[0134] QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0135] SEQ ID NO:14 (Humanized 8A [h8A] Variant VK3 Light Chain)

[0136] MKLPVRLLVLMFWIPASSSQSALTQPASVSGSPGQSITISCTGGRSDLGDSNFVSWYQQLP

[0137] GTAPKLLIYQVNKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYFCSSYTTTSTYVFGT

[0138] GTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG

[0139] VETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0140] SEQ ID NO:15 (Vaccinia Virus A33 Extracellular Domain with 6×His Tag)

[0141] VRLNQCMSANEAAITDAAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSCYILHS

[0142] DYQLFSDAKANCTAESSTLPNKSDVLITWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQE

[0143] VRKYFCVKTMNHHHHHH

[0144] SEQ ID NO:16 (Chimeric 6C [c6C] Heavy Chain)

[0145] MKCSWVIFFLMAVVTGVNSEVQLEQSGSEVKKPGASVKLSCKASGYTFTSYSLGWVRQAPG

[0146] QGLEWMGWINTKTGNPTYAQGFTGRFVFSLDTSVNTAYLQITSLKAEDTAVYFCAKGTFYY

[0147] GWGPYYNWFDPWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0148] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS

[0149] CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG

[0150] VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

[0151] PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

[0152] LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0153] SEQ ID NO:17(Humanized 6C [h6C] variant VH2 heavy chain)

[0154] MKCSWVIFFLMAVVTGVNSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYSLGWVRQAP

[0155] GQGLEWMGWINTKTGNPTYAQGFTGRFVFSLDTSVNTAYLQMNSLKTEDTAVYYCAKGTF

[0156] YYGWGPYYNWFDPWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

[0157] TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE

[0158] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

[0159] DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0160] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

[0161] SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0162] SEQ ID NO:18 (Chimeric 6C [c6C] light chain)

[0163] MKLPVRLLVLMFWIPASSSDIVLTQPPSVSAAPGQKITISCSGSGSNIGRHYVSWYQQFPGTAP

[0164] KILIYDNDKRPSGISDRFSGSKSGASATLDITGLQTGDEADYYCATWDTNLSGGVFGGGTKV

[0165] TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS

[0166] KQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0167] SEQ ID NO:19 (Humanized 6C [h6C] variant VK2 light chain)

[0168] MKLPVRLLVLMFWIPASSSQSVLTQPPSVSAAPGQKVTISCSGSGSNIGRHYVSWYQQLPGT

[0169] APKILIYDNDKRPSGIPDRFSGSKSGASATLGITGLQTGDEADYYCATWDTNLSGGVFGGGT

[0170] KLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT

[0171] PSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0172] SEQ ID NO:20 (Unmodified h7D11 Fc)

[0173] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0174] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0175] REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0176] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0177] SEQ ID NO:21 (h7D11 Fc YTE exchange modification)

[0178] GPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0179] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0180] REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0181] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0182] SEQ ID NO:22 (h7D11 Fc LS exchange modification)

[0183] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0184] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0185] REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0186] KSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0187] SEQ ID NO:23 (h7D11 Fc YTELS exchange modification)

[0188] GPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0189] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0190] REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0191] KSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0192] SEQ ID NO:24 (unmodified h8A Fc)

[0193] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0194] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0195] REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0196] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0197] SEQ ID NO:25 (h8A Fc YTE exchange modification)

[0198] GPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0199] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0200] REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0201] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0202] SEQ ID NO:26 (h8A Fc LS exchange modification)

[0203] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0204] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0205] REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0206] KSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0207] SEQ ID NO:27 (h8A Fc YTELS exchange modification)

[0208] GPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0209] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0210] REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0211] KSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0212] Definition

[0213] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It should be further understood that terms, such as those defined in commonly used dictionaries, should be understood as having a meaning that is consistent with their meaning in the relevant art and the context of this disclosure, and should not be understood in an idealized or overly formal sense unless expressly so defined herein. In case of conflict, the present document, including definitions, will prevail.

[0214] In describing this disclosure, it should be understood that numerous techniques and steps are disclosed. Each of these techniques has separate benefits, 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, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. Thus, for clarity, this description will avoid repeating every possible combination of individual steps. However, the specification and claims should be understood to encompass such combinations fully within the scope of this disclosure and the claims.

[0215] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit this disclosure. As used herein, the term "and / or" can include any combination and all combinations of one or more of the associated listed items. As used herein, the singular forms "a", "an", and "the" can include the plural forms as well as the singular forms, unless the context clearly dictates otherwise. It will also be understood that the terms "comprises" and / or "comprising", when used in this specification, specify the presence of the stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the terms "comprise(s)", "include(s)", "having", "has", "may", "contain(s)" and their variants can be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. Unless the context clearly dictates otherwise, all definitions included herein should also be understood to include the plural forms.

[0216] For the recitation of numerical ranges herein, each intermediate number therebetween having the same degree of precision is explicitly contemplated. For example, for the range of 6 - 9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range 6.0 - 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 explicitly contemplated.

[0217] As used herein, the term "about" or "approximately" can mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, in accordance with the practice in the art, "about" means plus or minus 10%. Optionally, "about" means 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 biological processes, the term means within an order of magnitude of the value, within 5-fold or within 2-fold. When a particular value is described in this application and the claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range of the particular value. In addition, in cases where a range and / or sub-range of values is provided, the range and / or sub-range can include the endpoints of the range and / or sub-range.

[0218] As used herein, the term "nucleic acid construct" can mean a linear polymer of nucleic acids. The polymer can contain a promoter sequence that drives the expression of one or more genes of interest. As used herein, the term "nucleic acid construct" can include, but is not limited to, oligonucleotides, RNA, linear DNA, closed-ended linear DNA, ministrings, transposons, dogbone DNA, GenWand DNA, and minimalistic, immunologically defined gene expression (MIDGE) DNA. As used herein, a nucleic acid construct can be DNA or RNA or a mixture of both, and can contain naturally occurring nucleotides or artificial (non-natural) nucleotides. As used herein, "naturally occurring nucleotides" can include adenine, guanine, cytosine, thymine, uracil, inosine, 2,6-diaminopurine, 5-hydroxymethylcytosine, N4-methylated cytosine, N6-methylated adenine, archaeosine, and other nucleotide modifications that occur in normal cell (eukaryotic, prokaryotic, or archaeal, including virus-induced or phage-induced) metabolism. As used herein, "artificial nucleotides" or "non-natural nucleotides" are nucleotide analogs or linkages that do not occur naturally, and can include, but are not limited to, peptide nucleic acids, morpholino nucleic acids, 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 ribose, threose nucleic acids, hexose nucleic acids, 2'-sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, 5-((3-indolyl)propionamide-N-allyl)-2'-deoxyuridine, substitution of 5-bromo or 5-iodo-uracil, and backbone modifications, anti-reverse cap analogs, substitution of pseudouridine, 5-methylcytidine, and / or N1-methyluridine, methylation and unusual base pairing combinations, including but not limited to isobases such as isocytidine and isoguanidine and (7-(2-thienyl)imidazo[4,5-b]pyridine, Ds).

[0219] As used herein, the term "plasmid" can refer to a circular DNA molecule physically separated from chromosomal DNA. As used herein, the term "plasmid" includes, but is not limited to, plasmids of bacterial origin, minicircles, episomal DNA, covalently closed circular DNA (cccDNA), extrachromosomal circular DNA (eccDNA), chromatin, chloroplast DNA, circular DNA of baculovirus origin, telomeres, nanoplasmids, and mitochondrial DNA. The term "plasmid" includes, but is not limited to, small circular double-stranded DNA molecules having a promoter sequence that drives the transcription of one or more genes of interest. Optionally, the plasmid may also contain an origin of replication (ori) site, a marker gene for selection and / or screening (e.g., an antibiotic resistance gene), enhancer elements, and restriction endonuclease (RE) sites to allow the insertion fragment to be cloned at specific sites. As used herein, plasmids can contain naturally occurring nucleotides and / or artificial nucleotides.

[0220] As used herein, "antibody" can refer to a protein that acts like an immunoglobulin or is an immunoglobulin or is designed to replace an immunoglobulin, including, but not limited to, polyclonal antibodies, monoclonal antibodies, single-chain variable fragments (scFv), Fab fragments, camel antibodies, nanobodies, designed ankyrin repeat proteins, monomers, anticalins, knottins, affimers, avimers, or an affinity clamp or affibody that specifically binds to a substance.

[0221] As used herein, "binds selectively", "bound selectively", "specifically binds", "specifically bound", "specifically recognizes", or "specifically recognized" can mean that one substance binds to another substance to the exclusion of other substances, and the binding generally has a dissociation constant (Kd) of less than or equal to one (1) micromolar per liter. Optionally, the binding is in an aqueous solution, optionally under specified or defined or stringent conditions, such as in a solution having a pH range of from about 5.5 to about 8, such as 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, 7.7, 7.8, 7.9, or 8.0, optionally at a defined salt (e.g., sodium chloride) concentration in the range of from about 0.0001 molar per liter to about 10 molar per liter, optionally in a buffer such as phosphate buffer, and in a temperature range of from about 23 degrees Celsius to about 37 degrees Celsius.

[0222] As used herein, the term "subject" or "patient" can refer to any living organism to which a composition or formulation according to the present disclosure can be administered, e.g., 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. A human can be greater than about: 1 year old, 2 years old, 5 years old, 10 years old, 20 years old, 30 years old, 40 years old, 50 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, 90 years old, 95 years old, 100 years old, 105 years old, 110 years old, 115 years old, or about 120 years old. A human can be a pediatric patient, a child, or an adult subject.

[0223] "Therapeutically effective amount" can mean an amount of a composition or pharmaceutical composition disclosed herein that effectively achieves its intended purpose, such as treating a disease, with or without additional agents. 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 age, health status, physical condition, sex, weight, degree of disease, frequency of treatment, and the nature and extent of the disease or condition of the recipient. For example, a therapeutically effective amount of a composition or pharmaceutical composition herein can range from about 0.0001 mg / kg to about 10,000 mg / kg, such as 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1.0 mg / kg, 10.0 mg / kg or 100.0 mg / kg, where mg is the mg of the composition or pharmaceutical composition and kg is the kg of the weight of the subject or patient.

[0224] As used herein, the term "treatment" or "treating" can mean a drug or other intervention regimen used to obtain a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic and / or prophylactic benefits. A therapeutic benefit refers to eradicating or ameliorating one or more symptoms of the underlying disorder being treated. Additionally, a therapeutic benefit can be achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder, such that an improvement can be observed in the subject, even though the subject may still be afflicted with the underlying disorder. Prophylactic effects include delaying, preventing or eliminating the onset 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 benefits, a subject at risk of developing a particular disease or a subject reporting one or more physiological symptoms of a disease can undergo a treatment disclosed herein, even if a diagnosis of the disease may not yet have been made.

[0225] As used herein, "transfection" can refer to the introduction of a nucleic acid construct or plasmid or engineered DNA (e.g., DNA) into a cell. Transfection can occur, for example, in vitro, ex vivo or in vivo.

[0226] As used herein, "adjuvant" can refer to any substance that promotes a stronger immune response in a subject receiving a vaccine. Examples of adjuvants include, but are not limited to, monophosphoryl lipid A (MPL), oligodeoxynucleotides containing unmethylated CpG motifs (ODN) (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.

[0227] As used herein, the term "drug package" can refer to a box, packet, bag, or plastic used to bundle together a Patient Package Insert (PPI), Medication Guide (MG), or Instructions for Use (IFU).

[0228] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0229] Detailed description

[0230] Overview

[0231] Compositions for preventing and treating orthopoxviruses are disclosed herein, as well as compositions comprising antibodies (having mono - or multi - epitope specificity) that bind to epitopes found on the intracellular mature virions or mature virion forms of orthopoxviruses and / or antibodies that bind to epitopes found on the extracellular enveloped virions or enveloped virion forms of orthopoxviruses. Compositions are also disclosed herein, including antibody compositions of non - blood origin, such as humanized antibodies and those that have been modified to reduce immunogenicity and / or extend serum half - life, and methods of using them in conferring passive immunity against orthopoxvirus infection (e.g., treating or preventing orthopoxvirus infection) in individuals at risk of orthopoxvirus infection or exhibiting vaccinia infection, including adverse events caused by certain smallpox vaccines, such as progressive vaccinia and vaccinia eczema, monkeypox infection, or smallpox infection.

[0232] Compositions or pharmaceutical compositions administered alone (simultaneously or sequentially), or in combination as a single composition or pharmaceutical composition of two or more altered and / or humanized monoclonal antibodies (mAbs) are also described herein. The monoclonal antibodies can target viral proteins on two major forms of the virus: the mature virion (MV), which is an unenveloped particle and is responsible for transmission, and the enveloped virion (EV), which is an MV particle that acquires an envelope when budding from an infected host cell and is responsible for inter - cellular spread within the infected host. When the MV particle is still within the infected host cell, it can be referred to as an intracellular mature virion or IMV. In some cases, the terms MV and IMV can be used interchangeably. When the EV particle is released from the infected host cell, it can be referred to as an extracellular enveloped virion or EEV. When the EV particle is initially budding from the infected host cell but has not been released from the cell, it can be referred to as a cell - associated enveloped virion or CEV.

[0233] Antibodies and their functional fragments

[0234] The present invention provides 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 orthopoxviruses in the IMV form. 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 a functional fragment thereof (e.g., mammalian, murine, rat, or rabbit) is selected or a composite engineered antibody is designed. One, two, three, four, five, or more sequence segments derived from variable regions of unrelated antibodies are used as building blocks to construct the composite engineered antibody. 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 the L1 protein of a poxvirus (e.g., vaccinia virus). In some embodiments, the initial antibody (e.g., 8A) is an antibody against the extracellular domain of the B5 protein of a poxvirus (e.g., vaccinia virus). In some embodiments, the initial antibody (e.g., 6C) is an antibody against the extracellular domain of the A33 protein of a poxvirus (e.g., vaccinia virus). In some embodiments, the engineered antibody or functional fragment thereof comprises a constant region (Fc region) having one or both of the CH2 and CH3 constant domains.

[0235] In some embodiments, the antibody or functional fragment thereof is altered, which can confer one or more desired characteristics in a subject, such as an increased half-life. In some embodiments, the antibody or functional fragment thereof is humanized, which can confer one or more desired characteristics in a human subject, such as reduced immunogenicity. In some embodiments, one or more sequences of the engineered composite antibody or functional fragment thereof are separately or individually humanized. In some cases, the non-human framework sequences can be in the Fc region of the antibody. In some embodiments, the antibody or functional fragment thereof comprises a light chain and / or heavy chain framework region (constant region), such as one or more framework regions (constant regions), including one or more IgG1, IgG2, IgG3, and / or IgG4 framework regions (constant regions).

[0236] Non-limiting examples of antibody fragments include the orthopoxvirus-binding region and / or effector region of an antibody (e.g., Fab, Fab’, F(ab’) 2 , Fv, scFv, (scFv) 2 , bispecific variable region antibodies, monospecific variable region antibodies, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab) 2, Fd, Fc, bispecific antibodies, di-diabodies, disulfide-linked Fvs (dsFvs), single domain antibodies (e.g., nanobodies), or other functional fragments). Generally, the variable (V) region domain can be any suitable arrangement of heavy (VH) chain and / or light (VL) chain variable domains. In some cases, each antibody can independently be an IgG, IgA, IgD, IgE, or IgM antibody, or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a bioactive fragment of any of these, such as a Fab fragment, or a light or heavy chain of any of these.

[0237] In some embodiments, the antibody or its functional fragment 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%, at least about 100% homologous or identical to the amino acid sequences listed in SEQ ID NOs: 1-27.

[0238] Methods for expressing and purifying antibodies

[0239] The antibodies disclosed herein can be prepared using a variety of methods. The antibodies of the invention and their functional fragments can be produced from host cells. A host cell refers to a vehicle that includes the essential cellular components (e.g., organelles) required for the expression of the polypeptides and constructs described herein from their corresponding nucleic acids. The nucleic acid can be contained in a nucleic acid vector, which can be introduced into the 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 to be used. Generally, preferred host cells are of prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

[0240] The nucleic acid sequences encoding the antibodies or their functional fragments of the invention can be prepared by a variety of methods. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. The nucleic acid molecules encoding the antibodies or their functional fragments of the invention can be obtained using standard techniques (e.g., gene synthesis). Alternatively, nucleic acid molecules encoding wild-type antibodies or their functional fragments can be altered using standard techniques in the art (e.g., mutagenesis) to contain specific amino acid substitutions. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.

[0241] The nucleic acid sequence encoding an antibody or a functional fragment thereof of the present invention can be inserted into a vector capable of replicating and expressing nucleic acid molecules 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, which can be adjusted and optimized to be compatible with a particular host cell. For example, vector components may include, but are not limited to, an origin of replication, 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. The vector can be linearized or supercoiled, exhibiting improved transfection efficiency.

[0242] In some embodiments, mammalian cells are used as the host cells of the present invention. In some embodiments, the glutamine auxotrophic and cholesterol auxotrophic phenotypes are induced by genetic manipulation of non-glutamine auxotrophic and non-cholesterol auxotrophic cells, including, for example, mutating or deleting genes necessary for endogenous glutamine biosynthesis, such as glutamine synthetase. Common methods of genetic engineering are well known to those skilled in the art, for example, site-directed mutagenesis, zinc finger nucleases, shRNA, transposons, see, for example, Cytotechnology. 2007 Apr; 53(1-3): 65-73. For example, the murine myeloma cell line called NS0 is known to be glutamine auxotrophic and cholesterol auxotrophic (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).

[0243] Additional examples of mammalian cell types that can be engineered to serve as host cells include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F), 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, Escherichia coli cells are used as the host cells of the present invention. Examples of E. coli strains include, but are not limited to, E. coli 294 E. coli λ1776 E. coli BL21(DE3) and E. coli RV308 Different host cells have characteristic 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 its functional fragment. Conventional techniques in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection, can be used to introduce the above expression vector into a suitable host cell. After introducing the vector into the host cell to produce the protein, the host cell is cultured in a conventional nutrient medium that is appropriately modified to induce the promoter, select transformants, or amplify the gene encoding the desired sequence. Methods for expressing antibodies or their functional fragments 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).

[0244] The host cells for producing the antibodies or their functional fragments of the present invention can be grown in media known in the art and suitable for culturing the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Expi 293 TM 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 selection agent, for example, ampicillin. The host cells are at a suitable temperature such as from about 20°C to about 39°C, for example, from 25°C to about 37°C, preferably 37°C, and CO 2Culture at a level such as 5% to 10% (preferably 8%). The pH of the culture medium is typically from about 6.8 to 7.4, for example, 7.0, mainly depending on the host organism. If 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 their functional fragments are known in the art. For example, see Butler, Cell Culture and Upstream Processing, Taylor & Francis; 1st Edition (May 25, 2007).

[0245] Protein recovery typically involves disrupting the host cells, usually by means such as osmotic shock, sonication, or lysis. After the cells are disrupted, cell debris can be removed by centrifugation or filtration. The protein can be further purified. The antibodies of the present invention can be purified by any method known in the art of protein purification, such as by protein A affinity chromatography, other chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. (See Process Scale Purification of Antibodies, Uwe Gottschalk (ed.), John Wiley & Sons, Inc., 2009). In some cases, the antibody or its functional fragment can be conjugated to a marker sequence (such as a peptide) to facilitate purification. Examples of marker amino acid sequences are hexahistidine peptides (His-tags), which bind to nickel-functionalized agarose affinity columns with micromolar affinity. Other peptide tags that can be used for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein.

[0246] In some embodiments, the antibody is designed empirically (i.e., improved by trial and error). In some embodiments, the antibody is designed computationally using one or more algorithms to optimize one or more characteristics. For example, a database can be created with antibody segments previously screened using immunogenetic assays (e.g., ex vivo T cell immunogenetic assays), with half-life information for each antibody segment, or with MHC class II binding information. One or more algorithms can then be used to evaluate the database and predict amino acid changes that improve one or more desired characteristics, such as increasing the half-life of the antibody in a subject, reducing the immunogenetic response 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 increase protein production. In some embodiments, the antibody is codon-optimized for expression in mammalian cells (e.g., murine cells).

[0247] Therapeutic methods

[0248] The present disclosure provides methods of treatment using the compositions or pharmaceutical compositions disclosed herein. In some embodiments, the methods described herein can be used to develop antibodies or functional fragments thereof that can be used to produce drugs. In some cases, cells can be used to manufacture biological products, including vaccines and antibodies or functional fragments thereof.

[0249] In some embodiments, the methods described herein can be used for in vivo delivery of an antibody or a functional fragment thereof. In some cases, the methods herein can be used to deliver an antibody or a functional fragment thereof to a subject in need thereof, such as a subject in need of treatment or prevention of a disease (e.g., a viral disease, orthopoxvirus infection). For example, orthopoxviruses can be treated, including abatino macacapox virus, akhmeta virus, alaskapoxvirus, camelpox virus, cowpox virus, ectromelia virus, monkeypox virus, raccoonpox virus, skunkpox virus, gerbilpox virus, vaccinia virus, variola virus, and volepox virus. In some embodiments, a vaccine comprising the compositions or pharmaceutical compositions disclosed herein can be administered to a subject to increase immunogenicity in the subject. In some embodiments, the compositions or pharmaceutical compositions can be used to prevent, ameliorate, and / or reduce the severity of orthopoxvirus infection.

[0250] In some embodiments, the methods, systems, and compositions can be used in animal models. For example, mice or any mammal can be generated by methods specifically designed for studying orthopoxviruses according to the present disclosure.

[0251] In some embodiments, a therapeutic method can comprise a dose (e.g., a unit dose) of a therapeutically effective amount of an antibody or a functional fragment thereof. In some cases, the compositions disclosed herein can be administered to a subject in need of treatment to effect treatment. In some cases, the treatment can be prophylactic and / or therapeutic, and it can be directed against any viral disease (e.g., orthopoxvirus). In some cases, the treatment regimen can be determined by a physician in each case based on factors such as the orthopoxvirus to be treated, the age and weight of the patient. 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 infusion, intracardiac, intravitreal, parenteral, vaginal, intracorporeal, intravesicular, rectal, transdermal, and perivascular.

[0252] In some embodiments, a method of treating a patient in need thereof with an antibody or a functional fragment thereof can include administering: a first antibody that binds to an epitope found on the mature virion (MV) form of a virus of the genus Orthopoxvirus; and a second antibody that binds to an epitope found on the enveloped virion (EV) form of a virus of the genus Orthopoxvirus.

[0253] In some embodiments, the present disclosure can include a method of treating a patient in need thereof with an antibody or a functional fragment thereof, the antibody or functional fragment thereof including: a first antibody that binds to an epitope found on the MV form of a virus of the genus Orthopoxvirus; a second antibody that binds to a first epitope found on the EV form of a virus of the genus Orthopoxvirus; and a third antibody that binds to a second epitope found on the EV form of a virus of the genus Orthopoxvirus, wherein the first epitope is different from the second epitope.

[0254] Vaccine applications

[0255] In some embodiments, the antibody or a functional fragment thereof can be delivered as a vaccine. In some embodiments, the present disclosure can include a method of conferring passive immunity against smallpox to a subject, the method including 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.

[0256] In some embodiments, the present disclosure can include a method of conferring passive immunity against monkeypox virus infection to a subject, the method including 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.

[0257] In some embodiments, the present disclosure can include a method of conferring passive immunity against smallpox to a subject, the method including 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; 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.

[0258] In some embodiments, the present disclosure can include a method of conferring passive immunity against monkeypox virus infection to 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 the monkeypox virus; 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.

[0259] Preparations and excipients, carriers, and diluents

[0260] In some embodiments, the compositions disclosed herein can comprise a pharmaceutical preparation that includes one or more of the antibodies or functional fragments thereof described herein. In some cases, the compositions herein can comprise a pharmaceutical composition. In some cases, the pharmaceutical composition can be in unit dose form. In some cases, a preparation containing a compound according to the present disclosure can take the form of a liquid, solid, semi-solid, or lyophilized powder form, such as, for example, a solution, suspension, emulsion, etc., preferably in a unit dosage form suitable for simple administration of an exact dose.

[0261] In some embodiments, the pharmaceutical composition can comprise conventional pharmaceutical carriers or excipients and can additionally comprise other medicaments, carriers, adjuvants, additives, etc. In some cases, the composition can be about 0.1% to about 85%, about 0.5% to about 75% by weight of an antibody or functional fragment thereof of the present disclosure, the remainder consisting essentially of suitable pharmaceutical excipients. In some embodiments, the amount of the active ingredient (e.g., one or more antibodies or functional fragments thereof of the present invention) contained in the pharmaceutical preparation is such that a suitable dose within a specified range is provided (e.g., a dose within the range of 0.01 mg / kg - 500 mg / kg body weight).

[0262] The acceptable carriers and excipients in the pharmaceutical composition are non-toxic to the recipient at the doses and concentrations used. Acceptable carriers and excipients can include buffering agents, antioxidants, preservatives, polymers, amino acids, and sugars. The pharmaceutical composition of the present invention can be administered parenterally in the form of an injectable preparation. A sterile solution or any pharmaceutically acceptable liquid can be used as a vehicle to formulate the pharmaceutical composition for injection (i.e., intravenous injection). Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle's Medium (α-MEM), F-12 medium). The formulation methods are known in the art, see, for example, Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd Edition) Taylor & Francis Group, CRC Press (2006). In some embodiments, the compositions herein can contain one or more of the following excipients: gum arabic, acesulfame potassium, glacial acetic acid, acetone, tributyl acetyl citrate, triethyl acetyl citrate, adipic acid, agar, albumin, alcohol, alginic acid, aliphatic polyesters, alitame, allantoin, almond oil, α-hydroxy acids, α-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, β-glucan, β-hydroxy acids, bentonite, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, boric acid, bronopol, tert-butylhydroxy alcohol, butylated hydroxyanisole, butylated hydroxytoluene, butanediol, butylparaben, calcium acetate, calcium alginate, calcium carbonate, calcium chloride, calcium hydroxide, calcium lactate, anhydrous calcium hydrogen phosphate, calcium hydrogen phosphate dihydrate, tricalcium phosphate, calcium silicate, calcium stearate, calcium sulfate, canola oil, decylene glycol, glyceryl tridecanoate, carbomer, carbon dioxide, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carrageenan, castor oil, hydrogenated castor oil, microcrystalline cellulose, microcrystalline cellulose and sodium carboxymethylcellulose, powdered cellulose, siliconized microcrystalline cellulose, cellulose acetate, cellulose acetate phthalate, ceramide, ceresin, cetearyl alcohol, cetrimonium bromide, cetyl alcohol, hexadecyl alcohol, cetylpyridinium chloride, chitosan, chlorhexidine, chlorobutanol, chlorocresol, hydrochlorofluorocarbon (HCFC), chlorofluorocarbon (CFC), chloroxylenol, cholesterol, citric acid monohydrate, coconut oil, collagen, colloidal silica, coloring agents, copper peptides, copovidone, corn oil, corn starch and pregelatinized starch,Cottonseed oil, cresol, croscarmellose sodium, crospovidone, cyclodextrin, cyclomethicone, denatonium benzoate, desitin, dextran, dextrin, dextrose, dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (hfc), dimethicone, dimethyl ether, dimethyl phthalate, dimethyl sulfoxide, dimethylacetamide, disodium edetate, sodium docusate, edetic acid, erythorbic acid, erythritol, ethyl acetate, ethyl lactate, ethyl maltol, ethyl oleate, ethyl vanillin, ethylcellulose, ethylene glycol distearate, ethylene-vinyl acetate copolymer, ethylparaben, fatty acid, ferulic acid, fructose, fumaric acid, gelatin, glucose solution, glycerin (glycerol), glyceryl behenate, glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate, glycine, polytetrahydrofuran glycol ether, glycolic acid, ethylene glycol stearate, guar gum, hectorite, heptafluoropropane (hfc), hexetidine, hydrocarbons (hc), hyaluronic acid, hydrochloric acid, hydrocortisone, hydrophobic colloidal silica, mesoporous silica, hydroquinone, hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl betacyclodextrin, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl starch, hydroxypropyl methylcellulose, succinate hydroxypropyl methylcellulose, phthalate hydroxypropyl methylcellulose, imidurea, inulin, iron oxide, isomalt, isoparaffin, isopropyl alcohol, isopropyl myristate, isopropyl palmitate, jojoba oil, kaolin, kojic acid, lactic acid, lactitol, anhydrous lactose, inhalable lactose, 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, aqueous lanolin, lanolin alcohol, lauric acid, lecithin, leucine, linoleic acid, polyethylene glycol 15 hydroxystearate, magnesium aluminum silicate, magnesium carbonate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, maleic acid, malic acid, maltitol, maltitol solution, maltodextrin, maltitol, maltose, mannitol, medium-chain triglycerides, meglumine, menthol, methionine, methyl cellulose, 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 alcohol and lanolin alcohol, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, phospholipid, phosphoric acid, phytic acid, phytosphingosine, potassium polacrilin, poloxamer, polycarbophil, polydextrose, poly(dl-lactic acid), polyethylene glycol, polyethylene oxide, polymethacrylate, poly(methyl vinyl ether / maleic anhydride), polyoxyethylene alkyl ether,Polyoxyl castor oil derivatives, polyoxyl sorbitan fatty acid esters, polyoxyl stearates, polyoxyl glyceryl esters, parabens, polysorbate 60, polysorbate 80, polyvinyl phthalate, polyvinyl alcohol, potassium alginate, potassium alum, potassium benzoate, potassium bicarbonate, potassium chloride, potassium citrate, potassium hydroxide, potassium metabisulfite, potassium sorbate, povidone, propionic acid, propyl gallate, acrylate, propylene glycol, propylene glycol alginate, propylparaben, sodium propylparaben, pyrrolidone, raffinose, tretinoin, retinol and retinoic acid derivatives, saccharin, sodium saccharin, safflower oil, salicylic acid, steatite, sesame oil, shellac, simethicone, sodium acetate, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium borate, sodium carbonate, sodium chloride, sodium citrate dihydrate, sodium cyclamate, sodium formaldehyde sulfoxylate, sodium hyaluronate, sodium hydroxide, sodium lactate, sodium lauryl sulfate, sodium metabisulfite, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium propionate, sodium carboxymethyl starch, sodium stearoyl fumarate, sodium ascorbyl phosphate, sodium deoxycholate, sodium hydroxide, sodium lauroyl lactate, sodium lauryl sulfate, sodium palmitate, sorbitan stearate, sodium sulfite (E221), spironolactone, sodium sulfite, sodium hyposulfite, sorbic acid, sorbitan esters (sorbitan fatty acid esters), sorbitan monostearate, sorbitol, soybean oil, sphingomyelin, starch, pregelatinized starch, sterilized corn starch, stearic acid, stearyl alcohol, squalene, aloinose, sucrose, sucrose octaacetate, compressible sugar, powdered sugar, sugar balls, sulfobutylether b-cyclodextrin, sulfur dioxide, sulfuric acid, sunflower oil, suppository base - hard fat, tagatose, talc, tartaric acid, tetrafluoroethane (HFC), thaumatin, thimerosal, thymol, titanium dioxide, tragacanth, trehalose, tretinoin, triacetin, tributyl citrate, glyceryl trioctanoate, triethanolamine, triethyl citrate, triethanolamine, glyceryl trioleate, 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.、

[0263] In some cases, compositions such as pharmaceutical compositions may contain a carrier or diluent. In some cases, the carrier or diluent may comprise water, an alcohol, a salt solution (e.g., saline), or a mixture thereof. In some cases, the carrier may comprise sugars, buffers, salts, pH regulators, or any combination thereof. In some cases, the compositions herein may contain buffering agents, polymers, antioxidants, preservatives, chelating agents, viscosity regulators, tonicity agents, coloring agents, odorants, opacifying agents, suspending agents, binders, fillers, plasticizers, lubricants, or any combination thereof.

[0264] In some embodiments, an injectable composition for parenteral administration (e.g., intravenous, intramuscular, or intrathecal) can comprise a compound in a suitable intravenous solution such as sterile saline solution. The composition can also be formulated as a suspension in an aqueous emulsion.

[0265] Administration

[0266] In some cases, a 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 infusion, intracardiac, intravitreal, parenteral, intravaginal, intracorporeal, intracapsular, rectal, topical, transdermal, inhalation, perivascular, ocular, otic, and any combination thereof.

[0267] In some cases, administration can include delivering the composition. In some cases, delivery 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 cases, a subject can administer the composition in the absence of supervision. In some cases, a subject can administer the composition under the supervision of a healthcare professional (e.g., a physician, nurse, physician assistant, paramedic, hospice worker). In some cases, a healthcare professional can administer the composition. In some cases, a subject can administer the composition.

[0268] In some embodiments, administration of the compositions herein can be carried out at least about: once daily, twice daily, three times daily, or more than four times daily. In some cases, administration can be carried out daily, weekly, monthly, or as needed. In some cases, administration or application of the compositions herein can be carried out for a treatment duration of at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days. In some cases, the treatment duration can be about: 1 to about 30 days, 1 to about 60 days, 1 to about 90 days, 30 days to about 90 days, 60 days to about 90 days, 30 days to about 180 days, 90 days to about 180 days, or 180 days to about 360 days. In some embodiments, administration of the compositions disclosed herein can be carried out for a treatment duration 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 for life. Administration can be repeated during the life cycle of the subject, for example, once monthly or once annually during the life cycle of the subject.

[0269] 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.

[0270] In some embodiments, a method of 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 a functional fragment thereof according to the present disclosure and / or at least one additional bioactive (e.g., antiviral) agent. In some cases, the therapeutically effective amount of the compositions herein is in the range of about 0.000001 mg / kg to about 1000 mg / kg, where mg can be the mg of the composition and kg can be the kg of the subject's body weight. For example, a composition of about the following can be administered to a subject in need thereof: 0.000001 mg / kg, 0.00001 mg / kg, 0.0001 mg / kg, 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1.0 mg / kg, 10 mg / kg, 100 mg / kg, or 1000 mg / kg.

[0271] Kit

[0272] In some embodiments, kits and their containers suitable for carrying out any of the methods disclosed herein are disclosed.

[0273] In some embodiments, such a kit may contain, in a container for transportation, a composition comprising an antibody or a functional fragment thereof, which may comprise: a first antibody that binds to an epitope found on the mature virion (MV) form of a virus of the genus Orthopoxvirus; and a second antibody that binds to an epitope found on the enveloped virion (EV) form of a virus of the genus Orthopoxvirus.

[0274] In some embodiments, such a kit may contain, in a container for transportation, a composition comprising an antibody or a functional fragment thereof, which comprises: a first antibody that binds to an epitope found on the MV form of a virus of the genus Orthopoxvirus; a second antibody that binds to a first epitope found on the EV form of a virus of the genus Orthopoxvirus; and a third antibody that binds to a second epitope found on the EV form of a virus of the genus Orthopoxvirus, wherein the first epitope is different from the second epitope.

[0275] In some embodiments, such a kit may contain, in a container for transportation, an antibody or a functional fragment thereof for delivery as a vaccine. In some embodiments, the kit comprises an effective amount of a composition, which comprises: 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.

[0276] In some embodiments, the kit comprises an effective amount of a composition, in a container for transportation, which comprises: 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 the monkeypox virus.

[0277] In some embodiments, the kit comprises an effective amount of a composition, in a container for transportation, which comprises: a first antibody that binds to an epitope found on the mature virion (MV) form of variola virus; a second antibody that binds to a first epitope found on the 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.

[0278] In some embodiments, the kit comprises an effective amount of a composition, in a container for transportation, which comprises: a first antibody that binds to an epitope found on the mature virion (MV) form of monkeypox virus; a second antibody that binds to a first epitope found on the 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.

[0279] In some cases, the kit may further comprise a suitable excipient, carrier, diluent, or any combination thereof. In some cases, the excipient, carrier, diluent, or any combination thereof may be any excipient, carrier, diluent, or any combination thereof known for storing an antibody or a functional fragment thereof. The storage device may be any device known in the art for storing an antibody or a functional fragment thereof, including but not limited to glass bottles, plastic bottles, pre-filled syringes, and ampoules. Examples

[0280] Example 1: Humanization of murine-human mAb c7D11

[0281] A series of nine (3 heavy chains and 3 light chains) humanized heavy chain V region sequences and light chain V region sequences were designed entirely from segments of human V region sequences with the aim of avoiding T cell epitopes. Variant designs were made using in silico tools for evaluating MHC class II binding and a database containing antibody segments previously screened using in vitro T cell immunogenicity assays. After design, the three heavy chain variable domain and three light chain variable domain sequences were codon-optimized for expression in murine cells, synthesized, and cloned into a cloning vector with flanking restrictions for subsequent cloning into an expression suitable vector.

[0282] The murine-human c7D11 mAb (SEQ ID NO:1) was cloned and expressed. Figure 1 The amino acid sequence of the fully humanized heavy chain h7D11 variant VH2 (SEQ ID NO:3) aligned with the original c7D11 heavy chain chimeric sequence (SEQ ID NO:2) is shown, with the amino acid changes occurring in the humanized variant VH2 marked. Figure 2 The amino acid sequence of a representative fully humanized light chain h7D11 variant VK3 (SEQ ID NO:5) aligned with the c7D11 light chain chimeric sequence (SEQ ID NO:4) is shown, with the amino acid changes occurring in the humanized variant VK3 marked.

[0283] Example 2: Variant c7D11 expression

[0284] A stable NS0 clone cell line was generated that expresses an appropriately folded and functionally intact IgG composed of two mature heavy chains with the sequence VH2 and two mature light chains with the sequence VK3, but without a signal peptide for each chain, which is cleaved during translocation in the cell.

[0285] Example 3: Humanization of chimpanzee-human chimeric mAb c8A

[0286] Full-size chimpanzee-human mAbs were prepared by fusing the heavy-chain variable domain with the human IgG1 constant sequence and the light-chain variable domain with the human lambda constant sequence. The chimeric mAbs demonstrated in vitro antiviral activity against both VACV and VARV in the comet-reduction assay. The mAbs also showed protective efficacy in a BALB / c mouse pneumonia model challenged lethally with VACV Western Reserve (WR).

[0287] The chimpanzee-human mAb c8A (SEQ ID NO:6) was cloned and expressed. Figure 3 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) is shown, with the amino acid changes occurring in the humanized variant VH3 marked. Figure 4 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) is shown, with the amino acid changes occurring in the humanized variant VK2 marked.

[0288] Example 4: Variant c8A Expression

[0289] A stable NS0 clone cell line designated AD2 was generated, which expressed an appropriately folded and functionally intact IgG having two mature heavy chains with the sequence VH3 and two mature light chains with the sequence VK2 but lacking the signal peptide, which was cleaved during translocation in the cell.

[0290] Example 5: Humanization of the Chimpanzee-Human Chimeric mAb c8A

[0291] Full-size chimpanzee-human mAbs were prepared by fusing the heavy-chain variable domain with the human IgG1 constant sequence and the light-chain variable domain with the human lambda constant sequence. The chimpanzee-human mAb c8A (SEQ ID NO:6) was cloned and expressed. Figure 5 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) is shown, with the amino acid changes occurring in the humanized variant VH1 marked. Figure 6 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) is shown, with the amino acid changes occurring in the humanized variant VK3 marked.

[0292] Example 6: Variant c8A Expression

[0293] A stable NS0 clone cell line designated BC9 was generated, which expresses properly folded and functionally intact IgG. The IgG consists of two mature heavy chains with the sequence VH1 and two mature light chains with the sequence VK3 but without a signal peptide, and the signal peptide is cleaved during the translocation process in the cell.

[0294] Example 7: Humanization of Chimpanzee-Human Chimeric mAb c6C

[0295] The variable domains were used to generate full-size chimpanzee-human mAbs by fusing the heavy-chain variable domain with the human IgG1 constant sequence and the light-chain variable domain with the 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 showed protective efficacy in a pneumonia model of BALB / c mice challenged lethally with VACV Western Reserve (WR).

[0296] Clone and express the chimpanzee-human mAb c6C (SEQ ID NO:15). Figure 7 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) is shown, and the amino acid changes occurring in the humanized variant VH2 are marked. Figure 8 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) is shown, and the amino acid changes in the humanized variant VK2 are marked.

[0297] Example 8: Half-Life of Engineered Enhanced Humanized mAbs

[0298] The neonatal Fc receptor (FcRn) belongs to the broad major histocompatibility complex (MHC) class of molecules, and its general function involves antigen presentation. However, FcRn itself has a narrow functional range because it cannot present antigens but plays a role in serum half-life via high-affinity binding to IgG at low pH. This interaction can be driven by the association of FcRn at the interface of the CH2 and CH3 domains in the Fc region of IgG. Engineering unmodified humanized 7D11 and 8A mAbs to increase serum half-life can result in a reduction in the effective dose and / or the number of doses, and can result in more sustained antiviral efficacy and protection against orthopoxvirus infection. Thus, the modifications herein can generate Fc variants with increased affinity for FcRn.

[0299] Modified to enhance the affinity of FcRn in the Fc region of the h7D11 heavy chain (SEQ ID NO:20). Figure 9 A- Figure 9 D shows the variation in the amino acid sequence in the Fc region of the h7D11 heavy chain (SEQ ID NO:21-23) compared to the unmodified iteration. The amino acid changes in the variation are marked.

[0300] Example 9: Modifications for enhancing the affinity of FcRn in the Fc region of the h8A heavy chain

[0301] Modified to enhance the affinity of FcRn in the Fc region of the h8A heavy chain (SEQ ID NO:24). Figure 10 A- Figure 10 D shows the variation in the amino acid sequence in the Fc region of the h8A heavy chain (SEQ ID NO:25-27) compared to the unmodified iteration. The amino acid changes in the variation are marked. It is also important to note that although the Fc regions are identical between h7D11 and h8A, due to 2 amino acid differences in the variable regions of the corresponding heavy chains, the amino acid changes, while the same, are in different positions.

[0302] Example 10: Pharmaceutical product composition

[0303] A combination of at least one anti-MV and one anti-EV can form an effective and potent pharmaceutical product. Combinations of mAb h7D11 and mAb h8A, mAb h7D11 and mAb h6C, or the triple combination of mAb h7D11, mAb h8A, and mAb h6C are disclosed, and such combinations can be in a pharmaceutical composition. Variants of each mAb component can be selected from those disclosed herein to include variants of mAb h7D11 and mAb h8A having modifications that confer an enhanced serum half-life as described herein. The pharmaceutical composition can be in the form of a liquid, a frozen composition, or a lyophilized composition. The amount of any antibody or fragment thereof can independently be present in the composition or pharmaceutical composition, for example, in an amount in the range of about 0.0001 mg to about 10,000 mg, such as 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 a functional fragment thereof can be administered to a subject in an amount in the range of about 0.001 mg / kg to about 200 mg / kg, where mg is the mg of the antibody or a functional fragment thereof and kg is the kg of the body weight of the subject. The antibody can be administered, for example, intravenously, subcutaneously, intramuscularly, as a separate formulation administered continuously or simultaneously, or as part of a pharmaceutical composition that includes a pharmaceutically acceptable: excipient, diluent, carrier, or any combination thereof. For example, the administration can be 1, 2, 3, 4, or 5 times per day, and the administration can be about: once a day, 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, when two or more antibodies (which can be monoclonal antibodies (mAbs)) are administered to a subject, each antibody or mAb can be mixed at the time of administration or given as a separate injection, intramuscularly, subcutaneously, or intravenously; given simultaneously or continuously. Any pharmaceutical or antibody composition or formulation can be in unit dose form. The subject can be a mammal, which can be a human. The subject can be a subject in need thereof. The composition, pharmaceutical composition, antibody, and its functional fragment can be contained in a container, such as a bag, for example, an intravenous bag or a syringe, thereby forming a kit.

[0304] Example 11: High-Concentration Pharmaceutical Product Formulation for Intramuscular Administration

[0305] To deliver an estimated dose of, for example, 10 mg - 200 mg total IgG / kg subject body weight at an intramuscular dose volume of 5 mL per injection, a concentration of >100 mg total IgG / mL can be effective. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Histidine, sucrose, and polysorbate-80 can be used alone or in any combination as components for monoclonal antibody formulations. The average pH of the monoclonal antibody formulation can be 6.0 ± 0.4. A stable formulation at pH 6.0 has been developed, which can have histidine (buffer), sucrose (stabilizer and excipient), and polysorbate-80 (stabilizer). In addition to liquid formulations, to minimize the injection volume, the composition can be filled with the target dose and lyophilized. Before administration, the lyophilized drug product can be reconstituted to half of the original fill volume. Similar formulations can be used for antibodies delivered subcutaneously or intravenously, functional fragments thereof, compositions comprising these, or pharmaceutical compositions comprising these.

[0306] Example 12: Use of the Composition

[0307] Indications for the antibodies and compositions herein can include: i) post-exposure prophylaxis of monkeypox in adult and pediatric patient individuals at high risk of progressing to symptomatic monkeypox; ii) treatment of monkeypox in adult and child patients with a positive monkeypox virus test result and at high risk of progressing to symptomatic monkeypox; and iii) pre-exposure prophylaxis for certain adult and pediatric patient individuals with no known monkeypox virus exposure, or with a moderately to severely impaired immune system or who are not recommended for any available monkeypox vaccination due to a history of severe adverse reactions to one or more monkeypox vaccines and / or one or more monkeypox vaccine components.

[0308] Medical conditions or treatments that can lead to moderate to severe immune impairment and an inadequate immune response to monkeypox vaccination include, but are not limited to: i) moderate or severe primary immunodeficiencies (e.g., DiGeorge syndrome, Wiskott-Aldrich syndrome); ii) active treatment of solid tumors and hematologic malignancies with chemotherapeutic agents classified as severely immunosuppressive; iii) receipt of solid organ transplantation and taking transplantation-related immunosuppressive drugs; iv) receipt of chimeric antigen receptor (CAR)-T cell or hematopoietic stem cell transplantation (within 2 years after transplantation or receiving immunosuppressive therapy); v) advanced or untreated HIV infection; or vi) active treatment with high-dose corticosteroids (e.g., ≥20 mg prednisone or equivalent per day when administered for ≥2 weeks), alkylating agents, tumor necrosis factor (TNF) blockers, and other immunosuppressive or immunomodulatory biologic agents (e.g., B cell depleting agents).

[0309] Example 13: Dosage and Administration

[0310] The dosages for pre-exposure prophylaxis, emergency use in the treatment of monkeypox in adult and pediatric patients, and post-exposure prophylaxis were determined in non-human animal studies and verified in a Phase 1 human trial in healthy adult volunteers. The dosage can be administered as two separate sequential intramuscular injections into each gluteal muscle, as a single intramuscular injection into one gluteal muscle containing both antibodies, or via intravenous infusion.

[0311] The injection can include a pharmaceutical composition comprising the active pharmaceutical ingredient (API) humanized mAb h7D11, with or without engineered half-life modification, as a liquid formulation in a single-dose vial or to be reconstituted from a lyophilized state. The injection can also include a pharmaceutical composition comprising the active pharmaceutical ingredient (API) humanized mAb h8A, with or without engineered half-life modification, as a liquid formulation in a single-dose vial or to be reconstituted from a lyophilized state. Two or more antibodies or functional fragments thereof can be combined into a single-dose vial as a liquid formulation or to be reconstituted from a lyophilized state.

[0312] The antibodies and compositions herein can be contraindicated in individuals with a history of severe hypersensitivity reactions (including anaphylaxis) to any of the antibodies or any of the components of the composition.

[0313] Other indications can include pre-exposure prophylaxis of monkeypox in individual adult and pediatric patients (12 years of age and older, weighing at least 40 kg): i) recent known exposure in individuals who are currently not infected with monkeypox and have no history of monkeypox infection; ii) individuals who are moderately to severely immunocompromised due to medical conditions or receipt of immunosuppressive medications or treatments and may not mount an adequate immune response to monkeypox vaccination; or iii) individuals who are not recommended for vaccination with any available approved or authorized monkeypox vaccine due to a history of severe adverse reactions (e.g., severe anaphylaxis) to one or more monkeypox vaccines and / or one or more monkeypox vaccine components.

[0314] Other indications can include post-exposure prophylaxis in individual adult and pediatric patients (12 years of age and older, weighing at least 40 kg) who are at high risk of progressing to severe monkeypox disease, including hospitalization or death.

[0315] Other indications can include the treatment of active monkeypox disease in individual adult and pediatric patients (12 years of age and older, weighing at least 40 kg), laboratory-confirmed monkeypox infection with a positive monkeypox virus test result, and who are at high risk of impending progression to symptomatic monkeypox, as well as symptomatic or asymptomatic patients for whom other forms of treatment are contraindicated or considered suboptimal.

[0316] A randomized, double-blind, placebo-controlled trial in adults established the pre-exposure prophylaxis dose. The primary endpoint confirmed that, compared with placebo, participants receiving a single IM or IV dose had a significantly lower incidence of monkeypox-positive symptomatic disease during the median follow-up time determined by the half-life of the drug product.

[0317] Pre-exposure prophylaxis authorization may require repeated dosing to ensure prophylactic efficacy in individuals for whom long-term protection is determined to be appropriate (e.g., immunocompromised or with comorbidities). Maintenance dosing to maintain a drug exposure similar to or slightly higher than that observed at the time point of near-determined single-dose efficacy was extrapolated from clinical trial data. The safety of repeated dosing may be different based on hypersensitivity reactions and the generation of anti-drug antibodies.

[0318] Example 14: Dose and Administration in Mice

[0319] Groups 1 and 2 of mice (10 male and 10 female BALBc mice; n = 20) were used as control groups. Group 1 was treated with vehicle, not challenged with ectromelia virus (ECTV), and Group 2 was treated with vehicle and challenged with 200 PFU of ECTV. Figure 11 It was shown that the average body weight of the mice in Group 1 did not change at the time of challenge, and the average body weight of the mice in Group 2 had a moderate change, indicating that the health of the animals was affected by the viral challenge but not by the vehicle treatment.

[0320] Groups 3, 4, 5, and 6 were challenged and dosed. The mice were challenged with 200 PFU of ECTV on Day 0. Groups 3, 4, 5, and 6 were treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture 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 It was shown that Group 3 had the least weight loss, followed by Groups 4, 5, and 6. This indicates that early treatment after exposure to ECTV has a more pronounced therapeutic benefit. Figure 17 It was shown that the survival probability at all treatment time points was greater than 90%, compared with a 30% survival probability in the control Group 2.

[0321] Groups 7, 8, 9, and 10 were attacked and administered drugs. Mice were attacked with 200 PFU of ECTV on day 0. Groups 7, 8, 9, and 10 were treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h8A (anti-B5 mAb), with a total mAb mixture dose of 10 mg / kg. Group 7 was treated on day 3 after the attack, Group 8 on day 4 after the attack, Group 9 on day 5 after the attack, and Group 10 on day 6 after the attack. Figure 13 It shows that Group 7 had the least weight loss, followed by Group 10, Group 8, and Group 9. This generally indicates that early treatment after exposure to ECTV has more obvious therapeutic benefits. Figure 18 It shows that compared with the 30% survival probability of the control in Group 2, the survival probabilities at the treatment time points of Groups 7 and 8 were greater than 90%, followed by 90% for Group 9 and 70% for Group 10.

[0322] Groups 11, 12, 13, and 14 were attacked and administered drugs. Mice were attacked with 200 PFU of ECTV on day 0. Groups 11, 12, 13, and 14 were treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb) and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture dose of 10 mg / kg. Group 11 was treated on day 3 after the attack, Group 12 on day 4 after the attack, Group 13 on day 5 after the attack, and Group 14 on day 6 after the attack. Figure 14 It shows that Group 13 had the least weight loss, followed by Groups 11, 12, and 14. This generally indicates that early treatment after exposure to ECTV has more obvious therapeutic benefits. Figure 19 It shows that compared with the 30% survival probability of the control in Group 2, the survival probabilities at all treatment time points were 100%.

[0323] Groups 15, 16, 17, and 18 were attacked and administered drugs. Mice were attacked with 200 PFU of ECTV on day 0. Groups 15, 16, 17, and 18 were treated with a mixture of the original chimeric form of the mAb, which was used as a comparator to the humanized form used in all other treatment groups, at 5 mg / kg c7D11 (anti-L1 mAb), 5 mg / kg c8A (anti-B5 mAb), and 5 mg / kg c6C (anti-A33 mAb), with a total mAb mixture dose of 15 mg / kg. Group 15 was treated on day 3 after the attack, Group 16 on day 4 after the attack, Group 17 on day 5 after the attack, and Group 18 on day 6 after the attack. Figure 15 It shows that Group 15 had the least weight loss, followed by Groups 16, 18, and 17. This generally indicates that early treatment after exposure to ECTV has more obvious therapeutic benefits. Figure 20It shows that the survival probability at all treatment time points is greater than 90% compared to the 30% survival probability of the control in Group 2.

[0324] Groups 19, 20, 21, and 22 were challenged and administered. Mice were challenged with 200 PFU of ECTV on Day 0. Groups 19, 20, 21, and 22 were treated with a mixture 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), with a total mAb mixture 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 It shows that Groups 19 and 20 had the least weight loss, followed by Groups 21 and 22. This generally indicates that early treatment after exposure to ECTV has more obvious therapeutic benefits. Figure 21 It shows that the survival probability at all treatment time points is greater than 90% compared to the 30% survival probability of the control in Group 2.

[0325] Groups 2, 3, 4, 5, and 6 were challenged and administered. 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 3, 4, 5, and 6 were treated with a mixture of 5 mg / kg h7D11 (anti-L1 mAb), 5 mg / kg h8A (anti-B5 mAb), and 5 mg / kg h6C (anti-A33 mAb), with a total mAb mixture 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.

[0326] Figure 22 It shows the comparison of the intervention on Day 3 of the composition compared to the control in Group 2. The survival probability of Groups 3, 7, 11, 15, and 19 treated on Day 3 was greater than 90% for all treatment groups, while the survival probability of the control in Group 2 was 30%.

[0327] Figure 23 It shows the comparison of the intervention on Day 4 of the composition compared to the control in Group 2. The survival probability of Groups 4, 8, 12, 16, and 20 treated on Day 4 was 100% for all treatment groups, while the survival probability of the control in Group 2 was 30%.

[0328] Figure 24Shows the comparison of the 5-day intervention of the composition compared to the control of Group 2. The survival probabilities of Groups 5, 9, 13, 17, and 21 treated on the 5th day are equal to or greater than 90% for all treatment groups, while the survival probability of the control of Group 2 is 30%.

[0329] Figure 25 Shows the comparison of the 6-day intervention of the composition compared to the control of Group 2. The survival probabilities of Groups 6, 10, 14, 18, and 22 treated on the 6th day are greater than 90% for all treatment groups except for Group 10 which is 70%, while the survival probability of the control of Group 2 is 30%.

[0330] Although embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and alternatives will now 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 present disclosure. The appended claims are intended to define the scope of the present disclosure and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

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

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

1.

4. The composition according to claim 3, wherein the first antibody is the humanized 7D11 antibody.

5. The composition according to 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. The composition according to claim 5, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTE exchange modification, and the h7D11 Fc YTE exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

21.

7. The composition according to claim 5, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc LS exchange modification, and the h7D11 Fc LS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

22.

8. The composition according to claim 5, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTELS exchange modification, and the h7D11 Fc YTELS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

23.

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

6.

11. The composition according to claim 10, wherein the second antibody is the humanized 8A antibody.

12. The composition according to 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. The composition according to claim 12, wherein the humanized 8A antibody variant comprises an h8A Fc YTE exchange modification, and the h8A Fc YTE exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

25.

14. The composition according to claim 12, wherein the antibody (B) is a humanized 8A antibody variant comprising an h8A Fc LS exchange modification, and the h8A Fc LS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

26.

15. The composition according to claim 12, wherein the antibody (B) is a humanized 8A antibody variant comprising an h8A Fc YTELS exchange modification, and the h8A Fc YTELS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

27.

16. The composition according to 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. The composition according to 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 according to claim 17, wherein the epitope of the A33 protein is within the domain of the A33 protein having the amino acid sequence of SEQ ID NO:

15.

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

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

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

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

1.

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

25. The composition according to 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. The composition according to claim 25, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTE exchange modification, and the h7D11 Fc YTE exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

21.

27. The composition according to claim 25, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc LS exchange modification, and the h7D11 Fc LS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

22.

28. The composition according to claim 25, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTELS exchange modification, and the h7D11 Fc YTELS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

23.

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

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

6.

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

32. The composition according to 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. The composition according to claim 32, wherein the humanized 8A antibody variant comprises an h8A Fc YTE exchange modification, and the h8A Fc YTE exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

25.

34. The composition according to claim 32, wherein the humanized 8A antibody variant comprises an h8A Fc LS exchange modification, and the h8A Fc LS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

26.

35. The composition according to claim 32, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS exchange modification, and the h8A Fc YTELS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

27.

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

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

15.

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

39. The composition according to 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. A method of conferring passive immunity against smallpox to 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 the variola virus; and (B) a second antibody that binds to an epitope found on the EV form of the variola virus.

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

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

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

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

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

1.

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

47. The method according to 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. The method according to claim 47, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTE exchange modification, and the h7D11 Fc YTE exchange modification comprises the Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

21.

49. The method according to claim 47, wherein the antibody (A) is a humanized 7D11 antibody variant comprising an h7D11 Fc LS exchange modification, and the h7D11 Fc LS exchange modification comprises the FcCH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

22.

50. The method according to claim 47, wherein the antibody (A) is a humanized 7D11 antibody variant comprising an h7D11 Fc YTELS exchange modification, and the h7D11 Fc YTELS exchange modification comprises the Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

23.

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

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

6.

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

54. The method according to 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. The method according to claim 54, wherein the humanized 8A antibody variant comprises an h8A Fc YTE exchange modification, and the h8A Fc YTE exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

25.

56. The method according to claim 54, wherein the humanized 8A antibody variant comprises an h8A Fc LS exchange modification, and the h8A Fc LS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

26.

57. The method according to claim 54, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS exchange modification, and the h8A Fc YTELS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

27.

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

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

15.

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

62. The method according to 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. A method of conferring passive immunity against monkeypox virus infection to 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 the monkeypox virus; and (B) a second antibody that binds to an epitope found on the EV form of the monkeypox virus.

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

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

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

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

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

1.

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

70. The method according to 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. The method according to claim 70, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTE exchange modification, the h7D11 Fc YTE exchange modification comprising Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

21.

72. The method according to claim 70, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc LS exchange modification, the h7D11 Fc LS exchange modification comprising Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

22.

73. The method according to claim 70, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTELS exchange modification, the h7D11 Fc YTELS exchange modification comprising Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

23.

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

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

6.

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

77. The method according to 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. The method according to claim 77, wherein the humanized 8A antibody variant comprises an h8A Fc YTE exchange modification, and the h8A Fc YTE exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

25.

79. The method according to claim 77, wherein the humanized 8A antibody variant comprises an h8A Fc LS exchange modification, and the h8A Fc LS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

26.

80. The method according to claim 77, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS exchange modification, and the h8A Fc YTELS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

27.

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

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

15.

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

85. The method according to 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. A method of conferring passive immunity against smallpox to 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 the 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.

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

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

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

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

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

1.

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

93. The method according to 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. The method according to claim 93, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTE exchange modification, and the h7D11 Fc YTE exchange modification comprises the Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

21.

95. The method according to claim 93, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc LS exchange modification, and the h7D11 Fc LS exchange modification comprises the Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

22.

96. The method according to claim 93, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTELS exchange modification, and the h7D11 Fc YTELS exchange modification comprises the Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

23.

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

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

6.

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

100. The method according to 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. The method according to claim 100, wherein the humanized 8A antibody variant comprises an h8A Fc YTE exchange modification, and the h8A Fc YTE exchange modification comprises the Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

25.

102. The method according to claim 100, wherein the humanized 8A antibody variant comprises an h8A Fc LS exchange modification, and the h8A Fc LS exchange modification comprises the Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

26.

103. The method according to claim 100, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS exchange modification, and the h8A Fc YTELS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

27.

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

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

15.

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

107. The method according to 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. A method of conferring passive immunity against monkeypox virus infection to 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 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.

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

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

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

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

1.

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

115. The method according to 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. The method according to claim 115, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTE exchange modification, and the h7D11 Fc YTE exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

21.

117. The method according to claim 115, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc LS exchange modification, and the h7D11 Fc LS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

22.

118. The method according to claim 115, wherein the humanized 7D11 antibody variant comprises an h7D11 Fc YTELS exchange modification, and the h7D11 Fc YTELS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

23.

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

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

6.

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

122. The method according to 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. The method according to claim 122, wherein the humanized 8A antibody variant comprises an h8A Fc YTE exchange modification, and the h8A Fc YTE exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

25.

124. The method according to claim 122, wherein the humanized 8A antibody variant comprises an h8A Fc LS exchange modification, and the h8A Fc LS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

26.

125. The method according to claim 122, wherein the humanized 8A antibody variant comprises an h8A Fc YTELS exchange modification, and the h8A Fc YTELS exchange modification comprises Fc CH2 and CH3 domains having the amino acid sequence of SEQ ID NO:

27.

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

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

15.

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

129. The method according to 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.

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