Antibodies and antigen-binding fragments that bind to monkeypox virus protein a35 and vaccinia virus protein a33 and methods of use

Abstract

Provided herein are antibodies and antigen-binding fragments thereof that bind to monkeypox virus (MPXV) protein A35 and / or to vaccinia virus (VACV) protein A33, nucleic acids and vectors encoding such antibodies and antigen-binding fragments, and methods of use. Provided are, for example, methods of reducing or blocking infection of a cell with MPXV or VACV. Also provided are methods of treating or preventing infection with MPXV or VACV.

Description

ANTIBODIES AND ANTIGEN-BINDING FRAGMENTS THAT BIND TO MONKEYPOX VIRUS PROTEIN A35 AND VACCINIA VIRUS PROTEIN A33 AND METHODS OF USESTATEMENT OF GOVERNMENT-SPONSORED RESEARCH

[0001] This invention was made with United States government support under grant No. U54CA267776 awarded by the National Institutes of Health. The United States government has certain rights in the invention.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety’. Said xml copy, created on August 20, 2025. is named SeqList-084284-00335.xml and is 44,103 bytes in size.FIELD

[0003] The present disclosure relates generally to the field of molecular biology' and medicine. More particularly, the methods and compositions herein are useful for, e.g., detecting monkeypox virus (MPXV) A35 or vaccinia virus (VACV) A33 and for the prevention or treatment of disease.BACKGROUND

[0004] For many decades, monkeypox (mpox) was restricted to African countries, with only sporadic cases reported in non-endemic settings. Ignored for too long, in 2022, monkeypox virus (MPXV) gained significant attention due to a global outbreak caused by Clade lib — one of the four currently recognized clades. This outbreak has affected 122 countries so far; 115 of which reported mpox cases for the first time. 100,000 cases and over 200 deaths have been registered to date, mostly in the Americas and Europe. This has led the World Health Organization (WHO) to declare the mpox epidemic as a Public Health Emergency of International Concern in mid-2022 and again in August 2024 (this time due to an upsurge of cases caused by Clade I MPXVs in Central Africa).

[0005] Poxviruses like MPXV are DNA-encoded viruses. Generally, DNA-encoded viruses have a lower mutational rate than RNA viruses. However, the recent switch in the clade lib176172084.1MPXV epidemiology, with viral spread sustained by human-to-human transmission, was accompanied by an upsurge of APOBEC-like mutations with implications for viral evolution and host adaptation.

[0006] JYNNEOS™ (smallpox / monkeypox vaccine) is an FDA-approved, live, attenuated smallpox / monkeypox vaccine. However, JYNNEOS™ vaccination does not promote a robust antibody response in humans at the gene level. In light of the lack of robust immune responses to MPXV induced by JYNNEOS™ and the unusual mutational pattern that has recently been observed for MPXV, new research and therapy tools for MPXV are urgently needed.SUMMARY

[0007] Provided herein are antibodies and antigen-binding fragments thereof that bind to MPXV protein A35 and / or to VACV protein A33 and methods of use.

[0008] Provided is an antibody or antigen-binding fragment thereof that binds to monkeypox vims (MPXV) A35 or to vaccinia virus (VACV) A33, the antibody or antigenbinding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1. CDR2, and CDR3, and wherein:(a) the sequence of CDR1H comprises SEQ ID NO: 1; the sequence of CDR2H comprises SEQ ID NO:2; the sequence of CDR3H comprises SEQ ID NO:3; the sequence of CDR1L comprises SEQ ID NO:4; the sequence of CDR2L comprises the sequence GAS; and the sequence of CDR3L comprises SEQ ID NO:6;(b) the sequence of CDR1H comprises SEQ ID NO:7; the sequence of CDR2H comprises SEQ ID NO:8; the sequence of CDR3H comprises SEQ ID NO:9; the sequence of CDR1L comprises SEQ ID NO: 10; the sequence of CDR2L comprises the sequence DAS; and the sequence of CDR3L comprises SEQ ID NO: 12; or(c) the sequence of CDR1H comprises SEQ ID NO: 13; the sequence of CDR2H comprises SEQ ID NO: 14; the sequence of CDR3H comprises SEQ ID NO: 15; the sequence of CDR1L comprises SEQ ID NO: 16; the sequence of CDR2L comprises the sequence KAS; and the sequence of CDR3L comprises SEQ ID NO: 18.

[0009] In some embodiments,(a) the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 19 and the sequence of the light chain variable region comprises a2176172084.1sequence that is least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:20;(b) the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:21 and the sequence of the light chain variable region comprises a sequence that is least at least 80%, at least 85%, at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:22; or(c) the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:23 and the sequence of the light chain variable region comprises a sequence that is least at least 80%, at least 85%, at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:24.

[0010] In some embodiments,(a) the sequence of the heavy chain variable region comprises SEQ ID NO: 19 and the sequence of the light chain variable region comprises SEQ ID NO:20;(b) the sequence of the heavy chain variable region comprises SEQ ID NO:21 and the sequence of the light chain variable region comprises SEQ ID NO:22; or(c) the sequence of the heavy chain variable region comprises SEQ ID NO:23 and the sequence of the light chain variable region comprises SEQ ID NO:24.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof is a multispecific or a bispecific antibody or antigen-binding fragment thereof.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof is an scFv, Fv, Fab’, Fab, F(ab’)2, or diabody.

[0014] In one embodiment, the antibody or antigen-binding fragment thereof has isotype IgGl.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to one or more of a cy to toxin, an anti-viral agent, a fluorescent label, and an imaging agent.

[0016] Provided is a nucleic acid or pair of nucleic acids encoding an antibody or antigenbinding fragment thereof disclosed herein.3176172084.1

[0017] Provided is a nucleic acid or pair of nucleic acids encoding an antibody or antigenbinding fragment thereof that binds to MPXV A35 or to VACV A33. the antibody or antigenbinding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:(a) the sequence encoding the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:43 and the sequence encoding the light chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:44;(b) the sequence encoding the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:45 and the sequence encoding the light chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:46; or(c) the sequence encoding the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:47 and the sequence encoding the light chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:48.

[0018] In some embodiments:(a) the sequence encoding the heavy chain variable region comprises SEQ ID NO:43 and the sequence encoding the light chain variable region comprises SEQ ID NO:44;(b) the sequence encoding the heavy chain variable region comprises SEQ ID NO:45 and the sequence encoding the light chain variable region comprises SEQ ID NO:46; or(c) the sequence encoding the heavy chain variable region comprises SEQ ID NO:47 and the sequence encoding the light chain variable region comprises SEQ ID NO:48.

[0019] The nucleic acid or pair of nucleic acids may be isolated.

[0020] Provided is a vector comprising a nucleic acid or pair of nucleic acids disclosed herein.

[0021] Provided is a cell comprising a vector disclosed herein. Provided is a cell comprising a nucleic acid or pair of nucleic acids disclosed herein. The cell may be a bacterial cell, a yeast cell, or a mammalian cell. The cell may be isolated.4176172084.1

[0022] Provided is a pharmaceutical composition comprising an antibody or antigenbinding fragment thereof disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0023] Provided is an assay device comprising an antibody or antigen-binding fragment thereof disclosed herein.

[0024] Provided is a kit for detecting the presence of MPXV A35 or VACV A33, or an antigenic fragment of MPXV A35 or VACV A33, in a sample comprising: (i) an antibody or antigen-binding fragment thereof disclosed herein, and (ii) a buffer. In one embodiment, the antibody or antigen-binding fragment thereof is bound to a substrate. In one embodiment, the antibody or antigen-binding fragment thereof is detectably labeled. In one embodiment, the kit further comprises a secondary antibody that specifically binds to the antibody or antigenbinding fragment thereof. In one embodiment, the secondary antibody is detectably labeled.

[0025] Provided is a method of making an antibody or antigen-binding fragment thereof that binds to MPXV A35 or to VACV A33, the method comprising:(i) providing a cell comprising one or more nucleic acid molecules encoding an antibody or antigen-binding fragment thereof disclosed herein;(ii) expressing in the cell the antibody or antigen-binding fragment thereof; and(iii) collecting the antibody or antigen-binding fragment thereof.

[0026] The antibody may be further purified.

[0027] Provided is a method of reducing or blocking infection of a cell with MPXV or VACV, the method comprising contacting the MPXV or VACV with an antibody or antigenbinding fragment thereof of disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cell is a macrophage, dendritic cell, natural killer (NK) cell, Langerhans cell, neutrophil, epithelial cell, keratinocyte. fibroblast, or endothelial cell. The cell may be a human cell.

[0028] Provided is a method of reducing or blocking transmission of MPXV or VACV from a first cell to a second cell, the method comprising contacting the MPXV or VACV with an antibody or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the first and / or the second cell is a macrophage, dendritic cell, natural killer (NK) cell, Langerhans cell, neutrophil, epithelial cell, keratinocyte, fibroblast, or endothelial cell. The cell may be a human cell.

[0029] Provided is a method of neutralizing MPXV or VACV, the method comprising contacting the MPXV or VACV with an antibody or antigen-binding fragment thereof5176172084.1disclosed herein or a pharmaceutical composition disclosed herein. In one embodiment, the method is performed in the presence of complement. Provided is a method of blocking the spread of MPXV or VACV in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition disclosed herein. Provided is a method of blocking the spread of MPXV or VACV in between two or more subjects in need thereof, the method comprising administering to at least one of the two or more subjects an antibody or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition disclosed herein. Provided is a method of treating or preventing an infection with MPXV or VACV in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition disclosed herein.

[0030] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.

[0031] The subject may be human.DESCRIPTION OF THE DRAWINGS

[0032] Fig. 1. Gating strategy to select A35R-specific B cells. Sorted memory B cells were gated as live, single cells, and excluded for CD3+, CD14+, CD16+, and CD56+(dump channel). Then, cells were gated to CD19+and CD20+, and CD27+(memory B cells). Cells binding to the empty probe (no antigen) were excluded and those binding to recombinant A35 were selected for sorting.

[0033] Figs. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 21. Binding characterization of the mAbs isolated from an MPXV-convalescent individual. Fig. 2A. PBMCs from an MPXV- convalescent participant were sorted for A35-specific single B cells. V(D)J was sequenced revealing eight human mAbs. Fig. 2B. Paired VH and VL genes of the eight isolated mAbs. Order of bar graphs from top to bottom as shown in the legend. Fig. 2C. Minimum binding concentration against both MPXV A35 Clades lb and lib and VACV A33. The Minimum binding concentration is defined as the lowest antigen concentration capable of eliciting a signal. The dashed line represents the limit of detection, which is set at twice the starting dilution (1 pg / mL). For each clade, bars from top to bottom represent the antibodies listed in in the figure legend from top to bottom. Figs. 2D, 2E, and 2F. Mean OD versus antigen concentration for MPXV A35 Clade lb (Fig. 2D), MPXV A35 Clade lib (Fig. 2E), and VACV A33 (Fig. 2F). The antigen initial concentration was 1 pg / mL, then serially diluted nine times6176172084.1to a final concentration of 0.001 pg / mL. The top three traces in Figs. 2D, 2E, and 2F belong to EV35-2, -6, and -7. The y axis in Figs. 2D, 2E, and 2F shows the mean OD at 450 nm. The x axis in Figs. 2D, 2E, and 2F shows the antibody concentration (Log scale; pg / mL). Fig. 2G, 2H, and 21. Affinity of the human EV mAbs to MPXV A35 Clade lb (bottom trace is EV35- 8) (Fig. 2G), MPXV A35 Clade lib (Fig. 2H), and VACV A33 (Fig. 21). The y axis in Figs. 2G, 2H, and 21 shows response (nm). The x axis in Figs. 2G, 2H, and 21 shows time (seconds). Top trace in Figs. 2G. 2H, and 21 is EV35-2. Fig. 2G: KD values listed in boxes from top to bottom: EV35-2, EV35-6, EV35-7, EV35-8. Figs. 2H and 21: KD values listed in boxes from top to bottom: EV35-2, EV35-6, EV35-7. Affinity ranged from 10'7to 10'12(nano and picomolar ranges) and was measured by Biolayer Interferometry (BLI). KD is the affinity constant, measuring how tightly the ligand binds to the antigens (A35 or A33).

[0034] Figs. 3A, 3B, and 3C. Epitope binning of anti-35 human mAbs against MPXV A35 (Clades lb and lib) and its ortholog VACV A33. After loading the antigen to the sensor surface, combinations of the EV35 mAbs were presented in tandem (50 pg / mL each) to verify the association with MPXV A35 Clade lb (Fig. 3A), MPXV A35 Clade lib (Fig. 3B), and VACV A33 (Fig. 3C). The y axis shows response (nm). The x axis shows time (seconds).

[0035] Figs. 4A, 4B, 4C, 4D, 4E, 4F, and 4G. In vitro neutralizing capacity of EV mAbs measured by a comet reduction assay (50 pg / mL). The neutralizing capacity7against VACV strain IHD-J was assessed using 50 pg / mL of each mAb. Figs. 4A, 4B, and 4C. Wells with no virus (Fig. 4A), virus-only (Fig. 4B), and in the presence of a polyclonal anti-VACV (Fig. 4C) were used as positive and negative controls. Figs. 4D, 4E, and 4F. Neutralizing capacity was defined by comet tail inhibition, the fewer comets, the more neutralizing activity7and assessed for EV35-2 (Fig. 4D), EV35-6 (Fig. 4E), and EV35-7 (Fig. 4F). Fig. 4G. Length, width, and number of comets for each mAb. Values were measured using ImageJ. The size of each circle indicates the number of comet formations, and the x and y-axis represent the mean width and length, respectively, of comet formation for each mAb / well. Virus only: circle in upper right. Remaining three circles: EV35-7 (top left), EV35-6 (bottom left), EV-35-2 (right).

[0036] Figs. 5A, 5B, 5C, 5D, 5E, 5F, and 5G. In vitro neutralizing capacity of EV mAbs measured by a comet reduction assay (5 pg / mL). The neutralizing capacity against VACV strain IHD-J was assessed using 5 pg / mL of each mAb. Figs. 5A, 5B, and 5C. Wells with no virus (Fig. 5A), virus-only (Fig. 5B), and in the presence of a polyclonal anti-VACV (Fig. 5C) were used as positive and negative controls. Figs. 5D, 5E, and 5F. Neutralizing capacity7was defined by comet tail inhibition, the fewer comets, the more neutralizing activity and assessed7176172084.1for EV35-2 (Fig. 5D), EV35-6 (Fig. 5E), and EV35-7 (Fig. 5F). Fig. 5G. Length, width, and number of comets for each mAh. Values were measured using ImageJ. The size of each circle indicates the number of comet formations, and the x and y-axis represent the mean width and length, respectively, of comet formation for each mAb / well. Circles from lower left to upper right: EV35-2; EV35-7; EV35-6; virus only.

[0037] Figs. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, and 61. In vivo evaluation of the protective efficacy of EV monoclonal antibodies. Fig. 6A. After the passive immunization with A35 mAbs, BALB / c mice (n = 5 per group) were infected intranasally with IxlO5PFU of VACV WRvFire and assessed for weight loss, survival, and viral load (bioluminescence). Figs. 6B and 6C. Weight loss (Fig. 6B) and survival (Fig. 6C) of mice infected with VACV WRvFire. Traces for Fig. 6B from top to bottom: EV35-7; EV356; EV35-2; control. Traces for Fig. 6C: bottom trace = control. Fig. 6D. Bioluminescence imaging of the control group (VACV WRvFire-infected mice that received PBS instead of the EV -mAbs prophylactical treatment). Figs. 6E, 6F, and 6G. Bioluminescence imaging of VACV WRvFire-infected mice treated prophylactically with EV35-2 (Fig. 6E). EV35-6 (Fig. 6F) and EV35-7 (Fig. 6G). Mice were imaged on days three, six. eight, and ten post-infections, capturing whole-body images. Luminescence intensity is represented by colors ranging from red (high) to blue and purple (low); Color scale = 3000-62000. Figs. 6H and 61. Total photon flux (average per day) in the head only (Fig. 6H) and in the whole body (Fig. 61). *p<0.05, **p<0.01, ***p<0.001.

[0038] Figs. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 71, 7J, 7K, and 7L. Competition assessment of antisera from vaccinees and MPXV-infected participants. Fig. 7A. Participants vaccinated with either vaccine (Dryvax or Jynneos; n=40),MPXV acute infection (n=20) and MPXV-conv alescent were assessed for circulating anti-A35. The positivity threshold (dashed line, OD30.300) was defined by the mean OD of control samples (from participants never exposed to orthopoxviruses) plus tw o standard deviations (95% CI). Fig. 7B. Neutralization against MPXV. Capacity was defined through logPRNT50 values, which represent the minimum concentration of sera needed to achieve 50 % plaque reduction. The closer to 1.0, the weaker the neutralization capacity. Fig. 7C. Experimental design of a competition ELISA (cELISA) used to assess competition between the EV35 mAbs and antibodies induced by either vaccination or natural infection. Figs. 7D, 7E, and 7F. Percentage and presence of competition in each group (Dryvax, Jynneos, MPXV-acute, and MPXV- convalescent) for EV35-2 (Fig. 7D), EV35-6 (Fig. 7E), and EV35-7 (Fig. 7F). Bars for Figs. 7D, 7E, and 7F are presented from left to right: Dryvax, Jynneos, MPXV-acute. and MPXV-8176172084.1convalescent. Fig. 7G. Percentage of competition between acute participants for each EV35 mAb. Fig. 7H. Correlation between competition percentage and anti-A35 levels among MPXV-acute participants. R values from top to bottom: EV35-2, EV35-6, EV35-7. Fig. 71. Correlation between competition percentage and neutralizing capacity (logPRNT50) among MPXV-acute participants. Fig. 7 J. Percentage of competition between convalescent participants for each EV35 mAb. Fig. 7K. Correlation between competition percentage and anti-A35 levels among MPXV -convalescent participants. R values from top to bottom: EV35- 2, EV35-6, EV35-7. Fig. 7L. Correlation between competition percentage and neutralizing capacity (logPRNT50) among MPXV-convalescent participants. R values from top to bottom: EV35-2, EV35-6, EV35-7. *p<0.05, **p<0.01, ***p<0.001.

[0039] Figs. 8A, 8B, 8C, 8D, 8E, and 8F. Fc-mediated activity of human A35 mAb. Figs.8A, 8B, 8C, and 8D. Plaque reduction assay using the mAbs against MPXV clade Ila was conducted using wells with no virus and virus-only as controls (Fig. 8A) and 50 pg / mL of EV35-2 (Fig. 8B), EV35-6 (Fig. 8C), and EV35-7 (Fig. 8D). Fig. 8E. In vitro reduction of viral spread (area in pixels2) by A35 mAbs with or without complement (5 % rabbit serum). The area was measured via ImageJ using a color-based threshold (RGB). Error bars plot the mean and standard deviation. Left bars: complement. Right bars: no complement. Fig. 8F. FcyRIIIa reporter activity of the A35 mAbs. An isotype-matched human anti-SARS-CoV-2 was used as a negative control, whereas MDCK cells were used as a technical positive control. The dotted line denotes LoD. The experiments were conducted in duplicates, and the error bars represent the variation between experiments.DETAILED DESCRIPTION

[0040] MPXV is a double-stranded DNA virus encoding around 180 proteins. Genes encoding the structural proteins of MPXV are located within the highly conserved central genomic region and expressed in different forms of MPXV. The two known MPXV infective forms are the Extracellular Enveloped Virion (EEV) and the Intracellular Mature Virion (IMV). Glycoprotein A35 is located on the surface of the EEV and an important factor for poxvirus virulence. “A35” and “A35R” refer to the same protein, with “R” signifying that it is a receptor protein.9176172084.1

[0041] The viruses from the orthopoxvirus genus, including MPXV, vaccinia virus (VACV). cowpox virus (CPXV), and variola virus (VARV). share significant genomic similarity (-96%). Further, antibodies targeting these viruses have proven to be immunologically cross-reactive and cross-protective. However, studies demonstrating the use of a single antibody, instead of cocktails of antibodies, as protective measure against orthopoxviruses is currently lacking. VACV protein A33 is an ortholog of MPXV protein A35.

[0042] Provided herein are antibodies and antigen-binding fragments thereof that bind to MPXV protein A35 and / or A33 VACV protein A33. These antibodies combine a variety of preferred characteristics. To start, antibodies disclosed herein were isolated from subjects recovering from infection with the strain of MPXV that caused the current outbreak and are therefore effective against circulating strains. Further, antibodies disclosed herein bind with high affinity to MPXV Clade lb, the virulent strain of MPXV currently causing a large number of deaths in Africa. Finally, antibodies disclosed herein protect mice against death caused by MPXV as a monotherapy (opposed to as part of a combination), an important advantage as compared to other MPXV therapies.

[0043] Antibodies and Antigen-Binding Fragments

[0044] Provided herein are antibodies and antigen-binding fragments thereof that bind to MPXV protein A35 and / or A33 VACV protein A33 as well as methods of using these antibodies and antigen-binding fragments for research or therapy. Antibodies and antigenbinding fragments thereof that bind to MPXV protein A35 and / or A33 VACV protein A33 are also referred to as “anti-MPXV A35 / VACV A33 antibodies and antigen-binding fragments thereof.”

[0045] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and antigen-binding fragments thereof (e.g, paratopes, CDRs), so long as they exhibit the desired biological activity and specificity. The terms “antigen-binding portion” or “antigen-binding fragment” as used herein may refer to a region on an antibody that binds to its antigen.

[0046] As used herein, “antibody variable domain” refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of Complementarity Determining Regions (CDRs; i.e., CDR1, CDR2, and CDR3), and Framework Regions (FRs).10176172084.1VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. The amino acid positions assigned to CDRs and FRs may be defined according to Kabat, Chothia, or IMGT. The term "‘framework regions” (FR) refers to those variable domain residues other than the CDR residues.

[0047] As used herein, the term “Complementarity Determining Regions” (CDRs) refers to portions of an antibody variable domain that are (typically) involved in antigen-binding. Each variable domain typically has three CDR regions identified as CDR1. CDR2 and CDR3. Each CDR can comprise amino acid residues from a CDR as defined by e g., Kabat (z.e., about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1987, 1991)). Each CDR can also comprise amino acid residues from a “hypervariable loop” ( / .<?., about residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia & Lesk, Canonical structures for the hypervariable regions of immunoglobulins, J Mol Biol. 1987 Aug 20; 196(4):901-17). In some instances, a CDR can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues (primary amino acid sequence). The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or CDR, of the basic variable domain structure. The Kabat numbering of residues may be determined for a given antibody or antigen-binding fragment thereof by alignment of residues of homology in the sequence of the antibody or antigen-binding fragment thereof with a “standard” Kabat numbered sequence. Alternatively, a CDR can be defined according to the ImMunoGeneTics (IMGT) system (Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Dev Comp Immunol. 2003 Jan;27(l):55-77).

[0048] In a preferred embodiment, the IMGT numbering system is used to identify the CDRs

[0049] Provided herein are also antibodies and antigen-binding fragments thereof that comprise CDR or variable chain sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with any of the sequences11176172084.1disclosed herein. Provided herein are also antibodies and antigen-binding fragments thereof that comprise CDR or variable chain sequences that have at least 1, 2, 3, 4, 5, 6, 7. 8, 9, or 10 amino acid substitutions as compared to any of the CDR or variable chain sequences disclosed herein.

[0050] As used herein, the term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. For example, when a position in the compared nucleotide sequence is occupied by the same base, then the molecules are identical at that position. A degree identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. For example, polypeptides having at least 85%, 90%, 95%, 98%, or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotides encoding such polypeptides, are contemplated. Methods and computer programs for determining both sequence identity and similarity are publicly available, including, but not limited to, the GCG program package (Devereux et al., A comprehensive set of sequence analysis programs for the VAX, Nucleic Acids Res. 1984 Jan 11:12(1 Pt 1):387- 95), BLASTP, BLASTN, FASTA (Altschul et al, Basic local alignment search tool, J Mol Biol. 1990 Oct 5;215(3):403-10), and the ALIGN program (version 2.0). The well-known Smith Waterman algorithm may also be used to determine similarity. The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al.. NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0. In comparing sequences, these methods account for various substitutions, deletions, and other modifications.

[0051] In some embodiments, the anti-MPXV A35 / V AC V A33 antibody or antigenbinding fragments thereof comprises 1, 2. 3, 4, 5, or 6 CDRs of the CDRs disclosed in Table 1.

[0052] In some embodiments, the anti-MPXV A35 / VACV A33 antibody or antigenbinding fragments thereof comprises 1, 2, 3, 4, 5, or 6 CDRs of the CDRs that are encoded by any of the CDR-encoding sequences in Table 2.

[0053] In some embodiments, the anti-MPXV A35 / VACV A33 antibody or antigenbinding fragments thereof comprises one or more sequences disclosed in Tables 1 or 2.Table 1. Amino acid sequences of selected anti-MPXV A35 / VACV A33 antibodies. Numbers in the table refer to SEQ ID NOs. IMGT numbering system is used to identify the CDRs.12176172084.1Table 2. Nucleic acid sequences encoding the CDRs and VH / VL chains of selected anti- MPXV A35 / VACV A33 antibodies. Numbers in the table refer to SEQ ID NOs

[0054] Provided is an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein:(a) the sequence of CDR1H comprises SEQ ID NO: 1; the sequence of CDR2H comprises SEQ ID NO:2; the sequence of CDR3H comprises SEQ ID NO: 3; the sequence of CDR1L comprises SEQ ID NO:4; the sequence of CDR2L comprises the sequence GAS; and the sequence of CDR3L comprises SEQ ID NO:6;(b) the sequence of CDR1H comprises SEQ ID NO:7; the sequence of CDR2H comprises SEQ ID NO:8; the sequence of CDR3H comprises SEQ ID NO:9; the sequence of CDR1L comprises SEQ ID NO: 10; the sequence of CDR2L comprises the sequence DAS; and the sequence of CDR3L comprises SEQ ID NO: 12; or(c) the sequence of CDR1H comprises SEQ ID NO: 13; the sequence of CDR2H comprises SEQ ID NO: 14; the sequence of CDR3H comprises SEQ ID NO: 15; the sequence of CDR1L comprises SEQ ID NO: 16; the sequence of CDR2L comprises the sequence KAS; and the sequence of CDR3L comprises SEQ ID NO: 18.

[0055] In some embodiments, the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any one of SEQ ID Nos: 19, 21, and 23.176172084.1

[0056] In some embodiments, the sequence of the light chain variable region comprises a sequence that is at least 80%, at least 85%. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any one of SEQ ID Nos:20, 22, and 24.

[0057] In some embodiments, the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 19 and the sequence of the light chain variable region comprises a sequence that is least at least 80%. at least 85%. at least 90%. at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:20.

[0058] In some embodiments, the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% to SEQ ID NO: 21 and the sequence of the light chain variable region comprises a sequence that is least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 22.

[0059] In some embodiments, the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%. at least 90%, at least 95%, at least 96%, at least 97%. at least 98%. or at least 99% to SEQ ID NO:23 and the sequence of the light chain variable region comprises a sequence that is least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:24.

[0060] In one embodiment, the sequence of the heavy chain variable region comprises SEQ ID NO: 19 and the sequence of the light chain variable region comprises SEQ ID NO:20.

[0061] In one embodiment, the sequence of the heavy chain variable region comprises SEQ ID NO:21 and the sequence of the light chain variable region comprises SEQ ID NO:22.

[0062] In one embodiment, the sequence of the heavy chain variable region comprises SEQID NO:23 and the sequence of the light chain variable region comprises SEQ ID NO:24.

[0063] Modifications of Anti-MPXV A35 / VACV A33 Antibodies and Antigen-Binding Fragments Thereof

[0064] In some embodiments of the aspects described herein, amino acid sequence modification(s) of the anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof described herein are contemplated. Amino acid sequence variants of the antibody or antigen-binding fragment thereof are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antibody or antigen-binding fragment thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into14176172084.1and / or substitutions of, residues within the amino acid sequences of the antibody or antigenbinding fragment thereof. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., binding specificity, and inhibition of biological activity.

[0065] One ty pe of antibody variant is a conservative amino acid substitution variant. These variants have at least one amino acid residue in the antibody or antigen-binding fragment thereof replaced by a different residue that has similar side chain properties. Amino acids can be grouped according to similarities in the properties of their side chains (see Lehninger, BIOCHEMISTRY (2nd ed„ Worth Publishers, New York, 1975)):(1) non-polar: Ala (A). Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M);(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C). Tyr (Y), Asn (N), Gin (Q):(3) acidic: Asp (D), Glu (E);(4) basic: Lys (K), Arg (R), His (H).

[0066] As such, a non-limiting example for a conservative amino acid substitution is one that replaces a non-polar amino acid with another non-polar amino acid.

[0067] Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties:(1) hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M);(2) neutral hydrophilic: Ser (S), Thr (T). Cys (C), Asn (N), Gin (Q);(3) acidic: Asp (D), Glu (E);(4) basic: Lys (K), Arg (R), His (H);(5) residues that influence chain orientation: Gly (G), Pro (P);(6) aromatic: Phe (F), Trp (W), Tyr (Y).

[0068] As such, a non-limiting example for a conservative amino acid substitution is one that replaces a hydrophobic ammo acid with another hydrophobic amino acid.

[0069] In some embodiments, one or more of the CDRs of an anti-MPXV A35 / V ACV A33 antibody or antigen-binding fragment disclosed herein has a conservative amino acid substitution.

[0070] Further contemplated are amino acid sequence insertions, which can include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody or antigen-binding fragment thereof with an N-terminal methionyl residue or the antibody or antigen-binding fragment15176172084.1thereof fused to a cytotoxic polypeptide. Other insertional variants of the antibody or antigenbinding fragment thereof include the fusion to the N- or C- terminus of the antibody or antigenbinding fragment thereof to an enzyme or a polypeptide which increases the serum half-life of the antibody or antigen-binding fragment thereof, such as, for example, biotin.

[0071] Any cysteine residue not involved in maintaining the proper conformation of the anti-MPXV A35 / V AC V A33 antibodies or antigen-binding fragments thereof also can be substituted, for example with a serine or an alanine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0072] Conversely, cysteine bond(s) can be added to the antibody or antigen-binding fragment thereof to improve its stability (particularly where the antibody or antigen-binding fragment thereof is an antibody fragment such as an Fv fragment).

[0073] In some embodiments, the antibodies or antigen-binding fragments thereof have amino acid alterations that alter the original glycosylation pattern of the antibody or antigenbinding fragment thereof. By “altering the original glycosylation pattern” is meant deleting one or more carbohydrate moieties found in the antibody or antigen-binding fragment thereof, and / or adding one or more glycosylation sites that are not present in the antibody or antigenbinding fragment thereof. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine. wherein X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine can also be used. Addition of glycosylation sites to the anti- MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof is accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by the addition of, or substitution by. one or more serine or threonine residues to the sequence of the original antibody or antigen-binding fragment thereof (for O-linked glycosylation sites).

[0074] In some embodiments, the anti-MPXV A35 / VACV A33 antibodies or antigenbinding fragments thereof provided herein are deglycosylated or aglycosylated.16176172084.1

[0075] Where the antibody or antigen-binding fragment thereof comprises an Fc region, the carbohydrate(s) attached thereto can be altered. For example, antibodies with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody or antigenbinding fragment thereof have been described. See, e.g., U.S. Patent Pubs. No. 2003 / 0157108; No. 2004 / 0093621. Antibodies with a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody or antigen-binding fragment thereof are referenced in WO 03 / 011878; U.S. PatentNo. 6.602.684. Antibodies with at least one galactose residue in the oligosaccharide attached to an Fc region of the antibody or antigen-binding fragment thereof are reported in WO 97 / 30087. See also WO 98 / 58964 and WO 99 / 22764 concerning antibodies with altered carbohydrate attached to the Fc region thereof.

[0076] The contemplated antibodies and antigen-binding fragments thereof also feature humanized frameworks for reduced immunogenicity. In certain embodiments, the CDRs of the contemplated antibody or antigen-binding fragment thereof are located in frameworks obtained from a human antibody or antigen-binding fragment thereof. In other embodiments, surface- exposed framework residues of the contemplated antibody or antigen-binding fragment thereof are replaced with framework residues of a human antibody or antigen-binding fragment thereof. The CDRs may also be located in rabbit or humanized frameworks linked to human constant regions (z.e., chimeric antibodies).

[0077] Techniques for humanization of antibodies are known to one of ordinary skill in the art and are generally reviewed in Safdari et al., Antibody humanization methods - a review and update, Biotechnol Genet Eng Rev. 2013;29: 175-86, hereby incorporated by reference in its entirety. Humanization of antibodies generally comprises grafting of CDRs (such as the CDRs disclosed herein) or conservative substituted variants thereof into an appropriate human variable region framework, for example, as disclosed in Jones et al., Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 1986 May 29-Jun 4;321(6069):522-5, hereby incorporated by reference in its entirety. Common methods used include, but are not limited to, framework-homology-based humanization, germline humanization, CDR-homology-based humanization and specificity determining residues (SDR) grafting.

[0078] In one embodiment, the CDRs of a contemplated antibody or antigen-binding fragment thereof are located in frameworks that are a composite of two or more human antibodies. In such embodiments, the contemplated antibodies or antigen-binding fragments thereof comprise two or more sequence segments (“composites’7) derived from V-regions of17176172084.1unrelated human antibodies that are selected to maintain monoclonal antibody sequences important for antigen-binding of the starting precursor anti-MPXV A35 / VACV A33 monoclonal antibody, and which have all been filtered for the presence of potential T cell epitopes using “in silico tools” (Holgate & Baker, Circumventing immunogenicity in the development of therapeutic antibodies, IDrugs. 2009 Apr;12(4):233-7). The close fit of human sequence segments with all sections of the starting antibody V regions and the elimination of CD4+T cell epitopes prior to synthesis of the antibody or antigen-binding fragment thereof allow this technology to circumvent immunogenicity while maintaining optimal affinity and specificity through the prior analysis of sequences necessary' for antigen-specificity (Holgate & Baker, 2009).

[0079] Antibodies with improved binding to the neonatal Fc receptor (FcRn), and increased half-lives, are described in WO 00 / 42072 and U.S. Patent Pub. No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. For example, the Fc region can have substitutions at one or more of positions 238, 250. 256, 265, 272, 286, 303, 305, 307. 311, 312, 314, 317, 340. 356, 360, 362, 376, 378, 380. 382, 413, 424. 428 or 434 (Eu numbering of residues). An Fc region may be preferred comprising an antibody variant with improved FcRn binding comprises amino acid substitutions at one, two or three of positions 307, 380 and 434 of the Fc region thereof (Eu numbering of residues). In one embodiment, the antibody or antigen-binding fragment thereof has mutatations at positions 307 and 434.

[0080] Antibody Fragments and Types

[0081] Provided herein are anti-MPXV A35 / VACV A33 antibodies and antigen-binding fragments thereof.

[0082] In some embodiments of the aspects described herein, the anti-MPXV A35 / VACV A33 antibody fragment is a Fab fragment, which comprises or consists essentially of a variable (VL) and constant (CL) domain of the light chain and a variable domain (Vn) and the first constant domain (CH I) of the heavy chain.

[0083] In some embodiments of the aspects described herein, the anti-MPXV A35 / VACV A33 antibody fragment is a Fab’ fragment, which refers to a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain.18176172084.1

[0084] In some embodiments of the aspects described herein, the anti-MPXV A35 / VACV A33 antibody fragment is an Fd fragment comprising or consisting essentially of VH and CHI domains.

[0085] In some embodiments of the aspects described herein, the anti-MPXV A35 / VACV A33 antibody portion is an Fd' fragment comprising VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain.

[0086] Single-chain Fv or scFv antibody fragments comprise or consist essentially of the VH and VL domains of antibody, such that these domains are present in a single polypeptide chain. Generally, an Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen-binding. See, for example. Pluckthun. 113 Pharmacology’ Monoclonal Antibodies 269 (Rosenburg & Moore, eds., Springer-Verlag, New York, 1994). Accordingly, in some embodiments of the aspects described herein, the anti-MPXV A35 / VACV A33 antibody fragment is a Fv fragment comprising or consisting essentially of the VL and VH domains of a single arm of an antibody.

[0087] In some embodiments of the aspects described herein, the anti-MPXV A35 / VACV A33 antibody portion is a diabody comprising two antigen-binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain.

[0088] In some embodiments of the aspects described herein, the anti-MPXV A35 / VACV A33 antibody portion is a dAb fragment comprising or consisting essentially of a VH domain.

[0089] In some embodiments of the aspects described herein, the anti-MPXV A35 / V ACV A33 antibody portion is a F(ab')2 fragment, which comprises a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region.

[0090] Linear antibodies refer to the antibodies as described in Zapata et al.. Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity. Protein Eng. 1995 Oct;8(10): 1057-62. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1), which, together with complementary’ light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. In some embodiments of the aspects described herein, the anti-MPXV A35 / VACV A33 antibody fragment is a linear antibody comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary’ light chain polypeptides, form a pair of antigen-binding regions.19176172084.1

[0091] Various techniques have been developed and are available for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies. See, e.g., Morimoto et al., 24 J. Biochem. Biophys. Meths. 107 (1992); Brennan et al., 229 Science 81 (1985). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed herein. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments. According to another approach. F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody fragment of choice is a single chain Fv fragment (scFv). See, for example. WO 93 / 16185.

[0092] Contemplated antibodies or antigen-binding fragments may have all types of constant regions, including IgAl, IgA2, IgM, IgG, IgD, and IgE, and any isotype, including IgGl, IgG2, IgG3. and IgG4. In one embodiment, the human isotype IgGl is used. In one embodiment, the human isotype IgGl is used. Light chain constant regions can be X or K. The antibody or antigen-binding fragment thereof may comprise sequences from more than one class or isotype.

[0093] In some embodiments, the anti-MPXV A35 / VACV A33 antibody or antigenbinding fragment thereof is an isolated antibody or antigen-binding fragment thereof. The terms “purified7’ or “isolated” antibody, peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein that has been separated from other proteins, lipids, and / or nucleic acids with which it may be naturally associated. The polypeptide / protein can constitute at least 10% (z.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%. 95%. and 99%) by dry weight of the purified preparation. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide / protein (e.g., anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof) described in herein can be produced by recombinant DNA techniques.

[0094] Binding of Anti-MPXV A35 / VACV A33 Antibodies and Antigen-Binding Fragments Thereof

[0095] As used herein, “binding” of an antibody or antigen-binding fragment thereof to MPXV A35 and / or VACV A33 or an epitope on the surface of MPXV A35 and / or VACV A3320176172084.1includes the selective interaction of the antibody or antigen-binding fragment thereof with MPXV A35 and / or VACV A33. Binding therefore includes, e.g., primary and secondary interactions including hydrogen bonds, ionic interactions, salt bridges, as well as hydrophilic and hydrophobic interactions.

[0096] As used herein, “affinity”, represented by the equilibrium constant for the dissociation (KD) of an antigen with an antigen-binding protein, is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein, such as an antibody or antibody fragment thereof. The smaller the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule. Alternatively, the affinity can also be expressed as the affinity constant (K ), which is 1 / KD). AS will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest.

[0097] In some embodiments, the anti-MPXV A35 / VACV A33 antibodies or antigenbinding fragments thereof described herein bind to MPXV A35 and / or VACV A33 with a KD of 10’5to 1012mol / 1, 10’6to 10’12mol / 1, 10’7to 10’12mol / 1, 10’8to 10’12mol / 1. 10'9to 10’12mol / 1, 10‘10to IO’12mol / 1, or 10'11to 10’13mol / 1. In other embodiments, the anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof described herein bind to MPXV A35 and / or VACV A33 with a KD of W5to 1 O'12mol / 1, IO’6to W12mol / 1, 10'7to 10’12mol / 1, 10'8to IO’12mol / 1. 10’9to 10'12mol / 1, 10'10to 10'12mol / l, 10'11to 10’12mol / l.

[0098] In embodiments, the antibodies and antigen-binding fragments thereof provided herein bind specifically to MPXV A35 and / or VACV A33. The term “specificity” herein refers to the ability of an antibody or antigen-binding fragment thereof, such as the anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof described herein, to bind to an MPXV A35 and / or VACV A33 epitope, while only having little or no detectable reactivity with other epitopes. Specificity can be relatively determined by competition assays or by epitope identification / characterization techniques described herein or their equivalents known in the art.

[0099] As used herein, an “epitope” can be formed both from contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary' folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a particular spatial conformation. An “epitope” includes the unit of structure21176172084.1conventionally bound by an immunoglobulin VH / VL pair. Epitopes define the minimum binding site for an antibody or antigen-binding fragment thereof, and thus represent the target of specificity of an antibody or antigen-binding fragment thereof. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation.

[0100] In one embodiment, the contemplated anti-MPXV A35 / V AC V A33 antibody or antigen-binding fragment specifically binds to the same epitope as antibody EV35-2. EV35-6, or EV35-7.

[0101] In one embodiment, the contemplated anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment specifically competes with antibody EV35-2, EV35-6, or EV35-7 for binding to MPXV A35 and / or VACV A33.

[0102] In some embodiments, an anti-MPXV A35 / V ACV A33 antibody or antigen-binding fragment disclosed herein blocks (partially or completely) binding of MPXV A35 and / or VACV A33 to an interaction partner.

[0103] As used herein, a "blocking" antibody or an antibody “antagonist'’ is one that inhibits or reduces biological activity of the antigen to which it binds. Inhibition of activity and inhibition of binding includes partial inhibition. Methods for the identification of anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof that block MPXV A35 and / or VACV A33 interaction with an interaction partner are described herein and are known to one skilled in the art. For instance, competing, cross-blocking, and cross-blocked antibodies can be identified using any suitable method known in the art, including competition ELISAs or BIACORE® assays where binding of the competing or cross-blocking antibody to MPXV A35 and / or VACV A33 prevents the binding of an antibody disclosed herein or vice versa.

[0104] Conjugates of Anti-MPXV A35 / VACV A33 Antibodies and Antigen-Binding Fragments Thereof

[0105] In some embodiments of the aspects described herein, the anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof are conjugated to a functional moiety. Examples of useful functional moieties include, but are not limited to, a blocking moiety, a detectable moiety, a diagnostic moiety, a targeting, and a therapeutic moiety.

[0106] Exemplary blocking moieties include moieties of sufficient steric bulk and / or charge such that reduced glycosylation occurs, for example, by blocking the ability7of a glycosidase to glycosylate the antibody or antigen-binding fragment thereof. The blocking moiety may additionally or alternatively, reduce effector function, for example, by inhibiting the ability of22176172084.1the Fc region to bind a receptor or complement protein. Blocking moieties may include cysteine adducts and PEG moieties.

[0107] In one embodiment, the blocking moiety is a cysteine, preferably a cysteine that has associated with a free cysteine, e.g. , during or subsequent to the translation of the Fc containing polypeptide, e.g., in cell culture. Other blocking cysteine adducts include cystine, mixed disulfide adducts, or disulfide linkages.

[0108] In one embodiment, the blocking moiety is a polyalkylene glycol moiety, for example, a PEG moiety and preferably a PEG-maleimide moiety. Pegylation moieties (or related polymers) can be, for example, polyethylene glycol (“PEG’’), polypropylene glycol (“PPG"’), polyoxyethylated glycerol C‘POG”) and other polyoxyethylated polyols, polyvinyl alcohol C‘PVA?’) and other polyalkylene oxides, polyoxyethylated sorbitol, or poly oxy ethylated glucose. The polymer can be a homopolymer, a random or block copolymer, a terpolymer based on the monomers listed above, straight chain or branched, substituted or unsubstituted as long as it has at least one active sulfone moiety. The polymeric portion can be of any length or molecular weight, but these characteristics can affect the biological properties. Polymer average molecular weights particularly useful for decreasing clearance rates in pharmaceutical applications are in the range of 2,000 to 35,000 Daltons. In addition, if two groups are linked to the polymer, one at each end, the length of the polymer can impact upon the effective distance, and other spatial relationships, between the two groups. Thus, one skilled in the art can vary the length of the polymer to optimize or confer the desired biological activity. PEG is useful in biological applications for several reasons. PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze, and is nontoxic. Pegylation can improve pharmacokinetic performance of a molecule by increasing the molecule's apparent molecular weight. The increased apparent molecular weight reduces the rate of clearance from the body following subcutaneous or systemic administration. In many cases, pegylation can decrease antigenicity and immunogenicity. In addition, pegylation can increase the solubility of a biologically active molecule.

[0109] Examples of detectable moieties which are useful in the methods and antibodies and antigen-binding fragments thereof contemplated herein include fluorescent moieties or labels, imaging agents, radioisotopic moieties, radiopaque moieties, and the like, e.g. , detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels. Exemplary fluorophores include fluorescent dyes (e.g. fluorescein, rhodamine, and the like) and other luminescent molecules (e.g. luminal). A fluorophore may be environmentally-sensitive such23176172084.1that its fluorescence changes if it is located close to one or more residues in the modified protein that undergo structural changes upon binding a substrate (e.g. dansyl probes). Exemplary radiolabels include small molecules containing atoms with one or more low sensitivity nuclei (13C,15N.2H,125I,123I, "TC,43K,52Fe,67Ga,68Ga,niIn and the like). Other useful moieties are known in the art.

[0110] Examples of diagnostic moieties which are useful in the methods and antibodies and antigen-binding fragments thereof contemplated herein include detectable moieties suitable for revealing the presence of a disease or disorder. Typically, a diagnostic moiety allows for determining the presence, absence, or level of a molecule, for example, a target peptide, protein, or proteins, that is associated with a disease or disorder. Such diagnostics are also suitable for prognosing and / or diagnosing a disease or disorder and its progression.

[0111] Techniques for conjugating moieties to antibodies are well known, see. e.g.. Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.). Robinson et al. (eds.). pp. 623-53 (Marcel Dekker. Inc. 1987); Thorpe. “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58 (1982).

[0112] To increase the half-life of the antibodies or polypeptide containing the amino acid sequences described herein, one can attach a salvage receptor binding epitope to the antibody or antigen-binding fragment thereof (especially an antibody fragment), as described, e.g., in U.S. Patent. No. 5,739,277. The term “salvage receptor binding epitope” may refer to an epitope of the Fc region of an IgG molecule (e.g. , IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule (e.g., Ghetie & Ward. Multiple roles for the major histocompatibility complex class I- related receptor FcRn, Annu Rev Immunol. 2000;18:739-66). Antibodies with substitutions in an Fc region thereof and increased serum half-lives are also described in WO 00 / 42072, WO 02 / 060919; Shields et al., High resolution mapping of the binding site on human IgGl for Fc gamma RI, Fc gamma RII. Fc24176172084.1gamma RIII, and FcRn and design of IgGl variants with improved binding to the Fc gamma R, J Biol Chem. 2001 Mar 2;276(9):6591-604; Hinton et al.. Engineered human IgG antibodies with longer serum half-lives in primates, J Biol Chem. 2004 Feb 20;279(8):6213-6. For example, a nucleic acid molecule encoding the salvage receptor binding epitope can be linked in frame to a nucleic acid encoding a polypeptide sequence described herein so that the fusion protein expressed by the engineered nucleic acid molecule comprises the salvage receptor binding epitope and a polypeptide sequence described herein. In another embodiment, the serum half-life can also be increased, for example, by attaching other polypeptide sequences. For example, antibodies or antigen-binding fragments thereof useful in the methods of the disclosure can be attached to serum albumin or a portion of serum albumin that binds to the FcRn receptor or a serum albumin binding peptide so that serum albumin binds to the antibody or antigen-binding fragment thereof, e.g, such polypeptide sequences are disclosed in WO 01 / 45746. In one embodiment, the half-life of a Fab is increased by these methods. See also, Dennis et al., Albumin binding as a general strategy for improving the pharmacokinetics of proteins, J Biol Chem. 2002 Sep 20;277(38):35035-43, for additional serum albumin binding peptide sequences.

[0113] In some embodiments, the therapeutic moiety is an anti-viral moiety.

[0114] Other types of functional moieties are known in the art and can be readily used in the methods and compositions of disclosed herein based on the teachings contained herein. The functional moiety may also have one or more of the above-mentioned functions.

[0115] Nucleic Acids

[0116] Also provided herein are nucleic acids encoding the anti-MPXV A35 / VACV A33 antibodies and antigen-binding fragments thereof disclosed herein, as well as vectors, host cells, and expression systems.

[0117] The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, mRNA, cDNA, DNA-RNA hybrids, or a polymer comprising punne and pynmidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0118] The nucleic acids encoding anti-MPXV A35 / VACV A33 antibodies and antigenbinding fragments thereof may be, e.g., DNA, cDNA, RNA, synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of those25176172084.1polynucleotides either alone or in combination. For example, provided is an expression vector comprising a polynucleotide sequence encoding an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof described herein operably linked to expression control sequences suitable for expression in a eukaryotic and / or prokaryotic host cell.

[0119] The term “vector’" refers to a vehicle capable of transporting another nucleic acid to which it has been linked. A “vector"’ includes, but is not limited to, a viral vector, a plasmid, an RNA vector or a linear or circular DNA or RNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. The vector can be a nucleic acid and or viral particle. In some embodiments, the employed vectors are those capable of autonomous replication (episomal vector) and / or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available. Viral vectors include retrovirus, adenovirus, parvovirus (e.g, adeno associated viruses, AAV), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e.g, measles and Sendai), positive strand RNA viruses such as picomavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g, Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C- type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, and spumavirus.

[0120] A variety of expression vectors have been developed for the efficient synthesis of antibodies and antigen-binding fragments thereof in prokaryotic cells such as bacteria and in eukaryotic systems, including but not limited to yeast and mammalian cell culture systems have been developed. The vectors can comprise segments of chromosomal, non-chromosomal and synthetic DNA sequences.

[0121] Also provided are cells comprising expression vectors for the expression of the contemplated anti-MPXV A35 / V AC V A33 antibodies or antigen-binding fragments thereof.

[0122] In one aspect, provided is a nucleic acid or set of nucleic acids encoding an anti- MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein. The sequences encoding the heavy chain variable region and the light chain variable region of an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein may be located on the same nucleic acid molecules or on different nucleic acid molecules.26176172084.1

[0123] In one embodiment, provided is a nucleic acid encoding the heavy chain variable region of an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein. In one embodiment, provided is a nucleic acid encoding the light chain variable region of an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein.

[0124] In one embodiment, provided is a vector or set of vectors comprising a sequence encoding the heavy chain variable region of an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein and the light chain variable region of an anti- MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein. In one embodiment, the heavy chain variable region is encoded by a first vector and the light chain variable region is encoded by a second vector.

[0125] In one embodiment, provided is a vector or set of vectors comprising a sequence encoding the heavy chain of an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein and the light chain of an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein. In one embodiment, the heavy chain is encoded by a first vector and the light chain is encoded by a second vector.

[0126] Provided herein are nucleic acids encoding the amino acid sequences disclosed in any one of Table 1, or variants of these sequences. Provided herein are nucleic acids encoding any of the anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof disclosed herein. Provided herein are nucleic acids comprising any of the nucleic acids in Table 2, or variants of such nucleic acids.

[0127] In some embodiments, provided is a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any one of SEQ ID Nos:25-48. In some embodiments, provided is a sequence comprising any one of SEQ ID Nos:25-48.

[0128] Provided is an isolated nucleic acid or pair of nucleic acids encoding an antibody or antigen-binding fragment thereof that binds to MPXV A35 or to VACV A33, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:(a) the sequence encoding the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:43 and the sequence encoding the light chain27176172084.1variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:44;(b) the sequence encoding the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:45 and the sequence encoding the light chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:46; or(c) the sequence encoding the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:47 and the sequence encoding the light chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:48.

[0129] Provided is an isolated nucleic acid or pair of nucleic acids encoding an antibody or antigen-binding fragment thereof that binds to MPXV A35 or to VACV A33, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:(a) the sequence encoding the heavy chain variable region comprises SEQ ID NO:43 and the sequence encoding the light chain variable region comprises SEQ ID NO:44;(b) the sequence encoding the heavy chain variable region comprises SEQ ID NO:45 and the sequence encoding the light chain variable region comprises SEQ ID NO:46; or(c) the sequence encoding the heavy chain variable region comprises SEQ ID NO:47 and the sequence encoding the light chain variable region comprises SEQ ID NO:48.

[0130] The nucleic acid sequences may be codon optimized. As used herein, codon optimization refers to an in vitro mutagenesis of a nucleic acid to increase or maximize expression of a gene (e.g. a transgene relative to the unmodified nucleic acid, without changing (or with minimal change) to the amino acid sequence of the synthesized protein, i.e. synonymous mutations. Codon optimization can affect protein expression rates up to 1,000 x fold, particularly by favoring efficient soluble protein expression. The codons changed are typically ones not generally used by the host cell translation system. Codon bias / codon usage frequency depends on the host organism, and is described, for example, in US patent 8,326,547, hereby incorporated by reference in its entirety'.28176172084.1

[0131] Antibody Preparation and Expression Systems

[0132] The antibodies or antigen-binding fragments thereof disclosed herein are typically produced by recombinant expression. Nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, are inserted into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the crossreacting antibodies.

[0133] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers (e g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362).

[0134] Signal peptides or signal sequence for the expression of antibodies and antigen-binding fragments are known in the art.

[0135] The expression of the antibodies and antigen-binding fragments contemplated herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insects, fungi, bird, and mammalian cells either in vivo, or in situ, or host cells of mammalian, insect, bird or yeast origin. The mammalian cell or tissue can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell may be used.

[0136] E. coli is one prokaryotic host particularly useful for cloning the polynucleotides (e.g. DNA sequences). Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.

[0137] Other microbes, such as yeast, are also useful for expression. Saccharomyces and Pichia are exemplary yeast hosts, with suitable vectors having expression control sequences29176172084.1(e.g., promoters), an origin of replication, termination sequences and the like as desired. Typical promoters include 3 -phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.

[0138] Further, by use of, for example, the yeast ubiquitin hydrolase system, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be accomplished. The fusion proteins so produced can be processed in vivo or purified and processed in vitro. allowing synthesis of an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein with a specified amino terminus sequence. Moreover, problems associated with retention of initiation codon-derived methionine residues in direct yeast (or bacterial) expression maybe avoided. Sabin et al., High-Level Expression and / / ? Vivo Processing of Chimeric Ubiquitin Fusion Proteins in Saccharomyces Cerevisiae, Nat Biotechnol 7, 705-709 (1989); Miller et al., Cloning and Expression of a Yeast Ubiquitin- Protein Cleaving Activity' in Escherichia Coli. Nat Biotechnol 7, 698-704 (1989).

[0139] Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in mediums rich in glucose can be utilized to obtain recombinant anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments disclosed herein. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

[0140] Production of anti-MPXV A35 / V ACV A33 antibodies or antigen-binding fragments thereof in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express a transmembrane polypeptide by methods known to those of skill. See AusubeL F.M., et al. (1993). Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York.

[0141] In addition to microorganisms, mammalian tissue culture may also be used to express and produce the antibodies or antigen-binding fragments thereof disclosed herein (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Eukaryotic cells might be preferred, because a number of suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, and include CHO cell lines, various COS cell lines. HeLa cells. 293 cells, myeloma cell lines, transformed B-cells. and hybridomas.30176172084.1Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Cell-type specific regulation of a kappa immunoglobulin gene by promoter and enhancer elements, Immunol Rev. 1986 Feb;89:49-68), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Expression control sequences may be promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al, Chimeric and humanized antibodies with specificity for the CD33 antigen. J Immunol. 1992 Feb 15;148(4): 1149-54.

[0142] Alternatively, nucleotide sequences encoding antibodies or antigen-binding fragments thereof can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g, Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489, and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and / or heavy' chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.

[0143] Additionally, plants have emerged as convenient, safe and economical alternative main-stream expression systems for recombinant antibody production, which are based on large scale culture of microbes or animal cells. Antibodies or antigen-binding fragments thereof can be expressed in plant cell culture, or plants grown conventionally. The expression in plants may be systemic, limited to sub-cellular plastids, or limited to seeds (endosperms). See, e.g., U.S. Patent Pub. No. 2003 / 0167531; U.S. Patent Nos. 6,080,560 and 6,512,162; and WO 0129242. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, e.g, Biolex, NC).

[0144] The vectors containing the polynucleotide sequences of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the ty pe of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection may be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., supra). For production of transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be31176172084.1incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

[0145] The antibodies and antigen-binding fragments thereof disclosed herein can be expressed using a single vector or two vectors. When the antibody heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms disclosed herein can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity can be preferred, and 98 to 99% or more homogeneity can be most preferred, for pharmaceutical uses.

[0146] Provided herein is a method of making an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof, the method comprising: (i) providing a cell comprising one or more nucleic acid molecules encoding an anti-MPXV A35 / VACV A33 antibody or antigenbinding fragment disclosed herein; (ii) expressing in the cell at least one of a heavy variable chain, a light variable chain, or combinations thereof; and (iii) collecting the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is further purified.

[0147] Kits and Methods of Use

[0148] Provided herein are kits for detecting MPXV A35 and / or VACV A33 present in a sample, including in a biological or clinical sample. These kits may comprise an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof disclosed herein and various reagents, for example, reagents that aid in detection of binding between the anti-MPXV A35 / VACV A33 antibody and an epitope present on MPXV A35 and / orVACV A33 or an antigenic fragment thereof.

[0149] The term “biological sample” as used herein may refer to a sample obtained from an organism (e.g.. patient) or from components (e.g, cells) of an organism. The sample may be of any biological tissue, cell(s) or fluid. The sample may be a “clinical sample” which is a sample derived from a subject, such as a human patient. Such samples include, but are not limited to, saliva, sputum, blood, blood cells (e.g, white cells), bodily fluids, lavages, pancreatic juices, gastric juices, discharges. CSF. lymph amniotic fluid, plasma, semen, bone32176172084.1marrow, and tissue or fine needle biopsy samples, urine, stool, peritoneal fluid, and pleural fluid, or cells therefrom, and any combinations thereof. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. A biological sample may also be referred to as a “patient sample.” A biological sample may also include a substantially purified or isolated protein, membrane preparation, or cell culture.

[0150] The kits may be used in vitro assays, such as immunoassays, e.g. enzyme immune assays (EIA), enzyme linked immunosorbent assay (ELISA). ELISPOT (enzyme-linked immunospot), radioimmunoassays (RIAs), immunofluorescence, and other assays known in the art, including but not limited to Western Blot analysis and / or immunoprecipitation methods. The in vitro assays may be competitive, or indirect, such as in a sandwich assay, or may be an antibody capture method. For example, in a direct ELISA, a buffered solution of an antigen, e.g.. a sample containing MPXV A35 and / or VACV A33 or an antigenic fragment thereof (e.g., a biological sample containing or suspected of containing MPXV A35 and / or VACV A33) is added to a well of a microtiter plate, e.g., a 96-well plate. A solution of non-reacting protein, e.g., bovine serum albumin or casein is then added to the well. The anti-MPXV A35 / VACV A33 antibody or antigen-binding fragments thereof conjugated to a reporter molecule enzyme is added, e.g., conjugated to horse-radish peroxidase, although that is not necessarily the enzyme, as other common enzymes include alkaline phosphatase, or P-D-galactosidase, although other enzymes are conceivable and considered embodied by the present disclosure. A substrate for the enzyme is then added, which leads to a detectable signal. For example, adding TMB to horseradish peroxidase leads to a colored product, in which case the ELISA is a colorimetric assay. ELIS As may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Sandwich ELISAs generally follow the following protocol. A capture anti-MPXV A35 / VACV A33 antibody or antigen-binding fragments thereof is bound to (i.e., “immobilized”) on a substrate, e.g., a mictotiter plate. Antigen-containing sample (i.e., sample containing MPXV A35 and / or VACV A33 or an antigenic fragment thereof, is then added to the substrate at which point it is captured by the anti-MPXV A35 / VACV A33 antibodies. The substrate is then washed to remove unbound antigen. A second anti-MPXV A35 / VACV A33 antibody or antigen-binding fragments thereof is added, which binds to a different epitope on MPXV A35 and / orVACV A33. The second anti-MPXV A35 / VACV A33 antibody or antigen-binding fragments thereof is bound to a reporter molecule, e.g., an enzyme, although the reporter molecule may be any33176172084.1molecule which leads to a detectable signal. The plate may be washed a second time, and in those instances where the reporter molecule is an enzyme, a substrate may be added, e.g., TMB, that results in a detectable signal (also a colorimetric assay). A third type of common ELISA is competitive ELISA. In these embodiments, unlabeled anti-MPXV A35 / VACV A33 antibody or antigen-binding fragments thereof is incubated in the presence of an antigen-containing sample (i.e., sample containing MPXV A35 and / or VACV A33 or an antigenic fragment thereof), which are then added to an antigen-coated well. The plate is washed to remove unbound antibodies. A secondary antibody is added that is specific to the primary antibody, e.g., a secondary antibody specific to anti-MPXV A35 / VACV A33 antibodies. The secondary antibody is bound to a reporter molecule, as described herein, such as an enzy me (or any other molecule that may lead to a detectable signal). Some competitive ELISA utilize labeled antigens rather than labeled antibodies; the less antigen in the sample, the more labeled antigen is retained and the stronger a detectable signal result.

[0151] Other forms of common in vitro assays include radioimmunoassays (RIAs). Typically, a known quantity of an antigen is linked to a radioactive tracer, e.g, 1-125 although others are suitable for use. which is then mixed with a known amount of antibody specific for the antigen, e.g, anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof. Then, a sample containing unknown quantity of an antigen is added, (e.g., a biological sample that contains or is suspected of containing MPXV A35 and / or VACV A33 an antigenic fragment thereof) is added. This is a direct competitive for specific binding; as the concentration of unlabeled antigen is increased, the binding between the anti-MPXV A35 / V ACV A33 antibodies and the labeled standard is decreased, which is directly measurable by measuring radioactivity. Other assays are known and a person of ordinary skill in the art would readily recognize their applicability.

[0152] In some embodiments, provided is a method of detecting MPXV A35 and / or VACV A33 or an antigenic fragment thereof in a sample. Such methods may utilize any' of the assays described herein, or others that are know n in the art. Several of the assays described herein are capable of quantifying the amount of antigen present in a sample, and so accordingly, in some embodiments, the present disclosure is directed to methods of quantifying the amount of MPXV A35 and / or VACV A33 or antigenic fragments thereof present in a sample, e.g., a biological sample.34176172084.1

[0153] Provided herein is a method of detecting MPXV A35 and / or VACV A33 in a sample, the method comprising contacting the sample with an anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment disclosed herein.

[0154] Pharmaceutical Compositions

[0155] Provided are pharmaceutically acceptable compositions (also referred to as pharmaceutical compositions) that comprise a therapeutically effective amount of anti-MPXV A35 / VACV A33 antibody or antigen-binding fragment thereof as described herein formulated together with one or more pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition further comprises complement.

[0156] The dosage of active agent(s) may vary, depending on the reason for use, the individual subject, and the mode of administration. The dosage may be adjusted based on the subject’s weight, the age and health of the subject, and tolerance for the compound(s) or composition. For example, depending on the disease, for an antibody or antigen-binding fragment thereof, this may require 0.1, 1.0, 3.0, 6.0, or 10.0 mg / Kg. For an IgG having a molecular mass of 150.000 g / mole (two binding sites), these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 pM, and 1.8 pM of binding sites for a 5 L blood volume.

[0157] The active agent and excipient(s) may be formulated into compositions and dosage forms according to methods known in the art. The pharmaceutical compositions of the present disclosure may be specially formulated in solid or liquid form, including those adapted for parenteral administration, for example, by subcutaneous, intratumoral, intramuscular or intravenous injection as, for example, a sterile solution or suspension.

[0158] Therapeutic compositions comprising anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof may formulated with one or more pharmaceutically- acceptable excipients, which can be a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), solvent or encapsulating material, involved in carrying or transporting the therapeutic compound for administration to the subject, bulking agent, salt, surfactant and / or a preservative. Some examples of materials which can serve as pharmaceutically-acceptable excipients include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil;35176172084.1glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents; water; isotonic saline; pH buffered solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0159] A bulking agent is a compound which adds mass to a pharmaceutical formulation and contributes to the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present disclosure include mannitol, glycine, polyethylene glycol and sorbitol.

[0160] The use of a surfactant can reduce aggregation of the reconstituted protein and / or reduce the formation of particulates in the reconstituted formulation. The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particulates after reconstitution. Suitable surfactants according to the present disclosure include polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octy l glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl-sarcosine; linoleyl-. myristyl-, or cetyl-betaine; lauroamidopropyl-. cocamidopropyl-, linolearmdopropyl- , myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68, etc.).

[0161] Preservatives may be used in formulations. Suitable preservatives for use in the formulation include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl-dimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical texts, e.g. in "‘Remington's Pharmaceutical Sciences7’, The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., (1995).

[0162] The compositions comprising an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier may comprise the anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof set forth herein at various concentrations. For36176172084.1example, the compositions may comprise an antibody or antigen-binding fragment thereof at 10 mg / ml to 200 mg / ml. 25 mg / ml to 130 mg / ml, 50 mg / ml to 125 mg / ml, 75 mg / ml to 110 mg / ml, or 80 mg / ml to 100 mg / ml. The compositions also may comprise an antibody or antigen-binding fragment thereof at about 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, or 150 mg / ml.

[0163] In some embodiments, the compositions comprising the antibody or antigen-binding fragment thereof and the pharmaceutically acceptable carrier are lyophilized and provided in a composition for reconstitution prior to administration or other use.

[0164] Methods

[0165] Provided herein are various methods that employ the anti-MPXV A35 / VACV A33 antibodies and antigen-binding fragments disclosed herein. The methods may be performed in vivo, ex vivo, or in vitro, as appropriate. In one aspect, provided are anti-MPXV A35 / VACV A33 antibodies and antigen-binding fragments thereof that are also useful for the treatment of subjects in need thereof or for the prevention of disease.

[0166] Provided is a method of reducing or blocking infection of a cell with MPXV or VACV, the method comprising contacting the MPXV or VACV with an antibody or antigenbinding fragment thereof of disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the cell is a macrophage, dendritic cell, natural killer (NK) cell, Langerhans cell, neutrophil, epithelial cell, keratinocyte, fibroblast, or endothelial cell. The cell may be a human cell.

[0167] As used herein, the terms “increasing” and “decreasing” (or “reducing”) are meant in reference to a control. For example, the increase or decrease could be by at least 1. 1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 9-fold, at least 8-fold, at least 10-fold, at least 15-fold, or at least 20-fold. A person skilled in the art can readily choose an appropriate control for a given application. For example, a control could refer to a sample, cell, tissue, or patient that is not exposed to an anti-MPXV A35 / VACV A33 antibody or antigen binding fragment thereof. A control could refer to the same sample, cell, tissue, or patient but at an earlier time. A control value may be derived from a single sample, cell, tissue, or patient. Alternatively, a control value may be calculated for a population of samples, cells, tissues, or patients. Depending on the37176172084.1application, the control may be a sample, cell, or tissue derived from a patient with a disease or disorder. The control patient may be a person who has a disease or disorder. The control may be a sample, cell, or tissue derived from a healthy patient. The control patient may be a healthy patient.

[0168] Provided is a method of reducing or blocking transmission of MPXV or VACV from a first cell to a second cell, the method comprising contacting the MPXV or VACV with an antibody or antigen-binding fragment thereof of disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the first and / or the second cell is a macrophage, dendritic cell, natural killer (NK) cell, Langerhans cell, neutrophil, epithelial cell, keratinocyte, fibroblast, or endothelial cell. The cell may be a human cell.

[0169] Provided is a method of neutralizing MPXV or VACV. the method comprising contacting the MPXV or VACV with an antibody or antigen-binding fragment thereof of disclosed herein or a pharmaceutical composition disclosed herein.

[0170] Provided is a method of blocking the spread of MPXV or VACV in a subject in need thereof, the method comprising administering to the subject with an antibody or antigenbinding fragment thereof of disclosed herein or a pharmaceutical composition disclosed herein.

[0171] Provided is a method of blocking the spread of MPXV or VACV in between two or more subjects in need thereof, the method comprising administering to at least one of the two or more subjects an antibody or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition disclosed herein.

[0172] Provided is method of treating or preventing an infection with MPXV or VACV in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof of disclosed herein or a pharmaceutical composition disclosed herein.

[0173] Provided is method of treating or preventing the spread of an infection with MPXV or VACV among different subjects in need thereof, the method comprising administering to the subjects an antibody or antigen-binding fragment thereof of disclosed herein or a pharmaceutical composition disclosed herein.

[0174] In embodiments, the antibodies and antigen-binding fragments disclosed herein may also be useful to treat or prevent infection of cells and subjects with cowpox virus or variola virus.38176172084.1

[0175] In some embodiments, the method further comprises administering to the subject a second therapeutic agent, e.g. , an anti-viral agent. In some embodiments, antibodies or antigenbinding fragments are used in the presence of complement.

[0176] By “subject” or “patient,” which are interchangeably used herein, is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, rodent (such as mouse, rat, rabbit) or feline, etc. Individuals are also subjects herein.

[0177] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to. alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (z.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total) or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0178] The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder, or slow its course of development.

[0179] In some embodiments, treatment with the antibodies or antigen-binding fragments disclosed herein prevents death of the subject and / or weight loss.

[0180] Provided herein are methods in which a therapeutically effective amount of an antibody or antigen-binding fragments thereof set forth herein is administered to a mammal in need thereof. Although antibodies or antigen-binding fragments thereof set forth herein are particularly useful for administration to humans, they may be administered to other mammals as well. The term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets and farm animals. “Therapeutically effective amount” means an amount of antibody or antigen-binding fragments thereof set forth herein that, when administered to a mammal, is effective in producing the desired therapeutic effect.39176172084.1

[0181] In some embodiments, the anti-MPXV A35 / VACV A33 antibodies or antigenbinding fragments thereof of the present disclosure are co-administered with one or more additional agents for the treatment of a disorder or disease.

[0182] Methods of Administration

[0183] Therapeutic compositions comprising the contemplated antibody or antigen-binding fragment thereof may be administered in any convenient manner, including by injection, or transfusion. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intracranially, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions disclosed herein are preferably administered by intravenous injection.

[0184] In some embodiments, the amount of antibody administered is in the range of about 0.001 mg / kg to about 1000 mg / kg of patient body weight, and any range in between. Depending on the type and severity of the infection, about 0. 1 mg / kg to about 50 mg / kg body weight (for example, about 0. 1-15 mg / kg / dose) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The anti-MPXV A35 / VACV A33 antibodies or antigen-binding fragments thereof can be delivered relatively low volume rates, for example but not necessarily from about 0.001 ml / day to 10 ml / day so as to minimize tissue disturbance or trauma near the site where the formulation is released. The formulation may be released at a rate of, depending on the specific biological agent(s), at a low dose, e.g. , from about 0.01 pg / hr or 0. 1 pg / hr, 0.25 pg / hr, 1 pg / hr, generally up to about 200 pg / hr, or the formulation is delivered at a low volume rate e.g., a volume rate of from about 0.001 ml / day to about 1 ml / day, for example, 0.01 micrograms per day up to about 20 milligrams per day. Dosage depends on a number of factors such as potency, bioavailability, and toxicity of the active ingredient used (e.g. the anti-MPXV A35 / V ACV A33 antibodies or antigen-binding fragments thereof) and the requirements of the subject. The progress of this therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0185] In certain embodiments, the antibody or antigen-binding fragment thereof is administered to the mammal by intravenous infusion, z.e., introduction of the antibody or40176172084.1antigen-binding fragment thereof into the vein of a mammal over a certain period of time. In certain embodiments, the period of time is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.

[0186] In certain embodiments, a dose of a compound or a composition is administered to a subject every day, every7other day, every couple of days, every7third day, once a week, twice a week, three times a week, once every two weeks, or once a month. In other embodiments, two, three or four doses of a compound or a composition is administered to a subject every' day. every7couple of days, every third day, once a week, once every two weeks or once a month. In some embodiments, a dose(s) of a compound or a composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days or 28 days. In certain embodiments, a dose of a compound or a composition is administered for 1 month, 1.5 months. 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.

[0187] It is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments disclosed herein. It is further to be understood that the disclosure includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments of the invention, and in the invention generally.

[0188] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).

[0189] All other referenced patents and applications, and scientific publications, including scientific articles and book chapters, are incorporated herein by reference in their entireties. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary' to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.41176172084.1

[0190] To facilitate a better understanding of the present invention, the following Examples of specific embodiments are given. The following Examples should not be read to limit or define the entire scope of the invention.EXAMPLES

[0191] Example 1: Materials and Methods for Examples 2-5

[0192] Participant and sample collection

[0193] The sample used in this study was from a single-site, observational study at Mount Sinai Hospital in New York (NY, USA). Peripheral blood mononuclear cells (PBMCs) were obtained from an MPXV convalescent participant 160 days after the onset of the symptoms.

[0194] Whole blood was collected through venipuncture and moved into serum separator and EDTA (edetic acid) tubes. Samples were coded before processing. PBMCs were isolated by density gradient centrifugation with SepMate tubes (STEMCELL Technologies, Cambridge, MA, USA). Serum and plasma samples were stored at -80 °C and PBMC samples were cryopreserved in liquid nitrogen until use. Protocols for clinical specimen collection from convalescent and post- vaccination individuals by the Personalized Virology Initiative were reviewed and approved by the Mount Sinai Hospital Institutional Review Board. The enrolled participant provided written informed consent before specimens and clinical information were collected. Permissions to store and share biospecimens were also obtained.

[0195] Tetramer construction, stainins., and sorting of human A35-speciftc B cells

[0196] Tetramers for the selection and sorting of A35-specific B cells are known in the art. Briefly, a biotinylated MPXV A35 protein (ACROBiosy stems, Newark, USA) was tetramerized with streptavidin-BV421 (Biolegend, catalog no. 405225) at a 2.2:1 (A35R:BV421) ratio for the selection of A35-specific cells. Cryopreserved PBMC samples were thawed in a water bath at 37 °C and resuspended in warmed Roswell Park Memorial Institute (RPMI) 1640 medium w / L-glutamine 25 mM 2-(4-(2-hy droxy ethyl)- 1-piperazinyl)- ethanesulfonic acid (HEPES) (Coming, catalog no. 10-041-CV) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio, catalog no.100-106) and 500 units of Benzonase Nuclease HC (MilliporeSigma, catalog no. 70664-3). Cells were then washed with chilled FACS buffer (phosphate-buffered saline (PBS) (Coming, catalog no. 21-040-CV); supplemented with 2% FBS) and resuspended in PBS for counting and viability assessment. Following counting and viability assessment, cells were stained with D-biotin to avoid cross-reactivity among probes, and the A35-BV421 tetramer plus the empty probe (streptavidin-PE-Cy-5.5) were diluted in Brilliant Buffer (BD Biosciences, catalog no. 566349) and incubated for 1 hour at 4 °C42176172084.1protected from light. After w ashing with FACS buffer, cells were stained with memory' B cell surface antibodies diluted in Brilliant Buffer for 30 minutes at 4 °C protected from light (Table 3).Table 3: Antibodies and fluorescent dyes used for flow cytometry and cell sorting (B-cell assay).

[0197] Following incubation, cells were washed with PBS, and dead cells were stained using LIVE / DEAD Fixable Blue Stain Kit (ThermoFisher, catalog no. L23105) diluted 1:100 in PBS for 30 minutes at 4°C protected from light. Subsequently, cells were washed with FACS buffer and resuspended for single-cell sorting into iPair Capture Plates (iRepertoire Inc.) on the Cytek AuroraCS. Analyses were performed using FlowJo V.10.9. The gating strategy used to sort A35-specific memory B cells is shown in Fig. 1.

[0198] Single-cell V(D) J sequencing of A35-specific human memory B cells

[0199] Following single-cell sorting, plates were shipped to iRepertoire Inc. (Huntsville, AL, USA) for BCR heavy and light chain amplification as described in Coelho et al., A human monoclonal antibody blocks malaria transmission and defines a highly conserved neutralizing epitope on gametes, Nat Commun. 2021 Mar 19; 12(1 ): 1750, incorporated herein by reference in its entirety. In summary, nested, multiplex primers including heavy, kappa, and lambda loci and partial Illumina adaptors were used to perform RT-PCR1. Following the first round of RT- PCR1, SPRISelect Beads (Beckman Coulter, Brea, USA) were used to rescue PCR1 products. A second PCR was performed using dual-indexed primers to provide plate positional information of the sequenced products and to complete the Illumina adaptors introduced during PCR1. After the second PCR. sequencing was performed using the MiSeq v2 500-cycle kit with 250 paired-end reads.176172084.1

[0200] A35 mAbs expression

[0201] Paired heavy chain and light chain sequences were cloned into the expression vectors, pTRIOZ-hlgGl (Invitrogen, ptrioz-higgl) for kappa light chains and pTRIOZ h!gGlL2 (Invitrogen, ptrioz-higgl 12) for lambda light chains, at Agel / Nhel and SgrAI / BsiWI (kappa) or Sgr Al / Avril (lambda) using an artificial mammalian leader sequence by GenScript. Plasmids were resuspended, transformed into competent E. coli, and plated overnight in Luria-Bertani (LB) agar plates containing the appropriate selective antibiotic (Zeocin, InvivoGen). Plasmidial DNA was extracted from E. coli using PureYield1MPlasmid Maxiprep System (Promega, A2393) or PureLink HiPure Plasmid DNA Purification (Thermo Fisher, K2100-02), followed by transfections of EXPI293 mammalian cells using the lipid-based Thermo Fisher Expi293™ Expression System Kit (Thermo Fisher, A14525) according to the manufacturer’s protocol. Briefly, plasmidial DNA, ExpiFectamine (transfection reagent), and the mammalian cell cultures (1 pg of DNA per mL of cell culture at 3 x 106) were incubated at 37 °C 8% CO2 for 18-22 hours on a platform shaker at 125 rpm. Enhancers 1 and 2 were then added and incubated on a platform shaker at 125 rpm at 37 °C 8% CO2 for an additional 5-6 days. Following incubation, cells were pelleted by centrifugation, and the culture cell media was filtered (0,45 mM) before mixing with protein A agarose resin (Gold Biotechnology, St. Louis, USA, P-400- 50) for 12 hours at 4 °C on a rotary shaker. On the next day, the entire volume was loaded into Poly -Prep chromatography columns (Bio-Rad Laboratories, Inc.. 7311550), and the IgG was eluted with glycine, IM Tris pH 8, and 5 M NaCl. Purified IgGs were pooled, concentrated using a 50 kDa protein concentrator, and stored at 4 °C short term or -80 °C long term.

[0202] Expression and purification of recombinant MPXV A35 and VAC A33 proteins

[0203] The extracellular segment of the MPXV A35 (Arg 58 - Thr 181, UniProtKB / Swiss- Prot: Q8V4U4) and VACV A33 (Vai 57 - Asn 185, UniProtKB / Swiss-Prot: P68616.1) EV proteins were expressed in mammalian human cells.

[0204] MPXV A35 sequence (UmProtKB / Swiss-Prot: Q8V4U4, SEQ ID NO:49):

[0205] MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSA FLIVRQNQCMSANEAAITDSAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSC YILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDY QDSDVSQEVRKYFCT

[0206] VACV A33 (UniProtKB / Swiss-Prot: P68616, SEQ ID NO:50)

[0207] MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSA FLIVRLNQCMSANEAAITDAAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSC44176172084.1YILHSDYQLFSDAKANCTAESSTLPNKSDVLITWLIDYVEDTWGSDGNPITKTTSDYQ DSDVSQEVRKYFCVKTMN

[0208] Briefly, gene fragments (Integrated DNA Technologies) containing the corresponding codon-optimized sequences were cloned into the pVRC vector (courtesy of the Vaccine Research Center, National Institutes of Health) and transformed into competent E. coll for expression. Transformed plasmids were selected based on kanamycin resistance and grown overnight to obtain plasmidial DNA that was later isolated and purified using a commercially available maxiprep kit (Thermo Fisher, Catalog# K210007). Plasmidial DNA was used to transfect Expi293F cells (Thermo Fisher, Catalog# Al 4527) at a concentration of Img per mL of cell culture. On the next day, transfected cell cultures were enhanced (Enhancers 1 and 2; Thermo Fisher, Catalog# A14527) and incubated at 37 °C 8% CO2 shaking at 125rpm for six days. After that, the cell culture was centrifuged at 4 °C, 3,000 x g to remove cell debris and the supernatant was filtered using a 0.45nm filter bottle. The recombinant proteins were purified using a Talon Metal Resin (Takara Bio, Catalog#635653).

[0209] Enzyme-linked immunosorbent assay (ELISA)

[0210] Recombinant proteins were coated on Coming 96-well microplates (Millipore Sigma) in 100 pL per well at a concentration of I pg / mL overnight at 4 °C. The following day, plates were washed three times using PBS containing 0.05% Tween-20 (PBST; Fisher Scientific) and blocked using PBS-Tween (PBST) supplemented with 3% non-fat milk (Life Technologies). Monoclonal antibodies were diluted to 1 pg / mL and then serially diluted at 2-fold seven times to a final concentration of 0.001 pg / mL (PBST- supplemented with 1 % non-fat milk; Life Technologies). Following blocking, mAb dilutions were added to the plates and incubated for 1 hour at room temperature. A total of 16 wells per plate were intentionally left blank to sen e as a measure of background. Plates were washed three times with PBST and lOOpL of goat anti-human IgG antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch) was diluted 1 :2000 in 1% non-fat milk PBST and added to each well. Following 1 hour of incubation at room temperature, plates were again washed three times with PBST and developed via the addition of lOOpL of 3.3',5,5'-tetramethylbenzidine substrate solution (TMB. Sigma- Aldrich). Plates were developed for 5-10 minutes at room temperature and then the reaction was stopped by adding 50pL 2M sulfuric acid solution (Sigma-Aldrich). Plates were read using a Synergy 4 (BioTek) plate reader at an optical density (OD) of 450 nanometers.

[0211] Bio-layer interferometry (BLI) to assess binding kinetics45176172084.1

[0212] Experiments were performed on BLItz system. For affinity assessment, Ni-NTA (Sartorius, #18-5101) biosensors were used to immobilize the antigens. Antigens were diluted in molecular biology grade water (Coming, #46-000-0) to a concentration of 100 pg / rnL, while IgG and Fabs were diluted in PBS IX and 10 mM Tris, pH 7.5,150 mM NaCl, respectively (2000 nM; 100 pg / mL). After immobilizing the antigen, each mAb was flown in for association and then dipped into a tube containing buffer alone to measure dissociation. The run steps were composed of an initial baseline (30 seconds), loading (90 seconds), baseline (30 seconds), association (90 seconds), and dissociation (90 seconds). Data was analyzed using the BLItz Pro™ software (version 1.3.1.3) (ForteBio) and affinity was assessed through KD values, obtained through the local fit of the curves by applying a 1 : 1 binding isotherm model using vendor-supplied software.

[0213] Epitope binning of anti-A35 mAbs using, BLI

[0214] Epitope binning was conducted in tandem format (BLItz system). Briefly, antigens were loaded and immobilized using Ni-NTA biosensors (Sartorius, catalog# 18-5101) and presented and saturated with the first mAb (100 pg / mL). Following, the sensor was presented to the second mAb, at the same concentration as the first. Binding to non-competing epitopes is suggested when saturation with the first antibody does not inhibit the binding of the second antibody, resulting in the formation of a second association peak. All measurements were repeated in subsequent independent experiments.

[0215] Cell lines and viruses

[0216] HeLa (ATCC®, catalog# CCL-2.2) and VERO E6 (ATCC®, catalog# CRL-1586) cells were cultured in DMEM supplemented with 1% sodium pyruvate and 10% FBS at 37 °C and 5% CO2; similarly, BS-C-1 cells (ATCC® CCL-26) were cultured in MEM supplemented with 10% FBS at 37 °C and 5% CO2. The cell lines were obtained from the ATCC® and tested negative for contamination with mycoplasma. VACV IHDJ (VACVIHDJ) was obtained from ATCC® (VR-156) and was expanded on VERO E6 cells. Similarly, MPXV (hMPXV / USA / MA001 / 2022; Lineage B.l, Clade lib) was obtained from BEI Resources (no. NR-58622) and expanded in BS-C-1 cells. The re-sequenced genomes were submitted to the National Center for Biotechnology Information (NCBL GenBank accession numbers: U94848 and ON563414, respectively). Cells were infected at a multiplicity’ of infection of 0.01 for 4 days to generate a working stock, and after incubation, the supernatant was clarified by centrifugation (450g for 5 min) and filtered through a 0.70pm filter. The pelleted virus was then resuspended in media respective to cell type and aliquoted for storage at -80 °C for46176172084.1predominantly mature virion (MV) stocks. For extracellular virion (EV) stocks, after cells infection, supernatant was harvested, clarified by centrifugation at (450 g for 10 min at 4°C), and the virus remaining in the supernatant was immediately tittered in the presence of MV neutralizing monoclonal antibody L1R (1=500). Clarified media was stored on ice at 4 °C and used within two weeks of isolation. Viral titers were measured by standard plaque assay using HeLa for VACV and BS-C-1 for MPXV. In brief, 300 pl of serial fold virus dilutions were used to infect HeLa or BS-C-1 cells in MEM supplemented with NaHCCh, 4% FBS and 0.6% Avicel RC-581. Plaques were resolved at 90 h post-infection by fixing in 10% formaldehyde for 1 h followed by 0.5% cry stal violet in 20% ethanol staining. Plates were rinsed in water to plaques enumeration. All in vitro and in vivo experiments with MPXV were performed in a biosafety level 3 laboratory.

[0217] Comet inhibition assay

[0218] BSC-1 cells were added in 12-well plates in minimum essential medium with Earl’s salts (EMEM) + 8 % FBS and checked for confluency in the following day (ideal confluency: around 80%). Following adsorption of ~30 PFU VACVIHDJ in a volume of 0.3mL into BS-C-1 monolayers for 1 hour at 37°C. After incubation, unbound virus was removed by washing with 0.5 mL of fresh medium. Cells were then overlaid with the mAbs diluted at 5 or 50 pg / rnL in a liquid medium (EMEM 2% FBS, 800pl / well) and incubated for an additional 36 - 40 hours. 5 or 50pg / mL of SARS2 and a 1 :50 dilution of anti-VACV polyclonal antibodies were used as negative and positive controls, respectively. Plates were fixed and stained with ethanol containing 0.02% crystal violet and assessed for inhibition of comet tail formation.

[0219] In vitro neutralization assays to determine complement-dependent activity) of mAbs

[0220] Six, threefold serially diluted mAbs, from 1:3 to 1:729, were incubated with VACV- VV.NP-S or MPXV- hMPXV / USA / MAOO 1 / 2022 (Lineage B L Clade lib) for 1 h at 37 °C. The mixture was subsequently incubated with HeLa (VACV) or BS-C-1 (MPXV) in a 12-well plate for Ih, for adsorption. Then, cells were overlayed with MEM supplemented with NaHCCh, 4% FBS, and 0.6% Avicel mixture. Plaques were resolved at 90 h post-infection byfixing in 10% formaldehyde for 1 h followed by staining in 0.5% crystal violet. All experiments were performed in parallel with sera from baseline controls, in an established viral concentration to generate 60-120 plaques per well. Direct neutralization experiments were conducted using frozen viral stocks containing predominantly MV particles, in the presence of 2% baby rabbit complement. Complement-mediated neutralization assays were performed using fresh viral particles stored on ice at 4 °C and used within two weeks of isolation to47176172084.1preserve both EV and MV particles (as described in the virus section above). The virus-mAbs mixtures were incubated in the presence of 7% baby rabbit complement. A range of complement concentrations (2%, 3%, 5%, 7%, 9%, and 10%) was tested to determine the optimal amount that did not block viral entry or interfere with the neutralization assay. Both direct and complement-mediated neutralization assays were always performed with the respective viral controls, using established viral concentrations to generate 60-120 plaques per well.

[0221] In vivo protection assays

[0222] BALB / c female mice (n = 20) were injected intraperitoneally with 150 pg of each mAb in 500 pl of PBS or with 500 pl of PBS alone as a control (5 per group). The next day. the mice were challenged intranasally with IxlO5PFU / mL of sucrose-gradient purified WRvFire (Towns ley & Moss, Two distinct low-pH steps promote entry of vaccinia virus. J Virol. 2007 Aug;81(16):8613-20, incorporated herein by reference in its entirety) in 20 pl of phosphate- buffered saline containing 0.05 BSA. For the imaging procedures, mice were anesthetized with isoflurane, and images were taken with the IVIS 200 series system on days 3, 6. 8, and 10 after the challenge. Mice were weighed daily for 10 days, and animals reaching 70% of their starting weight or 3 on the pain scale were sacrificed through CO2 euthanasia. The images were analyzed on Living Image Software with ROI being drawn around anatomic sites and light emission measured as photons / s / cm2 / sr for total photon flux comparisons. Weight and survival charts were produced using GraphPad Prism (V. 11)

[0223] Competition ELISA (cELISA) between EV35-2, EV35-6, EV35-7 and sera samples

[0224] Recombinant MPXV A35 and VACV A33 proteins were coated onto Coming 96-well microplates (Millipore Sigma, catalog# MSEHNFX40) by adding 100 pL per well at a concentration of 1 pg / mL and incubating overnight at 4 °C. The next day, the plates were washed three times with PBS containing 0.05% Tween-20 (PBST; Fisher Scientific) and then blocked with PBST supplemented with 3% non-fat milk (Life Technologies). Sera from participants vaccinated with either DRYVAX [REF] or JYNNEOS [REF], as well as sera from Mpox-convalescent individuals, were diluted at 1 :300 and incubated on the plates for 1 hour at 37 °C. Following, the biotinylated mAbs (EV35-2, EV35-6, and EV35-7). prepared using the EZ-Link NHS-PEG4-Biotin kit (Thermo Fisher, Catalog #90407, according to manufacturer’s instructions), w ere added at double the minimum binding concentration (previously determined by ELISA) and incubated for an additional hour at 37 °C._Following the incubation, the plates were washed with PBST, and 100 pL of Streptavidin-HRP (1 :4000 dilution) was added to each48176172084.1well. After a 1-hour incubation, the plates were washed again with PBST, developed with OPD substrate (o-phenylenediamine dihydrochloride), and the absorbance was measured at 490 nm using a multi-well plate reader (Synergy 4, BioTek). Competition between antibodies present in the sera and mAbs was calculated using the following formula: %inhibition = [(average absorbance (mAB only) - average absorbance (mAb + sera)] / (average absorbance absorbance (mAB only) x 100.

[0225] Example 2: Antibodies of the disclosure bind to MPXV A35 from clades I and II and to VACV A33 with high affinity

[0226] Eight VH and VL pairs of B cell receptor (BCR) sequences were obtained via amplification and sequencing of the V(D)J region from seventeen A35-specific single memory B cells (CD19+, CD20+, CD27+) sorted from a mpox convalescent individual (Fig. 2A, Table 4).

[0227] V gene usage analysis of the BCR sequences revealed five heavy-chain and seven lightchain unique genes, with IGHV3-30 (4 / 8, 50%) and IGKV3-20 (2 / 8, 25%) being the most VH and VL predominant genes, respectively (Fig. 2B). Most of the VL sequences used kappa chains (6 / 8, 75%).Table 4. Epidemiological and demographic characteristics of the MPXV-convalescent enrolled participant SPR-03.

[0228] The eight BCR pairs were expressed as IgGl .Three of the eight mAbs (EV35-2, EV35- 6, and EV35-7) bound to MPXV A35 clade lib, the strain that infected the convalescent person in this study and also to clade lb, a novel strain circulating in Africa. The mAbs were also cross-reactive to the vaccinia virus ortholog A33 (Figs. 2C-F). At the highest antigen49176172084.1concentrations (0.5 - Ipg / mL), a fourth mAh, EV35-8, bound to MPXV A35 Clade lb (Fig. 2E). While the minimum binding concentration was the same for EV35-6 and EV35-7 (0.001 pg / mL) across all proteins tested, EV35-2 bound to A33 at 0.0019pg / mL. OD values were two times higher for both MPXV A35 clade lib and VACV A33 when compared to MPXV A35 clade lb, which indicates differences within the binding site.

[0229] Potent therapeutic antibodies usually present intermediate to high affinity to the target antigen. EV35-2, EV35-6, and EV35-7 presented KD values in the nanomolar range, demonstrating high-affinity binding to both MPXV VACV A33 clade lb and lib (Figs. 2G-I). EV35-8 presented low affinity when tested against MPXV A35 Clade I (Fig. 2H), which explains binding activity only to high concentrations of the antigen (Fig. 2D).

[0230] Through an epitope binning analysis, it was demonstrated that EV35-2, EV35-6. EV35- 7, and EV35-8 bind to overlapping epitope regions of both MPXV A35 clades and VACV A33 (Figs. 3A-C), while presenting distinct combinations of heavy and light chain genes (Fig. 2B).

[0231] Example 3: Antibodies of the disclosure neutralize VACV in vitro

[0232] To assess the in vitro neutralizing capacity of EV35-2. EV35-6. and EV35-7 against orthopoxviruses, an anti-comet test was performed, measuring spread inhibition of released EVs (50 pg / mL) using the IHD-J VACV strain. While the EEVs of most VACV strains remain cell-associated, the IHD-J strain releases a higher percentage due to a single amino acid mutation in the A34 protein, a necessary feature to evaluate the in vitro neutralization capacity of anti-EEV mAbs.

[0233] To assess the viral particles’ infectivity7and spread, susceptible cells were incubated with the viruses only (no monoclonal antibodies added), and several comet-like plaque formations destroying the cell monolayer were observed (Fig. 4A). Comet formation was substantially reduced for EV35-2 (3 comets) (Fig. 4D). For EV35-6 and EV35-7, ten and eight comets were observed, respectively (Figs. 4E and 4F). Figs. 4B and 4C show controls.

[0234] To quantitively report the in vitro neutralization activity of the mAbs, a quantitative approach was developed, combining the measurement of the mean length and width of each comet. These metrics were considerably reduced for all wells containing the EEV mAbs when compared to controls (Fig. 4G).

[0235] The assay was performed with two concentrations (50 pg / mL (Fig. 4) and 5 pg / mL (Fig. 5). Even the lowest concentration (5 pg / mL), the antibodies blocked viral spread (Fig. 5). While the number of comets at the 5 pg / mL setting was higher when compared to 50 pg / mL50176172084.1(Figs. 5D, 5E, and 5F), no differences in length and width were observed (Fig. 5G). Figs. 5A, 5B, and 5C show controls.

[0236] Example 4: Antibodies of the disclosure confer full protection against VACV challenge

[0237] The protective efficacy of the identified antibodies was first tested through passive transfer followed by lethal challenge with WRvFire a VACV WR strain expressing luciferase).

[0238] BALB / c mice were inoculated with EV35-2, EV35-6, or EV35-7at 150 pg per mouse. On the next day, all groups were challenged intranasally with 1x105PFU of WRvFire, and following infection, mice were observed for weight loss and survival for ten days (Fig. 6A). Mice treated with the EEV mAbs lost 10 - 15% of their weight between 3 - 6 days postinfection, followed by a gradual weight recover}' up to 10 days post-infection, whereas the PBS-1X group kept losing weight until death (Fig. 6B). A survival rate of 20% was observed in the control group after nine days post-infection while 100% of the mAb-treated mice survived from infection (Fig. 6C).

[0239] To assess viral load and dissemination in vivo, bioluminescence to measure luciferase levels was recorded at three, six, eight-, and ten days post-infection (dpi). Time and gain settings were kept constant to prevent image saturation. All mice exhibited bioluminescence in the nasal area, the site of virus inoculation, but the intensity was lower in the passively immunized mice than in the PBS controls and was cleared between days 6 and 10 (Figs. 6D, 6E, 6F, and 6G). Furthermore, the spread of the infection to the chest was evident in the control mice (Fig. 6D) but was below detection in the immunized mice (Figs. 6E, 6F, and 6G). Photonflux measurements (a more sensitive and quantitative measure of luminescence) of head and body, the regions of interest, were determined separately, confirming low intensity in the head (day 3, p=0.0130; day 6, p=0.094; day 8, p=0.0123, Kruskal-Wallis) (Fig. 61) and negligible luminescence in the bodies of immunized mice (day 3, p=0.0079; day 6, p=0.0129; day 8, p=0.0044) (Fig. 6H) when compared to the PBS controls.

[0240] Example 5: Prevalence of antibodies targeting similar epitopes as EV35-2, EV35- 6, or EV35-7 is increased in infection compared to vaccinia immunization, but only levels of E V35-7-like antibodies in the sera correlate with neutralization against MPXV

[0241] To determine if the identified antibodies (which were produced by patients in response to natural MPXV infection) share paratope specificity with the EV35 mAbs, sera samples from51176172084.1(i) participants vaccinated with the smallpox first-generation vaccine (Dryvax, n=20), (ii) participants vaccinated with third generation and current vaccine strategy (Jynneos, n=20), (iii) participants with MPXV acute infection (n=20) and (iv) MPXV-convalescent participants were selected for further assays. Detailed epidemiological information on the samples is described in Table 5.Table 5. Epidemiological and Demographic characterization of Dryvax, Jynneos, MPXV- acute and MPXV-convalescent sera samples.

[0242] While among the groups, a similar number of individuals presented circulating antigenspecific antibodies (75%, 85%. 70% and 90% for Dry vax, Jynneos, MPXV-infected and MPXV-convalescent, respectively (p=>0.9999 for all comparisons; Kruskal -Wallis) (Fig. 7A), their neutralizing capacity' differed substantially between vaccinated vs. MPXV-acute and convalescent participants (Fig. 7B; Kruskal -Wallis, p=0.0089 for Dryvax vs. acute MPXV, p =0.0001 for Jynneos vs. acute MPXV and p=0.005 for Dryvax vs. convalescent; p=<0.0001 for Jynneos vs. convalescent). This indicates that infection by MPXV clade lib might elicit specific antibodies that target important neutralizing epitopes.

[0243] Next, a cELISA was developed to assess competition among the groups for binding to the MPXV antigen, A35 (Fig. 7C). Within the participants who received either vaccine, the percentage of competition ranged from 0 - 8%, whereas that for MPXV-acute and convalescent, it varied from 0 - 22% (Figs. 7D-F). As for the proportion of participants of each52176172084.1group presenting any competition, levels were significantly higher for the MPXV groups across all EV35 mAbs, with percentages ranging from 45 - 100%; while the vaccinated participants presented a frequency varying from 0 - 25%, or 0 - 15%, for Dryvax and Jynneos, respectively. An in-depth look into the MPXV groups revealed no differences in the percentage of competition nor the number of competing participants across the EV35 mAbs (Figs. 7G and 7J). However, the convalescent group had a higher number of participants exhibiting competition against all three mAbs (17 vs. 6) and their presence was consistent and showed strong correlations across all mAbs (data not shown). In contrast, the acute group exhibited a more restricted pattern, with a significant correlation observed only between participants competing with EV35-6 and EV35-7 (r = 0.728, Spearman). When correlating binding kinetics and functional performance, participants in the acute phase of MPXV infection presented no association (Fig. 7H and 71). Among convalescent individuals, while no correlation was observed with anti-A35 levels (Fig. 7K), a significant relationship was identified between neutralizing activity7and the level of paratope competition with EV35-7 (p = 0.0443, Spearman)(Fig. 7L)

[0244] Example 6: Anti-A35 mAbs exhibit enhanced in vitro neutralization in the presence of complement and interact with FcyRIIIa in the presence of orthopoxviruses

[0245] Complement-dependent cytotoxicity (CDC) mediates strong antibody inhibition of the cell-to-cell spread capacity of orthopox- viruses. To assess whether complement also enhances the in vitro neutralizing activity of anti-A35 mAbs disclosed herein, an MPXV plaque reduction assay was conducted using 50 pg / mL of each mAb in the presence or absence of 5% guinea pig serum.

[0246] While wells containing only virus also showed a reduction in the number of plaques in the presence of complement, plaque size remained unchanged, a key distinguishing feature observed only in the mAb-treated wells (Fig. 8A, Fig. SB, Fig. 8C, and Fig. 8D). In the presence of complement, EV35-2, EV35-6, and EV35-7 showed a visible reduction in the number of plaques and, more notably, a decrease in the size of viral foci compared with the control wells (Fig. 8B, Fig. 8C, and Fig. 8D). The total plaque formation area (in pixels2) decreased in the presence of complement (ranging from 51% to 56% reduction for all three mAbs), indicating increased activity7in the presence of complement (p = 0.036 for EV35-2 and p = 0.002 for both EV35-6 and-7; Mann- Whitney test) (Fig. 8E).53176172084.1

[0247] While complement enhanced the mAbs’ activity in vitro, a complete viral inhibition was not achieved in the presence of complement. Because of that, it was evaluated whether the mAbs could also elicit protection through other Fc-mediated functions, such as antibodydependent cell-mediated cytotoxicity (ADCC). To this end, the interaction between the mAbs’ Fc regions and FcyRIIIa was measured in vitro, using commercially available effector cells expressing human FcyRIIIa receptors and VACV-infected target cells.

[0248] All three mAbs demonstrated high potential to mediate protection through Fc- dependent effector functions (p = 0.0468 for EV35-2 and p = 0.0001 for both EV35-6 and EV35-7; unpaired t test) (Fig. 8F).

[0249] Taken together, these data demonstrate that the antibodies have the potential to function via direct neutralization (as shown in the comet assay) and Fc-mediated mechanisms (complement and / or ADCC).

[0250] In sum, a human anti-A35 repertoire of an MPXV-infected participant (clade lib), was screened at a single-cell level, to identify and characterize MPXV-elicited mAbs. The initial assessment revealed three clade lib MPXV-binding mAbs. These antibodies were confirmed to be cross-reactive to MPXV clade lb and their VACV ortholog, A33. Although not essential for viral replication, EEV proteins play an important role in assembling the EEV infective forms and facilitating the spread of viral particles within the host. Given that both species share a highly similar genomic central region (-93%), structural proteins such as A35 and A33 tend to share immunogenic epitopes.

[0251] Selected sequences can be found in Tables 6 and 7.Table 6. Selected amino acid sequences.54176172084.1Table 7. Selected nucleic acid sequences.55176172084.156176172084.157176172084.1176172084.1

Claims

CLAIMSWe claim:

1. An antibody or antigen-binding fragment thereof that binds to monkeypox virus (MPXV) A35 or to vaccinia virus (VACV) A33, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein each of the heavy chain and the light chain variable regions comprises a CDR1, CDR2, and CDR3, and wherein:(d) the sequence of CDR1H comprises SEQ ID NO: 1; the sequence of CDR2H comprises SEQ ID NO:2; the sequence of CDR3H comprises SEQ ID NO:3; the sequence of CDR1L comprises SEQ ID NO:4; the sequence of CDR2L comprises the sequence GAS; and the sequence of CDR3L comprises SEQ ID NO:6;(e) the sequence of CDR1H comprises SEQ ID NO:7; the sequence of CDR2H comprises SEQ ID NO:8; the sequence of CDR3H comprises SEQ ID NO:9; the sequence of CDR1L comprises SEQ ID NO: 10; the sequence of CDR2L comprises the sequence DAS; and the sequence of CDR3L comprises SEQ ID NO: 12; or(f) the sequence of CDR1H comprises SEQ ID NO: 13; the sequence of CDR2H comprises SEQ ID NO: 14; the sequence of CDR3H comprises SEQ ID NO: 15; the sequence of CDR1L comprises SEQ ID NO: 16; the sequence of CDR2L comprises the sequence KAS; and the sequence of CDR3L comprises SEQ ID NO: 18.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein:(a) the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO: 19 and the sequence of the light chain variable region comprises a sequence that is least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:20;(b) the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:21 and the sequence of the light chain variable region comprises a sequence that is least at least 80%, at least 85%. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:22; or59176172084.1(c) the sequence of the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:23 and the sequence of the light chain variable region comprises a sequence that is least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:24.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein:(a) the sequence of the heavy chain variable region comprises SEQ ID NO: 19 and the sequence of the light chain variable region comprises SEQ ID NO:20;(b) the sequence of the heavy chain variable region comprises SEQ ID NO:21 and the sequence of the light chain variable region comprises SEQ ID NO:22; or(c) the sequence of the heavy chain variable region comprises SEQ ID NO:23 and the sequence of the light chain variable region comprises SEQ ID NO:24.

4. The antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

5. The antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody or antigen-binding fragment thereof is a multispecific or a bispecific antibody or antigen-binding fragment thereof.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is an scFv, Fv. Fab’. Fab, F(ab’)2, or diabody.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof has isotype IgGl.

8. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more of a cytotoxin, an antiviral agent, a fluorescent label, and an imaging agent.60176172084.

19. An isolated nucleic acid or pair of nucleic acids encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-7.

10. An isolated nucleic acid or pair of nucleic acids encoding an antibody or antigen-binding fragment thereof that binds to MPXV A35 or to VACV A33, the antibody or antigenbinding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:(a) the sequence encoding the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:43 and the sequence encoding the light chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:44;(b) the sequence encoding the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:45 and the sequence encoding the light chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:46; or(c) the sequence encoding the heavy chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:47 and the sequence encoding the light chain variable region comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:48.1 1. The isolated nucleic acid or pair of nucleic acids of claim 10, the antibody or antigenbinding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:(d) the sequence encoding the heavy chain variable region comprises SEQ ID NO:43 and the sequence encoding the light chain variable region comprises SEQ ID NO:44;(e) the sequence encoding the heavy chain variable region comprises SEQ ID NO:45 and the sequence encoding the light chain variable region comprises SEQ ID NO:46; or61176172084.1(f) the sequence encoding the heavy chain variable region comprises SEQ ID NO:47 and the sequence encoding the light chain variable region comprises SEQ ID NO:48.

12. A vector comprising the nucleic acid or pair of nucleic acids of claim 10 or 11.

13. A cell comprising the vector of claim 12 or the nucleic acid or pair of nucleic acids of claim lO or 11.

14. The cell of claim 13, wherein the cell is a bacterial cell, a yeast cell, or an isolated mammalian cell.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-8 and a pharmaceutically acceptable carrier or excipient.

16. An assay device comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7.

17. A kit for detecting the presence of MPXV A35 or VACV A33, or an antigenic fragment of MPXV A35 or VACV A33. in a sample comprising: (i) the antibody or antigen-binding fragment thereof of any one of claims 1-7, and (ii) a buffer.

18. The kit of claim 17, wherein the antibody or antigen-binding fragment thereof is bound to a substrate.

19. The kit of claim 17 or 18, wherein the antibody or antigen-binding fragment thereof is detectably labeled.

20. The kit of any one of claims 17-19. the kit further comprising a secondary antibody that specifically binds to the antibody or antigen-binding fragment thereof.

21. The kit of claim 20, wherein the secondary antibody is detectably labeled.62176172084.

122. A method of making an antibody or antigen-binding fragment thereof that binds to MPXV A35 or to VACV A33. the method comprising:(i) providing a cell comprising one or more nucleic acid molecules encoding the antibody or antigen-binding fragment thereof of any one of claims 1-7;(ii) expressing in the cell the antibody or antigen-binding fragment thereof; and(iii) collecting the antibody or antigen-binding fragment thereof.

23. A method of reducing or blocking infection of a cell with MPXV or VACV, the method comprising contacting the MPXV or VACV with the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the pharmaceutical composition of claim 15.

24. The method of claim 23, wherein the cell is a macrophage, dendritic cell, natural killer (NK) cell, Langerhans cell, neutrophil, epithelial cell, keratinocyte, fibroblast, or endothelial cell.

25. The method of claim 23 or 24, wherein the cell is a human cell.

26. A method of reducing or blocking transmission of MPXV or VACV from a first cell to a second cell, the method comprising contacting the MPXV or VACV with the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the pharmaceutical composition of claim 15.

27. The method of claim 26, wherein the first and / or the second cell is a macrophage, dendritic cell, natural killer (NK) cell, Langerhans cell, neutrophil, epithelial cell, keratinocyte, fibroblast, or endothelial cell.

28. The method of claim 26 or 27, wherein the first and / or the second cell is a human cell.

29. A method of neutralizing MPXV or VACV, the method comprising contacting the MPXV or VACV with the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the pharmaceutical composition of claim 15, optionally, wherein the method is performed in the presence of presence of complement.63176172084.

130. A method of blocking the spread of MPXV or VACV in a subject in need thereof, the method comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the pharmaceutical composition of claim 15.

31. A method of treating or preventing an infection with MPXV or VACV in a subject in need thereof, the method comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the pharmaceutical composition of claim 1 .

32. The method of claim 30 or 31, the method further comprising administering to the subject a second therapeutic agent.

33. The method of any one of claim 30-32, wherein the subject is human.64176172084.1

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