Treatment of herpes simplex virus infection

Abstract

The present invention relates to the treatment of herpes simplex virus (HSV) infection using anti-HSV antibodies. Specifically, the anti-HSV antibody specifically binds to glycoprotein D (gD) of herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2). The treatment of the invention is effective against drug resistant and / or recurrent HSV infection.

Description

[0001] Related Applications

[0002] This application claims the contents of U.S. Provisional Patent Application No. 63 / 403,408 filed on September 2, 2022, the contents of which are hereby incorporated by reference in their entirety. Technical Field

[0003] The present invention relates to the treatment of herpes simplex virus (HSV) infection using anti-HSV antibodies. Specifically, the anti-HSV antibodies specifically bind to glycoprotein D (gD) of herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2). The treatment of the present invention is effective against drug-resistant and / or recurrent HSV infections. Background Art

[0004] Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) are members of the herpes virus family and are highly prevalent pathogens worldwide, causing recurrent oral and genital ulcers. Generally speaking, HSV infection does not cause serious symptoms and complications, but it can cause severe symptoms in some cases of infection in immunocompromised patients and newborns. Although the symptoms of ordinary patients are not too severe, many patients may still relapse. HSV recurrences may plague these patients for many years and reduce their quality of life.

[0005] Although there are several licensed antiviral agents for the treatment of primary and recurrent HSV infections, these antiviral agents are not always effective and may have significant side effects. In addition, drug-resistant HSV strains (e.g., acyclovir-resistant) significantly reduce the efficacy of these traditional antiviral agents.

[0006] Patients with high recurrence rates or those infected by drug-resistant strains lack good alternative treatment options. Better therapies are needed to treat recurrent HSV infections, especially those with drug-resistant HSV infections. Therefore, there is still an urgent need for novel interventional therapies for effective prevention and treatment purposes. Summary of the invention

[0007] The present invention is based on the discovery that anti-HSV antibodies can effectively target drug-resistant HSV strains and reduce the recurrence of HSV infection. Specifically, anti-HSV antibodies specifically bind to glycoprotein D (gD) of herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2). Anti-HSV antibodies can neutralize drug-resistant HSV strains and / or inhibit viral transmission. Treatment with anti-HSV antibodies can effectively alleviate symptoms caused by drug-resistant HSV infection and reduce or delay the recurrence of HSV infection.

[0008] Thus, in one aspect, the present invention provides a method for treating drug-resistant and / or recurrent herpes simplex virus (HSV) infection, comprising administering to a subject in need thereof an anti-HSV antibody, wherein the anti-HSV antibody specifically binds to glycoprotein D (gD) of herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2).

[0009] In some embodiments, the anti-HSV antibodies neutralize drug-resistant HSV strains.

[0010] In some embodiments, the anti-HSV antibodies inhibit viral transmission.

[0011] In some embodiments, the anti-HSV antibody comprises

[0012] (a) a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 6; and

[0013] (b) a light chain variable region (VL), comprising a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO:9, a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO:11, and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO:13.

[0014] In some embodiments, the anti-HSV antibody comprises

[0015] The VH comprises the amino acid sequence of SEQ ID NO: 15; and / or

[0016] The VL comprises the amino acid sequence of SEQ ID NO:16.

[0017] In some embodiments, the anti-HSV antibody comprises

[0018] (a) a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 20, and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 22; and

[0019] (b) a light chain variable region (VL), comprising a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO:24, a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO:26, and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO:28.

[0020] In some embodiments, the anti-HSV antibody comprises

[0021] The VH comprises the amino acid sequence of SEQ ID NO: 30; and / or

[0022] The VL comprises the amino acid sequence of SEQ ID NO:31.

[0023] In some embodiments, the anti-HSV antibody comprises

[0024] (a) a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 33, and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 6; and

[0025] (b) a light chain variable region (VL), comprising a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO:35, a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO:11, and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO:38.

[0026] In some embodiments, the anti-HSV antibody comprises

[0027] The VH comprises the amino acid sequence of SEQ ID NO: 40; and / or

[0028] The VL comprises the amino acid sequence of SEQ ID NO:41.

[0029] In some embodiments, the antibody is an antigen-binding fragment thereof.

[0030] In some embodiments, the antibody is humanized.

[0031] In some embodiments, the individual is susceptible to or already infected with a drug-resistant strain of HSV.

[0032] In some embodiments, the subject is an immunocompromised patient.

[0033] In some embodiments, the anti-HSV antibody is administered to a subject as a single dose.

[0034] In some embodiments, the antibody is administered in an amount effective to reduce symptoms caused by HSV infection.

[0035] In some embodiments, the antibody is administered in an amount effective to delay the incidence of relapse and / or reduce the frequency of relapse.

[0036] In some embodiments, anti-HSV antibodies are administered to a subject after symptoms develop.

[0037] The present invention also provides an anti-HSV antibody as described herein or a composition comprising the anti-HSV antibody for use in treating drug-resistant and / or recurrent HSV infection in an individual in need thereof. Further disclosed is the use of an anti-HSV antibody as described herein in the manufacture of a medicament for treating drug-resistant and / or recurrent HSV infection in an individual in need thereof.

[0038] The details of one or more specific embodiments of the present invention are described in the following description. Other features or advantages of the present invention will be apparent from the following detailed description of several specific embodiments and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The above summary of the invention and the following embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the presently preferred specific embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the precise arrangements and means shown.

[0040] In the following figure:

[0041] Figure 1 The binding affinity of UB-621 to recombinant gD was shown as determined by surface plasmon resonance (SPR) analysis.

[0042] Figure 2 The results show that UB-621 inhibits the efficacy of laboratory HSV-1 and HSV-2 strains on Vero cells, IC 50 Value and IC 90 value.

[0043] Figure 3A and 3B A single SC dose of UB-621 was shown to have an inhibitory effect on genital symptoms in mice with HSV-2 vaginal infection ( Figure 3A ) and mortality rate( Figure 3B )’s therapeutic effect.

[0044] Figure 4A and4B The efficacy of UB-621 was demonstrated when mice were given UB-621 after the onset of infection symptoms. Figure 4A As shown, and the survival rate (mortality rate) is as Figure 4B shown.

[0045] Figure 5A and 5B UB-621 and acyclovir (ACV) showed that ACV-resistant artificial HSV mutant virus strains, HSV-1RE TKnull ( Figure 5A ) and HSV-2 333TKnull( Figure 5B ) inhibitory efficacy, IC 50 The results showed that UB-621 was more effective against infection by ACV-resistant HSV-1RE TKnull (A) and HSV-2 333TKnull (B) mutant virus strains.

[0046] Figures 6A-6C Shows the inhibitory efficacy, IC 50 Value and IC 90 The isolates included HSV-1 and HSV-2 from Asia. Fig. 6A ), HSV-2 from Asian origin ( Figure 6B ), and ACV-resistant HSV-2 ( Figure 6C ). The results showed that UB-621 was more effective than ACV in inhibiting clinically derived HSV isolates of both American and Asian origin as well as ACV resistance.

[0047] Figure 7 The susceptibility of clinical HSV isolates resistant to ACV (Acyclovir), PFA (Foscarnet), or CDV (Cidofovir) to inhibition by UB-621 was shown.

[0048] Figure 8 The therapeutic efficacy of a single dose of UB-621 is shown against infection with an ACV-resistant HSV-1 clinical isolate in a murine model.

[0049] Fig. 9A and 9B The effect of UB-621 on the survival rate after infection with the HSV-2IC clinical isolate virus strain is shown. Fig. 9AA single dose of UB-621 increases survival against infection with the ACV-resistant HSV-2IC strain. The effect of UB-621 on the severity of vaginitis caused by infection with the HSV-2IC clinical isolate is shown in Fig. 9B As shown. A single dose of UB-621 reduces clinical vaginitis in response to infection with the ACV-resistant HSV-2 IC strain. * Statistical significance, p value < 0.05; ** p value < 0.01.

[0050] Fig. 10A and 10B The therapeutic effect of UB-621 against infection with an ACV-resistant HSV-2 clinical isolate in genitally infected mice is shown. The effect of UB-621 on survival after infection with an HSV-2 clinical isolate pol-mut is shown. Fig. 10A A single dose of UB-621 increases survival against infection with ACV-resistant HSV-2 pol-mut strains. The effect of UB-621 on the severity of vaginitis caused by infection with HSV-2 pol-mut clinical isolates is shown in Fig. 10B As shown. A single dose of UB-621 reduces clinical vaginitis in response to infection with an ACV-resistant HSV-2 pol-mut strain. *Statistically significant, p value < 0.05.

[0051] Fig.11A and 11B UB-621 showed the effect of HSV-1( Fig.11A ) and HSV-2( Fig. 11B ) of the cell-to-cell spread.

[0052] Fig.12 Shows the inhibitory effect of UB-621 on anterograde interneuronal spread of HSV-1 in BALB / c mice. **Statistical significance, p value < 0.01.

[0053] Fig.13A and 13B The results showed that UB-621 has a significant effect on primary HSV-2 infection in guinea pigs. The effect of UB-621 on the severity of primary infection vaginitis is as follows Fig.13A UB-621 administration can alleviate the symptoms of vaginitis in guinea pigs infected with genital HSV-2. The effect of UB-621 on the cumulative number of relapses is shown in Fig. 13B UB-621 administration delayed the incidence and reduced the frequency of relapses in genital HSV-2 infected guinea pigs.

[0054] Fig.14A and 14BThe results showed that UB-621 has a significant effect on recurrent HSV-2 infection in guinea pigs. The effect of UB-621 on the severity of vaginitis after the first HSV recurrence is as follows Fig.14A UB-621 administration after the first HSV relapse can reduce the symptoms of vaginitis in guinea pigs infected with genital HSV-2. The effect of UB-621 on the cumulative number of relapses is shown in Fig. 14B UB-621 administration after a first HSV relapse reduces the frequency of relapses in genital HSV-2 infected guinea pigs. DETAILED DESCRIPTION

[0055] The following description is intended only to illustrate various specific embodiments of the present invention. Therefore, the specific specific embodiments or modifications discussed herein should not be interpreted as limiting the scope of the present invention. It is obvious to those skilled in the art that various changes or equivalents can be made without departing from the scope of the present invention.

[0056] In order to provide a clear and understandable understanding of the present invention, specific terms are first defined. Additional definitions are set forth throughout the embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0057] I. Definitions

[0058] In order to provide a clear and understandable understanding of the present invention, specific terms are first defined. Additional definitions are set forth throughout the embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0059] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a component" includes a plurality of such components and equivalents thereof known to those skilled in the art.

[0060] The term "comprising" or "including" is generally used in the sense of including / comprising, which means that one or more features, ingredients or components are allowed to be present. The term "comprising" or "including" encompasses the term "consisting of" or "consisting of".

[0061] As used herein, "herpesvirus" means a member of the herpetoviridae family, including herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). The present invention is particularly useful for alleviating symptoms caused by HSV infection. HSV can infect many parts of the body, including the mouth, genital area, eyes, skin and brain. Generally speaking, HSV-1 mainly infects the mouth, and HSV-2 mainly infects the genital area. Normally, HSV is transmitted to uninfected individuals by direct contact with the infected area of ​​the infected person. The clinical symptoms associated with HSV infection are well known in the art. HSV infection may be asymptomatic or symptomatic, which may include painful blisters or ulcers, and may recur over time. HSV recurrence can be caused by several factors, such as stress, immunodeficiency, sunburn and injury. HSV infection can be fatal, especially for immunodeficient individuals. Typical HSV infectious diseases include herpetic gingivostomatitis, herpes labialis, herpetic eye infection (herpes keratitis), genital herpes, herpetic whitlow, herpes gladiatorum, herpesviral encephalitis, herpesviral meningitis, and herpes esophagitis. The initial symptoms of orolabial herpes include painful vesicular eruptions with distinct blisters on the lips, tongue, or buccal mucosa. The blisters may coalesce and rupture to form shallow ulcers covered with necrotic material. The typical clinical symptoms of genital herpes include small clusters of blisters on an erythematous base. Blisters may slough spontaneously or form ulcers through direct abrasion. Ulcers typically form a crust and then undergo re-epithelialization.

[0062] See, for example, US Publication Nos. US20020147210A1 and US6599945B2, the contents of which are hereby incorporated by reference in their entirety.

[0063] As used herein, the term "polypeptide" means a polymer composed of amino acid residues linked by peptide bonds. The term "protein" generally refers to a relatively large polypeptide. The term "peptide" generally refers to a relatively short polypeptide (e.g., containing up to 100, 90, 70, 50, 30, 20 or 10 amino acid residues).

[0064] As used herein, the term "approximately" or "about" means a degree of acceptable deviation that will be understood by those of ordinary skill in the art, which may vary to some extent depending on the context in which it is used. Specifically, "approximately" or "about" may refer to a numerical value having a range of ±10% or ±5% or ±3% around the cited value.

[0065] As used herein, the term "substantially identical" means that two sequences have 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more homology.

[0066] As used herein, the term "antibody" (used interchangeably with the plural form of antibodies) means an immunoglobulin molecule that has the ability to specifically bind to a specific target antigen molecule. As used herein, the term "antibody" includes not only complete (i.e., full-length) antibody molecules, but also antigen-binding fragments (e.g., Fab, Fab', F(ab')2, and Fv) that retain antigen-binding ability. Such fragments are well known in the art and are often used in vitro and in vivo. The term "antibody" also includes chimeric antibodies, humanized antibodies, human antibodies, bivalent antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules (including antigen recognition sites of desired specificity), including antibody amino acid sequence variants, antibody glycosylation variants, and covalently modified antibodies.

[0067] A complete or full antibody comprises two heavy chains and two light chains. Each heavy chain comprises a variable region (V H ) and the first, second and third constant regions (C H 1. C H 2 and C H 3); and each light chain contains a variable region (V L ) and constant region (C L). Antibodies are in the shape of a "Y", where the backbone of the Y consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y includes the variable region and the first constant region of a single heavy chain, which is bound to the variable region and constant region of a single light chain. The variable regions of the light chain and the heavy chains are responsible for antigen binding. The variable regions of the two chains are usually responsible for antigen binding, each of which contains three highly variable regions, called complementary determining regions (CDRs); that is, the heavy (H) chain CDRs, including HC CDR1, HC CDR2, HC CDR3, and the light (L) chain CDRs, including LC CDR1, LC CDR2 and LC CDR3. The three CDRs are separated by framework regions (FR1, FR2, FR3 and FR4), which are more highly conserved than the CDRs and form a skeleton that supports the highly variable regions. The constant regions of the heavy and light chains are not responsible for antigen binding, but are involved in various effector functions. Immunoglobulins can be divided into different classes according to the antibody amino acid sequence of the constant domain of its heavy chain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0068] As used herein, the term "antigen binding fragment" or "antigen binding domain" means a portion or region of an intact antibody molecule that is responsible for antigen binding. An antigen binding fragment can bind to the same antigen as the parent antibody. Examples of antigen binding fragments include, but are not limited to: (i) a Fab fragment, which can be composed of V H -C H -1 chain with V L -C L (ii) F(ab′)2 fragment, which can be a bivalent fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fv fragment, which is composed of the V and V of antibody molecules bound together by non-covalent interactions. H With V L (iv) single-chain Fv (scFv), which can be composed of V H Domain and V L The domains are connected via a peptide linker to form a single polypeptide chain; and (v) (scFv) 2 , which may contain two V H Domain with two V L domain, the two V L The domain is linked to two V H The domains are connected.

[0069] As used herein, the term "chimeric antibody" refers to an antibody containing polypeptides from different sources (e.g., different species). In some embodiments, in a chimeric antibody, the variable regions of both the light chain and the heavy chain may mimic the variable regions of an antibody derived from a mammalian species (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant region may be homologous to an antibody sequence derived from another mammal (e.g., human).

[0070] As used herein, the term "humanized antibody" refers to an antibody comprising a framework region derived from a human antibody and one or more CDRs from a non-human (usually mouse or rat) immunoglobulin.

[0071] As used herein, the term "human antibody" means an antibody in which substantially the entire sequence of the light and heavy chain sequences, including the complementarity determining regions (CDRs), are derived from human genes. In some cases, a human antibody may include one or more amino acid residues not encoded by human germline immunoglobulin sequences, for example, by mutation of one or more of the CDRs, or mutation of one or more of the FRs, for example, to reduce possible immunogenicity, increase affinity, and remove cysteine ​​that may cause undesirable folding.

[0072] As used herein, the term "specific binding" or "specifically binds" means a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope of its target antigen. An antibody that "specifically binds" to a target antigen or epitope is a term familiar in the art, and methods for determining such specific binding are also familiar in the art. An antibody is said to "specifically bind" to a target antigen if it binds to it with greater affinity / affinity, more easily and / or for a longer duration than it binds to other substances. In other words, it can also be understood by reading this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, the affinity of a binding can be expressed in terms of a dissociation constant (K D ) definition. Generally, when applied to antibodies, specifically binding can refer to binding with less than about 10 -8 M, for example about 10 -9 M or smaller, about 10 -10 M or smaller, about 10 -11 M or smaller, about 10 -12 M or smaller, or even smaller K D The present invention relates to an antibody that specifically binds to (recognizes) its target with a K value corresponding to an affinity that is at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower or at least 10,000 times lower than binding to a non-specific antigen (e.g., BSA or casein). DBinds to a specific target.

[0073] As used herein, the term "nucleic acid" or "polynucleotide" may refer to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs, including those with non-naturally occurring nucleotides. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. It will be understood that when a nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences (i.e., A, U, G, C), wherein "U" replaces "T". The term "cDNA" means a DNA that is complementary or identical to an mRNA, whether in single-stranded or double-stranded form.

[0074] As used herein, the term "complementary" means the topological compatibility or matching together of the interaction surfaces of two polynucleotides. A first polynucleotide is complementary to a second polynucleotide when the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Thus, a polynucleotide with a sequence of 5'-ATATC-3' is complementary to a polynucleotide with a sequence of 5'-GATAT-3'.

[0075] As used herein, the term "encoding" refers to the natural property of a particular nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) that can be used as a template for the synthesis of other polymers and macromolecules in a biological process having a given RNA transcript (i.e., rRNA, tRNA, and mRNA) sequence or a given amino acid sequence, and the biological properties resulting therefrom. Thus, if transcription and translation of the mRNA produced by the gene produces the protein in a cell or other biological system, the gene encodes the protein. It will be appreciated by those skilled in the art that, due to the degenerate nature of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. It will also be appreciated that those skilled in the art can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed. Thus, unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" encompasses all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.

[0076] As used herein, the term "recombinant nucleic acid" means a polynucleotide or nucleic acid having sequences that are not naturally linked together. Recombinant nucleic acids can exist in the form of vectors. A "vector" can contain a given nucleotide sequence of interest and regulatory sequences. A vector can be used to express a given nucleotide sequence (expression vector) or to maintain a given nucleotide sequence to replicate it, manipulate it, or transfer it between different locations (e.g., between different organisms). The vector can be introduced into an applicable host cell for the above purposes. "Recombinant cell" means a host cell into which a recombinant nucleic acid has been introduced. "Transformed cell" means a cell into which a DNA molecule encoding a protein of interest has been introduced through recombinant DNA technology.

[0077] The vector may be of various types, including plasmids, cosmids, episomes, F-type cohesin plasmids, artificial chromosomes, phages, viral vectors, and the like. Typically, in a vector, a given nucleotide sequence is operably linked to a regulatory sequence so that when the vector is introduced into a host cell, the given nucleotide sequence can be expressed in the host cell under the control of the regulatory sequence. The regulatory sequence may include, for example but not limited to, a promoter sequence (e.g., a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a T7 promoter, and an alcohol oxidase gene (AOX1) promoter), a start codon, an origin of replication, an enhancer, a secretion signal sequence (e.g., an α mating factor signal), a stop codon, and other control sequences (e.g., a Shine-Dalgarno sequence and a stop sequence). Preferably, the vector may further contain a marker sequence (e.g., an antibiotic resistance marker sequence) for subsequent screening / selection procedures. For the purpose of protein production, in a vector, a given nucleotide sequence of interest may be linked to another nucleotide sequence other than the above-mentioned regulatory sequence, so that a fused polypeptide is produced and is conducive to subsequent purification procedures. The fusion polypeptide includes a tag (eg, a His tag) for purification purposes.

[0078] As used herein, the term "treat," ...

[0079] II. Antibodies against HSV

[0080] According to the present invention, as used herein, anti-HSV antibodies specifically bind to gD of HSV-1 and HSV-2 and are capable of neutralizing drug-resistant HSV strains. When the antibody binds to the virus, virus neutralization typically occurs, thereby preventing infection of susceptible cells. It has been found that the anti-HSV antibodies of the present invention exhibit high efficacy in neutralizing drug-resistant HSV strains. Neutralization efficacy can be measured by methods known in the art. For example, in a viral plaque assay, the anti-HSV antibodies of the present invention may have an IC50 value of less than 100nM, for example, about 1nM to about 99nM, such as about 1nM to about 80nM, about 1nM to about 70nM, about 1nM to about 60nM, about 1nM to about 50nM, about 1nM to about 40nM, about 1nM to about 30nM, about 1nM to about 20nM, or about 1nM to about 10nM.

[0081] It has also been found that, as used herein, anti-HSV antibodies inhibit viral spread, including cell-to-cell and / or neuronal spread.

[0082] Exemplary anti-HSV antibodies include monoclonal antibodies (mAbs) E317 (ie, UB-621 mAb described in the Examples below), E425, and Y571 as described in U.S. Pat. No. 8,252,906, the contents of which are hereby incorporated by reference in their entirety.

[0083] According to U.S. Pat. No. 8,252,906, mAbs E317, E425, and Y571 each comprise a heavy chain variable region (V H ) (HC CDR1, HC CDR2 and HC CDR3) and a light chain variable region (V L )(LC CDR1, LC CDR2 and LC CDR3), as described in Table 1.

[0084] Table 1. Amino acid sequences of mAbs E317, E425 and Y571.

[0085]

[0086]

[0087]

[0088] In some specific embodiments, the anti-HSV antibody of the present invention is a functional variant of mAb E317, characterized by comprising: (a) VH, which comprises HC CDR1 of SEQ ID NO: 2, HC CDR2 of SEQ ID NO: 4, and HC CDR3 of SEQ ID NO: 6; and (b) V L, which comprises LC CDR1 of SEQ ID NO:9, LC CDR2 of SEQ ID NO:11, and HC CDR3 of SEQ ID NO:13, or an antigen-binding fragment thereof.

[0089] In some specific embodiments, the anti-HSV antibodies of the present invention have: (a) a VH comprising a HC CDR1 of SEQ ID NO: 2, a HC CDR2 of SEQ ID NO: 4, and a HC CDR3 of SEQ ID NO: 6; and (b) a V L , which comprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2 of SEQ ID NO: 11, and the HC CDR3 of SEQ ID NO: 13, and may comprise V H , which comprises SEQ ID NO: 15 or an amino acid sequence substantially identical thereto, and V L , which comprises SEQ ID NO: 16 or an amino acid sequence substantially identical thereto. Specifically, the anti-HSV antibody of the present invention comprises V H , which comprises an amino acid sequence that is at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identical to SEQ ID NO:15, and V L , which comprises an amino acid sequence that is at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identical to SEQ ID NO:16.

[0090] In some specific embodiments, the anti-HSV antibody of the present invention is a functional variant of mAb E425, characterized by comprising: (a) VH, which comprises HC CDR1 of SEQ ID NO: 18, HC CDR2 of SEQ ID NO: 20, and HC CDR3 of SEQ ID NO: 22; and (b) V L , which comprises LC CDR1 of SEQ ID NO:24, LC CDR2 of SEQ ID NO:26, and HC CDR3 of SEQ ID NO:28, or an antigen-binding fragment thereof.

[0091] In some specific embodiments, the anti-HSV antibodies of the present invention have: (a) VH comprising HC CDR1 of SEQ ID NO: 18, HC CDR2 of SEQ ID NO: 20, and HC CDR3 of SEQ ID NO: 22; and (b) V L, which comprises LC CDR1 of SEQ ID NO: 24, LC CDR2 of SEQ ID NO: 26, and HC CDR3 of SEQ ID NO: 28, and may comprise V H , which comprises SEQ ID NO: 30 or an amino acid sequence substantially identical thereto, and V L , which comprises SEQ ID NO: 31 or an amino acid sequence substantially identical thereto. Specifically, the anti-HSV antibodies of the present invention include V H , which comprises an amino acid sequence that is at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identical to SEQ ID NO:30, and V L , which comprises an amino acid sequence that is at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identical to SEQ ID NO:31.

[0092] In some specific embodiments, the anti-HSV antibody of the present invention is a functional variant of mAb Y571, characterized by comprising: (a) VH, which comprises HC CDR1 of SEQ ID NO: 2, HC CDR2 of SEQ ID NO: 33, and HC CDR3 of SEQ ID NO: 6; and (b) V L , which comprises LC CDR1 of SEQ ID NO:35, LC CDR2 of SEQ ID NO:11, and HC CDR3 of SEQ ID NO:38, or an antigen-binding fragment thereof.

[0093] In some specific embodiments, the anti-HSV antibodies of the present invention have: (a) a VH comprising a HC CDR1 of SEQ ID NO: 2, a HC CDR2 of SEQ ID NO: 33, and a HC CDR3 of SEQ ID NO: 6; and (b) a V L , which comprises LC CDR1 of SEQ ID NO: 35, LC CDR2 of SEQ ID NO: 11, and HC CDR3 of SEQ ID NO: 38, and may comprise V H , which comprises SEQ ID NO:40 or an amino acid sequence substantially identical thereto, and V L , which comprises SEQ ID NO: 41 or an amino acid sequence substantially identical thereto. Specifically, the anti-HSV antibody of the present invention comprises V H, which comprises an amino acid sequence that is at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identical to SEQ ID NO:40, and V L , which comprises an amino acid sequence that is at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identical to SEQ ID NO:41.

[0094] The anti-HSV antibodies of the present invention also include those encoding the relevant V H or V L Any recombinantly derived (engineered) antibody encoded by a polynucleotide sequence of an amino acid sequence.

[0095] The term "substantially identical" may refer to related amino acid sequences of variants (e.g., in FRs, CDRs, V H or V L(in) there is little difference compared to the reference antibody, so that the variant has substantially similar binding activity (e.g., affinity, specificity or both) and biological activity relative to the reference antibody. This variant may include minor amino acid changes. It is understood that the polypeptide may have a limited number of changes or modifications, which may be made in a portion of the polypeptide that is unrelated to its activity or function, but will still produce a variant with an acceptable level of equivalent or similar biological activity or function. In some examples, the amino acid residue change is a conservative amino acid substitution, meaning that the amino acid residue has a similar chemical structure to another amino acid residue, and has little or substantially no effect on the function, activity or other biological effect of the polypeptide. Generally, compared with the CDR region, the FR region can be substituted relatively more, as long as it does not adversely affect the binding function and biological activity of the antibody (for example, the binding affinity is reduced by more than 50% compared to the original antibody). In some embodiments, the sequence identity between the reference antibody and the variant may be about 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99% or more. Variants may be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those referenced in conjunction with such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook et al., 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. For example, conservative substitutions of amino acids include substitutions between amino acids within the following groups: (i) A, G; (ii) S, T; (iii) Q, N; (iv) E, D; (v) M, I, L, V; (vi) F, Y, W; and (vii) K, R, H.

[0096] The antibodies described herein may be animal antibodies (e.g., mouse-derived antibodies), chimeric antibodies (e.g., mouse-human chimeric antibodies), humanized antibodies, or human antibodies. The antibodies described herein may also include antigen-binding fragments thereof, such as Fab fragments, F(ab')2 fragments, Fv fragments, single-chain Fv (scFv), and (scFv) 2 Antibodies or antigen-binding fragments thereof can be prepared by methods known in the art.

[0097] III. Preparation of Antibodies

[0098] Many conventional methods in the art can be used to obtain antibodies or antigen-binding fragments thereof.

[0099] In some embodiments, the antibodies provided herein can be prepared by conventional hybridoma technology. Generally, a target antigen optionally coupled to a carrier protein and / or mixed with an adjuvant can be used to immunize a host animal to produce antibodies that bind to the antigen. Lymphocytes secreting monoclonal antibodies are collected and fused with myeloma cells to produce hybridomas. Subsequently, hybridoma strains formed in this manner are screened to identify and select those hybridoma strains that secrete the desired monoclonal antibodies.

[0100] In some embodiments, the antibodies provided herein can be prepared by recombinant technology. In related aspects, isolated nucleic acids encoding the disclosed amino acid sequences, vectors containing such nucleic acids, and host cells transformed or transfected with the nucleic acids are also provided.

[0101] For example, nucleic acids comprising nucleotide sequences encoding the heavy and light chain variable regions of such antibodies can be cloned into expression vectors (e.g., bacterial vectors, such as E. coli vectors, yeast vectors, viral vectors, or mammalian vectors) via conventional techniques, and any vector can be introduced into applicable cells (e.g., bacterial cells, yeast cells, plant cells, or mammalian cells) to express antibodies. Examples of mammalian host cell lines are human embryonic kidney cell lines (293 cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (VERO cells), and human liver cells (Hep G2 cells). Recombinant vectors for expressing antibodies described herein typically contain nucleic acids encoding antibody amino acid sequences, which are operably linked to constitutive or inducible promoters. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of nucleic acids encoding antibodies. The vector optionally contains selection markers for prokaryotic and eukaryotic systems. In some examples, heavy and light chain coding sequences are all included in the same expression vector. In other examples, each heavy and light chain of an antibody is cloned into a separate vector and produced separately, which can then be cultured under suitable conditions for antibody assembly.

[0102] Recombinant vectors for expressing antibodies described herein generally contain nucleic acids encoding antibody amino acid sequences, which are operably linked to constitutive or inducible promoters. Recombinant antibodies can be produced in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, insects, and mammalian cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of nucleic acids encoding antibodies. The vector optionally contains selection markers for prokaryotic and eukaryotic systems. The antibody protein produced can be further separated or purified to obtain a substantially homogeneous preparation for further testing and application. Applicable purification procedures, for example, can include separation on immunoaffinity or ion exchange columns, ethanol precipitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), ammonium sulfate precipitation, and gel filtration.

[0103] When a full-length antibody is desired, any of the V H and V L The coding sequences of the heavy and light chains can be linked to the coding sequences of the Fc region of an immunoglobulin, so that the resulting genes encoding full-length antibody heavy and light chains can be expressed and assembled in suitable host cells (e.g., plant cells, mammalian cells, yeast cells, or insect cells).

[0104] Antigen-binding fragments can be prepared by conventional methods. For example, F(ab') 2 Fragments can be generated by pepsin digestion of full-length antibody molecules, while Fab fragments can be generated by reducing F(ab') 2 Alternatively, such fragments can also be prepared by recombinant technology, by expressing heavy chain and light chain fragments in suitable host cells and assembling them in vivo or in vitro to form the desired antigen-binding fragment. Single-chain antibodies can be prepared by recombinant technology, by connecting the nucleotide sequence encoding the heavy chain variable region with the nucleotide sequence encoding the light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions.

[0105] IV. Composition

[0106] According to the present invention, the anti-HSV antibody can be formulated into a composition together with a pharmaceutically acceptable carrier for delivery and absorption.

[0107] As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition, and preferably can stabilize the active ingredient and is safe for the receiving individual. The carrier may be a diluent, a vehicle, an excipient, or a matrix for the active ingredient. Typically, a composition comprising an anti-HSV antibody as described herein as an active ingredient may be in the form of a solution, such as an aqueous solution (e.g., a saline solution, or it may be provided in powder form). The composition may further contain the desired pharmaceutically acceptable auxiliary substances to approximate physiological conditions, for example, pH regulators and buffers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The composition may be in the form of a suspension, a lotion, a solution, a sterile injection, and a packaged powder. The composition of the present invention may be delivered via any physiologically acceptable route, such as parenteral (e.g., intramuscular, intravenous, subcutaneous, and intraperitoneal) and intranasal methods. In certain specific embodiments, the composition of the present invention is administered as a liquid injectable formulation, which may be provided as a ready-to-use dosage form or as a reconstituted stable powder.

[0108] V. Treatment

[0109] The present invention provides a method for treating drug-resistant and / or recurrent HSV infection by administering an anti-HSV antibody as described herein. The method of the present invention can effectively alleviate the symptoms caused by HSV infection, delay the recurrence rate, and / or reduce the frequency of recurrence. The method of the present invention can effectively alleviate the symptoms caused by HSV infection, delay the recurrence rate, and / or reduce the frequency of recurrence.

[0110] As used herein, the term "drug resistance" may indicate that a virus is able to survive exposure to one or more antiviral drugs. Specifically, resistance to antiviral drugs may indicate that viral replication is not inhibited by normal amounts of antiviral drugs, or that clinical efficacy of the drug against the virus has not been demonstrated. The term "multidrug resistance" may refer to a virus that is resistant to more than one antiviral drug.

[0111] In some embodiments, the drug-resistant HSV strain has a mutation in tyrosine kinase (TK) or DNA polymerase. In some embodiments, the strain is resistant to at least one selected from the group consisting of acyclovir (ACV), famciclovir (FCV), penciclovir (PCV), valacyclovir (VCV), trifluridine (TFD), foscarnet (phosphono-formic acid, PFA) and cidofovir (CDV). In some specific embodiments, the virus strain includes clinically isolated virus strains, such as HSV-1RH (originating in the United States), HSV-1 Bethesda (originating in the United States), HSV-2MO (originating in the United States), HSV-2 Bethesda (originating in the United States), HSV-2JA-1 (originating in Asia), HSV-2JA-2 (originating in Asia), HSV-2JA-3 (originating in Asia), HSV-2poly-mut (polymerase mutant), HSV-2C7 (TK mutant), C8 (TK mutant), IC (ACV resistance). In some specific embodiments, the virus strain includes clinically isolated virus strains (originating in Germany), such as HSV-1R2, R4, R7, R8, R9, R10, R11 and R13, and HSV-2R5, R6 and R14.

[0112] The methods of the present invention using the anti-HSV antibodies described herein can be effective against drug-resistant HSV infections. In some embodiments, the anti-HSV antibodies described herein can achieve low IC against several drug-resistant HSV strains. 50 Values ​​(such as 1 nM to 30 nM) with a potency 3-fold or more (e.g., 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold, 2,500-fold, 5,000-fold or more) higher than acyclovir, which provides a high IC against several drug-resistant HSV strains. 50 Values ​​(such as 100 nM to 35,000 or more). In some embodiments, administration of an anti-HSV antibody as described herein in a single dose of 15 to 50 mg / kg is effective to protect animals with drug-resistant HSV infection from death.

[0113] As used herein, the term "relieve symptoms" or "relieve symptoms" may refer to an active agent that reduces or eliminates one or more symptoms of a disease or other abnormal state. The severity of the symptoms of a disease can be determined by any applicable index or score known in the art. In general, a higher level of the index or a higher score indicates a higher severity of the disease. In some specific embodiments, the clinical disease and clinical symptoms observed in animals with HSV infection can be scored as follows: 0 points for no lesions; 1 point for erythema only; 2 points for a single or a few blisters; 3 points for ulcerative lesions; and 4 points for abnormal movement except for lesions of 3 points. In some specific embodiments, the clinical disease and clinical symptoms observed in animals with HSV infection can be scored as follows: 0 points for normal; 1 point for slight redness of the external genitalia; 2 points for redness and swelling of the external genitalia, and / or pus / mucus; 3 points for severe swelling of the external genitalia, accompanied by pus / mucus, and some hair loss (alopecia) in the surrounding area; 4 points for genital tissue ulcers, redness and swelling; 5 points for aggravated ulcers, redness and swelling, and paralysis of the hind limbs; and 6 points for death. In some embodiments, symptom reduction may include a decrease in the level or score of an index or score of disease level compared to what one of ordinary skill in the art and / or a medical professional (e.g., a physician) would expect for a diseased individual or a group of individuals with similar physical characteristics and medical history, such as 5%, 10%, 15%, 20%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, symptom reduction may refer to a reduction in one or more perceived symptoms of a disease or other abnormal state to a normal state.

[0114] As used herein, "recurrent infection" may refer to a recurrence or recurrence of infection in a patient after a previous / primary infection event is considered cured, such as a second, third, or subsequent episode of infection. Normally, the first onset of symptoms is called a preliminary or initial attack, and subsequent attacks are called relapses. Clinically, a recurrent HSV infection may indicate that the reactivation of HSV in the lesion is the same as the antibodies in the serum. A patient experiencing a recurrent infection may refer to the virus reacting again. In certain embodiments, some patients with genital herpes may experience one or more relapses per month; some relapse every two to four months, and some relapse less than once every four months. In certain embodiments, some patients with oral herpes relapse one or more times per month, some relapse every two to four months, and some relapse less than once every four months.

[0115] As used herein, the term "treating recurrent HSV infection" may refer to a reduction in the severity, frequency, duration, and / or number of one or more recurrent viral symptoms in an infected individual, or a reduction in the mean or median severity, frequency, duration, and / or number of one or more recurrent viral symptoms in a group of individuals. In some embodiments, administration of an anti-HSV antibody as described herein is effective in delaying the incidence of relapses. In some embodiments, administration of an anti-HSV antibody as described herein is effective in reducing the number or frequency of relapses. In certain instances, when the anti-HSV antibodies described herein are administered to a patient population with herpes lesions, clinical symptoms do not occur within a median time of at least one month after cessation of treatment. Preferably, the relapse-free time is greater than 2-3 months.

[0116] The term "effective amount" as used herein means the amount of active ingredient that imparts the desired biological effect in the treated individual or cell. For example, as described herein, an effective amount may be the amount of an anti-HSV antibody as an active agent, which can provide relief from the symptoms of HSV infection and / or delay the recurrence of HSV infection. The effective amount may vary for various reasons, such as the route and frequency of administration, the weight and species of the individual receiving the medicine, and the purpose of administration. Those skilled in the art can determine the dosage in each case based on the disclosure herein, established methods, and their own experience.

[0117] In some specific embodiments, the anti-HSV antibody as described herein is administered in a dosage range of 0.01 to 100 mg, particularly 0.1 to 100 mg, more particularly 1 to 80 mg, and even more particularly 10 to 50 mg per kg body weight of the individual. In certain examples, the anti-HSV antibody as described herein is administered in a dosage range of 1 to 10 mg / kg, for example 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0118] In some embodiments, the antibody is included in the composition as a whole dose and is administered to the individual in a single dose. In general, the earlier the antibody is administered, the better. In some embodiments, the antibody is administered early in the infection process, for example, within 10 days (e.g., 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day) after infection. Preferably, the antibody is administered within 5 days (e.g., 5 days, 4 days, 3 days, 2 days or 1 day) after infection.

[0119] The individual to be treated by the treatment methods described herein may be a mammal, preferably a human. Mammals include, but are not limited to, farm animals, sports animals, pets, primates, horses, dogs, cats, mice and rats. The human individual in need of treatment may be a human patient suffering from, at risk of, or suspected of having a target disease / disorder. An individual suspected of having any such target disease / disorder may exhibit one or more symptoms of the disease / disorder. An individual at risk of a disease / disorder may be an individual with one or more risk factors for the disease / disorder. In some embodiments, the individual is a patient infected with HSV or a patient with a low immune system, such as an infant, a pregnant woman, a cancer patient, an organ transplant recipient, and a carrier of the human immunodeficiency virus (HIV). In some embodiments, the individual is a patient infected with HSV and poorly controlled by a small molecule drug.

[0120] In some embodiments, an anti-HSV antibody as described herein is administered to an individual in need during the incubation period (early / asymptomatic stage). Typically, the incubation period for HSV-1 and HSV-2 is 2 to 12 days before symptoms appear. Thus, in certain instances, an anti-HSV antibody as described herein is administered to an individual in need within 10 days (such as 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day) after infection.

[0121] In some embodiments, an anti-HSV antibody as described herein is administered to an individual in need after the onset of symptoms. In some instances, an anti-HSV antibody as described herein is administered to an individual in need after the onset of symptoms during the initial or primary attack. In some instances, an anti-HSV antibody as described herein is administered to an individual in need after the onset of symptoms during the relapse period. In some instances, an anti-HSV antibody as described herein is administered to an individual in need 10 days or later (such as 12 days, 15 days, 20 days, 25 days, 30 days or more) after infection.

[0122] In some embodiments, an anti-HSV antibody as described herein is administered to an individual in need at a low frequency. In some embodiments, an anti-HSV antibody as described herein is administered weekly or less frequently (e.g., every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or less frequently). In some embodiments, an anti-HSV antibody as described herein is administered once within a period of 1 to 3 months, for example, once a month, once every two months, or once every three months.

[0123] In some embodiments, administration of an anti-HSV antibody as described herein is effective in reducing the number or frequency of relapses. In some instances, the number of relapses 30 days after administration of a single dose of an anti-HSV antibody during the relapse period is reduced to 3 or less, compared to more than 3 (e.g., 4 or 5) relapses after administration of multiple doses of a small molecule antiviral drug (see Fig. 14B ).

[0124] The present invention is further illustrated by the following examples, which are provided for illustration and not limitation. In view of this disclosure, it will be appreciated by those skilled in the art that many changes may be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention and still obtaining similar or similar results.

[0125] Example

[0126] Example 1: Determination of Binding Affinity of UB-621 to Recombinant gD by Surface Plasmon Resonance (SPR) Analysis Kinetic analysis of the binding of UB-621 (anti-HSV-gD mAb) to recombinant gD was performed by surface plasmon resonance (SPR) technology using a Biacore X100 instrument. Anti-human IgG Fc fragment antibodies were first immobilized on the surface of a CM5 gold sensor chip by amine coupling. UB-621 was diluted to 5 mg / mL in running buffer (two-fold serial dilutions, 6.25 to 100 nM) and captured by anti-FC antibodies on the CM5 chip. A gD sample diluted in HBS-EP buffer was injected, including a reference pool. Dissociation rate constants (kd), association rate constants (ka), and equilibrium dissociation rate constants (KD) were automatically calculated using a 1:1 model, and steady-state affinity was calculated using Biacore evaluation software.

[0127] The mean KD of UB-621 was estimated to be 1.35 x 10 -10 M( Figure 1 ).

[0128] Example 2: Neutralization of HSV-1 and HSV-2 Laboratory Virus Strains Infected by UB-621

[0129] The efficacy of UB-621 against HSV infection was first investigated in Vero cells by cell viability assay and IC 50 and IC 90The four laboratory HSV strains tested were HSV-1KOS, HSV-1RE, HSV-2333, and HSV-2G. The virus strains were mixed with a series of concentrations of UB-621 at room temperature for one hour. The mixture was then aliquoted into 96-well plates seeded with Vero cells. After 48 hours of incubation at 37°C, the plates were washed once with PBS, and live cells were incubated with 1% Alamar blue for 1 hour. Fluorescence was measured at 530 nm and 590 nm to calculate the percentage of cell death.

[0130] UB-621 completely neutralizes all laboratory HSV-1 and HSV-2 strains with high potency ( Figure 2 ), such as low levels of IC 50 and IC 90 As shown (Table 2).

[0131] Table 2: UB-621 with low IC 50 Value and IC 90 The value of inhibiting the infection of HSV-1 and HSV-2 virus strains in the laboratory on Vero cells

[0132]

[0133] Example 3: Therapeutic effect of UB-621 on genital HSV-2 infection

[0134] 3.1 Post-infection treatment before symptoms appear

[0135] Female BALB / c mice aged 6-8 weeks were used in the HSV-2 intravaginal infection model (Marshak JO et al., 2014). The mice were randomly assigned to the hIgG control group and the UB-621 treatment group (n = 6). On the 4th day and the 1st day before HSV-2 intravaginal infection, the mice were subcutaneously injected with 2.5 mg of progesterone in the upper back. Subsequently, the mice were inoculated intravaginally with 10 μL of virus suspension (HSV-2 strain 333, 1×10 5 PFU). On day 1 after infection, 300 μg of UB-621 (UB-621 treatment group) or an equivalent amount of human IgG (hIgG control group) was administered to each mouse by subcutaneous injection. Clinical signs of genital infection were scored on days 1, 2, 3, 4, 5, 6, 7, 8, 10 and 12 after infection, which were scored from 0 to 5 according to a comprehensive scale (Gill N et al., 2005): 0 for no signs of infection; 1 for slight redness of the external genitalia; 2 for redness and swelling of the external genitalia, and / or pus / mucus; 3 for severe swelling of the external genitalia, accompanied by pus / mucus, and some alopecia in the surrounding area; 4 for genital tissue ulceration, redness and swelling; 5 for aggravated ulceration, redness and swelling, and hind limb paralysis; 6 for death.

[0136] Compared with a score of 5 in hIgG-treated mice on day 7 after HSV-2 infection, a single 300 μg dose of UB-621 controlled genital symptoms and signs to within 2 points ( Figure 3A UB-621 treated mice survived 5.5 days longer compared to hIgG treated mice (12 days vs. 6.5 days) ( Figure 3B ).

[0137] 3.2 Post-infection treatment after the onset of infection symptoms

[0138] Mice were randomly assigned to hIgG control group and UB-621 treatment group (n=3). On the 4th day and the 1st day before HSV-2 intravaginal infection, mice were subcutaneously injected with 2.5 mg of progesterone in the upper back. Subsequently, mice were intravaginally inoculated with 10 μL of virus suspension (HSV-2 strain 333, 1×10 5 On day 4 after infection, 300 μg of UB-621 (UB-621 treatment group) or an equivalent amount of human IgG (hIgG control group) was administered to each mouse by subcutaneous injection.

[0139] Compared with scores of 5 to 6 in hIgG-treated mice on day 12 after HSV-2 infection, a single 300 μg dose of UB-621 controlled genital symptoms and signs to 3 ( Figure 4A ). Compared to the median survival of 8 days for mice treated with hIgG, 100% of mice treated with UB-621 survived ( Figure 4B ).

[0140] Example 4: Neutralization of HSV-1RE TKnull and HSV-2 333TKnull mutant virus strains by UB-621 in Vero cells

[0141] Comparative studies of UB-621 and acyclovir (ACV) were performed using Vero cells infected with two HSV laboratory mutant strains, and the inhibitory activity was analyzed by plaque assay. The wild-type HSV-1RE and HSV-2 333 strains were mutated by inserting enhanced green fluorescent protein (EGFP) in the TK gene, making the resulting HSV-1RE TKnull and HSV-2 333TKnull mutants resistant to ACV. Vero cells seeded in 12-well plates were infected with HSV-1RE mutants or HSV-2 mutants (MOI = 0.01) at 37°C for 75 minutes. Subsequently, the infected cells were cultured in culture medium with or without a series of concentrations of UB-621 or ACV for 24 hours. Virus plaques were counted and the half-maximal inhibitory activity was calculated as IC 50 express.

[0142] UB-621 neutralized both virus strains with much higher potency than ACV ( Figure 5A and 5B UB-621 uses low level IC 50 The inhibitory activity of UB-621 was 11.3 nM against HSV-1 RE mutant and 28.1 nM against HSV-2 333 mutant (Table 3). 50 A value could represent 1000 times more potent than ACV.

[0143] Table 3: UB-621 has a much lower IC value than ACV (acyclovir) 50 The value inhibited two HSV-1RE TKnull and HSV-2333TKnull virus strains

[0144]

[0145] Example 5: Neutralization of UB-621 against clinically derived HSV-1 and HSV-2 isolates

[0146] 5.1 American and Asian origins and ACV resistance.

[0147] The efficacy of UB-621 and ACV in inhibiting clinically derived HSV-1 and HSV-2 isolates was compared with 11 isolates of U.S., Asian origin, and acyclovir resistance. Four U.S. isolates (HSV-1 Bethesda and HSV-1RH; HSV-2 Bethesda and HSV-2RH), three Asian isolates (HSV-2JA-1, HSV-2JA-2, and HSV-2JA-3), and four ACV-resistant isolates (poly-mut, C7, C8, and IC) were investigated via a cell viability assay. The strains were mixed with a range of concentrations of UB-621 or ACV at room temperature for one hour. The mixture was then aliquoted into 96-well plates seeded with Vero cells. After 48 hours of incubation at 37°C, the plates were washed once with PBS, and live cells were incubated with 1% Alamar blue for 1 hour. Fluorescence was measured at 530nm and 590nm to calculate the percentage of cell death.

[0148] UB-621 has high efficiency (low IC 50 and IC 90 ) neutralizes all clinical HSV-1 and HSV-2 strains ( Fig. 6A , 6B and 6C), which was at least 500-fold higher than ACV (Table 4).

[0149] Table 4: UB-621 inhibits clinically derived HSV isolates of American and Asian origin and ACV resistance with IC50 and IC90 values ​​much lower than ACV (acyclovir)

[0150]

[0151] 5.2 German origin and other resistance.

[0152] Serial dilutions of UB-621 from 250 to 0 nM were mixed with a viral load of 100 TCID 50 The drug-resistant HSV-1 or HSV-2 isolates were cultured in cell culture medium at 37°C for 1 hour. Subsequently, the antibody virus inoculum was applied to Vero cell monolayers grown in 96-well plates. After 48 hours of culture, cytopathic effect (CPE) was scored by optical microscopy. The antibody concentration required for complete neutralization of the virus was defined as the neutralizing titer.

[0153] Compared with these IC 50 UB-621 can completely neutralize all tested drug-sensitive or single or multi-resistant clinical HSV isolates (HSV-1, including R2, R4, R7, R8, R9, R10, R11 and R13) and HSV-2 isolates (R5, R6 and R14) at very low concentrations (ranging from 7.8 to 31.3 nM) Figure 7 ). The neutralization efficacy of UB-621 is independent of the virus strain source and drug resistance status.

[0154] Example 6: Efficacy study of UB-621 against infection with ACV-resistant HSV-1 clinical isolates in mice.

[0155] BALB / c mice were divided into 3 groups, with 6 mice in each group. After each mouse was anesthetized, the cornea of ​​the right eye of each mouse was scratched with a needle, and 5 μL of virus inoculum containing 5×10 6 pfu of ACV-resistant HSV-1 clinical isolate virus strain. On the first day after infection, one dose of UB-621 (50 mg / kg) or human IgG (50 mg / kg) was administered to each designated mouse by subcutaneous injection. On the first and second days after infection, 125 mg / kg of acyclovir (ACV) was administered to each designated mouse twice a day by oral gastric tube gavage. All mice were sacrificed on the third day after infection, and the right eyes, trigeminal ganglia and brain of the mice were collected, and the viral load in the tissues was determined by plaque lysis assay.

[0156] Compared with human IgG (control) treatment, UB-621 at 50 mg / kg significantly reduced viral replication in the eye (virus inoculation site) ( Figure 8 ). There were no significant differences between the ACV-treated and control groups. However, this HSV clinical isolate has difficulty spreading and replicating in the neural tissues (trigeminal ganglia and brain) of all mice, so differences in efficacy in these tissues were not evaluated.

[0157] Example 7: Efficacy study of UB-621 against infection with ACV-resistant HSV-2 clinical isolates in mice

[0158] 7.1 HSV-2 Clinical Isolate IC Virus Strain

[0159] As previously reported, 6-8 week old female BALB / c mice were given 0.1 ml of a suspension containing 2.5 mg of progesterone by subcutaneous injection on days 4 and 1 before challenge to increase susceptibility to vaginal HSV infection. Subsequently, the animals were inoculated intravaginally with 10 μL of a suspension containing 1×10 6 pfu suspension of HSV-2 clinical isolate IC strain (Figure 9). On the first day after infection, animals were administered a single dose of UB-621 by subcutaneous injection, or multiple doses of ACV were orally administered on days 1-5. Subsequently, animals were tracked daily for at least 15 days. The severity of vaginitis was scored on a comprehensive scale of 0 to 6 points.

[0160] UB-621 at dose levels of 15 mg / kg to 50 mg / kg completely protected mice from death caused by viral infection ( Fig. 9A A single dose of UB-621 significantly controlled genital symptoms in a dose-dependent manner compared with saline control mice and ACV-treated mice ( Fig. 9B ).

[0161] 7.2 HSV-2 clinical isolates pol-mut virus strains

[0162] Separate studies were performed using other clinical isolates of pol-mut virus strains. Briefly, animals were inoculated intravaginally with 10 μL of a 1×10 6 pfu suspension of HSV-2 clinical isolate pol-mut strain (Figure 10). On day 1 after infection, animals were administered a single dose of UB-621 by subcutaneous injection, or multiple doses of ACV were orally administered on days 1-5. Subsequently, animals were tracked daily for at least 15 days.

[0163] Similar results were observed in the efficacy evaluation study of UB-621 against clinical isolates of the pol-mut virus strain. UB-621 completely protected mice from death at dose levels of 15 mg / kg to 50 mg / kg ( Fig. 10A A single dose of UB-621 significantly controlled genital symptoms in a dose-dependent manner compared to saline control mice ( Fig. 10B ).

[0164] Example 8: Inhibition of HSV-1 and HSV-2 Cell-to-Cell Transmission by UB-621

[0165] The inhibition of cell-to-cell spread by UB-621 was investigated as previously described (Krawczyk A, et al., 2013) with modifications. Briefly, 1 x 10 5 Vero cells were seeded in 24-well plates at 200 TCID 50 Confluent cell cultures were infected with HSV-1- or HSV-2-ΔgE-GFP reporter viruses. After 2 h of culture, the inoculation medium was removed and the cell cultures were incubated with serial dilutions of UB-621 (0–1000 nM). After 2 days of culture, plaque formation was examined by fluorescence microscopy. 2% DMEM alone was used as a negative control, and anti-gB mAb H1817 (Pereira L et al., 1989) was used as a positive control. Plaque formation was assessed by fluorescence microscopy.

[0166] Plaque formation of HSV-1 and HSV-2 decreased with increasing UB-621 concentration. Complete neutralization of HSV-1 was observed at 1000 nM (150 μg / mL) UB-621 concentration ( Fig.11A UB-621 clearly showed a plaque-reducing effect against HSV-2, which is evidence of cell-to-cell spread inhibitory activity ( Fig. 11B ).

[0167] Example 9: Inhibition of HSV-1 Anterograde Interneuronal Transmission in BALB / c Mice by UB-621

[0168] Female BALB / c mice were inoculated with HSV-1RE strain via the cornea to infect the right eye. The dose was 5 μL per mouse (containing 1×10 6 pfu). On day 1 post infection, animals received a single intraperitoneal dose (15 mg / kg) of UB-621. Animals (5 animals per group (n=5)) were sacrificed on day 5 post infection to analyze the viral load in the right eye, right trigeminal ganglion, and whole brain.

[0169] The results showed that the presence of UB-621 significantly inhibited the transmission of interneurons from the eye to the trigeminal ganglion and the brain ( Fig.12 ).

[0170] Example 10: Study on the efficacy of UB-621 against recurrent HSV-2 infection in guinea pigs

[0171] Unlike the mouse model, the guinea pig genital model exhibits acute disease with spontaneous relapses. On day 0, female Hartley strain guinea pigs (200-300 g) were infected with 10 6 PFU of HSV-2 strain 333. In order to study the efficacy of UB-621 and its effect on recurrence ( Fig.13A and 13B ,as well as Fig.14A and 14B ), when administered in HSV primary and recurrent infections, a single dose of UB-621 (30 or 60 mg / kg) was administered subcutaneously on day 1 or day 20 after infection. Multiple doses of acyclovir (125 mg / kg, twice daily) were administered orally on days 1-7 or 20 to 26 after infection. Control animals were administered saline subcutaneously and orally. The severity of post-infection symptoms was determined by direct examination of the external genital skin and animal vitality. Each animal was observed daily for 55 days (dose during the acute infection period; Fig.13A ) or observe for 40 days after treatment (administered during the recurrent infection period; Fig.14A ), which uses a clinical score: 0 for no lesions; 1 for erythema only; 2 for a single or a few blisters; 3 for ulcerated lesions; and 4 for abnormal movement except for lesions scored at 3. When the symptom score was 4, the animal was humanely sacrificed. Relapse was determined when the clinical symptoms were more severe than the previous day.

[0172] The results showed that a single dose of UB-621 can effectively relieve the clinical symptoms of vaginitis, regardless of the primary infection ( Fig.13A ) or after first HSV recurrence ( Fig.14A ) were administered to infected animals. Compared with saline or ACV treatment, UB-621 treatment not only delayed the incidence of relapses, but also reduced the cumulative number of relapses ( Fig. 13B and 14B ).

[0173] 3. Conclusion

[0174] UB-621 effectively neutralizes wild-type and drug-resistant HSV-1 and HSV-2 (Examples 2, 4, and 5), and a single dose of UB-621 can reduce disease symptoms and improve survival in HSV-infected mice and guinea pigs (Examples 3, 6, 7, 9, and 10).

[0175] UB-621 effectively inhibited HSV replication in vitro in all 23 drug-resistant HSV clinical isolates, including isolates resistant to several small molecule drugs (e.g., ACV, PFA) (Example 5).

[0176] Many patients are infected with drug-resistant HSV and require higher doses of small molecule drugs to treat the disease. At the same time, a single dose of UB-621 can reduce disease symptoms and improve survival in primary HSV infection with drug-resistant strains (Examples 6 and 7).

[0177] When the patient's immune system weakens or small molecule drug treatment is interrupted, some patients may experience recurrent symptoms of HSV. In the HSV recurrent guinea pig model, a single dose of UB-621 at the primary HSV infection stage showed a strong therapeutic effect (Example 10).

[0178] The significant therapeutic effect of UB-621 in animals may be related to the neutralization of HSV gD and the inhibition of HSV cell-to-cell transmission (Examples 8 and 9).

[0179] Currently, there are no mAb drugs available for HSV treatment, and small molecule drugs have many limitations and side effects. UB-621 has shown effective therapeutic efficacy in animal models and may benefit patients infected with drug-resistant HSV strains whose symptoms are poorly controlled by small molecule drugs.

[0180] References

[0181] Boado RJ, Zhou QH, Lu JZ, et al. Pharmacokinetics and brain uptake ofagenetically engineered bifunctional fusion antibody targeting the mousetransferrin receptor. Mol Pharmacol 7:237–244(2010).

[0182] Eddie C hang,Laurence Galle,David Maggs,D.Mark Estes,and WilliamJ.Mitchell.Pathogenesis of Herpes Simplex Virus Type 1Induced CornealInflammation in Perforin Deficient Mice.(2000)Journal of Virology.74:1183211840.

[0183] Gill N,Rosenthal KL,Ashkar AA.NK and NKT cell-independentcontribution of interleukin-15to innate protection against mucosal viralinfection.J.Virol.79:4470–4478(2005).

[0184] Krawczyk A,Arndt MA,Grosse-Hovest L,Weichert W,Giebel B,Dittmer U,Hengel H,Jager D,Schneweis KE,Eis-Hubinger AM,Roggendorf M,KraussJ.Overcomingdrug-resistant herpes simplex virus(HSV)infection by ahumanizedantibody.PNAS,USA 110:6760-6765(2013).

[0185] Marshak JO,Dong L,Koelle DM,et al.The Murine Intravaginal HSV-2ChallengeModel for Investigation.Methods Mol Biol.1144:305–327(2014).

[0186] Pereira L,Ali M,Kousoulas K,Huo B,Banks T.Domain structure ofherpessimplex virus 1 glycoprotein B:neutralizing epitopes map in regions ofcontinuous anddiscontinuous residues.Virology 172:11-24(1989).

[0187] Pepinsky RB,Shao Z,Ji B,et al.Wang,Q.,Meng,G.,Walus,L.,Lee,X.,Hu,Y.,Graff,C.,Garber,E.,Meier,W.,and Mi,S.Exposure levels of anti-LINGO-1Li81antibody in the central nervous system and dose-efficacy relationships inrat spinal cordremyelination models after systemic administration.J.Pharmacol.Exp.Ther.339:519–529(2011).

Claims

1. A method for treating drug-resistant and / or recurrent herpes simplex virus (HSV) infection, comprising administering an anti-HSV antibody to a subject in need thereof, wherein the anti-HSV antibody specifically binds to glycoprotein D (gD) of herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2).

2. The method of claim 1, wherein the anti-HSV antibody can effectively neutralize drug-resistant HSV strains and / or inhibit viral transmission.

3. The method of claim 1 or 2, wherein the anti-HSV antibody comprises (a) a heavy chain variable region (VH), comprising a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region (VL), which comprises a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO:9, a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO:11, and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO:

13.

4. The method of claim 3, wherein The VH comprises the amino acid sequence of SEQ ID NO: 15; and / or The VL comprises the amino acid sequence of SEQ ID NO:

16.

5. The method of claim 1 or 2, wherein the anti-HSV antibody comprises (a) a heavy chain variable region (VH), which comprises a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 20, and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 22; and (b) a light chain variable region (VL), which comprises a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO:24, a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO:26, and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO:

28.

6. The method of claim 5, wherein The VH comprises the amino acid sequence of SEQ ID NO: 30; and / or The VL comprises the amino acid sequence of SEQ ID NO:

31.

7. The method of claim 1 or 2, wherein the anti-HSV antibody comprises (a) a heavy chain variable region (VH), comprising a heavy chain complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementary determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 33, and a heavy chain complementary determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region (VL), which comprises a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO:35, a light chain complementary determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO:11, and a light chain complementary determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO:

38.

8. The method of claim 7, wherein The VH comprises the amino acid sequence of SEQ ID NO:40; and / or The VL comprises the amino acid sequence of SEQ ID NO:

41.

9. The method of any one of claims 1 to 8, wherein the antibody is an antigen-binding fragment thereof.

10. The method of any one of claims 1 to 9, wherein the antibody is humanized.

11. The method of any one of claims 1 to 10, wherein the individual is susceptible to or infected with a drug-resistant HSV strain.

12. The method of any one of claims 1 to 11, wherein the individual is an immunocompromised patient.

13. The method of any one of claims 1 to 12, wherein the anti-HSV antibody is administered to the subject in a single dose.

14. The method of any one of claims 1 to 13, wherein the anti-HSV antibody is administered to the subject at an early stage of infection.

15. The method of any one of claims 1 to 14, wherein the antibody is administered in an amount effective to reduce symptoms caused by HSV infection.

16. The method of any one of claims 1 to 15, wherein the antibody is administered in an amount effective to delay the incidence of relapse and / or reduce the frequency of relapse.

17. The method of any one of claims 1 to 16, wherein the anti-HSV antibody is administered to the individual after the onset of symptoms.

18. Use of an anti-HSV antibody or a composition thereof as defined in any one of claims 1 to 10 for treating drug-resistant and / or recurrent HSV infection in an individual in need thereof.

19. Use of an anti-HSV antibody as defined in any one of claims 1 to 10 in the manufacture of a medicament for treating drug-resistant and / or recurrent HSV infection in an individual in need thereof.

20. Use of an anti-HSV antibody or a composition thereof as claimed in claim 18 or use of an anti-HSV antibody as claimed in claim 19, wherein The individual is susceptible to or infected with a drug-resistant HSV strain; The individual is an immunocompromised patient; The anti-HSV antibody is administered to the subject in a single dose; The anti-HSV antibody is administered to the subject at an early stage of infection; wherein the antibody is administered in an amount effective to alleviate symptoms caused by HSV infection; wherein the antibody is administered in an amount effective to delay the incidence of relapse and / or reduce the frequency of relapse; and / or wherein the anti-HSV antibody is administered to the individual after symptoms develop.

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