Treatment of herpes simplex virus infections

Abstract

The present invention relates to the treatment of herpes simplex virus (HSV) infection using anti-HSV antibodies. In particular, 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 methods of the present invention are effective against drug-resistant and / or recurrent HSV infections.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 403,408, filed September 2, 2022, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present invention relates to the treatment of herpes simplex virus (HSV) infection using anti-HSV antibodies. In particular, 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 methods of the present invention are effective against drug-resistant and / or recurrent HSV infections. [Background technology]

[0003] Background of the Invention Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are members of the herpesvirus family and are highly prevalent pathogens worldwide, causing recurrent oral and genital ulcers. While HSV infection generally does not cause severe symptoms or complications, it can cause severe symptoms in immunocompromised patients and newborns. While symptoms are less severe in the average patient, HSV can still recur in many patients. Recurrence of HSV can plague patients for years, reducing their quality of life.

[0004] Although several antiviral drugs are approved for the treatment of primary and recurrent HSV infections, these drugs are not always effective and can be associated with significant side effects. Furthermore, drug-resistant HSV strains (e.g., acyclovir resistance) significantly reduce the effectiveness of these conventional antiviral drugs.

[0005] "Patients with a high recurrence rate or infected with drug-resistant strains lack good alternative treatments. There is a need to provide better therapies for the treatment of patients with recurrent HSV infection, especially those infected with drug-resistant HSV. Therefore, new effective preventive and therapeutic interventions remain urgently needed." Summary of the Invention

[0006] Summary of the Invention The present invention is based on the discovery that anti-HSV antibodies are effective against drug-resistant HSV strains and suppress recurrence of HSV infection. In particular, 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 effectively alleviates symptoms caused by drug-resistant HSV infection and reduces or delays recurrence of HSV infection.

[0007] Accordingly, 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).

[0008] In some embodiments, the anti-HSV antibodies neutralize drug-resistant HSV strains.

[0009] In one embodiment, the anti-HSV antibodies inhibit viral spread.

[0010] In some embodiments, the anti-HSV antibody (a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 9, a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 11, and a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 13; Includes:

[0011] In some embodiments, the anti-HSV antibody comprises the following VH and VL: VH comprises the amino acid sequence of SEQ ID NO: 15; and / or VL comprises the amino acid sequence of SEQ ID NO:16.

[0012] In some embodiments, the anti-HSV antibody (a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 20, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 22; and (b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 24, a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 26, and a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 28. Includes:

[0013] In some embodiments, the anti-HSV antibody comprises the following VH and VL: VH comprises the amino acid sequence of SEQ ID NO: 30; and / or VL comprises the amino acid sequence of SEQ ID NO:31.

[0014] In some embodiments, the anti-HSV antibody (a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 33, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 35, a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 11, and a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO: 38. Includes:

[0015] In some embodiments, the anti-HSV antibody comprises the following VH and VL: VH comprises the amino acid sequence of SEQ ID NO: 40; and / or VL comprises the amino acid sequence of SEQ ID NO:41.

[0016] In some embodiments, the antibody is an antigen-binding fragment thereof.

[0017] In some embodiments, the antibody is humanized.

[0018] In some embodiments, the subject is susceptible to or infected with a drug-resistant HSV strain.

[0019] In some embodiments, the subject is a patient with a weakened immune system.

[0020] In some embodiments, the anti-HSV antibody is administered to the subject in a single dose.

[0021] In some embodiments, the antibody is administered in an amount effective to reduce symptoms caused by HSV infection.

[0022] In some embodiments, the antibody is administered in an amount effective to delay relapse and / or reduce the frequency of relapse.

[0023] In some embodiments, the anti-HSV antibody is administered to the subject after the onset of symptoms.

[0024] The present invention also provides an anti-HSV antibody described herein or a composition comprising the same for use in treating drug-resistant and / or recurrent HSV infection in a subject in need thereof. Further, the present invention discloses the use of an anti-HSV antibody described herein for the manufacture of a medicament for treating drug-resistant and / or recurrent HSV infection in a subject in need thereof.

[0025] The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention may be apparent from the following detailed description of some embodiments, and from the appended claims. [Brief explanation of the drawings]

[0026] The foregoing summary, as well as the following detailed description of the invention, can be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0027] [Figure 1] FIG. 1 shows the determination of the binding affinity of UB-621 to recombinant gD by surface plasmon resonance (SPR) analysis. [Figure 2] FIG. 2 shows the potency, IC50 and IC90 values ​​of UB-621 in inhibiting experimental HSV-1 and HSV-2 strains on Vero cells. [Figure 3] Figures 3A and 3B show the therapeutic effect of a single SC dose of UB-621 on genital symptoms (Figure 3A) and mortality (Figure 3B) in HSV-2 vaginally infected mice. [Figure 4] Figures 4A and 4B show the therapeutic effect of UB-621 administered to mice after the onset of infection symptoms. Figure 4A shows the severity of vaginitis (genital symptoms), and Figure 4B shows the survival rate (mortality rate). [Figure 5] Figures 5A and 5B show the potency and IC50 values ​​of UB-621 and ACV in inhibiting the infection of artificial acyclovir (ACV)-resistant HSV mutants, HSV-1 RE TKnull (Figure 5A) and HSV-2 333 TKnull (Figure 5B), in Vero cells. The results show that UB-621 is more potent against infection of both the ACV-resistant HSV-1 RE TKnull mutant (A) and the HSV-2 333 TKnull mutant (B). [Figure 6] Figures 6A-6C show the potency, IC50, and IC90 values ​​of UB-621 and acyclovir (ACV) in inhibiting clinical HSV-1 and HSV-2 isolates, including HSV-1 and HSV-2 from America (Figure 6A), HSV-2 from Asia (Figure 6B), and HSV-2 ACV-resistant (Figure 6C). The results show that UB-621 is more potent than ACV in inhibiting clinical HSV isolates from America and Asia, and ACV-resistant isolates. [Figure 7] FIG. 7 shows the susceptibility of clinical HSV isolates resistant to ACV (acyclovir), PFA (foscarnet), or CDV (cidofovir) to inhibition by UB-621. [Figure 8] FIG. 8 shows the therapeutic effect of a single dose of UB-621 against infection with an ACV-resistant HSV-1 clinical isolate in a mouse model. [Figure 9] Figures 9A and 9B show the therapeutic effect of UB-621 on infection with an ACV-resistant HSV-2 clinical isolate in genital-infected mice. Figure 9A shows the effect of UB-621 on survival after IC infection with an HSV-2 clinical isolate. A single dose of UB-621 increased survival after infection with an ACV-resistant HSV-2 IC strain. Figure 9B shows the effect of UB-621 on the severity of vaginitis induced by infection with an IC clinical isolate of HSV-2. A single dose of UB-621 reduced clinical vaginitis after infection with an ACV-resistant HSV-2 IC strain. *Statistical significance: p-value < 0.05; **p-value < 0.01. [Figure 10]Figures 10A and 10B show the therapeutic effect of UB-621 on infection with an ACV-resistant HSV-2 clinical isolate in genital-infected mice. Figure 10A shows the effect of UB-621 on survival after infection with an HSV-2 clinical isolate, pol-mut. A single dose of UB-621 increased survival after infection with an ACV-resistant HSV-2 pol-mut strain. Figure 10B shows the effect of UB-621 on the severity of vaginitis induced by infection with a clinically isolated HSV-2 pol-mut strain. A single dose of UB-621 reduced clinical vaginitis after infection with an ACV-resistant HSV-2 pol-mut strain. *Statistical significance indicated a p-value of <0.05. [Figure 11] Figures 11A and 11B show the inhibition of cell-to-cell spread of HSV-1 (Figure 11A) and HSV-2 (Figure 11B) by UB-621. [Figure 12] Figure 12 shows the inhibitory effect of UB-621 on anterograde interneuronal spread of HSV-1 in BALB / c mice. ** indicates statistical significance with a p-value <0.01. [Figure 13] Figures 13A and 13B show the significant effect of UB-621 on primary HSV-2 infection in guinea pigs. The effect of UB-621 on the severity of vaginitis in primary infection is shown in Figure 13A. Administration of UB-621 reduced 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 Figure 13B. Administration of UB-621 delayed relapse and reduced the frequency of relapse in guinea pigs infected with genital HSV-2. [Figure 14] Figures 14A and 14B show the significant effect of UB-621 on recurrent HSV-2 infection in guinea pigs. The effect of UB-621 on the severity of vaginitis after the first HSV recurrence is shown in Figure 14A. Administration of UB-621 after the first HSV recurrence reduced the symptoms of vaginitis in guinea pigs infected with genital HSV-2. The effect of UB-621 on the cumulative number of recurrences is shown in Figure 14B. Administration of UB-621 after the first HSV recurrence reduced the frequency of recurrences in guinea pigs infected with genital HSV-2. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description of the Invention The following description is merely for the purpose of illustrating various aspects of the present invention. As such, the specific aspects or modifications described herein should not be construed as limiting the scope of the present invention. It may be apparent to those skilled in the art that various modifications or equivalents may be made without departing from the scope of the present invention.

[0029] For a clear and ready understanding of the present invention, certain terms are first defined. Additional definitions are found in many places in the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0030] I. Definition For a clear and ready understanding of the present invention, certain terms are first defined. Additional definitions are provided throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] 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.

[0032] The terms "comprise" or "comprising" are generally used in the sense of include / including, meaning to permit the presence of one or more features, components or ingredients. The terms "comprise" or "comprising" encompass the terms "consists" or "consisting of."

[0033] As used herein, "herpes virus" refers to members 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 advantageous for use in alleviating symptoms caused by HSV infection. HSV can infect many parts of the body, including the oral cavity, genitals, eyes, skin, and brain. Generally, HSV-1 primarily infects the oral cavity, while HSV-2 primarily infects the genitals. HSV is usually transmitted to uninfected individuals through direct contact with the infected area of ​​an infected individual. Clinical symptoms associated with HSV infection are well known in the art. HSV infection may be asymptomatic or symptomatic and may present with painful blisters or ulcers. HSV recurrence can be triggered by several factors, including stress, immunodeficiency, sunburn, and injury. HSV infection can be fatal, especially in immunocompromised individuals. Typical HSV infections include herpetic gingivostomatitis, herpes labialis, herpetic eye infection (herpes keratitis), genital herpes, herpetic vitiligo, herpes gladiatorum, herpes viral encephalitis, herpes viral meningitis, and herpes esophagitis. Initial symptoms of herpes labialis include a painful vesicular rash with distinct vesicles on the lips, tongue, or buccal mucosa. The vesicles may coalesce and rupture, forming shallow ulcers covered with necrotic material. A common clinical symptom of genital herpes is a cluster of small vesicles on an erythematous base. The vesicles ulcerate spontaneously or upon direct abrasion. The ulcers usually form crusts and subsequently re-epithelialize.

[0034] See, for example, U.S. Publication Nos. US20020147210A1 and US6599945B2, the entire contents of which are incorporated herein by reference.

[0035] As used herein, the term "polypeptide" refers to a polymer of amino acid residues linked through peptide bonds. The term "protein" typically refers to a relatively large polypeptide. The term "peptide" typically refers to a relatively short polypeptide (e.g., containing up to 100, 90, 70, 50, 30, 20, or 10 amino acid residues).

[0036] As used herein, the term "approximately" or "about" refers to an acceptable degree of deviation that would be understood by one 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" can refer to a numerical value having a range of ±10%, ±5%, or ±3% around the cited value.

[0037] As used herein, the term "substantially identical" means that two sequences have a homology of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more.

[0038] As used herein, the term "antibody" (which may be used interchangeably in the plural) refers to an immunoglobulin molecule capable of specifically binding to a particular target antigenic molecule. As used herein, the term "antibody" encompasses not only intact (i.e., full-length) antibody molecules, but also antigen-binding fragments of antibodies (e.g., Fab, Fab', F(ab')2, and Fv) that retain antigen-binding ability. Such fragments are well known in the art and are routinely used in vitro and in vivo. The term "antibody" also encompasses chimeric antibodies, humanized antibodies, human antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and other modified constructs of immunoglobulin molecules that comprise an antigen-recognition site of the required specificity, including amino acid sequence variants of antibodies, glycosylation variants of antibodies, and covalently modified antibodies.

[0039] An intact or whole antibody contains two heavy chains and two light chains. Each heavy chain contains a variable region (V H) and the first, second and third constant regions (C H 1. C H 2 and C H 3), each light chain comprising a variable region (V L ) and constant region (C L ). Antibodies are "Y" shaped, with the stem of the Y consisting of the second and third constant regions of two heavy chains connected via disulfide bonds. Each arm of the Y contains the variable and first constant regions of a single heavy chain bound to the variable and constant region of a single light chain. The light and heavy chain variable regions are responsible for antigen binding. The variable regions of both chains generally are responsible for antigen binding and each contain three highly variable regions called complementarity-determining regions (CDRs): the heavy (H) chain CDRs, which include heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and the light (L) chain CDRs, which include light chain (LC) CDR1, LC CDR2, and LC CDR3. The three CDRs are flanked by framework regions (FR1, FR2, FR3, and FR4), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable regions. The constant regions of the heavy and light chains are not involved in antigen binding but are involved in various effector functions. Immunoglobulins are classified into different classes depending on the antibody amino acid sequence of the constant domain of the 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 alpha, delta, epsilon, gamma, and mu, respectively.

[0040] As used herein, the term "antigen-binding fragment" or "antigen-binding domain" refers to the 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: (i) V H -C H 1 chain and V L -C L(ii) a Fab fragment, which may be a monovalent fragment consisting of two Fab chains; (iii) a F(ab')2 fragment, which may be a bivalent fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region; or (iv) a V fragment of an antibody molecule non-covalently linked to the V chain. H Domains and V L (iv) an Fv fragment consisting of V domains linked via a peptide linker; H Domains and V L (v) a single-chain Fv (scFv), which may be a single peptide chain consisting of two V domains linked by a peptide linker; and H domain and two V via disulfide bridges H Two Vs bound to the domain L These include, but are not limited to, (scFv)2, which may comprise a domain.

[0041] As used herein, the term "chimeric antibody" refers to an antibody comprising polypeptides derived from different sources, e.g., different species. In some embodiments, in a chimeric antibody, the variable regions of both the light and heavy chains may mimic the variable regions of an antibody derived from a certain mammal (e.g., a non-human mammal such as a mouse, rabbit, or rat), while the constant regions may be homologous to the sequences of an antibody derived from another mammal, such as a human.

[0042] As used herein, the term "humanized antibody" refers to an antibody that contains a framework region derived from a human antibody and one or more CDRs derived from a non-human (usually mouse or rat) immunoglobulin.

[0043] As used herein, the term "human antibody" refers to an antibody in which essentially the entire light and heavy chain sequences, including the complementarity-determining regions (CDRs), are derived from human genes. In some circumstances, a human antibody may contain one or more amino acid residues not encoded by human germline immunoglobulin sequences (e.g., multiple mutations in one or more of the CDRs) or one or more of the FRs, for example, to reduce immunogenicity, increase affinity, or remove cysteines that may cause undesired folding.

[0044] As used herein, the terms "specific binding" or "specifically binds" refer to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope of a target antigen. An antibody that "specifically binds" to a target antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. An antibody "specifically binds" to a target antigen if it binds with higher affinity / avidity, more readily, and / or for a longer period of time than it binds to other substances. In other words, it is 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. Thus, "specific binding" or "preferential binding" does not necessarily require exclusive binding (although it can include it). Generally, the affinity of binding is measured by the dissociation constant (K D ) Generally, specific binding, when used in reference to antibodies, can be defined as about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 It can mean an antibody that specifically binds (recognizes) its target with a KD value, such as M or less, and that has an affinity that is at least 10-fold lower, e.g., at least 100-fold lower, at least 1,000-fold lower, or at least 10,000-fold lower, than its affinity for binding to a non-specific antigen (such as BSA or casein).

[0045] As used herein, the term "nucleic acid" or "polynucleotide" can 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 containing non-naturally occurring nucleotides. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. When a nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), it can be understood to also include an RNA sequence in which "U" is substituted for "T" (i.e., A, U, G, C). The term "cDNA" refers to DNA that is complementary to or identical to mRNA, in single- or double-stranded form.

[0046] As used herein, the term "complementary" refers to the topological compatibility or correspondence of the interacting 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 the sequence 5'-ATATC-3' is complementary to a polynucleotide with the sequence 5'-GATAT-3'.

[0047] As used herein, the term "encoding" refers to the natural property, and biological properties resulting therefrom, of a particular sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) serving as a template for the synthesis of other polymers and macromolecules in biological processes having either a predetermined sequence of RNA transcripts (i.e., rRNA, tRNA, mRNA) or a predetermined sequence of amino acids. Thus, a gene encodes a protein when a protein is produced in a cell or other biological system by transcription and translation of the mRNA produced by that gene. Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. Those skilled in the art will also understand that, using routine techniques, nucleotide substitutions that do not affect the polypeptide sequence encoded by a described polynucleotide may be made to reflect the codon usage of the particular host organism in which the polypeptide is expressed. Thus, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.

[0048] As used herein, the term "recombinant nucleic acid" refers to a polynucleotide or nucleic acid having a sequence not associated with it in nature. Recombinant nucleic acid may be in the form of a vector. A "vector" may contain a given nucleotide sequence of interest and regulatory sequences. Vectors may be used to express a given nucleotide sequence (expression vector) or to maintain a given nucleotide sequence for replication, manipulation, or transfer between different locations (e.g., between different organisms). Vectors can be introduced into an appropriate host cell for the above purposes. "Recombinant cell" refers to a host cell into which a recombinant nucleic acid has been introduced. "Transformed cell" refers to a cell into which a DNA molecule encoding a protein of interest has been introduced by recombinant DNA techniques.

[0049] Vectors can be of various types, including plasmids, cosmids, episomes, fosmids, artificial chromosomes, phages, and viral vectors. Generally, in a vector, a given nucleotide sequence is operably linked to a regulatory sequence such that, when the vector is introduced into a host cell, the given nucleotide sequence is expressed in the host cell under the control of the regulatory sequence. Regulatory sequences may include, but are not limited to, promoter sequences (e.g., cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, T7 promoter, and alcohol oxidase gene (AOX1) promoter), initiation codons, replication origins, enhancers, secretion signal sequences (e.g., α-mating factor signal), stop codons, and other regulatory sequences (e.g., Shine-Dalgarno sequence and termination sequence). Preferably, the vector further includes a marker sequence (e.g., antibiotic resistance marker sequence) for subsequent screening / selection procedures. For the purpose of protein production, the desired nucleotide sequence in the vector may be linked to another nucleotide sequence other than the above-mentioned regulatory sequence, so that a fusion polypeptide is produced and is useful for subsequent purification procedures, and the fusion polypeptide contains a tag for purification (e.g., a His tag).

[0050] As used herein, the term "treatment" means the application or administration of one or more active agents to a subject having a disorder, a symptom or condition of a disorder, a disorder caused by a disorder, or the progression or predisposition to a disorder, with the intent to cure, heal, alleviate, mitigate, alter, relieve, ameliorate, improve or affect the disorder, a symptom or condition of a disorder, or the progression of a disorder.

[0051] II. Antibodies to HSV According to the present invention, the anti-HSV antibodies used herein specifically bind to gD of HSV-1 and HSV-2 and can neutralize drug-resistant HSV strains. Viral neutralization typically occurs when the antibody binds to the virus and prevents it from infecting susceptible cells. The anti-HSV antibodies of the present invention have been found to exhibit high potency in neutralizing drug-resistant HSV strains. Neutralization potency can be measured using methods known in the art. For example, the anti-HSV antibodies of the present invention may have an IC50 of less than 100 nM in a viral plaque assay, e.g., about 1 nM to about 99 nM, e.g., about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 30 nM, about 1 nM to about 20 nM, or about 1 nM to about 10 nM.

[0052] The anti-HSV antibodies used herein have also been found to inhibit viral spread, including cell-to-cell and / or neuronal spread.

[0053] Exemplary anti-HSV antibodies include monoclonal antibody (mAb) E317 (i.e., UB-621 mAb described in the Examples below), E425, and Y571, described in U.S. Pat. No. 8,252,906, the entire contents of which are incorporated herein by reference.

[0054] According to U.S. Pat. No. 8,252,906, mAbs E317, E425, and Y571 each comprise a heavy chain variable region (V) with its complementarity determining regions (HC CDR1, HC CDR2, HC CDR3) as shown in Table 1. H ) and its complementarity-determining regions (LC CDR1, LC CDR2, LC CDR3). L ) is included.

[0055] [Table 1-1] [Table 1-2] [Table 1-3]

[0056] In some embodiments, the anti-HSV antibody of the invention comprises (a) a V CDR1 comprising an HC CDR1 of SEQ ID NO: 2, an HC CDR2 of SEQ ID NO: 4, and an HC CDR3 of SEQ ID NO: 6. H and (b) a V comprising an LC CDR1 of SEQ ID NO: 9, an LC CDR2 of SEQ ID NO: 11, and an LC CDR3 of SEQ ID NO: 13. L or an antigen-binding fragment thereof.

[0057] In some embodiments, (a) a V comprising an HC CDR1 of SEQ ID NO: 2, an HC CDR2 of SEQ ID NO: 4, and an HC CDR3 of SEQ ID NO: 6. H and (b) a V comprising an LC CDR1 of SEQ ID NO: 9, an LC CDR2 of SEQ ID NO: 11, and an LC CDR3 of SEQ ID NO: 13. L The anti-HSV antibody of the present invention has a V comprising an amino acid sequence of SEQ ID NO: 15 or substantially identical thereto. H and V comprising an amino acid sequence of SEQ ID NO: 16 or substantially identical thereto. L Specifically, the anti-HSV antibodies of the present invention may comprise a V comprising an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99%) identity to SEQ ID NO: 15. H and V comprising an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99%) identity to SEQ ID NO: 16. L Includes.

[0058] In some embodiments, the anti-HSV antibody of the invention comprises (a) a V CDR1 comprising an HC CDR1 of SEQ ID NO: 18, an HC CDR2 of SEQ ID NO: 20, and an HC CDR3 of SEQ ID NO: 22. Hand (b) a V comprising an LC CDR1 of SEQ ID NO: 24, an LC CDR2 of SEQ ID NO: 26, and an LC CDR3 of SEQ ID NO: 28. L A functional variant of mAb E425, or an antigen-binding fragment thereof, characterized in that it comprises:

[0059] In some embodiments, (a) a V comprising an HC CDR1 of SEQ ID NO: 18, an HC CDR2 of SEQ ID NO: 20, and an HC CDR3 of SEQ ID NO: 22. H and (b) a V comprising an LC CDR1 of SEQ ID NO: 24, an LC CDR2 of SEQ ID NO: 26, and an HC CDR3 of SEQ ID NO: 28. L The anti-HSV antibody of the present invention has a V comprising an amino acid sequence of SEQ ID NO: 30 or substantially identical thereto. H and V comprising an amino acid sequence of SEQ ID NO: 31 or substantially identical thereto. L Specifically, the anti-HSV antibodies of the present invention may comprise a V comprising an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99%) identity to SEQ ID NO: 30. H and V comprising an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99%) identity to SEQ ID NO: 31. L Includes:

[0060] In some embodiments, the anti-HSV antibody of the invention comprises (a) a VHV CDR1 comprising SEQ ID NO: 2, a HC CDR2 comprising SEQ ID NO: 33, and a HC CDR3 comprising SEQ ID NO: 6. H and (b) a V comprising an LC CDR1 of SEQ ID NO: 35, an LC CDR2 of SEQ ID NO: 11, and an HC CDR3 of SEQ ID NO: 38. L or an antigen-binding fragment thereof.

[0061] In some embodiments, (a) a V comprising an HC CDR1 of SEQ ID NO: 2, an HC CDR2 of SEQ ID NO: 33, and an HC CDR3 of SEQ ID NO: 6.H and (b) a V comprising an LC CDR1 of SEQ ID NO: 35, an LC CDR2 of SEQ ID NO: 11, and an HC CDR3 of SEQ ID NO: 38. L The anti-HSV antibody of the present invention has a V comprising an amino acid sequence of SEQ ID NO: 40 or substantially identical thereto. H and V comprising an amino acid sequence of SEQ ID NO: 41 or substantially identical thereto. L Specifically, the anti-HSV antibodies of the present invention may comprise a V comprising an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99%) identity to SEQ ID NO: 40. H and V comprising an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99%) identity to SEQ ID NO: 41. L Includes:

[0062] The anti-HSV antibodies of the present invention also include the related VHV antibodies described herein. H or V L Recombinantly (engineered) derived antibodies encoded by a polynucleotide sequence that encodes an amino acid sequence are also included.

[0063] The term "substantially identical" refers to the sequence of a variant (e.g., FR, CDR, V H or V LThis may mean that the relevant amino acid sequence (in the FR region) is not substantially different from the reference antibody, and the variant has substantially similar binding activity (e.g., affinity, specificity, or both) and biological activity to the reference antibody. Such variants may include minor amino acid mutations. It is understood that a polypeptide may have a limited number of mutations or modifications that can be made within a certain portion of the polypeptide, regardless of its activity or function, and still result in a variant with an acceptable level of equivalent or similar biological activity or function. In some examples, the amino acid residue mutation is a conservative amino acid substitution, which refers to an amino acid residue with a similar chemical structure to another amino acid residue, and has little or no substantial other biological effect on the function, activity, or properties of the polypeptide. Generally, in the FR region, as opposed to the CDR region, a relatively large number of substitutions can be made as long as they do not adversely affect the binding function and biological activity of the antibody (e.g., a 50% or greater decrease in binding affinity 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 can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, such as those described in references that summarize such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. For example, conservative amino acid substitutions include substitutions made 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.

[0064] 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 Fvs (scFvs), and (scFv)2 fragments. Antibodies or antigen-binding fragments thereof can be prepared by methods known in the art.

[0065] III. Antibody Preparation Numerous methods are available for obtaining antibodies or antigen-binding fragments thereof, which are conventional in the art.

[0066] In some embodiments, the antibodies provided herein can be produced by conventional hybridoma technology. Generally, a target antigen, optionally conjugated to a carrier protein and / or mixed with an adjuvant, can be used to immunize a host animal to produce antibodies that bind to that antigen. Lymphocytes secreting monoclonal antibodies are collected and fused with myeloma cells to produce hybridomas. The hybridoma clones thus formed are then screened to identify and select those secreting the desired monoclonal antibody.

[0067] In some embodiments, the antibodies provided herein can be prepared by recombinant techniques. In a related aspect, isolated nucleic acids encoding the disclosed amino acid sequences are also provided, along with vectors containing such nucleic acids and host cells transformed or transfected with the nucleic acids.

[0068] For example, nucleic acids containing 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) using conventional techniques, and any of the vectors can be introduced into appropriate cells (e.g., bacterial cells, yeast cells, plant cells, or mammalian cells) for antibody expression. Examples of mammalian host cell lines include human embryonic kidney (293) cells, baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO) cells, African green monkey kidney (Vero) cells, and human hepatocytes (Hep G2) cells. Recombinant vectors for antibody expression described herein typically contain a nucleic acid encoding the antibody amino acid sequence operably linked to either a constitutive or inducible promoter. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for controlling expression of the antibody-encoding nucleic acid. Vectors, if desired, contain selectable markers for both prokaryotic and eukaryotic systems. In some instances, the coding sequences for both the heavy and light chains are contained in the same expression vector, while in other instances, the heavy and light chains of an antibody are each cloned into separate vectors, which are produced separately and incubated under conditions suitable for antibody assembly.

[0069] Recombinant vectors for expressing antibodies described herein typically contain a nucleic acid encoding an antibody amino acid sequence operably linked to either a constitutive or inducible promoter. Recombinant antibodies can be produced in prokaryotic or eukaryotic expression systems, such as bacterial, yeast, insect, or mammalian cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for controlling expression of the antibody-encoding nucleic acid. Vectors also contain selectable markers for both prokaryotic and eukaryotic systems, as needed. The produced antibody protein can be further isolated or purified to obtain substantially homogeneous preparations for further assays and uses. Suitable purification procedures include, for example, fractionation 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.

[0070] When full-length antibodies are desired, the V H Chain and V L The coding sequence for either chain can be linked to the coding sequence for the Fc region of an immunoglobulin, and the resulting genes encoding full-length antibody heavy and light chains can be expressed and assembled in a suitable host cell, such as a plant cell, a mammalian cell, a yeast cell, or an insect cell.

[0071] Antigen-binding fragments can be prepared by conventional methods. For example, F(ab')2 fragments can be produced by pepsin digestion of full-length antibody molecules, and Fab fragments can be produced by reducing the disulfide bonds of F(ab')2 fragments. Alternatively, such fragments can be prepared recombinantly by expressing heavy and light chain fragments in suitable host cells and assembling them to form the desired antigen-binding fragment either in vivo or in vitro. Single-chain antibodies can be prepared recombinantly by linking a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions.

[0072] IV. Composition In accordance with the present invention, anti-HSV antibodies may be formulated with a pharmaceutically acceptable carrier into compositions for delivery and absorption.

[0073] As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition, preferably capable of stabilizing the active ingredient, and safe for the recipient. The carrier may be a diluent, vehicle, excipient, or matrix for the active ingredient. Generally, compositions containing the anti-HSV antibody described herein as an active ingredient may be in the form of a solution, such as an aqueous solution (e.g., physiological saline), or may be provided in powder form. The composition may further contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. Composition forms include suspensions, lotions, solutions, sterile injectable solutions, and packaged powders. The compositions of the present invention can be delivered via any physiologically acceptable route, such as parenteral (e.g., intramuscular, intravenous, subcutaneous, and intraperitoneal) and intranasal. In certain embodiments, the compositions of the present invention are administered as liquid injectable formulations, which may be provided as ready-to-use formulations or as stable powders that can be reconstituted.

[0074] V. Treatment The present invention provides methods for treating drug-resistant and / or recurrent HSV infection by administering anti-HSV antibodies described herein, which are effective in alleviating symptoms caused by HSV infection, delaying recurrence, and / or reducing the frequency of recurrence.

[0075] As used herein, the term "drug resistance" may refer to a virus's ability to survive exposure to one or more antiviral drugs. Specifically, drug resistance to an antiviral drug indicates that normal doses of the antiviral drug do not inhibit viral replication or that the drug does not demonstrate clinical efficacy against the virus. The term "multidrug resistance" may refer to a virus that is resistant to multiple antiviral drugs.

[0076] In some embodiments, the drug-resistant HSV strain has a mutation in the tyrosine kinase (TK) enzyme or DNA polymerase enzyme. 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 (phosphonoformic acid (PFA)), and cidofovir (CDV). In some embodiments, the strains include clinical isolates such as HSV-1 RH (US origin), HSV-1 Bethesda (US origin), HSV-2 MO (US origin), HSV-2 Bethesda (US origin), HSV-2 JA-1 (Asia origin), HSV-2 JA-2 (Asia origin), HSV-2 JA-3 (Asia origin), HSV-2 poly-mut (polymerase mutant), HSV-2 C7 (TK mutant), C8 (TK mutant), IC (ACV resistant), etc. In some embodiments, the strains include clinical isolates (German origin) such as HSV-1 R2, R4, R7, R8, R9, R10, R11, and R13, and HSV-2 R5, R6, and R14.

[0077] The methods of the invention using the anti-HSV antibodies described herein are effective against drug-resistant HSV infections. In some embodiments, the anti-HSV antibodies described herein have low IC against some drug-resistant HSV strains. 50 (e.g., 1 nM to 30 nM) and high IC against several drug-resistant HSV strains 50 and are 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) more potent than acyclovir (e.g., 100 nM to 35,000 nM or more). In some embodiments, administration of the anti-HSV antibodies described herein at a single dose of 15-50 mg / kg is effective in protecting animals infected with drug-resistant HSV from death.

[0078] As used herein, the term "relieving symptoms" or "symptom reduction" can refer to an effective agent that reduces or eliminates one or more symptoms of a disease or other abnormal condition. The severity of disease symptoms can be determined by any suitable index or score known in the art. Generally, a higher level of the index or a higher score indicates a more severe disease. In some embodiments, animals with HSV infection are observed for clinical disease, where clinical symptoms can be scored as 0 for no lesions; 1 for erythema only; 2 for one or a few blisters; 3 for ulcerated lesions; and 4 for lesions scored 3 plus abnormal movement. In some embodiments, animals with HSV infection observed for clinical disease can be scored as follows: normal (0), slight redness of the external genitalia (1), swelling and redness of the external genitalia and / or pus / mucous (2), severe swelling of the external genitalia with pus / mucous and some surrounding hair loss (3), ulceration, redness, and swelling of the genital tissues (4), ulceration, redness, swelling, and increasing hind leg paralysis (5), and death (6). In some embodiments, a reduction in symptoms can include, for example, a 5%, 10%, 15%, 20%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more reduction in the level or score of such indicators compared to the disease level or disease score of such indicators that a skilled artisan and / or medical professional, e.g., a physician, would expect a diseased individual or population with similar physical characteristics and medical history to have. In some embodiments, symptomatic relief can mean that one or more subjective symptoms of a disease or other abnormal condition are alleviated to normal.

[0079] As used herein, "recurrent infection" refers to a recurrence or recurrence of infection, e.g., a second, third, or subsequent episode of infection in a patient after the previous / initial infection episode has been deemed cured. The initial onset of symptoms is typically referred to as primary disease, and subsequent occurrences are referred to as recurrent disease. Clinically, recurrent HSV infection may indicate reactivation of the same HSV in the lesions, as indicated by serum antibodies. A patient with recurrent infection may indicate that the virus has reactivated. In certain embodiments, patients with genital herpes experience one or more recurrences per month, some patients experience one recurrence per 2-4 months, and some patients experience less than one recurrence per 4 months. In certain embodiments, patients with oral herpes experience one or more recurrences per month, some patients experience one recurrence per 2-4 months, and some patients experience less than one recurrence per 4 months.

[0080] As used herein, the term "treating recurrent HSV infection" can refer to a reduction in the severity, frequency, duration, and / or amount of one or more recurrent viral symptoms in an infected individual, or a reduction in the mean or median severity, frequency, duration, and / or amount of one or more recurrent viral symptoms in a population of individuals. In certain embodiments, administration of an anti-HSV antibody described herein is effective in delaying the occurrence of relapses. In certain embodiments, administration of an anti-HSV antibody described herein is effective in reducing the number or frequency of relapses. In some examples, administration of an anti-HSV antibody described herein to a patient population with herpetic lesions results in a median absence of clinical symptoms for at least one month after treatment is discontinued. Preferably, the relapse-free period is 2-3 months or longer.

[0081] As used herein, the term "effective amount" refers to the amount of an active ingredient that provides a desired biological effect to a treated subject or cell. For example, the effective amount described herein may be the amount of an anti-HSV antibody as an active agent that can alleviate symptoms of HSV infection and / or delay recurrence. The effective amount may vary depending on various factors, such as the route and frequency of administration, the weight and species of the individual receiving the drug, and the purpose of administration. Those skilled in the art can determine the dosage for each case based on the disclosure herein, established methods, and their own experience.

[0082] In some embodiments, the anti-HSV antibodies described herein are administered at a dose range of 0.01-100 mg / kg, particularly 0.1-100 mg / kg, more particularly 1-80 mg / kg, and even more particularly 10-50 mg / kg of the subject's body weight. In some examples, the anti-HSV antibodies described herein are administered at a dose range of 1-10 mg / kg, e.g., 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.

[0083] In some embodiments, the antibody is administered in a single dose in the composition at full dose. Generally, the sooner the antibody is administered, the better. In some embodiments, the antibody is administered early in the course of infection, for example, within 10 days (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day) after infection. Preferably, the antibody is administered within 5 days (e.g., 5, 4, 3, 2, or 1 day) after infection.

[0084] The subject treated by the treatment methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, livestock, sport (race) animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment is a human patient who has, is at risk for, or is suspected of having a disease / disorder of interest. A subject suspected of having any of these disease / disorders may exhibit one or more symptoms of the disease / disorder. A subject at risk of a disease / disorder may have one or more risk factors for the disease / disorder. In some embodiments, the subject is a patient infected with HSV or a patient with a weakened immune system, such as an infant, pregnant woman, cancer patient, organ transplant recipient, or human immunodeficiency virus (HIV) carrier. In some embodiments, the subject is a patient with an HSV infection that is inadequately controlled by small molecule drugs.

[0085] In some embodiments, the anti-HSV antibodies described herein are administered to a subject in need thereof during the incubation period (early / asymptomatic period). Typically, the incubation period for HSV-1 and HSV-2 symptoms is 2-12 days. Thus, in some examples, the anti-HSV antibodies described herein are administered to a subject in need thereof within 10 days after infection, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day after infection.

[0086] In some embodiments, the anti-HSV antibodies described herein are administered to a subject in need thereof after the onset of symptoms. In some examples, the anti-HSV antibodies described herein are administered to a subject in need thereof after the onset of primary or primary symptoms. In some examples, the anti-HSV antibodies described herein are administered to a subject in need thereof after the onset of symptoms during a recurrent phase. In some examples, the anti-HSV antibodies described herein are administered to a subject in need thereof 10 days or more after infection, for example, 12, 15, 20, 25, 30 or more days after infection.

[0087] In certain embodiments, the anti-HSV antibodies described herein are administered infrequently to a subject in need thereof. In some embodiments, the anti-HSV antibodies described herein are administered weekly or less frequently, e.g., every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or less frequently. In some embodiments, the anti-HSV antibodies described herein are administered once every 1 to 3 months, e.g., once per month, once every 2 months, or once per 3 months.

[0088] In certain embodiments, administration of an anti-HSV antibody described herein is effective in reducing the number or frequency of relapses. In some instances, administration of a single dose of an anti-HSV antibody during a relapse period reduces the number of relapses to three or fewer 30 days after administration, compared to three or more (e.g., four or five) relapses resulting from multiple doses of a small molecule antiviral agent (see Figure 14B).

[0089] The present invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. Those skilled in the art will, in light of the description herein, appreciate that many changes can be made in the specific embodiments described and still obtain like or similar results without departing from the spirit and scope of the invention. [Example]

[0090] Example Example 1: Determination of the binding affinity of UB-621 to recombinant gD by surface plasmon resonance (SPR) analysis Kinetic binding assays of UB-621 (anti-HSV-gD mAb) with recombinant gD were performed using surface plasmon resonance (SPR) technology on a Biacore X100 instrument. First, an anti-human IgG Fc fragment antibody was immobilized on the surface of a CM5 gold sensor chip via amine coupling. UB-621 was diluted to 5 mg / mL in running buffer (2-fold serial dilutions, 6.25–100 nM) and captured with the anti-FC antibody on the CM5 chip. A gD sample diluted in HBS-EP buffer was injected, including a reference cell. The dissociation rate constant (kd), association rate constant (ka), and dissociation equilibrium constant (KD) were automatically calculated using the 1:1 model and steady-state affinity using Biacore Evaluation software.

[0091] The average KD for UB-621 is 1.35 x 10 -10 It was estimated to be M (Figure 1).

[0092] Example 2: Neutralizing effect of UB-621 on HSV-1 and HSV-2 laboratory strain infections The efficacy of UB-621 against HSV infection was first investigated in Vero cells by cell viability assay, and IC 50 and IC 90 The four laboratory HSV strains tested were HSV-1 KOS, HSV-1 RE, HSV-2 333, and HSV-2 G. The virus strains were mixed with serial concentrations of UB-621 for 1 hour at room temperature. The mixture was then dispensed into a 96-well plate seeded with Vero cells. After 48 hours of incubation at 37°C, the plate was washed once with PBS, and viable cells were incubated with 1% Alamar Blue for 1 hour. Fluorescence at 530 nm and 590 nm was measured, and the cell death rate was calculated.

[0093] UB-621 is an IC 50 and IC 90 As shown by the low levels of β-glucan (Table 2), all HSV-1 and HSV-2 strains in the laboratory were completely neutralized with high efficacy (Fig. 2).

[0094] [Table 2]

[0095] Example 3: Therapeutic effect of UB-621 on genital HSV-2 infection 3.1 Post-infection treatment before symptoms appear Six- to eight-week-old female BALB / c mice were used in an HSV-2 intravaginal infection model (Marshak JO, et al., 2014). Mice were randomly assigned to an hIgG control group and an UB-621-treated group (n = 6). Four and one days before HSV-2 intravaginal infection, mice were subcutaneously injected with 2.5 mg of progesterone in the upper back. Then, mice were inoculated with 10 μL of virus suspension (HSV-2 strain 333, 1 × 10 5 PFU) were inoculated intravaginally. On day 1 postinfection, each mouse received either 300 μg of UB-621 (UB-621 treatment group) or the same amount of human IgG (hIgG control group) via subcutaneous injection. Clinical signs of genital infection were scored on days 1, 2, 3, 4, 5, 6, 7, 8, 10, and 12 postinfection according to a scale of 0 to 5 (Gill N, et al., 2005): 0 = no signs of infection; 1 = slight redness of the external genitalia; 2 = swelling and redness of the external genitalia and / or pus / mucus; 3 = severe swelling and pus / mucus of the external genitalia and surrounding hair loss; 4 = ulceration, redness, and swelling of the genital tissues; 5 = ulceration, redness, increasing swelling, and paralysis of the hind legs; and 6 = death.

[0096] A single 300 μg dose of UB-621 suppressed genital symptoms and signs to within a score of 2 compared with a score of 5 in hIgG-treated mice on day 7 post-HSV-2 infection (Fig. 3A). Survival of UB-621-treated mice was extended by 5.5 days compared with hIgG-treated mice (12 days vs. 6.5 days) (Fig. 3B).

[0097] 3.2 Post-infection treatment after the onset of infection symptoms Mice were randomly divided into an hIgG control group and an UB-621-treated group (n = 3). Four days and one day before intravaginal HSV-2 infection, mice were subcutaneously injected with 2.5 mg of progesterone in the upper back. Then, mice were injected with 10 μL of a virus suspension (HSV-2 strain 333, 1 × 10 5 Four days after infection, each mouse was subcutaneously injected with 300 μg of UB-621 (UB-621 treatment group) or the same amount of human IgG (hIgG control group).

[0098] A single 300 μg dose of UB-621 suppressed genital symptoms and signs to a score of 3 on day 12 post-HSV-2 infection, compared with scores of 5–6 in hIgG-treated mice (Figure 4A). Mice treated with UB-621 survived 100% longer than mice treated with hIgG, with a median survival of 8 days (Figure 4B).

[0099] Example 4: Neutralizing effect of UB-621 on HSV-1 RE TKnull mutant strain and HSV-2 333 TKnull mutant strain in Vero cells A comparative study of UB-621 versus acyclovir (ACV) was performed using Vero cells infected with two HSV laboratory mutants, analyzing their inhibitory activity by plaque assay. Wild-type HSV-1 RE strain and HSV-2 333 strain were mutated by inserting enhanced green fluorescent protein (EGFP) into the TK gene, resulting in HSV-1 RE TKnull mutants and HSV-2 333 TKnull mutants resistant to ACV. Vero cells seeded in 12-well plates were infected with HSV-1 RE mutants or HSV-2 mutants (MOI = 0.01) at 37°C for 75 minutes. The infected cells were then cultured for 24 hours in medium supplemented with or without serial concentrations of UB-621 or ACV. Viral plaques were counted, and IC values ​​were calculated. 50 The half-maximal inhibitory activity, expressed as .times. ...

[0100] UB-621 neutralized both virus strains with much higher potency than ACV (Figures 5A and 5B). UB-621 neutralized both virus strains with low IC values ​​of 11.3 nM for the HSV-1 RE mutant and 28.1 nM for the HSV-2 333 mutant. 50 The IC of UB-621 inhibited the infection (Table 3). 50 The value may indicate a 1000-fold increase in potency over ACV.

[0101] [Table 3]

[0102] Example 5: Neutralizing activity of UB-621 against clinically derived HSV-1 and HSV-2 isolates 5.1 Strains originating from the US and Asia and ACV-resistant strains The inhibitory efficacy of UB-621 and ACV against clinical HSV-1 and HSV-2 isolates was compared against 11 strains from the United States, Asia, and acyclovir resistance. Four isolates from the United States (HSV-1 Bethesda and HSV-1 RH; HSV-2 Bethesda and HSV-2 RH), three isolates from Asia (HSV-2 JA-1, HSV-2 JA-2, HSV-2 JA-3), and four ACV-resistant isolates (poly-mut, C7, C8, and IC) were tested by cell viability assay. The virus isolates were mixed with UB-621 or ACV at serial concentrations for 1 hour at room temperature. The mixtures were then dispensed into 96-well plates seeded with Vero cells. After 48 hours of incubation at 37°C, the plates were washed once with PBS, and viable cells were incubated with 1% Alamar Blue for 1 hour. Fluorescence at 530 nm and 590 nm was measured, and cell death was calculated.

[0103] UB-621 is at least 500 times more potent than ACV (lower IC 50 Value and IC 90 values) neutralized all clinical HSV-1 and HSV-2 strains (Figs. 6A, 6B, and 6C) (Table 4).

[0104] [Table 4]

[0105] 5.2 Strains from Germany and other drug-resistant strains UB-621 serially diluted from 250 to 0 nM was used in a viral load of 100 TCID 50 The antibody was incubated with drug-resistant HSV-1 or HSV-2 isolates in cell culture medium for 1 hour at 37°C. The antibody virus was inoculated onto Vero cell monolayers grown in 96-well plates. Cytopathic effect (CPE) was scored by light microscopy after 48 hours of incubation. The antibody concentration required to completely neutralize the virus was defined as the neutralization titer.

[0106] UB-621 is an IC 50 UB-621 was able to completely neutralize all tested drug-susceptible or single- or multidrug-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 extremely low concentrations of 7.8–31.3 nM, compared with small molecule antivirals that inhibit HSV at low- to high-μM levels (Figure 7). The neutralizing effect of UB-621 was independent of the virus strain origin and resistance status.

[0107] Example 6: Efficacy test of UB-621 against infection with ACV-resistant HSV-1 clinical isolates in mice BALB / c mice were divided into three groups, with six mice per group. After anesthetizing each mouse, the cornea of ​​the right eye of each mouse was incised with a needle, and 5 × 10 6 Five microliters of virus inoculum containing pfu of an ACV-resistant HSV-1 clinical isolate was applied. UB-621 (50 mg / kg) or human IgG (50 mg / kg) was administered subcutaneously to each designated mouse once on day 1 postinfection. Acyclovir (ACV) was administered orally at 125 mg / kg twice daily on days 1 and 2 postinfection. All mice were sacrificed on day 3 postinfection, and the right eye, trigeminal ganglion, and brain were harvested to measure the viral load in the tissues by plaque assay.

[0108] Compared with human IgG (control), 50 mg / kg UB-621 significantly reduced viral replication in the eye (the site of viral inoculation) (Figure 8). There was no significant difference between the ACV-treated and control groups. However, because this HSV clinical isolate had difficulty infecting and replicating in all mouse neural tissues (trigeminal ganglion and brain), differences in efficacy in these tissues were not assessed.

[0109] Example 7: Efficacy test of UB-621 against infection with ACV-resistant HSV-2 clinical isolates in mice 7.1 HSV-2 clinical isolate IC strain Six- to eight-week-old female BALB / c mice were susceptible to vaginal HSV infection by subcutaneous injection of 0.1 ml of a suspension containing 2.5 mg of progesterone 4 and 1 days before challenge, as previously described. 6 Mice were inoculated intravaginally with 10 μL of a suspension containing pfu of an HSV-2 clinical isolate (IC strain) (Figure 9). A single subcutaneous dose of UB-621 was administered on day 1 postinfection, or multiple oral doses of ACV were administered on days 1–5. Animals were then followed daily for at least 15 days. The severity of vaginitis was scored on a composite scale of 0 to 6.

[0110] UB-621 completely protected mice from viral infection at dose levels between 15 mg / kg and 50 mg / kg (Fig. 9A). A single dose of UB-621 significantly suppressed genital symptoms in a dose-dependent manner compared with saline control and ACV-treated mice (Fig. 9B).

[0111] 7.2 HSV-2 clinical isolate pol-mut strain Another study was performed using a different clinical isolate, the pol-mut strain. Briefly, animals were treated with 1 × 10 6 Ten microliters of a suspension containing pfu of a clinical HSV-2 isolate, pol-mut, was inoculated intravaginally (Figure 10). A single subcutaneous dose of UB-621 was administered on day 1 post-infection, or multiple oral doses of ACV were administered on days 1–5. Animals were then followed daily for at least 15 days.

[0112] Similar results were observed in a clinical isolate of UB-621 against the pol-mut strain. UB-621 completely protected mice from death at dose levels of 15 mg / kg to 50 mg / kg (Figure 10A). A single dose of UB-621 significantly suppressed reproductive symptoms in a dose-dependent manner compared with saline control mice (Figure 10B).

[0113] Example 8: Inhibition of cell-to-cell spread of HSV-1 and HSV-2 by UB-621 Inhibition of cell-to-cell spreading by UB-621 was tested using a modified method previously described (Krawczyk A, et al., 2013). Briefly, Vero cells were cultured in 24-well plates at 1x10 5 Cells were seeded at 1000 cells / well. Confluent cell cultures were infected with 200 TCID50 HSV-1 or HSV-2-ΔgE-GFP reporter virus / well. After 2 hours of incubation, the inoculation medium was removed, and the cell cultures were incubated with serial dilutions of UB-621 (0–1000 nM). After 2 days of incubation, plaque formation was observed under a fluorescent microscope. 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 under a fluorescent microscope.

[0114] Plaque formation by HSV-1 and HSV-2 decreased with increasing UB-621 concentration. Complete neutralization of HSV-1 was observed at a UB-621 concentration of 1000 nM (150 μg / mL) (Fig. 11A). UB-621 clearly reduced plaque formation against HSV-2, evidence of its inhibitory activity against cell-to-cell spread (Fig. 11B).

[0115] Example 9: Inhibition of HSV-1 anterograde interneuronal spread in BALB / c mice by UB-621 Female BALB / c mice were inoculated with 1 × 10 HSV-1 RE strain. 6pfu, 5 μL per mouse was inoculated into the cornea, and the right eye was infected. One day after infection, a single dose of 15 mg / kg of UB-621 was administered intraperitoneally. Five mice (n = 5) per group were sacrificed on day 5 after infection for analysis of viral load in the right eye, right trigeminal ganglion, and whole brain.

[0116] The results showed that the presence of UB-621 could significantly inhibit interneuronal transmission from the eyeball to the trigeminal ganglion and brain (Figure 12).

[0117] Example 10: Efficacy study of UB-621 against recurrent HSV-2 infection in guinea pigs The guinea pig reproductive model differs from the mouse model in that it exhibits both acute disease and spontaneous recurrence. Female Hartley guinea pigs (200-300 g) were given 10 6 Mice were infected genitally with PFU of HSV-2 strain 333. To examine the efficacy of UB-621 when administered during primary and recurrent HSV infections and its effect on recurrence (Figures 13A and 13B, and Figures 14A and 14B), a single subcutaneous dose of UB-621 (30 or 60 mg / kg) was administered on days 1 or 20 postinfection. Multiple oral doses of acyclovir (125 mg / kg, twice daily) were administered on days 1–7 or 20–26 postinfection. Control animals received saline subcutaneously and orally. The severity of symptoms after infection was assessed by direct examination of the external genital skin and by the animal's activity. Each animal was observed daily for 55 days (acute infection treatment; Figure 13A) or 40 days after treatment (relapse treatment; Figure 14A). A clinical scale was used, with no lesions scored as 0, erythema alone scored as 1, one or a few blisters scored as 2, ulcerated lesions scored as 3, and lesions scored as 3 plus abnormal movement scored as 4. Animals were humanely sacrificed when symptoms reached a score of 4. Relapse was defined as clinical signs more severe than the previous day.

[0118] The results showed that a single dose of UB-621 effectively alleviated clinical symptoms of HSV vaginitis, whether administered to infected animals at the time of primary infection (Figure 13A) or after the first HSV recurrence (Figure 14A). Treatment with UB-621 not only delayed the onset of recurrence but also reduced the cumulative number of recurrences compared with treatment with saline or ACV (Figures 13B and 14B).

[0119] 3. Conclusion UB-621 is effective in neutralizing wild-type and drug-resistant HSV-1 and HSV-2 (Examples 2, 4, and 5), and a single dose of UB-621 reduces disease symptoms and increases survival in HSV-infected mice and guinea pigs (Examples 3, 6, 7, 9, and 10).

[0120] UB-621 effectively inhibits in vitro replication of HSV in all 23 drug-resistant HSV clinical isolates that are resistant to several small molecule drugs (eg, ACV, PFA) (Example 5).

[0121] Many patients suffer from drug-resistant HSV infections, necessitating the use of higher doses of small molecule drugs for treatment. A single dose of UB-621 can reduce disease symptoms and increase survival rates in primary HSV infections with drug-resistant strains (Examples 6 and 7).

[0122] Some patients experience recurrence of HSV symptoms when their immune system weakens or when small molecule drug therapy is discontinued. A single dose of UB-621 administered at the primary HSV infection stage shows potent therapeutic effects in a guinea pig model of recurrent HSV (Example 10).

[0123] The significant therapeutic effect of UB-621 in animals may be related to the neutralization of HSV gD and the inhibition of cell-to-cell spread of HSV (Examples 8 and 9).

[0124] Currently, there are no approved mAb drugs for the treatment of HSV, and small molecule drugs have many limitations and side effects. UB-621 has shown remarkable therapeutic efficacy in animal models and may be effective in patients infected with drug-resistant HSV strains whose symptoms are insufficiently controlled with small molecule drugs.

[0125] References Boado RJ, Zhou QH, Lu JZ, et al. Pharmacokinetics and brain uptake of a genetically engineered bifunctional fusion antibody targeting the mouse transferrin receptor. Mol Pharmacol 7:237244 (2010). Eddie C hang, Laurence Galle, David Maggs, D. Mark Estes, and William J. Mitchell. Pathogenesis of Herpes Simplex Virus Type 1 Induced Corneal Inflammation in Perforin Deficient Mice. (2000) Journal of Virology. 74: 11832 11840. Gill N, Rosenthal KL, Ashkar AA. NK and NKT cell-independent contribution of interleukin-15 to innate protection against mucosal viral infection. J. Virol. 79:4470-4478 (2005). Krawczyk A, Arndt MA, Grosse-Hovest L, Weichert W, Giebel B, Dittmer U, Hengel H, Jager D, Schneweis KE, Eis-Hubinger AM, Roggendorf M, Krauss J.Overcoming drug-resistant herpes simplex virus (HSV) infection by a humanized antibody. PNAS, USA 110:6760-6765 (2013). Marshak JO, Dong L, Koelle DM, et al. The Murine Intravaginal HSV-2 Challenge Model for Investigation. Methods Mol Biol. 1144:305-327 (2014). Pereira L, Ali M, Kousoulas K, Huo B, Banks T. Domain structure of herpes simplex virus 1 glycoprotein B: neutralizing epitopes map in regions of continuous and discontinuous residues. Virology 172:11-24 (1989). 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-1 Li81 antibody in the central nervous system and dose-efficacy relationships in rat spinal cord remyelination 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 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).

2. 10. The method of claim 1, wherein the anti-HSV antibody is effective in neutralizing drug-resistant HSV strains and / or inhibiting viral spread.

3. Anti-HSV antibodies (a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO:2, a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO:4, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO:6; and (b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 9, a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 11, and a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO:

13.

3. The method of claim 1 or 2, comprising:

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

16.

5. Anti-HSV antibodies (a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 20, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 22; and (b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 24, a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 26, and a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO:

28.

3. The method of claim 1 or 2, comprising:

6. VH comprises the amino acid sequence of SEQ ID NO: 30; and / or VL comprises the amino acid sequence of SEQ ID NO: 31; The method of claim 5.

7. (a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence of SEQ ID NO: 33, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 35, a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence of SEQ ID NO: 11, and a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence of SEQ ID NO:

38.

3. The method of claim 1 or 2, comprising:

8. VH comprises the amino acid sequence of SEQ ID NO: 40; and / or VL comprises the amino acid sequence of SEQ ID NO: 41; The method of claim 7.

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

12. 12. The method of any one of claims 1 to 11, wherein the subject is a patient with a weakened immune system.

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

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

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

18. 11. An anti-HSV antibody or composition thereof according to any one of claims 1 to 10 for use in treating drug-resistant and / or recurrent HSV infection in a subject in need thereof.

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

20. the subject is susceptible to or infected with drug-resistant HSV; the subject is a patient with a weakened immune system; A single dose of anti-HSV antibody is administered to the subject; Anti-HSV antibodies are administered to the subject at an early stage of infection; The anti-HSV antibody is administered in an amount effective to reduce symptoms caused by HSV infection; The anti-HSV antibody is administered in an amount effective to delay recurrence and / or reduce the frequency of recurrence; and / or The anti-HSV antibody is administered to the subject after the onset of symptoms.

20. An anti-HSV antibody or composition thereof for use according to claim 18, or use of an anti-HSV antibody according to claim 19.

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