Novel anti-HSV antibody

Abstract

(A first) anti-HSV antibody or an antigen-binding fragment thereof that binds to glycoprotein B (gB) of HSV-1 and / or HSV-2, wherein the antibody comprises complementarity-determining regions V each containing the sequences defined in the claims H CDR1, V H CDR3, V L CDR1, V L CDR2, and V L CDR3, and wherein the antibody or its antigen-binding fragment has a maximum dissociation rate k -4 s -1 of at most 5.0 x 10 -4 s -1 preferably at most 1.0 x 10 -5 s -1 most preferably at most 2.9 x 10 -5 s -1 is described. Further, (A) the (first) anti-HSV antibody or its antigen-binding fragment, and (B) a second anti-HSV antibody or its antigen-binding fragment that recognizes / binds to glycoprotein B (gB) of HSV-1 and / or HSV-2, wherein the antibody comprises complementarity-determining regions V each containing the sequences defined in the claims dis CDR1, V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, and V LA combination of a second anti-HSV antibody or its antigen-binding fragment is described, comprising CDR3, wherein the second antibody has a dissociation constant Kd of up to 40 nM, preferably up to 30 nM, more preferably up to 20 nM, even more preferably up to 15 nM, up to 13 nM, and up to 10 nM. Furthermore, a pharmaceutical composition is described comprising an effective amount of the anti-HSV antibody or its antigen-binding fragment, or the combination of the antibodies, and at least one pharmaceutically acceptable excipient. Furthermore, the anti-HSV antibody or its antigen-binding fragment or the combination of the antibodies is described for use in a method for the prophylactic or therapeutic treatment of a disorder or disease as defined in the claims. Furthermore, (A) V, which is the complementarity-determining region of the first antibody described above. H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, and V L (B) The first binding domain containing CDR3 and (B) the complementarity determining region of the second antibody described above. H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, and V L A bispecific antibody that binds to glycoprotein B(gB) of HSV-1 and / or HSV-2, comprising a second binding domain containing CDR3, with a maximum capacity of 5.0 x 10⁻¹⁴. -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 A bispecific antibody having a low dissociation rate of kdis is described. Finally, a triplicate antibody is described that includes a third binding domain in addition to the first and second binding domains described for the bispecific antibody.

Description

Technical Field

[0001] The present invention relates to a (first) anti-HSV antibody or an antigen-binding fragment thereof that binds to glycoprotein B (gB) of HSV-1 and / or HSV-2, wherein the antibody comprises complementarity-determining regions V dis CDR1, V H CDR3, V L CDR1, V L CDR2, and V L CDR3, and the antibody or antigen-binding fragment thereof has a low dissociation rate k -4 s -1 , preferably at most 1.0×10 -4 s -1 , at most 5.0×10 -5 s -1 , most preferably at most 2.9×10 -5 s -1 of, (first) anti-HSV antibody or antigen-binding fragment thereof. Further, the present invention relates to (A) the (first) anti-HSV antibody or antigen-binding fragment thereof, and (B) a second anti-HSV antibody or antigen-binding fragment thereof that recognizes / binds to glycoprotein B (gB) of HSV-1 and / or HSV-2, wherein the antibody comprises complementarity-determining regions V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, and V L ​​The present invention relates to a combination of a second anti-HSV antibody or its antigen-binding fragment, comprising CDR3, wherein the second antibody has a dissociation constant Kd of up to 40 nM, preferably up to 30 nM, more preferably up to 20 nM, even more preferably up to 15 nM, up to 13 nM, and up to 10 nM. Furthermore, the present invention relates to a pharmaceutical composition comprising a combination of the anti-HSV antibody or its antigen-binding fragment or an effective amount of the antibody and at least one pharmaceutically acceptable excipient. Furthermore, the present invention relates to the anti-HSV antibody or its antigen-binding fragment or the combination of the antibody for use in a method for prophylactic or therapeutic treatment of a disorder or disease as defined in the claims. Finally, the present invention relates to (A) V, which is the complementarity-determining region of the first antibody described above. H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, and V L (B) The first binding domain containing CDR3 and (B) the complementarity determining region of the second antibody described above. H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, and V L A bispecific antibody that binds to glycoprotein B(gB) of HSV-1 and / or HSV-2, comprising a second binding domain containing CDR3, with a maximum capacity of 5.0 x 10⁻¹⁴. -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis The present invention relates to a bispecific antibody having the above characteristics. Finally, the present invention relates to a triplicate antibody comprising a third binding domain in addition to the first and second binding domains described for the bispecific antibody. [Background technology]

[0002] Herpes simplex virus (HSV) refers to two closely related members of the herpesviridae family, herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2), which accounted for approximately 67% and 13% of people aged 15–49 worldwide in 2016, respectively.[1] HSV-1 and HSV-2 are among the most common viral infections worldwide, transmitted through close contact and resulting in lifelong infections. HSV-1 infection is often acquired in infancy as an asymptomatic infection, while some cases present with severe illness. HSV-2 is usually acquired through sexual contact and can cause recurrent chronic lesions in the genital area. Herpes simplex virus infections are classified into one of several different disorders based on the site of infection. Oral herpes (herpes simplex labialis), whose visible symptoms are colloquially called cold sore or fever blister, is an infection of the face or mouth. Oral herpes is the most common form of infection. Genital herpes (herpes simplex vulva) is the second most common form of herpes. Herpetic paronychia, xiphoid herpes, eczema herpeticum, herpetic folliculitis, ocular herpes (herpes simplex retinitis, herpes simplex conjunctivitis, herpes simplex keratitis, or herpes simplex keratitis), herpes simplex encephalitis, Moraley's meningitis, neonatal herpes simplex, and possibly Bell's palsy are all caused by HSV-1 or HSV-2 infection, but both viruses can also infect and affect organs such as the lungs, kidneys, and liver. Serological studies have correlated HSV-1 infection with a high risk of developing Alzheimer's disease (AD) or dementia. This suggests that persistent HSV-1 infection may influence the development of neurological disorders, and that anti-HSV therapy may be used to prevent these neurological disorders or to extend the time until they develop.

[0003] Outcomes of HSV-1 or HSV-2 infection are mostly asymptomatic, but can range from mild symptoms to life-threatening conditions, with a wide range of pathological outcomes. After primary infection, HSV spreads from infected epithelial cells to the axons of sensory neurons, primarily in the peripheral nervous system, supplying the primary infection site. It is then transported retrogradely to the respective dorsal root ganglia, where the virus establishes a lifelong latent storage site. While this is associated with high morbidity and mortality in central nervous system infections, HSV has also been found to establish latent infection in brain organoids in vitro. This suggests that a similar process may occur in vivo. Like all herpesviruses, HSV-1 and HSV-2 have a lytic phase and a latent phase, and reactivation from latent infection to lytic replication can be triggered by a variety of factors, such as immunosuppression, which can lead to retrograde viral transport from neurons to epithelial cells, inducing vesicle and lesion formation. Therefore, intermittent HSV reactivation leads to the production of infectious HSV from latent infected neurons. Initial reactivation events from latent infection to lytic replication occur more frequently than expected, and these events may only occasionally lead to symptomatic disease. Suppression of HSV reactivation and transmission under an immune response state may be a result of tissue-resident immune cells that act immediately at the site and time of HSV reactivation, thereby blocking transmission. Viral reactivation can be triggered by widespread stress stimuli acting on neurons, peripheral sites innervated by infected ganglia, or systemically (e.g., immunodeficiency, illness, trauma, fever, menstruation, UV light, and sexual intercourse).

[0004] The development of HSV infection can be caused by the direct cytopathic effect of the virus, which leads to cell lysis and local necrosis of the infected area, or by an indirect immune response, such as in the case of HSV interstitial keratitis or encephalitis [2]. Herpes simplex is mostly transmitted by direct contact with lesions or bodily fluids of an infected individual. Oral herpes is easily diagnosed if the patient presents with visible sores or ulcers. Transmission can also occur through skin-to-skin contact during periods of asymptomatic viral shedding. Barrier protection methods are the most reliable way to stop herpes transmission, but they only reduce the risk, not eliminate it.

[0005] There is currently no cure for herpes. Once infected, the virus remains in the body for life. Occasionally, symptomatic relapses (acute outbreaks) occur. However, after several years, the severity of the acute outbreaks decreases, they become sporadic, and some people become permanently asymptomatic, never experiencing acute outbreaks again, although they may still be able to infect others. Treatment with antiviral drugs can reduce viral shedding and lessen the severity of symptomatic episodes. Oral herpes simplex (also known as labial herpes, recurrent labial herpes, or oral herpes) is a type of herpes simplex disease that occurs on the lips, an infection caused by HSV-1 or HSV-2. Acute outbreaks typically cause small blisters or sores on or around the mouth, commonly known as labial herpes or febrile herpes. While sores typically heal within 2-3 weeks, the herpes virus remains dormant in the facial nerve after an oral-facial infection, reactivating periodically (in symptomatic individuals) to create sores in the same area of ​​the mouth or face where the initial infection occurred. Oral herpes has a frequency rate that varies from rare episodes to more than 12 relapses per year. Individuals with this condition typically experience 1-3 acute outbreaks per year. The frequency and severity of acute outbreaks generally decrease over time.

[0006] Genital herpes simplex (or genital herpes) is a genital infection caused by HSV-1 or HSV-2. A 1998 study showed that, by case count, HSV infection is the most common sexually transmitted infection. Most individuals carrying the herpes simplex virus are unaware they are infected, and many suffer no symptoms of an outbreak with blisters similar to oral herpes. While most individuals infected with genital herpes are asymptomatic, severe clinical symptoms can occur, especially in populations with underlying immune deficiencies. Surprisingly, in contrast to HSV-2, HSV-1 infection does not appear to lead to chronically recurrent genital HSV-1 outbreaks, although an increase in the number of primary genital herpes infections caused by HSV-1 has been observed. Oral sex is considered the primary means of HSV-1 transmission that causes primary genital herpes lesions. In symptomatic cases, the typical symptoms of primary herpetic genital infection are clusters of genital sores consisting of inflammatory papules and vesicles on the outer surface of the genitals, similar to oral herpes. These usually first appear 4 to 7 days after exposure to HSV. HSV-2 is usually responsible for chronically recurrent genital herpes disease, but the differences between it and HSV-1 in terms of chronic persistence and reactivation are not understood. There is no cure for genital herpes disease, but symptoms usually become progressively milder over time, and the frequency of acute outbreaks gradually decreases in older populations. HSV-2 infection increases the risk of HIV acquisition and transmission by 2 to 3 times in co-infected individuals [3].

[0007] Herpes simplex keratitis is an ocular inflammation primarily caused by HSV infection of the cornea. HSV infection of the eye can cause a range of eye diseases of varying severity, from conjunctivitis and dendritic keratitis to interstitial edema and necrotizing interstitial keratitis. HSV-1 accounts for over 90% of HSV infections of the eye and is the leading cause of virus-induced blindness in developed countries.

[0008] Furthermore, there are other, fairly rare HSV infections of mucosal or epidermal tissues, which are briefly discussed below. Chronic or disseminated cutaneous herpes simplex infections, not limited to the lips or genitals, are known. Immunocompromised patients are most often affected by this disease, such as those with hypogammaglobulinemia or cutaneous T-cell lymphoma. Chronic cutaneous herpes simplex is a characteristic clinical manifestation of HSV in immunocompromised hosts. This infection can be defined as chronically active, destructive skin lesions that can potentially progress to a disseminated (systemic) form. While most HSV infections present as episodes that show healing within a week or two, the lesions of chronic cutaneous herpes simplex have a slowly progressive course that can last for several months. Chronic cutaneous herpes simplex is common in immunocompromised patients and is characterized by atypical, chronic, and persistent lesions that complicate and delay diagnosis. This can lead to death due to associated complications. When evaluating long-term chronic ulcers, especially in children, it is extremely important to consider the possibility of chronic herpes simplex virus infection.

[0009] Herpes xinode refers to a herpes skin infection that occurs in adolescents among wrestlers, but is also common in other contact sports. Herpes xinode usually occurs on the head, but most commonly on the chin, neck, chest, face, stomach, and legs. Herpetic eczema, also known as Kaposi's varicelliform eruption, is a widespread vesicular rash caused by a pre-existing skin condition, usually atopic dermatitis, and is caused by a viral infection, usually herpes simplex virus (HSV). Children with atopic dermatitis are at high risk of developing herpetic eczema, and HSV type 1 (HSV-1) is the most common pathogen in herpetic eczema. If left untreated, herpetic eczema can become severe and progress to disseminated infection and death. Diseases caused by HSV, particularly herpes simplex labialis and herpes simplex vulva, are the most common skin infections.

[0010] Genital herpes is transmitted perinatally and can cause life-threatening neonatal herpesvirus infections (HSV). Neonatal herpes is a rare event, estimated to occur in about 10 cases per 100,000 births.[4] Neonatal HSV infections can be caused by HSV-1, the most prevalent cause of neonatal infections in the Americas, Europe, and the Western Pacific, and HSV-2, the most prevalent cause of neonatal herpes in Africa, Southeast Asia, and the eastern Mediterranean.[4] Overall, there are about 14,000 cases each year, of which 10,000 are caused by HSV-2 and 4,000 by HSV-1.[4]

[0011] The serum prevalence of HSV-1 has also been linked to the development of neurological disorders such as dementia and Alzheimer's disease (AD). While the contribution of asymptomatic viral reactivation in the central nervous system remains debatable, the idea that increased aggregation of Aβ protein acts as a defense mechanism against viral reactivation, thereby inhibiting viral transmission, is intriguing.

[0012] Currently, the use of antiviral agents in anti-HSV therapy is widely known. The most common antiviral agents (e.g., acyclovir, penciclovir, foscarnet, idoxuridine) are nucleoside or pyrophosphate analogs whose common principle of activity is based on the inhibition of DNA synthesis in virus-infected cells. One of the most important therapeutic agents for treating HSV infection is the purine nucleoside analog acyclovir. Acyclovir is phosphorylated by viral thymidine kinase, which then interferes with viral DNA replication. In contrast, human DNA polymerase is less affected by acyclovir, at 1 / 50 to 1 / 30, and for this reason, only very few side effects are observed. In a double-blind, placebo-controlled trial in patients with herpes simplex labialis infection, acyclovir (in the form of Zovirax cream) was shown to reduce the infection by only 0.5 days (e.g., from 4.8 days to 4.3 days) compared to placebo-treated patients [5].

[0013] Currently, foscarnet, a pyrophosphate analog, is used, particularly in immunocompromised patients, against acyclovir-resistant herpesviruses. This drug directly inhibits viral DNA polymerase and does not affect thymidine kinase. However, the use of foscarnet can lead to severe and undesirable side effects such as renal failure and cardiac problems, and it is toxic to the bone marrow and can also cause skin ulceration. Foscarnet is also teratogenic and may not be administered during pregnancy. Furthermore, the formation of cross-resistant strains has been observed, making the development of alternative treatments extremely necessary. Passive or active immunological prophylaxis is not currently available. Currently used antiviral agents are only effective against infected cells in which the virus is actively replicating. Moreover, treatment with current antiviral agents has the disadvantage of not reducing or preventing the risk of relapse, and only treating symptoms with minimal clinical effect.

[0014] Humoral immunity plays a crucial role in the management of HSV and other viral infections. Circulating serum antibodies can bind to the viral envelope glycoprotein necessary for viral entry and are produced during infection. The presence of HSV-specific maternal serum antibodies has been shown to reduce neonatal transmission of HSV-2 [6]. HSV-1 seropositive individuals have a lower incidence of symptomatic genital HSV-2 infection, suggesting some degree of cross-protection [6]. Indeed, an HSV-2 subunit vaccine targeting glycoprotein D (gD) induced 82% protection against culture-positive HSV-1 but showed no protection against HSV-2 acquisition in young women participating in the HerpeVac trial [7, 8]. The discrepancies in protection against HSV-1 and HSV-2 infections from this vaccine trial remain unclear to date. Several attempts to develop a protective vaccine against chronic relapsing HSV-2 infection have failed to date [7-9]. A therapeutic vaccine candidate, GEN-003, containing the HSV-2 gD deletion variant, HSV-2 ICP4.2, and the Matrix-M2 adjuvant, was tested at various doses in HSV-2 seropositive individuals with genital herpes and showed reductions in viral shedding, lesion size, relapse rate, and relapse duration at specific doses, but was not advanced to clinical development

[10]

[11] . Antibody and cellular immune responses were stimulated. However, long-term persistence was questionable

[12]

[13] . A phase 1 clinical study using the replication-deficient HSV-2 vaccine HSV529 elucidated safety and serum neutralizing antibody responses, as well as moderate CD4+ T cell responses, in 78% of seronegative vaccine recipients

[14] . This vaccine also induced antibodies that mediated HSV-2-specific natural killer cell activation, and vaccine-induced antibodies could be detected in cervical-vaginal fluid

[15] . However, long-term prevention of relapse was uncertain.

[0015] Neutralizing serum antibodies can bind to HSV in vitro in the space between neuronal terminals and epithelial cells, limiting bidirectional viral transfer between these tissues

[16] . Clearly, serum antibodies or systemically applied antibodies can limit the extent of HSV infection and block transmission. Several approaches have been proposed and investigated for antibody-based treatment of HSV infection

[17] . Notably, some antibody-dependent cytotoxicity (ADCC)-mediated anti-HSV antibodies have been associated with excellent protection. Low levels of ADCC-mediated antibodies have been suggested to play a significant role in vaccine-mediated absent protection against HSV-2, because in preclinical studies, gD-deficient HSV-2 vaccine candidates induced strongly protective ADCC-mediated antibodies with little neutralizing ability [18,19]. However, the role of antibody-mediated phagocytosis (ADCP) and subsequent immune cell activation, particularly T cell activation, has been ignored

[17] .

[0016] E317 is the original clone of UB-621, a fully human antibody targeting HSV glycoprotein D (gD)

[20] , and has been shown to reduce mortality in an intraperitoneal HSV-1 exposure model in adult mice. Phase 1 clinical trials for subcutaneous administration demonstrated safety and tolerability in healthy individuals, and Phase 2 clinical trials for the treatment of recurrent genital HSV infections have been approved in the United States and China.

[0017] HSV8 is a fully human gD-specific IgG1 isolated using a phage display library that reduced mortality in intraperitoneal HSV-1 adult mouse models and intranasal HSV-1 / HSV-2 neonatal mouse models

[21] . HSV8 was also tested in a phase 1 clinical trial in combination with a broadly neutralizing anti-HIV antibody, demonstrating the safety of using the antibody in reducing vaginal transmission of HSV and HIV using MB66 vaginal film

[22] . Topically applied HSV8 also protected mice from HSV-2 vaginal transmission

[23] .

[0018] Glycoprotein B (gB) is an attractive target because it mediates the interaction between the virus and its cell receptor. However, other glycoproteins are essential for viral entry and therefore inevitably become attractive targets for therapeutic intervention. Glycoprotein B (gB) constitutes the viral fusion protein and thus plays a central role in the virus's entry into target cells. Currently, there are no approved anti-HSV gB monoclonal antibodies for therapeutic purposes.

[0019] Recently, a mouse monoclonal antibody (MAb) 2c that specifically recognizes the glycoprotein B (gB) of HSV-1 and HSV-2 has been described. HSV-gB is an essential component of the multicomponent fusion mechanism required for viral entry and intercellular fusion. HSV-gB constitutes a viral fusion protein that enables the fusion of the virus with the target cell membrane during the initial infection phase. Although the exact mechanism underlying the induction of viral-cell membrane fusion is not yet fully understood, it is hypothesized that glycoprotein D, which binds to a receptor on the target cell, induces gB activation via glycoprotein H / L, mediating fusion and the transfer of gB from the pre-fusion to the post-fusion conformation. MAb2c has been shown to neutralize cell-free viruses and inhibit intercellular transmission of the virus, a key mechanism by which HSV-1 or HSV-2 evades humoral immune surveillance independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) [24-27], WO2011 / 038933 (Patent Document 1), WO2015 / 197763 (Patent Document 2). MAb2c was obtained from mice immunized with inactivated HSV-1 strain 342 hv

[24] .

[0020] The corresponding humanized IgG1 MAb2c (HDIT101) is a novel antibody that has been therapeutically tested. HDIT101 protected immune-responsive mice from lethal intravaginal exposure to HSV-1 or HSV-2 and protected mice from the development of herpetic interstitial keratitis [26-28]. In immunodeficient mice, HDIT101 provides better protection from lethal infection by HSV-1 than by HSV-2

[29] . HDIT101 inhibits intercellular transmission, a characteristic of anti-HSV antibodies, which has recently been shown to correlate with increased protection from HSV-1 reactivation

[30] . In vitro, HDIT101 does not exert a measurable effect via induction of CDC or ADCC, but it can mediate antibody-dependent phagocytosis (ADCP) via the binding of its Fc fragment to antigen-presenting cells (APCs), and subsequently translocate the antibody-HSV complex into cells, inducing a robust anti-HSV T cell response. This function may play an important role in preventing recurrent HSV reactivation.

[31]

[0021] In a recent Phase 1 dose-escalation clinical trial conducted in healthy volunteers, intravenous HDIT101 demonstrated a superior safety profile even at excessively high doses of >12 g

[32] . In the Phase II clinical trial MATCH-2 ("Monoclonal Antibody Therapy for Chronic Herpesvirus 2 Infection," ClinicalTrials.gov identifier NCT04165122), which compared a single intravenous dose of HDIT101 with standard treatment of an episode with valacyclovir in patients with chronic recurrent anogenital HSV-2 infection, patients treated with HDIT101 showed a longer recurrence-free period after administration compared to the control group.

[0022] Furthermore, WO2023 / 003951 (Patent Document 3) describes several anti-HSV gB antibodies with neutralizing activity that have recently been isolated from seropositive human subjects (see, for example, Figures 5A and 5B of WO2023 / 003951).

[0023] While the antibodies described above, which specifically recognize the glycoprotein B (gB) of HSV-1 and HSV-2, have been found to have beneficial effects enabling the treatment and / or prevention of diseases caused by HSV-1 or HSV-2, further improvements are still needed, particularly in terms of specificity and / or binding ability, to provide further means and methods to the known "toolbox" of known anti-HSV therapies.

[0024] Furthermore, despite positive observations using HDIT101 in the MATCH-2 trial, the emergence of HDIT101-resistant HSV-1 and HSV-2 escape variants in vitro at suboptimal HDIT101 concentrations suggests that long-term monotherapy with HDIT101 for recurrent HSV infections may potentially lead to the evolution of HDIT101-resistant mutant virus strains.

[0025] Therefore, considering the prior art, there is a need to provide further means and / or improvements for the effective treatment or prevention of infections caused by HSV. [Prior art documents] [Patent Documents]

[0026] [Patent Document 1] WO2011 / 038933 [Patent Document 2] WO2015 / 197763 [Patent Document 3] WO2023 / 003951 [Overview of the project]

[0027] This invention is partly based on the surprising discovery that newly identified antibodies that specifically recognize glycoprotein B (gB) of HSV-1 and HSV-2 exhibit unexpected properties, as shown in the attached examples.

[0028] More specifically, the present invention provides a novel antibody (HDIT102(H4)) derived from a phage library of head and neck cancer patients and therefore being fully human IgG1. HDIT102(H4)Fab has a very low dissociation rate (2.9 x 10⁻¹⁰). -5 s -1 k dis It possesses the unique characteristic of being extremely potent and specific in its binding to its target protein gB. This represents an advancement over the current closest control drug, HDIT101, and thus its antiviral effect is unique and highly efficient. HDIT102(H4) did not show tissue cross-reactivity in human tissue immunohistochemistry experiments using a diverse set of tissues from three unrelated donors, conducted in accordance with the latest drug discovery guidelines.

[0029] Furthermore, while WO2023 / 003951 discloses several anti-HSV gB antibodies with neutralizing activity isolated from serologically positive human subjects (see, for example, Figures 5A and 5B of WO2023 / 003951), the present invention is partly based on the surprising discovery that newly identified antibodies that specifically recognize glycoprotein B (gB) of HSV-1 and HSV-2 exhibit unexpected properties compared to the antibodies disclosed in WO2023 / 003951, as shown in the attached examples. More specifically, the binding properties of the antibodies of the present invention have been tested compared to those of the antibodies in WO2023 / 003951. As shown in Example 26, the antibodies of the present invention bind to different epitopes compared to the antibodies in WO2023 / 003951.

[0030] Furthermore, as shown in Example 26, the antibody of WO2023 / 003951 exhibited a slower association rate compared to the antibody of the present invention. Therefore, considering the low dissociation rate of the antibody of the present invention, the antibody of the present invention has unexpected properties compared to the prior art antibody, in that its dissociation constant KD is superior overall to that of the prior art antibody.

[0031] Furthermore, while the antibody tested in WO2023 / 003951 was shown to possess neutralizing ability, this neutralizing ability is accompanied by activity that induces antibody-dependent cytotoxicity (ADCC). In contrast, the antibody of the present invention potently neutralizes HSV-1 and HSV-2 without activating ADCC or CDC, and therefore avoids nonspecific toxicity in patients, thus possessing unique properties particularly relevant to its therapeutic use.

[0032] Furthermore, the present invention defines the HDIT102(H4) epitope on the HSV-1 gB protein. This epitope is located nearby and partially overlaps with the HDIT101 epitope. Indeed, in vitro competition studies have demonstrated that HDIT102(H4) can compete with HDIT101 for binding to the fused recombinant gB protein and to fused gB expressed ectopically on the cell surface.

[0033] This finding, in part, opens the way to addressing the aforementioned disadvantages of (long-term) monotherapy for recurrent HSV infection (the risk of HDIT101-resistant HSV-1 and HSV-2 escape variants emerging, and the risk of HDIT101-resistant mutant virus strains evolving, particularly at suboptimal HDIT101 concentrations). Indeed, this invention is partly based on the surprising finding that, as expected for competing antibodies, no synergistic effect was observed in vitro with the combination of HDIT101 and HDIT102(H4), but a synergistic effect was observed in the survival of immune-responsive Balb / c mice after vaginal infection with a lethal dose of HSV-2G when using the combination compared to monotherapy with the same total amount of IgG. This is particularly surprising and unexpected given that both antibodies compete in recombinant gB binding. This finding leads to the suggestion that the formation of irregularly formed immune complexes by asymmetric cross-linking with the two competing antibodies results in improved signaling that recognizes the antibody-opsonized immune complexes. In the following examples, HDIT101 or HDIT102(H4) in the combination cocktail binds to individual gB protomers within the gB trimer, thereby creating an asymmetrically crosslinked immune complex. Structural binding analysis of HDIT101 and HDIT102(H4)Fab demonstrated the vertical orientation of HDIT101 and HDIT102(H4)Fab when bound to recombinant gB.

[0034] Therefore, in one aspect of the present invention, as part of a combination therapeutic formulation, the directions of two or more antibodies competing at a single binding site of an antigen are opposite, so that an irregularly formed immune complex of the opsonized multimeric antigen (trimer in the case of gB) is formed, which leads to a greater immune response. In the case of the combination of HDIT101 and HDIT102(H4), this leads to better protection of mice infected with a lethal dose of HSV-2G, possibly due to enhanced immune stimulation.

[0035] In fact, surprisingly, the present invention demonstrates that a combination of two therapeutic anti-HSV antibodies that compete in recombinant gB binding in vitro shows a considerably better therapeutic response in an in vivo infection model at doses equivalent to monotherapy using the individual antibodies. The present invention also demonstrates that a bispecific molecule using scFv targeting HDIT101 and HDIT102(H4)gB, fused to the IgG Fc domain at the carboxyl terminus, has improved characteristics in neutralizing cell-free HSV while retaining the low kdis observed for HDIT102(H4)IgG. Thus, this compound provides the superior characteristics of the HDIT101 and HDIT102(H4) combination in a single molecule.

[0036] Accordingly, the present invention partially solves the problem of the emergence of HDIT101-resistant HSV-1 and HSV-2 escape variants in (long-term) monotherapy for recurrent HSV infection by providing a combination of the prior art antibody HDIT101 and HDIT102(H4) (a fully human antibody with superior characteristics) in either combination therapy or a single bispecific molecule, as described below. In fact, when HSV-1 or HSV-2 was grown in vitro in the presence of suboptimal concentrations of HDIT101 or HDIT102(H4) alone, resistant virus strains emerged due to single amino acid substitutions, but the emergence of resistant viruses was prevented when HDIT101 and HDIT102(H4) were combined. As usefully demonstrated in the attached examples, when the combination of HDIT101 and HDIT102(H4) was applied, bispecific viruses could not be grown in vitro. Furthermore, when either the HDIT101-resistant virus or the HDIT102(H4)-resistant virus was grown in the presence of the other antibody, no development of resistance to the antibody used was observed. That is, the HDIT101-resistant virus did not become resistant to HDIT102(H4), and the HDIT102(H4)-resistant virus did not become resistant to HDIT101. This is particularly surprising because both antibodies target partially overlapping epitopes and compete for binding to recombinant gB and ectopically expressed gB on cells in vitro.

[0037] In fact, a combination of monoclonal anti-HSV antibodies for treating HSV infection has never been suggested. Combinations of monoclonal antibodies have been described as a possible treatment strategy for other viral infections. For example, two monoclonal antibodies, AX290 and AX677, targeting non-overlapping epitopes on the SARS-CoV-2 spike protein, have been shown to efficiently neutralize the virus and prevent the emergence of resistance escape variants in vitro

[33] . Similarly, Baum et al. reported that a non-competitive antibody cocktail against SARS-CoV-2 did not induce the emergence of resistance variants, but a cocktail with competitive antibodies did

[34] . Therefore, the results provided in the presented invention, that two competitive antibodies against HSV-1 / 2gB with partially overlapping epitopes prevented the emergence of resistant viruses and further acted synergistically in an in vivo infection model, are unexpected and novel findings. A synergistic effect was observed in vivo for the combination of two neutralizing antibodies, but not in vitro. However, these targeted distinct epitopes on the E1 and E2 structural proteins of the chikungunya virus

[35] . Human monoclonal antibodies that broadly neutralize hepatitis C virus were also found to work synergistically when applied in combination. However, even in this case, antibodies with overlapping epitopes did not work synergistically in vitro when combined

[36] . Combination therapy approaches using monoclonal antibodies targeting different non-overlapping sites on the Env protein have also been tested for human immunodeficiency virus (HIV) [37,38]. Antibody combinations have also been proposed for non-infectious treatments, such as cancer treatment

[39] .

[0038] However, considering prior art, the synergistic therapeutic effect of combining two or more monoclonal antibodies that compete for target binding in vitro, i.e., have at least partially overlapping epitopes, is remarkable. In this invention, the inventors demonstrate that in an experimental in vivo model, they enhanced the synergistic therapeutic effect on survival using two HSV gB-targeting antibodies that have partially overlapping epitopes that compete for recombinant gB binding in vitro. Combining equal amounts of the two antibodies resulted in significantly superior protection compared to using the individual antibodies alone.

[0039] Accordingly, in consideration of the prior art, the fundamental technical problem underlying the present invention is to provide further means and / or methods for treating or preventing infections caused by HSV, in particular to provide improved means and methods for treating herpes simplex infections that facilitate known dosing regimens in the art and prevent local transmission of infections and the emergence of resistance variants.

[0040] The technical problems are solved by providing the embodiments characterized in the claims.

[0041] Therefore, Phase 1 Therefore, the present invention relates to an anti-HSV antibody or its antigen-binding fragment that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, The complementarity determination region is V, which includes SEQ ID NO:1. H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V containing CDR3, SEQ ID NO:4 L V, including CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L The antibody contains CDR3, The antibody or its antigen-binding fragment may be up to 5.0 x 10 -4 s -1 Preferably a maximum of 1.0 x 10 -4 s-1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis It has, Preferably, the present invention relates to an anti-HSV antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment can neutralize HSV.

[0042] Furthermore, related to the first phase described above Phase 2 So, the present invention is, (A) An anti-HSV antibody or its antigen-binding fragment as defined in the context of the first phase, (B) Anti-HSV antibody or antigen-binding fragment that binds to glycoprotein B(gB) of HSV-1 and / or HSV-2 And, The complementary determination area is SEQ ID NO: 11 V including H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L The antibody contains CDR3, The antibody is an anti-HSV antibody or its antigen-binding fragment that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, having a dissociation constant Kd of up to 40 nM, preferably up to 30 nM, more preferably up to 20 nM, even more preferably up to 15 nM, up to 13 nM, and up to 10 nM. Regarding combinations.

[0043] Furthermore, related to the second phase mentioned above Third phase So, the present invention is, (A) The complementarity determination region, including V, which contains SEQ ID NO:1 H V, including CDR1 and SEQ ID NO:2 HV containing CDR2 and SEQ ID NO:3 H V containing CDR3 and SEQ ID NO:4 L V containing CDR1 and SEQ ID NO:5 L V containing CDR2 and SEQ ID NO:6 L A first binding domain containing CDR3, and (B) V containing SEQ ID NO:11, which is a complementarity-determining region H V containing CDR1 and SEQ ID NO:12 H V containing CDR2 and SEQ ID NO:13 H V containing CDR3 and SEQ ID NO:14 L V containing CDR1 and SEQ ID NO:15 L V containing CDR2 and SEQ ID NO:16 L A second binding domain containing CDR3 and A bispecific antibody or an antigen-binding fragment thereof that binds to glycoprotein B (gB) of HSV-1 and / or HSV-2, comprising The bispecific antibody has a maximum dissociation rate of 5.0 x 10 -4 s -1 , preferably a maximum of 1.0 x 10 -4 s -1 , a maximum of 5.0 x 10 -5 s -1 , most preferably a maximum of 2.9 x 10 -5 s -1 low dissociation rate k dis of a bispecific antibody or an antigen-binding fragment thereof.

[0044] Furthermore, Phase 4 In this regard, the present invention provides (A) V containing SEQ ID NO:1, which is a complementarity-determining region H V containing CDR1 and SEQ ID NO:2 H V containing CDR2 and SEQ ID NO:3 H V containing CDR3 and SEQ ID NO:4 L V containing CDR1 and SEQ ID NO:5 L V containing CDR2 and SEQ ID NO:6 LA first binding domain comprising a CDR3, and (B) a V comprising SEQ ID NO:11, which is a complementarity determining region H CDR1, a V comprising SEQ ID NO:12 H CDR2, a V comprising SEQ ID NO:13 H CDR3, a V comprising SEQ ID NO:14 L CDR1, a V comprising SEQ ID NO:15 L CDR2, and a V comprising SEQ ID NO:16 L a second binding domain comprising a CDR3, and (C) a V comprising SEQ ID NO:31, which is a complementarity determining region H CDR1, a V comprising SEQ ID NO:32 H CDR2, a V comprising SEQ ID NO:33 H CDR3, a V comprising SEQ ID NO:34 L CDR1, a V comprising SEQ ID NO:35 L CDR2, and a V comprising SEQ ID NO:36 L a third binding domain comprising a CDR3 and a trispecific antibody or an antigen-binding fragment thereof that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, comprising the trispecific antibody having a maximum dissociation rate k of 5.0x10 -4 s -1 , preferably a maximum of 1.0x10 -4 s -1 , a maximum of 5.0x10 -5 s -1 , most preferably a maximum of 2.9x10 -5 s -1 and having a low dissociation rate k -1 such that the trispecific antibody at a concentration of up to 10 nM, preferably up to 5 nM, more preferably up to 2 nM, up to 1 nM, up to 0.8 nM, up to 0.6 nM, and most preferably up to 0.5 nM can neutralize an amount of HSV defined as the 100 TCID50. Regarding the trispecific antibody or an antigen-binding fragment thereof.

Mode for Carrying Out the Invention

[0045] In the following, Phase 1 This will be explained in more detail.

[0046] The antibody or its antigen-binding fragment used in accordance with the first aspect of the present invention is not particularly limited, as long as it is an "anti-HSV antibody or its antigen-binding fragment" and specifically binds to or interacts with the glycoprotein B(gB) of HSV-1 and / or HSV-2. The glycoprotein B(gB) of HSV-1 and / or HSV-2 is a domain or antigen of HSV-1 and / or HSV-2, as will be described in more detail below.

[0047] As used in the context of the present invention, the term "binding" defines the binding (interaction) of at least two "antigen interaction sites" to each other. The term "antigen interaction site" defines, according to the present invention, a portion of the antibody or its antigen-binding fragment that exhibits the ability to specifically interact with a polypeptide motif, i.e., a specific antigen or group of antigens of HSV, in particular a specific antigen or group of antigens of the glycoprotein B(gB) of HSV-1 and / or HSV-2. The term "binding" means, according to the present invention, that the antibody can specifically interact with and / or bind to at least two amino acids of the glycoprotein B(gB) of HSV-1 and / or HSV-2, as defined herein.

[0048] In preferred embodiments, the binding / interaction is also understood to preferably define “specific recognition,” particularly when binding to a specific epitope is involved, as will be described in more detail below. The term “specifically recognize” means, according to the present invention, that the antibody can specifically interact with and / or bind to at least two amino acids of a defined epitope of glycoprotein B(gB) of HSV-1 and / or HSV-2.

[0049] Antibodies can recognize different epitopes on HSV gB. This term "recognize" is particularly related to the specificity of the antibody molecule, i.e., its ability to distinguish specific regions, in particular, specific epitopes of glycoprotein B(gB) of HSV-1 and / or HSV-2.

[0050] The term “specific interaction” as used in accordance with the present invention means that the antibody or its antigen-binding fragment of the present invention does not cross-react with (poly)peptides of similar structure, or does not essentially cross-react with them. Accordingly, the antibody or its antigen-binding fragment of the present invention specifically binds to / interacts with the structure of glycoprotein B(gB), preferably the glycoprotein B(gB) of HSV-1 and / or HSV-2. Specific examples of such molecules are given herein.

[0051] The cross-reactivity of a group of antibodies or antigen-binding fragments under study may be tested, for example, by evaluating the binding of the antibody or antigen-binding fragments to the (poly)peptide of interest, as well as to a number of (poly)peptides that are more or less closely related (structurally and / or functionally), under conventional conditions (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)). Only constructs (i.e., antibodies, their antigen-binding fragments, etc.) that bind to a specific structure of HSV, e.g., a specific epitope or (poly)peptide / protein of HSV (preferably, a specific epitope of glycoprotein B(gB) of HSV-1 and / or HSV-2), but do not bind to any other epitopes or (poly)peptides of the same glycoprotein B(gB) of HSV-1 and / or HSV-2, or do not bind in any meaningful way, are considered specific to the epitope or (poly)peptide / protein of interest and are selected for further study according to the methods provided herein. These methods may, among other things, include binding studies, blocking and competition studies using molecules that are closely related in terms of structure and / or function. These binding studies also include FACS analysis, surface plasmon resonance (SPR, e.g., SPR using BIAcore®), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence polarization spectroscopy, fluorescence spectroscopy, or radiolabeled ligand binding assays.

[0052] The term "binding" may relate not only to linear epitopes but also to conformational epitopes, structural epitopes, or discontinuous epitopes, or parts thereof, consisting of two regions of a human target molecule. In the context of this invention, a conformational epitope is defined as two or more distinct amino acid sequences that are separated in the primary sequence but come together on the surface of the molecule when the polypeptide folds into a native protein (Sela, Science 166 (1969), 1365 and Laver, Cell 61 (1990), 553-536).

[0053] Therefore, specificity can be experimentally confirmed by methods known in the art and by methods described herein. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, and peptide scanning.

[0054] In a preferred embodiment, the anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention is a monoclonal antibody or a polyclonal antibody.

[0055] In a more preferred embodiment, the anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention is a humanized antibody or a fully human antibody.

[0056] In a more preferred embodiment, the anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention is a mouse antibody.

[0057] As outlined above in the context of the first aspect of the present invention, the term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., individual antibodies constituting a population that are identical except for possible innate variations that may be present in trace amounts. Monoclonal antibodies are highly specific and are produced against a single antigen site. Monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture without being essentially contaminated by other immunoglobulins. The modifier “monoclonal” indicates that the antibody is part of a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method. As stated above, monoclonal antibodies used in accordance with the present invention can be produced by the hybridoma method described in Kohler, Nature 256 (1975), 495.

[0058] In the context of this disclosure, the term “polyclonal antibody” as used herein refers to an antibody produced between or in the presence of one or more other non-identical antibodies. Generally, polyclonal antibodies are produced from B lymphocytes in the presence of several other B lymphocytes that produce non-identical antibodies. Polyclonal antibodies are typically obtained directly from immunized animals.

[0059] As used herein, the term “fully human antibody” refers to an antibody containing only human immunoglobulin protein sequences. Fully human antibodies may contain mouse carbohydrate chains if produced in mice, mouse cells, or hybridomas derived from mouse cells. Similarly, “mouse antibody” or “murine antibody” refers to an antibody containing only mouse / murine immunoglobulin protein sequences. Alternatively, “fully human antibodies” may contain rat carbohydrate chains if produced in rats, rat cells, or hybridomas derived from rat cells. Similarly, the term “rat antibody” refers to an antibody containing only rat immunoglobulin sequences. Fully human antibodies may also be produced, for example, by phage display, a widely used screening technique that enables the production and screening of fully human antibodies. Phage antibodies can also be used in the context of this invention. Phage display methods are described, for example, in US5,403,484, US5,969,108, and US5,885,793. Another technique that enables the development of fully human antibodies involves improvements to mouse hybridoma techniques. Mice are transgenicized to include the human immunoglobulin locus by exchanging their own mouse genes (see, for example, US5,877,397).

[0060] The term "chimeric antibody" refers to an antibody comprising a variable region of the present invention that is fused with or chimeric with an antibody region (e.g., a constant region) derived from another human or non-human species (e.g., mouse, horse, rabbit, dog, cattle, chicken).

[0061] The term antibody also refers to recombinant human antibodies, heterologous antibodies, and heterohybrid antibodies. The term “recombinant human antibody” includes all human sequence antibodies prepared, expressed, produced, or isolated by recombinant means, e.g., antibodies isolated from transgenic animals (e.g., mice) for human immunoglobulin genes; antibodies expressed using recombinant expression vectors introduced into host cells by transfection; antibodies isolated from recombinant combinatorial human antibody libraries; or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences against other DNA sequences. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. However, such antibodies may be subjected to in vitro mutagenesis (or, when transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis), and therefore the amino acid sequences of the VH and VL regions of recombinant antibodies are derived from and related to human germline VH and VL sequences, but may not naturally exist within the human antibody germline repertoire in vivo.

[0062] The term "heterogeneous antibody" is defined in relation to transgenic non-human organisms that produce such antibodies. This term corresponds to antibodies found in organisms that are not transgenic non-human animals, and generally refers to antibodies that have an amino acid sequence or coding nucleic acid sequence derived from a species other than a transgenic non-human animal.

[0063] The term "heterohybrid antibody" refers to an antibody that has light and heavy chains of different biological origins. For example, an antibody with a human heavy chain bound to a mouse light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric antibodies and humanized antibodies.

[0064] The term antibody also refers to humanized antibodies. The "humanized" form of a non-human (e.g., mouse or rabbit) antibody is a chimeric immunoglobulin, which is an immunoglobulin chain containing a minimal sequence derived from a non-human immunoglobulin. Often, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's complementarity-determining region (CDR) are replaced with residues derived from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, resulting in desired specificity, affinity, and capability. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient antibody or the transferred CDR or framework sequence. These modifications are made to further refine and optimize the antibody's performance. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the FR region is the FR region of the human immunoglobulin consensus sequence. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, see Jones Nature 321 (1986), 522-525; Reichmann Nature 332 (1998), 323-327, and Presta Curr Op Struct Biol 2 (1992), 593-596.

[0065] A popular method for antibody humanization involves CDR grafting, in which a functional antigen-binding site derived from a non-human "donor" antibody is grafted onto a human "acceptor" antibody. CDR grafting methods are publicly known in the art and are described, for example, in US5,225,539, US5,693,761, and US6,407,213. Another related method is the production of humanized antibodies from transgenic animals genetically engineered to contain one or more humanized immunoglobulin loci that can undergo gene rearrangement and gene conversion (see, for example, US7,129,084).

[0066] Accordingly, in the context of anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention, the term “antibody” refers to a complete immunoglobulin molecule and a portion of such an immunoglobulin molecule (i.e., “its antigen-binding fragment”). Furthermore, the term, in general terms, refers to modified and / or altered antibody molecules. The term also refers to antibodies produced / synthesized by recombination or synthesis. The term also refers to intact antibodies. The term “antibody” also includes, but is not limited to, fully human antibodies, chimeric antibodies, humanized antibodies, CDR graft antibodies, and antibody constructs such as single-chain Fv(scFv) or antibody-fusion proteins.

[0067] In the context of the first aspect of the present invention, the term “the antigen-binding fragment” means up to 5.0 x 10¹⁶, which will be further described below. -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis Insofar as it is mentioned that it possesses the aforementioned capabilities, it is not particularly limited.

[0068] As mentioned, according to the first aspect of the present invention, a V containing SEQ ID NO:1, which binds to glycoprotein B(gB) of HSV-1 and / or HSV-2 and is a complementarity-determining region. H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V containing CDR3, SEQ ID NO:4 L V, including CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L Anti-HSV antibodies containing CDR3 or their antigen-binding fragments are up to 5.0 x 10 -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis It holds.

[0069] In this context, "low dissociation rate k" dis "Extraordinarily strong and specific" means that the binding of the antibody to its target is exceptionally strong and specific. More specifically, "low" in this context means that the antibody has a maximum binding strength of 5.0 x 10⁶. -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis It means having

[0070] In a more preferred embodiment, the anti-HSV antibody or its antigen-binding fragment may be up to 5.0 x 10 -4 s -1 Preferably a maximum of 4.0 x 10 -4 s -1 , more preferably up to 3.0x10 -4 s -1, more preferably, up to 2.0x10 -4 s -1 , up to 1.0x10 -4 s -1 , up to 9.0x10 -5 s -1 , up to 8.0x10 -5 s -1 , up to 7.0x10 -5 s -1 , up to 6.0x10 -5 s -1 , up to 5.0x10 -5 s -1 , up to 4.0x10 -5 s -1 , up to 3.0x10 -5 s -1 , most preferably, up to 2.9x10 -5 s -1 , up to 2.0x10 -5 s -1 , up to 1.5x10 -5 s -1 , up to 1.0x10 -5 s -1 , up to 5.0x10 -1 s -1 , up to 2.0x10 -6 s -1 , up to 1.0x10 -6 s -1 , up to 5.0x10 -7 s -1 , up to 2.0x10 -7 s -1 , up to 1.0x10 -7 s -1 , up to 1.0x10 -8 s -1 , up to 1.0x10 -9 s -1 with a low dissociation rate k dis having.

[0071] As shown above and below in this specification, the anti-HSV antibody of the present invention, surprisingly, when bound to recombinant gB when using the biolayer interferometry (BLI) method, most preferably, 2.9x10 -5 s -1 of k disHas a very low dissociation rate (kdis) equal to or lower than that.

[0072] Dissociation rate k dis Can be determined by methods known to those skilled in the art. In one embodiment, this dissociation rate k dis Is determined as described in the examples added at the end of this specification. In a particular embodiment, this dissociation rate k dis Is determined using recombinant gB and the biolayer interferometry (Octet).

[0073] In a preferred embodiment, this can be determined as outlined below.

[0074] To produce recombinant HSV-1 gB protein, a codon-optimized DNA sequence encoding the HSV-1 gB extracellular domain (aa 30 - 729; UniProtKB P06436.1) or HSV-2 gB extracellular domain (aa 22 - 724; GenBank: QAU10948.1), containing a signal peptide and a C-terminal tag, can be cloned into a mammalian expression vector. Then, the HSV gB recombinant protein can be transiently expressed in HEK293-E6 suspension cells cultured in culture medium. HEK293-E6 cells are transfected with the gB-encoding plasmid. Five days after transfection, the supernatant is collected by a centrifugation step. Next, the pH of the supernatant is adjusted by adding 1 ml of 2M Tris buffer pH9 / 100 ml of supernatant. Then, the gB protein is purified by tag-affinity gravity flow purification. Next, the tag of the gB protein is removed by thrombin digestion and dialyzed against 50 mM Tris, 150 mM NaCl, pH8. Then, the thrombin-digested gB protein is purified three times by tag-specific affinity chromatography to deplete the sample from any remaining tagged gB protein. Next, the gB protein is concentrated and further purified via size exclusion chromatography. The peak fractions are pooled and concentrated.

[0075] The anti-HSV gB antibody Fab may be prepared by digestion with papain (as done for HDIT101) followed by purification, or by recombination by transfection of plasmids encoding the light chain and cleaved heavy chain into 293T-E6 cells (as done for HDIT102(H4)). The HSV gB recombinant protein is biotinylated, and then any remaining biotin is removed. An initial loading scout is performed to find the best biosensor loading concentration. Various concentrations of biotinylated gB are loaded into a streptavidin biosensor (Octet), and the absorption kinetics of the test antibody Fab fragment are measured. The dilution series of the Fab fragment is then analyzed to determine the dissociation rate (kdis).

[0076] More specifically, the dissociation rate k is as outlined below. dis It is possible to find this.

[0077] To produce recombinant HSV-1 gB protein, a codon-optimized DNA sequence encoding either the HSV-1F gB external domain (aa 30-729; UniProtKB P06436.1) or the HSV-2G gB external domain (aa 22-724; GenBank: QAU10948.1), including the BM40 signal peptide and a C-terminal double Strep-tag, can be cloned into a pCAGGS mammalian expression vector. The recombinant HSV gB protein can then be transiently expressed in HEK293-E6 suspension cells cultured in serum-free medium, e.g., F17 medium (ThermoFisher) supplemented with 0.1% Kolliphor (Sigma) and 4 mM glutamine. HEK293-E6 cells are cultured with 1 μg of gB coding plasmid and 2 μg of PeiMax / ml culture medium at a rate of 1.5–2.0 x 10⁶ 6Transfect cells using PeiMax (Polysciences) at a cell density of cells / ml. Add Tryptone N1 feeder (Organi Technie) to the culture 24 hours after transfection. On day 5 after transfection, collect the supernatant by two centrifugation steps: first at 1200 rpm to remove cells, and then at 3600 rpm to remove cell debris. Next, adjust the pH of the supernatant by adding 1 ml of 2 M Tris buffer pH 9 / 100 ml of supernatant. Then, purify the gB protein by gravity flow purification using Strep-Tactin XT (IBA, Germany) according to the manufacturer's protocol. Next, remove the Strep-tag from the gB protein by thrombin digestion (Serva) and dialyze against 50 mM Tris, 150 mM NaCl, pH 8. Next, the thrombin-digested gB proteins are purified three times by Strep-Tactin XT affinity chromatography to deplete the sample from any remaining Strep-tagged gB proteins. Then, the gB proteins are concentrated on an Amicon spin column (cutoff 30k) and further purified using a Superdex 200 10 / 300 GL SEC column and Akta Pure FPLC. The peak fractions are pooled and concentrated using an Amicon spin column.

[0078] The anti-HSV gB antibody Fab may be prepared by digestion with papain (as done for HDIT101) followed by purification, or by recombination by transfection of plasmids encoding the light chain and cleaved heavy chain into 293T-E6 cells (as done for HDIT102(H4)). The HSV gB recombinant protein is biotinylated at a ratio of 3:1 at room temperature for 30 minutes (NHS-PEG4-biotin (Thermo Fischer Scientific, A39259)), and the remaining biotin is then removed using a desalting column and centrifugation at 1000 g for 2 minutes (Zeba Spin Desalting Columns; 7K MWCO, 2 ml (Thermo Scientific)). (UE285726). An initial loading scout is performed to find the best biosensor loading concentration. Various concentrations of biotinylated gB are loaded into the streptavidin biosensor (Octet), and the absorption kinetics of the test antibody Fab fragment are measured. The optimal gB concentration for loading into the biosensor was determined to be 5 μg / ml. Biotinylated gB (wt) [5 μg / ml] is used to load into the biosensor. The binding kinetics of the antibody Fab fragment (100 nM) to immobilized gB can be analyzed using a global 1:1 fit model. Subsequently, a dilution series of the Fab fragment is analyzed to determine the dissociation rate (kdis).

[0079] An anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention is not particularly limited, as long as it is "an anti-HSV antibody or its antigen-binding fragment that binds to the glycoprotein B(gB) of HSV-1 and / or HSV-2". Accordingly, the antibody may be any antibody that specifically binds to, specifically recognizes, or interacts with the glycoprotein B(gB) of HSV-1 and / or HSV-2, i.e., the domain, antigen of the glycoprotein B(gB) of HSV-1 and / or HSV-2. A person skilled in the art can easily produce such an antibody made against a particular domain (i.e., an antigen, preferably the surface antigen of the glycoprotein B(gB) of HSV-1 and / or HSV-2) and, based on the common sense of a person skilled in the art and the method described above, determine whether each antibody can detect / bind to a particular domain, antigen, preferably the surface antigen of the glycoprotein B(gB) of HSV-1 and / or HSV-2.

[0080] An anti-HSV antibody or its antigen-binding fragment according to a first aspect of the present invention binds to / recognizes "glycoprotein B (gB) of HSV-1 and / or HSV-2". Viral antigen glycoproteins D, B, C, H, L, E, or I (i.e., gD, gB, gC, gH, gL, gE, gI) are well-characterized and known surface or envelope proteins of HSV-1 and / or HSV-2. These proteins may be found not only on the surface of HSV-1 and / or HSV-2, or on the envelope structure, i.e., on the surface of released infected particles (i.e., the envelope of free virions), but also on the surface of infected cells, i.e., on the cell surface. However, in a more preferred embodiment, the antibody of the present invention binds to / recognizes viral surface antigen glycoprotein B (i.e., gB) of the HSV-1 and / or HSV-2 envelope.

[0081] Accordingly, in a preferred embodiment, an anti-HSV antibody or its antigen-binding fragment for use according to the present invention binds to surface glycoprotein B(gB) of the HSV-1 and / or HSV-2 envelope in the features of the present invention as defined above, and preferably recognizes its epitope in the features of the present invention and as defined above.

[0082] The glycoprotein B of HSV-1 and / or HSV-2 is well-characterized. Without being constrained to a specific sequence, example sequences of various HSV-1 and HSV-2 strains are shown in SEQ ID NO: 9, 10, and 21-24, and 40, respectively. SEQ ID NO: 9 shows the sequence of glycoprotein B of HSV-1 strain F, SEQ ID NO: 10 shows the sequence of glycoprotein B of HSV-1 strain KOS, SEQ ID NO: 21 shows the sequence of glycoprotein B of HSV-1 strain gC-39-R6, SEQ ID NO: 22 shows the sequence of glycoprotein B of HSV-2 strain HG52, SEQ ID NO: 23 shows the sequence of glycoprotein B of HSV-2 strain 333, SEQ ID NO: 24 shows the sequence of glycoprotein B of HSV-2 strain MMA, and SEQ ID NO: 40 shows the sequence of glycoprotein B of HSV-2 strain G. The sequence alignment of these glycoprotein B amino acid sequences shows that while the overall amino acid homology, preferably identity, of gB in HSV-1 and HSV-2 is 85%, the sequences are least conserved in the N-terminal and C-terminal regions of the protein.

[0083] As mentioned, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention comprises a complementarity-determining region containing SEQ ID NO:1. H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V containing CDR3, SEQ ID NO:4 L V, including CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L Includes CDR3.

[0084] As used herein, the term "CDR" refers to the "complementarity-determining region" as is well known in the art. The CDR is the immunoglobulin portion that determines the specificity of the molecule and contacts a specific ligand. The CDR is the most variable part of the molecule and contributes to the diversity of these molecules. Each V domain has three CDR regions: CDR1, CDR2, and CDR3. CDR-H refers to the CDR region of the variable heavy chain, and CDR-L refers to the CDR region of the variable light chain. VH means variable heavy chain, and VL means variable light chain. The CDR region of the Ig-derived region can be determined by various means and methods known in the art.

[0085] For example, the CDR region of the Ig-derived region can be determined as described in Kabat, "Sequences of Proteins of Immunological Interest," 5th edit. NIH Publication no. 91-3242 US Department of Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987), 901-917, or Chothia Nature 342 (1989), 877-883.

[0086] In the context of the present invention, the CDR region (and framework region (FR)) is determined according to Martin's numbering scheme as described in Norman, RA, F. Ambrosetti, A. Bonvin, LJ Colwell, S. Kelm, S. Kumar and K. Krawczyk (2020). "Computational approaches to therapeutic antibody design: established methods and emerging trends." Brief Bioinform 21(5): 1549-1567.

[0087] Therefore, in the context of the present invention, references to amino acid residues follow Martin's numbering scheme.

[0088] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention is located at positions 1-25 (V) of the following framework region: SEQ ID NO: 7 H FR1), 36~49(V H FR2), 67~98(V H FR3), 112~122(V H FR4), SEQ ID NO: 81~22(V L FR1), 34~48(V L FR2), 56~87(V L FR3), and 97~106(V L Each of the amino acid residues shown in FR4) contains an amino acid sequence having at least 70% sequence identity.

[0089] As described above, in the context of the present invention, the CDR region (and framework region (FR)) is determined according to Martin's numbering scheme, as described in Norman, RA, F. Ambrosetti, A. Bonvin, LJ Colwell, S. Kelm, S. Kumar and K. Krawczyk (2020). "Computational approaches to therapeutic antibody design: established methods and emerging trends." Brief Bioinform 21(5): 1549-1567.

[0090] Therefore, in the context of the present invention, references to amino acid residues follow Martin's numbering scheme.

[0091] In a more preferred embodiment, an anti-HSV antibody or antigen-binding fragment according to the first aspect of the present invention contains an amino acid sequence having at least 75%, at least 80%, more preferably at least 85%, at least 90%, even more preferably at least 95%, most preferably 98% or 99% total sequence identity in the framework region compared to one of each of the amino acid residues indicated at positions 1-30, 38-51, 68-99, and 112-122 of SEQ ID NO: 7, and positions 1-23, 41-55, 63-94, and 104-114 of SEQ ID NO: 8. The antibody or antigen-binding fragment has a maximum size of 5.0 x 10¹⁶ as described above and below in this specification. -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis Such antibodies are suitable for the purposes of the present invention as long as they bind to the gB of HSV-1 or HSV-2, and / or can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission) as described above and below herein, and / or can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0092] Accordingly, in a preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention comprises the above-mentioned variable regions of the light chain and heavy chain (i.e., the CDR defined above, i.e., V containing SEQ ID NO:1). H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V containing CDR3, SEQ ID NO:4 L V, including CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 Lcomprises an amino acid sequence having a CDR3), but this amino acid sequence has a variation in the framework region with at least 75%, at least 80%, more preferably at least 85%, at least 90%, even more preferably at least 95%, most preferably 98% or 99% sequence identity compared to each of one of the amino acid residues shown at positions 1 to 30, 38 to 51, 68 to 99, and 112 to 122 of SEQ ID NO:7, and positions 1 to 23, 41 to 55, 63 to 94, and 104 to 114 of SEQ ID NO:8.

[0093] In this context, SEQ ID NO:7 or SEQ ID NO:8 is aligned with the most closely matching sequence of the polypeptide of interest, and if the amino acid identity between the two aligned sequences is at least X% over positions 1 to 30, 38 to 51, 68 to 99, and 112 to 122 of SEQ ID NO:7, and positions 1 to 23, 41 to 55, 63 to 94, and 104 to 114 of SEQ ID NO:8, the polypeptide has "at least X% sequence identity" to SEQ ID NO:7 or 8 in the framework region. Further, as outlined in more detail below, such amino acid sequence alignments can be performed using publicly available computer homology programs, such as the "BLAST" program provided on the National Centre for Biotechnology Information (NCBI) homepage, using the default settings provided therein. Further methods for calculating the percent sequence identity of a set of amino acid or nucleic acid sequences are known in the art.

[0094] In a further preferred embodiment, the anti-HSV antibody or antigen-binding fragment thereof according to the first aspect of the invention is the V of SEQ ID NO:7 H and the V of SEQ ID NO:8 L and comprises.

[0095] Accordingly, in a preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention is an antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, comprising, or consisting of, a VH domain (heavy chain variable region) and a VL domain (light chain variable region), i.e., the amino acid sequence of the variable region of the heavy chain of the antibody shown in SEQ ID NO:7 and the amino acid sequence of the variable region of the light chain of the antibody shown in SEQ ID NO:8.

[0096] However, the antibodies used in the present invention are not particularly limited to such variable heavy and light chain variable regions, and the antibodies may be up to 5.0 x 10 as described above and below in this specification. -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis Insofar as it has such a characteristic, it may also be an antibody or antigen-binding fragment that binds to glycoprotein B (gB) of the HSV-1 and / or HSV-2 envelope, comprising or consisting of VH domains and VL domains having, most preferably 100%, at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 75%, 70%, 65%, 60%, 55%, or 50% sequence homology, preferably identity, with the sequences of SEQ ID NO: 7 and 8, respectively. Furthermore, the antibody may be up to 5.0 x 10⁻¹⁶ as described above and below in this specification. -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k disInsofar as it has the following characteristics, the antibody or its antigen-binding fragment is a molecule containing VH and VL domains having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conserved amino acid substitutions based on the sequences of SEQ ID NO: 7 and 8. Furthermore, the antibody or its antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFV, F(ab')2, and diabody.

[0097] To determine whether an amino acid sequence has a certain degree of identity with sequences SEQ ID NO: 7 and 8, a person skilled in the art can use means and methods known in the art, such as alignment, either manually or using a computer program known to the art. Such alignment can be performed, for example, using means and methods known to the art, such as the Lipman-Pearson method (Science 227 (1985), 1435) or a known computer algorithm such as the CLUSTAL algorithm. In such alignment, it is preferable that maximum homology is assigned to conserved amino acid residues present in the amino acid sequence. In a preferred embodiment, ClustalW2 is used for comparing amino acid sequences. For pairwise comparison / alignment, the following settings are preferably selected: Protein weight matrix: BLOSUM62; gap open: 10; gap extension: 0.1. For multiple comparisons / alignments, the following settings are preferably selected: protein weight matrix: BLOSUM62; gap open: 10; gap extension: 0.2; gap distance: 5; end gap: none.

[0098] According to the present invention, the terms “identical” or “percent identical” in the context of two or more nucleic acids or amino acid sequences mean that, when compared or aligned to the greatest extent possible across a comparison window or a specified region, as measured using sequence comparison algorithms as known in the art, or by manual alignment and visual inspection, two or more sequences or subsequences, or gBs of HSV-1 or HSV-2, and as described above and below herein, up to 5.0 x 10⁻¹⁶. -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k disThis refers to two or more sequences or subsequences having a specified percentage (e.g., 60% or 65% identity, preferably 70-95% identity, more preferably at least 95% identity) of amino acid residues or nucleotides that are identical to the nucleic acid sequence or amino acid sequence described above, and / or can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission), or can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). For example, sequences having 60-95% or greater sequence identity are considered substantially identical. Such definitions also apply to complementary strands of test sequences. Preferably, the described identity exists over a region of at least about 15-25 amino acids or nucleotides in length, more preferably over a region of about 50-100 amino acids or nucleotides in length. Those skilled in the art know, for example, how to determine the percentage identity between sequences using algorithms, such as the CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or algorithms based on FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as are well known in the art.

[0099] The FASTDB algorithm typically does not consider internal mismatched deletions or additions, i.e., gaps, in the sequence in its calculations, but this can be manually corrected to avoid overestimating % identity. However, CLUSTALW does take sequence gaps into account in its identity calculations. The BLAST and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol. Biol. 215:403-410) are also available to those skilled in the art. The BLASTN program for nucleic acid sequences uses, by default, a word length (W) of 11, an expected value (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3 and an expected value (E) of 10. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89:10915) uses 50 alignments (B), an expected value (E) of 10, M=5, N=4, and comparison of both strands.

[0100] Preferably, amino acid substitutions are “conservative substitutions” that refer to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, backbone conformation, and stiffness) so that changes can be frequently made without altering the biological activity of the protein. Those skilled in the art will generally recognize that substituting a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co. 4th Ed. (1987), 224). Furthermore, substitutions of amino acids that are structurally and / or functionally similar are less likely to disrupt biological activity. In the context of the present invention, the conjugate compound / antibody of the present invention comprises a polypeptide chain having a sequence that, when compared to a specific amino acid sequence disclosed herein, for example, SEQ ID NO: 7 (referring to the variable region of the antibody heavy chain) and 8 (referring to the variable region of the antibody light chain), includes 0 (unchanged), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conserved amino acid substitutions. As used herein, the conserved amino acid substitutions in the phrase "up to X" include 0 substitutions, up to 10, and any number of substitutions including 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions.

[0101] Such exemplary substitutions are preferably made according to the substitutions shown in Table 1 below.

[0102] (Table 1) Exemplary Conservative Amino Acid Substitutions TIFF2026520211000001.tif167128

[0103] The specificity of the antibody or antigen-binding fragment of the first aspect of the present invention is not only represented by the amino acid sequence of the antibody or antigen-binding fragment as defined above, but may also be represented by the epitopes to which the antibody can bind. Accordingly, in a preferred embodiment, the present invention utilizes an anti-HSV antibody or antigen-binding fragment according to the present invention that recognizes the same epitopes as the antibody described above.

[0104] Therefore, in a more preferred embodiment, the anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention is not limited to the above-described anti-HSV antibody or its antigen-binding fragment, but may be an anti-HSV antibody or its antigen-binding fragment that recognizes the same epitope as the antibody defined above.

[0105] More specifically, in a preferred embodiment, the present invention relates to an anti-HSV antibody or its antigen-binding fragment that recognizes the same epitopes as defined above herein, wherein the epitopes are located at amino acids D199, A203, K204, Y303, R304, K320, Q321, V322, D323, Y326, R335, and T337 of glycoprotein B of HSV-1 strain F, and at contact amino acid residues D191, A195, K196, Y295, R296, K312, Q313, V314, D315, Y318, R327, and T329 of glycoprotein B of HSV-2 strain G, Preferably, the epitope relates to an anti-HSV antibody or its antigen-binding fragment, wherein the epitope consists of contact amino acid residues D199, A203, K204, Y303, R304, K320, Q321, V322, D323, Y326, R335, and T337 of glycoprotein B (SEQ ID NO: 9) of HSV-1 strain F, and contact amino acid residues D191, A195, K196, Y295, R296, K312, Q313, V314, D315, Y318, R327, and T329 of glycoprotein B (SEQ ID NO: 40) of HSV-2 strain G.

[0106] In certain embodiments, epitopes can be identified by cryo-electron microscopy (Cryo-EM).

[0107] Whether an anti-HSV antibody or its antigen-binding fragment recognizes the same epitope as the (reference) antibody can be determined by routine methods known in the art. Appropriate methods are described below in more general terms, but further down, more detailed methods are described that lead to the identification of the amino acid residues D199, A203, K204, Y303, R304, K320, Q321, V322, D323, Y326, R335, and T337 of glycoprotein B of HSV-1 strain F, and the contact amino acid residues D191, A195, K196, Y295, R296, K312, Q313, V314, D315, Y318, R327, and T329 of glycoprotein B of HSV-2 strain G, respectively, according to the present invention.

[0108] Generally, epitopes may be contained in gB proteins, but may also be contained in their degradation products, or may be chemically synthesized peptides, as will be understood by those skilled in the art. The amino acid positions are shown only to demonstrate the position of the corresponding amino acid sequence in the gB protein sequence. The present invention encompasses all peptides containing epitopes. The peptides may be portions of polypeptides with a length of more than 100 amino acids, or small peptides with a length of less than 100, preferably less than 50, more preferably less than 25 amino acids, and even more preferably less than 16 amino acids. The amino acids of such peptides may be natural amino acids, non-natural amino acids (e.g., β-amino acids, γ-amino acids, D-amino acids), or combinations thereof. Furthermore, the present invention may encompass retro-inverso peptides of each epitope. The peptides may or may not be bound. The peptide may be bound to, for example, a low molecular weight (e.g., a drug or fluorophore), a high molecular weight polymer (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI), hydroxypropyl methacrylate (HPMA), etc.), or a protein, fatty acid, or sugar moiety, or inserted into a membrane. To test whether the antibody in question and the antibody of the present invention recognize the same epitope, the following competitive study may be performed: Vero cells infected with 3 moi (multiple levels of infection) are incubated for 20 hours with various concentrations of the antibody in question as a competitor for 1 hour. In the second incubation step, the antibody of the present invention is applied at a constant concentration of 100 nM, and its binding is detected by flow cytometry using a fluorescently labeled antibody made against the constant domain of the antibody of the present invention. Anti-proportional binding relative to the concentration of the antibody in question indicates that both antibodies recognize the same epitope. However, many other assays that can be used are known in the art.

[0109] The antibody or antigen-binding fragment of the present invention is not limited to antibodies that detect the above-mentioned epitopes of glycoprotein B of HSV-1 and HSV-2. In fact, other antibodies that detect other epitopes of glycoprotein B, or even other protein or polypeptide epitopes of HSV-1 and HSV-2, may also be used, as such antibodies are described above and below herein, up to 5.0 x 10⁻¹⁶. -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis This is assumed insofar as it has the ability to inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission), as described above and below herein, and / or insofar as it can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0110] Using the ordinary techniques and routine methods of those skilled in the art, relevant epitopes (and functional fragments) of HSV polypeptides useful in the production of antibodies such as polyclonal and monoclonal antibodies can be readily determined from the sequences provided herein. However, those skilled in the art are also readily in a position to provide engineered antibodies such as CDR graft antibodies, or humanized antibodies and fully human antibodies, etc.

[0111] Monoclonal antibodies are particularly preferred in the context of the present invention. Any technique that provides antibodies produced by serial cell line culture can be used to prepare monoclonal antibodies. Examples of such techniques include the hybridoma method, trioma method, human B-cell hybridoma method, and EBV-hybridoma method for producing human monoclonal antibodies (Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press; Goding and Goding (1996), Monoclonal Antibodies: Principles and Practice - Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, Academic Pr Inc, USA).

[0112] Antibody derivatives can also be produced by peptidomimetic methods. Furthermore, techniques described for the production of single-chain antibodies (see, in particular, U.S. Patent 4,946,778) can be combined to produce single-chain antibodies that specifically recognize the HSV antigen. In addition, transgenic animals may be used to express humanized antibodies against HSV polypeptides.

[0113] The present invention also envisions the production of specific antibodies against native and recombinant polypeptides of glycoprotein B, or against any other protein or polypeptide of HSV-1 and HSV-2. This production is based on immunization of animals such as mice. However, other animals are also envisioned in the present invention for antibody / antiserum production. For example, monoclonal and polyclonal antibodies can be produced by rabbits, mice, goats, donkeys, etc. Polynucleotides encoding a accordingly selected polypeptide of HSV-1 or HSV-2 can be subcloned into a suitable vector, where the recombinant polypeptide is expressed in an organism capable of expression, such as bacteria. Thus, the expressed recombinant protein can be injected intraperitoneally into mice, and the resulting specific antibodies can be obtained, for example, from mouse serum obtained by intracardiac blood aspiration. The present invention also envisions the production of specific antibodies against native and recombinant polypeptides using DNA vaccine strategies, as illustrated in the appended examples. DNA vaccine strategies are well known in the art and include delivery via liposomes, gene guns or jet injections, and intramuscular or intradermal injections. Therefore, antibodies produced against HSV-1 and HSV-2 polypeptides, proteins, or epitopes can be obtained by directly immunizing animals with a vector expressing the desired HSV-1 and HSV-2 polypeptide, protein, or epitope, particularly the gB epitope, by direct intramuscular injection. The amount of specific antibody obtained can be quantified using ELISA, which is also described below herein. Further methods for antibody production are well known in the art. For example, see Harlow and Lane, "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor, 1988.

[0114] As used herein, the term “specifically bind” refers to a binding reaction that determines the presence of an antibody in the presence of a desired polypeptide or protein or epitope of HSV-1 and HSV-2, particularly the gB epitope, as well as a heterogeneous population of proteins and other biologics.

[0115] Therefore, under specified assay conditions, the specified antibody and the corresponding polypeptides or proteins or epitopes of HSV-1 and HSV-2, particularly the gB epitope, bind to each other but not to other components present in the sample in significant amounts. Specific binding to the target analyte under such conditions may require a binding site selected for specificity to the particular target analyte. Various immunoassay formats may be used to select antibodies that specifically react with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immune to an analyte. For a description of immunoassay formats and conditions that can be used to confirm specific immunoreactivity, see Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press, and / or Howard and Bethell (2000) Basic Methods in Antibody Production and Characterization, Crc. Pr. Inc. Typically, specific or selective reactions have a background signal at least twice as loud as the noise, and more typically 10 to 100 times louder than the background. Those skilled in the art are in a position to provide and construct specific binding molecules for novel polypeptides. For specific binding assays, specific binding molecules can be readily used to avoid undesirable cross-reactivity. For example, polyclonal antibodies can be readily purified and selected by known methods (see Shepherd and Dean, previously cited).

[0116] The term “anti-HSV antibody or its antigen-binding fragment” means, in accordance with the present invention, that the antibody molecule or its antigen-binding fragment can specifically recognize, specifically interact with, and / or bind to at least two amino acids of a desired polypeptide or protein or epitope of HSV-1 and HSV-2, particularly the gB epitope. The term relates to the specificity of the antibody molecule, i.e., its ability to distinguish a specific region of a desired polypeptide or protein or epitope of HSV-1 and HSV-2, particularly the gB epitope. Thus, specificity can be experimentally confirmed by methods known in the art and by methods disclosed and described herein. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA tests and peptide scans. Such methods also include determining the Kd value, as exemplified in the appended examples. Peptide scans (pepspot assays) are routinely used to map linear epitopes in polypeptide antigens. The primary sequence of a polypeptide is synthesized sequentially on activated cellulose using peptides that overlap with each other. The recognition of a specific peptide by an antibody, whose ability to detect or recognize a particular antigen / epitope is to be tested, is scored by a chemiluminescent reaction or by routine color development using similar means known in the art (secondary antibody with horseradish peroxide, 4-chloronaphthol, and hydrogen peroxide). In particular, the reaction can be quantified in the case of a chemiluminescent reaction. If the antibody reacts with a particular set of overlapping peptides, the minimum amino acid sequence required for the reaction can be estimated. The same assay may reveal two separate clusters of reactive peptides. This indicates that discontinuous epitopes, i.e., conformational epitopes, in the antigenic polypeptide have been recognized (Geysen (1986), Mol. Immunol. 23, 709-715).

[0117] In a preferred embodiment, the present invention relates to an anti-HSV antibody or its antigen-binding fragment that recognizes the same epitope as defined above herein, wherein the recognition of the epitope can be confirmed by cryo-electron microscopy (Cryo-EM).

[0118] As mentioned above, the antibody of the present invention binds to specific epitopes on gB, including the following contact amino acid residues: D199, A203, K204, Y303, R304, Q321, V322, D323, K320, Y326, R335, and T337 in HSV-1 gB, or D191, A195, K196, Y295, R296, K312, Q313, V314, D315, Y318, R327, and T329 in HSV-2 gB. These epitopes were confirmed by cryo-electron microscopy (CryoEM) analysis.

[0119] In a preferred embodiment, the antibody of the present invention binds to a specific epitope on gB, consisting of the following contact amino acid residues: D199, A203, K204, Y303, R304, Q321, V322, D323, K320, Y326, R335, and T337 in HSV-1 gB, or D191, A195, K196, Y295, R296, K312, Q313, V314, D315, Y318, R327, and T329 in HSV-2 gB. This epitope has been confirmed by cryo-electron microscopy (CryoEM) analysis. Therefore, in a particular embodiment, the epitope can be confirmed by cryo-electron microscopy (CryoEM).

[0120] As outlined above, whether an antibody binds to the same epitope as a reference antibody can be determined by methods known to those skilled in the art. However, in one embodiment, this can be determined as described in the examples added to the end of this specification. In certain embodiments, the epitope can be determined by cryo-electron microscopy (Cryo-EM).

[0121] In a preferred embodiment, the epitope can be confirmed as outlined below. HSV-1 gB and HDIT102(H4)Fab are mixed in an appropriate ratio. Aliquots of the mixture are adsorbed onto an appropriate grid, wiped off with filter paper, and vitrified in liquid ethane at -180°C. Data of the HSV-1 gB and HDIT102(H4)-Fab composite are acquired using a transmission electron microscope. Microscope video is recorded in counting mode at an appropriate magnification and dose.

[0122] Data processing and model building are performed using specific software for image processing steps such as dose splitting and gain-corrected video dose weighting and motion correction, as well as contrast transfer function (CTF) parameter estimation. Electron microscope images exhibiting strong drift, astigmatism, and maximum CTF resolution worse than 8 Å are excluded from further processing. A total of 1 million to 10 million particles are picked. The particle dataset is cleaned by three reference-free 2D classifications. A novel initial 3D model is created from the 2D particles using a probabilistic algorithm. The particle dataset is further cleaned by three unsupervised 3D classifications. The remaining particles are subjected to Bayesian particle polishing, CTF and aberration refinement, and final high-resolution 3D refinement to obtain the final map.

[0123] The HSV-1 gB X-ray structure (PDB-ID: 2GUM) can be manually mutated at positions T313S, Q443L, and V553A and placed into the final map using specific software. For HDIT102(H4)Fab, the crystal structure of the humanized recombinant Fab fragment (PDB-ID 7PHU) is mutated in silico based on sequence alignment. Three HDIT102(H4)Fab fragments are placed in the final map in silico. Specialized software is used for the initial fitting of HSV-1 gB and the three HDIT102(H4)Fab fragments into the final map. The final protein model is obtained by repeating manual model construction, refinement, and model validation several times. Statistical analysis of data acquisition, refinement, and validation can be performed as outlined in the data processing workflow in Table 1 and Figure 8D.

[0124] In a more preferable embodiment, and more specifically, the epitope can be identified as outlined below.

[0125] HSV-1 gB and HDIT102(H4)Fab are mixed in a ratio of 1 to 3.5. A 4 μl aliquot of the mixture is adsorbed onto a glow-discharged Quantifoil Cu-R2 / 1-300 mesh holey carbon coated grid (Quantifoil, Germany), wiped with Whatman 1 filter paper, and vitrified in liquid ethane at -180°C using a Leica EM GP2 plunger (Leica microsystems, Austria) operated at 10°C and 85% humidity. Data on the HSV-1 gB and HDIT102(H4)-Fab complex are acquired using a Titan Krios G1 TEM (ThermoFisher, USA) operated at 300 kV and equipped with a Gatan Energy Filter (Ametek, USA) and a K2 Summit direct electron detector (Ametek, USA). 40 frames of microscope video are recorded at a magnification of 165,000 × (pixel size 0.82 Å) and 1.15 e. - / Å 2Recorded in counting mode with dose per frame, approximately 46e per irradiation. - / Å 2 The total accumulated dose at the specimen level is obtained.

[0126] Data processing and model building were performed as follows: All image processing steps were performed using Relion v4.0 (Kimanius, Dong et al., 2021, Biochem J, Vol. 478 (24)). Dose splitting and gain-corrected video dose weighting and motion correction were performed using the UCSF motioncor2 program's Relion implementation. Contrast transfer function (CTF) parameters were estimated using ctffind 4.1.14 (Rohou and Grigorieff, 2015, J Struct Biol, Vol. 192 (2)). Electron micrographs exhibiting strong drift, astigmatism greater than 1000 Å, and maximum CTF resolution worse than 8 Å were excluded from further processing. A total of 1 million particles are picked using a Laplacian-of-Gaussian (LoG) filter in Relion 4.0 (Kimanius, Dong et al., 2021, Biochem J, Vol. 478 (24)). The particle dataset is further refined with three reference-free 2D classifications to obtain 277,376 particles. A new 3D initial model is created from the 2D particles using Relion's Stochastic Gradient Descent (SGD) algorithm. The particle dataset is further refined with three unsupervised 3D classifications. The remaining 208,280 particles were subjected to Bayesian particle polishing, CTF and aberration refinement, and final high-resolution 3D refinement, resulting in a final map with an FSC of 0.143 and a total resolution of 3.44 Å according to the gold standard Fourier shell correlation (FSC).

[0127] The HSV-1 gB X-ray structure (PDB-ID: 2GUM) can be manually mutated at positions T313S, Q443L, and V553A and placed in the final map using coot (Casanal, Lohkamp et al., 2020, Protein Sci, Vol. 29 (4)). In the case of HDIT102(H4)Fab, the crystal structure of the humanized recombinant Fab fragment (PDB-ID 7PHU) is mutated using coot (Casanal, Lohkamp et al., 2020, Protein Sci, Vol. 29 (4)) based on the sequence alignment created by Needle EMBOSS (Rice, Longden et al., 2000, Trends Genet, Vol. 16 (6)). Three HDIT102(H4)Fabs are placed into the final map using coot (Casanal, Lohkamp et al., 2020, Protein Sci, Vol. 29 (4)). Molrep from CCP-EM software suite v1.6 (Nicholls, Tykac et al., 2018, Acta Crystallogr D Struct Biol, Vol. 74 (Pt 6)) is used for the initial fitting of HSV-1 gB and the three HDIT102(H4)Fabs into the final map. The final protein model is obtained by repeatedly performing manual model construction using coot (Casanal, Lohkamp et al., 2020, Protein Sci, Vol. 29 (4)), Refmac-Servalcat refinement, and model validation using CCP-EM software suite v1.6 (Nicholls, Tykac et al., 2018, Acta Crystallogr D Struct Biol, Vol. 74 (Pt 6)). Statistical analysis of data acquisition, refinement, and validation can be performed as outlined in the data processing workflow in Table 1 and Figure 8D.

[0128] In a preferred embodiment, the present invention relates to an anti-HSV antibody or its antigen-binding fragment as defined above and below herein, wherein the anti-HSV antibody is a humanized antibody or a fully human antibody.

[0129] The “humanization approach” is well known in the art and is described in particular with respect to antibody molecules, e.g., Ig-derived molecules. The term “humanized” refers to a humanized form of a non-human (e.g., mouse) antibody or fragment thereof (e.g., Fv, Fab, Fab', F(ab'), scFv, or other antigen-binding subsequences of an antibody) that contains a portion of a sequence derived from a non-human antibody. Humanized antibodies include human immunoglobulins in which residues derived from the complementarity-determining region (CDR) of human immunoglobulin are replaced with residues derived from CDRs of non-human species such as mouse, rat, or rabbit, which have the desired binding specificity, affinity, and ability.

[0130] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies are created by introducing one or more amino acids from a non-human source, either to retain the original binding activity of the antibody or to further improve it. Methods for humanizing antibodies / antibody molecules are described in detail in Jones et al., Nature 321 (1986), 522-525; Reichmann et al., Nature 332 (1988), 323-327; and Verhoeyen et al., Science 239 (1988), 1534-1536, as well as in patent documents, e.g., EP-B1 451216. Specific examples of humanized antibodies, e.g., antibodies made against EpCAM, are known in the art. See, for example, LoBuglio, Proceedings of the American Society of Clinical Oncology Abstract (1997), 1562, and Khor, Proceedings of the American Society of Clinical Oncology Abstract (1997), 847.

[0131] In the context of humanization, the operating principle is to improve the fit between CDR and FR in the antibody. To improve this fit, amino acid substitutions can be made in either FR or CDR.

[0132] Humanized antibodies may be based on "chimeric antibodies" or on "CDR" graft antibodies. The term "chimeric" generally refers to an antibody in which part of the heavy chain and / or light chain originates from a particular source or species, but the remainder of the heavy chain and / or light chain originates from a different source or species.

[0133] To create antibodies that are more human-like, antibody chimeras are typically created (by replacing the constant region of a mouse antibody with a constant region derived from a human), but simple chimeras are usually not called "humanized." More precisely, the amino acid sequence of a humanized antibody is usually further modified so that the protein sequence of the humanized antibody is essentially identical to that of the human variant, even though part of the complementarity-determining region (CDR) segment responsible for the antibody's ability to bind to its target antigen is derived from a non-human.

[0134] More specifically, the "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing the minimal sequence derived from the non-human antibody. Mostly, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's hypervariable region (i.e., CDR) are replaced with residues derived from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, resulting in desirable antibody specificity, affinity, and capability. Thus, in certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR corresponds to that of the non-human antibody, and all or substantially all of the framework corresponds to that of the human antibody.

[0135] When "humanizing" an antibody, further improvements can be made, namely modifications to the amino acid sequence of the antibody. For example, modifications can be made to the amino acid sequence of the antibody to reduce its immunogenicity in humans while retaining or substantially retaining the antigen-binding properties of the parent antibody.

[0136] Various modifications and adaptations to address these variations are known to those skilled in the art.

[0137] Therefore, the "humanization" of antibodies may also include further improvements that, in certain embodiments, result in humanized antibodies that contain residues not found in either the recipient or donor antibody. These modifications are made to further improve antibody performance.

[0138] In some cases, framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues.

[0139] Humanized antibodies also optionally include, at least, a portion of the antibody / immunoglobulin constant region (Fc), typically a portion of the antibody / immunoglobulin constant region (Fc) of human immunoglobulins. The humanized form of an antibody, e.g., a non-human antibody, refers to a humanized antibody. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0140] Accordingly, in the context of the present invention and in preferred embodiments, an antibody molecule that is humanized and can be successfully used in a pharmaceutical composition is provided.

[0141] In a preferred embodiment, the anti-HSV antibody according to the first aspect of the present invention is a full-length antibody, that is, a fully immunoglobulin molecule, often also called a complete antibody.

[0142] The present invention also provides anti-HSV antigen-binding fragments of anti-HSV antibodies according to a first aspect of the present invention. Preferred antigen-binding fragments are described below.

[0143] In the context of this invention, a "single-chain Fv" or "scFv" antibody fragment is the V of the antibody. H Domain and V L It has domains, and these domains are present on a single polypeptide chain. Generally, scFv polypeptides allow scFv to form structures desirable for antigen binding. H Domain and V L It further includes a polypeptide linker between the domains. Techniques for producing single-chain antibodies are described, for example, in Pluckthun in The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer-Verlag, NY (1994), 269-315.

[0144] As used herein, "Fab fragment" refers to a C123 fragment consisting of one light chain and one heavy chain. H It consists of a 1 and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0145] The "Fc" region is the C of the antibody. H 2 and C H It contains two heavy chain fragments, each containing three domains. The two heavy chain fragments have two or more disulfide bonds and C H The three domains are bound together by hydrophobic interactions.

[0146] A "Fab' fragment" has one light chain and V such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule. H Domain and C H It contains 1 domain, C H 1 domain and C H It contains a single heavy chain portion that also includes the region between the two domains.

[0147] The "F(ab')2 fragment" consists of two light chains and C, such that an interchain disulfide bond is formed between the two heavy chains. H 1 domain and C H It contains two heavy chains, each containing a constant region between the two domains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments linked by a disulfide bond between the two heavy chains.

[0148] The "Fv region" includes variable regions derived from both heavy and light chains, but lacks a steady region.

[0149] Antibodies, antibody constructs, antibody fragments, antibody derivatives (all derived from Ig), or their corresponding immunoglobulin chains used in accordance with the present invention may be further modified using conventional techniques known in the art, for example, by using alone or in combination, amino acid deletions, insertions, substitutions, additions, and / or recombinations and / or any other modifications known in the art. Methods for introducing such modifications into the DNA sequence underlying the amino acid sequence of the immunoglobulin chain are well known to those skilled in the art. See, for example, Sambrook (1989), previously cited. The term “Ig-derived domain” particularly refers to (poly)peptide constructs comprising at least one CDR. The listed fragments or derivatives of Ig-derived domains define (poly)peptides that are part of the antibody molecules described above and / or modified by chemical / biochemical or molecular biological methods. The corresponding methods are publicly known in the art and, in particular, are described in the laboratory manual (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd edition (2001); Gerhardt et al., Methods for General and Molecular Bacteriology ASM Press (1994); Lefkovits, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press (1997); Golemis, Protein-Protein Interactions: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press (2002)).

[0150] Accordingly, in the context of the present invention, the antibody molecules described above herein are selected from the group consisting of full-length antibodies (immunoglobulins such as IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE), chimeric antibodies, CDR graft antibodies, fully human antibodies, bivalent antibody constructs, antibody fusion proteins, synthetic antibodies, bivalent single-chain antibodies, trivalent single-chain antibodies, and polyvalent single-chain antibodies.

[0151] Furthermore, as outlined above, the present invention also relates to antigen-binding fragments of the antibody of the present invention, preferably selected from the group consisting of F(ab)-, Fab'-SH-, Fv-, Fab'-, and F(ab')2- fragments.

[0152] Therefore, in a preferred embodiment, the anti-HSV antibody according to the first aspect of the present invention is a human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE antibody.

[0153] In a more preferred embodiment, the antigen-binding fragment of the anti-HSV antibody according to the first aspect of the present invention is a human F(ab)-, Fab'-SH-, Fv-, Fab'-, or F(ab')2- fragment.

[0154] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention has a dissociation constant Kd of up to 10 nM, preferably up to 8 nM, more preferably up to 4 nM, even more preferably up to 2 nM, up to 1 nM, up to 0.8 nM, up to 0.4 nM, up to 0.2 nM, up to 0.1 nM, up to 0.09 nM, up to 0.08 nM, up to 0.07 nM, up to 0.06 nM, up to 0.05 nM, up to 0.04 nM, and most preferably up to 0.03 nM.

[0155] Kd represents the dissociation constant as a measure of the tendency of a complex to reversibly dissociate into its components (i.e., the affinity of the antibody to the antigen), and is the reciprocal of the association constant. Kd can be calculated from the Scatchard formula, and methods for determining Kd are well known in the art.

[0156] As described above and below in this specification, the anti-HSV antibody of the present invention provides a fully human antibody or antigen-binding fragment (HDIT102(H4)) that binds to a conserved epitope of glycoprotein B(gB) of herpes simplex virus type 1 and type 2 with a Kd of about 0.09 to 0.03 nM.

[0157] The dissociation constant Kd can be determined by methods known to those skilled in the art. In one embodiment, the dissociation constant Kd is determined as described in the examples added to the end of this specification.

[0158] Therefore, in a preferred embodiment, Kd can be determined as outlined below.

[0159] To produce recombinant HSV-1 gB protein, a codon-optimized DNA sequence encoding the HSV-1 gB extradomain (aa 30-729; UniProtKB P06436.1) or the HSV-2 gB extradomain (aa 22-724; GenBank: QAU10948.1), including a signal peptide and C-terminal tag, can be cloned into a mammalian expression vector. The recombinant HSV gB protein can then be transiently expressed in HEK293-E6 suspension cells cultured in culture medium. HEK293-E6 cells are transfected with the gB coding plasmid. On day 5 after transfection, the supernatant is collected by centrifugation. The pH of the supernatant is then adjusted by adding 1 ml of 2 M Tris buffer pH9 / 100 ml of supernatant. The gB protein is then purified by tag-affinity gravity flow purification. Next, the gB protein tag is removed by thrombin digestion and dialyzed against 50 mM Tris, 150 mM NaCl, pH 8. Then, the thrombin-digested gB protein is purified three times by tag-specific affinity chromatography to deplete the sample from any remaining tagged gB protein. Next, the gB protein is concentrated and further purified by size exclusion chromatography. The peak fraction is pooled and concentrated.

[0160] The anti-HSV gB antibody Fab may be prepared by digestion with papain (as done for HDIT101) followed by purification, or by recombination by transfection of plasmids encoding the light chain and cleaved heavy chain into 293T-E6 cells (as done for HDIT102(H4)). The HSV gB recombinant protein is biotinylated, and then any remaining biotin is removed. An initial loading scout is performed to find the best biosensor loading concentration. Various concentrations of biotinylated gB are loaded into a streptavidin biosensor (Octet), and the absorption kinetics of the test antibody Fab fragment are measured. The diluted series of the Fab fragments are then analyzed to determine the association rate (ka), dissociation rate (kdis), and dissociation constant Kd (Kd = kdis / ka).

[0161] More specifically, Kd can be calculated as outlined below.

[0162] To produce recombinant HSV-1 gB protein, a codon-optimized DNA sequence encoding either the HSV-1F gB external domain (aa 30-729; UniProtKB P06436.1) or the HSV-2G gB external domain (aa 22-724; GenBank: QAU10948.1), including the BM40 signal peptide and a C-terminal double Strep-tag, can be cloned into a pCAGGS mammalian expression vector. The recombinant HSV gB protein can then be transiently expressed in HEK293-E6 suspension cells cultured in serum-free medium, e.g., F17 medium (ThermoFisher) supplemented with 0.1% Kolliphor (Sigma) and 4 mM glutamine. HEK293-E6 cells are cultured with 1 μg of gB coding plasmid and 2 μg of PeiMax / ml culture medium at a rate of 1.5–2.0 x 10⁻¹⁴. 6Transfect cells at a cell density of cells / ml using PeiMax (Polysciences). Add Tryptone N1 feeder (Organi Technie) to the culture 24 hours after transfection. On day 5 after transfection, collect the supernatant by two centrifugation steps: first at 1200 rpm to remove cells, and then at 3600 rpm to remove cell debris. Next, adjust the pH of the supernatant by adding 1 ml of 2 M Tris buffer pH 9 / 100 ml of supernatant. Then, purify the gB protein by gravity flow purification using Strep-Tactin XT (IBA, Germany) according to the manufacturer's protocol. Next, remove the Strep-tag from the gB protein by thrombin digestion (Serva) and dialyze against 50 mM Tris, 150 mM NaCl, pH 8. Next, the thrombin-digested gB proteins are purified three times by Strep-Tactin XT affinity chromatography to deplete the sample from any remaining Strep-tagged gB proteins. Then, the gB proteins are concentrated on an Amicon spin column (cutoff 30k) and further purified using a Superdex 200 10 / 300 GL SEC column and Akta Pure FPLC. The peak fractions are pooled and concentrated using an Amicon spin column.

[0163] The anti-HSV gB antibody Fab may be prepared by digestion with papain (as done for HDIT101) followed by purification, or by recombination by transfection of plasmids encoding the light chain and cleaved heavy chain into 293T-E6 cells (as done for HDIT102(H4)). The HSV gB recombinant protein is biotinylated at a ratio of 3:1 at room temperature for 30 minutes (NHS-PEG4-biotin (Thermo Fischer Scientific, A39259)), and the remaining biotin is then removed using a desalting column and centrifugation at 1000 g for 2 minutes (Zeba Spin Desalting Columns; 7K MWCO, 2 ml (Thermo Scientific)). (UE285726). An initial loading scout is performed to find the best biosensor loading concentration. Various concentrations of biotinylated gB are loaded into the streptavidin biosensor (Octet), and the absorption kinetics of the test antibody Fab fragment are measured. The optimal gB concentration for loading into the biosensor was determined to be 5 μg / ml. Biotinylated gB (wt) [5 μg / ml] is used to load into the biosensor. The binding kinetics of the antibody Fab fragment (100 nM) to immobilized gB can be analyzed using a global 1:1 fit model. The dilution series of the Fab fragment is then analyzed to determine the association rate (ka), dissociation rate (kdis), and Kd (Kd = kdis / ka).

[0164] Other methods for determining the dissociation constant Kd are also known to those skilled in the art. Not bound by theory, as a further example, the dissociation constant Kd can also be roughly determined, for example, by using infected cells.

[0165] Therefore, the dissociation constant Kd can be determined as outlined below.

[0166] To determine the dissociation constant Kd, Vero cells (approximately 80% confluent) are infected with HSV-1F or HSV-2G at a multiple of infection (MOI) of 1. After a 16-20 hour incubation period, the virus-containing medium is discarded, the cells are washed once with PBS, resuspended, and collected. The cells are then pelleted at 300 × g for 5 minutes and refrigerated in PBS at a rate of 5.0 × 10⁶. 6 Resuspend the cells at a cell density of / ml cells. Distribute the suspension uniformly into 96-well plates at a volume of 100 μl / well. To determine the equilibrium dissociation constant, incubate HSV-infected Vero cells with a 1:2 dilution series (0.03–500 nM) of unbound primary antibody three times at room temperature for 45 minutes. After two washes, add FITC-conjugated anti-human Fc detection antibody (Fcγ IgG, polyclonal rabbit anti-human FITC, Jackson ImmunoResearch (309-096-008)) that targets the Fc domain of the primary antibody. After a 30-minute incubation period, wash the cells twice and transfer them to 500 μl of PBS. Then, determine the mean fluorescence intensity (MFI) of each sample by flow cytometry. As a negative control for staining, infected cells are incubated with secondary antibody alone. As a negative control for specificity, uninfected Vero cells are stained in the same manner. The equilibrium dissociation constant (maximum half-capacity binding or maximum half-capacity saturation of the antibody) is calculated using Graph Pad Prism and applying the one-site specific binding method for nonlinear regression. The calculation principle is as follows: Y = Bmax × X / (Kd + X), which is based on a curve known as a rectangular hyperbola, binding isotherm, or saturation binding curve. Y starts at 0 and increases to the maximum plateau value Bmax. This equation represents the equilibrium binding between ligand and receptor as a function of gradually increasing ligand concentration. X is the ligand concentration, Y is the specific binding, and Bmax is the maximum number of binding sites, expressed in the same units as the Y axis. Kd is the equilibrium dissociation constant, expressed in the same units (concentration) as the X axis. When the drug concentration is equal to KD, half of the binding sites are occupied in equilibrium, i.e., 50% of binding is observed for a normalized Y value (EC50).

[0167] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention can neutralize HSV. In a still more preferred embodiment, the antibody or its antigen-binding fragment can neutralize HSV-1 and / or HSV-2.

[0168] Accordingly, in a more preferred embodiment, an anti-HSV antibody or antigen-binding fragment according to the first aspect of the present invention at a concentration of up to 20 nM, preferably up to 16 nM, more preferably up to 12 nM, up to 10 nM, up to 8 nM, up to 6 nM, and most preferably up to 4 nM can neutralize a defined amount of HSV of 100 TCID50.

[0169] In this specification, "neutralization" means that an antibody opsonizes the virus so that it can no longer infect cells. For example, an antibody at a maximum concentration of 20 nM opsonizes a defined amount, e.g., 100 TCID 50 An assay for testing whether HSV can be neutralized. Eis-Hubinger et al., Intervirology 32:351-360 (1991); Eis-Hubinger et al., Journal of General Virology 74:379-385 (1993), and Examples 1 and 2 of WO2011 / 038933A2. Accordingly, in a preferred embodiment, the antibody of the present invention at a concentration of up to 20 nM, preferably up to 16 nM, more preferably up to 12 nM, up to 10 nM, up to 8 nM, up to 6 nM, and most preferably up to 4 nM is used in 100 TCID 50 The defined amount of HSV can be neutralized to more than 50%, preferably more than 60%, more preferably more than 80%, more preferably more than 90%, for example more than 95%, more preferably more than 96%, for example more than 97%, most preferably more than 98%, for example more than 99%, or even up to 100%.

[0170] As described above and below in this specification, the anti-HSV antibodies of the present invention inhibit cell-free HSV-1 infection of Vero cells at IC50 concentrations of up to 30 nM, up to 20 nM, more preferably up to 10 nM, up to 8 nM, and most preferably up to 6 nM.

[0171] Preferably, IC50 is determined as follows. IC50 may be determined by methods known to those skilled in the art. In one embodiment, IC50 is determined as described in the examples added to the end of this specification. In a particular embodiment, IC50 is determined using infected cells.

[0172] In a preferred embodiment, the IC50 is determined as follows. The following method can be used to investigate the antiviral activity of anti-HSV gB antibodies against cell-free viruses. Various antibody dilutions are incubated with a constant viral dose (100 TCID50 HSV-1 or HSV-2). The antibody-virus mixture is applied to Vero cells with 80-90% confluenza at a volume of 100 μl / well in a 96-well plate. As a control, Vero cells are infected with a viral dose of 100 TCID50 without prior incubation with a neutralizing antibody. The degree of cytopathic effect is examined by light microscopy three days after infection. The neutralization concentration is determined to be the maximum antibody dilution at which the virus is still completely neutralized and the formation of CPE in the inoculated cell culture is completely inhibited. Furthermore, the neutralizing antibody concentration (IC50) at which 50% of the cell culture wells are protected from infection is calculated.

[0173] More specifically, the IC50 can be determined as outlined below. To investigate the antiviral activity of anti-HSV gB antibodies against cell-free viruses, the following method can be used: Various antibody dilutions are incubated with a constant viral dose (100 TCID50 HSV-1F or HSV-2G) at 37°C for 1 hour. The antibody-virus mixture is then placed in a 96-well plate (2.0 x 10⁶). 4The solution is applied to Vero cells containing 80-90% confluenza at a volume of 100 μl / well (cells / well). As a control, Vero cells are infected with a viral dose of 100 TCID50 without prior incubation with neutralizing antibody. The degree of cytopathic effect is examined by light microscopy 3 days after infection. The neutralizing titer is determined to be the maximum antibody dilution at which the virus is still completely neutralized and the formation of CPE in the inoculated cell culture is completely inhibited. Furthermore, the neutralizing antibody concentration (IC50) at which 50% of the cell culture wells are protected from infection is calculated using the 50% neutralizing titer formula (Krawczyk, Krauss et al., 2011, J Virol, Vol. 85 (4)): T=x+((b / 10)·x). T = neutralizing antibody titer at which 50% of the infected cell culture is protected from infection. x = minimum antibody dilution at which at least 50% of the cell culture is infected. b = the number of infected cell cultures with an infection rate exceeding 50% at dilution x.

[0174] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission).

[0175] Recently, the ability of antibodies to block intercellular transmission has been correlated with the ability of antibodies to block recurrence of oral HSV-1 outbreaks (Alt, Wolf et al., 2023, Front Immunol, Vol. 14).

[0176] Intercellular transmission is the ability of herpesviruses to spread to adjacent, uninfected cells without releasing cell-free particles. Reducing or eliminating the ability of herpesviruses to spread to adjacent cells has the beneficial effect of preventing the development of lesions. Methods well known to those skilled in the art can be used to investigate whether antibodies can inhibit the transmission of HSV from infected cells to adjacent, uninfected cells (intercellular transmission).

[0177] As an example, the following assay can be used: Vero cells grown to confluence on a coverslip in a 24-well tissue culture plate are subjected to 400 TCID. 50 Infect a well with a fixed viral load at 37°C for 4 hours. The median tissue culture infective dose (TCID) is calculated as follows: 50 ) is the amount of cytopathogenic substance (agent), such as a virus, that produces a cytopathic effect in 50% of the inoculated cell culture. Subsequently, the viral inoculum is removed, the cells are washed twice with PBS, and then further incubated at 37°C for 2 days in 1 ml DMEM, 2% FCS, Pen / Strep containing an excess amount of various anti-HSV antibodies or polyclonal anti-HSV control serum to inhibit viral transmission via the supernatant. Viral antigens of HSV-infected cells are detected with fluorescently labeled polyclonal goat-anti-HSV serum (e.g., BETHYL Laboratories, Montgomery, TX USA, catalog number A190-136F, Lot No. A190-136F-2). Preferably, if less than 20% of adjacent cells are infected, the antibody inhibits intercellular transmission. In the above assay, less than 15%, less than 10%, less than 5%, more preferably less than 3%, and most preferably less than 1% of adjacent cells are infected.

[0178] Intercellular transmission can also be assayed as follows: The presence of neutralizing antibodies does not necessarily prevent intercellular transmission of herpesviruses. To compare antibodies during disruption of HSV-1 and HSV-2 intercellular transmission, this unique mode of spread can be mimicked in vitro using standard assay methods. For example, to infect individual cells, confluent Vero cell monolayers are first incubated with HSV-1 or HSV-2 at a low MOI (e.g., 100 TCID50), respectively. After adsorption at 37°C for 4 hours, the viral inoculum is removed. To promote direct intercellular transmission from individually infected cells, but to prevent viral transmission via viral particles across the cell culture supernatant, Vero cell monolayers are treated with an excess of neutralizing anti-GB antibody, a control, or the culture medium alone. After 48 hours, viral transmission can be detected by immunostaining using a mouse monoclonal antibody specific to a common epitope on glycoprotein D of HSV-1 and HSV-2 (e.g., Acris Antibodies, San Diego, CA, USA) and a fluorescently conjugated secondary antibody. Immunofluorescence images can be acquired at 100x or 400x magnification using a fluorescence microscope.

[0179] The ability to inhibit the transmission of HSV from infected cells to adjacent uninfected cells (intercellular transmission) can also be confirmed as described in the examples added to the end of this specification. In certain embodiments, this intercellular transmission can be confirmed using infected cells.

[0180] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention exhibits its antiviral or neutralizing activity independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), preferably the antibody can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0181] Since the above assay for testing whether an antibody can inhibit intercellular transmission does not contain complement and / or cytotoxic effector cells, the same assay may be used to determine whether an antibody can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0182] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention is conjugated to an effector moiety, a therapeutic moiety, or a detectable label.

[0183] In this context, the term “conjugated” refers to any method known in the art for functionally linking protein domains, including but not limited to recombination fusion with or without an intervening domain, intein-mediated fusion, non-covalent association, and covalent bonding, such as disulfide-peptide bonds, hydrogen bonds, electrostatic bonds, and structural bonding, such as biotin-avidin association. Conjugation with an effector moiety may be by chemical or recombinant means. Chemical means refer to a reaction between an antibody and an effector moiety such that there is a covalent bond formed between the two molecules to form a single molecule.

[0184] The term "effector portion" refers to the compound intended to affect the target cells of an antibody. The effector portion may be, for example, a therapeutic portion or a detectable portion.

[0185] The "therapeutic portion" is a compound intended to act as a therapeutic agent, such as a cytotoxic agent or cytotoxic drug. Examples of compounds for pharmaceutical compositions are shown below.

[0186] "Detectable labels" include any compound or protein-tag that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means, such as fluorescent labels.

[0187] In the following, Phase 2 This will be explained in more detail.

[0188] As described above, in the second aspect relating to the first aspect described above, the present invention comprises (A) an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, (B) Anti-HSV antibody or antigen-binding fragment that binds to glycoprotein B(gB) of HSV-1 and / or HSV-2 The antibody is, The complementarity determination region is V, which includes SEQ ID NO:11. H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L Includes CDR3, The antibody is an anti-HSV antibody or its antigen-binding fragment that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, having a dissociation constant Kd of up to 40 nM, preferably up to 30 nM, more preferably up to 20 nM, even more preferably up to 15 nM, up to 13 nM, and up to 10 nM. Regarding combinations.

[0189] A first component (A), namely an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention, has already been described above. With respect to a preferred embodiment of the "anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention," the same applies to this first component (A), with modifications as necessary, as described above in the context of the first aspect of the present invention as defined above.

[0190] The second component (B) is an anti-HSV antibody or its antigen-binding fragment that binds to the glycoprotein B(gB) of HSV-1 and / or HSV-2, wherein the antibody is The complementarity determination region is V, which includes SEQ ID NO:11. H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L Includes CDR3, The antibody has a dissociation constant Kd of up to 40 nM, preferably up to 30 nM, more preferably up to 20 nM, even more preferably up to 15 nM, up to 13 nM, and up to 10 nM.

[0191] A method for determining that the dissociation constant Kd is at most 40 nM, preferably at most 30 nM, more preferably at most 20 nM, even more preferably at most 15 nM, at most 13 nM, and at most 10 nM has already been described in the context of the first aspect of the present invention. The same applies to the second component (B) of the second aspect of the present invention, with modifications as necessary.

[0192] The term "CDR" as used in relation to the second component (B) of the second aspect of the present invention relates to the "complementarity-determining region" well known in the art. The CDR is an immunoglobulin portion that determines the specificity of the molecule and contacts a specific ligand. The CDR is the most variable part of the molecule and contributes to the diversity of these molecules. Each V domain has three CDR regions: CDR1, CDR2, and CDR3. CDR-H refers to the CDR region of the variable heavy chain, and CDR-L refers to the CDR region of the variable light chain. VH means variable heavy chain, and VL means variable light chain. The CDR region of the Ig-derived region can be determined as described in Kabat, "Sequences of Proteins of Immunological Interest," 5th edit. NIH Publication no. 91-3242 US Department of Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987), 901-917, or Chothia Nature 342 (1989), 877-883.

[0193] In the context of the present invention, the CDR region (and framework region (FR)) is determined according to Martin's numbering scheme as described in Norman, RA, F. Ambrosetti, A. Bonvin, LJ Colwell, S. Kelm, S. Kumar and K. Krawczyk (2020). "Computational approaches to therapeutic antibody design: established methods and emerging trends." Brief Bioinform 21(5): 1549-1567.

[0194] Therefore, in the context of the present invention, references to amino acid residues follow Martin's numbering scheme.

[0195] Accordingly, in the context of the second component (B) of the second aspect of the present invention, the antibody molecules described herein above are selected from the group consisting of full-length antibodies (immunoglobulins such as IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE), chimeric antibodies, CDR graft antibodies, fully human antibodies, bivalent antibody-constructs, antibody-fusion proteins, synthetic antibodies, bivalent single-chain antibodies, trivalent single-chain antibodies, and polyvalent single-chain antibodies. Furthermore, preferably, antigen-binding fragments of antibody molecules according to the second component (B) of the second aspect of the present invention are also envisioned, selected from the group consisting of F(ab)-, Fab'-SH-, Fv-, Fab'-, and F(ab')2- fragments.

[0196] Furthermore, the antibody of the second component (B) of the second aspect of the present invention is an antibody or its antigen-binding fragment that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2.

[0197] In a preferred embodiment, the antibody of the second component (B) of the second aspect of the present invention is an antibody or an antigen-binding fragment comprising, or consisting of, a VH domain (heavy chain variable region) and a VL domain (light chain variable region), i.e., the amino acid sequence of the variable region of the heavy chain of the antibody shown in SEQ ID NO:19 and the amino acid sequence of the variable region of the light chain of the antibody shown in SEQ ID NO:20.

[0198] Therefore, in a preferred embodiment, the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention is V of SEQ ID NO:19 H and V of SEQ ID NO:20 L It is an antibody or its antigen-binding fragment, which contains [the specified substance].

[0199] However, the antibodies or antigen-binding fragments used in the present invention are not particularly limited to such variable heavy and light chain variable regions, and as described above and below herein, as long as they have a dissociation constant Kd of up to 40 nM, preferably up to 30 nM, more preferably up to 20 nM, even more preferably up to 15 nM, up to 13 nM, or up to 10 nM, or as long as they can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission), or as long as they can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), SEQ ID The antibodies or antigen-binding fragments thereof that bind to glycoprotein B(gB) of the HSV-1 and / or HSV-2 envelope, comprising or consisting of VH domains and VL domains having at least 95%, 90%, 85%, 75%, 70%, 65%, 60%, 55%, or 50% sequence homology to the sequences of NO:19 and 20, respectively.

[0200] Furthermore, the antibody or its antigen-binding fragment is a molecule containing VH and VL domains having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conserved amino acid substitutions based on the sequences of SEQ ID NO: 19 and 20. Furthermore, the antibody or its antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, FV, scFV, F(ab')2, and diabody.

[0201] To determine whether the amino acid sequences have a certain degree of identity with the sequences of SEQ ID NO: 19 and 20, a person skilled in the art can use means and methods known in the art, such as alignment, either manually or using a computer program known to the art. Such alignment can be performed, for example, using means and methods known to the art, such as the Lipman-Pearson method (Science 227 (1985), 1435) or a known computer algorithm such as the CLUSTAL algorithm. In such alignment, it is preferable that maximum homology is assigned to conserved amino acid residues present in the amino acid sequences. In a preferred embodiment, ClustalW2 is used for comparing amino acid sequences. For pairwise comparison / alignment, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.1. For multiple comparisons / alignments, the following settings are preferably selected: protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gap distance: 5; no end gap.

[0202] According to the present invention, in the context of two or more nucleic acid sequences or amino acid sequences, the terms “identical” or “percent identical” mean that, when compared or aligned to the greatest extent possible across a comparison window or a specified region, as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection, two or more sequences or subsequences, or as described above and below herein, can bind to gB of HSV-1 or HSV-2, and have a maximum density of 40 nM, preferably 30 nM, more preferably 20 nM, and even more Preferably, the nucleic acid sequence or amino acid sequence can have a dissociation constant Kd of up to 15 nM, up to 13 nM, or up to 10 nM, or can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission), or can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), and refers to two or more sequences or subsequences having a specified percentage (e.g., 60% or 65% identity, preferably 70-95% identity, more preferably at least 95% identity) of amino acid residues or nucleotides, which are the same as the aforementioned nucleic acid sequence or amino acid sequence. For example, sequences with 60-95% or greater sequence identity are considered substantially identical. Such definitions also apply to complementary strands of test sequences. Preferably, the described identity exists over a region of at least about 15-25 amino acids or nucleotides in length, more preferably over a region of about 50-100 amino acids or nucleotides in length. Those skilled in the art know, for example, how to determine the percentage identity between sequences using algorithms, such as the CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or algorithms based on FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as are well known in the art.

[0203] The FASTDB algorithm typically does not consider internal mismatched deletions or additions, i.e., gaps, in the sequence in its calculations, but this can be manually corrected to avoid overestimating % identity. However, CLUSTALW does take sequence gaps into account in its identity calculations. The BLAST and BLAST2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul (1990) J. Mol. Biol. 215:403-410) are also available to those skilled in the art. The BLASTN program for nucleic acid sequences uses, by default, a word length (W) of 11, an expected value (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3 and an expected value (E) of 10. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89:10915) uses 50 alignments (B), an expected value (E) of 10, M=5, N=4, and comparison of both strands.

[0204] Preferably, amino acid substitutions are “conservative substitutions” that refer to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, backbone conformation, and stiffness) so that changes can be frequently made without altering the biological activity of the protein. Those skilled in the art will generally recognize that substituting a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co. 4th Ed. (1987), 224). Furthermore, substitutions of amino acids that are structurally and / or functionally similar are less likely to disrupt biological activity. In the context of the present invention, the conjugate compound / antibody of the present invention comprises a polypeptide chain having a sequence that, when compared to a specific amino acid sequence disclosed herein, for example, SEQ ID NO: 19 (referring to the variable region of the antibody heavy chain) and 20 (referring to the variable region of the antibody light chain), includes 0 (unchanged), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conserved amino acid substitutions. As used herein, the conserved amino acid substitutions in the phrase "up to X" include 0 substitutions, up to 10, and any number of substitutions including 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions.

[0205] Such exemplary substitutions are preferably made in accordance with the substitutions shown in Table 1, as already indicated above, in the context of the first aspect of the invention. The same applies to the second aspect of the invention, with modifications as necessary.

[0206] Furthermore, in a preferred embodiment, the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention is located in the following framework region: SEQ ID NO:17 Position 1~30(V) H FR1), 38~51(V H FR2), 68~99(V HFR3), and 112~122(V H FR4), SEQ ID NO: 18, 1-23 (V L FR1), 41~55(V L FR2), 63~94(V L FR3), and 104~114(V L Amino acid sequences having at least 70% sequence identity for each of the amino acid residues shown in FR4) It is an antibody containing or an antigen-binding fragment thereof.

[0207] As described above, in the context of the present invention, the CDR region (and framework region (FR)) is determined according to Martin's numbering scheme, as described in Norman, RA, F. Ambrosetti, A. Bonvin, LJ Colwell, S. Kelm, S. Kumar and K. Krawczyk (2020). "Computational approaches to therapeutic antibody design: established methods and emerging trends." Brief Bioinform 21(5): 1549-1567.

[0208] Therefore, in the context of the present invention, references to amino acid residues follow Martin's numbering scheme.

[0209] In a more preferred embodiment, the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention contains an amino acid sequence having at least 75%, at least 80%, more preferably at least 85%, at least 90%, even more preferably at least 95%, most preferably 98% or 99% total sequence identity in the framework region compared to one of the amino acid residues shown at positions 1-30, 38-51, 68-99, and 112-122 of SEQ ID NO:17 and positions 1-23, 41-55, 63-94, and 104-114 of SEQ ID NO:18. Such an antibody or its antigen-binding fragment is suitable for the medical use of the present invention as long as it binds to the gB of HSV-1 or HSV-2 as described above and below herein and has a dissociation constant Kd of up to 40 nM, preferably up to 30 nM, more preferably up to 20 nM, even more preferably up to 15 nM, up to 13 nM, or up to 10 nM, or as long as it can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission), or as long as it can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0210] Accordingly, in a preferred embodiment, the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention comprises the above-mentioned variable regions of the light chain and heavy chain (i.e., the CDR as defined above, i.e., V including SEQ ID NO:11). H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 LThe amino acid sequence contains CDR3), but this amino acid sequence has at least 75%, at least 80%, more preferably at least 85%, at least 90%, even more preferably at least 95%, and most preferably 98% or 99% overall sequence identity and variability in the framework region compared to one of the amino acid residues shown at positions 1-30, 38-51, 68-99, and 112-122 of SEQ ID NO:17, and positions 1-23, 41-55, 63-94, and 104-114 of SEQ ID NO:18.

[0211] In this context, if SEQ ID NO:17 or SEQ ID NO:18 is aligned with the best-matching sequence of the polypeptide of interest, and the amino acid identity between the two aligned sequences is at least X% across positions 1-30, 38-51, 68-99, and 112-122 of SEQ ID NO:17 and positions 1-23, 41-55, 63-94, and 104-114 of SEQ ID NO:18, then the polypeptide has "at least X% sequence identity" with respect to SEQ ID NO:17 or 18 in the framework region. As mentioned above, such amino acid sequence alignment can be performed using publicly available computer homology programs, such as the "BLAST" program provided on the National Center for Biotechnology Information (NCBI) website, using the default settings provided therein. Further methods for calculating the sequence identity percentage of an amino acid sequence or set of nucleic acid sequences are known in the art.

[0212] Furthermore, in a preferred embodiment, the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention is V of SEQ ID NO:19 H and V of SEQ ID NO:20 L Includes.

[0213] The specificity of the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention is expressed not only by the content of the amino acid sequence of the antibody or antigen-binding fragment as defined above, but may also be expressed by the epitope to which the antibody can bind. Accordingly, in a preferred embodiment, the present invention utilizes an antibody or antigen-binding fragment that recognizes the same epitope as the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention as described above. As shown in the Examples section and illustrated in Figures 13A and 13B of WO2011 / 038933A2, this epitope is a discontinuous, or more precisely, pseudocontinuous, epitope located at amino acids 172-195 and 295-313 of glycoprotein B of HSV-1 and HSV-2, exhibiting partial resistance to denaturation. In the context of this application, the epitope of the mAb 2c antibody may be located inside the first 487 amino-terminal residues of the gB protein. Preferably, the epitope may include at least one amino acid sequence located within the amino acid sequence between positions 172 and 307 of the gB protein.

[0214] In a preferred embodiment, the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention is an antibody that recognizes the same epitope as the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention as defined above.

[0215] In a more preferred embodiment, the epitopes are located at amino acids Y301-E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B of HSV-1, and at the corresponding sites Y293-E297, and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B of HSV-2, Preferably, the epitopes consist of contact amino acid residues Y301-E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B (SEQ ID NO:9) of HSV-1 strain F, and contact amino acid residues Y293-E297, and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B (SEQ ID NO:40) of HSV-2 strain G.

[0216] In certain embodiments, the epitope can be identified by cryo-electron microscopy (Cryo-EM).

[0217] Previously, the epitope to which the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention binds was described, for example, in WO2011 / 038933. The epitope was confirmed by using a duplicate 15-merpeptide spanning the gB region from amino acids 31 to 505, as also described in Daumer et al., Med Microbiol Immunol 2011 (200):85-97. This epitope recognized by mAb 2c (i.e., the epitope to which the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention binds) was previously mapped using a high-resolution 13-merpeptide microarray in Krawczyk et al., Journal of Virology 2011 (85):1793-1803.

[0218] However, in the context of the present invention and as shown in the appended examples, the epitopes to which the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention binds were further characterized in detail using cryo-electron microscopy (Cryo-EM). As outlined in further detail above and below in this specification, the epitopes are located at amino acids Y301-E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B of HSV-1, and at the corresponding sites Y293-E297, and 300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B of HSV-2. This epitope characterization is consistent with those previously shown as described in the literature cited above, but the present invention uses this further refined epitope characterization.

[0219] Before further detailing the epitopes of the present invention, as generally derived by Cryo-EM, it will be understood by those skilled in the art that epitopes may be contained in gB proteins, but may also be contained in their degradation products, or may be chemically synthesized peptides. The amino acid positions are shown solely to demonstrate the location of the corresponding amino acid sequence in the gB protein sequence. The present invention encompasses all peptides containing epitopes. The peptides may be portions of polypeptides with a length greater than 100 amino acids, or small peptides with a length of less than 100, preferably less than 50, more preferably less than 25 amino acids, and even more preferably less than 16 amino acids. The amino acids of such peptides may be natural amino acids, non-natural amino acids (e.g., β-amino acids, γ-amino acids, D-amino acids), or combinations thereof. Furthermore, the present invention may encompass each retroinversopeptide of an epitope. The peptides may or may not be bound. The peptide may be bound to, for example, a low molecular weight (e.g., a drug or fluorophore), a high molecular weight polymer (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI), hydroxypropyl methacrylate (HPMA), etc.), or a protein, fatty acid, or sugar moiety, and may be inserted into the membrane. Generally, as outlined above in the context of the first aspect of the present invention, whether an antibody binds to the same epitope as a reference antibody can be determined by methods known to those skilled in the art. This applies, with modifications as necessary, to the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention as stated above in the context of the first aspect of the present invention.

[0220] Accordingly, in the context of the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention, the following competitive study may be performed to test whether the antibody in question and the antibody of the present invention recognize the same epitope: Vero cells infected with 3 moi (multiple levels of infection) are incubated for 20 hours with various concentrations of the antibody in question as a competitor for 1 hour. In the second incubation step, the antibody of the present invention is applied at a constant concentration of 100 nM, and its binding is detected by flow cytometry using a fluorescently labeled antibody prepared against the constant domain of the antibody of the present invention. Binding that is inversely proportional to the concentration of the antibody in question indicates that both antibodies recognize the same epitope. However, many other assays that can be used are known in the art.

[0221] Therefore, generally, as described in Daumer et al., Med Microbiol Immunol 2011 (200):85-9, the epitopes to which antibodies bind can be determined by using duplicate 15-merpeptides spanning the gB region from amino acids 31 to 505. Furthermore, as described in Krawczyk et al., Journal of Virology 2011 (85):1793-1803, the epitopes of HSV-1 and HSV-2 glycoprotein B recognized by mAb 2c can also be determined using high-resolution 13-merpeptide microarrays.

[0222] The sequences of glycoprotein B in HSV-1 and / or HSV-2 are well-characterized, and, as defined above, without being constrained to specific sequences, example sequences of various HSV-1 and HSV-2 strains are shown in SEQ ID NO: 9, 10, and 21-24, respectively. As previously mentioned, the epitopes recognized by the mAb 2c antibody are highly conserved among various HSV strains, as well as between HSV-1 and HSV-2.

[0223] Using the ordinary techniques and routine methods of those skilled in the art, relevant epitopes (and functional fragments) of HSV polypeptides useful in the production of antibodies such as polyclonal and monoclonal antibodies can be readily determined from the sequences provided herein. However, those skilled in the art are also readily in a position to provide engineered antibodies such as CDR graft antibodies, or humanized antibodies and fully human antibodies, etc.

[0224] In the context of the present invention (and especially in the context of the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention), monoclonal antibodies are particularly preferred. To prepare monoclonal antibodies, any technique that provides antibodies produced by serial cell line culture can be used. Examples of such techniques include the hybridoma method, trioma method, human B-cell hybridoma method, and EBV-hybridoma method for producing human monoclonal antibodies (Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press; Goding and Goding (1996), Monoclonal Antibodies: Principles and Practice - Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, Academic Pr Inc, USA).

[0225] The antibody derivative of the second component (B) of the second aspect of the present invention can also be produced by peptidomimetic. Furthermore, the techniques described for the production of single-chain antibodies (see, in particular, U.S. Patent 4,946,778) can be combined to produce single-chain antibodies that specifically recognize the HSV antigen. In addition, transgenic animals may be used to express humanized antibodies against HSV polypeptides.

[0226] The present invention also envisions the production of specific antibodies against native and recombinant polypeptides of glycoprotein B, or any other protein or polypeptide of HSV-1 and HSV-2, in the context of the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention. This production is based on immunization of an animal such as a mouse. However, other animals are also envisioned in the present invention for the production of antibodies / antisera. For example, monoclonal and polyclonal antibodies can be produced by rabbits, mice, goats, donkeys, etc. Polynucleotides encoding a accordingly selected polypeptide of HSV-1 or HSV-2 can be subcloned into a suitable vector, where the recombinant polypeptide is expressed in an organism capable of expression, such as bacteria. Thus, the expressed recombinant protein can be injected intraperitoneally into a mouse, and the resulting specific antibody can be obtained, for example, from mouse serum obtained by intracardiac blood aspiration. The present invention also envisions the production of specific antibodies against native and recombinant polypeptides using a DNA vaccine strategy, as illustrated in the appendix examples. DNA vaccine strategies are well known in the art and include liposome-mediated delivery, gene gun or jet injection, and intramuscular or intradermal injection. Therefore, antibodies produced against HSV-1 and HSV-2 polypeptides, proteins, or epitopes can be obtained by directly immunizing animals by directly intramuscular injection of a vector expressing the desired HSV-1 and HSV-2 polypeptide, protein, or epitope, particularly the gB epitope. The amount of specific antibody obtained can be quantified using ELISA, which is also described below herein. Further methods for antibody production are well known in the art. See, for example, Harlow and Lane, "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor, 1988.

[0227] As used herein, the term “specifically bind” refers to a binding reaction that determines the presence of an antibody in the presence of a desired polypeptide or protein or epitope of HSV-1 and HSV-2, particularly the gB epitope, as well as a heterogeneous population of proteins and other biologics.

[0228] Therefore, under specified assay conditions, the specified antibody and the corresponding polypeptides or proteins or epitopes of HSV-1 and HSV-2, particularly the gB epitope, bind to each other and do not bind to other components present in the sample in significant amounts. Specific binding to the target analyte under such conditions may require a binding site selected for specificity to the particular target analyte. Various immunoassay formats may be used to select antibodies that react specifically with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immune to an analyte. For a description of immunoassay formats and conditions that can be used to confirm specific immunoreactivity, see Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press and / or Howard and Bethell (2000) Basic Methods in Antibody Production and Characterization, Crc. Pr. Inc. Typically, specific or selective reactions have a background signal at least twice as loud as noise, and more typically 10 to 100 times louder than the background. Those skilled in the art are in a position to provide and construct specific binding molecules for novel polypeptides. For specific binding assays, these specific binding molecules can be readily used to avoid undesirable cross-reactivity. For example, polyclonal antibodies can be readily purified and selected by known methods (see Shepherd and Dean, previously cited).

[0229] In the context of the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention, the term “anti-HSV antibody or its antigen-binding fragment” means that the antibody molecule or its antigen-binding fragment can specifically recognize, or specifically interact with, and / or bind to, at least two amino acids of the desired polypeptide or protein or epitope of HSV-1 and HSV-2, particularly the gB epitope. The term relates to the specificity of the antibody molecule, i.e., its ability to distinguish specific regions of the desired polypeptide or protein or epitope of HSV-1 and HSV-2, particularly the gB epitope. Thus, specificity can be experimentally confirmed by methods known in the art and by methods disclosed and described herein. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA tests and peptide scans. Such methods also include determining the Kd value, as exemplified in the appended examples. Peptide scans (PEPSpot assays) are routinely used to map linear epitopes in polypeptide antigens. The primary sequence of a polypeptide is synthesized sequentially on activated cellulose using peptides that overlap with each other. The recognition of a particular peptide by an antibody, whose ability to detect or recognize a specific antigen / epitope is to be tested, is scored by a chemiluminescent reaction or by routine color development using similar means known in the art (secondary antibody with horseradish peroxide, 4-chloronaphthol, and hydrogen peroxide). In particular, the reaction can be quantified in the case of a chemiluminescent reaction. If the antibody reacts with a particular set of overlapping peptides, the minimum amino acid sequence required for the reaction can be estimated. The same assay may reveal two separate clusters of reactive peptides. This indicates that discontinuous epitopes, i.e., conformational epitopes, in the antigenic polypeptide have been recognized (Geysen (1986), Mol. Immunol. 23, 709-715).

[0230] In a preferred embodiment, the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention is an mAb 2c antibody (or its antigen-binding fragment). This monoclonal antibody MAb 2c has been described elsewhere and has been shown to neutralize the virus by inhibiting intercellular transmission of the virus, which is an important mechanism by which HSV-1 / 2 evades humoral immune surveillance independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Eis-Hubinger et al., Intervirology 32:351-360 (1991); Eis-Hubinger et al., Journal of General Virology 74:379-385 (1993); WO2011 / 038933 A2; Krawczyk A, et al., Journal of virology (2011);85(4):1793-1803; Krawczyk A, et al., Proc Natl Acad Sci USA (2013);110(17):6760-6765.

[0231] As described above in the context of the present invention and as shown in the appended examples, the epitopes to which the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention binds were further characterized in detail using cryo-electron microscopy (Cryo-EM). As outlined in further detail above and below in this specification, the epitopes are located at amino acids Y301-E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B of HSV-1, and at the corresponding sites Y293-E297, and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B of HSV-2. This epitope characterization is consistent with those previously shown as described in the literature cited above, but the present invention uses this further refined epitope characterization.

[0232] Accordingly, in a preferred embodiment, the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention is an antibody that recognizes the same epitope as the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention as defined above, wherein the epitope is located at amino acids Y301-E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B of HSV-1, and the corresponding sites Y293-E297, and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B of HSV-2, wherein the recognition of the epitope is confirmed by cryo-electron microscopy (Cryo-EM), and preferably, the epitope is the same as that of glycoprotein B (SEQ ID) of HSV-1 strain F. It consists of contact amino acid residues Y301-E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of NO:9), and contact amino acid residues Y293-E297, and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B (SEQ ID NO:40) of HSV-2 G strain.

[0233] In certain embodiments, the epitope can be identified by cryo-electron microscopy (Cryo-EM).

[0234] Accordingly, the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention binds to a specific epitope on gB, which includes the following contact amino acid residues Y301-E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B of HSV-1, and the corresponding sites Y293-E297, and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B of HSV-2. This epitope was confirmed by cryo-electron microscopy (CryoEM) analysis.

[0235] As outlined above in the context of the first aspect of the present invention, whether an antibody binds to the same epitope as a reference antibody can be determined by methods known to those skilled in the art. However, in one embodiment, this is determined as described in the examples added to the end of this specification. In certain embodiments, the epitope is determined by cryo-electron microscopy (Cryo-EM). This applies, with modifications as necessary, to the antibody or antigen-binding fragment of the second component (B) of the second aspect of the present invention, as described above in the context of the first aspect of the present invention.

[0236] Therefore, more specifically, the epitope can be identified as outlined below.

[0237] Cryo-EM grid preparation and data acquisition are performed as follows: HSV-1 gB and HDIT101 Fab are mixed in an optimal ratio. Aliquots of the mixture are adsorbed onto a suitable grid, wiped with filter paper, and vitrified in liquid ethane at -180°C using a plunger operated at 8–12°C and 75–95% humidity. Data of the HSV-1-gB and HDIT101-Fab composite are acquired using a suitable transmission electron microscope. 40 frames of microscope video are recorded in counting mode at appropriate magnification and dose. For data processing and model building, the following is performed: All image processing steps are performed using appropriate software. Dose weighting and motion correction of dose-splitting and gain-corrected video are performed using appropriate software. Contrast transfer function (CTF) parameters are estimated using appropriate software. Electron micrographs showing strong drift, astigmatism greater than 1000 Å, and maximum CTF resolution worse than 8 Å are excluded from further processing. A total of 1 million to 10 million particles are picked. The particle dataset is further refined by three reference-free 2D classifications. A new initial 3D model is created from the 2D particles using an appropriate algorithm. The particle dataset is further refined by three unsupervised 3D classifications. The remaining particles are subjected to Bayesian particle polishing, CTF and aberration refinement, and final high-resolution 3D refinement to obtain the final map. The HSV1 gB X-ray structure (PDB-ID: 2GUM) is manually mutated at positions T313S, Q443L, and V553A and placed in the final map using appropriate software. For HDIT101 Fab, the crystal structure of the humanized recombinant Fab fragment of the mouse antibody (PDB-ID 3AAZ) is mutated based on sequence alignment using appropriate software. The three HDIT101 Fabs are placed in the final map. Appropriate software is used for the initial fitting of HSV-1 gB and three HDIT101 Fabs to the final map. The final protein model is obtained by repeating manual model building, refinement, and model validation several times.

[0238] In a more preferable embodiment, and more specifically, the epitope can be identified as outlined below.

[0239] Cryo-EM grid preparation and data acquisition are performed as follows: HSV-1 gB and HDIT101 Fab are mixed in a ratio of 1 to 3.5. A 4 μl aliquot of the mixture is adsorbed onto a glow-discharged Quantifoil Cu-R1.2 / 1.3-300 mesh Holley carbon coated grid (Quantifoil, Germany), wiped with Whatman 1 filter paper, and vitrified in liquid ethane at -180°C using a Leica EM GP2 plunger (Leica microsystems, Austria) operated at 10°C and 85% humidity. Data on the HSV-1-gB and HDIT101-Fab composite are acquired using a Glacios TEM (ThermoFisher) equipped with a Quantum K3 direct electron detector (Gatan) and operated at 200 kV. 40 frames of microscope video are recorded at a magnification of 45,000 × (pixel size 0.878 Å) and 1.25 e - / Å 2 Recorded in counting mode with dose per frame, approximately 50e per irradiation. - / Å 2The total accumulated dose at the sample level is obtained. For data processing and model building, the following is done: All image processing steps are performed using Relion v4.0 (Kimanius, Dong et al., 2021, Biochem J, Vol. 478 (24)). Dose splitting and gain-corrected video dose weighting and motion correction are performed using the UCSF motioncor2 program to run Relion. Contrast transfer function (CTF) parameters are estimated using ctffind 4.1.14 (Rohou and Grigorieff, 2015, J Struct Biol, Vol. 192 (2)). Electron micrographs showing strong drift, astigmatism greater than 1000 Å, and maximum CTF resolution worse than 8 Å are excluded from further processing. A total of 3 million particles were picked using a Laplacian-of-Gaussian (LoG) filter in Relion 4.0 (Kimanius, Dong et al., 2021, Biochem J, Vol. 478 (24)). The particle dataset was cleaned by three reference-free 2D classifications to obtain 714,565 particles. A novel 3D initial model was created from the 2D particles using Relion's stochastic gradient descent (SGD) algorithm. The particle dataset was further cleaned by three unsupervised 3D classifications. The remaining 233,330 particles were subjected to Bayesian particle polishing, CTF and aberration refinement, and final high-resolution 3D refinement, resulting in a final map with an FSC of 0.143 and a full resolution of 3.27 Å according to the gold standard Fourier shell correlation (FSC). The HSV1 gB X-ray structure (PDB-ID:2GUM) was manually mutated at positions T313S, Q443L, and V553A, and then placed into the final map using coot (Casanal, Lohkamp et al., 2020, Protein Sci, Vol. 29 (4)).In the case of HDIT101 Fab, the crystal structure of the humanized recombinant Fab fragment of the mouse antibody (PDB-ID 3AAZ) is mutated in coot (Casanal, Lohkamp et al., 2020, Protein Sci, Vol. 29 (4)) based on the sequence alignment created by Needle EMBOSS (Rice, Longden et al., 2000, Trends Genet, Vol. 16 (6)). Three HDIT101 Fabs are then placed in the final map using coot (Casanal, Lohkamp et al., 2020, Protein Sci, Vol. 29 (4)). Molrep from CCP-EM software suite v1.6 (Nicholls, Tykac et al., 2018, Acta Crystallogr D Struct Biol, Vol. 74 (Pt 6)) was used for the initial fitting of HSV1 gB and three HDIT101 Fabs to the final map. The final protein model was obtained by repeating several manual model constructions in coot (Casanal, Lohkamp et al., 2020, Protein Sci, Vol. 29 (4)), Refmac-Servalcat refinements, and model validations in CCP-EM software suite v1.6 (Nicholls, Tykac et al., 2018, Acta Crystallogr D Struct Biol, Vol. 74 (Pt 6)). Statistics for data collection, refinement, and validation were performed as summarized in Table 2, and the data processing workflow is shown in Figure 30D.

[0240] In the context of the second aspect of the present invention, the term “combination,” that is, the combination of the two components (A) and (B) described above, refers to the following:

[0241] In a preferred embodiment, it is assumed that they are applied simultaneously. However, the combination also includes the possibility that the two components are applied later. Thus, one of these components may be administered before the other of the combination, simultaneously with the other of the combination, after the other of the combination, or vice versa.

[0242] Accordingly, as used herein in the context of the second aspect of the present invention, “in combination” does not limit the timing between the administration of the two components. Thus, when the two components are not administered simultaneously / concurrently, the administrations may be at intervals of 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours, and may be separated by any appropriate time difference readily verifiable by those skilled in the art and / or described herein. In a preferred embodiment, when the two components are not administered simultaneously / concurrently, the administrations may be at intervals of 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours, and may be separated by any appropriate time difference readily verifiable by those skilled in the art and / or described herein.

[0243] In a preferred embodiment, in the context of a second aspect of the present invention, the present invention relates to a combination of the anti-HSV antibody or its antigen-binding fragment as defined above, wherein the anti-HSV antibody according to (B) above is a humanized antibody or a fully human antibody.

[0244] The term “humanized antibody or fully human antibody” has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the term “humanized antibody or fully human antibody,” the same applies to the second component (B) as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0245] In a preferred embodiment, in the context of a second aspect of the present invention, the present invention relates to a combination of the anti-HSV antibody or its antigen-binding fragment as defined above, wherein the anti-HSV antibody according to (B) above is a full-length antibody.

[0246] The term "full-length antibody" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the term "full-length antibody," the same applies to the second component (B) as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0247] In a preferred embodiment, in the context of a second aspect of the present invention, the present invention relates to a combination of the anti-HSV antibody or its antigen-binding fragment as defined above, wherein the anti-HSV antibody according to (B) above is a human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE antibody.

[0248] The terms “human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, and IgE antibodies” have already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the terms “human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, and IgE antibodies”, the same applies to the second component (B) as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0249] In a preferred embodiment, in the context of a second aspect of the present invention, the present invention relates to a combination of the anti-HSV antibody or its antigen-binding fragment as defined above, wherein the antigen-binding fragment according to (B) is an F(ab)-, Fab'-SH-, Fv-, Fab'-, or F(ab')2- fragment.

[0250] The terms “F(ab)-, Fab'-SH-, Fv-, Fab'-, and F(ab')2-fragments” have already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the terms “F(ab)-, Fab'-SH-, Fv-, Fab'-, and F(ab')2-fragments”, the same applies to the second component (B) as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0251] In a preferred embodiment, in the context of a second aspect of the present invention, the present invention relates to a combination of anti-HSV antibodies or antigen-binding fragments as defined above, wherein an antibody according to (B) at a concentration of up to 20 nM, preferably up to 16 nM, more preferably up to 12 nM, up to 10 nM, up to 8 nM, up to 6 nM, and most preferably up to 4 nM, can neutralize a defined amount of HSV of 100 TCID50.

[0252] The phrase "can neutralize a defined amount of HSV of 100 TCID50" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the phrase "can neutralize a defined amount of HSV of 100 TCID50," the same applies to the second component (B) as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0253] In a preferred embodiment, in the context of a second aspect of the present invention, the present invention relates to a combination of the anti-HSV antibody or its antigen-binding fragment as defined above, wherein the anti-HSV antibody according to (B) above can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission).

[0254] The phrase "can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission)" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the phrase "can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission)," the same applies to the second component (B) as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0255] In a preferred embodiment, in the context of a second aspect of the present invention, the present invention relates to a combination of the anti-HSV antibody or its antigen-binding fragment as defined above, wherein the anti-HSV antibody according to claim (B) exerts its antiviral or neutralizing activity independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), preferably, the antibody can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0256] The phrase "can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the phrase "can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)," the same applies to the second component (B) as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0257] In a preferred embodiment, in the context of a second aspect of the present invention, the present invention relates to a combination of the anti-HSV antibody or its antigen-binding fragment as defined above, wherein the anti-HSV antibody according to (B) above is conjugated to an effector moiety, a therapeutic moiety, or a detectable label.

[0258] The term “conjugated to an effector portion, therapeutic portion, or detectable label” has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the term “conjugated to an effector portion, therapeutic portion, or detectable label,” the same applies to the second component (B) as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0259] In the following, Third phase This will be explained in more detail.

[0260] Therefore, as stated above, in the third aspect, the present invention is (A) The complementarity determination region, including V, which contains SEQ ID NO:1 H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V containing CDR3, SEQ ID NO:4 L V, including CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L The first binding domain, which includes CDR3, (B) The complementarity determination region, including SEQ ID NO:11 H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L The second binding domain, including CDR3, A bispecific antibody or its antigen-binding fragment that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, comprising: The aforementioned bispecific antibody has a maximum output of 5.0 x 10 -4 s -1 Preferably a maximum of 1.0 x 10 -4 s-1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis This relates to a bispecific antibody or its antigen-binding fragment having the same properties.

[0261] The complementarity determination region is V, which includes SEQ ID NO:1. H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V containing CDR3, SEQ ID NO:4 L V, including CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L The sequence of part (A) of the first binding domain of the bispecific antibody, which includes CDR3, corresponds to the CDR sequence of the antibody according to the first aspect of the present invention, and has already been described in detail in the context of the first aspect of the present invention.

[0262] With respect to this definition and preferred embodiments, the same applies, with modifications as necessary, to bispecific antibodies as described above in the context of the first aspect of the present invention as defined above.

[0263] The complementarity determination region is V, which includes SEQ ID NO:11. H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L The sequence of part (B) of the binding domain of a bispecific antibody, including CDR3, corresponds to the CDR sequence of an antibody according to the second aspect of the present invention (part (B) thereof), which has already been described in detail in the context of the second aspect of the present invention (part (B) thereof).

[0264] With respect to this definition and preferred embodiments, the same applies to bispecific antibodies as described above in the context of the second aspect of the present invention as defined above (part (B) thereof).

[0265] Maximum 5.0x10 -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis With regard to the capabilities and preferred embodiments of bispecific antibodies having the same, the same applies to bispecific antibodies as described above in the context of the first aspect ( relating to Part (A)) and the second aspect ( relating to Part (A)) of the present invention, respectively, as defined above, with modifications as necessary.

[0266] Therefore, in a more preferred embodiment, the bispecific antibody has a maximum of 5.0 x 10 -4 s -1 Preferably a maximum of 4.0 x 10 -4 s -1 , more preferably up to 3.0x10 -4 s -1 More preferably, up to 2.0x10 -4 s -1 , up to 1.0x10 -4 s -1 , up to 9.0x10 -5 s -1 , up to 8.0x10 -5 s -1 , up to 7.0x10 -5 s -1 , up to 6.0x10 -5 s -1 , up to 5.0x10 -5 s -1 , up to 4.0x10 -5 s -1 , up to 3.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10-5 s -1 , up to 2.0x10 -5 s -1 , up to 1.5x10 -5 s -1 , up to 1.0x10 -5 s -1 , up to 5.0x10 -6 s -1 , up to 2.0x10 -6 s -1 , up to 1.0x10 -6 s -1 , up to 5.0x10 -7 s -1 , up to 2.0x10 -7 s -1 , up to 1.0x10 -7 s -1 , up to 1.0x10 -8 s -1 , up to 1.0x10 -9 s -1 Low dissociation rate k dis It holds.

[0267] In a more preferred embodiment, the bispecific antibody that binds to the HSV-1 and / or HSV-2 glycoprotein B (gB) has the amino acid sequence SEQ ID NO:27.

[0268] Generally, bispecific antibodies have been well-known in the art for several decades and relate to artificial proteins that can simultaneously bind to two different types of antigens, or to two different epitopes on the same antigen.

[0269] The bispecific antibody molecule according to the present invention is a (monoclonal) bispecific antibody having binding specificity to at least two different sites or epitopes (which may overlap), and may be in any format. A wide variety of recombinant antibody formats, such as bivalent, trivalent, or tetravalent bispecific antibodies, have been developed some time ago. Examples include the fusion of IgG antibody format and single-chain domains (for different formats, see, for example, Coloma, MJ, et al., Nature Biotech 15 (1997), 159-163; WO 2001 / 077342; Morrison, SL, Nature Biotech 25 (2007), 1233-1234; Holliger, P., et. al, Nature Biotech. 23 (2005), 1126-1136; Fischer, N., and Leger, O., Pathobiology 74 (2007), 3-14; Shen, J., et. al., J. Immunol. Methods 318 (2007), 65-74; Wu, C., et al., Nature Biotech. 25 (2007), 1290-1297). The bispecific antibodies or fragments described herein also include the bivalent, trivalent, or tetravalent bispecific antibodies described in WO2009 / 080251;WO2009 / 080252;WO2009 / 080253;WO2009 / 080254;WO2010 / 112193;WO2010 / 115589;WO2010 / 136172;WO2010 / 145792;WO2010 / 145793, and WO2011 / 117330.

[0270] Accordingly, in the context of the third aspect of the present invention, the “antibody” of the present invention has two or more binding domains and is bispecific. That is, even if there are more than two binding domains (i.e., the antibody is trivalent or polyvalent), the antibody may still be bispecific. The bispecific antibodies of the present invention include, for example, polyvalent single-chain antibodies, diabodies and triabodies, as well as antibodies having a constant domain structure of a full-length antibody in which further antigen-binding domains (e.g., single-chain Fv, VH domain and / or VL domain, Fab, or (Fab)2) are linked via one or more peptide linkers. The antibody may be full-length and derived from a single species, may be chimeric, or may be humanized. In the case of an antibody with more than two antigen-binding domains, some of the binding domains may be identical, as long as the protein has binding domains for two different antigens.

[0271] As outlined, in certain embodiments, the bispecific antibody of the present invention comprises two main modules, namely a first binding domain (A) and a second binding domain (B). The arrangement of the first binding domain (A) and the second binding domain (B) within the bispecific antibody is not particularly limited. Thus, the first binding domain (A) and the second binding domain (B) may be located at either end (i.e., at the N-terminus or C-terminus in the case of a bispecific antibody). Therefore, the bispecific antibody may have an (A)-(B) or (B)-(A) arrangement. However, the bispecific antibody preferably has the (B)-(A) arrangement.

[0272] A bispecific antibody according to the present invention may include modules other than the two main modules described above, namely the first binding domain (A) and the second binding domain (B). More precisely, it may be desirable to have (a) one linker portion / multiple linker portions between the individual modules, for example, to facilitate the construction of the construct.

[0273] The contents and length of the linker are not particularly limited. In a preferred embodiment, the linker between the first binding domain (A) and the second binding domain (B) contains one or more amino acids. These further one or more amino acids may include polypeptide chains of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably up to 20 amino acids, or more preferably up to 30 amino acids.

[0274] Furthermore, in addition to the first binding domain (A) and the second binding domain (B), the bispecific antibody preferably includes one or more additional amino acids that may be adjacent to or interspersed with the first binding domain (A) and the second binding domain (B), respectively. Thus, one or more additional amino acids may be added to the N-terminus and / or C-terminus of the first binding domain (A) and the second binding domain (B). The additional amino acids comprise a polypeptide chain of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably up to 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids, or even more preferably up to 130, 150, 200, 300, 400, or 500 amino acids.

[0275] Bispecific antibodies may exist in the form of fusion proteins, i.e., proteins formed by the expression of a hybrid gene created by combining at least two gene sequences. Typically, this is achieved by cloning cDNA in-frame with an existing gene into an expression vector. Thus, the construct may also be a fusion protein, i.e., a chimeric molecule formed by linking two or more polypeptides via a peptide bond between the amino terminus of one module and the carboxyl terminus of another molecule. In this way, the first binding domain (A) and the second binding domain (B) described above are linked together in the form of a fusion protein. Once cloned in-frame, the fusion protein is then recombinantly expressed by the corresponding nucleic acid sequence encoding the fusion protein.

[0276] Various methods for constructing fusion proteins are known, including nucleic acid synthesis, hybridization, and / or amplification to generate synthetic double-stranded nucleic acids encoding the fusion protein of interest. Such double-stranded nucleic acids may be inserted into expression vectors for fusion protein generation using standard molecular biology techniques (see, for example, Sambrook et al., Molecular Cloning, A laboratory manual, 2nd Ed, 1989).

[0277] Alternatively, at least one of the two modules of the bispecific antibody may be covalently bonded by a chemical conjugate, preferably both modules. Thus, the modules of the bispecific antibody may be chemically bonded by covalent bonds.

[0278] The term "covalently chemically bonded" relates to conjugate methods well known to those skilled in the art. Many methods for producing conjugates with proteins or peptides by covalent or noncovalent bonds are known in the art, and any such known method can be utilized. Although not bound by theory, constructs according to the present invention can be prepared using heterobifunctional crosslinkers such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for such conjugates are well known in the art. For example, see Wong, Chemistry of Protein Conjugation and Cross-linking (CRC Press 1991); Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), pp. 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pp. 60-84 (Cambridge University Press 1995). Therefore, given that methods for covalently linking portions of each other, preferably proteins / peptides, are well known to those skilled in the art, the examples shown herein are not limiting.For an overview of methods for binding pigments to proteins (by covalent bonds), see, for example, the review in Brinkley M. Bioconjug Chem. 1992 Jan-Feb; 3(1): 2-13, and Chapter 16 titled "Vinyl Sulfone: A Multi-Purpose Function in Proteomics" in the book Biochemistry, Genetics and Molecular Biology "Integrative Proteomics" edited by Hon-Chiu Eastwood Leung, Subject editors: Tsz-Kwong Man and Ricardo J. Flores, ISBN 978-953-51-0070-6, published February 24, 2012.

[0279] Accordingly, in one embodiment, both modules (i.e., the first binding domain (A) and the second binding domain (B)) may be synthesized individually (chemically or by recombinant technology), optionally purified, and then chemically bonded together by covalent bonds.

[0280] In certain embodiments, the antibody according to the third aspect of the present invention is a multispecific antibody, for example, a bispecific antibody. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites.

[0281] Techniques for producing multispecific antibodies, particularly bispecific antibodies, include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, WO93 / 08829, and Traunecker, A., et al., EMBO J. 10 (1991) 3655-3659), as well as "knob-in-hole" engineering (see, e.g., US 5,731,168). Multispecific antibodies, particularly bispecific antibodies, can also be produced by manipulating the electrostatic steering effect to create antibody Fc-heterodimer molecules (WO2009 / 089004); by crosslinking two or more antibodies or fragments (see, e.g., US4,676,980, and Brennan, M., et al., Science 229 (1985) 81-83); by generating bispecific antibodies using a leucine zipper (see, e.g., Kostelny, SA, et al., J. Immunol. 148 (1992) 1547-1553); or by using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90 (1993)). See 6444-6448; as well as by using single-chain Fv(sFv) dimers (see, for example, Gruber, M., et al., J. Immunol. 152 (1994) 5368-5374); and by preparing triplicate antibodies as described, for example, Tutt, A., et al., J. Immunol. 147 (1991) 60-69.

[0282] Preferred embodiments of bispecific antibodies that bind to glycoprotein B (gB) of HSV-1 and / or HSV-2, according to a third aspect of the present invention, are described in further detail below.

[0283] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention is a bispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, Part (A) covers the following framework areas: SEQ ID NO:7 Position 1~25(V) H FR1), 36~49(V H FR2), 67~98(V H FR3), 112~122(V H FR4), SEQ ID NO: 81~22(V L FR1), 34~48(V L FR2), 56~87(V L FR3), and 97~106(V L Each of the amino acid residues shown in FR4) contains an amino acid sequence having at least 70% sequence identity, Part (B) covers the following framework areas: SEQ ID NO:17 Position 1~30(V) H FR1), 38~51(V H FR2), 68~99(V H FR3), and 112~122(V H FR4), SEQ ID NO: 18, 1-23 (V L FR1), 41~55(V L FR2), 63~94(V L FR3), and 104~114(V L This relates to a bispecific antibody containing an amino acid sequence having at least 70% sequence identity with the amino acid residue shown in FR4).

[0284] With respect to this definition and preferred embodiments, the same applies, with modifications as necessary, to bispecific antibodies as described above in the context of the first aspect ( relating to Part (A)) and the second aspect ( relating to Part (B)) of the present invention as defined above.

[0285] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention is a bispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, Part (A) is V of SEQ ID NO:7 H and V of SEQ ID NO:8 L Includes, Part (B) is V of SEQ ID NO:19 H and V of SEQ ID NO:20 L including, Regarding bispecific antibodies.

[0286] With respect to this definition and preferred embodiments, the same applies, with modifications as necessary, to bispecific antibodies as described above in the context of the first aspect ( relating to Part (A)) and the second aspect ( relating to Part (B)) of the present invention as defined above.

[0287] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention is a bispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, Part (A) recognizes the same epitope as the antibody, The aforementioned epitopes are located at contact amino acid residues D199, A203, K204, Y303, R304, K320, Q321, V322, D323, Y326, R335, and T337 of glycoprotein B in HSV-1 strain F, and at contact amino acid residues D191, A195, K196, Y295, R296, K312, Q313, V314, D315, Y318, R327, and T329 of glycoprotein B in HSV-2 strain G, respectively. Preferably, the epitopes consist of the contact amino acid residues D199, A203, K204, Y303, R304, K320, Q321, V322, D323, Y326, R335, and T337 of glycoprotein B (SEQ ID NO: 9) of HSV-1 strain F, and the contact amino acid residues D191, A195, K196, Y295, R296, K312, Q313, V314, D315, Y318, R327, and T329 of glycoprotein B (SEQ ID NO: 40) of HSV-2 strain G. Part (B) recognizes the same epitope as the antibody, The epitopes are located at amino acids Y301-E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B of HSV-1, and at the corresponding sites Y293-E297, and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B of HSV-2. Preferably, the invention relates to a bispecific antibody in which the epitopes consist of contact amino acid residues Y301-E305 and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B (SEQ ID NO:9) of HSV-1 strain F, and contact amino acid residues Y293-E297 and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B (SEQ ID NO:40) of HSV-2 strain G.

[0288] In certain embodiments, the epitope can be identified by cryo-electron microscopy (Cryo-EM).

[0289] With respect to this definition and preferred embodiments, the same applies, with modifications as necessary, to bispecific antibodies as described above in the context of the first aspect of the present invention ( relating to Part (A)) and the second aspect of the present invention ( relating to Part (B)), respectively, as defined above.

[0290] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention relates to a bispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, wherein the bispecific antibody is a humanized antibody or a fully human antibody.

[0291] The term “humanized antibody or fully human antibody” has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the term “humanized antibody or fully human antibody,” the same applies to bispecific antibodies as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0292] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention relates to a bispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, wherein the bispecific antibody is a full-length antibody.

[0293] The term "full-length antibody" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the term "full-length antibody," the same applies to bispecific antibodies as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0294] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention relates to a bispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, wherein the bispecific antibody is a human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE antibody.

[0295] The terms “human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, and IgE antibodies” have already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the terms “human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, and IgE antibodies”, the same applies, with modifications as necessary, to the bispecific antibodies as described above in the context of the first aspect of the present invention as defined above.

[0296] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention relates to a bispecific antibody that binds to the glycoprotein B(gB) of HSV-1 and / or HSV-2 as defined above, wherein the bispecific antibody is an F(ab)-, Fab'-SH-, Fv-, Fab'-, or F(ab')2- fragment.

[0297] The terms “F(ab)-, Fab'-SH-, Fv-, Fab'-, and F(ab')2- fragments” have already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the terms “F(ab)-, Fab'-SH-, Fv-, Fab'-, and F(ab')2- fragments,” the same applies to bispecific antibodies as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0298] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention relates to a bispecific antibody that binds to glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, wherein the bispecific antibody at a concentration of 20 nM, preferably up to 16 nM, more preferably up to 13 nM, up to 11 nM, up to 9 nM, up to 7 nM, up to 6 nM, up to 5 nM, and most preferably up to 4 nM, can neutralize a defined amount of HSV of 100 TCID50.

[0299] The phrase "capable of neutralizing a defined amount of HSV of 100 TCID50" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the phrase "capable of neutralizing a defined amount of HSV of 100 TCID50," the same applies to bispecific antibodies as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0300] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention relates to a bispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, wherein the bispecific antibody can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission).

[0301] The phrase "can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission)" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the phrase "can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission)," the same applies to bispecific antibodies as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0302] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention relates to a bispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, wherein the bispecific antibody exerts its antiviral or neutralizing activity independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), preferably, the antibody can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0303] The phrase "capable of inhibiting intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the phrase "capable of inhibiting intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)," the same applies, with modifications as necessary, to bispecific antibodies as described above in the context of the first aspect of the present invention as defined above.

[0304] In a preferred embodiment, in the context of a third aspect of the present invention, the present invention relates to a bispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, wherein the bispecific antibody is conjugated to an effector moiety, a therapeutic moiety, or a detectable label.

[0305] The terms “conjugated to an effector portion, therapeutic portion, or detectable label” have already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the terms “conjugated to an effector portion, therapeutic portion, or detectable label,” the same applies, with modifications as necessary, to bispecific antibodies as described above in the context of the first aspect of the present invention as defined above.

[0306] In the following, Phase 4 This will be explained in more detail.

[0307] Accordingly, as stated above, in the fourth aspect, the present invention relates to a trispecific antibody or antigen-binding fragment thereof that binds to glycoprotein B (gB) of HSV-1 and / or HSV-2, comprising the following:

[0308] moreover, Phase 4 So, the present invention is, (A) The complementarity determination region, including V, which contains SEQ ID NO:1H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V containing CDR3, SEQ ID NO:4 L V, including CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L The first binding domain, which includes CDR3, (B) The complementarity determination region, including SEQ ID NO:11 H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L The second binding domain, which includes CDR3, (C) V, which is the complementary determination region, including SEQ ID NO:31 H V containing CDR1, SEQ ID NO:32 H V, containing CDR2, SEQ ID NO:33 H V containing CDR3, SEQ ID NO:34 L V containing CDR1, SEQ ID NO:35 L V including CDR2 and SEQ ID NO:36 L A third binding domain including CDR3 and A triplicate antibody or antigen-binding fragment that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, comprising: The aforementioned triplicate antibody has a maximum output of 5.0 x 10 -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis It has, The present invention relates to a trispecific antibody or its antigen-binding fragment that binds to glycoprotein B (gB) of HSV-1 and / or HSV-2, at concentrations of 10 nM, preferably up to 8 nM, more preferably up to 6 nM, up to 4 nM, up to 2 nM, up to 1 nM, up to 0.9 nM, up to 0.7 nM, and most preferably up to 0.5 nM, which can neutralize a defined amount of HSV of 100 TCID50.

[0309] The complementarity determination region is V, which includes SEQ ID NO:1. H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V containing CDR3, SEQ ID NO:4 L V, including CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L The sequence of part (A) of the first binding domain of a triplicate antibody containing CDR3 corresponds to the CDR sequence of an antibody according to the first aspect of the present invention, which has already been described in detail in the context of the first aspect of the present invention.

[0310] With respect to this definition and preferred embodiments, the same applies, with modifications as necessary, to the triplicate antibodies as described above in the context of the first aspect of the present invention as defined above.

[0311] The complementarity determination region is V, which includes SEQ ID NO:11. H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 LThe sequence of part (B) of the binding domain of a triplicate antibody containing CDR3 corresponds to the CDR sequence of an antibody according to the second aspect of the present invention (part (B) thereof), which has already been described in detail in the context of the second aspect of the present invention (part (B) thereof).

[0312] With respect to this definition and preferred embodiments, the same applies, with modifications as necessary, to the triplicate antibodies as described above in the context of the second aspect of the present invention as defined above (part (B) thereof).

[0313] With regard to the ability of a triplicate antibody to neutralize a defined amount of HSV, namely 100 TCID50, at concentrations of 10 nM, preferably up to 8 nM, more preferably up to 6 nM, up to 4 nM, up to 2 nM, up to 1 nM, up to 0.9 nM, up to 0.7 nM, and most preferably up to 0.5 nM, and with regard to preferred embodiments, the same applies, with modifications as necessary, to the triplicate antibody as described above in the context of the first aspect ( relating to Part (A)) and the second aspect ( relating to Part (A)) of the present invention as defined above.

[0314] Maximum 5.0x10 -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis With regard to the capabilities and preferred embodiments of the triplicate antibody having the same, the same applies to the triplicate antibody as described above in the context of the first aspect (related to Part (A)) and the second aspect (related to Part (A)) of the present invention, respectively, as defined above, with modifications as necessary.

[0315] Therefore, in a more preferred embodiment, the triplicate antibody has a maximum of 5.0 x 10 -4 s -1Preferably a maximum of 4.0 x 10 -4 s -1 , more preferably up to 3.0x10 -4 s -1 More preferably, up to 2.0x10 -4 s -1 , up to 1.0x10 -4 s -1 , up to 9.0x10 -5 s -1 , up to 8.0x10 -5 s -1 , up to 7.0x10 -5 s -1 , up to 6.0x10 -5 s -1 , up to 5.0x10 -5 s -1 , up to 4.0x10 -5 s -1 , up to 3.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 , up to 2.0x10 -5 s -1 , up to 1.5x10 -5 s -1 , up to 1.0x10 -5 s -1 , up to 5.0x10 -6 s -1 , up to 2.0x10 -6 s -1 , up to 1.0x10 -6 s -1 , up to 5.0x10 -7 s -1 , up to 2.0x10 -7 s -1 , up to 1.0x10 -7 s -1 , up to 1.0x10 -8 s -1 , up to 1.0x10 -9 s -1 Low dissociation rate k dis It holds.

[0316] In a more preferred embodiment, the triplicate antibody that binds to glycoprotein B(gB) of HSV-1 and / or HSV-2 has the amino acid sequence SEQ ID NO:30.

[0317] Generally, tripspecific antibodies have been well-known in the art for several decades and relate to artificial proteins that can simultaneously bind to three different types of antigens, or to three different epitopes on the same antigen.

[0318] The triplicate antibody molecule according to the present invention is a (monoclonal) bispecific antibody having binding specificity to at least three different sites or epitopes (which may overlap), and may be in any format. A wide variety of recombinant antibody formats, such as trivalent or tetravalent bispecific antibodies, have been developed some time ago. Examples include the fusion of IgG antibody format and single-chain domains (for different formats, see, for example, Coloma, MJ, et al., Nature Biotech 15 (1997), 159-163; WO 2001 / 077342; Morrison, SL, Nature Biotech 25 (2007), 1233-1234; Holliger, P., et. al, Nature Biotech. 23 (2005), 1126-1136; Fischer, N., and Leger, O., Pathobiology 74 (2007), 3-14; Shen, J., et. al., J. Immunol. Methods 318 (2007), 65-74; Wu, C., et al., Nature Biotech. 25 (2007), 1290-1297). The bispecific antibodies or fragments described herein also include the bivalent, trivalent, or tetravalent bispecific antibodies described in WO2009 / 080251;WO2009 / 080252;WO2009 / 080253;WO2009 / 080254;WO2010 / 112193;WO2010 / 115589;WO2010 / 136172;WO2010 / 145792;WO2010 / 145793 and WO2011 / 117330.

[0319] Accordingly, in the context of the fourth aspect of the present invention, the “antibody” of the present invention has three or more binding domains and is triply specific. That is, the antibody may be triply specific even if it has more than three binding domains. The triply specific antibodies of the present invention include, for example, antibodies having a constant domain structure of a multivalent single-chain antibody, diabody and tribody, and a full-length antibody, to which further antigen-binding domains (e.g., single-chain Fv, VH domain and / or VL domain, Fab, or (Fab)2) are linked via one or more peptide linkers. The antibody may be full-length and derived from a single species, and may be chimeric or humanized. In the case of an antibody having more than two antigen-binding domains, some of the binding domains may be identical, as long as the protein has binding domains for two different antigens.

[0320] In certain embodiments, the antibody according to the fourth aspect of the present invention is a multispecific antibody, for example, a triplicate antibody. A multispecific antibody is a monoclonal antibody having binding specificity to at least three different sites.

[0321] Techniques for producing multispecific antibodies, particularly tripspecific antibodies, include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, WO93 / 08829, and Traunecker, A., et al., EMBO J. 10 (1991) 3655-3659), as well as "knob-in-hole" engineering (see, e.g., US 5,731,168). Multispecific antibodies, particularly bispecific antibodies, can also be produced by manipulating the electrostatic steering effect to create antibody Fc-heterodimer molecules (WO2009 / 089004); by crosslinking two or more antibodies or fragments (see, e.g., US4,676,980, and Brennan, M., et al., Science 229 (1985) 81-83); by generating bispecific antibodies using a leucine zipper (see, e.g., Kostelny, SA, et al., J. Immunol. 148 (1992) 1547-1553); by using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and by using single-chain Fv(sFv) dimers (see, e.g., Gruber, M., et al. See al., J. Immunol. 152 (1994) 5368-5374; and triplicate antibodies can be prepared, for example, as described in Tutt, A., et al., J. Immunol. 147 (1991) 60-69).

[0322] As outlined, in certain embodiments, the triplicate antibody of the present invention comprises three main modules, namely a first binding domain (A), a second binding domain (B), and a third binding domain (C).

[0323] The arrangement of the first binding domain (A), the second binding domain (B), and the third binding domain (C) within a bispecific antibody is not particularly limited. Therefore, the first binding domain (A), the second binding domain (B), and the third binding domain (C) may each be located at either end (i.e., at the N-terminus or C-terminus in the case of a trispecific antibody), and the remaining binding domain may be located between the other two binding domains. Thus, modules (C), (A), and (B) may each be located between other modules (B) and (A), (B) and (C), (C) and (B), and (C) and (A), respectively. Therefore, triplicate antibodies may have the configurations (A)-(B)-(C), (A)-(C)-(B), (B)-(A)-(C), (B)-(C)-(A), (C)-(A)-(B), or (C)-(B)-(A). However, bispecific antibodies preferably have the configuration (B)-(A)-(C).

[0324] A triplicate antibody according to the present invention may include, for example, (a) one linker portion / multiple linker portions between two or three of the individual modules, which may facilitate the construction of the construct.

[0325] The contents and length of the linker are not particularly limited. In a preferred embodiment, the linker between the first binding domain (A) and / or the second binding domain (B) and / or the third binding domain (C) contains one or more amino acids. These further one or more amino acids may include polypeptide chains of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably up to 20 amino acids, or more preferably up to 30 amino acids.

[0326] Furthermore, in addition to the first binding domain (A), the second binding domain (B), and the third binding domain (C), the triplicate antibody preferably includes one or more additional amino acids that may be adjacent to or interspersed with each of the first binding domain (A), the second binding domain (B), and the third binding domain (C). Thus, one or more additional amino acids may be added to the N-terminus and / or C-terminus of the first binding domain (A) and / or the second binding domain (B) and / or the third binding domain (C). The additional amino acids comprise a polypeptide chain of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, preferably up to 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids, or even more preferably up to 130, 150, 200, 300, 400, or 500 amino acids.

[0327] Triple-specific antibodies may exist in the form of fusion proteins, i.e., proteins formed by the expression of a hybrid gene created by combining at least three gene sequences. Typically, this is achieved by cloning cDNA in-frame with an existing gene into an expression vector. Thus, the construct may also be a fusion protein, i.e., a chimeric molecule formed by linking two or more polypeptides via peptide bonds between the amino terminus of one module and the carboxyl terminus of another molecule. In this way, the first binding domain (A), the second binding domain (B), and the third binding domain (C) described above are linked together in the form of a fusion protein. Once cloned in-frame, the fusion protein is then recombinantly expressed by the corresponding nucleic acid sequence encoding the fusion protein.

[0328] Various methods for constructing fusion proteins are known, including nucleic acid synthesis, hybridization, and / or amplification to generate synthetic double-stranded nucleic acids encoding the fusion protein of interest. Such double-stranded nucleic acids may be inserted into expression vectors for fusion protein generation using standard molecular biology techniques (see, for example, Sambrook et al., Molecular Cloning, A laboratory manual, 2nd Ed, 1989).

[0329] Alternatively, at least one, preferably two, or more preferably three of the three modules of the triplicate antibody may also be covalently bonded by a chemical conjugate. Thus, the modules of the triplicate antibody may be chemically bonded by covalent bonds.

[0330] The term "covalently chemically bonded" has already been stated in the context of bispecific antibodies in the third aspect of the present invention. The same applies to triplicate antibodies in the fourth aspect of the present invention, with modifications as necessary.

[0331] In one embodiment, all three modules (i.e., the first binding domain (A), the second binding domain (B), and the third binding domain (C)) may be synthesized individually (chemically or by recombinant technology), optionally purified, and then chemically bonded together by covalent bonds.

[0332] Therefore, the triplicate antibody according to the present invention may be a triplicate antibody in which the first binding domain (A), the second binding domain (B), and the third binding domain (C) are chemically bonded by covalent bonds. Alternatively, modules (A) and (B) may be a fusion protein in which module (C) is chemically bonded by covalent bonds to the fusion protein containing modules (A) and (B). In another option, modules (A) and (C) may be a fusion protein in which module (B) is chemically bonded by covalent bonds to the fusion protein containing modules (A) and (C). In yet another option, modules (B) and (C) may be a fusion protein in which module (A) is chemically bonded by covalent bonds to the fusion protein containing modules (B) and (C).

[0333] While the two modules may take the form of a fusion protein, it is also conceivable that a third module could be chemically bonded via covalent bonds.

[0334] Preferred embodiments of a triplicate antibody that binds to glycoprotein B (gB) of HSV-1 and / or HSV-2 according to a fourth aspect of the present invention are described in further detail below.

[0335] In a preferred embodiment, in the context of the fourth aspect of the present invention, the present invention is a trispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, Part (A) covers the following framework areas: SEQ ID NO:7 Position 1~25(V) H FR1), 36~49(V H FR2), 67~98(V H FR3), 112~122(V H FR4), SEQ ID NO: 81~22(V L FR1), 34~48(V L FR2), 56~87(V L FR3), and 97~106(V LEach of the amino acid residues shown in FR4) contains an amino acid sequence having at least 70% sequence identity, Part (B) covers the following framework areas: SEQ ID NO:17 Position 1~30(V) H FR1), 38~51(V H FR2), 68~99(V H FR3), and 112~122(V H FR4), SEQ ID NO: 18, 1-23 (V L FR1), 41~55(V L FR2), 63~94(V L FR3), and 104~114(V L It includes an amino acid sequence having at least 70% sequence identity with the amino acid residue shown in FR4), Part (C) covers the following framework areas: SEQ ID NO:37 Position 1~25(V) H FR1), 36~49(V H FR2), 67~98(V H FR3), and 112~122(V H FR4), SEQ ID NO: 38, 1-23 (V L FR1), 35~49(V L FR2), 57~88(V L FR3), and 98~107(V L This relates to a triplicate antibody containing an amino acid sequence having at least 70% sequence identity with the amino acid residue shown in FR4).

[0336] With respect to this definition and preferred embodiments, the same applies, with modifications as necessary, to the triplicate antibodies described above in the context of the first aspect of the present invention ( relating to Part (A)) and the second aspect of the present invention ( relating to Part (B)), respectively, as defined above.

[0337] In a preferred embodiment, in the context of the fourth aspect of the present invention, the present invention is a trispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, Part (A) is V of SEQ ID NO:7 H and V of SEQ ID NO:8 L Includes, Part (B) is V of SEQ ID NO:19 H and V of SEQ ID NO:20 L Includes, Part (C) is V of SEQ ID NO:37 H and V of SEQ ID NO:38 L This concerns triplicate antibodies, including those mentioned above.

[0338] With respect to this definition and preferred embodiments, the same applies, with modifications as necessary, to the triplicate antibodies described above in the context of the first aspect of the present invention ( relating to Part (A)) and the second aspect of the present invention ( relating to Part (B)), respectively, as defined above.

[0339] In a preferred embodiment, in the context of a fourth aspect of the present invention, the present invention relates to a tripspecific antibody that is a humanized antibody or a fully human antibody and binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above.

[0340] The term “humanized antibody or fully human antibody” has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the term “humanized antibody or fully human antibody,” the same applies to the trispecific antibody as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0341] In a preferred embodiment, in the context of a fourth aspect of the present invention, the present invention relates to a full-length antibody, a trispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above.

[0342] The term "full-length antibody" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the term "full-length antibody," the same applies to the triplicate antibody as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0343] In a preferred embodiment, in the context of a fourth aspect of the present invention, the present invention relates to a trispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, which is a human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE antibody.

[0344] The terms “human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, and IgE antibodies” have already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the terms “human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, and IgE antibodies”, the same applies, with modifications as necessary, to the trispecific antibodies as described above in the context of the first aspect of the present invention as defined above.

[0345] In a preferred embodiment, in the context of a fourth aspect of the present invention, the present invention relates to a tripspecific antibody that binds to the glycoprotein B(gB) of HSV-1 and / or HSV-2 as defined above, which is a F(ab)-, Fab'-SH-, Fv-, Fab'-, or F(ab')2- fragment.

[0346] The terms “F(ab)-, Fab'-SH-, Fv-, Fab'-, and F(ab')2- fragments” have already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the terms “F(ab)-, Fab'-SH-, Fv-, Fab'-, and F(ab')2- fragments,” the same applies to the tripspecific antibodies as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0347] In a preferred embodiment, in the context of a fourth aspect of the present invention, the present invention relates to a trispecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, wherein the trispecific antibody at a concentration of 10 nM, preferably up to 8 nM, more preferably up to 6 nM, up to 4 nM, up to 2 nM, up to 1 nM, up to 0.9 nM, up to 0.7 nM, and most preferably up to 0.5 nM can neutralize a defined amount of HSV of 100 TCID50.

[0348] The phrase "capable of neutralizing a defined amount of HSV of 100 TCID50" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the phrase "capable of neutralizing a defined amount of HSV of 100 TCID50," the same applies to the triplicate antibody as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0349] In a preferred embodiment, in the context of a fourth aspect of the present invention, the present invention relates to a tripspecific antibody that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, which can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission).

[0350] The phrase "can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission)" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the phrase "can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission)," the same applies to the trispecific antibody as described above in the context of the first aspect of the present invention as defined above, with modifications as necessary.

[0351] In a preferred embodiment, in the context of a fourth aspect of the present invention, the present invention relates to a tripspecific antibody that binds to glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, wherein the tripspecific antibody exerts its antiviral or neutralizing activity independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), preferably, the antibody can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0352] The phrase "capable of inhibiting intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)" has already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the phrase "capable of inhibiting intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)," the same applies, with modifications as necessary, to the trispecific antibodies described above in the context of the first aspect of the present invention as defined above.

[0353] In a preferred embodiment, in the context of a fourth aspect of the present invention, the present invention relates to a tripspecific antibody conjugated to the glycoprotein B (gB) of HSV-1 and / or HSV-2 as defined above, which is conjugated to an effector portion, a therapeutic portion, or a detectable label.

[0354] The terms “conjugated to an effector portion, therapeutic portion, or detectable label” have already been defined above in the context of the first aspect of the present invention. With respect to this definition, and preferred embodiments of the terms “conjugated to an effector portion, therapeutic portion, or detectable label,” the same applies, with modifications as necessary, to the tripspecific antibodies as described above in the context of the first aspect of the present invention as defined above.

[0355] Anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above are particularly useful in medical settings.

[0356] Accordingly, in a preferred embodiment, the present invention relates to a pharmaceutical composition comprising an effective amount of an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a triplicate antibody according to the fourth aspect of the present invention as defined above, and at least one pharmaceutically acceptable excipient.

[0357] Accordingly, in a preferred embodiment, the present invention relates to an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above, for use as a drug.

[0358] The terms “treatment” and / or “prevention” are used herein to mean, in general, obtaining a desired pharmacological and / or physiological effect. Therefore, the treatments of the present invention may relate to the treatment of a particular (acute) state of a disease, but may also relate to prophylactic treatment in that they completely or partially prevent the disease or its symptoms. Preferably, the term “treatment” should be understood to be therapeutic in that it partially or completely cures the disease and / or the side effects and / or symptoms resulting from it. In this regard, “acute” means that the subject exhibits symptoms of the disease. In other words, the subject to be treated actually needs treatment, and in the context of the present invention, the term “acute treatment” relates to measures taken to actually treat the disease after the onset or sudden onset of the disease. Treatment may also be a preventive treatment, i.e., measures taken for disease prevention, for example, to prevent infection and / or the onset of the disease.

[0359] The pharmaceutical composition or drug of the present invention may be administered via a wide class of dosage forms known to those skilled in the art. These dosage forms may include, but are not limited to, tablets, needle injections, inhalers, creams, foams, gels, lotions, and ointments, and may be systemic, topical, or oral, or via aerosols.

[0360] Preferably, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above are administered intravenously, locally, intradermally, subcutaneously, intra-cutanously, intramuscularly, and / or subarachnoidally. These routes of administration, i.e., intravenously, locally, intradermally, subcutaneously, intradermally, intramuscularly, and / or subarachnoidally, are known to those skilled in the art.

[0361] Excipients or carriers are inert substances formulated with the active ingredient, i.e., the antibody of the present invention, for the purpose of increasing the volume of a formulation containing a potent active ingredient. Excipients are often also called “volume extenders,” “fillers,” or “diluents.” Volume extension allows for the easy and accurate preparation of the active ingredient when producing the dosage form. They can also serve various therapeutic enhancement purposes, such as enhancing drug absorption or solubility, or other pharmacokinetic considerations. Excipients may also be useful in the manufacturing process by promoting powder flowability or non-stick properties, in addition to aiding in vitro stability, such as preventing denaturation over the expected shelf life, to assist in the handling of the active ingredient in question. The selection of an appropriate excipient also depends on the route of administration and dosage form, as well as the active ingredient and other factors.

[0362] Accordingly, as described above, a pharmaceutical composition or drug comprising an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above, may be in solid, liquid, or gaseous form, and in particular may be in powder, tablet, solution, or aerosol form. The pharmaceutical composition preferably optionally contains a pharmaceutically acceptable carrier and / or diluent.

[0363] These pharmaceutical compositions can be administered to a subject in an appropriate dose. The administration of an appropriate composition may be influenced by various methods, such as intravenous, intraperitoneal, subcutaneous, intramuscular, local, intradermal, intranasal, or intrabronchial administration. Such administration is particularly preferred by injection and / or delivery, for example, to a site in the pulmonary artery or by direct injection and / or delivery to the lung. The compositions of the present invention may also be administered directly to a target site, for example, by biorhythmic delivery to an external target site or internally, such as the lung. The administration plan is determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any patient depends on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, duration and route of administration, overall health, and other drugs being administered concurrently. The proteinaceous pharmaceutically active substance may be present in amounts of 1 ng to 10 mg / kg body weight per dose. However, doses lower or higher than this exemplary range are assumed, particularly considering the factors mentioned above. If the regimen involves continuous infusion, this should also be in the range of 1 μg to 10 mg per kilogram of body weight per minute.

[0364] Examples of suitable pharmaceutical carriers, excipients, and / or diluents are well known in the art and include phosphate-buffered saline solutions, water, emulsions, e.g., oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject in an appropriate dose, i.e., an "effective dose" that can be readily determined by those skilled in the art by methods known in the art. The administration plan is determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any patient depends on many factors, including the size of the patient or subject, body surface area, age, the specific compound to be administered, sex, duration and route of administration, overall health, and other drugs being administered concurrently.

[0365] Accordingly, preferably, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a triplicate antibody (one or more) according to the fourth aspect of the present invention as defined above are each included in an effective amount. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is administered. Accordingly, the content of the antibody of the present invention in the pharmaceutical composition is not limited as long as it is useful for the treatment as described above, but preferably contains 0.0000001 to 10% by weight of the total composition. Furthermore, the antibody (one or more) described herein is preferably used in a carrier. Generally, an appropriate amount of pharmaceutically acceptable salt is used in a carrier to make the composition isotonic. Examples of carriers include, but are not limited to, saline solution, Ringer's solution, and dextrose solution.Preferably, buffers, e.g., citric acid, phosphoric acid, and other organic acids; salt-forming counterions, e.g., sodium and potassium; low molecular weight (>10 amino acid residues) polypeptides; proteins, e.g., serum albumin or gelatin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates including glucose, mannose, or dextrin; monosaccharides; disaccharides; other sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; chelating agents, e.g., EDTA; nonionic surfactants, e.g., Twee Acceptable excipients, carriers, or stabilizers, including n, Pluronics, or polyethylene glycol; antioxidants including methionine, ascorbic acid, and tocopherol; and / or preservatives, e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkylparabens, e.g., methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol), are nontoxic at the dosages and concentrations used. Suitable carriers and their formulations are described in further detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co.

[0366] Regular evaluations allow for monitoring of treatment progress.

[0367] The pharmaceutical composition / drug of the present invention may be a sterile aqueous or non-aqueous solution, suspension, and emulsion, as well as a cream and suppository. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, e.g., olive oil, and organic esters, e.g., ethyl oleate. Aqueous carriers include water, alcohol solutions / aqueous solutions, emulsions, or suspensions, including saline solution and buffered media. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. Furthermore, the pharmaceutical composition of the present invention may contain additional agents depending on the intended use of the pharmaceutical composition. These agents may be, for example, polyoxyethylene sorbitan monolaurate, propylene glycol, EDTA, citrate, sucrose, and other agents well known to those skilled in the art and suitable for the intended use of the pharmaceutical composition.

[0368] According to the present invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient.

[0369] As outlined above, a second aspect of the present invention relates to combinations of the two types of antibodies and / or antibody-binding fragments defined above.

[0370] Therefore, the following applies in particular in the context of the second aspect of the present invention.

[0371] In the context of the second aspect of the present invention, the term “combination” means a combination of the following two components (as well as preferred embodiments described herein in the context of the first and second aspects of the present invention, respectively): (A) In the context of the first aspect of the present invention, an anti-HSV antibody or its antigen-binding fragment as defined herein, i.e., an anti-HSV antibody or its antigen-binding fragment that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, The complementarity determination region is V, which includes SEQ ID NO:1. HV, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V containing CDR3, SEQ ID NO:4 L V, including CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L CDR3 is an anti-HSV antibody or its antigen-binding fragment as defined herein in the context of the first aspect of the present invention, comprising the antibody, (B) In the context of the second aspect of the present invention, an anti-HSV antibody or its antigen-binding fragment as defined herein, i.e., an anti-HSV antibody or its antigen-binding fragment that binds to the glycoprotein B(gB) of HSV-1 and / or HSV-2, The complementarity determination region is V, which includes SEQ ID NO:11. H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L The antibody contains CDR3, In the context of the first aspect of the present invention, the antibody or its antigen-binding fragment has a dissociation constant Kd of up to 40 nM, preferably up to 30 nM, more preferably up to 20 nM, even more preferably up to 15 nM, up to 13 nM, and up to 10 nM, as defined herein. It refers to.

[0372] In a preferred embodiment, it is assumed that they are applied simultaneously. However, the combination also includes the possibility that the two components are applied later. Thus, one of these components may be administered before the other of the combination, simultaneously with the other of the combination, after the other of the combination, or vice versa.

[0373] Therefore, as used herein, “in combination” does not limit the timing between the administration of the two components. Thus, when the two components are not administered simultaneously / concurrently, the administrations may be spaced at intervals of 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours, and may be spaced at any appropriate time difference readily verifiable by those skilled in the art and / or described herein. In a preferred embodiment, when the two components are not administered simultaneously / concurrently, the administrations may be spaced at intervals of 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours, and may be spaced at any appropriate time difference readily verifiable by those skilled in the art and / or described herein.

[0374] In a more preferred embodiment, the present invention relates to herpes simplex labialis, herpes simplex vulva, chronic or disseminated cutaneous herpes simplex infection, herpes xiphoid, herpetic eczema, herpetic keratoconjunctivitis, neonatal herpes (Herpes neonatorum), Alzheimer's disease (AD), HSV pneumonia, Bell's palsy, herpetic esophagitis, herpes viral encephalitis and herpetic meningitis, herpetic folliculitis, herpetic paronychia, herpetic gingivostomatitis, the presence of recurrent or recurrent oral herpes, the presence of recurrent or recurrent genital herpes, and herpetic eczema. The present invention relates to an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, for use in methods for preventive or therapeutic treatment of disorders or diseases selected from the group consisting of herpeticatum, neonatal herpes, immunodeficiency, immunocompromised patients, resistance to viral suppressants, encephalitis, meningitis, meningoencephalitis, ocular infections, and / or generalized HSV infection, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above.

[0375] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above are each for use in preventive or therapeutic treatment of an HSV-related disease, the disease being caused by HSV-1 or HSV-2, and more preferably, the HSV-related disease being selected from the group consisting of herpes simplex labialis, herpes simplex vulva, chronic or disseminated cutaneous herpes simplex infection, herpes xiphoid, and herpetic eczema.

[0376] HSV infection can cause several different diseases. Common infections of the skin or mucous membranes can affect the face and mouth (orofacial herpes), genitals (genital herpes), or hands (herpetic whiting). More severe disabilities can occur when the virus infects and damages the eyes (herpetic keratitis), or when it invades the central nervous system and damages the brain (herpetic encephalitis). Patients with immature or suppressed immune systems, such as newborns, transplant recipients, or AIDS patients, are more susceptible to severe complications from HSV infection. HSV-related diseases also include impairments related to cognitive impairment in Alzheimer's disease, such as xiphoid herpes, Moraley's meningitis, possibly Bell's palsy, bipolar disorder (also known as manic depressive disorder or bipolar affective disorder), and Alzheimer's disease. Recent scientific publications have demonstrated that herpes simplex virus type 1 DNA is prominently localized within β-amyloid plaques in relation to Alzheimer's disease. This suggests that the virus may be a cause of these plaques. Furthermore, serological analysis has correlated HSV seropositivity with a high risk of dementia or Alzheimer's disease.

[0377] Finally, if the emergence of resistant strains to common chemotherapeutic viral suppressants is observed, for example, during prolonged prophylactic and therapeutic treatments in immunocompromised patients, the use of anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and trispecific antibodies according to the fourth aspect of the present invention as defined above, respectively, is useful. Accordingly, in a preferred embodiment, HSV-related diseases are characterized by one or more of the following: the presence of recurrent or recurrent oral herpes, the presence of recurrent or recurrent genital herpes, herpetic eczema, neonatal herpes, immunodeficiency (immunocompromised patients), immunosuppression, encephalitis, meningitis, meningoencephalitis, ocular infections, generalized HSV infection, and / or resistance to viral suppressants.

[0378] In a more preferred embodiment, the present invention relates to an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above for use, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above for use, a bispecific antibody according to the third aspect of the present invention as defined above for use, and a triplicate antibody according to the fourth aspect of the present invention as defined above for use, wherein the antibody is administered intravenously, locally, intradermally, subcutaneously, intradermally, intramuscularly, and / or subarachnoidally.

[0379] The present invention also relates to a method for treating or preventing a disorder or disease as defined herein in a subject, wherein each of the following is administered to the subject, preferably in a therapeutically effective amount as defined above: an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above; a combination of the anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above; a bispecific antibody according to the third aspect of the present invention as defined above; and a trispecific antibody according to the fourth aspect of the present invention as defined above.

[0380] With regard to preferred embodiments of the treatment method, the same applies, with modifications as necessary, in the context of antibodies or pharmaceutical compositions for use as defined above, as described above.

[0381] In the present invention, the subjects are, in a preferred embodiment, mammals such as dogs, cats, pigs, cattle, sheep, horses, rodents such as rats, mice, and guinea pigs, or primates such as gorillas, chimpanzees, and humans. In the most preferred embodiment, the subjects are humans.

[0382] In a preferred embodiment, in the medical setting described above, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a triplicate antibody according to the fourth aspect of the present invention as defined above may each be administered in combination with a viral suppressant.

[0383] Preferably, such combination therapies exhibit a synergistic effect on the treatment according to the present invention.

[0384] The term “combination” as used herein is described above. With respect to preferred embodiments of such combination therapies, the same applies, with modifications as necessary, in the context of pharmaceutical compositions for use as defined above.

[0385] Viral inhibitors are well known to those skilled in the art and are also commonly called antiviral drugs, a class of drugs used to treat viral infections. Specific antiviral agents are used against specific viruses. Unlike most antibiotics, antiviral drugs do not destroy the target pathogen; instead, they inhibit its development and / or infection and / or replication.

[0386] With respect to HSV infection, those skilled in the art are in a position to select a suitable viral inhibitor suitable for inhibiting the development of the virus as defined above in accordance with the present invention. For example, the viral inhibitor may be selected from the group consisting of nucleoside analogs, pyrophosphate analogs, nucleotide analogs, amantadine derivatives, and helicase-primase inhibitors. Accordingly, the present invention relates to an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above, each administered in combination with a viral inhibitor selected from the group consisting of nucleoside analogs, pyrophosphate analogs, nucleotide analogs, and helicase-primase inhibitors.

[0387] Nucleoside analogs are well known in the art and relate to molecules that act like nucleosides in DNA synthesis. Nucleoside analogs include a variety of antiviral products used to inhibit viral replication in infected cells. When phosphorylated, nucleoside analogs act as antimetabolites by being similar enough to nucleotides to be incorporated into growing DNA strands, but they also act as chain terminaters, stopping viral DNA polymerase. Nucleosides, nucleotides, and pyrophosphate analogs are generally known to inhibit viral nucleic acid synthesis and block viral replication. Nucleosides and nucleotide analogs are antimetabolites. Pyrophosphate analogs (e.g., foscarnet) structurally mimic anionic pyrophosphate and exert antiviral activity by selectively inhibiting pyrophosphate binding sites on virus-specific DNA polymerase at concentrations that do not affect cellular DNA polymerase. Nucleosides and pyrophosphate analogs do not require initial activation (phosphorylation) by thymidine kinase or other kinases before being taken up by cells. Helicase-primase inhibitors are non-nucleoside inhibitors that target viral helicase-primase.

[0388] Preferably, commonly known and approved antiviral agents may be used, as summarized below. As nucleoside analogs, compounds selected from the group consisting of acyclovir, penciclovir, valacyclovir, and famaciclovir may be exemplified and used in the above combination therapy. Foscarnet may be used as a pyrophosphate analog. Sidofovir may be used as a nucleotide analog. Pritelivir is exemplified as a helicase-primase inhibitor. Tromantandine may be used as an amantadine derivative.

[0389] Acyclovir, also known as acycloguanosine (ACV) or 2-amino-9-(2-hydroxyethoxymethyl)-3H-purine-6-one, is a guanosine analog antiviral drug marketed under trade names such as ACERPES®, Acic®, Aciclobeta®, AcicloCT®, Aciclostad®, Aciclovir, Acic®, Ophtal®, Acivir®, AciVision, Acyclovir®, Aviral®, Cyclovir, Helvevir®, Herpex, Supraviran®, Virucalm®, Virupos®, Virzin, Zoliparin®, Zovir, and Zovirax®.

[0390] Penciclovir (2-amino-9-[4-hydroxy-3-(hydroxymethyl)butyl]-6,9-dihydro-3H-purine-6-one) is a guanine analog antiviral drug marketed under trade names such as Denavir and Fenistil.

[0391] Famciclovir (2-[(acetyloxy)methyl]-4-(2-amino-9H-purine-9-yl)butyl acetate) is a prodrug of penciclovir with improved oral bioavailability.

[0392] Foscarnet is a conjugate base of compounds having the formula H02CP03H2 and is sold under the trade names Foscavir® and Triapten®.

[0393] Valacyclovir, also known as (S)-2-[(2-amino-6-oxo-6,9-dihydro-3H-purine-9-yl)methoxy]ethyl-2-amino-3-methylbutanoate, is a prodrug of the guanosine analog antiviral drug ACV, which is marketed under the name Valtrex®.

[0394] Cidovovir (CDV), also known as (S)-1-[3-hydroxy-2-(phosphonylmethoxypropyl)]cytosine, is a nucleotide analog antiviral drug marketed under the name Visitde®.

[0395] Pritelevir, also known as AIC-316 or BAY 57-1293, is a thiazolylamide helicase-primase inhibitor currently in Phase II clinical trials for the treatment of genital HSV-2 infections.

[0396] The topical treatment drug tromantadine (Viru-Merz Serol Gel) is explicitly used for the topical treatment of HSV skin infections. Tromantadine is an amantadine derivative. Griffin's U.S. Patent No. 4,351,847 discloses that amantadine derivatives are effective against herpes simplex virus.

[0397] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a triplicate antibody according to the fourth aspect of the present invention as defined above are each administered locally.

[0398] Accordingly, in a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above are each for use in preventive or therapeutic treatment of an HSV-related disease, the disease being caused by HSV-1 or HSV-2, and more preferably, the HSV-related disease being selected from the group consisting of herpes simplex labialis, herpes vulva, chronic or disseminated cutaneous herpes simplex infection, herpes xiphoid, and herpetic eczema, and one type of antibody / multiple types of antibodies being administered topically.

[0399] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a triplicate antibody according to the fourth aspect of the present invention as defined above are for use in the treatment of acute infections of mucosal or epidermal tissue in a subject caused by HSV-1 or HSV-2, selected from the group consisting of herpes simplex labialis, herpes simplex vulva, chronic or disseminated cutaneous herpes simplex infection, herpes xiphoid, and herpetic eczema, and the antibody is administered topically.

[0400] As described in WO2015 / 197763(A1), it was surprisingly demonstrated that local administration of humanized anti-HSV antibodies to acute lip tissue infections during HSV infection inhibited local transmission of herpes infection via intercellular transmission, thereby preventing the development of lesions, and the infection rapidly cleared within 24 hours.

[0401] In particular, infections of mucosal or epidermal tissues are relevant not only in the context of more specific aspects of local administration, but also include infections selected from the group consisting of herpes simplex labialis, herpes vulvar herpes, chronic or disseminated cutaneous herpes simplex infection, herpes xiphoid, and herpetic eczema, which are well known to those skilled in the art and have been described in detail. Herpes simplex labialis (also called herpes labialis, herpes simplex labialis, recurrent herpes labialis, or herpes labialis) is a type of herpes simplex that occurs on the lips, namely an infection caused by the herpes simplex virus (HSV). An acute outbreak typically causes small blisters or sores on or around the mouth, commonly known as herpes labialis or febrile herpes. The sores typically heal within 2-3 weeks, but the herpes virus remains dormant in the facial nerve after the oral-facial infection and (in symptomatic individuals) periodically reactivates to create sores in the same part of the mouth or face where the initial infection occurred. Oral herpes has a wide range of incidence, from rare episodes to more than 12 relapses per year. People with this condition typically experience 1 to 3 attacks per year. The frequency and severity of attacks generally decrease over time.

[0402] Genital herpes simplex virus (HSV) is a genital infection caused by the herpes simplex virus. A 1998 study showed that, by case count, genital herpes simplex virus is the most common sexually transmitted infection. Most individuals carrying herpes are unaware they are infected, and many suffer little to no acute outbreaks with blisters similar to oral herpes. There is no cure for herpes, but symptoms gradually become milder over time, and the frequency of acute outbreaks gradually decreases. HSV is classified into two distinct categories: HSV-1 and HSV-2. Genital herpes was traditionally caused primarily by HSV-2, but genital HSV-1 infections are increasing and now account for up to 80% of infections. In symptomatic cases, the typical symptoms of primary HSV-1 or HSV-2 genital infection are clusters of genital ulcers consisting of inflammatory papules and vesicles on the outer surface of the genitals, similar to oral herpes. These usually first appear 4 to 7 days after genital exposure to HSV. Genital HSV-1 infections recur at about one-sixth the rate of genital HSV-2.

[0403] Chronic or disseminated cutaneous herpes simplex infections, not limited to the lips or genitals, are known. Immunocompromised patients are most susceptible to this disease, such as those with hypogammaglobulinema or cutaneous T-cell lymphoma. Chronic cutaneous herpes simplex is a characteristic clinical manifestation of herpes simplex virus (HSV) in immunocompromised hosts. The infection can be defined as a chronic, active, destructive cutaneous lesion that can potentially progress to a disseminated (systemic) form. While most HSV infections present as episodes that show healing within one or two weeks, the lesions of chronic cutaneous herpes simplex have a slowly progressive course that can last for several months. Chronic cutaneous herpes simplex is common in immunocompromised patients and is characterized by atypical, chronic, and persistent lesions that complicate and delay diagnosis. This can lead to death due to associated complications. When evaluating long-term chronic ulcers, it is crucial to consider the possibility of chronic herpes simplex virus infection, especially in children.

[0404] Herpes xinode is a herpes skin infection that commonly occurs in adolescents among wrestlers, but is also common in other contact sports. Herpes xinode usually occurs on the head, but most commonly on the jaw, neck, chest, face, stomach, and legs.

[0405] Herpetic eczema, also known as Kaposi's varicelliform eruption, is a widespread vesicular rash caused by a viral infection, usually herpes simplex virus (HSV), and typically arises from a pre-existing skin condition, usually atopic dermatitis (AD). Children with AD are at high risk of developing herpetic eczema, and HSV type 1 (HSV-1) is the most common pathogen in herpetic eczema. If left untreated, herpetic eczema can become severe and progress to disseminated infection and even death.

[0406] Accordingly, in a preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above are each applied topically to infected mucosal or epidermal tissue of skin areas of the lips, genitals, nose, ears, eyes, fingers, toes, and / or the entire body, preferably of skin areas of the head, jaw, neck, chest, face, stomach, and / or legs.

[0407] In a more preferred embodiment, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a triplicate antibody according to the fourth aspect of the present invention as defined above are each locally applied to the portion surrounding the infected mucosal or epidermal tissue.

[0408] This is particularly relevant in the context of more specific embodiments of local administration, and preferably, the treatment also relates to the treatment of acute infections.

[0409] The term "treatment" is used herein to generally mean obtaining a desired pharmacological and / or physiological effect. However, in the context of topical administration, the treatment is preferably related to the treatment of an acute infection, and therefore excludes cases where the effect is preventive in that it completely or partially prevents the disease or its symptoms. More precisely, in this context, the term "treatment" should be understood to be therapeutic in that it partially or completely cures the disease of acute HSV infection as defined above, and / or the side effects and / or symptoms resulting from this disease. Thus, the treatment of the present invention relates to the treatment of an acute infection. In this regard, "acute" means that the subject exhibits symptoms of the disease. In other words, the subject to be treated actually needs the treatment, and in the context of the present invention, the term "acute treatment" relates to measures taken to actually treat the disease after the onset or occurrence of the disease. The term "acute" as referred to in the context of the present invention is in contrast to preventive or protective measures, i.e., measures taken for disease prevention, for example, to prevent the onset / sudden occurrence of an infection and / or disease. More specifically, preventive measures can be understood as preventing free viral particles (from outside the body) from attaching to target cells, thereby inhibiting viral replication. In contrast, in acute infections (which may be primary or recurrent infections), progeny viruses are raced during HSV replication. Therefore, the “acute measures” referred to in this invention do not expressly relate to preventive or prophylactic measures for infections caused by HSV-1 or HSV-2.

[0410] Mucosal tissues that can show signs of acute infection refer to mucosal tissues that are primarily of endodermal origin, covered with epithelium, and involved in absorption and secretion. These line cavities that are exposed to the external environment and internal organs. They are found in several places adjacent to the skin, such as the nostrils, lips of the mouth, eyelids, ears, genitals, and anus.

[0411] The epidermal tissue that can show signs of acute infection refers to the epidermis, the outermost layer of cells in the skin, which together with the dermis to form the skin. The epidermis is stratified squamous epithelium composed of proliferating basal keratinocytes and differentiated epibasal keratinocytes, which act as the body's primary barrier against unsuitable environments by preventing the invasion of pathogens and making the skin a natural barrier against infection. The epidermis also regulates the amount of water released from the body into the atmosphere through transepidermal water loss.

[0412] Local administration according to the present invention relates to a drug or application or administration applied to a body surface such as the skin or mucous membrane to treat the infections mentioned above, via a wide variety of classes of dosage forms, including but not limited to creams, foams, gels, lotions, and ointments. In a preferred embodiment, local administration is understood to be epicutaneous, meaning that an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above are applied directly to the skin. Not bound by theory, but to provide some further non-limiting examples, local application may also be an inhaled drug such as an asthma drug, or may be applied to a surface of tissue other than the skin, such as eye drops applied to the conjunctiva, or ear drops placed in the ear, or a drug applied to the tooth surface. As a route of administration, local administration is contrasted with enteral administration (in the gastrointestinal tract) and intravascular / intravenous administration (injection into the circulatory system). In its broadest sense, local action can be understood in a pharmacodynamic sense as relating to a drug's local target rather than its systemic effect.

[0413] The embodiments of topical administration according to the present invention, namely, drugs, pharmaceutical compositions, or applications or administrations applied to a body surface such as the skin or mucous membrane to treat an acute infection of mucosal or epidermal tissue in a subject, caused by HSV-1 or HSV-2 selected from the group consisting of herpes simplex labialis, herpes simplex vulva, chronic or disseminated cutaneous herpes simplex infection, herpes xiphoid, and herpetic eczema, are not particularly limited, and those skilled in the art know of many forms and preparations that may be suitable for topical administration. Without being bound by theory and without limitation, the following examples are given: There are many general classes where the boundaries between similar formulations suitable for topical administration are not clearly defined. As an example, topical solutions may be used. Topical solutions are generally low-viscosity solutions and often use water or alcohol as the base.

[0414] As another example, lotions are sometimes used for topical administration of anti-HSV antibodies. While lotions resemble solutions, they are thicker and, in reality, tend to soften the skin more than solutions. Lotions are typically oils mixed with water and often contain no less alcohol than solutions.

[0415] As another example, creams may be used for topical administration of anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above. Creams are typically emulsions of oil and water in approximately equal ratios. Creams penetrate well into the outer layers of the stratum corneum of the skin. Creams are thicker than lotions and maintain their shape when removed from their containers. Creams tend to be moderate in terms of moisturizing properties.

[0416] As another example, ointments may be used for topical administration of anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above. Ointments are generally homogeneous, viscous, semi-solid preparations, most commonly high-viscosity, smooth, dense oils (80% oil - 20% water) intended for external application to the skin or mucous membranes. Ointments have a water number that specifies the maximum amount of water the ointment can contain. Ointments may be used as emollients to apply anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above, to the skin for the purpose of protection, treatment, or prevention of disease development, and where some degree of occlusion is desirable. The vehicle of the ointment is known as the ointment base. The choice of base depends on the clinical indication of the ointment and is appropriately selected based on the knowledge of those skilled in the art. Various types of ointment bases may include hydrocarbon bases, e.g., hard paraffin, soft paraffin, microcrystalline wax, and ceresin; absorbent bases, e.g., lanolin fat, beeswax; water-soluble bases, e.g., macrogol 200, 300, 400; emulsifying bases, e.g., emulsifying wax, cetrimide; and vegetable oils, e.g., olive oil, coconut oil, sesame oil, tonsil oil, and peanut oil. Typically, the medicinal drug is dispersed in the base and then separated after the drug has penetrated the living cells of the skin. Ointments are usually formulated with hydrophobic, hydrophilic, or water-emulsifying bases to provide a preparation that is immiscible, miscible, or emulsifiable with skin secretions. Ointments may also be derived from hydrocarbon (fatty) bases, absorbent bases, water-removable bases, or water-soluble bases.

[0417] As another example, gels may be used for local administration of anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above. Gels are usually more concentrated than solutions. Gels are often semi-solid emulsions dissolved in an alcohol base. Some melt at body temperature. Gels tend to undergo cellulose cut using alcohol or acetone.

[0418] As another example, foam may be used for local administration of an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above.

[0419] As another example, transdermal patches may be used for local administration of anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above. Transdermal patches can be a highly precise time-release method for drug delivery. The release of the active ingredient from the transdermal delivery system (patch) may be controlled by diffusion through an adhesive covering the entire patch, or by diffusion through a membrane where the adhesive is only present at the edges of the patch. Alternatively, drug release may be controlled by release from a polymer matrix.

[0420] As another example, powders may be used for topical administration of anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above. The powders may be pure drugs themselves (talc powder) or drugs mixed in a carrier such as corn starch or corn cob powder (Zeosorb AF-miconazole powder).

[0421] As another example, a solid form may be used for topical administration of anti-HSV antibodies. Thus, anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above may each be arranged in solid form. Examples include deodorants, antiperspirants, astringents, and hemostatic agents. In preferred embodiments, particularly in the treatment of acute infections of mucosal or epidermal tissue caused by HSV-1 or HSV-2 of genital herpes simplex, anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above, each may be administered topically, in which case the anti-HSV antibodies may be administered in the form of suppositories. The suppository is a drug delivery system that, in the context of treating vulvar herpes simplex, contains an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a triplicate antibody according to the fourth aspect of the present invention as defined above, and can be inserted into the vagina (i.e., in the form of a vaginal suppository) to dissolve or melt and release the anti-HSV antibody therein, thereby assisting in the local delivery of the anti-HSV antibody.

[0422] As another example, a vaporizer may be used to locally administer an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above. Therefore, an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above, may each be applied as an ointment or gel and can reach the mucous membrane via vaporization.

[0423] As another example, a paste preparation may be used for topical administration of an anti-HSV antibody or its antigen-binding fragment according to the first aspect of the present invention as defined above, a combination of an anti-HSV antibody or its antigen-binding fragment according to the second aspect of the present invention as defined above, a bispecific antibody according to the third aspect of the present invention as defined above, and a trispecific antibody according to the fourth aspect of the present invention as defined above. The paste preparation combines three types of drugs—oil, water, and powder. It is an ointment in which the powder is suspended.

[0424] As a final, non-limiting example, tinctures may be used for topical administration of anti-HSV antibodies or their antigen-binding fragments according to the first aspect of the present invention as defined above, combinations of anti-HSV antibodies or their antigen-binding fragments according to the second aspect of the present invention as defined above, bispecific antibodies according to the third aspect of the present invention as defined above, and triplicate antibodies according to the fourth aspect of the present invention as defined above. A tincture is a skin preparation having a high percentage of alcohol.

[0425] Other aspects and advantages of the present invention are described in the following examples. The following examples are provided for illustrative purposes only and not for limiting purposes. Each publication, patent, patent application or other document cited herein is incorporated herein by reference in its entirety. [Brief explanation of the drawing]

[0426] [Figure 1A] Biolayer interferometry (BLI) analysis of the interaction between recombinant HSV gB and HDIT101 or HDIT102(H4). Octet sensor gram demonstrating the binding of HSV gB-specific IgG HDIT101(MAb hu2c) (as control) (A) or HDIT102(H4) (B) to immobilized wild-type recombinant glycoprotein B of HSV-1F. Biotinylated gB was immobilized on a streptavidin (SA) biosensor chip and incubated with 100 nM HDIT101 or HDIT102(H4)Fab. (C) Measurement of association (ka) and dissociation (kdis) rates and calculation of binding affinity (Kd) using a diluted series of HDIT101 or HDIT102(H4)Fab for HSV-1 or HSV-2 recombinant gB. [Figure 1B] Refer to the explanation in Figure 1A. [Figure 1C] Refer to the explanation in Figure 1A. [Figure 2] Determination of the dissociation constant (Kd) using biolayer interferometry. The binding affinity of HDIT101 or HDIT102(H4)Fab to recombinant gB derived from HSV-1 or HSV-2 was determined using biolayer interferometry (Octet). The Kd value was calculated using the formula Kd = kdis / ka. [Figure 3]Specific recognition of HSV-1F or HSV-2G gB by HDIT102(H4). Binding of several concentrations of HDIT102(H4) dilution series to HSV-1 / 2, VZV, HCMV, and EBV antigens was analyzed by ELISA using microplates (Enzygnost, Siemens) coated with the respective viral antigens. Absorbance at 450 nm was measured. Anti-HSV-ELISA does not distinguish between the detection of anti-HSV-1 and anti-HSV-2 IgG. Binding was detected using HRP-conjugated anti-human γFc-specific IgG. Cytotect (anti-CMV polyclonal antibody preparation) was used as a positive control for all studies. [Figure 4] In vitro neutralization of cell-free HSV-1F or HSV-2G with HDIT102(H4). The antibody concentration required to reduce virus-induced cytopathic effects (CPE) by 100% or 50% was determined by an endpoint dilution assay. Serial dilutions of the antibody were incubated with 100 TCID50 HSV-1F or HSV-2G dissolved in cell culture medium at 37°C for 1 hour. The antibody-virus inoculant was applied to a monolayer of Vero cells grown in a microtiter plate, incubated at 37°C for 72 hours, and then the cytopathic effects (CPE) were scored. The mean and error bars showing the standard deviation of the mean were calculated based on three independent experiments (biological replicas on three different days). 100% neutralization experiments yielded identical EC50 and therefore could not show error bars. [Figure 5]HDIT102(H4) protects NOD / SCID mice from lethal HSV-1 infection. (A) The therapeutic activity of HDIT102(H4) was evaluated by injection (intravaginal inoculation of 5.0 x 10⁵ TCID50) into 8-week-old female NOD / SCID mice (treatment group N=9, control N=6). Four hours after infection, the mice were treated with ip (intravenous injection) with 600 μg, 300 μg, or 150 μg of HDIT102(H4). For 55 days post-infection, the mice were monitored daily for weight loss, symptoms of HSV infection, e.g., vaginal hair loss, redness, swelling, and lesions, as well as survival. Differences between survival curves were calculated using the log-rank Mantel-Cox test. ****p value < 0.0001. (B) The viral genome copy number of vaginal swabs obtained on days 1, 3, and 6 post-infection was evaluated by qPCR. The average value from two technical replicates is shown. The error bars represent the standard deviation of the mean. [Figure 6] Survival of immune-responsive Balb / c mice treated with HDIT102(H4) after HSV-2G infection. (A) In vivo protection of 8-week-old immune-responsive Balb / c mice from lethal HSV-2G infection by HDIT102(H4) was investigated. Mice were vaginally infected with a lethal dose of HSV-2G (5.0 x 10⁴ TCID50), and 4 hours later, 600 μg or 300 μg of HDIT102(H4) (10 mice / group) was intraperitoneally injected. In contrast, the control group was given PBS. Statistical differences between survival curves were calculated using the log-rank-Mantelcox test. **p value < 0.05, ***p value < 0.001. (B) Vaginal swabs were obtained on days 1, 2, 4, and 7 post-infection, and HSV-2G copy number was measured by qPCR. Mean values ​​are from two technical replicates. Error bars represent the standard deviation of the mean. [Figure 7]Inhibition of intercellular transmission of HSV-1F or HSV-2G by HDIT102(H4). Fluorescence microscopy images of Vero cells infected with HSV-1F or HSV-2G, treated with HDIT102(H4) or human polyclonal anti-HSV antibody, or left untreated, are shown. Cells were infected with HSV-1F or HSV-2G, incubated with 500 nM (75 μg) test antibody for 48 hours, washed, stained with anti-HSV-FITC (5 μg / ml) and Hoechst (1:5000), then fixed with 5% paraformaldehyde and imaged (20X, inverted microscope, Leica). One representative image per condition is shown. Arrows indicate plaques, or in the case of HDIT102(H4), early infected cells. [Figure 8A] Cryo-electron microscopy was used to analyze the costructure of HDIT102(H4)Fab bound to HSV-1F gB. The structure of HDIT102(H4)Fab (black) bound to the recombinant trimer HSV-1F gB (gray) was analyzed at a resolution of approximately 3.44 Å using cryo-electron microscopy. Side views (A), top views (B), and bottom views (C) of the trimer gB and the three bound Fab molecules are shown. (D) Single-particle cryo-EM data processing workflow. A graph of the processing steps is shown. The calculations were performed using one of the following software: Relion (Kimanius, Dong et al., 2021, Biochem J, Vol. 478 (24)), CCP-EM (Nicholls, Tykac et al., 2018, Acta Crystallogr D Struct Biol, Vol. 74 (Pt 6)), or coot (Casanal, Lohkamp et al., 2020, Protein Sci, Vol. 29 (4)). [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 8D] See the explanation in Figure 8A. [Figure 9A]Structural analysis of the variable fragment and CDR of HDIT102(H4). Schematic diagram (A) and surface structure (B) of the HDIT102(H4) variable fragment (Fv) derived from the Cryo-EM construct of HDIT102(H4) bound to HSV-1F gB, illustrating the heavy chain (HC), light chain (LC), and complementarity-determining region (CDR). (C) Sequences of the HDIT102(H4) CDR in the HC and LC, and their respective numbering according to the Martin numbering scheme. (D) Location of the residue defining CDR1 in the HC. (E) Location of the residue defining CDR2 in the HC. (F) Location of the residue defining CDR3 in the HC. (G) Location of the residue defining CDR1 in the LC. (H) Location of the residue defining CDR2 in the LC. (I) Location of the residue defining CDR3 in the LC. [Figure 9B] See the explanation in Figure 9A. [Figure 9C] See the explanation in Figure 9A. [Figure 9D] See the explanation in Figure 9A. [Figure 9E] See the explanation in Figure 9A. [Figure 9F] See the explanation in Figure 9A. [Figure 9G] See the explanation in Figure 9A. [Figure 9H] See the explanation in Figure 9A. [Figure 9I] See the explanation in Figure 9A. [Figure 10] Structural analysis of the HDIT102(H4) epitope in HSV-1 gB using key residues. (A) Schematic diagram and surface structure model of the region of the HSV-1F gB protein (light gray) analyzed by cryo-EM analysis, using black gB amino acid residues located immediately adjacent to the CDR residue of HDIT102(H4) to mediate polar or nonpolar interactions. (B) Electrostatic map of HDIT102(H4)Fv using nearby black HSV-1F gB residues to mediate interactions. Positively charged HSV-1F gB residues K204 and R335 are located near the negatively charged region (circle) of HDIT102(H4)Fv. [Figure 11]Analysis of the interaction between HSV-1 gB residue D323 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with recombinantly produced HSV-1F gB. HSV-1 gB D323 is located in proximity to HDIT102(H4)HC CDR1 H27 and HC CDR1 T31 to form contact. [Figure 12] Analysis of the interaction between HSV-1 gB residue Y303 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with recombinantly produced HSV-1F gB. HSV-1 gB Y303 is located in proximity to HDIT102(H4)HC CDR1 R30 and HC CDR2 N53 to form contact. [Figure 13] Analysis of the interaction between HSV-1 gB residue R304 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with HSV-1F gB produced by recombination. HSV-1 gB R304 is located in proximity to HDIT102(H4)HC CDR1 H27 to form contact. [Figure 14] Analysis of the interaction between HSV-1 gB residue Q321 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with HSV-1F gB produced by recombination. HSV-1 gB Q321 is located in proximity to HDIT102(H4) HC CDR1 T31 to form contact. [Figure 15] Analysis of the interaction between HSV-1 gB residue V322 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with HSV-1F gB produced by recombination. HSV-1 gB V322 is located in proximity to HDIT102(H4)HC CDR3 T99 to form contact. [Figure 16]Analysis of the interaction between HSV-1 gB residue D199 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with HSV-1F gB produced by recombination. HSV-1 gB D199 is located in proximity to HDIT102(H4)HC CDR3 T100a and T100b to form polar contact. [Figure 17] Analysis of the interaction between HSV-1 gB residue A203 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with recombinantly produced HSV-1F gB. HSV-1 gB A203 is located nearby to form nonpolar contacts with HDIT102(H4)HC CDR3 T100b and LC CDR1 S32. [Figure 18] Analysis of the interaction between HSV-1 gB residue K320 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with recombinantly produced HSV-1F gB. When HSV-1 gB K320 is in a specific rotational isomer conformation, it is located in proximity to HDIT102(H4)HC CDR3 T100b and LC CDR1 S32 to form polar contact. [Figure 19] Analysis of the interaction between HSV-1 gB residue Y326 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with HSV-1F gB produced by recombination. HSV-1 gB Y326 is located nearby to form a polar contact with HDIT102(H4)HC CDR3 D101. [Figure 20] Analysis of the interaction between HSV-1 gB residue K204 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with recombinantly produced HSV-1F gB. HSV-1 gB K320 is located nearby to form polar contact with the peptide backbone located at HDIT102(H4)LC CDR2 D51, as well as LC CDR1 G29, S30, and K31. [Figure 21] Analysis of the interaction between HSV-1 gB residue R335 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with HSV-1F gB produced by recombination. HSV-1 gB R335 is located nearby to form polar contact with HDIT102(H4)LC CDR2 D53 and nonpolar contact with LC CDR2 Y50. [Figure 22] Analysis of the interaction between HSV-1 gB residue T337 and HDIT102(H4) CDR residues, derived from the Cryo-EM structure of HDIT102(H4)Fab complexed with HSV-1F gB produced by recombination. HSV-1 gB T337 is located in proximity to HDIT102(H4)LC CDR2 S56 to form contact. [Figure 23]Alanine scanning mutation analysis of epitope residues in HSV-1F gB and analysis of HDIT102(H4) binding. Using structural information from Cryo-EM analysis, specific HSV-1F gB amino acid residues immediately adjacent to the HDIT102(H4) CDR residue were selected and substituted with alanine to investigate whether this contributes to HDIT102(H4) binding. In the case of R304, substitution with Q304 was tested. This is an HDIT101 resistance mutation that evolves in vitro at suboptimal HDIT101 concentrations. In the case of R335, both A335 and Q335 were tested. R335Q is an observed HDIT102(H4) resistance escape mutant that evolves in vitro at suboptimal HDIT102(H4) concentrations. HEK293T cells were transfected with either a plasmid encoding wild-type HSV-1F gB or a plasmid encoding the indicated single amino acid point mutant. HEK293T cells were stained 2 days later with HDIT102(H4), HDIT101, or anti-gB control IgG, or without primary antibody. Secondary staining was performed using FITC-labeled anti-human Fc antibody. To include information regarding possible differences in cell surface presentation of the mutant gB protein, integrated mean fluorescence intensity (iMFI) was calculated by multiplying the percentage of HDIT102(H4)-positive stained cells by the geometric MFI of the positively stained cells. [Figure 24]Analysis of alanine scanning mutations of epitope residues in HSV-1F gB and analysis of HDIT102(H4)-induced inhibition of intercellular fusion. (A) In cryo-EM costructures, we investigated whether substituting specific HSV-1F gB amino acid residues adjacent to the HDIT102(H4) CDR residue with alanine contributed to HDIT102(H4)-mediated inhibition of gB-induced intercellular fusion. Plasmids encoding wild-type HSV-1F gB or single amino acid point mutants were introduced into a 1:1 mixture of HEK293T-GFP and HEK293T-E2C fluorescent reporter cells by cotransfection with plasmids encoding HSV-1F gD, gH, and gL. After incubation of the transfected cells at 37°C and 5% CO2 for 5 hours, HDIT102(H4) (75 μg / ml) was added. No antibody was added to the control sample. Cell fusion was determined by the presence of GFP+E2C+ double-positive cells by flow cytometry two days later. Changes in fusion inhibition by HDIT102(H4) were calculated for each mutant by comparing HDIT102(H4)-treated cells with untreated cells, normalized to the mean value of wild-type gB (WT gB), and shown for at least three independent biological replicas (n=3). Statistical analysis was performed using independent t-tests. (B) Fusion activity as a combined indicator of cell surface expression and membrane fusion ability (fusogenicity) of gB mutants was analyzed by comparing it with wild-type gB (WT gB) in the absence of any antibody, as i...

Claims

1. An anti-HSV antibody or its antigen-binding fragment that binds to glycoprotein B(gB) of HSV-1 and / or HSV-2, The complementarity determination region is V, which includes SEQ ID NO:

1. H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V, including CDR3, SEQ ID NO:4 L V containing CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L The antibody contains CDR3, The antibody or its antigen-binding fragment has a dissociation rate k of at most 5.0x10 -4 s -1 , preferably at most 1.0x10 -4 s -1 , at most 5.0x10 -5 s -1 , most preferably at most 2.9x10 -5 s -1 and has a low dissociation rate k dis of Preferably, an anti-HSV antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment can neutralize HSV.

2. The aforementioned antibody is located in the following framework region: SEQ ID NO:7 Position 1-25 (V) H FR1), 36-49 (V H FR2), 67-98 (V H FR3), 112-122 (V H FR4), SEQ ID NO: 81-22 (V L FR1), 34-48 (V L FR2), 56-87 (V L FR3), and 97-106 (V L Amino acid sequences having at least 70% sequence identity for each of the amino acid residues shown in FR4) The anti-HSV antibody or antigen-binding fragment according to claim 1, comprising:

3. The aforementioned antibody is V with SEQ ID NO:7 H and V of SEQ ID NO:8 L An anti-HSV antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising:

4. An anti-HSV antibody or its antigen-binding fragment that recognizes the same epitope as the antibody described in any one of claims 1 to 4, These epitopes are located at contact amino acid residues D199, A203, K204, Y303, R304, K320, Q321, V322, D323, Y326, R335, and T337 of glycoprotein B in HSV-1 strain F, and at contact amino acid residues D191, A195, K196, Y295, R296, K312, Q313, V314, D315, Y318, R327, and T329 of glycoprotein B in HSV-2 strain G, respectively. Preferably, the epitope comprises contact amino acid residues D199, A203, K204, Y303, R304, K320, Q321, V322, D323, Y326, R335, and T337 of glycoprotein B (SEQ ID NO: 9) of HSV-1 strain F, and contact amino acid residues D191, A195, K196, Y295, R296, K312, Q313, V314, D315, Y318, R327, and T329 of glycoprotein B (SEQ ID NO: 40) of HSV-2 strain G, wherein the anti-HSV antibody or its antigen-binding fragment is an anti-HSV antibody.

5. The anti-HSV antibody or antigen-binding fragment according to claim 4, wherein the recognition of the epitope can be confirmed by cryo-electron microscopy (Cryo-EM).

6. The anti-HSV antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein the anti-HSV antibody is a humanized antibody or a fully human antibody.

7. The anti-HSV antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein the anti-HSV antibody is a full-length antibody.

8. An anti-HSV antibody according to any one of claims 1 to 7, which is a human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE antibody.

9. An antigen-binding fragment according to any one of claims 1 to 8, which is a human F(ab)-, Fab'-SH-, Fv-, Fab'-, or F(ab')2- fragment.

10. The anti-HSV antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody has a dissociation constant Kd of up to 10 nM, preferably up to 8 nM, more preferably up to 4 nM, even more preferably up to 2 nM, up to 1 nM, up to 0.8 nM, up to 0.4 nM, up to 0.2 nM, up to 0.1 nM, up to 0.09 nM, up to 0.08 nM, up to 0.07 nM, up to 0.06 nM, up to 0.05 nM, up to 0.04 nM, and most preferably up to 0.03 nM.

11. The anti-HSV antibody or antigen-binding fragment according to any one of claims 1 to 10, wherein the antibody at a concentration of up to 20 nM, preferably up to 16 nM, more preferably up to 12 nM, up to 10 nM, up to 8 nM, up to 6 nM, and most preferably up to 4 nM can neutralize a defined amount of HSV of 100 TCID50.

12. The anti-HSV antibody or antigen-binding fragment according to any one of claims 1 to 11, wherein the antibody can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission).

13. The anti-HSV antibody or antigen-binding fragment according to any one of claims 1 to 12, wherein the antibody exerts its antiviral or neutralizing activity independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), preferably, the antibody can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

14. The anti-HSV antibody or antigen-binding fragment according to any one of claims 1 to 13, wherein the antibody is conjugated to an effector portion, a therapeutic portion, or a detectable label.

15. (A) an anti-HSV antibody or its antigen-binding fragment as defined in any one of claims 1 to 14, (B) Anti-HSV antibody or antigen-binding fragment that recognizes / binds to the glycoprotein B(gB) of HSV-1 and / or HSV-2 And, The complementarity determination region is V, which includes SEQ ID NO:

11. H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L The antibody contains CDR3, The antibody is an anti-HSV antibody or its antigen-binding fragment that recognizes / binds to the glycoprotein B(gB) of HSV-1 and / or HSV-2, having a dissociation constant Kd of up to 40 nM, preferably up to 30 nM, more preferably up to 20 nM, even more preferably up to 15 nM, up to 13 nM, and up to 10 nM. A combination.

16. The antibody according to claim 15(B) is located in the following framework region: SEQ ID NO:17 Position 1-30 (V) H FR1), 38-51(V H FR2), 68-99 (V H FR3), and 112-122(V H FR4), SEQ ID NO: 18, 1-23 (V L FR1), 41-55(V L FR2), 63-94 (V L FR3), and 104-114(V L Amino acid sequences having at least 70% sequence identity with the amino acid residues shown in FR4) The combination according to claim 15, including the combination described in claim 15.

17. The anti-HSV antibody according to claim 15(B) is V of SEQ ID NO:19 H and V of SEQ ID NO:20 L A combination of the above, including the combination of the above, according to claim 15 or claim 16.

18. The anti-HSV antibody according to claim 15(B) is an antibody that recognizes the same epitope as the antibody according to any one of claims 15(B) to 17, The epitopes are located at amino acids Y301–E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B of HSV-1, and at the corresponding sites Y293–E297, and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B of HSV-2. Preferably, the epitope consists of contact amino acid residues Y301 to E305, and H308, K320, D323, Y326, P339, T341, W356, and P358 of glycoprotein B (SEQ ID NO: 9) of HSV-1 F strain, and contact amino acid residues Y293 to E297, and H300, K312, D315, Y318, P331, T333, W348, and P350 of glycoprotein B (SEQ ID NO: 40) of HSV-2 G strain, according to any one of claims 15 to 17.

19. The combination according to claim 18, wherein the recognition of the epitope is confirmed by cryo-electron microscopy (Cryo-EM).

20. The combination according to any one of claims 15 to 19, wherein the anti-HSV antibody according to claim 15(B) is a humanized antibody or a fully human antibody.

21. The combination according to any one of claims 15 to 20, wherein the anti-HSV antibody according to claim 15(B) is a full-length antibody.

22. The combination according to any one of claims 15 to 21, wherein the anti-HSV antibody according to claim 15(B) is a human IgG1, IgG2, IgG2a, IgG2b, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE antibody.

23. The combination according to any one of claims 15 to 22, wherein the antigen-binding fragment according to claim 15(B) is an F(ab)-, Fab'-SH-, Fv-, Fab'-, or F(ab')2- fragment.

24. A combination according to any one of claims 15 to 23, wherein the antibody according to claim 15(B) can neutralize a defined amount of HSV, namely 100 TCID50, at a concentration of up to 20 nM, preferably up to 16 nM, more preferably up to 12 nM, up to 10 nM, up to 8 nM, up to 6 nM, and most preferably up to 4 nM.

25. The combination according to any one of claims 15 to 24, wherein the anti-HSV antibody according to claim 15(B) can inhibit the transmission of HSV from an infected cell to another adjacent uninfected cell (intercellular transmission).

26. A combination of any one of claims 15 to 25, wherein the anti-HSV antibody according to claim 15(B) exhibits its antiviral or neutralizing activity independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), preferably the antibody can inhibit intercellular transmission independently of antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

27. The combination according to any one of claims 15 to 26, wherein the anti-HSV antibody according to claim 15(B) is conjugated to an effector moiety, a therapeutic moiety, or a detectable label.

28. A pharmaceutical composition comprising an effective amount of an anti-HSV antibody or its antigen-binding fragment according to any one of claims 1 to 14, or a combination of antibodies according to any one of claims 15 to 27, and at least one pharmaceutically acceptable excipient.

29. An anti-HSV antibody according to any one of claims 1 to 14 or an antigen-binding fragment thereof, or a combination of the antibody according to any one of claims 15 to 27, for use as a drug.

30. Oral herpes simplex, genital herpes simplex, chronic or disseminated cutaneous herpes simplex infection, xiphoid herpes, herpetic eczema (Eczema herpeticum), herpetic keratoconjunctivitis, neonatal herpes, Alzheimer's disease (AD), HSV pneumonia, Bell's palsy, herpetic esophagitis, herpesvirus encephalitis and herpesvirus meningitis, herpetic folliculitis, herpetic paronychia, herpetic gingivostomatitis, presence of recurrent or recurrent oral herpes, presence of recurrent or recurrent genital herpes, herpetic eczema (Eczema An anti-HSV antibody according to any one of claims 1 to 14 or an antigen-binding fragment thereof, or a combination of antibodies according to any one of claims 15 to 27, for use in a method for the prophylactic or therapeutic treatment of a disorder or disease selected from the group consisting of herpeticatum, neonatal herpes, immunodeficiency, immunocompromised patients, resistance to antiviral agents, encephalitis, meningitis, meningoencephalitis, ocular infections, and / or generalized HSV infection.

31. The anti-HSV antibody or its antigen-binding fragment or combination of the antibody for use according to claim 30, wherein the antibody is administered intravenously, locally, intradermally, subcutaneously, intradermally, intramuscularly, and / or subarachnoidally.

32. (A) The complementarity determination region, including V, which contains SEQ ID NO:1 H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V, including CDR3, SEQ ID NO:4 L V containing CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L The first binding domain, which includes CDR3, (B) The complementarity determination region, including SEQ ID NO:11 H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L The second binding domain, including CDR3, A bispecific antibody or its antigen-binding fragment that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, comprising: This bispecific antibody has a maximum output of 5.0 x 10 -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis A bispecific antibody or its antigen-binding fragment having the same properties.

33. (A) The complementarity determination region, including V, which contains SEQ ID NO:1 H V, including CDR1 and SEQ ID NO:2 H V, including CDR2, SEQ ID NO:3 H V, including CDR3, SEQ ID NO:4 L V containing CDR1, SEQ ID NO:5 L V including CDR2 and SEQ ID NO:6 L The first binding domain, which includes CDR3, (B) The complementarity determination region, including SEQ ID NO:11 H V containing CDR1, SEQ ID NO:12 H V, including CDR2, SEQ ID NO:13 H V containing CDR3, SEQ ID NO:14 L V containing CDR1, SEQ ID NO:15 L V including CDR2 and SEQ ID NO:16 L A second binding domain, including CDR3, (C) V, which is the complementary determination region, including SEQ ID NO:31 H V containing CDR1, SEQ ID NO:32 H V containing CDR2, SEQ ID NO:33 H V, including CDR3, SEQ ID NO:34 L V containing CDR1, SEQ ID NO:35 L V including CDR2 and SEQ ID NO:36 L A third binding domain including CDR3 and A triplicate antibody or antigen-binding fragment that binds to the glycoprotein B (gB) of HSV-1 and / or HSV-2, comprising: This trispecific antibody has a maximum output of 5.0 x 10 -4 s -1 Preferably a maximum of 1.0 x 10 -4 s -1 , up to 5.0x10 -5 s -1 Most preferably a maximum of 2.9 x 10 -5 s -1 Low dissociation rate k dis It has, A trispecific antibody or its antigen-binding fragment, wherein the trispecific antibody at a concentration of up to 10 nM, preferably up to 8 nM, more preferably up to 6 nM, up to 4 nM, up to 2 nM, up to 1 nM, up to 0.9 nM, up to 0.7 nM, and most preferably up to 0.5 nM, can neutralize a defined amount of HSV of 100 TCID50.

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