TOPICAL APPLICATION FOR AN ANTI-HSV ANTIBODY

Abstract

Described is an anti-HSV antibody or an antigen-binding fragment thereof for use in treating 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 genitalis, chronic or disseminated cutaneous herpes simplex infection, Herpes gladiatorum and Eczema herpeticum, wherein said antibody is to be topically administered as well as to a pharmaceutical composition comprising an effective amount of said antibody or antigen-binding fragment thereof and at least one pharmaceutically acceptable excipient.

Claims

1-18. (canceled)

19. A method of treating an acute infection of mucosal or epidermal tissue in a subject caused by HSV-1 or HSV-2, wherein the method comprises administering to the subject a neutralizing full-length anti-HSV antibody, wherein the subject is suffering from an acute infection of one or more of Herpes simplex labialis, Herpes simplex genitalis, chronic or disseminated cutaneous herpes simplex infection, Herpes gladiatorum or Eczema herpeticum, wherein said antibody inhibits cell-to-cell spread, and wherein said antibody is topically administered to mucosal or epidermal tissue.

20. The method of claim 19, wherein said anti-HSV antibody is a monoclonal or a polyclonal antibody.

21. The method of claim 19, wherein said anti-HSV antibody is a humanized or fully human antibody.

22. The method of claim 19, wherein said anti-HSV antibody recognizes the glycoprotein B (gB) of the HSV-1 and/or HSV-2.

23. The method of claim 19, wherein the antibody is capable of inhibiting cell-to-cell spread independent from antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).

24. The method of claim 19, wherein the antibody is capable of inhibiting the spreading of HSV from an infected cell to an adjacent second non-infected cell (cell to cell spread).

25. The method of claim 19, wherein the antibody comprises an amino acid sequence with at least 70% sequence identity to the amino acid residues shown in positions 1 to 30, 38 to 51, 68 to 99, and 112 to 122 of SEQ ID NO: 7 and in positions 1 to 23, 41 to 55, 63 to 94, and 104 to 114 of SEQ ID NO: 8.

26. The method of claim 19, wherein said antibody comprises the V.sub.H of SEQ ID NO:9 and the V.sub.L of SEQ ID NO:10.

27. The method of claim 19, wherein said antibody recognizes the same epitope as mAb 2c, wherein said epitope is located at the amino acids 172-195 and 295-313 of glycoprotein B of HSV-1 and HSV-2.

28. The method of claim 19, wherein said antibody is the mAb 2c antibody.

29. The method of claim 19, wherein said antibody is to be topically applied to infected mucosal or epidermal tissue of the lips, genitals, nose, ears, eyes, fingers, toes and/or skin areas throughout the body, preferably on the head, the jaw area, neck, chest, face, stomach and/or legs.

30. The method of claim 19, wherein said antibody is to be topically applied to areas surrounding the infected mucosal or epidermal tissue.

31. The method of claim 19, wherein said antibody is to be administered in combination with a virostatic agent.

32. The method of claim 31, wherein said virostatic agent is selected from the group consisting of the drug classes of nucleoside analogues, pyrophosphate analogues, nucleotide analogues, an amantadin derivative, and helicase-primase inhibitors.

33. The method of claim 32, wherein said nucleoside analogue is selected from the group consisting of Acyclovir, Penciclovir, Valacyclovir and Famaciclovir; wherein said pyrophosphate analogue is Foscarnet; wherein said nucleotide analogue is Cidofovir; wherein said amantadin derivative is Tromantandin; and wherein said helicase-primase inhibitor is Pritelevir.

34. The method of claim 19, wherein the antibody is part of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

35. The method of claim 19, wherein said anti-HSV antibody comprises SEQ ID NO:9.

36. The method of claim 19, wherein said anti-HSV antibody comprises SEQ ID NO:10.

37. The method of claim 19, wherein symptoms of said acute infection include itching of the lips, burning or tingling near the lips or mouth area, or blisters.

Description

[0157] Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.

[0158] FIG. 1: compares the survival of immunodeficient mice with acute genital HSV-2 infection after topical treatment either with the humanized monoclonal antibody hu2c or acyclovir. Female mice (NOD.CB17-Prkdc.sup.scid/NCrHsd) were treated with a long-acting progestin (Depo-Clinovir, Pharmacia) 7 days prior to viral challenge to increase susceptibility to HSV-2 infection and to eliminate differences caused by the estrous cycle. Anesthetized mice were vaginally challenged with a lethal dose of 5×10.sup.5 PFU of HSV-2 G (20 μl). Mice displaying visible infection (perineal hair loss, reddening, swelling) were treated one day after viral challenge (A) once with 40 μl hu2c (5 mg/ml) (.circle-solid.) or 40 μl control IgG (5 mg/ml) (□) or (B) twice daily for 4 days with 40 μl ACV (25 mg/ml) (Δ) or 40 μl PBS (x). Drug solutions or PBS were applied topically to the outer genital epithelium. Mice were monitored for 36 days after viral inoculation. Mice displaying sever systemic signs and/or severe lesions/zoster were killed. Surviving mice were sacrificed at day 36. Test groups contained eight animals each, control groups contained five animals each. Kaplan-Meier survival curves were analyzed by log-rank (Mantel-Cox) test. Two-tailed significance tests were used to compare the significance level between two groups. All protocols were approved by the Animal Care and Use Committee.

[0159] FIG. 2: shows the clinical scoring of acute genital HSV-2 infection after topical treatment with the humanized monoclonal antibody hu2c or acyclovir. Mice displaying visible infection (perineal hair loss, reddening, swelling) one day after intravaginal challenge with a lethal dose of 5×105 PFU of HSV-2 G (20 μl) were treated (A & C) twice daily for 4 days with (A) 40 μl PBS or (C) 40 μl ACV (25 mg/ml), or treated (B & D) once at 24 h post infection with (B) 40 μl hu2c (5 mg/ml) or (D) 40 μl control IgG (5 mg/ml). Drug solutions or PBS were applied topically to the outer genital epithelium.

[0160] Infected animals were observed daily and their clinical status was scored as follows: 0, lack of symptoms, no lesions; 1-2, redeness and/or swelling (erosion); 3, localized lesion<1 mm; 4-5, localized lesion 2-3 mm; 6-7 localized lesion 4-5 mm; 8-9, severe hyperemia, destruction of the epithelium and stroma with necrosis; 10, systemic signs, death. Animals with grading >8 were killed to prevent undue suffering. Test groups contained eight animals each, control groups contained five animals each. Arrows indicate time points of treatment.

EXAMPLES

Example 1: Topical Application of a Humanized Anti-HSV Antibody

[0161] 1. Subjects, Materials, Methods

[0162] 1.1 Generation and Production of a HSV Neutralizing Humanized Monoclonal Antibody.

[0163] Recently, it has been demonstrated that cross-linking of a highly conserved glycoprotein B epitope of HSV-1/2 through the murine monoclonal antibody mAb 2c does not only result in highly efficient neutralization of free virions but also in inhibition of direct virus spread from infected to non-infected cells (Krawczyk A, et al., Journal of virology 2011; 85(4):1793-1803). To exploit these unique properties for therapeutic use in humans, we generated a humanized derivative of mAb 2c.

[0164] For the vast majority of humanized antibodies retention of a set of potentially immunogenic murine residues within the human frameworks is usually required for maintaining the structural integrity of the grafted antigen binding loops. In order to generate a humanized antibody with the lowest possible immunogenic potential any framework manipulations had been avoided by careful selection of appropriate human germline sequences and simultaneous employment of our previously described sequence multi-alignment approach (Krauss J, et al., Protein Eng 2003; 16(10):753-759).

[0165] To identify appropriate human germline acceptor scaffolds for grafting the mAb 2c complementarity determining regions (CDRs), variable domain framework sequences of mAb 2c were aligned to corresponding human sequences of the V Base database (http://vbase.mrc-cpe.cam.ac.uk/). The highest framework sequence identities to the corresponding murine mAb 2c variable light (V.sub.L) and variable heavy (V.sub.H) chain sequence showed the human germline sequences DP28 (88.5%) and DPK13 (88.9%), respectively. Hence, CDR coding gene segments of the murine donor-antibody 2c (i.e. 2c V.sub.L-CDR1/2/3 and 2c V.sub.H-CDR1/2/3) were grafted into acceptor frameworks coding for DP28 and DPK13, respectively. Variable domain encoding genes of the chimeric and humanized V.sub.L chain and V.sub.H chain were subsequently cloned into immunoglobulin expression vectors containing a human constant heavy γ1 chain, and a human constant K chain, respectively. The humanized antibody was either produced from stably transfected Sp2/0 mouse myeloma cell lines or transient transfected HEK293 cells under serum-free conditions and purified from culture supernatants to homogeneity by protein A chromatography. Purity was assessed by gel filtration chromatography (Superdex 200GL, GE Healthcare) as ≥95% (Krawczyk A, et al., Proc Natl Acad Sci USA 2013; 110(17):6760-6765).

[0166] 1.2 Trial Description

[0167] Between 2010-2013, twelve healthy 30-59 year old volunteers (7 female, 5 male) with an acute recurrence of oral herpes infection (cold sores) were treated. Volunteers presented themselves when the onset of initial HSV symptoms (itching of the lips, burning or tingling near the lips or mouth area) occurred or had progressed to visible skin disorders on the outer lips. Observed skin disorders included small to large blisters filled with clear yellowish fluid or external herpetic lesions including leaking red blisters.

[0168] Oral herpes infection of the mouth area is mainly caused by the herpes simplex virus type 1 (HSV-1). However, sometimes HSV-2 is spread to the mouth during oral sex, causing oral herpes. The type of HSV infection (HSV-1 or HSV-2) was not analyzed.

[0169] The antibody was packaged as sterile solutions either in PBS or PBS/ash crème (1:1) at concentrations of 0.7-1 mg/ml. Participants applied approx. 10 μl of the antibody topically once, once per day for two days or for a total of three times maximum.

[0170] 2. Results ZOVIRAX Cream had been evaluated in 2 double-blind, randomized, placebo (vehicle)-controlled trials (see Zovirax N-. Zovirax Prescribing Information. http://wwwaccessdatafdagov/drugsatfda_docs/label/2002/21478_zovirax_Iblpdf#page=1&zoom=auto,0,792).

[0171] In the Zovirax studies, subjects were instructed to initiate treatment within 1 hour of noticing signs or symptoms and continue treatment for 4 days, with application of study medication 5 times per day. In both studies, the mean duration of the recurrent herpes labialis episode was approximately one-half day shorter in the subjects treated with ZOVIRAX Cream (n=682) compared with subjects treated with placebo (n=703) (approximately 4.5 days versus 5 days, respectively). No significant difference was observed between subjects receiving ZOVIRAX Cream or vehicle in the prevention of progression of cold sore lesions.

[0172] Compared to previous HSV outbreaks that have been treated with aciclovir (Zovirax crème) all participants using the antibody solution reported a fast symptom and pain relief within 24 h after application of the antibody. In contrast to the experiences with aciclovir therapy active blisters regressed and did not turn into weeping blisters when treated topically with the antibody. When antibody treatment was started at the stage of visible external herpetic lesions, participants reported a rapid healing and disappearance of crusted areas. All participants reported in contrast to their experience with Zovirax that the infected area did not spread upon antibody treatment.

[0173] One volunteer experienced Herpes labialis at the upper and lower lip at the same time and started antibody treatment for the upper lip (three times) and Zovirax treatment for the lower lip (3-4 times a day for 3 days). At time of treatment several small blisters were visible. For the antibody treated HSV infection a quick recovery was observed. Blisters of the upper lip disappeared within 24 h, the swelling subsided within 48 h and no lesions occurred. The infection of lower lip treated with Zovirax remained painful for 3 days, blisters grew together into larger blister which eventually broke open. The occurred lesions took two weeks to heal.

[0174] Efficacy of the treatment seemed to be independent from the antibody formulation (PBS or PBS/ash crème).

[0175] Interestingly the participants have the impression that the overall rates of clinical reactivation tend to be reduced.

Example 2: Topical Application of Anti-HSV mAb hu2c in Animal Experiments

[0176] The ability of the humanized monoclonal antibody hu2c to alter the clinical course of acute genital HSV-2 infection in immunodeficient mice following a single topical treatment with 200 μg mAb hu2c (5 mg/ml) was investigated. To infect 100% of mice as assessed by visible lesions and culture of vaginal lavage, a viral inoculum of 5×105 PFU of HSV-2 G was delivered to the vagina of anesthetized mice.

[0177] Although acute HSV-1 or HSV-2 infections result in a fatal outcome in 100% of mice with severe combined immunodeficiency when compared to 70-90% mortality in immunocompetent mice, the immunodeficient model has nevertheless been chosen to discriminate a possible clinical efficacy of the therapy from an elimination of the viral infection due to immune effector cells of the mouse (Minagawa et al., Arch Virol 103, 73-82 (1988); Nagafuchi et al., J Gen Virol 44, 715-723 (1979)). Within genital mucosa the expansion rates of HSV-2 are extremely rapid. At 24 h after viral challenge infected mice received topically at the infected area either twice per day for 4 days 1 mg acyclovir (ACV) (25 mg/ml) or 40 μl buffer (PBS) or a single treatment of 200 μg mAb hu2c (5 mg/ml) or 200 μg control mAb (5 mg/ml). Clinical efficacy of the HSV-specific mAb was compared to the irrelevant mAb (isotypcontrol), ACV and PBS treatment by means of Kaplan-Meier survival curves and daily assessment of the clinical status of the mucous membranes of the genital and anal area. Results from mice displaying visible infection (perineal hair loss, reddening, swelling) and detectable peripheral replication 24 h after infection were evaluated.

[0178] As expected, no significant differences in overall survival were observed in control groups treated either with an irrelevant mAb vs PBS and all mice were dead by day 7 after infection (FIGS. 1A & B). Surprisingly, a single topical application of mAb hu2c to the outer genital infected epithelium resulted in a statistically significant difference in survival curves (P=0.003) when compared to the control groups (FIG. 1 A) and even prevented the lethal outcome of the infection in one mouse. Survival curves of the antibody hu2c treated group (FIG. 1 A) and the ACV treated group (FIG. 1 B) showed no significant differences (P>0.5) although ACV was applied twice daily for 4 days.

[0179] The medical advantage of the topical antibody therapy over the standard therapy with ACV became even more apparent when evaluating the clinical status of the acute genital infection over a period of 14 days (FIG. 2). A clinical score grading can be applied to investigate if the clinical course of an infection can be altered upon treatment (Minagawa et al., Arch Virol 103, 73-82 (1988); Sanna et al., Virology 215, 101-106 (1996)).

[0180] The clinical status of vaginitis/vulvitis was scored as follows: 0, lack of symptoms, no lesions; 1-2, redeness and/or swelling (erosion); 3, localized lesion<1 mm; 4-5, localized lesion 2-3 mm; 6-7 localized lesion 4-5 mm; 8-9, severe hyperemia, destruction of the epithelium and stroma with necrosis; 10, systemic signs, death.

[0181] Acute genital HSV-2 infection resolved in 7 out of 8 mice (88%) within 48 h post single topical treatment with anti-HSV mAb hu2c (FIG. 2D).

[0182] In contrast, animals treated with ACV displayed an extremely heterogeneous clinical grading. At 48 h under ongoing treatment with ACV (twice per day, for 4 days) local genital symptoms resolved only in 1 out of 8 mice (13%), and 72 h after commencement of ACV treatment only 5 out of 8 mice (63%) had no local signs (FIG. 2C). Although both, the hu2c antibody and ACV were applied only topically, HSV-2 lethal encephalitis could be prevented in 1 out of 8 mice in both cases.

[0183] Mice either treated with buffer or an irrelevant control mAb had progressive local HSV-2 infections spreading across the genital and anal areas and systemic dissemination of the virus resulted in the death of all animals at day 7 (FIGS. 2 A & B).