WO2019044926A1 - 抗HSV gBモノクローナル抗体又はその抗原結合断片 - Google Patents
抗HSV gBモノクローナル抗体又はその抗原結合断片 Download PDFInfo
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates to anti-HSV gB monoclonal antibodies or antigen binding fragments thereof.
- the herpes simplex virus (Herpes simplex virus; HSV) is a neurotropic pathogen, and after primary infection in mucosal epithelia, it translocates to the sensory nerve and becomes a latent infection in the trigeminal ganglia or sacral ganglia.
- the latent HSV sometimes reactivates and causes various pathological conditions (Non-patent Document 1).
- HSV-1 and HSV-2 Two serotypes (HSV-1 and HSV-2) are known for HSV, HSV-1 mainly causes labial and corneal herpes, and HSV-2 mainly causes genital herpes.
- HSV-1 often causes genital herpes and HSV-2 causes cold sores due to diversification of sexual behavior.
- the percentage of antibody-positive (pre-infected) patients in Japan is 60 to 80% for HSV-1 and 10% for HSV-2, and even if it is limited to HSV-2, the potential demand for vaccine is estimated to be 10 million.
- Non-Patent Document 3 the percentage of antibody-positive persons (pre-infected persons) in the United States is 57% for HSV-1 and 20% for HSV-2 (of which approximately 10% is manifest genital herpes)
- Non-Patent Document 3 the percentage of antibody-positive persons (pre-infected persons) in the United States is 57% for HSV-1 and 20% for HSV-2 (of which approximately 10% is manifest genital herpes
- Infection with HSV cells is known to involve five envelope glycoproteins (glycoproteins) at two stages: adsorption and invasion.
- the five envelope glycoproteins are called envelope glycoprotein B (gB), envelope glycoprotein C (gC), envelope glycoprotein D (gD), envelope glycoprotein H (gH), and envelope glycoprotein L (gL), respectively.
- gB envelope glycoprotein B
- gC envelope glycoprotein C
- gD envelope glycoprotein D
- gH envelope glycoprotein H
- gL envelope glycoprotein L
- Non-patent Documents 5 and 6 Non-patent Documents 5 and 6
- This process is not essential for HSV entry into cells, but is thought to be involved in more efficient entry.
- gB and gD bind to their respective host cell receptors and are initiated by fusion of the viral envelope and the host cell membrane.
- gB receptors and gD receptors are known.
- NM-IIA Non-patent Documents 7 and 8
- MAG Non-patent Document 9
- Nectin 1 Non-patent Document 10
- HVEM Non-patent Document 11
- 3-O-sulfated heparan sulfate Non-patent document 12
- gH / gL heterodimers are known to interact with gB and gD and play an important role in membrane fusion (Non-patent Document 13).
- Non-patent Document 14 the structure of gB is similar to that of VSV (Vesicular stomatitis virus), which is known as a membrane fusion protein, and this confirms that gB is a membrane fusion protein of HSV.
- VSV Vesicular stomatitis virus
- GB is also highly conserved in other herpesviruses, and its function is considered to be common to herpesviruses.
- antiviral drugs such as acyclovir are used for the treatment of HSV, but the virus can not be completely removed, and when it is discontinued, the virus is reactivated. This is due to HSV taking a special form of infection called latent infection to ganglia. Therefore, it is desirable to develop a preventive vaccine that protects HSV infection itself or a therapeutic vaccine that reduces or alleviates the symptoms of infection / reactivation, but there is currently no effective vaccine, and its unmet need is high.
- Non-patent Document 19 There is also concern that the frequent or long-term use of antiviral agents such as acyclovir or foscarnet will result in an increase in resistant virus strains. Furthermore, these antiviral agents have various side effects such as liver dysfunction, spermatogenic dysfunction, digestive tract disorder, renal dysfunction and so on, and in some cases reduction or withdrawal may be unavoidable. .
- HSV is known to be one of the pathogens that can not acquire sufficient protective immunity in conventional vaccines and pathogen infection history. It is believed that this is because HSV has a variety of immune evasion mechanisms and cleverly bypasses the host's immune response.
- PILR alpha is a herpes simplex virus-1 entry coreceptor that associates with glycoprotein B.
- the present invention provides a novel anti-HSV gB monoclonal antibody or an antigen-binding fragment thereof that exhibits a neutralizing effect on HSV, suppresses intercellular transmission of HSV, and can be used for the prevention and / or treatment of HSV infection. To be a task.
- the present inventors obtained 44 types of anti-gB antibodies by exhaustive search of anti-HSV gB antibodies performed using a human antibody library.
- strains designated as monoclonal antibody D48 have potent virus neutralization activity (plaque number reduction activity) against HSV-1 and HSV-2 and cell-to-cell spread (Cell to It showed the suppression activity of cell infection spread, and found that the activity intensity overwhelmed other antibodies.
- the present inventors conducted further detailed analysis on the monoclonal antibody D48 to complete the present invention.
- an anti-HSV gB monoclonal antibody or an antigen-binding fragment thereof which specifically binds to envelope glycoprotein B (gB) of herpes simplex virus (HSV),
- a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 3
- a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 4
- a heavy chain variable region comprising heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 5
- An anti-HSV gB monoclonal antibody or an antigen-binding fragment thereof comprising:
- a pharmaceutical composition for preventing or treating HSV infection comprising the anti-HSV gB monoclonal antibody of the above (1) or (2) or an antigen-binding fragment thereof.
- the pharmaceutical composition according to (8) above, wherein the HSV infection is HSV-1 infection or HSV-2 infection.
- HSV infection is selected from the group consisting of cold sores, corneal herpes, genital herpes, systemic neonatal herpes, and stomatitis due to HSV, skin diseases, encephalitis, meningitis, and myelitis
- a heavy chain variable comprising heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 3, heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 5 Area, A light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 6, a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 8
- the anti-HSV gB monoclonal antibody or the antigen-binding fragment thereof according to (11) above which comprises (13) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1; A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; The anti-HSV gB monoclonal antibody or the antigen-binding fragment thereof according to (12) above, which
- the anti-HSV gB monoclonal antibody or antigen-binding fragment thereof of the present invention has potent virus neutralization activity (plaque number reduction activity) against HSV-1 and HSV-2 in vitro and also potent cell-to-cell infection spread ( It has the inhibitory activity between cells) and shows a significant protective effect against not only prophylactic administration in vivo but also therapeutic administration under the situation where HSV infection has already been established in the living body. .
- HSV infection can be applied for prevention and / or treatment / reactivation prevention, and in particular immunocompromised patients at high risk of HSV infection, or bone marrow transplantation, blood stem cell transplantation or organ transplantation patients who are administering an immunosuppressant It can be used as a preventive agent for H. pylori or as a therapeutic agent for patients who repeatedly reactivate HSV, as a substitute for or in combination with existing antiviral agents.
- FIG. 7 shows the results of reactivity analysis of anti-HSV gB antibody (Fab) by ELISA of Example 2.
- FIG. 7 shows the results of the reactivity analysis of anti-HSV gB antibody (human-mouse chimeric IgG2a) by ELISA of Example 2.
- FIG. 8 shows the results of the reactivity analysis of anti-HSV gB antibodies by the immunoblot of Example 3.
- FIG. 16 shows the results of epitope analysis by hydrogen-deuterium exchange mass spectrometry (HDX-MS) of Example 6. It is a figure which shows the comparison result of the survival rate of the mouse infection protection test by the antibodies D48, F67, and E31 of Example 8.
- FIG. 16 shows the results of survival rates of prophylactic administration to mice by the antibody D48 of Example 8.
- FIG. 16 shows the results of symptom scores of prophylactic administration to mice by the antibody D48 of Example 8.
- FIG. 16 shows the results of survival rates of therapeutic administration of the antibody D48 of Example 8 to mice.
- FIG. 16 shows the results of symptom scores of therapeutic administration to mice by the antibody D48 of Example 8.
- FIG. 16 shows the results of symptom scores of prophylactic administration to guinea pigs by the antibody D48 of Example 9.
- FIG. 16 shows the results of symptom scores of therapeutic administration to guinea pigs by the antibody D48 of Example 9.
- FIG. 16 shows the results of HSV release in vaginal swab of therapeutic administration to guinea pigs with antibody D48 of Example 9.
- FIG. 7 shows the comparison result of multiple alignment of the amino acid sequence of HSV-1 derived gB (SEQ ID NO: 10) and the amino acid sequence of HSV-2 derived gB (SEQ ID NO: 11), wherein the italics indicate the leader sequence and underlined Denotes amino acid residues 383 to 388 (I383-R388) of domain II of HSB-1 derived gB and amino acids residue 386 to 391 (I386-R391) of domain II of HSB-2 derived gB .
- the anti-HSV gB monoclonal antibody or antigen-binding fragment thereof of one embodiment of the present invention specifically binds to envelope glycoprotein B (gB) of herpes simplex virus (HSV).
- the anti-HSV gB monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and A heavy chain variable region containing heavy chain CDR3 consisting of an amino acid sequence, a light chain CDR1 consisting of the amino acid sequence as shown in SEQ ID NO: 6, a light chain CDR2 consisting of the amino acid sequence as shown in SEQ ID NO: 7, and And a light chain variable region comprising a light chain CDR3 consisting of an amino acid sequence.
- the present inventors obtained 44 types of anti-gB antibodies by exhaustive search of anti-HSV-2 gB antibodies performed using a human antibody library. As a result of subjecting these antibodies to a virus neutralization test, monoclonal antibody D48 showed a strong plaque number reducing activity and a cell to cell infection spread inhibitory activity.
- Antibody D48 has a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 1 shown in Table 1 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 2 And a heavy chain variable region comprising heavy chain CDR1 and heavy chain CDR2 and heavy chain CDR3 and a light chain comprising light chain CDR1 and light chain CDR2 and light chain CDR3 consisting of amino acid sequences of SEQ ID NOs: 6 to 7 And a chain variable region.
- VH heavy chain variable region
- VL light chain variable region
- the anti-HSV gB monoclonal antibody or antigen-binding fragment thereof of the present invention comprises the CDR sequences of monoclonal antibody D48, it has potent virus neutralizing activity (plaque number reducing activity) against HSV-1 and HSV-2. It has potent Cell to cell infection spread (intercellular propagation) inhibitory activity.
- the anti-HSV gB monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2. It is also good.
- the anti-HSV gB monoclonal antibody is an antibody comprising at least a heavy chain variable domain and a light chain variable domain, and may be a complete antibody.
- a complete antibody has two full-length light chains and two full-length heavy chains, each light chain and heavy chain being linked by a disulfide bond.
- Intact antibodies include IgA, IgD, IgE, IgM and IgG, and IgG includes IgG 1 , IgG 2 , IgG 3 and IgG 4 as subtypes.
- the antigen binding fragment is also referred to as an antibody fragment, and means a fragment having a function of binding to an antigen (antigen binding fragment).
- Antigen binding fragments include, for example, single chain variable region fragments (scFv), Fab, Fab ', F (ab') 2 , scAbs, scFvFc and Fv.
- the Fab has one antigen binding site as a structure having the variable regions of the light chain and the heavy chain and the constant region of the light chain and the first constant region (CH1 domain) of the heavy chain.
- F (ab ') 2 antibodies are generated by disulfide bonds between cysteine residues in the hinge region of Fab'.
- Fv (variable fragment) means the smallest antigen-binding fragment with only heavy and light chain variable regions.
- the double-chain Fv (dsFv) is linked to the light-chain variable site by a heavy-chain variable site and a disulfide bond, and the single-chain Fv (scFv) Covalently linked to the region.
- the scAb is obtained by binding a part of the constant domain of L chain or H chain (C domain) to scFv.
- the scFv Fc is a combination of scFv with CH1 and CH2 of H chain.
- Antigen binding fragments can be obtained, for example, from whole antibodies using proteolytic enzymes. For example, a complete antibody can be cleaved with papain to obtain a Fab fragment, and cleavage with pepsin can yield an F (ab ′) 2 fragment. Antigen binding fragments can also be produced using genetic engineering techniques.
- the variable regions of the light chain and the heavy chain comprise three multiply variable regions called complementarity determining regions (hereinafter referred to as "CDRs").
- the CDRs are primarily responsible for binding to an epitope of the antigen.
- the three CDRs are referred to as CDR1, CDR2 and CDR3, respectively, from the N-terminus.
- the anti-HSV gB monoclonal antibody may be a human antibody (a fully human antibody), a humanized antibody, or a chimeric antibody, and particularly preferably a human monoclonal antibody or a humanized monoclonal antibody.
- HSV gB envelope glycoprotein B of herpes simplex virus
- HSV gB contains HSV-1 derived gB and HSV-2 derived gB, but Figure 13 shows the amino acid sequence of HSV-1 derived gB (SEQ ID NO: 10) and the amino acid sequence of HSV-2 derived gB (SEQ ID NO: 11) Shows a comparison result of multiple alignment. According to FIG.
- the anti-HSV gB monoclonal antibody or the antigen-binding fragment thereof of the present embodiment in which the sequence identity between the two is 87% specifically binds to either HSV-1 derived gB or HSV-2 derived gB.
- the anti-HSV gB monoclonal antibody or the antigen-binding fragment thereof of the present embodiment is considered to have two merits as (1) less side effects and (2) low risk of emergence of resistant virus strains as merits to existing antiviral agents.
- the anti-HSV gB monoclonal antibody or antigen-binding fragment thereof of this embodiment is specific for an epitope sequence on surface glycoprotein B (gB) of HSV-1 and HSV-2 coats integral to the virus replication cycle. Appearance of HSV resistance which is an antibody that binds to the antibody and therefore may be due to a mutation of the relevant epitope sequence in the gB protein, as it is believed that the mutation itself results in a loss of viral infectivity. Sex is considered to be low.
- the anti-HSV gB monoclonal antibody or the antigen-binding fragment thereof is the 383-388 th of envelope glycoprotein B (gB) of herpes simplex virus-1 (HSV-1) described in SEQ ID NO: 10 At least one amino acid residue in the region consisting of amino acid residues and / or the remaining amino acid residues 386-391 of herpes simplex virus-2 (HSV-2) envelope glycoprotein B (gB) described in SEQ ID NO: 11 It specifically binds to at least one amino acid residue in the region consisting of groups.
- the anti-HSV gB monoclonal antibody or antigen-binding fragment thereof includes, for example, a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 4, and And a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and the SEQ ID NO: 8 And a light chain variable region comprising a light chain CDR3 consisting of the amino acid sequence of
- a polynucleotide encoding the monoclonal antibody or an antigen-binding fragment thereof, an expression vector comprising the polynucleotide and one or more regulatory sequences operably linked to the polynucleotide, and the expression vector There is provided a transformant comprising
- the polynucleotide may be a polynucleotide encoding a complete antibody, as well as a polynucleotide encoding an antigen-binding fragment comprising the amino acid sequence set forth in SEQ ID NO: 1-8, and one polynucleotide encoding a plurality of CDRs. It may be incorporated into a polynucleotide. Such polynucleotides can be obtained, for example, by chemical synthesis.
- An expression vector comprising a polynucleotide encoding the anti-HSV gB monoclonal antibody or an antigen-binding fragment thereof of the present embodiment and one or more regulatory sequences operably linked to the polynucleotide is not particularly limited. In cells, it may be a vector capable of replicating and / or expressing a polynucleotide. For example, pCAG, pET and the like can be mentioned.
- the regulatory sequence is a sequence (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.) which controls the expression of a polynucleotide in a host cell, and can be appropriately selected depending on the type of host.
- the expression vector is operably linked to an appropriate promoter so that the polynucleotide can be expressed in a host cell, and may be a vector containing a selection marker. Furthermore, polynucleotides encoding each antigen binding fragment comprising the amino acid sequence set forth in SEQ ID NO: 1 to 8, for example, a polynucleotide encoding a heavy chain variable region and a polynucleotide encoding a light chain variable region are separately provided. It may be expressed in a vector.
- the host cell is not particularly limited as long as it can express an antibody, for example, mammalian cells such as human, Chinese hamster, mouse, rat, rabbit, pig, monkey, goat, and horse, plant cells, yeast cells And insect cells or bacterial cells.
- mammalian cells such as human, Chinese hamster, mouse, rat, rabbit, pig, monkey, goat, and horse
- plant cells yeast cells And insect cells or bacterial cells.
- the method of transformation of host cells is not particularly limited, and conventionally known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method and the like can be suitably used.
- a monoclonal antibody or an antigen binding fragment thereof can be produced, for example, by a known genetic engineering method.
- the monoclonal antibody or antigen binding fragment thereof can be produced, for example, by expressing in a host cell a vector containing a polynucleotide encoding the above-described anti-HSV gB monoclonal antibody or antigen binding fragment thereof.
- the polynucleotide can be appropriately designed by those skilled in the art by human antibody, humanized antibody or chimeric antibody.
- the heavy chain and light chain variable regions shown in SEQ ID NOS: 1 and 2, or heavy chain CDRs 1-3 and light chain CDRs 1-3 shown in SEQ ID NOs: 3-8. can further comprise at least a portion of a polynucleotide encoding the amino acid sequence of a portion other than these portions of the human antibody.
- a complete antibody it can be obtained by functionally linking the above-described variable region or polynucleotides encoding CDRs 1 to 3 and polynucleotides encoding other portions in the correct order.
- the polynucleotides encoding heavy and light chains may be incorporated into one polynucleotide or may be incorporated into separate polynucleotides. In addition, it may further contain a promoter for expression and a polynucleotide encoding a tag necessary for confirmation of expression. In the case of a chimeric antibody or an antigen-binding fragment thereof, the heavy chain and light chain variable regions shown in SEQ ID NOS: 1 and 2, or heavy chain CDRs 1-3 and light chain CDRs 1-3 shown in SEQ ID NOs: 3-8.
- the portion other than the encoding polynucleotide may include a polynucleotide encoding an amino acid sequence derived from an animal such as mouse or guinea pig.
- a further embodiment provides a pharmaceutical composition for preventing or treating HSV infection comprising an anti-HSV gB monoclonal antibody or an antigen-binding fragment thereof.
- HSV infections include infections due to HSV-1 and HSV-2, and include, for example, herpes labialis, corneal herpes, genital herpes, generalized neonatal herpes, and stomatitis due to HSV, skin disease, encephalitis, meninges And inflammation.
- Prevention prevents the onset of infection caused by administration to subjects who are at risk of HSV infection, in particular immunocompromised patients at high risk of HSV infection, or bone marrow transplant / blood stem cell transplant / organ transplant patients receiving immunosuppressants. Or it means to delay or alleviate the symptoms.
- treatment means alleviation of symptoms by administration to a subject who has already had HSV infection in the living body, improvement or complete cure, and also includes prevention of recurrence by administration to a subject who has recurrence of HSV recurrence.
- the pharmaceutical composition for treating HSV infection can be used as a substitute for or in combination with existing antiviral agents.
- the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like. These carriers may be used alone or in combination of two or more.
- compositions can be tablets, granules, capsules, injections, eye drops, inhalants, poultices.
- formulation agents therefor for example, lubricants, emulsifiers, preservatives, buffers, stabilizers, etc., may be added, as well as small amounts of auxiliary substances that increase the preservation or effectiveness of the antibody or antibody portion.
- Example 1 Preparation of Anti-HSV gB Monoclonal Antibody D48 scFv-hFc type, Fab type, for antibody D48 obtained by comprehensive search of anti-HSV-2 gB (HSV gB2) antibodies performed using a human antibody library Four molecular forms of antibody D48, human-mouse chimeric IgG2a type and human-guinea pig chimeric IgG22 type, were prepared and further analyzed.
- ⁇ ScFv-hFc> An isolated antibody D48 scFv (single chain Fv) gene encoding the amino acid sequence shown in SEQ ID NO: 9 is linked with an Fc gene (CH2-CH3) derived from human IgG1 and cloned into a pCAG vector to obtain an scFv-Fc expression plasmid Built.
- Fc gene CH2-CH3 derived from human IgG1
- the VH region of the isolated antibody D48 scFv gene was ligated with the CH1 gene derived from mouse IgG2a, and cloned into a pCAG vector to construct an H chain expression plasmid.
- the VL region of antibody D48 scFv gene was linked to a mouse CL gene, and cloned into a pCAG vector to construct an L chain expression plasmid.
- His tag gene is linked after CH1 gene (C-terminal side).
- FreeStyle293 or Expi293 expression system was used for expression. The expression plasmid was transfected into cells, and the culture supernatant was recovered in 4 to 6 days. The culture supernatant was purified using Ni NTA Agarose (QIAGEN) to obtain Fab.
- VH region of the isolated antibody D48 scFv gene was ligated with a heavy chain constant region gene (CH1-CH2-CH3) derived from mouse IgG2a, and cloned into a pCAG vector to construct a heavy chain expression plasmid.
- VL region of antibody D48 scFv gene was linked to a mouse CL gene, and cloned into a pCAG vector to construct an L chain expression plasmid.
- FreeStyle293 or Expi293 expression system was used for expression.
- the expression plasmid was transfected into cells, and the culture supernatant was recovered in 4 to 6 days. The culture supernatant was purified using Hi Trap Protein A HP Column (GE Healthcare) to obtain human-mouse chimeric IgG2a.
- the VH region of the isolated antibody D48 scFv gene was ligated with an H chain constant region gene (CH1-CH2-CH3) derived from guinea pig IgG2, and cloned into a pCAG vector to construct an H chain expression plasmid.
- the VL region of antibody D48 scFv gene was linked to a mouse CL gene, and cloned into a pCAG vector to construct an L chain expression plasmid.
- FreeStyle293 or Expi293 expression system was used for expression.
- the expression plasmid was transfected into cells, and the culture supernatant was recovered in 4 to 6 days. The culture supernatant was purified using Hi Trap Protein A HP Column to obtain human-guinea pig chimeric IgG2 ⁇ .
- the Fab was diluted to 2 ⁇ g / mL in PBS, 100 ⁇ L was placed in a maxi sorp plate (Nunc), and the Fab was immobilized by incubating for 2 hours at room temperature. After immobilization, the plate is washed with PBS, serially diluted from 1 ⁇ g / mL to 3.16 times 0.316 ng / mL, and 100 ⁇ L is added to the wells of the recombinant HSV-1 gB (gB1-705-strep) plate And incubated at 37 ° C.
- the cells were washed with PBST, and 100 ⁇ L of detection antibody anti-strep-Tactin / HRP (Funakoshi) was added to the wells of the plate and incubated at 37 ° C. After 1 hour, it was washed with PBST and developed by adding 100 ⁇ L of TMB (3,3 ', 5,5'-tetramethylbenzidine) to the wells of the plate. After 30 minutes, the reaction was stopped with 1N sulfuric acid, and the color development value (OD 450 nm / 650 nm) was measured with a microplate reader (Molecular Device).
- the recombinant gB (gB1-705-strep) used was a Strep tag II at the C-terminus of the wild type gB ectodomain from strain 333 of HSV-2 consisting of amino acid residues 1 to 705 of the amino acid sequence shown in SEQ ID NO: 11 These tags were used for purification and detection with ELISA.
- recombinant gB (gB1-705-strep) showed specific binding to antibody D48 Fab.
- ⁇ Reactivity analysis of human-mouse chimeric IgG2a> The binding activity of the obtained human-mouse chimeric IgG2a was evaluated by ELISA.
- the recombinant gB (gB1-705-strep) was immobilized by diluting the recombinant gB (gB1-705-strep) to 1 ⁇ g / mL with PBS, adding 100 ⁇ L to a MaxiSorp plate and incubating for 1 hour at room temperature.
- the plate was washed with PBS, serially diluted from 1 ⁇ g / mL to 3.16 times 0.316 ng / mL, and 100 ⁇ L of human-mouse chimeric IgG2a was added to the plate wells and incubated at 37 ° C. .
- the cells were washed with PBST, and 100 ⁇ L of detection antibody rabbit anti-mouse IgG / HRP (invitrogen) was added to the wells of the plate and incubated at 37 ° C.
- the cells were washed with PBST and developed by adding 100 ⁇ L of TMB to the wells of the plate. After 30 minutes, the reaction was stopped with 1N sulfuric acid, and the color development value (OD 450 nm / 650 nm) was measured with a microplate reader.
- human-mouse chimeric IgG2a showed specific binding to recombinant gB (gB1-705-strep).
- Example 3 Reactivity Analysis of Anti-HSV gB Antibody by Immunoblot gB1-705-strep was electrophoresed by injecting 2 ⁇ g / lane into a gel for 8-16% by weight SDS-PAGE. After electrophoresis, the gel was transferred to a nitrocellulose membrane (Immobilon-P, MILLIPORE) and blocked with 2% skimmed milk (Wako) -PBST.
- a nitrocellulose membrane Immobilon-P, MILLIPORE
- the blocked nitrocellulose membrane was reacted with 2% skimmed milk-PBST and 10 ⁇ g / mL of scFv-hFc, Fab, human-mouse chimeric IgG2a, or human-guinea pig chimeric IgG2 ⁇ for 60 minutes at room temperature after washing with PBST .
- the nitrocellulose membrane is anti-hIgG (H + L) / HRP (BIORAD), anti-His tag / HRP (R & D), anti-mouse IgG (H + L) / HRP, or anti in IgG 2% skimmed milk-PBST, respectively.
- Native gB1-705-strep, modified gB1-705-strep, and reduced / modified gB1-705-strep were prepared.
- the reduced / modified gB1-705-strep was obtained by adding 1 M DTT and boiling at 96 ° C. for 5 minutes.
- Denatured gB1-705-strep was obtained by boiling at 96 ° C. for 5 minutes.
- Native gB1-705-strep has not performed these operations.
- the prepared native gB1-705-strep, modified gB1-705-strep and reduced / denatured gB1-705-strep were directly injected into the gel.
- Lanes 1 to 3 are, in order, native gB1-705-strep, modified gB1-705-strep, and reduced / modified gB1-705-strep, and lanes M and M 'for BenchMark prestained Ladder and Magic Western standard, respectively. there were.
- the photograph on the left is SDS-PAGE, and CBB staining was performed for band staining.
- the four photos on the right were the results of Western blot (WB). As can be seen from FIG.
- the original scFv-hFc had an interesting feature of strongly reacting to the reduced / modified gB1-705-strep as compared to the native gB1-705-strep, but the molecule This feature was also inherited in the form of modified forms of Fab, human-mouse chimeric IgG2a and human-guinea pig chimeric IgG2 ⁇ .
- An antibody that reacts strongly to a denatured antigen is considered to be highly likely to recognize an epitope of linear sequence. Therefore, from this result, it is inferred that antibody D48 recognizes a linear sequence of gB.
- the sensor chip was CM5 (GE Healthcare), and gB1-705-strep was immobilized for about 100 resonance units (RU).
- the measurements are performed in single cycle kinetics mode and are 128 nM, 64 nM, 32 nM, 16 nM, 8 nM for scFv-hFc, 64 nM, 32 nM, 16 nM, 8 nM, 4 nM for Fab, human-mouse chimeric IgG2a, human-guinea pig chimeric IgG2 kappa
- the monoclonal antibody E31 and the monoclonal antibody F67 were used after diluting 256 times, 128 times, 64 times, 32 times and 16 times. All samples were measured only once.
- the monoclonal antibody E31 and the monoclonal antibody F67 are both antibodies that recognize domain IV of gB.
- Example 5 Epitope Analysis by Alanine Scanning
- 187 clones in which charged amino acids contained in gB1-705 were substituted with alanine were prepared, and the reactivity of antibody D48 with scFv-hFc was confirmed.
- a gene in which each of the charged amino acid residues (187 points) in gB1-705 was changed to alanine was constructed by PCR and cloned into pCAGGS1-dhfr-neo.
- FreeStyle293 or Expi293 expression system was used for expression.
- the expression amount of the obtained gB alanine substitution product and the binding activity of the alanine substitution product and the antibody fragment were evaluated by ELISA.
- the culture supernatant containing the gB alanine substitution was placed in a MaxiSorp plate, and the gB alanine substitution was immobilized by incubating for 1 hour at room temperature. After immobilization, the plate was washed with PBST, and 100 ⁇ L of detection antibody StrepTactin / HRP (IBA) was added to the plate wells and incubated at room temperature. After 1 hour, the cells were washed with PBST and developed by adding 100 ⁇ L of TMB to the wells of the plate. After 30 minutes, the reaction was stopped with 1N sulfuric acid, and the color development value (OD 450 nm / 650 nm) was measured with a microplate reader to determine the expression level.
- IBA detection antibody StrepTactin / HRP
- the culture supernatant containing the gB alanine substitute was put in a Streptactin (IBA) -immobilized MaxiSorp plate, and the gB alanine substitute was immobilized by incubating at room temperature for 1 hour. After immobilization, the plate was washed with PBST, and 100 ⁇ L of antibody fragment was added to the plate wells and incubated at room temperature. After 1 hour, the cells were washed with PBST, and 100 ⁇ L of detection antibody anti-human Fc / HRP (Cosmo Bio) was added to the wells of the plate and incubated at room temperature.
- IBA Streptactin
- the cells were washed with PBST and developed by adding 100 ⁇ L of TMB to the wells of the plate. After 30 minutes, the reaction was stopped with 1 N sulfuric acid, and the color development value (OD 450 nm / 650 nm) was measured with a microplate reader to determine the binding activity. Epitope candidates were selected depending on whether the reactivity per expression level was changed as compared to wild type gB1-705 not substituted with alanine.
- R391 on domain II of gB is an important amino acid residue as an epitope.
- Example 6 Epitope Analysis by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Alanine scanning is analysis targeting only hydrophilic residues, and it is possible that other non-hydrophilic residues are epitopes. Well expected.
- HDX-MS hydrogen / deuterium exchange mass spectrometry
- HDX-MS has become a well-established and established method that has been widely applied in various fields, and in particular, in epitope analysis of antibodies, it is important in that conformational epitopes can be analyzed while maintaining the three-dimensional structure in solution.
- the experimental method was as follows. Equal amounts of 120 ⁇ M gB1-705 and 400 ⁇ M antibody D48 Fab were mixed to prepare gB1-705 / antibody D48 Fab complex. This complex was prepared in PBS (pH 7.2) and subjected to H / D exchange reaction at 20 ° C. using PBS (pD 7.2) as a deuterium buffer. After the reaction, Quench buffer (100 mM sodium phosphate, 150 mM sodium chloride, 4 M guanidine hydrochloride, 150 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP)) was added to the complex to adjust to pH 2.4. .
- TCEP (2-carboxyethyl) phosphine hydrochloride
- the complex was converted to peptide fragments using a pepsin column and analyzed by LC-MS. Cleavage and elution conditions in this case were: Injected sample: 75 pmol, pepsin digestion + desalting time: 6 minutes, LC gradient condition: 9 minutes. Under these conditions, the peptide common to three determinations of gB1-705 and gB1-705 / antibody D48 Fab complex was 192 and the sequence coverage was 99.2%.
- the region consisting of I383-R388 of HSV-1 gB described in SEQ ID NO: 10 corresponding to I386-R391 of HSV-2 gB described in SEQ ID NO: 11 is highly likely to be an epitope of antibody D48. It is considered high.
- I386-R391 forms an ⁇ -helix located on the solvent surface of gB domain II, which is a site accessible to antibodies. Also, although the hit criteria could not be achieved, a difference was also detected in the deuterium exchange rate for L416-L422. This L deuterium exchange rate difference is considered to be affected by the structural change, since L416-L422 is located at the inner side of the structure from I386-R391 and is present at a site that is difficult to be exposed to the solvent.
- Non-patent Document 18 Non-patent Document 18
- Example 7 Virus Neutralization Test ⁇ Culturing of Cells and Virus> Vero cells (CCL. 81) purchased from ATCC were used for virus culture, infectivity titer measurement, and neutralizing antibody titer measurement. Vero cells were cultured at 37 ° C., 5% CO 2 conditions. At the time of expansion, maintenance, and analysis plate preparation, MEM medium containing 10% FBS was used, and at the time of infectivity measurement and neutralizing antibody titer measurement, MEM medium containing 2% FBS was used.
- the neutralization test was performed using two methods of plaque number reduction activity (plaque reduction activity) measurement and cell to cell infection spread suppression activity measurement. Two target viruses were used: HSV-2 MS strain and HSV-1 KOS strain.
- the test antibody was prepared to a predetermined concentration, mixed with about 100 PFU of HSV-2 MS strain or HSV-1 KOS strain, and then reacted at 37 ° C. for 1 hour.
- the reaction solution is seeded on a full sheet of Vero cells in a 48-well plate, adsorbed for 1 hour at 30 ° C., cultured for 24 hours in 1% methylcellulose-containing MEM (2% FBS) medium, 50% methanol / 50% ethanol ( Inactivation and fixation were carried out at -20 ° C for 30 minutes at -20 ° C. Then, an anti-HSV gB monoclonal antibody is reacted at 37 ° C.
- Cell-to-cell infection expansion inhibitory activity measurement is performed by inoculating approximately 100 PFU of HSV-2 MS strain or HSV-1 KOS strain into a 48-well plate in full sheet Vero cells, and adsorbing at 30 ° C for 1 hour, and then predetermined concentration 1% methylcellulose-containing MEM (2% FBS) medium (antibody concentrations are 5, 25 and 125 ⁇ g / mL) containing the test antibodies of the following test, HSV-2 MS strain is about 40 hours, HSV-1 KOS strain is about After 48 hours of culture, inactivation and fixation were carried out with 50% methanol / 50% ethanol ( ⁇ 20 ° C.) at ⁇ 20 ° C. for 30 minutes.
- the self-prepared anti-HSV gB monoclonal antibody is reacted at 37 ° C. for 1 hour, immunostained with anti-mouse IgG / HRP and TMBH, the image of each well is taken up with ELISPOT analyzer, and the average plaque size is analyzed with analysis software It analyzed.
- the results are shown in Table 3.
- the scFv-hFc of antibody D48, Fab, human-mouse chimeric IgG2a, and human-guinea pig chimeric IgG2k are all less than or equal to 1 nM to both strains of KOS (HSV-1) and MS (HSV-2). It showed a strong 50% plaque number reducing activity.
- scFv-hFc of antibody D48 and human-mouse chimeric IgG2a exhibited potent 50% cell to cell infection spread-suppressing activity of 40 nM and 48 nM, respectively, against MS (HSV-2) strain.
- Example 8 Mouse Infection Protection Test ⁇ Test Method> Using a mouse genital herpes infection model, an infection protection test was carried out for preventive administration and therapeutic administration of anti-HSV gB monoclonal antibody (human-mouse chimeric IgG2a).
- BALB / c mice (5 weeks old, female) were used. A predetermined amount of antibody is dissolved in saline for injection and the antibody is administered to mice.
- prophylactic administration it was intraperitoneally administered at a volume of 200 ⁇ L / animal 24 hours before virus inoculation.
- therapeutic administration it was intraperitoneally administered at a volume of 200 ⁇ L / animal 48 hours after virus inoculation.
- the number of cases of N 10 was set per group.
- Depo-Provera was subcutaneously inoculated at 2 mg / animal 6 days before virus inoculation. Under anesthesia, 5 ⁇ 10 5 PFU / 20 ⁇ L of HSV-2 MS strain was intravaginally inoculated, and 21 days follow-up was performed. The ability to protect against infection was shown using the survival days (survival rate) and symptom scores as indicators. The symptom score defined the score by the presence or absence and the degree of the vaginal lesion symptom, and showed the average value in each group.
- the scoring method was 0: no change, 1: partial erythema / swelling, 2: extensive swelling / edema, 3: ulcer / bleeding, 4: death. If there are serious systemic symptoms with no hope of recovery (such as pipils, paralysis, tremors, convulsions, etc.), score that day as 3.5, sacrifice to death, and treat it as death the next day. It was four.
- antibody D48 showed a remarkable effect of prolonging the survival day at 30 ⁇ g / animal / times and 3 ⁇ g / animal / times, and even 0.3 ⁇ g / animal / times showed a significant effect of survival day In contrast, both F67 and E31 showed only limited effects. From this result, antibody D48 has remarkable infection protective activity not only in vitro but also in vivo as compared to other anti-HSV gB2 antibodies, and its superiority may be based on the difference in epitope region and affinity. Was suggested.
- the results of prophylactic administration are shown in Table 6 (survival days by dose), FIG. 6 (survival rate) and FIG. 7 (symptom score).
- the results of therapeutic administration are shown in Table 7 (survival days by dose), FIG. 8 (survival rate) and FIG. 9 (symptom score).
- Prophylactic administration at all set doses (10 mg / kg, 3 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.3 mg / kg) compared to the negative control group (saline administration group) It showed a significant survival time extension effect.
- two doses of 10 mg / kg and 3 mg / kg, which are high dose ranges, showed remarkable improvement in survival rate and symptom score.
- HSV has been reported to migrate to ganglia within 48 hours of entry into the body from a local infection.
- therapeutic administration of antibody D48 at 48 hours after infection also showed remarkable improvement in survival rate and symptom score at two doses of 10 mg / kg and 3 mg / kg. From these results, it was confirmed that the antibody D48 exhibits a strong infection protective effect not only in prophylactic administration but also in therapeutic administration.
- Example 9 Guinea Pig Infection Protection Test ⁇ Test Method> The guinea pig genital herpes infection model was used to perform an infection protection test in prophylactic and therapeutic administration of anti-HSV gB monoclonal antibody D48 (human-guinea pig chimeric IgG2k). Hartley guinea pigs (3-5 weeks old, female) purchased from SLC were used. A predetermined amount of antibody is dissolved in saline for injection (saline), and 1 mg / kg each for 24 hours before virus inoculation for prophylactic administration and 4 days after virus inoculation for therapeutic administration. It was administered intraperitoneally at a volume of ⁇ 30 mg / kg.
- vaginal swab vaginal swab
- the vaginal Swab was collected by wiping the mucous membrane on the inner wall of the vagina after inserting a swab moistened with MEM medium into the vagina.
- vaginal Swab was suspended in MEM medium in 1 mL aliquots in siliconized tubes, and stored frozen until use.
- Vaginal swab was diluted 10 times, 100 times and 1000 times with the stock solution at the time of measurement of virus release, and 100 ⁇ L / well was used to inoculate Vero cells in 96 or 48 wells as full sheet.
- virus adsorption was performed at 37 ° C. for 1 hour, and after culturing for 24 to 72 hours in 2% FBS MEM medium containing 1% methyl cellulose, the number of plaques was counted by a predetermined method.
- HSV infection can be applied for prevention and / or treatment, and in particular, as a preventive drug for immunocompromised patients at high risk of HSV infection or bone marrow transplant / blood stem cell transplant / organ transplant patients receiving immunosuppressant, or It can be used as a therapeutic agent for patients with recurrent HSV recurrence as a substitute for or in combination with existing antiviral agents.
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Abstract
Description
(1)単純ヘルペスウイルス(HSV)のエンベロープ糖タンパク質B(gB)に特異的に結合する、抗HSV gBモノクローナル抗体又はその抗原結合断片であって、
配列番号3に記載のアミノ酸配列からなる重鎖CDR1、配列番号4に記載のアミノ酸配列からなる重鎖CDR2、及び配列番号5に記載のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域と、
配列番号6に記載のアミノ酸配列からなる軽鎖CDR1、配列番号7に記載のアミノ酸配列からなる軽鎖CDR2、及び配列番号8に記載のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域と
を含む、抗HSV gBモノクローナル抗体又はその抗原結合断片。
(2)配列番号1に記載のアミノ酸配列を含む重鎖可変領域と、配列番号2に記載のアミノ酸配列を含む軽鎖可変領域と、を含む、上記(1)の抗HSV gBモノクローナル抗体又はその抗原結合断片。
(3)HSVが、HSV-1又はHSV-2である、上記(1)又は(2)の抗HSV gBモノクローナル抗体又はその抗原結合断片。
(4)上記(1)~(3)のいずれかの抗HSV gBモノクローナル抗体又はその抗原結合断片をコードするポリヌクレオチド。
(5)上記(4)のポリヌクレオチドと、当該ポリヌクレオチドに作動可能に連結された1又は複数の調節配列とを含む発現ベクター。
(6)上記(5)の発現ベクターが導入された形質転換体。
(7)上記(4)のポリヌクレオチド、上記(5)の発現ベクター、上記(6)に記載の形質転換体を用いて、上記(1)~(3)のいずれかの抗HSV gBモノクローナル抗体又はその抗原結合断片を製造する方法。
(8)上記(1)又は(2)の抗HSV gBモノクローナル抗体又はその抗原結合断片を含む、HSV感染症を予防又は治療するための医薬組成物。
(9)HSV感染症が、HSV-1感染症又はHSV-2感染症である、上記(8)の医薬組成物。
(10)HSV感染症は、口唇ヘルペス、角膜ヘルペス、性器ヘルペス、全身性の新生児ヘルペス、並びに、HSVに起因する口内炎、皮膚疾患、脳炎、髄膜炎、及び脊髄炎からなる群より選択されるものである、上記(8)又は(9)の医薬組成物。
(11)配列番号10に記載の単純ヘルペスウイルス-1(HSV-1)のエンベロープ糖タンパク質B(gB)の383-388番目のアミノ酸残基からなる領域における少なくとも1つのアミノ酸残基、及び/又は、配列番号11に記載の単純ヘルペスウイルス-2(HSV-2)のエンベロープ糖タンパク質B(gB)の386-391番目のアミノ酸残基からなる領域における少なくとも1つのアミノ酸残基に特異的に結合する、抗HSV gBモノクローナル抗体又はその抗原結合断片。
(12)配列番号3に記載のアミノ酸配列からなる重鎖CDR1、配列番号4に記載のアミノ酸配列からなる重鎖CDR2、及び配列番号5に記載のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域と、
配列番号6に記載のアミノ酸配列からなる軽鎖CDR1、配列番号7に記載のアミノ酸配列からなる軽鎖CDR2、及び配列番号8に記載のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域と
を含む、上記(11)の抗HSV gBモノクローナル抗体又はその抗原結合断片。
(13)配列番号1に記載のアミノ酸配列を含む重鎖可変領域と、
配列番号2に記載のアミノ酸配列を含む軽鎖可変領域と、
を含む、上記(12)の抗HSV gBモノクローナル抗体又はその抗原結合断片。
(14)上記(11)~(13)のいずれかの抗HSV gBモノクローナル抗体又はその抗原結合断片をコードするポリヌクレオチド。
(15)上記(14)のポリヌクレオチドと、当該ポリヌクレオチドに作動可能に連結された1又は複数の調節配列とを含む発現ベクター。
ヒト抗体ライブラリーを用いて実施した抗HSV-2 gB(HSV gB2)抗体の網羅的探索によって、取得した抗体D48について、scFv-hFc型、Fab型、ヒト-マウスキメラIgG2a型及びヒト-モルモットキメラIgG2κ型の4種類の分子形態の抗体D48を作製し、さらに解析を行った。
配列番号9に示すアミノ酸配列をコードする単離した抗体D48 scFv(single chain Fv)遺伝子をヒトIgG1に由来するFc遺伝子(CH2-CH3)と連結し、pCAGベクターにクローニングし、scFv-Fc発現プラスミドを構築した。発現には、FreeStyle293又はExpi293発現システム(ライフテクノロジー社)を用いた。発現プラスミドを細胞にトランスフェクションし、4~6日で培養上清を回収した。培養上清をAb-Rapid PuRe 10(ProteNova)又はAb-Rapid PuRe Ex(ProteNova)を用いて精製し、scFv-hFcを得た。
単離した抗体D48 scFv遺伝子のVH領域をマウスIgG2aに由来するCH1遺伝子と連結し、pCAGベクターにクローニングし、H鎖発現プラスミドを構築した。また、抗体D48 scFv遺伝子のVL領域をマウスCL遺伝子と連結し、pCAGベクターにクローニングし、L鎖発現プラスミドを構築した。精製を容易にするため、CH1遺伝子の後(C末端側)にHis tag遺伝子を連結している。発現には、FreeStyle293又はExpi293発現システムを用いた。発現プラスミドを細胞にトランスフェクションし、4~6日で培養上清を回収した。培養上清をNi NTA Agarose(QIAGEN)を用いて精製し、Fabを得た。
単離した抗体D48 scFv遺伝子のVH領域をマウスIgG2aに由来するH鎖定常領域遺伝子(CH1-CH2-CH3)と連結し、pCAGベクターにクローニングし、H鎖発現プラスミドを構築した。また、抗体D48 scFv遺伝子のVL領域をマウスCL遺伝子と連結し、pCAGベクターにクローニングし、L鎖発現プラスミドを構築した。発現には、FreeStyle293又はExpi293発現システムを用いた。発現プラスミドを細胞にトランスフェクションし、4~6日で培養上清を回収した。培養上清をHi Trap ProteinA HP Column(GEヘルスケア)を用いて精製し、ヒト-マウスキメラIgG2aを得た。
単離した抗体D48 scFv遺伝子のVH領域をモルモットIgG2に由来するH鎖定常領域遺伝子(CH1-CH2-CH3)と連結し、pCAGベクターにクローニングし、H鎖発現プラスミドを構築した。また、抗体D48 scFv遺伝子のVL領域をマウスCL遺伝子と連結し、pCAGベクターにクローニングし、L鎖発現プラスミドを構築した。発現には、FreeStyle293又はExpi293発現システムを用いた。発現プラスミドを細胞にトランスフェクションし、4~6日で培養上清を回収した。培養上清をHi Trap ProteinA HP Columnを用いて精製し、ヒト-モルモットキメラIgG2κを得た。
<Fabの反応性解析>
取得したFabの結合活性はELISAによって評価した。FabをPBSで2μg/mLに希釈し、MaxiSorp plate(Nunc)に100μL入れ、2時間室温でインキュベートすることによってFabを固相化した。固相化後、プレートをPBSで洗浄し、1μg/mLから3.16倍ずつ0.316ng/mLまで段階希釈して組換えHSV-1 gB(gB1-705-strep)プレートのウェルに100μL加え、37℃でインキュベーションした。1時間後、PBSTで洗浄し、検出抗体抗strep-Tactin/HRP(フナコシ)をプレートのウェルに100μL加え、37℃でインキュベーションした。1時間後、PBSTで洗浄し、TMB(3,3’,5,5’-テトラメチルベンジジン)をプレートのウェルに100μL加えることによって発色させた。30分後、1N硫酸で反応を停止させ、マイクロプレートリーダー(モレキュラーデバイス)で発色値(O.D.450nm/650nm)を測定した。使用した組換えgB(gB1-705-strep)は、配列番号11に示すアミノ酸配列の1~705アミノ酸残基からなるHSV-2の333株由来の野生型gBのエクトドメインのC末端にStrep tagIIを結合したものであり、このタグを用いて精製やELISAでの検出を行った。
取得したヒト-マウスキメラIgG2aの結合活性はELISAによって評価した。組換えgB(gB1-705-strep)をPBSで1μg/mLに希釈し、MaxiSorp plateに100μL入れ、1時間室温でインキュベートすることによって組換えgB(gB1-705-strep)を固相化した。固相化後、プレートをPBSで洗浄し、1μg/mLから3.16倍ずつ0.316ng/mLまで段階希釈して、ヒト-マウスキメラIgG2aをプレートのウェルに100μL加え、37℃でインキュベーションした。1時間後、PBSTで洗浄し、検出抗体ウサギ抗マウスIgG/HRP(invitrogen)をプレートのウェルに100μL加え、37℃でインキュベーションした。1時間後、PBSTで洗浄し、TMBをプレートのウェルに100μL加えることによって発色させた。30分後、1N硫酸で反応を停止させ、マイクロプレートリーダーで発色値(O.D.450nm/650nm)を測定した。
gB1-705-strepを8-16重量%SDS-PAGE用ゲルに2μg/レーン注入し電気泳動した。電気泳動後、ゲルをニトロセルロース膜(Immobilon-P、MILLIPORE)へ転写し、2%スキムミルク(Wako)-PBSTを用いてブロッキングした。ブロッキングしたニトロセルロース膜は、PBSTによる洗浄後、2%スキムミルク-PBST、並びに、10μg/mLのscFv-hFc、Fab、ヒト-マウスキメラIgG2a、又はヒト-モルモットキメラIgG2κと室温で60分間反応させた。再度の洗浄の後、ニトロセルロース膜は、2%スキムミルク-PBST中でそれぞれ抗hIgG(H+L)/HRP(BIORAD)、抗Hisタグ/HRP(R&D)、抗マウスIgG(H+L)/HRP、又は抗モルモット(H+L)/HRP(Invitrogen)と反応させ、Immobilon Western Detection Regent(Millipore)で発色させた。未変性gB1-705-strep、変性gB1-705-strep、及び、還元・変性gB1-705-strepを作成した。還元・変性gB1-705-strepは1M DTTを加え、96℃で5分間煮沸させることで得た。変性gB1-705-strepは、96℃で5分間煮沸することで得た。未変性gB1-705-strepはこれらの操作を行っていない。作成した未変性gB1-705-strep、変性gB1-705-strep、及び、還元・変性gB1-705-strepを直接ゲルに注入した。
Biacore T200(GE Healthcare)を使用して行われた。すべての実験においてHBS-EP+bufferを使用し、温度は25℃、流速は30μL/分に設定した。センサーチップはCM5(GE Healthcare)を使用し、gB1-705-strepを約100レゾナンスユニット(RU)固相化した。測定はシングルサイクルカイネティクスモードで実施し、scFv-hFcについては128nM、64nM、32nM、16nM、8nM、Fabについては64nM、32nM、16nM、8nM、4nM、ヒト-マウスキメラIgG2a、ヒト-モルモットキメラIgG2κ、モノクローナル抗体E31、モノクローナル抗体F67については256倍、128倍、64倍、32倍、16倍に希釈して使用した。すべてのサンプルは一回のみ測定した。モノクローナル抗体E31とモノクローナル抗体F67はともにgBのドメインIVを認識する抗体である。
抗体D48のエピトープを同定するためにgB1-705に含まれる荷電アミノ酸をアラニンに置換した187クローンを作製し、抗体D48のscFv-hFcとの反応性を確認した。
アラニンスキャングは親水性残基のみをターゲットとした解析であり、その他の非親水性残基がエピトープであるという可能性が十分に予想された。
<細胞及びウイルスの培養>
ウイルスの培養、感染価測定、中和抗体価測定にはATCCから購入したVero細胞(CCL.81)を使用した。Vero細胞は、37℃、5%CO2条件下で培養した。拡張、維持、解析プレート作製時は、10%FBS含有MEM培地を使用し、感染価測定及び中和抗体価測定時は、2%FBS含有MEM培地を使用した。中和試験及び感染防御能解析に用いるウイルスバンクはATCCから購入した、Human herpesvirus 2(HSV-2)MS株(VR-540)及びHuman herpesvirus 1(HSV-1)KOS株(VR-1493)をm.o.i=0.01~1でフルシートのVero細胞に接種し、2~3日間2%FBS含有MEM培地で培養した。回収した感染細胞培養ボトルを3回凍結融解して細胞を破砕後、TOMY遠心器で室温にて3500rpm、10分遠心し、上清をHSV-2ウイルスバンク及びHSV-1ウイルスバンクとした。
中和試験はプラーク数減少活性(プラークリダクション活性)測定とcell to cell感染拡大抑制活性測定の2種類の方法を用いて行った。対象とするウイルスはHSV-2 MS株とHSV-1 KOS株の2種を用いた。
<試験方法>
マウス性器ヘルペス感染モデルを用いて、抗HSV gBモノクローナル抗体(ヒト-マウスキメラIgG2a)の予防的投与及び治療的投与における感染防御試験を実施した。BALB/cマウス(5週齢、メス)を用いた。所定量の抗体を注射用生理食塩水(saline)に溶解してマウスに抗体を投与する。予防的投与の場合にはウイルス接種24時間前に200μL/匹の容量にて腹腔内投与した。治療的投与の場合にはウイルス接種48時間後に、200μL/匹の容量にて腹腔内投与した。1群あたりN=10の例数を設定した。ウイルス接種時の感染効率を向上させるために、ウイルス接種6日前にDepo-Proveraを2mg/匹で皮下接種した。麻酔下で5×105PFU/20μLのHSV-2 MS株を経腟接種し、21日間経過観察を行った。生存日数(生存率)及び症状スコアを指標に感染防御能を示した。症状スコアは、膣病変症状の有無及び程度によってスコアを定義し各群における平均値を示した。スコアの付け方として、0:変化なし、1:部分的な紅斑・腫脹、2:広範囲の腫脹・浮腫、3:潰瘍・出血、4:死亡、とした。回復の見込みのない重篤な全身症状(立毛、麻痺、震戦、痙攣など)が認められた場合、その日はスコアを3.5とし、犠牲死させ、次の日に死亡として扱ってスコアを4とした。
表4に示す3種類の抗HSV gB2抗体D48、F67及びE31(何れもヒト-マウスキメラIgG2a)に関して、それぞれのマウス感染防御能を評価し比較した。抗体D48がHSV gBのドメインII領域にエピトープを有し強力なウイルス中和活性(50%プラーク数減少活性)を有する抗体であるのに対し、F67及びE31は共にドメインIV領域にエピトープを有し、前者がウイルス中和活性を有さず、後者は中程度のウイルス中和活性を有していた。
マウス性器ヘルペス感染モデル(n=10)を用いて、抗体D48(ヒト-マウスキメラIgG2a)の予防的投与及び治療的投与における感染防御試験結果を示す。予防的投与の結果は、表6(投与量別の生存日数)、図6(生存率)及び図7(症状スコア)に示す。治療的投与の結果は、表7(投与量別の生存日数)、図8(生存率)及び図9(症状スコア)に示す。
<試験方法>
モルモット性器ヘルペス感染モデルを用いて、抗HSV gBモノクローナル抗体D48(ヒト-モルモットキメラIgG2k)の予防的投与及び治療的投与における感染防御試験を実施した。SLC社から購入したHartleyモルモット(3~5週齢、メス)を用いた。所定量の抗体を注射用生理食塩水(saline)に溶解し、予防的投与の場合にはウイルス接種24時間前に、また治療的投与の場合にはウイルス接種4日間後に、何れも1mg/kg~30mg/kgの容量にて腹腔内投与した。治療的投与の場合は、投与前に症状観察を行い、膣症状を呈している個体を選別し、各群の平均スコアに偏りが生じない様にランダマイズした。各群の動物例数を予防的投与ではN=9に設定し、治療的投与ではN=15に設定した。ウイルス接種は麻酔下で5×105PFU/50μLのHSV-2 MS株を経腟接種し、急性期症状を接種後2~3週間観察した。症状スコアは、0:明確な病変なし、0.5-1:紅斑、1.5-2:限局的な水泡、2.5-3:限局的な潰瘍又は痂皮、3-5:広範に及ぶ水泡・潰瘍又は痂皮、3-7:失禁を伴う広範な潰瘍又は痂皮、7.5:重篤な症状による安楽殺、8:死亡、とした。また、ウイルス接種後7日目に、膣拭い液(膣swab)を採取し、プラーク法によってウイルス放出量を測定した。膣Swabは、MEM培地で湿潤させた綿棒を膣内に挿入後、膣内壁の粘膜を拭い取るようにして採取した。採取した膣Swabは、シリコナイズドチューブに1mLずつ分注したMEM培地にて懸濁し、使用時まで凍結保存した。ウイルス放出量の測定時に膣swabを原液、10倍、100倍、1000倍希釈し、100μL/ウェルで96ウェル又は48ウェルにフルシートになったVero細胞に接種した。膣Swab接種後37℃で1時間ウイルス吸着を行い、1%メチルセルロース含有2%FBS MEM培地で24~72時間培養した後に、所定の方法でプラーク数を計測した。
モルモット性器ヘルペス感染モデル(急性期)を用いて、抗体D48(ヒト-モルモットキメラIgG2κ)の予防的投与の感染防御試験の症状スコアの結果を図10に、治療的投与感染防御試験の症状スコアの結果を図11に、治療的投与感染防御試験の膣拭い液中のHSV放出量を図12に示す。
Claims (15)
- 単純ヘルペスウイルス(HSV)のエンベロープ糖タンパク質B(gB)に特異的に結合する、抗HSV gBモノクローナル抗体又はその抗原結合断片であって、
配列番号3に記載のアミノ酸配列からなる重鎖CDR1、配列番号4に記載のアミノ酸配列からなる重鎖CDR2、及び配列番号5に記載のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域と、
配列番号6に記載のアミノ酸配列からなる軽鎖CDR1、配列番号7に記載のアミノ酸配列からなる軽鎖CDR2、及び配列番号8に記載のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域と
を含む、抗HSV gBモノクローナル抗体又はその抗原結合断片。 - 配列番号1に記載のアミノ酸配列を含む重鎖可変領域と、
配列番号2に記載のアミノ酸配列を含む軽鎖可変領域と、
を含む、請求項1に記載の抗HSV gBモノクローナル抗体又はその抗原結合断片。 - 前記HSVが、HSV-1又はHSV-2である、請求項1又は2に記載の抗HSV gBモノクローナル抗体又はその抗原結合断片。
- 請求項1~3のいずれか一項に記載の抗HSV gBモノクローナル抗体又はその抗原結合断片をコードするポリヌクレオチド。
- 請求項4に記載のポリヌクレオチドと、当該ポリヌクレオチドに作動可能に連結された1又は複数の調節配列とを含む発現ベクター。
- 請求項5に記載の発現ベクターが導入された形質転換体。
- 請求項4に記載のポリヌクレオチド、請求項5に記載の発現ベクター、又は請求項6に記載の形質転換体を用いて、請求項1~3のいずれか一項に記載の抗HSV gBモノクローナル抗体又はその抗原結合断片を製造する方法。
- 請求項1~3のいずれか一項に記載の抗HSV gBモノクローナル抗体又はその抗原結合断片を含む、HSV感染症を予防又は治療するための医薬組成物。
- 前記HSV感染症が、HSV-1感染症又はHSV-2感染症である、請求項8に記載の医薬組成物。
- 前記HSV感染症は、口唇ヘルペス、角膜ヘルペス、性器ヘルペス、全身性の新生児ヘルペス、並びに、HSVに起因する口内炎、皮膚疾患、脳炎、髄膜炎、及び脊髄炎からなる群より選択されるものである、請求項8又は9に記載の医薬組成物。
- 配列番号10に記載の単純ヘルペスウイルス-1(HSV-1)のエンベロープ糖タンパク質B(gB)の383-388アミノ酸残基からなる領域における少なくとも1つのアミノ酸残基、及び/又は、配列番号11に記載の単純ヘルペスウイルス-2(HSV-2)のエンベロープ糖タンパク質B(gB)の386-391アミノ酸残基からなる領域における少なくとも1つのアミノ酸残基に特異的に結合する、抗HSV gBモノクローナル抗体又はその抗原結合断片。
- 配列番号3に記載のアミノ酸配列からなる重鎖CDR1、配列番号4に記載のアミノ酸配列からなる重鎖CDR2、及び配列番号5に記載のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域と、
配列番号6に記載のアミノ酸配列からなる軽鎖CDR1、配列番号7に記載のアミノ酸配列からなる軽鎖CDR2、及び配列番号8に記載のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域と
を含む、請求項11に記載の抗HSV gBモノクローナル抗体又はその抗原結合断片。 - 配列番号1に記載のアミノ酸配列を含む重鎖可変領域と、
配列番号2に記載のアミノ酸配列を含む軽鎖可変領域と、
を含む、請求項12に記載の抗HSV gBモノクローナル抗体又はその抗原結合断片。 - 請求項11~13のいずれか一項に記載の抗HSV gBモノクローナル抗体又はその抗原結合断片をコードするポリヌクレオチド。
- 請求項14に記載のポリヌクレオチドと、当該ポリヌクレオチドに作動可能に連結された1又は複数の調節配列とを含む発現ベクター。
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WO2020191181A1 (en) * | 2019-03-19 | 2020-09-24 | Albert Einstein College Of Medicine | Monoclonal antibodies for prevention and treatment of herpes simplex viral infections |
WO2022087149A2 (en) | 2020-10-22 | 2022-04-28 | Gilead Sciences, Inc. | Interleukin-2-fc fusion proteins and methods of use |
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US11421002B2 (en) * | 2017-08-30 | 2022-08-23 | Km Biologics Co., Ltd. | Modified HSV gB protein and HSV vaccine including same |
WO2023003951A2 (en) * | 2021-07-20 | 2023-01-26 | Albert Einstein College Of Medicine | Compositions and methods for the treatment of herpes simplex virus infection |
CN117122676A (zh) * | 2022-05-27 | 2023-11-28 | 江苏瑞科生物技术股份有限公司 | Hsv免疫原性组合物及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6156313A (en) * | 1994-01-04 | 2000-12-05 | The Scripps Research Institute | Human monoclonal antibodies to herpes simplex virus and methods therefor |
JP2013506403A (ja) * | 2009-10-01 | 2013-02-28 | ユニバーシタット ダイズバーグ−エッセン | 抗hsv抗体 |
JP2017520572A (ja) * | 2014-06-26 | 2017-07-27 | ハイデルべルク イミュノセラピューティクス ゲーエムベーハー | 抗hsv抗体の外用適用 |
-
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- 2018-08-29 JP JP2019539595A patent/JP7145862B2/ja active Active
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6156313A (en) * | 1994-01-04 | 2000-12-05 | The Scripps Research Institute | Human monoclonal antibodies to herpes simplex virus and methods therefor |
JP2013506403A (ja) * | 2009-10-01 | 2013-02-28 | ユニバーシタット ダイズバーグ−エッセン | 抗hsv抗体 |
JP2017520572A (ja) * | 2014-06-26 | 2017-07-27 | ハイデルべルク イミュノセラピューティクス ゲーエムベーハー | 抗hsv抗体の外用適用 |
Non-Patent Citations (29)
Title |
---|
"Decision Resources", EMERGING VACCINES, 2008 |
ARII, J.: "Non-muscle myosin IIA is a functional entry receptor for herpes simplex virus-1", NATURE, vol. 467, 2010, pages 859 - 62 |
BENDER FC ET AL.: "Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions", J. VIROL., vol. 81, 2007, pages 3827 - 3841, XP002571808, DOI: 10.1128/JVI.02710-06 * |
BENDER FC: "Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions", J VIROL., vol. 81, no. 8, April 2007 (2007-04-01), pages 3827 - 41, XP002571808, DOI: 10.1128/JVI.02710-06 |
CAIRNS TM ET AL.: "Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans", J. VIROL., vol. 88, 2014, pages 12612 - 12622, XP055581609 * |
CHAUHAN V ET AL.: "Identification of broadly reactive epitopes targeting major glycoproteins of Herpes simplex virus (HSV) 1 and 2 - An immunoinformatics analysis", INFECT. GENET. EVOL., vol. 61, 3 July 2018 (2018-07-03), pages 24 - 35, XP055581611 * |
EISENBERG RJ: "Herpes virus fusion and entry: a story with many characters", VIRUSES, vol. 4, 2012, pages 800 - 832 |
GERAGHTY, R. J.: "Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor", SCIENCE, vol. 280, 1998, pages 1618 - 20, XP002114163, DOI: 10.1126/science.280.5369.1618 |
HASHIDO ML: "An epidemiologic study of herpes simplex virus type 1 and 2 infection in Japan based on type-specific serological assays", EPIDEMIOL INFECT., vol. 120, no. 2, March 1998 (1998-03-01), pages 179 - 86 |
HELDWEIN EE ET AL.: "Crystal structure of glycoprotein B from herpes simplex virus 1", SCIENCE, vol. 313, 2006, pages 217 - 220, XP002571810, DOI: 10.1126/science.1126548 * |
HEROLD, B. C.: "Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity", J VIROL, vol. 65, 1991, pages 1090 - 8 |
HEROLD, B. C.: "Glycoprotein C-independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein", B. J GEN VIROL, vol. 75, 1994, pages 1211 - 22 |
KRAWCZYK A: "mpact of valency of a glycoprotein B-specific monoclonal antibody on neutralization of herpes simplex virus", J VIROL., vol. 85, no. 4, February 2011 (2011-02-01), pages 1793 - 803, XP055372640, DOI: 10.1128/JVI.01924-10 |
MONTGOMERY, R. I.: "Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family", CELL, vol. 87, 1996, pages 427 - 36, XP002062424, DOI: 10.1016/S0092-8674(00)81363-X |
NAVARRO D ET AL.: "Domains of herpes simplex virus I glycoprotein B that function in virus penetration , cell -to- cell spread, and cell fusion", VIROLOGY, vol. 186, 1992, pages 99 - 112, XP023049309, DOI: 10.1016/0042-6822(92)90064-V * |
PEREIRA L ET AL.: "Domain structure of herpes simplex virus 1 glycoprotein B: neutralizing epitopes map in regions of continuous and discontinuous residues", VIROLOGY, vol. 172, 1989, pages 11 - 24, XP023047051, DOI: 10.1016/0042-6822(89)90102-5 * |
POTTAGE JC JR: "Herpes simplex virus resistance to acyclovir: clinical relevance", INFECT AGENTS DIS., vol. 4, no. 3, September 1995 (1995-09-01), pages 115 - 24 |
QADRI I ET AL.: "Mutations in conformation-dependent domains of herpes simplex virus 1 glycoprotein B affect the antigenic properties, dimerization, and transport of the molecule", VIROLOGY, vol. 180, 1991, pages 135 - 152, XP023046842, DOI: 10.1016/0042-6822(91)90017-6 * |
ROIZMAN, B.: "Herpes simplex viruses", LIPPINCOTT WILLIAMS &WILKINS, article "Fields Virology", pages: 2501 - 2602 |
SANCHEZ-PESCADOR L ET AL.: " "Epitopes of herpes simplex virus type 1 glycoprotein B that bind type-common neutralizing antibodies elicit type-specific antibody-dependent cellular cytotoxicity"", THE JOURNAL OF INFECTIOUS DISEASES, vol. 166, no. 3, 1 September 1992 (1992-09-01), pages 623 - 627, XP009519759, DOI: 10.1093/infdis/166.3.623 * |
SATOH, T.: "PILRalpha is a herpes simplex virus-1 entry coreceptor that associates with glycoprotein B", CELL, vol. 132, 2008, pages 935 - 44 |
SCIENCE, vol. 313, no. 14, 2006, pages 217 - 220 |
See also references of EP3677677A4 |
SHUKLA, D.: "A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry", CELL, vol. 99, 1999, pages 13 - 22, XP000982864, DOI: 10.1016/S0092-8674(00)80058-6 |
SUENAGA, T.: "Myelin-associated glycoprotein mediates membrane fusion and entry of neurotropic herpesviruses", PROC NATL ACAD SCI U S A, vol. 107, 2010, pages 866 - 71 |
VIROLOGY, vol. 172, no. 1, 1989, pages 11 - 24 |
VIROLOGY, vol. 180, no. 1, 1991, pages 135 - 152 |
VIROLOGY, vol. 186, no. 1, 1992, pages 99 - 112 |
VIRUS, vol. 60, no. 2, 2010, pages 187 - 196 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020191181A1 (en) * | 2019-03-19 | 2020-09-24 | Albert Einstein College Of Medicine | Monoclonal antibodies for prevention and treatment of herpes simplex viral infections |
CN113661176A (zh) * | 2019-03-19 | 2021-11-16 | 阿尔伯特爱因斯坦医学院 | 用于预防和治疗单纯疱疹病毒感染的单克隆抗体 |
WO2022087149A2 (en) | 2020-10-22 | 2022-04-28 | Gilead Sciences, Inc. | Interleukin-2-fc fusion proteins and methods of use |
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US20200239549A1 (en) | 2020-07-30 |
AU2018323503A1 (en) | 2020-03-19 |
JPWO2019044926A1 (ja) | 2020-08-13 |
JP7145862B2 (ja) | 2022-10-03 |
CN111148832A (zh) | 2020-05-12 |
EP3677677A4 (en) | 2021-09-22 |
EP3677677A1 (en) | 2020-07-08 |
US11472867B2 (en) | 2022-10-18 |
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