Monoclonal antibodies for the prevention and treatment of herpes simplex virus infection

Abstract

Antibodies and antigen-binding fragments that bind to herpes simplex virus type 2 (HSV-2), methods of use using the antibodies and / or antigen-binding fragments, and pharmaceutical compositions comprising the antibodies and / or antigen-binding fragments are provided.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 820,495, filed Mar. 19, 2019, the content of which is incorporated herein by reference in its entirety.

[0002] Description of government support<00000(...)​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ IRSSGSS (Sequence ID 8), and ARGGGLRHYFDY (Sequence ID 9) Heavy chain variable region including; and / or GNIHNY (Sequence ID 10), HAE (SEQ ID NO: 11), and QHFWSTPYT(Sequence ID 12) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0006] An antibody or antigen-binding fragment, GFTFTDYY (Sequence ID 13), IRNKANGYTT (SEQ ID NO: 14), and ACGNYVGYAMDY (Sequence ID 15) Heavy chain variable region including; and / or QSLLNSRTRKNY (Sequence ID 16), WAS (SEQ ID NO: 17), and KQSYNLYT (Sequence ID 18) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0007] An antibody or antigen-binding fragment, GFSLSRHD (Sequence ID 79), IWGDGST (Sequence ID 80), and AKEDYGIFPY (Sequence ID 81) Heavy chain variable region including; and / or QDISSY (Sequence ID 82), RAN (Sequence ID 83), and LQYDEFPLT(Sequence ID 84) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0008] An antibody or antigen-binding fragment, GYTFTNYD (Sequence ID 87), IYPRDGST (Sequence ID 88), and ARGIFYVNYDVY (SEQ ID NO: 89) comprising a heavy chain variable region; and / or DHINNW (SEQ ID NO: 90), GAA (SEQ ID NO: 91), and QQYWSSPLT (SEQ ID NO: 92) comprising a light chain variable region An antibody or antigen-binding fragment comprising the same is provided.

[0009] An antibody or antigen-binding fragment, EYEFPSHD (SEQ ID NO: 93), INSDGGST (SEQ ID NO: 94), and ARHSSGYVLDY (SEQ ID NO: 95) comprising a heavy chain variable region; and / or DHINHW (SEQ ID NO: 96), GAT (SEQ ID NO: 97), and QQYWSTPYT (SEQ ID NO: 98) comprising a light chain variable region An antibody or antigen-binding fragment comprising the same is provided.

[0010] An antibody or antigen-binding fragment, EYEFPSHD (SEQ ID NO: 85), INSDGGST (SEQ ID NO: 80), and ARHSSGYVLDY (SEQ ID NO: 81) comprising a heavy chain variable region; and / or DHINHW (SEQ ID NO: 86), GAT (SEQ ID NO: 83), and QQYWSTPYT (SEQ ID NO: 84) comprising a light chain variable region An antibody or antigen-binding fragment comprising the same is provided.

[0011] A pharmaceutical composition comprising an antibody or antigen-binding fragment described herein and a pharmaceutically acceptable carrier or excipient.

[0012] A nucleic acid encoding the heavy chain variable region of an antibody, wherein the heavy chain variable region is GYSFTTYD (Sequence ID 1), IYPREGST (SEQ ID NO: 2), and ATYGSSRYYTMDY(Sequence ID 3); or GYSITNGNH(array_7); IRSSGSS (Sequence ID 8), and ARGGGLRHYFDY(sequence number 9); or GFTFTDYY (Sequence ID 13), IRNKANGYTT (SEQ ID NO: 14), and ACGNYVGYAMDY(sequence number 15); or GFSLSRHD (Sequence ID 79), IWGDGST (Sequence ID 80), and AKEDYGIFPY(sequence number 81); or GFSLNNYD (Sequence ID 85), IWGDGST (Sequence ID 80), and AKEDYGIFPY(sequence number 81); or GYTFTNYD (Sequence ID 87), IYPRDGST (Sequence ID 88), and ARGIFYVNYDVY(sequence number 89); or EYEFPSHD (SEQ ID NO: 93), INSDGGST (SEQ ID NO: 94), and ARHSSGYVLDY (Sequence ID 95) A nucleic acid having an amino acid sequence containing [specific amino acids].

[0013] A nucleic acid that encodes the light chain variable region of an antibody, the light chain variable region is ESVDNFGISF(Sequence 4); AAS(sequence number 5); and QQSKEVPLT (sequence number 6), or GNIHNY(Sequence ID 10); HAE(Sequence ID 11); and QHFWSTPYT (sequence number 12), or QSLLNSRTRKNY(Sequence ID 16); WAS(sequence code 17); and KQSYNLYT(sequence code 18); or QDISSY (Sequence ID 82), RAN (Sequence ID 83), and LQYDEFPLT(sequence number 84); or QDINSY (Sequence ID 86), RAN (Sequence ID 83), and LQYDEFPLT(sequence number 84); or DHINNW (Sequence ID 90), GAA (SEQ ID NO: 91), and QQYWSSPLT(sequence code 92); or DHINHW (Sequence ID 96), GAT (SEQ ID NO: 97), and QQYWSTPYT (sequence number 98) A nucleic acid having an amino acid sequence containing [specific amino acids].

[0014] Nucleic acids encoding the light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 of the light chain of the antibody described herein.

[0015] Nucleic acids encoding the heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 of the heavy chain of the antibody described herein.

[0016] A host cell containing one or more of the nucleic acids described herein.

[0017] An antibody or antigen-binding fragment as described herein, conjugated to or attached to a therapeutic agent.

[0018] A method for inhibiting herpes simplex virus type 2 (HSV-2) activity in a subject, comprising administering an antibody or antigen-binding fragment described herein in an amount effective for inhibiting HSV-2 activity in the subject.

[0019] A method for activating antibody-dependent cytotoxicity (ADCC) in cells infected with herpes simplex virus type 2 (HSV-2), comprising contacting the cells with an isolated antibody or antigen-binding fragment described herein in an amount that induces cellular ADCC.

[0020] Use of an effective amount of the antibody or antigen-binding fragment described herein or the pharmaceutical composition described herein for treating or preventing a disease or condition associated with herpes simplex virus type 2 (HSV-2) infection in a subject.

[0021] Those skilled in the art will understand that the following drawings are for illustrative purposes only. The drawings are not intended to limit the scope of this instruction.

[0022] A patent or application document shall include at least one colored drawing. A copy of the published patent or patent application containing the colored drawing shall be provided by the Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0023] [Figure 1] ELISA results showing the binding of culture supernatant to HSV-2G infected cell lysates and uninfected Vero cell lysates. Germ center (GC) B cells from the inguinal lymph nodes of mice vaccinated with ΔgD-2 were directly sorted into 96-well plates (1 cell / well). These GC B cells were then cultured in the presence of NB-21.2D9 feeder cells. After culturing, the presence of IgG was first determined by ELISA, and then the reactivity of culture supernatant IgG to HSV-2G infected cell lysates (HSV-2G) and uninfected Vero lysates (control) was tested by ELISA. [Figure 2] Distribution of VH gene segments obtained from GC B cells on days 16-17. [Figure 3A-B]Retention of HSV-2G lysate binding by recombinant antibodies (22D10, 32H6, 33B8, and 35H7). Binding of recombinant antibodies to HSV-2G lysates was tested by ELISA. Both heavy and light chain V(D)J rearrangements were selected and amplified by standard RT-PCR from single B cell culture wells (22D10, 32H6, 33B8, and 35H7). These cloned V(D)J rearrangements were introduced into expression vectors. Recombinant antibodies were obtained by simultaneous translocation of their expression vectors into Expi293F cells. ≥4 mg of purified IgG was obtained (not shown). These antibodies did not bind to control uninfected Vero lysates. [Figure 4] FcγRIV activation by IgG1 and IgG2 isotype of individual mAbs. FcγRIV activation was assayed at a concentration of 1 mg / mL using the Promega FcγRIV ADCC Reporter Bioassay. Statistical analysis was performed by ANOVA, **P<0.01, ****P<0.0001. [Figure 5A-B] This shows the binding of recombinant antibodies (BMPC-23 and 19G7) cloned from single myeloid plasma cells and GC B cells, respectively, to HSV-2G lysates. Binding of recombinant antibodies to HSV-2G-infected and uninfected cell lysates was tested by ELISA. [Figure 6A-B] Passive transmission in mice using cloned HSV-reactive IgG2c antibodies (BMPC-23, 33B8, and 22D10) provides protection against lethal HSV-2 challenge in overall survival (Figure 6A) and epithelial (Figure 6B) and neurological assays (Figure 6C). [Figure 6C] Passive transmission in mice using cloned HSV-reactive IgG2c antibodies (BMPC-23, 33B8, and 22D10) provides protection against lethal HSV-2 challenge in overall survival (Figure 6A) and epithelial (Figure 6B) and neurological assays (Figure 6C). [Figure 7A-B]Immunoprecipitation-mass spectrometry (IP-MS) was performed to identify the binding targets of 22D10, 33B8, and BMPC-23 antibodies. The results identified human herpesvirus glycoprotein B (gB) as the target. [Figure 8] This shows the binding of recombinant antibodies to HSV-1 glycoprotein B (gB-1) as measured by the Luminex multiplex assay. [Figure 9A] This graph shows the median fluorescence intensity (MFI) against IgG concentration (ng / ml), indicating the binding of various recombinant antibodies to gB-1 as measured by the Luminex multiplex assay. [Figure 9B] This graph shows the median fluorescence intensity (MFI) against IgG concentration (ng / ml), indicating the binding of various recombinant antibodies to gB-1 as measured by the Luminex multiplex assay. [Figure 10A-D] This demonstrates the ability of a gB-1-specific mouse monoclonal IgG antibody to inhibit the binding of BMPC-23 human IgG antibody to gB-1. [Figure 10E-G] This demonstrates the ability of a gB-1-specific mouse monoclonal IgG antibody to inhibit the binding of BMPC-23 human IgG antibody to gB-1. [Modes for carrying out the invention]

[0024] Prophylactic treatment with the anti-respiratory syncytial virus (RSV) F neutralizing monoclonal antibody (mAb) palivizumab has proven effective in preventing severe complications of RSV infection in high-risk infants and young children, and is currently approved for this purpose and used in accordance with regulations. Commercially available anti-HSV-2 or anti-HSV-1 antibody treatments are extremely useful but do not currently exist. Such products would be particularly useful in immunocompromised patients or immature patients with underdeveloped immune responses.

[0025] Antibodies, antigen-binding fragments, and nucleic acids encoding antibodies and antigen-binding fragments are disclosed herein. The antibodies or antigen-binding fragments bind to the herpes simplex virus type 2 (HSV-2) antigen. The antibodies or antigen-binding fragments include a heavy chain variable region having an amino acid sequence containing three heavy chain complementarity-determining regions (HCDRs), a light chain variable region having an amino acid sequence containing three light chain complementarity-determining regions (LCDRs), or a combination thereof. The antibodies or antigen-binding fragments include a heavy chain variable region having an amino acid sequence containing heavy chain HCDR1, HCDR2, and HCDR3; a light chain variable region having an amino acid sequence containing light chain LCDR1, LCDR2, and LCDR3; or a combination thereof.

[0026] An antibody or antigen-binding fragment, GYSFTTYD (Sequence ID 1), IYPREGST (SEQ ID NO: 2), and ATYGSSRYYTMDY (Sequence ID 3) Heavy chain variable region including; and / or ESVDNFGISF (Sequence ID 4), A AS (Sequence ID 5), and QQSKEVPLT(Sequence ID 6) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0027] An antibody or antigen-binding fragment, GYSITNGNH (Sequence ID 7), IRSSGSS (Sequence ID 8), and ARGGGLRHYFDY (Sequence ID 9) Heavy chain variable region including; and / or GNIHNY (Sequence ID 10), HAE (SEQ ID NO: 11), and QHFWSTPYT(Sequence ID 12) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0028] An antibody or its antigen-binding fragment, GFTFTDYY (Sequence ID 13), IRNKANGYTT (SEQ ID NO: 14), and ACGNYVGYAMDY (Sequence ID 15) Heavy chain variable region including, and / or QSLLNSRTRKNY (Sequence ID 16), WAS (SEQ ID NO: 17), and KQSYNLYT (Sequence ID 18) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0029] An antibody or antigen-binding fragment, GFSLSRHD (Sequence ID 79), IWGDGST (Sequence ID 80), and AKEDYGIFPY (Sequence ID 81) Heavy chain variable region including; and / or QDISSY (Sequence ID 82), RAN (Sequence ID 83), and LQYDEFPLT(Sequence ID 84) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0030] An antibody or antigen-binding fragment, GFSLNNYD (Sequence ID 85), IWGDGST (Sequence ID 80), and AKEDYGIFPY (Sequence ID 81) Heavy chain variable region including; and / or QDINSY (Sequence ID 86), RAN (Sequence ID 83), and LQYDEFPLT(Sequence ID 84) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0031] An antibody or antigen-binding fragment, GYTFTNYD (Sequence ID 87), IYPRDGST (Sequence ID 88), and ARGIFYVNYDVY (Sequence ID 89) Heavy chain variable region including; and / or DHINNW (Sequence ID 90), GAA (SEQ ID NO: 91), and QQYWSSPLT(sequence number 92) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0032] An antibody or antigen-binding fragment, EYEFPSHD (SEQ ID NO: 93), INSDGGST (SEQ ID NO: 94), and ARHSSGYVLDY (Sequence ID 95) Heavy chain variable region including; and / or DHINHW (Sequence ID 96), GAT (SEQ ID NO: 97), and QQYWSTPYT (sequence number 98) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0033] An antibody or antigen-binding fragment, GYTFTNYW (Sequence ID 99), IHPNIGIT (SEQ ID NO: 100), and ARGSDSGSAWFAY(Sequence ID 101) Heavy chain variable region including; and / or DHINNW (Sequence ID 90), GAT (SEQ ID NO: 97), and QQYWSTPLT(Sequence ID 102) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0034] An antibody or antigen-binding fragment, GYTFTSYW (Sequence ID 103), IHPNSGIT (SEQ ID NO: 104), and ARGSNSGSAWFAY(Sequence ID 105) Heavy chain variable region including; and / or DHINNW (Sequence ID 90), GAT (SEQ ID NO: 97), and QQYWSTPLT(Sequence ID 102) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0035] An antibody or antigen-binding fragment, GYTFTSYW (Sequence ID 103), IHPNSGIT (SEQ ID NO: 104), and ARGSNSGSAWFAY(Sequence ID 105) Heavy chain variable region including; and / or DHINNW (Sequence ID 90), GAA (SEQ ID NO: 91), and QQYWSSPLT(sequence number 92) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0036] An antibody or antigen-binding fragment, GYTFTTYG (Sequence ID 107), INTYSGVS (Sequence ID 108), and AQLNYGMDY. (Sequence ID 109) Heavy chain variable region including; and / or QDVSTS (Sequence ID 110), SAS (SEQ ID NO: 111), and HQHYSIPRT (Sequence ID 112) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0037] An antibody or antigen-binding fragment, GYTFTTYG (Sequence ID 107), INTYSGVS (Sequence ID 108), and AQVNYAMDY (Sequence ID 113) Heavy chain variable region including; and / or QDVSTA (Sequence ID 114), SAS (SEQ ID NO: 111), and QQHYSTPRT (Sequence ID 115) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0038] An antibody or antigen-binding fragment, GYSITSGYD (Sequence ID 116), ISYSGLT (SEQ ID NO: 117), and ARGPPWYFDV (Sequence ID 118) Heavy chain variable region including; QIVRAT(Sequence ID 119), LAS (Sequence ID 120), and LQYWNYPYT (Sequence ID 121) Light chain variable region including An antibody or antigen-binding fragment containing the above is provided.

[0039] In the embodiment, the antibody or its antigen-binding fragment is an IgG antibody. In the embodiment, the antibody or its antigen-binding fragment is an IgG antibody having the IgG2c isotype.

[0040] In the embodiment, the antibody or fragment thereof is a humanized antibody.

[0041] In embodiments, an antibody is provided. In embodiments, an antigen-binding fragment is provided. In embodiments, the antigen is herpes simplex virus type 2 (HSV-2) antigen. For example, the HSV-2 antigen is glycoprotein B (gB).

[0042] In an embodiment, the antibody or fragment thereof includes an Fc region that binds to the Fc gamma receptor RIII (FcγRIII). In an embodiment, the antibody or fragment thereof includes an Fc region that activates human FcγRIII when bound thereto. In an embodiment, the human FcγRIII receptor is FcγRIIIa.

[0043] In the embodiment, the antibody or a fragment thereof binds to the Fc gamma receptor RIV (FcγRIV).

[0044] In the embodiment, the antibody or a fragment thereof binds to the HSV-2 antigen.

[0045] In the embodiment, the framework region of the light chain and / or heavy chain is a human framework region or has 85% or more identity with it.

[0046] In one embodiment, the heavy chain V(D)J rearrangement is derived from the germline VH of IGHV1-85, the D of IGHD1-1, and the JH of IGHJ4. In another embodiment, the heavy chain V(D)J rearrangement has 95% or more identity with the germline VH of IGHV1-85, the D of IGHD1-1, and the JH of IGHJ4. In yet another embodiment, the light chain is derived from the germline VL of IGKV3-2 and the JL of IGKJ5. In yet another embodiment, the light chain has 95% or more identity with the germline VL of IGKV3-2 and the JL of IGKJ5.

[0047] In one embodiment, the heavy chain V(D)J rearrangement is derived from the germline VH of IGHV3-4, the D of IGHD2, and the JH of IGHJ4. In another embodiment, the heavy chain V(D)J rearrangement has 95% or more identity with the germline VH of IGHV3-4, the D of IGHD2, and the JH of IGHJ4. In yet another embodiment, the light chain is derived from the germline VL of IGKV12-41 and the JL of IGKJ2. In yet another embodiment, the light chain has 95% or more identity with the germline VL of IGKV12-41 and the JL of IGKJ2.

[0048] In one embodiment, the heavy chain V(D)J rearrangement is derived from the germline VH of IGHV7-3, the D of IGHD2-1, and the JH of IGHJ4. In another embodiment, the heavy chain V(D)J rearrangement has 95% or more identity with the germline VH of IGHV7-3, the D of IGHD2-1, and the JH of IGHJ4. In yet another embodiment, the light chain is derived from the germline VL of IGKV8-21 and the JL of IGKJ2. In yet another embodiment, the light chain has 95% or more identity with the germline VL of IGKV8-21 and the JL of IGKJ2.

[0049] In one embodiment, the heavy chain V(D)J rearrangement is derived from germline VH of IGHV2-3, D of IGHD2-1, and JH of IGHJ3. In another embodiment, the heavy chain V(D)J rearrangement has 95% or more identity with germline VH of IGHV2-3, D of IGHD2-1, and JH of IGHJ3. In yet another embodiment, the light chain is derived from germline VL of IGKV14-111 and JL of IGKJ5. In yet another embodiment, the light chain has 95% or more identity with germline VL of IGKV14-111 and JL of IGKJ5.

[0050] In one embodiment, the heavy chain V(D)J rearrangement is derived from the germline VH of IGHV1-85, the D of IGHD2-1, and the JH of IGHJ3. In another embodiment, the heavy chain V(D)J rearrangement has 95% or more identity with the germline VH of IGHV1-85, the D of IGHD2-1, and the JH of IGHJ3. In yet another embodiment, the light chain is derived from the germline VL of IGKV13-85 and the JL of IGKJ5. In yet another embodiment, the light chain has 95% or more identity with the germline VL of IGKV13-85 and the JL of IGKJ5.

[0051] In one embodiment, the heavy chain V(D)J rearrangement is derived from the germline VH of IGHV5-2, the D of IGHD3-2, and the JH of IGHJ4. In another embodiment, the heavy chain V(D)J rearrangement has 95% or more identity with the germline VH of IGHV5-2, the D of IGHD3-2, and the JH of IGHJ4. In yet another embodiment, the light chain is derived from the germline VL of IGKV13-85 and the JL of IGKJ2. In yet another embodiment, the light chain has 95% or more identity with the germline VL of IGKV13-85 and the JL of IGKJ2.

[0052] In one embodiment, the heavy chain V(D)J rearrangement is derived from germline VH of IGHV1-64, D of IGHD3-2, and JH of IGHJ3. In another embodiment, the heavy chain V(D)J rearrangement has 95% or more identity with germline VH of IGHV1-64, D of IGHD3-2, and JH of IGHJ3. In yet another embodiment, the light chain is derived from germline VL of IGKV13-85 and JL of IGKJ5. In yet another embodiment, the light chain has 95% or more identity with germline VL of IGKV13-85 and JL of IGKJ5.

[0053] In one embodiment, the heavy chain V(D)J rearrangement is derived from the germline VH of IGHV9-3, the D of IGHD2-12, and the JH of IGHJ4. In another embodiment, the heavy chain V(D)J rearrangement has 95% or more identity with the germline VH of IGHV9-3, the D of IGHD2-12, and the JH of IGHJ4. In yet another embodiment, the light chain is derived from the germline VL of IGKV6-17 and the JL of IGKJ1. In yet another embodiment, the light chain has 95% or more identity with the germline VL of IGKV6-17 and the JL of IGKJ1.

[0054] In one embodiment, the heavy chain V(D)J rearrangement is derived from the germline VH of IGHV3-1, the D of IGHD6-3, and the JH of IGHJ1. In another embodiment, the heavy chain V(D)J rearrangement has 95% or more identity with the germline VH of IGHV3-1, the D of IGHD6-3, and the JH of IGHJ1. In yet another embodiment, the light chain is derived from the germline VL of IGKV6-14 and the JL of IGKJ2. In yet another embodiment, the light chain has 95% or more identity with the germline VL of IGKV6-14 and the JL of IGKJ2.

[0055] Pharmaceutical compositions comprising an effective amount of the antibody or antigen-binding fragment described herein and a pharmaceutically acceptable carrier or excipient are also provided. In embodiments, the pharmaceutical composition further comprises an antiviral small molecule drug. The antiviral small molecule includes famciclovir, penciclovir, valacyclovir, or a combination thereof. In embodiments, the small molecule antiviral drug is acyclovir.

[0056] The following are exemplary nucleotide and amino acid sequences of anti-HSV antibodies. The corresponding heavy or light chain CDRs (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) are underlined in order. A summary of the nucleotide and amino acid sequences, including the corresponding HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-HSV antibodies, is provided in Table 4.

[0057] BMPC-23_IgH nucleotide: [ka] amino acid: [ka]

[0058] BMPC-23_IgL nucleotide [ka] amino acid: [ka]

[0059] 22D10_IgH nucleotide [ka] amino acid: [ka]

[0060] 22D10_IgL nucleotide [ka] amino acid: [ka]

[0061] 33B8_IgH nucleotide [ka] amino acid: [ka]

[0062] 33B8_IgL nucleotide [ka] amino acid: [ka]

[0063] HSV010-4_IgH nucleotide [ka] amino acid [ka]

[0064] HSV010-4_IgL nucleotide [ka] amino acid [ka]

[0065] HSV010-7_IgH nucleotide [ka] amino acid [ka]

[0066] HSV010-7_IgL nucleotide [ka] amino acid [ka]

[0067] HSV010-34_IgH nucleotide [ka] amino acid [ka]

[0068] HSV010-34_IgL nucleotide [ka] amino acid [ka]

[0069] HSV010-6_IgH nucleotide [ka] amino acid [ka]

[0070] HSV010-6_IgL nucleotide [ka] amino acid [ka]

[0071] HSV010-9_IgH nucleotide [ka] amino acid [ka]

[0072] HSV010-9_IgL nucleotide [ka] amino acid [ka]

[0073] HSV010-13_IgH nucleotide [ka] amino acid [ka]

[0074] HSV010-13_IgL nucleotide [ka] amino acid [ka]

[0075] HSV010-14_IgH nucleotide [ka] amino acid [ka]

[0076] HSV010-14_IgL nucleotide [ka] amino acid [ka]

[0077] HSV010-28_IgH nucleotide [ka] amino acid [ka]

[0078] HSV010-28_IgL nucleotide [ka] amino acid [ka]

[0079] HSV010-20_IgH nucleotide [ka] amino acid [ka]

[0080] HSV010-20_IgL nucleotide [ka] amino acid [ka]

[0081] HSV010-8_IgH nucleotide [ka] amino acid [ka]

[0082] HSV010-8_IgL nucleotide [ka] amino acid [ka]

[0083] HSV010-15_IgH nucleotide [ka] amino acid [ka]

[0084] HSV010-15_IgL nucleotide [ka] amino acid [ka]

[0085] HSV010-16_IgH nucleotide [ka] amino acid [ka]

[0086] HSV010-16_IgL nucleotide [ka] amino acid [ka]

[0087] In one embodiment, an antibody is provided having one heavy chain HCDR1, HCDR2, and HCDR3 of the heavy chain amino acid sequences listed above, and one light chain LCDR1, LCDR2, and LCDR3 of any one of the light chain amino acid sequences listed above. In one embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chain HCDR and light chain LCDR of BMPC-23. In another embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chain HCDR and light chain LCDR of 33B8. In yet another embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chain HCDR and light chain LCDR of 22D10. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-4. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-8. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-7. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-34. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-6. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSC010-9. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-13.In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-14. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-15. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-16. In the embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3 are the same as the heavy chains HCDR and light chains LCDR of HSV010-28. In this embodiment, both the heavy chains HCDR1, HCDR2, and HCDR3, as well as the light chains LCDR1, LCDR2, and LCDR3, are the same as the heavy chain HCDR and light chain LCDR of HSV010-20.

[0088] A nucleic acid encoding an antibody or its antigen-binding fragment is provided. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment is also provided.

[0089] In embodiments, nucleic acids encoding the light chain of an antibody are provided. In particular, the nucleic acid encodes the variable region of the antibody's light chain. The light chain is a lambda chain or a kappa chain. In embodiments, the nucleic acid also encodes the human framework region. The nucleic acid may be an isolated nucleic acid molecule.

[0090] In one embodiment, the nucleic acid encodes the light chain variable region of an antibody having a nucleic acid sequence including SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 69, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 61, or SEQ ID NO: 66. In another embodiment, the nucleic acid encodes the light chain variable region of an antibody having a light chain having a nucleic acid sequence that is 95% or more identical to SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 69, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 61, or SEQ ID NO: 66.

[0091] In the embodiment, nucleic acids are ESVDNFGISF(Sequence 4); AAS(sequence number 5); and QQSKEVPLT (sequence number 6), or GNIHNY(Sequence ID 10); HAE(Sequence ID 11); and QHFWSTPYT (sequence number 12), or QSLLNSRTRKNY(Sequence ID 16); WAS(sequence code 17); and KQSYNLYT (Sequence ID 18), or QDISSY(sequence number 82); RAN(sequence number 83); and LQYDEFPLT (SEQ ID NO: 84), or QDINSY(array sequence 86); RAN(sequence number 83); and LQYDEFPLT (SEQ ID NO: 84), or DHINNW(sequence number 90); GAA (SEQ ID NO: 91); and QQYWSSPLT (sequence number 92), or DHINHW (Sequence ID 96) GAT (Sequence ID 97) QQYWSTPYT (sequence number 98), or DHINNW (Accession No. 90); GAT (Accession No. 97); and QQYWSTPLT (Accession No. 102), or DHINNW (Accession No. 90); GAT (Accession No. 97); and QQYWSSPLT (Accession No. 92), or QDVSTS (Accession No. 110); SAS (Accession No. 111); and HQHYSIPRT (Accession No. 112), or QDVSTA (Accession No. 114); SAS (Accession No. 111); and QQHYSTPRT (Accession No. 115), or QIVRAT (Accession No. 119); LAS (Accession No. 120); and LQYWNYPYT (Accession No. 122) (listing LCDR1, LCDR2, and LCDR3 in that order) encoding the light chain variable region of an antibody having an amino acid sequence comprising<x

[0092] In embodiments, the nucleic acid also encodes a human framework region. In embodiments, the light chain is a lambda chain or a kappa chain.

[0093] There is provided a nucleic acid encoding the heavy chain of an antibody. In particular, the nucleic acid encodes the light chain variable region of the antibody. In embodiments, the nucleic acid also encodes a human framework region. The nucleic acid can be an isolated nucleic acid molecule.

[0094] In one embodiment, the nucleic acid encodes the heavy chain variable region of an antibody, wherein the heavy chain has a nucleic acid sequence including SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 67, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 59, or SEQ ID NO: 63. In another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody, wherein the heavy chain has a nucleic acid sequence that is 95% or more identical to SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 67, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 59, or SEQ ID NO: 63.

[0095] In the embodiment, nucleic acids are GYSFTTYD(sequence number 1); IYPREGST(array_2); and ATYGSSRYYTMDY (SEQ ID NO: 3), or GYSITNGNH (Sequence ID 7), IRSSGSS(Sequence ID 8); and ARGGGLRHYFDY (Sequence ID 9), or GFTFTDYY(sequence number 13); IRNKANGYTT(SEQ ID NO: 14); and ACGNYVGYAMDY (Sequence ID 15), or GFSLSRHD(sequence number 79); IWGDGST(Sequence ID 80); and AKEDYGIFPY (Sequence ID 81), or GFSLNNYD(sequence number 85); IWGDGST(Sequence ID 80); and AKEDYGIFPY (Sequence ID 81), or GYTFTNYD(sequence number 87); IYPRDGST(sequence number 88); and ARGIFYVNYDVY (sequence number 89), or EYEFPSHD(sequence number 93); INSDGGST(sequence number 94); and ARHSSGYVLDY (Sequence ID 95), or GYTFTNYW(sequence code 99); IHPNIGIT(SEQ ID NO: 100); and ARGSDSGSAWFAY (Sequence ID 101), or GYTFTSYW(sequence number 103); IHPNSGIT(Sequence ID 104); and ARGSNSGSAWFAY(SEQ ID NO: 105), or GYTFTTYG(Sequence code 107); INTYSGVS(sequence number 108); and AQLNYGMDY (Sequence ID 109), or GYTFTTYG(Sequence code 107); INTYSGVS(sequence number 108); and AQVNYAMDY (Sequence ID 113), or GYSITSGYD(sequence number 116); ISYSGLT(sequence code 117); and ARGPPWYFDV (Sequence ID 118) (List HCDR1, HCDR2, and HCDR3 in that order.) This encodes the heavy chain variable region of an antibody having an amino acid sequence containing the following.

[0096] In one embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 19 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 21. In another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 23 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 25. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 27 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 29. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 31 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 33. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 67 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 69. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 35 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 37. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 39 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 41. In one embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 43 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 45. In another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 47 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 49. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 51 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 53. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 55 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 57. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 71 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 73. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 75 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 77. In yet another embodiment, the nucleic acid encodes the heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 59 and the light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 61.In an embodiment, the nucleic acid encodes a heavy chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 63 and a light chain variable region of an antibody having the nucleic acid sequence of SEQ ID NO: 65.

[0097] In an embodiment, the nucleic acid also encodes a human Fc region. In an embodiment, the nucleic acid also encodes a human Fc region that binds to FcγRIII. In an embodiment, the antibody or antigen-binding fragment comprises an Fc region that activates human FcγRIII when bound thereto. In an embodiment, the human FcγRIII receptor is FcγRIIIa. In an embodiment, the heavy chain is a gamma chain.

[0098] In an embodiment, the nucleic acid of the present disclosure encodes a polypeptide that is less than 500 amino acids in length, less than 250 amino acids in length, or less than 120 amino acids in length. In an embodiment, the amino acid of the present disclosure encodes a polypeptide that is 70 to 100 amino acids in length.

[0099] In an embodiment of the nucleic acid, the nucleic acid is DNA. In an embodiment, the nucleic acid is cDNA. In an embodiment, the nucleic acid is RNA.

[0100] The nucleic acids disclosed herein are suitable for the recombinant production of the antibodies or antigen-binding fragments disclosed herein. Accordingly, the present invention also relates to vectors and host cells containing nucleic acid sequences encoding antibodies or antigen-binding fragments.

[0101] Vectors encoding the nucleic acid molecules described herein are provided. Host cells comprising one or more of the nucleic acids described herein or the vectors described herein are also provided.

[0102] The antibody or antigen-binding fragment can be bound or conjugated to a heterologous molecule. The heterologous molecule can be a therapeutic agent, such as a cytotoxic drug, a radioisotope, an immunomodulator, a secondary antibody, an anti-viral small molecule, or a combination thereof. In an embodiment, the antibody or antigen-binding fragment can be bound or conjugated to a therapeutic agent.

[0103] A method for inhibiting HSV-2 in a subject includes administering an antibody or antigen-binding fragment in an amount effective to inhibit HSV-2 activity in the subject. The antibody or antigen-binding fragment may inhibit HSV-2 activity in the subject by removing the virus from the subject, preventing the dissemination of HSV-2 in the subject, preventing the establishment of latency in the subject, and / or preventing infection of the subject by HSV-2.

[0104] In one embodiment, the subject is infected with HSV-2. In another embodiment, the subject is not yet infected with HSV-2. In one embodiment, the subject is immunocompromised. In another embodiment, the immunocompromised patient has cancer, has undergone a transplant, or is receiving immunosuppressive medication. In another embodiment, the subject is pregnant. In another embodiment, the subject is a newborn.

[0105] In the embodiments, the subject is currently receiving or has previously received antiviral therapy. The antiviral therapy includes administration of antiviral small molecules to the subject. The antiviral small molecules include famciclovir, penciclovir, valacyclovir, or a combination thereof. In the embodiments, the antiviral therapy includes administration of acyclovir to the subject.

[0106] A method for activating antibody-dependent cytotoxicity (ADCC) in cells infected with HSV-2 comprises contacting the cells with an isolated antibody or antigen-binding fragment in an amount that induces ADCC in the cells.

[0107] In one embodiment, the cells are present in a subject infected with HSV-2.

[0108] The following are provided for use in the treatment or prevention of diseases or conditions associated with herpes simplex virus type 2 (HSV-2) infection in subjects: an antibody or antigen-binding fragment described herein, or a pharmaceutical composition containing an antibody or antigen-binding fragment.

[0109] Use of an effective amount of the antibody or antigen-binding fragment described herein for the manufacture of a pharmaceutical product for treating or preventing a disease or condition associated with HSV-2 infection in a subject.

[0110] The one or more antibodies described herein may be one or more isolated antibodies. Similarly, the antigen-binding fragments described herein may be isolated antigen-binding fragments.

[0111] In the embodiments, the antibody or its antigen-binding fragment is chimeric or humanized. In the embodiments, the antibody or its antigen-binding fragment has a human sequence Fc region.

[0112] In the embodiment, the antibody or antigen-binding fragment includes a monoclonal antibody, scFv, Fab fragment, Fab' fragment, F(ab')2 fragment, or a combination thereof.

[0113] In embodiments, isolated antibodies, isolated antigen-binding fragments, antibodies, or antigen-binding fragments include monoclonal antibodies, scFv, Fab fragments, Fab' fragments, F(ab')2 fragments, or combinations thereof. Note that while scFv is strictly speaking a fusion protein rather than an antibody fragment, the antigen-binding fragments of antibodies used herein include scFv unless otherwise specified.

[0114] In embodiments, the method is for the treatment and prevention of neurological HSV-2 infection symptoms in subjects.

[0115] In embodiments, the method is for the treatment and prevention of epithelial HSV-2 infection symptoms.

[0116] In the embodiment, the antibody or its antigen-binding fragment comprises a VH framework containing a human germline framework sequence named in Table 2 or Table 4, and / or (ii) a VL framework containing a human germline framework sequence named in Table 2 or Table 4.

[0117] In the embodiment, the antibody or fragment thereof binds to the HSV-2 surface antigen at a binding affinity (KD) of about 1 × 10⁻⁹ molar concentration (M) to about 1 × 10⁻¹² M.

[0118] A method for producing an antibody or its antigen-binding fragment is provided herein, comprising culturing host cells under conditions in which the antibody or its antigen-binding fragment is produced by the host cells. The term “host cells” refers to cells into which an exogenous nucleic acid encoding the antibody or antigen-binding fragment has been introduced, and includes offspring of such cells. Examples of host cells include “transformers,” “transformed cells,” “transferred organisms,” “transferred cells,” and “transduced cells,” which include primary transformers / transferred / transduced cells and their offspring, regardless of passage number. The offspring do not have to be exactly identical in nucleic acid content to the parent cells and may contain mutations. This also includes mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells.

[0119] The host cell may be a mammalian host cell. Examples of mammalian host cells for expressing the antibodies or antigen-binding fragments provided herein include Chinese hamster ovary (CHO) cells, e.g., CHO-K1, CHO-S, CHO-K1SV, NSO myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells, SP2 cells, and Expi293F cells. In embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In embodiments, the host cell is an Expi293F cell.

[0120] As used herein, the term "antibody" refers to an intact antibody, i.e., an antibody having complete Fc and Fv regions. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions are further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains or binding moieties that interact with the antigen.

[0121] As used herein, the terms “antigen-binding fragment” or “antigen-binding portion” refer to any portion of an antibody that is less than a complete antibody but retains the ability to specifically bind to an antigen, or a portion of an antibody bound together with it. The antigen-binding fragment competes with the intact antibody, which is the fragment for specific binding. In this case, the antigen is the HSV-2 antigen. The antigen may be the HSV-1 antigen. “Antibody” or its “fragment” may include any class of immunoglobulin, e.g., IgG, IgM, IgA, IgD, and IgE. An exemplary class is IgG, and an exemplary isotype is IgG2c.

[0122] Examples of antibody terms that include "antigen-binding fragment" or "antigen-binding moiety" include: Fab fragment (a monovalent fragment consisting of VL, VH, CL, and CH1 domains), F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by disulfide crosslinks in the hinge region), Fab' fragment (a monovalent fragment produced by reduction of the F(ab')2 fragment, possessing a free sulfhydryl group), fragments consisting of the VL and VH domains of a single arm of the antibody, Fv fragment (a fragment consisting of the VL and VH domains of a single arm of the antibody), and single-chain fragments (s Examples include cFv, variable domain light chain (VL) and variable domain heavy chain (VH) linked via a peptide linker, dAb fragments (consisting of the VH domain); isolated complementarity-determining regions (CDRs); nanobodies (heavy chain variable regions containing a single variable domain and two constant domains); their mutants, naturally occurring variants; fusion proteins containing antibody moieties with the required specificity of antigen recognition sites; humanized antibodies; chimeric antibodies; and any other modified stereochemistry of immunoglobulin molecules containing the required specificity of antigen recognition sites.

[0123] Antigen-binding fragments can be prepared, for example, by cleaving intact antibodies or by recombinant means. See, in general, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), incorporated herein by reference in whole. Antigen-binding fragments can be produced by recombinant DNA technology, by enzymatic or chemical cleavage of intact antibodies, or by molecular biology techniques. In some embodiments, the fragments are Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, or single-chain antibodies (scFv). In the case of scFv fragments, VL and VH are encoded by separate genes and are linked together via recombinant means by a synthetic linker that allows the VL and VH regions to pair and form a monovalent molecule as a single protein chain. In embodiments, scFv includes a variable domain framework sequence having the same sequence as human variable domains FR1, FR2, FR3, or FR4. In embodiments, scFv comprises a synthetic linker which is a peptide with a length of 5 to 30 amino acid residues. For example, scFv comprises a linker peptide which contains one or more glycine, serine, and threonine residues. In embodiments, the linker of scFv is 10 to 25 amino acid residues long. In embodiments, the peptide linker comprises glycine, serine, and / or threonine residues (see, for example, Bird et al., Science, 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988), which are incorporated herein by reference as a whole).

[0124] The antigen-binding fragment may be a polypeptide, for example a diabody, containing at least a portion of an antibody sufficient to confer HSV-2 antigen-specific binding to the polypeptide. From the N-terminus to the C-terminus, both the mature light chain and heavy chain variable domains include regions FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each domain follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987), or Chothia et al. (Nature 342:878-883 (1989)), respectively, which are incorporated herein by reference as a whole. As used herein, the term “polypeptide” encompasses natural or artificial proteins, protein fragments, and / or polypeptide analogs of protein sequences. Polypeptides may be monomers or polymers. As used herein, the Fd fragment refers to an antibody fragment consisting of VH and CH1 domains; the Fv fragment consists of the V1 and VH domains of a single arm of the antibody; and the dAb fragment (incorporated herein as a whole by reference, Ward et al., Nature 341:544-546 (1989)) consists of the VH domain. In some embodiments, the fragment is at least 5, 6, 8, or 10 amino acid long. In other embodiments, the antigen-binding fragment is at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, or 200 amino acid long.

[0125] As used herein, the terms “monoclonal antibody” or “mAb” refer to an antibody member of a substantially homogeneous population of antibodies; that is, the individual antibodies in the population are identical except for possible mutations that may exist in trace amounts, such as spontaneous mutations. Therefore, the modifier “monoclonal” indicates the property of the antibody that it is not a mixture of distinct antibodies. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed to different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Monoclonal antibody preparations are advantageous in that, in addition to their specificity, they are typically not contaminated with other immunoglobulins. Therefore, identified monoclonal antibodies, once their sequences are identified, can be produced by non-hybridoma techniques, for example, by appropriate recombination methods.

[0126] In the embodiments of the present invention described herein, the antibody is isolated. As used herein, the term “isolated antibody” means an antibody having at least one (e.g., 1, 2, 3, or 4) in terms of its origin or source of origin: (1) it does not have any naturally associated components in its natural state; (2) it does not have other proteins from the same species; (3) it is expressed by cells from a different species; or (4) it does not occur naturally unless it is artificial.

[0127] In embodiments, the antibody is humanized. As used herein, “humanized antibody” refers to a form of antibody that contains sequences from both human and non-human (e.g., mouse, rat) antibodies. The humanized form of a non-human (e.g., mouse) antibody is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulin. In one embodiment, the humanized antibody is an antibody that has the sequence of human immunoglobulin (recipient antibody) in addition to residues from the mouse hypervariable region (HVR) (or CDR). In embodiments, the framework (FR) residues of the mouse mAb are replaced with the corresponding human immunoglobulin variable domain framework (FR) residues. These humanized antibodies can be further modified to refine antibody performance. The humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. Alternatively, the humanized antibody may not contain residues not found in either the recipient antibody or the donor antibody. Generally, humanized antibodies include at least one, typically two, variable domains where all or substantially all of the hypervariable loop corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR is that of a human immunoglobulin sequence. Optionally, humanized antibodies also include at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For example, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409, the contents of which are incorporated herein by reference as a whole.In one embodiment, the humanized antibody comprises residues not found in either the recipient antibody or the donor antibody, and the Fc region of the humanized antibody is modified as described in International Publication No. 99 / 58572, the contents of which are incorporated herein by reference in their entirety.

[0128] Techniques for humanizing monoclonal antibodies are well known and are described, for example, in U.S. Patents 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370, the contents of which are incorporated herein by reference as a whole. Numerous "humanized" antibody molecules containing antigen-binding sites derived from non-human immunoglobulins are described, for example, antibodies having rodent or modified rodent V regions fused to human constant domains and associated complementarity-determining regions (CDRs). For example, see Winter et al. Nature 349:293-299 (1991), Lobuglio et al. Proc.Nat.Acad.Sci.USA 86:4220-4224 (1989), Shaw et al. J.Immunol.138:4534-4538 (1987), and Brown et al. Cancer Res.47:3577-3583 (1987), the contents of which are incorporated herein by reference as a whole. Other references describe rodent hypervariable regions or CDRs grafted into human support framework regions (FRs) before fusion with appropriate human antibody constant domains. For example, see Riechmann et al. Nature 332:323-327 (1988), Verhoeyen et al. Science 239:1534-1536 (1988), and Jones et al. Nature 321:522-525 (1986), the contents of which are incorporated herein by reference as a whole. Another reference is the description of a rodent CDR supported by a recombinant rodent framework region - European Patent Application Publication No. 0519596 (incorporated herein by reference as a whole). These “humanized” molecules are designed to minimize unwanted immunological responses to rodent anti-human antibody molecules that limit the duration and effectiveness of therapeutic application of their portions in human recipients.The constant region of an antibody can be engineered to be immunologically inactive (e.g., not trigger complement lysis). See, for example, International Publication No. 99 / 58572; and UK Patent Application No. 9809951.8. Other methods for humanizing antibodies that can be similarly utilized are disclosed by Daugherty et al., Nucl. Acids Res. 19:2471-2476 (1991), and U.S. Patent Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and International Publication No. 01 / 27160 (each incorporated as a whole by reference).

[0129] In embodiments, the antibodies or fragments described herein can be produced by recombination. For example, antibodies can be expressed using a recombinant expression vector that is translocated into host cells, antibodies can be isolated from a recombinant combinatorial human antibody library, and antibodies can be isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes.

[0130] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domain of the antibody’s heavy or light chain. The variable domain of the heavy chain can be referred to as “VH,” and the variable domain of the light chain can be referred to as “VL.” These domains are generally the most variable parts of the antibody and contain the antigen-binding site. The term “variable” refers to the fact that certain parts of the variable domain differ significantly in sequence between antibodies, and this is used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. In both the light and heavy chain variable domains, variability is concentrated in three segments called hypervariable regions (HVRs) (or CDRs). The more highly conserved parts of the variable domain are called frameworks (FRs). The natural heavy and light chain variable domains each contain four FR regions, which take a beta-sheet configuration, mostly connected by three CDRs, and the CDRs form loops that connect to the beta-sheet structure and, in some cases, form part of the beta-sheet structure. The CDRs in each chain are held together in close proximity to the FR region and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody binding to the antigen, but exhibits various effector functions, such as the antibody's involvement in antibody-dependent cytotoxicity.

[0131] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0132] "Framework" or "FR" residues are variable domain residues other than the HVR residues as defined herein.

[0133] In embodiments, the antibodies described herein have a human Fc region. The term “Fc region” as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, the intact antibodies used herein may or may not have other C-terminal lysine.

[0134] Compositions or pharmaceutical compositions comprising antibodies, scFv, or antibody fragments disclosed herein preferably include stabilizers to prevent loss of protein activity or structural integrity due to denaturation, oxidation, or aggregation effects over a period of time during storage and transport prior to use. The compositions or pharmaceutical compositions may include one or more combinations of salts, surfactants, pH, and isotonic agents, such as sugars, which may contribute to overcoming aggregation problems. When the compositions or pharmaceutical compositions of the present invention are used as injections, it is desirable that they have a pH value in an approximately neutral pH range, and it is also advantageous to minimize surfactant levels to avoid bubbles in the formulation that would be detrimental to injection into the subject. In embodiments, the compositions or pharmaceutical compositions are in liquid form, stably supporting high concentrations of bioactive antibodies in solution, and are suitable for inhalation or parenteral administration. In embodiments, the compositions or pharmaceutical compositions are formulated for administration to mammalian subjects, particularly human subjects. In embodiments, the compositions or pharmaceutical compositions are formulated for intravenous, intramuscular, intraperitoneal, intradermal, and / or subcutaneous injection. In embodiments, the composition or pharmaceutical composition may be in liquid form and have a minimized risk of bubbling and anaphylactic-like side effects. In embodiments, the composition or pharmaceutical composition is isotonic. In one embodiment, the composition or pharmaceutical composition has a pH of 6.8 to 7.4.

[0135] In embodiments, the scFv or antibody fragments disclosed herein are lyophilized and / or freeze-dried and reconstituted before use.

[0136] Examples of pharmaceutically acceptable carriers include, but are not limited to, phosphate-buffered saline solutions, sterile water (e.g., water for injection, USP), emulsions, such as oil / water emulsions, and various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are phosphate-buffered saline or normal (0.9%) saline, such as 0.9% sodium chloride solution, USP. Compositions containing such carriers are formulated by well-known and conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000, the contents of which are incorporated herein by reference). In non-limiting examples, this may include dibasic sodium phosphate, potassium chloride, monobasic potassium phosphate, polysorbate 80 (e.g., 2-[2-[3,5-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl(E)-octadec-9-enoate), disodium edetate dehydrate, sucrose, monobasic sodium phosphate monohydrate, and dibasic sodium phosphate dihydrate.

[0137] The antibodies or antibody fragments, or compositions, or pharmaceutical compositions described herein may be provided lyophilized or in any preferred form, including, but not limited to, injection solutions or inhalation solutions, gels, and tablets.

[0138] Antibody substitution variants are encompassed by this invention. A substitution variant has at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place. The most interesting sites for substitutional mutagenesis are the hypervariable regions, but framework changes are also intended. Conservative substitutions are shown in Table 1 under the heading "Conservative Substitutions". If such substitutions result in changes in biological activity, they are designated as "Exemplary Substitutions" in Table 1, or more substantial changes may be introduced, as further described below with reference to amino acid classes, and the product may be screened.

[0139] [Table 1]

[0140] Substantial modification of the biological properties of an antibody is achieved by selecting substitutions that differ significantly in (a) the structure of the polypeptide backbone in the substitution region, e.g., β-sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the maintenance of the bulkiness of the side chain. Spontaneously occurring residues are grouped based on common side-chain properties: (1) Nonpolar: norleucine, Met, Ala, Val, Leu, Ile; (2) Non-charged polarities: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) Residues that affect chain direction: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His.

[0141] Non-conservative substitutions are created by replacing one member of one of these classes with one of another.

[0142] For example, one type of substitution that can be made is to replace one or more cysteine ​​residues in an antibody that may be chemically reactive with another residue, such as, but not limited to, alanine or serine. For example, substitutions of non-canonical cysteine ​​may exist. Substitutions can be made in the CDR or framework region or constant region of the variable domain of the antibody. In some embodiments, the cysteine ​​is canonical. Any cysteine ​​residue that does not participate in maintaining the proper three-dimensional structure of the antibody can also generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, especially if the antibody is an antibody fragment, such as an Fv fragment, its stability can be improved by adding cysteine ​​bonds to the antibody.

[0143] Antibodies can be modified, for example, in the variable domains of the heavy and / or light chains to alter their binding properties. Changes in the variable regions can alter binding affinity and / or specificity. In some embodiments, one to five or fewer conserved amino acid substitutions are produced within the CDR domain. In other embodiments, one to three or fewer conserved amino acid substitutions are produced within the CDR domain.

[0144] The antibodies described herein can be recombinantly produced. Antibodies produced in a eukaryotic expression system include glycosylation at a residue on the Fc portion corresponding to Asn297.

[0145] In embodiments, compositions or pharmaceutical compositions comprising the antibody or antigen-binding fragment described herein are substantially pure with respect to the antibody or antigen-binding fragment. A composition or pharmaceutical composition comprising the antibody or antigen-binding fragment described herein is "substantially pure" with respect to the antibody or fragment if at least 60% to 75% of a sample of the composition or pharmaceutical composition exhibits a single type of antibody or antigen-binding fragment. A substantially pure composition or pharmaceutical composition comprising the antibody or antigen-binding fragment described herein may contain 60%, 70%, 80%, or 90%, more typically about 95%, preferably more than 99%, of a single type of antibody or antigen-binding fragment in the portion that is the antibody or antigen-binding fragment. Purity or homogeneity can be tested by a number of methods well known in the art, such as polyacrylamide gel electrophoresis or HPLC.

[0146] In the embodiment of the method, the subject is a mammal.

[0147] In this embodiment, the mammal is a human.

[0148] All combinations of the various elements described herein are within the scope of the present invention unless otherwise specifically stated herein or unless otherwise clearly contradicted by the context.

[0149] The terms used herein are for the purpose of describing specific embodiments only and are not limiting. The singular forms “a,” “an,” and “it” as used herein include the plural form, for example, “at least one,” unless otherwise specified. “At least one” should not be construed as limiting “a” or “an.” “Or” means “and / or.” The term “and / or” as used herein includes any and all combinations of one or more of the enumerated items. The terms “include,” “contain,” and “have” are comprehensive and mean that additional elements other than the enumerated elements may exist.

[0150] Pharmacopoeia-acceptable: As used herein, the term "pharmacopoeia-acceptable" means that, in addition to other formulations that are safe for use in animals, more specifically humans and / or non-human mammals, it is approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. Pharmacopoeia-acceptable carrier: As used herein, the term "pharmacopoeia-acceptable carrier" refers to excipients, diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or vehicles administered with the antibody or fragment. Such carriers may be sterile liquids, e.g., water and oils, e.g., petroleum, animal, plant or synthetic origins, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.; polyethylene glycol, glycerin, propylene glycol or other synthetic solvents. Antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfate; chelating agents, e.g., ethylenediaminetetraacetic acid; and agents for adjusting tonicity, e.g., sodium chloride or dextrose may also be carriers. Methods for producing compositions in combination with carriers are known to those skilled in the art. In some embodiments, the term “pharmaceutically acceptable carrier” includes any and all solvents, dispersions, coatings, isotonic and absorption retarders, etc., that are compatible with the pharmacopoeia. The use of such carriers and agents of pharmaceutically active substances is well known in the art. See, for example, Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Such use in a composition is intended unless any conventional carrier or agent is incompatible with the active compound.

[0151] The aspects and embodiments of the present invention described herein are understood to include "consisting of" and / or "essentially consisting of" aspects and embodiments.

[0152] Throughout this disclosure, various aspects of the invention may be presented in scope form. It should be understood that scope form is merely for convenience and brevity and should not be interpreted as an irrevocable limitation to the scope of the invention. Therefore, scope forms should be considered to specifically disclose all possible partial ranges and the individual numbers within those ranges. For example, a scope form, e.g., 1–6, should be considered to specifically disclose partial ranges, e.g., 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, etc., as well as the individual numbers within those ranges, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0153] Other objects, advantages, and features of the present invention will become apparent from the following specification, as interpreted in conjunction with the accompanying drawings.

[0154] While this disclosure has been described with reference to various embodiments, it will be understood by those skilled in the art that various modifications can be made without departing the scope of this disclosure, and equivalents can be substituted for its elements. Furthermore, many modifications can be made to adapt specific situations or materials to the teachings of this disclosure without departing the essential scope. Therefore, this disclosure is not limited to any particular embodiment disclosed as the best way contemplated for the implementation of this disclosure, and is intended to include all embodiments that fall within the scope of the appended claims.

[0155] All cited patents, patent applications, and other references are incorporated herein by reference as a whole. However, if any terminology in this application conflicts with or is inconsistent with any terminology in an incorporated reference, the terminology from this application shall prevail over the conflicting terminology from the incorporated reference.

[0156] This invention can be better understood from the following experimental details. [Examples]

[0157] Experiment Details A candidate HSV-2 single-cycle virus vaccine lacking immunodominant envelope glycoprotein D (named ΔgD-2) induces high-titer antibodies that can actively or passively protect mice from HSV disease in a mouse model. These antibodies are weakly neutralizing but potentially activate mouse Fc gamma receptor IV (FcγRIV), thereby activating antibody-dependent cell-mediated cytotoxicity (ADCC). This disclosure analyzes, clones, sequences, and tests monoclonal antibodies (mAbs) isolated from germinal center B cells of immunized mice to identify several that passively protect mice from viral disease. Some mAbs were sufficiently active for use in the prophylaxis and treatment of patients with HSV (e.g., in humanized forms) or patients at risk for disseminated disease (e.g., neonates, immunocompromised hosts).

[0158] Mice were prime-boosted immunized with ΔgD-2, and spleen, influx region lymph nodes, or bone marrow were isolated. Germ center (GC) B cells were then cultured on NB-21.2D9 cells (Nojima culture). After culturing, the supernatant was screened by ELISA for the presence of HSV lysate-bound IgG. V(D)J rearrangements amplified from selected GC B cell cultures were sequenced for B cell antigen receptor (BCR) repertoire analysis in GC B cell clones between individual mice. Antibodies rearranged from selected single GC B cell cultures and single bone marrow plasma cells were then evaluated for their ability to activate mFcγRIV in vitro and protect mice in a passive transmission model.

[0159] In short, mice were vaccinated with ΔgD-2, and then 16 days after vaccination, inguinal lymph nodes in the influx region were harvested. GC B cells were isolated from these tissues and cultured in Nojima cultures. The overall cloning efficiency of GC B cells from these (transport) tissues was lower than that from freshly isolated tissues (6% vs. 20%), but 50 GC clones specific to HSV-2G strain (HSV-2G or HSV2-G) lysates were obtained. ELISA was performed on HSV-2G lysates and uninfected Vero lysates (control), and the results are shown in Figure 1. GC B cells from the inguinal lymph nodes of ΔgD-2-vaccinated mice were directly sorted into 96-well plates (1 cell / well). These GC B cells were then cultured in the presence of NB-21.2D9 feeder cells. After culturing, the presence of IgG was first determined by ELISA, and then the reactivity of the culture supernatant IgG to HSV-2G lysate (HSV-2G) and non-infected Vero lysate was tested by ELISA.

[0160] The molecular genetic characteristics of GC B cells were analyzed (Figure 2). V(D)J rearrangements were amplified from cDNA of HSV-2G lysate-reactive IgG+ cultures, then cloned and sequenced. The rearranged V, D, and J gene segments were determined using IMGT V-QUEST (see the World Wide Web at imgt.org / ). A diverse set of VH gene segments was used by HSV2-specific GC B cells. VH1-64 and VH1-26 appeared more frequently (approximately 10%) than other gene segments.

[0161] Based on the V(D)J sequences obtained from each B cell culture, paired heavy and light chains were reexpressed in Expi293F cells (as recombinant mouse IgG1 and IgG2c antibodies) (Kuraoka et al. Immunity 2016; DiLillo et al. Nature Medicine 2013). Four independent HSV-2G lysate-reactive IgG+ monocellular B cell cultures were then selected from 16-day GC B cells. As expected, all recombinant antibodies reacted specifically with HSV-2G lysates (Figure 3). Clone 32H6 bound most strongly to the lysate, followed by 35H7, 22D10, and 33B8. Figure 3 shows the retention of HSV-2G lysate binding by recombinant antibodies. Binding of recombinant antibodies to HSV-2G lysates was tested by ELISA. Both heavy and light chain V(D)J rearrangements were amplified by standard RT-PCR from selected single B cell culture wells (22D10, 32H6, 33B8, and 35H7). These cloned V(D)J rearrangements were introduced into expression vectors. Recombinant antibodies were obtained by simultaneous translocation of their expression vectors into Expi293F cells. ≥4 mg of purified IgG was obtained (not shown). These antibodies did not bind to control uninfected Vero lysates. These recombinant antibodies (≥4 mg each) were tested for reaction antigen identification in an in vitro FcγRIV activation assay (Promega FcγRIV ADCC Reporter Bioassay) (Figure 4) and an in vivo protection assay.

[0162] ΔgD-2 Prime Boost vaccination establishes a long-term HSV-reactive (and protective) serum IgG response in mice, as evidenced by results showing that mice were still protected from HSV challenge 8 months after boosting. These results suggest that vaccination successfully establishes long-term viable plasma cells. It was hypothesized that HSV-reactive plasma cells producing protective IgG could be isolated from the bone marrow of Prime Boost-vaccinated mice. Compared to naive mice, ΔgD-2-vaccinated mice contained four times higher frequency of surface IgM-CD138hiB220lo / -FSChi plasma cells in the bone marrow in experiments in which bone marrow plasma cells were isolated from ΔgD-2-vaccinated mice.

[0163] To analyze the molecular genetic characteristics of bone marrow plasma cells, V(D)J rearrangements were amplified from the cDNA of a single sIgM-CD138hiB220lo / -FSChi plasma cell, and the amplification products were then cloned and sequenced. The rearranged V, D, and J gene segments were determined using IMGT V-QUEST (available on the World Wide Web at imgt.org / ). From 96 individual cells, the inventors recovered 21 pairs of heavy and light chains, as well as 4 heavy chain-only clones (Table 1). The 25 heavy chain sequences consisted of 4 IgG3 (16%), 9 IgG1 (36%), 7 IgG2b (28%), 4 IgG2c (16%), and 1 IgA (4%) isotypes. Twenty individual clones (80%) carried at least one nucleotide substitution in the VH gene segment (average 4.4 VH mutations). Table 2 below provides a summary of V(D)J rearrangements recovered from bone marrow plasma cells of ΔgD:HSV-vaccinated mice. As shown in Table 2, CDR3 exhibits conserved residues (C and W for HDCR and C and F for LCDR). With the exception of BMPC-7, IgG2b or IgG2c clones (gray rows in Table 2) carry VH and VL mutations, suggesting that their plasma cell clones are generated via the GC response.

[0164] [Table 2]

[0165] [Table 3]

[0166] Protection by serum antibodies induced by ΔgD-2 vaccination depends on the activation of FcγRIV. We focused on IgG2c antibodies that preferentially bind to activated FcγR. Based on the V(D)J sequences obtained from each plasma cell, the paired heavy and light chains were reexpressed in Expi293F cells (as recombinant mouse IgG1 and IgG2c antibodies) as described above. Nine clones carrying the constant regions of IgG2b and IgG2c were selected (BMPC-1, BMPC-12, BMPC-7, BMPC-8, BMPC-9, BMPC-23, BMPC-26, BMPC-57, and BMPC-61). Of these nine antibodies, BMPC-23 reacted with HSV-2G lysate, but the others did not (Figures 5A and 5B and data are not shown). The binding of BMPC-23 was equivalent to that of another HSV-2G lysate-reactive IgG cloned from GC B cells of 19G7, ΔgD-2 vaccinated mice. BMPC-23 IgG1 and IgG2c isotypes were analyzed in an in vitro FcγR-activation assay (Figure 4) and an in vivo protection assay (see description below, Figures 6A-6C). As shown in Figure 4, BMPC-23 IgG2c showed the highest level of FcγR activation.

[0167] Both IgG1 and IgG2c forms of the 22D10, 33B8, and BMPC-23 antibodies (≥4 mg each) were evaluated for reproducibility in in vivo protection assays and any dependence of protection on Ig isotype. Representative experiments are shown in Figures 6A–6C. Briefly, the overall results from two independent experiments demonstrate that the IgG2c version of BMPC-23 protected 8 / 10 mice, 22D10 protected 5 / 10 mice, and 33B8 protected 4 / 10 mice from lethal challenge. The three monoclonal antibodies BMPC-23, 22D10, and 33B8 (as IgG2c) provided significant protection against HSV-2. To test the role of Ig isotype in the ability to provide protection, IgG1 and IgG2c isotypes of these three antibodies were generated and evaluated for their ability to activate mFcγRIV signaling (as a measure for ADCC) and in in vivo protection assays. The results are shown in Figure 4.

[0168] IP-MS was performed to identify the targets of the 22D10, 33B8, and BMPC-23 antibodies. The results are shown in Figures 7A and 7B, and human herpesvirus glycoprotein B (gB) was identified as the target.

[0169] BMPC-23 binds to HSV-1 gB (gB-1). The binding of the antibody to gB-1 was determined by a Luminex multiplex assay. A mixture of antigen (gB-1) conjugate microspheres (1,000 counts each) was added to each well of a 96-well filter plate (Millipore). After washing with PBS containing 1% BSA, 0.05% Tween 20, and 0.05% NaN3, serially diluted recombinant antibodies (5E7, 19G7, 18G4, 22E10, 3A2, 17G3, 22D10, 32H6, 33B8, 25H7, BMPC-23, and S5V2-29) were added to the plate, and the plate was incubated at room temperature for 2 hours with gentle agitation. The samples were diluted in PBS containing 1% milk, 1% BSA, 0.05% Tween 20, and 0.05% NaN3. After washing, PE goat anti-mouse IgG was added to the plates and incubated at room temperature for 1 hour with gentle agitation. After washing, the microspheres were resuspended in PBS containing 1% BSA, 0.05% Tween 20, and 0.05% NaN3, and the fluorescence signals from each microsphere were measured using a Bio-Plex® 3D instrument. As shown in Figure 8, BMPC-23 was the only antibody that demonstrated substantial binding to gB-1.

[0170] Discovery of gB-1-specific HSV antibodies. After Nojima culture (single B cell culture), HSV-specific antibodies were cloned from germinal center B cells and tested for binding to HSV-1 gB-1 using the Luminex multiplex assay as described above. Figures 9A and 9B show the gB-1 binding results for 10 antibodies compared to BMPC-23. Table 3 shows the cell origin and isolation methods for the tested antibodies. Except for BMPC-23, the antibodies in Table 3 were derived from germinal center B cells (lymph nodes) collected 15-17 days after initial vaccination with ΔgD-2. BMPC-23 was derived from bone marrow plasma cells collected 2 weeks after ΔgD-2 boost. Table 4 shows the nucleotide and amino acid sequences of the heavy and light chains, which are the amino acid sequences of the light chain and heavy chain CDR1, CDR2, and CDR3.

[0171] Discovery of BMPC-like antibodies. Nine of the ten gB-1-specific mouse monoclonal IgG antibodies shown in Figure 3 were tested for their ability to inhibit the binding of BMPC-23 human IgG antibody to gB-1. Serially diluted gB-1-specific mouse monoclonal antibodies or unrelated control mouse antibody (H33Lγ1) were incubated with gB-1 conjugate Luminex beads. After incubation, a fixed concentration (2 ng / ml) of BMPC-23 human IgG antibody was added to each well. After washing, mouse anti-human IgG-PE was added to detect binding of human BMPC-23 antibody. The y-axis shows the MFI percentage of the maximum binding, determined as the mean MFI in the presence of control IgG, H33Lγ1, which does not bind to gB-1. Figure 10A shows that BMPC-23 mouse IgG (at 222 ng / ml) inhibited human BMPC by >99%. The potent inhibition by HSV010-4 (>95% at 222 ng / ml), HSV010-7 (>90% at 222 ng / ml), and HSV010-34 (>80% at 222 ng / ml), shown in Figures 10B, 10F, and 10G respectively, indicates that these antibodies bind to the gB-1 epitopes in close overlap with those recognized by BMPC-23. Therefore, similar protective efficacy to that of BMPC-23 is predicted from these antibodies (experiments in progress). The mild inhibition by HSV010-28 (30% inhibition at 222 ng / ml in Figure 10D) and HSV010-6, 9, 13, and 14 (32%-48% at 222 ng / ml in Figure 10E) indicates that the gB-1 epitopes recognized by these antibodies partially overlap with those recognized by BMPC-23. The lack of inhibition by HSV010-20, shown in Figure 10C, indicates that the epitopes recognized by this antibody do not overlap with those recognized by BMPC-23. The protective efficacy of these antibodies needs to be tested (experiments are ongoing). The above data is summarized in Table 3 below.

[0172] Additional experiments. Target antigen determination for antibodies 22D10 and 33B8 was performed using a method similar to that used for antigen determination of the BMPC-23 antibody. For example, methods such as mass spectrometry of the protein conjugated by the 22D10 or 33B8 antibody and / or screening of 22D10 or 33B8 against other non-gB purified antigens from HSV-1 can be used.

[0173] The ability of the 12 non-test antibodies listed in Table 3 to protect against HSV-1-induced eye infections is evaluated using the method described above.

[0174] The epitope structures of the HSV-1 binding antibodies shown in Table 3 were determined. Methods for this include cocrystallization of Fab fragments of the antibody with the HSV-1 protein (e.g., gB-1), and analysis by X-ray diffraction and XDS (see, for example, Watanabe et al, 2019, Cell, 177, 1124-1135).

[0175] [Table 4]

[0176] [Table 5]

[0177] [Table 6]

[0178] [Table 7]

Claims

1. A herpes simplex virus type 2 (HSV-2) glycoprotein B (gB) conjugated antibody or antigen-binding fragment, wherein the heavy chain CDR is sequentially: GYSFTTYD (Sequence ID 1), IYPREGST (Sequence ID 2), and ATYGSSRYYTMDY (Sequence ID 3) The heavy chain variable region includes the complementarity determination region (CDR); and The light chain CDRs are in order: ESVDNFGISF (Sequence ID 4), AAS (Sequence ID 5), and QQSKEVPLT (Sequence ID 6) This is the light chain variable region, which includes the complementarity determination region (CDR). An antibody or antigen-binding fragment comprising, where the antibody is an Fc gamma receptor (FcγR) activating antibody.

2. The antibody or antigen-binding fragment according to claim 1, having an IgG2c isotype.

3. The antibody or antigen-binding fragment according to claim 1 or 2, which is a humanized antibody.

4. The antibody or antigen-binding fragment according to claim 1 or 2, comprising an Fc region that binds to the human Fc gamma receptor RIII (FcγRIII).

5. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the light chain framework region, the heavy chain framework region, or a combination thereof is a human framework region or has 85% or more identity with the human framework region.

6. A nucleic acid encoding the heavy chain variable region of an antibody, wherein the heavy chain variable region is comprised of the following heavy chain CDRs in order: GYSFTTYD (Sequence ID 1), IYPREGST (Sequence ID 2), and ATYGSSRYYTMDY (Sequence ID 3) A nucleic acid having an amino acid sequence that includes a complementarity-determining region (CDR).

7. The nucleic acid according to claim 6, further comprising a nucleic acid sequence encoding a human heavy chain framework region.

8. The nucleic acid according to claim 6 or 7, further comprising a nucleic acid sequence encoding a human Fc region.

9. A nucleic acid encoding the light chain variable region of an antibody, wherein the light chain variable region is comprised of the light chain CDRs in the following order: ESVDNFGISF (Sequence ID 4), AAS (Sequence ID 5), and QQSKEVPLT (Sequence ID 6) A nucleic acid having an amino acid sequence that includes a complementarity-determining region (CDR).

10. The nucleic acid according to claim 9, further comprising a nucleic acid sequence encoding a human light chain framework region.

11. A host cell comprising one or more nucleic acids according to any one of claims 6 to 10.

12. The antibody is an anti-HSV glycoprotein B antibody or antigen-binding fragment containing the variable heavy chain nucleic acid sequence of SEQ ID NO: 19 and the light chain nucleic acid sequence of SEQ ID NO:

21.

13. The antibody is an anti-HSV glycoprotein B antibody or antigen-binding fragment containing the variable heavy chain amino acid sequence of SEQ ID NO: 20 and the light chain amino acid sequence of SEQ ID NO:

22.

14. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 5, 12, or 13 in the manufacture of a pharmaceutical product for inhibiting the activity of herpes simplex virus type 2 (HSV-2) in a target.

15. The use according to claim 14, wherein the subject is infected with HSV-2.

16. The use according to claim 14, wherein the subject is not yet infected with HSV-2.

17. The use according to any one of claims 14 to 16, wherein the subject is immunodeficient.

18. The use according to any one of claims 14 to 17, wherein the subject is pregnant or a newborn.

19. The use according to any one of claims 14 to 18, wherein the subject is currently receiving or has previously received an antiviral drug.

20. The use according to claim 19, wherein the antiviral agent comprises acyclovir.

21. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 5, 12, or 13, and a pharmaceutically acceptable carrier or excipient.

22. The pharmaceutical composition according to claim 21, further comprising a small molecule antiviral agent.

23. The pharmaceutical composition according to claim 22, wherein the small molecule antiviral drug is acyclovir.

24. The pharmaceutical composition according to any one of claims 21 to 23, wherein an antibody or antigen-binding fragment is present in an effective amount for treating or preventing a disease or condition associated with herpes simplex virus type 2 (HSV-2) infection in a subject.

25. Use of an effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 5, 12, or 13 for the manufacture of a pharmaceutical product for treating or preventing a disease or condition associated with herpes simplex virus type 2 (HSV-2) infection in a subject.

26. Use of an antibody or antigen-binding fragment encoded by a nucleic acid according to any one of claims 6 to 10 for the manufacture of a pharmaceutical product for treating or preventing a disease or condition associated with herpes simplex virus type 2 (HSV-2) infection in a subject.

27. ​​Use of an antibody or antigen-binding fragment encoded by a nucleic acid according to any one of claims 6 to 10 in the manufacture of a pharmaceutical product for inhibiting herpes simplex virus type 2 (HSV-2) activity in a subject.

28. The use according to claim 27, wherein the subject is infected with HSV-2.

29. The use according to claim 27, wherein the subject is not yet infected with HSV-2.

30. The use according to any one of claims 27 to 29, wherein the subject is immunodeficient.

31. The use according to any one of claims 27 to 30, wherein the subject is pregnant or a newborn.

32. The use according to any one of claims 27 to 31, wherein the subject is receiving or has received an antiviral drug.

33. The use according to claim 32, wherein the antiviral agent comprises acyclovir.

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