Antibodies and antigen-binding fragments against CD155, and their methods of use.

Abstract

We provide antibody-based therapeutics against CD155 that can treat CNS cancers and systemic cancers. [Solution] Humanized antibodies or antigen-binding fragments specific to the poliovirus receptor (PVR) are used to prepare antibody-drug conjugates (ADCs) that target nucleic acids, peptides, proteins, drugs, and radiopharmaceuticals to cancer cells. These antibodies can be used for checkpoint blockade, blocking PVR (CD155) from binding to TIGIT. Humanized antibodies are used either alone or in combination with other checkpoint inhibitors known in the art. Humanized antibodies modulate the PVR-DNAM-1 axis to upregulate DNAM1 (CD226) expression on T cells or NK cells, thereby restoring the immune surveillance mechanism. The antibodies are incorporated into CAR-T cells or CAR NK cells.

Description

Technical Field

[0001] Reference to Sequence Listing This application contains an electronic sequence listing in a file named CD155 Sequence Listing_ST25, created on August 19, 2021, and containing 27 KB, which is incorporated herein by reference.

[0002] The present disclosure generally relates to the treatment of diseases using monoclonal antibodies. In particular, the present disclosure relates to targeting cells expressing either CD155 (PVR) or nectin 4 (or both) using the anti-CD155 antibodies or antigen-binding fragments (antibody fragments) disclosed herein. The antibodies to CD155 disclosed in this application bind to the poliovirus receptor and also bind to nectin 4. CD155 and nectin 4 are receptors that are overexpressed in many primary and metastatic tumors. CD155 and nectin 4 have also been identified as ligands for the checkpoint molecule, T cell immunoglobulin and ITIM domain (TIGIT).

Background Art

[0003] The CD155 / PVR and receptor family are present in various tissues of humans and other animals. In mice, the mouse ortholog of CD155 is known as Tage4. CD155 and nectin 4 have been described as novel checkpoints. The CD155 / PVR receptor may play a role in normal development, but this receptor is overexpressed in certain pathological conditions including glioblastoma multiforme (GBM), breast cancer including triple-negative breast cancer (TNBC), leukemia (including AML), sarcoma, ovarian cancer, colon cancer, pancreatic cancer, lung cancer, and rare but highly lethal cancers such as malignant peripheral nerve sheath tumors. CD155 / PVR plays roles in cell adhesion, motility, apoptosis, proliferation, and metastasis. Modified polioviruses have been utilized as vectors for treating cancers that overexpress CD155 / PVR by direct local administration to the tumor.

[0004] Since the vast majority of the world's population has been vaccinated against poliovirus, and poliovirus-based therapies are likely to be neutralized by the immune system when administered systemically, systemic administration of modified poliovirus therapies for cancer is not feasible.

[0005] It would be advantageous to have antibody-based therapeutics against CD155 that can treat CNS cancers and systemic cancers. Such CD155 antibodies can also enter the central nervous system by crossing the blood-brain barrier, or by any other route, either as a therapeutic agent itself or as a delivery agent for another therapeutic agent. This disclosure shows that monoclonal antibodies targeting mouse CD155 / PVR can enter the central nervous system by systemic administration and can cross the blood-brain barrier in vivo in mice with GL261 tumors in the brain. In embodiments, it is also shown that monoclonal antibodies targeting human and mouse CD155 / PVR are internally translocatable and can be used to deliver and express payloads such as mRNA. Antibodies and peptides that cross the human blood-brain barrier are known. For example, antibodies or antigen-binding fragments against the transferrin receptor have been shown to cross the blood-brain barrier in animal models and, more recently, in humans. Sequences of single-domain antibodies or polypeptides such as FC5 and FC44 are also known in the art, and FC5 and FC44 cross the blood-brain barrier. Therapeutic agents incorporating blood-brain barrier-penetrating molecules are in or will be in clinical trials. Antibodies or ligands against insulin receptors and insulin growth factor 1 receptors are yet another example of peptides and proteins that can cross the blood-brain barrier and fuse with the anti-CD155 antibodies described in this invention. In embodiments, the anti-CD155 antibody is fused with a protein or peptide either chemically or through recombinant molecular biology to enable the anti-CD155 antibody to cross the blood-brain barrier. Anti-CD155 antibodies, including D171, Ab825, and any humanized versions thereof, can be fused or chemically ligated with peptides or proteins (or antibodies) such as FC5, FC44, anti-insulin-like growth factor receptor type 1 (IGF1R), and anti-transferrin receptor antibodies (against transferrin receptor 1 or transferrin receptor 2), and antigen-binding fragments, or produced by recombinant technology. Such constructs facilitate the crossing of the anti-CD155 antibody across the blood-brain barrier. [Overview of the project]

[0006] CD155 and nectin 4 have been described as novel checkpoints, and the roles of these novel checkpoints are of great importance in immuno-oncology. When CD155 binds to T cell immunoglobulin and the ITIM domain (TIGIT), T cells and NK cells are inhibited. Recently, it has also been shown that nectin 4 binds to TIGIT, and thus tumors expressing nectin 4 are thought to inhibit immune cells, particularly T cells. Furthermore, soluble CD155 levels are generally higher in tumor patients who overexpress CD155. Elevated levels of membranous CD155 and soluble CD155 (sCD155) may have at least partially immunosuppressive effects by downregulating DNAM1 (CD226). The antibodies against CD155 described in this invention can bind to CD155 (membranous or soluble CD155) and result in upregulation of DNAM1 (CD226). Furthermore, antibodies that bind to both CD155 and nectin 4 may be used to target cancers that overexpress either CD155 or nectin 4 (or cancers that express both CD155 and nectin 4). Anti-CD155 antibodies can also be used to block checkpoint pathways, or they can be used to produce bispecific antibodies that bind to CD155 (or nectin 4) and also to T cells or NK cells, thereby enabling T cells or NK cells to kill tumor cells. To date, no CD155-targeted ADCs are in clinical trials and none have been approved by the FDA. However, enfortumab vedontin, which targets nectin 4, is an ADC approved by the FDA for the treatment of bladder cancer. Antibody-drug conjugates may be prepared using the humanized antibodies described herein to treat cancer patients that overexpress CD155 (or nectin 4). As part of this disclosure, it is shown that an antibody against CD155 delivers the payload. In this case, mRNA was delivered to cancer cells that overexpress CD155, and expression occurred.Furthermore, antigen-binding fragments that bind to CD155 or nectin 4 can be incorporated into chimeric antigen receptors of immune cells (i.e., CAR T cells and CAR NK cells) for the treatment of tumors expressing either nectin 4 or CD155. Antibodies or antigen-binding fragments against CD155 can also be used in combination with other small molecule therapeutics such as tyrosine kinase inhibitors and PARP inhibitors, as well as in combination with antibodies against known checkpoint targets. The antibodies of this disclosure can be used in combination with one or more checkpoint inhibitors targeting CTLA4, PD1, PDL1, CD112R, OX40, TIGIT, NKG2A, CEACAM1, B7H3, B7-H4, VISTA, LAG3, CD137, KIR, TIM1, TIM3, LAIR1, HVEM, BTLA, CD160, CD200, CD200R, and A2r, as well as other checkpoints well known in the art.

[0007] In embodiments, the antibodies and antigen-binding fragments against CD155 and nectin 4 described herein may be developed as theranostics, wherein the antibody or antigen-binding fragment against CD155 includes one of the following radiolabels: (a) I-124, (b) gallium 68, or (c) lutetium 177, for either the diagnosis or treatment of cancer.

[0008] This disclosure relates to the treatment of various diseases using monoclonal antibodies against CD155 and nectin 4. Embodiments of this disclosure are methods for treating systemic or neurological disorders in mammals, comprising administering to a patient a composition comprising a nonviral ligand capable of binding to CD155, the ligand being conjugated to the therapeutic agent for the treatment of the neurological or systemic disorder. In embodiments, the neurological disorder is a primary or metastatic brain tumor. In embodiments, the neurological or systemic disorder is at least one from the group consisting of primary or metastatic cancers (i) glioblastoma multiforme; (ii) neuroblastoma; (iii) oligodendroglioma; (iv) glioma; (v) astrocytoma; (vi) anaplastic astrocytoma; and (vii) meningioma; and (viii) primary or metastatic cancers of breast cancer, lung cancer, kidney cancer, pancreatic cancer, and metastatic melanoma. In embodiments, the disorder is at least one from the group consisting of (i) breast cancer (including TNBC); (ii) lung adenocarcinoma; (iii) melanoma; (iv) ovarian cancer; (v) AML; (vi) sarcoma; (vii) leukemia; (viii) bladder cancer; (ix) pancreatic cancer; (x) cervical cancer; (xi) colorectal cancer; (xii) epidermal carcinoma; (xiii) hepatocellular carcinoma; (xiv) malignant glioma; (xv) malignant peripheral nerve sheath tumor and melanoma, and in embodiments, the tumor overexpresses CD155. In embodiments, the tumor overexpresses nectin 4. In embodiments, the tumor expresses both CD155 and nectin 4.

[0009] In embodiments, the ligand is at least one from the group consisting of: (i) an antibody against CD155 or nectin 4; (ii) an antigen-binding fragment against CD155 or nectin 4. In embodiments, the ligand is transmissible across the blood-brain barrier. In embodiments, the fusion protein comprises a first segment that binds to CD155 and a second segment that is a therapeutic agent. In embodiments, the antibody or antigen-binding fragment is conjugated to a therapeutic agent, which is at least one from the group consisting of: (ii) a prodrug; (iii) siRNA; (iv) antisense DNA or RNA; (v) messenger RNA; and (vi) guide RNA for CRISPR; (vii) plasmid; (vii) single-stranded or double-stranded oligonucleotide (RNA or DNA); (viii) peptide; (ix) protein; (x) gene; and (xi) toxin. In the embodiments, the ligand can bind to nectin, nectin 2 (CD112), nectin 4, Neel, PVRL1, Necl-1, Necl-4, poliovirus receptor-associated protein 1, or poliovirus receptor-associated protein 2. In the embodiments, the antibody or antigen-binding fragment drug conjugate further comprises liposomes or lipid nanoparticles (LNPs) that encapsulate the therapeutic agent. In the embodiments, the liposomes are pegylated liposomes crosslinked to the ligand. In the embodiments, the ligand is a D171 antibody, or an antibody having a heavy chain variable region sequence or a light chain variable region sequence having more than 75% homology to the D171 heavy chain variable region sequence or the D171 light chain variable region sequence.

[0010] In the embodiment, the anti-CD155 antibody specifically binds to intracellular, transmembrane, or extracellular epitopes of CD155. In the embodiment, D171 specifically binds to amino acids 35-50 of CD155. In the embodiment, the antibody or antigen-binding fragment against CD155 binds to a tumor and induces cancer growth or an immune response against the tumor. In the embodiment, at least one antibody selected from the group consisting of a complete antibody, an antigen-binding fragment, Fab, Fab2, Fc, and scFv, the variable regions of the light chain and / or heavy chain, comprises a first component that binds to CD155 for binding to T cells or NK cells or other immune cells, or for stimulating antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), and a second component that binds to effector cells. In the embodiment, at least one is selected from monoclonal antibodies, polyclonal antibodies, humanized antibodies, and chimeric antibodies. In the embodiment, the effector cell is at least one from the group consisting of lymphocytes, including natural killer cells and cytotoxic T cells. In embodiments, the antibody is administered by at least one method from the group consisting of intravenous, intraarterial, intratumoral, subarachnoid, intramuscular, subcutaneous, intravesical (i.e., bladder), intraperitoneal, and via convective delivery.

[0011] Embodiments include at least one antibody selected from the group comprising a complete antibody, an antigen-binding fragment, Fab, Fab2, Fc, and scFv, or other antigen-binding fragments containing a first component that binds to CD155, and a second component that binds to effector cells to stimulate antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity (CDC). Embodiments of the present disclosure are methods for delivering a therapeutic agent to the central nervous system by either systemic or topical administration. In one embodiment, the anti-CD155 antibody or antigen-binding fragment is capable of crossing the mammalian blood-brain barrier, and the administration of a CD155-targeting nonviral ligand into the mammalian bloodstream comprises the nonviral ligand being conjugated with the therapeutic agent. In the embodiment, the mammal has a brain tumor. In the embodiment, the mammal suffers from a non-tumor-related adverse brain condition. In one embodiment, the anti-CD155 antibody or antigen-binding fragment is either therapeutic in itself or delivers a therapeutic agent for the treatment of systemic diseases such as kidney cancer, pancreatic cancer, ovarian cancer, lung cancer, colon cancer, and sarcomas such as malignant peripheral nerve sheath tumors (MPNSTs).

[0012] In embodiments, the nucleic acid-based drug is conjugated to an anti-CD155 antibody or antigen-binding fragment. In embodiments, the nucleic acid-based drug is conjugated to an anti-nectin 4 antibody that also binds to CD155. Embodiments of the present disclosure are compositions comprising a humanized D171 antibody or humanized Ab825. Embodiments of the present disclosure are humanized anti-CD155 antibodies, such as humanized Ab825, which contain more than 10 selenocysteine ​​residues from the C-terminus.

[0013] Embodiments of this disclosure include a humanized D171 antibody or humanized Ab825 antibody containing an Fc region optimized for enhancing ADCC. Embodiments of this disclosure include a humanized D171 antibody or humanized Ab825 antibody optimized for CDC. Embodiments of this disclosure include a humanized D171 antibody or humanized Ab825 antibody conjugated to a contrast agent for MRI / CT. Embodiments of this disclosure include a humanized D171 antibody or humanized Ab825 antibody conjugated to a diagnostic agent for PET scans. Embodiments of this disclosure include a humanized D171 antibody or humanized Ab825 antibody conjugated to a fluorescent probe such as IR800. Embodiments of this disclosure include a humanized D171 antibody or humanized Ab825 antibody conjugated to a radiosensitizer. Embodiments of this disclosure include a humanized D171 antibody or humanized Ab825 antibody conjugated to a chelator. Embodiments of this disclosure are humanized D171 antibody or humanized Ab825 antibody conjugated to a radioisotope. Embodiments of this disclosure are humanized D171 antibody or humanized Ab825 antibody conjugated to nucleic acid therapeutics such as mRNA, siRNA, RNA or DNA oligonucleotide, or single-stranded or double-stranded DNA. Embodiments of this disclosure are humanized D171 antibody or humanized Ab825 antibody, where the humanized D171 antibody or humanized Ab825 antibody is a therapeutic agent. Embodiments of this disclosure are humanized D171 antibody or humanized Ab825 antibody, where the humanized D171 antibody or humanized Ab825 antibody blocks the binding of CD155 (poliovirus receptor) to TIGIT. Embodiments of this disclosure are humanized D171 antibody or humanized Ab825 antibody, where the humanized D171 antibody or humanized Ab825 antibody blocks the binding of nectin 4 to TIGIT.

[0014] Embodiments of the present disclosure are humanized D171 antibody or humanized Ab825 antibody, which block CD155 (poliovirus receptor) and subsequently upregulate DNAM1 expression on NK cells or T cells (or both). Embodiments of the present disclosure are humanized D171 antibody or humanized Ab825 antibody, which block CD155 (poliovirus receptor) and subsequently upregulate DNAM1 expression on T cells. All of the above also applies to antigen-binding fragments for CD155 and nectin 4.

[0015] Embodiments of the present disclosure are methods for identifying the extent of tumors in a CD155-overexpressing mammal, comprising the step of administering an antibody or antigen-binding fragment that binds to CD155, the antibody or antigen-binding fragment being conjugated with a label. In embodiments, the label is at least one selected from the group consisting of radioisotopes, chromophores, fluorophores, and enzymes. In embodiments, the label is selected from the group consisting of gamma-ray or positron-emitting radioisotopes, magnetic resonance imaging contrast agents, X-ray contrast agents, and ultrasound contrast agents. In embodiments, selenocysteine ​​residues are used to conjugate the label with an antibody or antigen-binding fragment against CD155. In embodiments, imaging is performed by at least one technique selected from the group consisting of CT, ultrasound, MRI, SPECT, and PET. In embodiments, imaging is performed during at least one period selected from the group consisting of preoperative, intraoperative, and postoperative. Embodiments of the present disclosure are methods for delivering a therapeutic agent across the blood-brain barrier, the therapeutic agent being conjugated with an anti-CD155 antibody or antigen-binding fragment. The above provides a fairly broad overview of the features of this disclosure in order to better understand the detailed explanation below. Additional features and advantages of this disclosure are described below, and these form the subject matter of the claims. In embodiments, the primary or metastatic tumor overexpresses CD155. In embodiments, the primary or metastatic tumor overexpresses nectin 4.

[0016] In embodiments, the anti-CD155 antibody or antigen-binding fragment described herein is expressed as part of therapeutic chimeric antigen receptor T cells (CART) or therapeutic chimeric antigen receptor NK cells for the treatment of CD155-positive tumors. In embodiments, the humanized antibody or antigen-binding fragment described herein is expressed as part of therapeutic chimeric antigen receptor T cells (CART) or therapeutic chimeric antigen receptor NK cells for the treatment of nectin 4-positive tumors.

[0017] In the embodiment, the anti-CD155 antibody or antigen-binding fragment can cross the blood-brain barrier. In the embodiment, the fusion protein comprises at least two segments, the first segment binding to CD155 or nectin 4, and the second segment facilitating crossing of the blood-brain barrier. In the embodiment, the cell therapy comprises T cells expressing an antigen-binding fragment that binds to CD155 and / or nectin 4. In the embodiment, the method further comprises liposomes or polyethyleneimine (PEI) polymers or lipid nanoparticles (LNPs). In the embodiment, a humanized D171 antibody or an antibody having at least 90% homology to the D171 antibody specifically binds to amino acids 35-50 of CD155. In the embodiment, the anti-CD155 antibody or antigen-binding fragment binds to a primary or metastatic tumor expressing CD155 and / or nectin 4, and after binding, induces an immune response against the primary or metastatic tumor.

[0018] In embodiments, at least one antibody or fragment selected from the group consisting of complete antibodies, antigen-binding fragments, Fab, Fab2, Fc, and scFv comprises a first component that binds to CD155 and a second component that binds to effector cells. In embodiments, at least one selected from the group consisting of complete antibodies, antigen-binding fragments, Fab, Fab2, Fc, and scFv is selected from monoclonal antibodies, humanized antibodies, and chimeric antibodies. In embodiments, a patient may be treated by injecting the patient with an antibody or antigen-binding fragment, or DNA encoding any one of the above antibodies (or antigen-binding fragment), the injection of which is at least one of intravenous, intra-arterial, intramuscular, intrathecal, intraventricular, intratumoral, subcutaneous, or intralymphatic, intravesicular (i.e., bladder cancer or ventricular tumor of the CNS), intratumoral, and convective delivery.

[0019] Embodiments of the present disclosure are methods for delivering a therapeutic agent across the mammalian blood-brain barrier, comprising administering a CD155-targeting nonviral ligand into the mammalian bloodstream, wherein the nonviral ligand is conjugated with the therapeutic agent. In embodiments, the nonviral ligand is a monoclonal antibody or antigen-binding fragment as described in this application. In embodiments, the therapeutic agent is encapsulated in liposomes, which are conjugated with a monoclonal antibody or antigen-binding fragment that binds to CD155 and / or nectin 4. In embodiments, the mammal has a brain tumor. In embodiments, the mammal has a cerebrovascular condition, and the cerebrovascular condition is not a tumor. In embodiments, the therapeutic agent is conjugated with an anti-CD155 antibody or antigen-binding fragment. In embodiments, the therapeutic agent is conjugated with an anti-CD155 antibody or anti-CD155 antigen-binding fragment and administered to a mammal for the treatment of motor neurons in the brain, brainstem, spinal cord, or other CD155-expressing cells within the brain. Embodiments of the present disclosure are methods for delivering an antibody to the mammalian brain by fusing an antibody or antigen-binding fragment of another therapeutic antibody to an anti-CD155 antibody or antigen-binding fragment. In embodiments, the therapeutic antibody or therapeutic antigen-binding fragment is fusing to the anti-CD155 antibody or antigen-binding fragment by a method selected from the group consisting of chemical, antibody manipulation, or avidin-biotin-based crosslinking.

[0020] In embodiments, an anti-CD155 antibody or antigen-binding fragment that binds to CD155 is itself a therapeutic agent. In embodiments, an anti-nectin 4 antibody or antigen-binding fragment is itself a therapeutic agent. Embodiments of the present disclosure are methods for identifying the extent of tumors in a CD155-overexpressing mammal, comprising the steps of: administering a nonviral ligand targeting CD155 conjugated with a label; and imaging the brain tumor. In embodiments, the label is at least one selected from the group consisting of radioisotopes, chromophores, fluorophores, and enzymes. In embodiments, the label is selected from the group consisting of gamma-ray or positron-emitting radioisotopes, magnetic resonance imaging contrast agents, X-ray contrast agents, and ultrasound contrast agents. In embodiments, a selenocysteine ​​residue in the antibody or antigen-binding fragment is utilized to conjugate the label with the nonviral ligand. In embodiments, imaging is performed by at least one technique selected from the group consisting of CT, ultrasound, MRI, SPECT, and PET. In the embodiment, imaging is performed during at least one period selected from the group consisting of preoperative, intraoperative, and postoperative.

[0021] In one embodiment, a human antibody or humanized antibody against the human poliovirus receptor is modified with a selenocysteine ​​residue to facilitate stoichiometric conjugate to a reporter molecule as a therapeutic agent for the diagnosis or treatment of a disease. In another embodiment, a human antibody or humanized antibody against the human poliovirus receptor is conjugated with a therapeutic agent, such as a small drug molecule directly bound to or encapsulated in nanoparticles delivered to target cells that overexpress CD155, such as tumor cells. In yet another embodiment, a human antibody or humanized antibody against the human poliovirus receptor is conjugated to a fluorophore or other reporter molecule for non-surgical, pre-operative, intra-operative, or post-operative diagnosis or treatment of target cells that overexpress the CD155 receptor, such as tumor cells.

[0022] In one embodiment, a human antibody or humanized antibody against the human poliovirus receptor is fused with a therapeutic peptide either through antibody manipulation or chemical bonding. In another embodiment, a human antibody or humanized antibody against the human poliovirus receptor is fused with another therapeutic antibody (or antigen-binding fragment) either chemically or through antibody manipulation to produce a bispecific antibody, one of which is a human antibody or humanized antibody (or antigen-binding fragment) against the human poliovirus receptor. In yet another embodiment, a human antibody or humanized antibody against the human poliovirus receptor binds to tumor cells that overexpress CD155.

[0023] In embodiments, a human antibody or humanized antibody against the human poliovirus receptor also binds to tumor cells overexpressing nectin 4. Embodiments of the Disclosure include a humanized D171 antibody or humanized Ab825, or an antibody having a sequence similar to that of the D171 antibody, comprising at least one selected from the group consisting of: selenocysteine ​​residues more than 10 amino acids from the C-terminus; an Fc region optimized to enhance ADCC; CDC optimization; binding to contrast agents for MRI / CT; binding to diagnostic agents for PET scans; binding to fluorescent probes; binding to radiosensitizers; binding to chelating agents; binding to radioisotopes; and binding to therapeutic agents, where the humanized anti-CD155 antibody is a therapeutic agent. Embodiments of the Disclosure include a humanized anti-CD155 antibody or antigen-binding fragment disclosed herein that binds to the poliovirus receptor and inhibits the binding of the poliovirus receptor to TIGIT. Another embodiment includes a humanized anti-CD155 antibody and antigen-binding fragment disclosed herein that binds to nectin 4 and inhibits the binding of nectin 4 to TIGIT.

[0024] The above provides a fairly broad overview of the features of this disclosure in order to better understand the detailed explanation below. Additional features and advantages of this disclosure are described below, and these form the subject matter of the claims. [Brief explanation of the drawing]

[0025] To obtain the above and other enhancements and ways of achieving the objectives, a more specific description of the present disclosure briefly described above will be given by referring to its specific embodiments shown in the accompanying drawings. It should be understood that these drawings only show typical embodiments of the present disclosure and should not be considered as limiting the scope of the present disclosure.

[0026] [Figure 1] Mouse anti-IL13 alpha 2 antibody and mouse anti-CD155 antibody are shown to pass through the BBB in GL261 tumors in vivo and bind to mouse IL13 alpha 2 (mIL13 alpha 2) and mouse CD155 (mCD155). Fluorophore-labeled mouse anti-IL13 alpha 2 receptor antibody and fluorophore-labeled anti-CD155 antibody were injected into test mice. The fluorophore-labeled antibodies passed through the blood-brain barrier and accumulated in the GL261 tumors of C57B16 mice. The GL261 tumors express both receptors for mouse CD155 and mouse IL13 alpha 2 receptor. [Figure 2] It is shown that the D171 monoclonal antibody conjugated to eGFP mRNA encapsulated in liposomes internalizes in vitro and GFP is expressed in U87 tumors. U87 tumors overexpress human CD155. After exposing U87 cells to 10 ng / well of eGFP-encoding RNA containing liposomes functionalized with the D171 antibody conjugated to liposome-encapsulated eGFP mRNA for 24 hours, eGFP fluorescence in U87 cells was recorded. Three cell densities were investigated. [Figure 3] It is shown that fluoroscein-labeled mRNA encapsulated in liposomes and the D171 antibody against human CD155 conjugated to fluorescein were detected in approximately 80% of U87 cells. The D171 conjugate internalizes in U87 cells in vitro. U87 cells are known to overexpress CD155. [Figure 4]Comparison of tumor bioluminescence and pigment fluorescence imaging 72 hours after injection of Ab825-VT680 and control VT680 in mice bearing U87MG tumors is shown. The Ab825-VT680 conjugate binds to and accumulates in U87MG tumors in vivo. Control mice were either untreated or treated with only the VT680 pigment. As demonstrated by fluorescence imaging, mice treated with the fluorescently labeled anti-CD155 antibody showed accumulation within the tumors. Bioluminescence showed that the fluorescence accumulation region correlated with the tumor region seen by bioluminescence imaging. [Figure 5] Shows the potential mechanisms of action of anti-CD155 antibodies and antigen-binding fragments. CD155-TIGIT interactions and CD155-CD96 interactions (not shown) are targets for checkpoint blockade. Anti-CD155 antibodies and antigen-binding fragments can be used for (a) ADCC, (c) checkpoint blockade, (d) radioimmunotherapy, (e) antibody-drug conjugates (ADCs), (f) bispecific antibodies where one binds to CD155 and the other binds to a second receptor such as CD3 on T cells, and (g) preparation of anti-CD155 CAR T or CAR NK cells. Anti-CD155 antibodies and antigen-binding fragments also bind to nectin 4, and the constructs described herein can also target nectin 4. [Figure 6] DC T cell assays in 50 patient samples using humanized monoclonal antibodies VH3 N54S D56G / Vk2 N92Q (Sample 2) and VH3 N54S D56G / Vk3N92Q (Sample 3) and VH4 N54S D56G / Vk3N92Q (Sample 4) are shown. The frequency of proliferation (% of patient samples with proliferation) and stimulation index (SI) for each of the tested antibodies and controls are displayed. [Figure 7]This report presents the results of a binding assay demonstrating potent and specific binding of a humanized antibody against CD155 (PVR) to the cell membrane receptors PVR and Nectin 4. Cell fixation was not performed. Inverse hits were observed with CD226 (DNAM1) and TIGIT, which are natural ligands for PVR. The assay was performed on over 5000 membrane receptors. Strong and specific binding was observed between PVR and Nectin 4. Inverse hits (white spots) were observed with TIGIT and CD226 because these receptors are ligands for PVR and bind to endogenous PVR in the assay, generating inverse hit spots. [Figure 8] Cell binding studies of humanized monoclonal antibodies against CD155 using EC50 in HAP1 cells, Vero cells, and U87 cells. Compared with variants having N54Q D56E or N54S D56E in the HCVR or N92E in the LCVR, variants with N54S D56G in the heavy chain variable region (HCVR) and variants with N92Q in the light chain variable region (LCVR) showed higher binding affinity to CD155. [Modes for carrying out the invention]

[0027] The details provided herein are intended solely to illustrate preferred embodiments of the Disclosure and to provide the most useful and readily understandable explanation of the principles and conceptual aspects of the various embodiments of the Disclosure. In this regard, no attempt has been made to provide structural details of the Disclosure in any way that would be unnecessary for a basic understanding of the Disclosure, and the accompanying drawings will make it clear to those skilled in the art how some forms of the Disclosure can actually be materialized.

[0028] The following definitions and explanations are intended, or will be intended, to control any future constructions unless explicitly and unambiguously altered in the following examples, or unless the application of the meaning renders the construction meaningless or essentially meaningless. Where the construction of a term renders it meaningless or essentially meaningless, the definition must be quoted from Webster's Dictionary, Third Edition.

[0029] As used herein, “conjugated” means any mode of binding of a ligand to a therapeutic agent, including, but not limited to, the creation of a ligand-therapeutic fusion construct using any chemical, electrostatic, or molecular biological tool. The phrase “all molecular biological tools” includes, but is not limited to, the conventional manufacture of chimeric antigen receptors, where an antibody or antigen-binding fragment is incorporated into a chimeric antigen receptor in CAR T-cell therapy for the elimination of diseases including, but not limited to, cancer.

[0030] Antibodies (Abs), also known as immunoglobulins (Ig), are large, Y-shaped proteins produced by plasma cells and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. Antibodies recognize specific parts of foreign targets called antigens. Each end of the antibody's "Y" contains a paratope (a tablet-like structure) that is specific to one particular epitope (a key-like structure) on the antigen, allowing these two structures to bind precisely.

[0031] Bispecific monoclonal antibodies (BsMAb, BsAb) are artificial proteins composed of fragments of two different monoclonal antibodies, resulting in them binding to two different types of antigens. The most widely used application of this approach is cancer immunotherapy, where BsMAb are designed to simultaneously bind to receptors on cytotoxic cells (such as CD3 receptors on T cells) and receptors on tumor cells to kill tumor cells.

[0032] The blood-brain barrier (BBB) ​​is a highly selective permeable barrier in the central nervous system (CNS) that separates circulating blood from extracellular fluid (BECF). The blood-brain barrier is formed by capillary endothelial cells, which are connected by tight junctions with extremely high electrical resistivity of at least 0.1 Dm. The blood-brain barrier not only allows the passage of water, some gases, and lipid-soluble molecules by passive diffusion, but also enables the selective transport of molecules essential for nerve function, such as glucose and amino acids.

[0033] Conjugation includes, but is not limited to, molecular, chemical, and electrostatic bonds. An example of molecular bonding is antibody engineering. An example of chemical bonding is covalent bonding. Examples of electrostatic bonding are biotin and streptavidin.

[0034] CD155 is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. Because CD155 is involved in primate cell poliovirus infection, it is commonly known as the poliovirus receptor (PVR), and its normal cellular function is the establishment of intercellular adhesion junctions between epithelial cells. CD155 is a transmembrane protein with three extracellular immunoglobulin-like domains D1-D3, with D1 being recognized by the virus. Synonyms include PVR, CD155, HVED, NECL5, Necl-5, PVS, and TAGE4.

[0035] A fusion protein, or chimeric protein, is a protein produced by the fusion of two or more genes that originally encoded separate proteins. Translation of this fusion gene yields one or more polypeptides with functional properties derived from each of the original proteins.

[0036] Fusion proteins are produced through molecular biology techniques or through the chemical bonding of two antibody or antigen-binding fragments.

[0037] In embodiments, the antibody has functional groups that can be used for labeling, crosslinking, or modification by covalent modification. In embodiments, the functional groups on the antibody are incorporated primary amines, sulfhydryl groups, carbohydrates, selenocysteine, or unnatural amino acids.

[0038] In the embodiment, a crosslinking agent can be used to link genes, polypeptides, or small molecules to antibodies. In the embodiment, the crosslinking agent may be a heterobifunctional crosslinking agent. In the embodiment, the heterobifunctional crosslinking agent is succinimidylacetylthioacetate (SATA), succinimidyltrans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), N-((2-pinyldithio)ethyl)-4-azidosacilamide (PEAS;AET), 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester (ATFB, SE), benzophenone-4-maleimide, benzophenone-4-isothiocyanate, 4-benzoylbenzoic acid, succinimidyl ester, iodoacetamide azide, iodoacetamide alkyne, Click-iT maleimide The crosslinking agent is a DIBO alkyne, azide (PEO) 4-propionic acid, succinimidyl ester, alkyne, succinimidyl ester, or Click-iT succinimidyl ester DIBO alkyne. In another embodiment, the crosslinking agent may be a peptide crosslinking agent-sensitive cathepsin cleavage agent. In another embodiment, the crosslinking agent may be used for site-specific crosslinking, such as an azide crosslinking agent having a BCN for click chemistry. Using the available linkers, antibodies can be crosslinked to the payload based on click chemistry using azide (N3). The reactive functional group is the same at each end. The reactive ends often target primary amines and sulfhydryl groups. In an embodiment, the azide reacts with the alkyne via a copper-catalyzed azide-alkyne cycloaddition reaction. In another embodiment, a metal-free azide-alkyne reaction can be used as the conjugate. In an embodiment, the crosslinking agent reacts non-specifically with available sites upon UV irradiation.

[0039] Human antibodies are antibodies produced in human or other mammalian systems, or using yeast or phage technology. Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants naturally produced in humans. The "humanization" process is typically applied to monoclonal antibodies developed for administration to humans (e.g., antibodies developed as anticancer agents). Humanization may be necessary if the process of developing a particular antibody involves production in a non-human immune system (such as that in mice). Because the protein sequences of antibodies produced in this way are partially different from homologous antibodies naturally present in humans, they may exhibit immunogenicity when administered to human patients.

[0040] Antibody humanization involves removing potentially immunogenic sequences within non-human antibodies. Less immunogenic sequences may be inserted in place of the immunogenic ones. There are many methods for humanizing a particular antibody. In embodiments, humanizing a particular non-human antibody can take more than a year. In embodiments, a human antibody is humanized by: 1) examining its structure to determine which sequences are immunogenic to humans; 2) removing sequences that are immunogenic to humans; 3) maintaining or improving the antibody's ability to bind to its target receptor; 4) evaluating the antibody's binding to the target receptor; 5) screening for poor immunogenic responses; and 6) removing sequences that cause poor immunogenic responses. In embodiments, software is used to identify immunogenic sequences that may be removed. In embodiments, poor immunogenic responses may still exist even after removing sequences identified by the software, indicating the software is not accurate. In embodiments, in vitro studies are performed to determine which sequences to remove. If different parties humanize a particular antibody, the sequences of that particular antibody may differ. Mutations in the CDR may be necessary to remove potential aspartate isomerization and deamidation-prone sites. Because CDR mutations can affect binding, antibody humanization is often limited to framework residues. The goal of humanization and mutation of unstable sites is to stabilize potential deamidation and isomerization sites within the CDR while minimizing injection reaction risk and immunogenicity risk. Even after humanization, different humanized antibodies may have different dissociation constants (Kd), different profiles in cytokine release assays, and different immunogenicity profiles based on Episcreen and DC TCell Episcreen assays. Humanization of antibodies D171 or Ab825 has not been previously taught and is taught for the first time in this invention. Ab825 was found to have the highest injection reaction risk compared to humanized variants.

[0041] In embodiments, an anti-CD155 antibody may be fused to another therapeutic antibody to navigate the therapeutic antibody across the blood-brain barrier. In embodiments, the approved therapeutic antibody cannot cross the blood-brain barrier on its own. In embodiments, the anti-CD155 antibody travels retrogradely through nerves to enter the CNS. In embodiments, the anti-CD155 antibody travels across the blood-CSF barrier.

[0042] A ligand is a substance (usually a small molecule) that forms a complex with a biomolecule to perform a biological purpose. In protein-ligand binding, the ligand is typically a signal trigger molecule that binds to a site on the target protein. In many examples described herein, the target protein to which the ligand binds is a receptor on a cell. Antibodies or antigen-binding fragments that bind to CD155 are considered ligands for CD155.

[0043] Non-viral means any polypeptide containing DNA / RNA segments and proteins that lacks the structure necessary to be considered a virus. Non-viral further excludes any complex polypeptides that begin as a virus and are produced through a process of cleaving parts of the virus to generate polypeptides.

[0044] Polypeptides are linear chains of amino acid residues. Proteins contain at least one long polypeptide. Short polypeptides, containing approximately 20-30 residues, are rarely considered proteins and are generally called peptides, or sometimes oligopeptides. Individual amino acid residues are linked by peptide bonds and adjacent amino acid residues.

[0045] Proteins are large biomolecules, or macromolecules, consisting of long chains of one or more amino acid residues. Proteins differ primarily in their amino acid sequences, which are determined by the nucleotide sequences of their genes. Typically, the amino acid sequence determines how a protein folds into a specific three-dimensional structure and thus its activity.

[0046] Receptors are protein molecules typically embedded within the cell membrane surface of cells that receive chemical signals from outside the cell. When such chemical signals bind to receptors, some form of cell / tissue response is triggered, such as a change in the cell's electrical activity. In this sense, receptors are protein molecules that recognize and respond to endogenous chemical signals. CD155 is also known as the poliovirus receptor.

[0047] The "therapeutic dose" is the amount that eliminates or reduces the patient's tumor burden, or prevents, delays, or inhibits metastasis. The dose varies depending on many parameters, including the nature of the tumor, the patient's medical history, the patient's condition, the possibility of concomitant use of cytotoxic drugs, and the method of administration. Methods of administration include injection (e.g., parenteral, subcutaneous, intravenous, intraperitoneal, subarachnoid, convection-enhanced, etc.), in which case the molecule or complex that binds to PVR is provided in a non-toxic, pharmaceutically acceptable carrier.

[0048] In both mouse (against mouse CD155) and human (against human CD155) cell lines, monoclonal antibodies against the respective poliovirus receptors of these cell lines were used to deliver (for example) the mRNA or gene of the reporter molecule EGFP to CD155-expressing cells via anti-CD155 conjugated in nucleic acid-encapsulated liposomes. Using the D171 antibody, liposome-encapsulated mRNA was delivered to U87 tumors expressing CD155. Upon delivery of the D171 mRNA conjugate, GFP mRNA was expressed. This delivery system is extendable to other nucleic acids and drugs based on the results. The anti-CD155 monoclonal antibody unexpectedly entered the central nervous system and coated the tumor. Potential applications of this finding include the use of CD155 to facilitate the transcytosis of ligands and ligand conjugates across the blood-brain barrier or other barriers that prevent entry into the CNS, providing a potential alternative to the transferrin receptor. The fusion of therapeutic peptides or proteins with antibodies against CD155 or antigen-binding fragments against CD155 may provide a pathway to cross the blood-brain barrier or other barriers that prevent entry into the CNS. Antibodies against CD155 bind to CD155 in human and mouse cell lines and are internalized by mouse and human cells in vitro. Antibodies against mouse CD155 bind to tumors in vivo and cross the blood-brain barrier. Anti-CD155 antibodies have in vivo diagnostic potential for diagnosing CD155 tumors and for defining the boundaries of CD155 tumors preoperatively, intraoperatively, and postoperatively. Anti-CD155 antibodies may have therapeutic potential, either alone or fused with other peptides, polypeptides, or antibodies, inducing immune system responses such as antibody-targeted cytotoxicity. Various cargoes include toxins, proteins, peptides, drug small molecules, genes, messenger RNA, and RNA or DNA-based drugs such as oligonucleotides.Each of the above can be directly conjugated to an anti-CD155 antibody, or antibodies against CD155 can be conjugated to nanoparticles / liposomes that encapsulate RNA or DNA-based drugs such as toxins, proteins, peptides, drug small molecules, genes, messenger RNA, and oligonucleotides. Ligand-PVR interactions, such as anti-CD155 antibody-PVR interactions, can function as transcytosis pathways for macromolecules crossing the blood-brain barrier and may prove to be an alternative to transferrin. Any ligand that binds to an anti-CD155 antibody or PVR crossing the blood-brain barrier can also function as a transporter for drugs, proteins, peptides, and polypeptides fused to or conjugated to the anti-CD155 antibody or other PVR ligand.

[0049] Ligands that may be used in this disclosure include, but are not limited to, anti-CD155 AB, and further include fusion proteins of antibodies and antigen-binding fragments that bind to CD155 (and / or nectin 4), as well as such antibody and antigen-binding fragment fusion proteins. Furthermore, this includes ligand conjugates of liposomes and encapsulation of RNA or DNA or modified nucleic acids or other therapeutic agents, as well as ligand conjugates of nucleic acids or other therapeutic agents that do not involve liposomes.

[0050] In embodiments, antibodies against CD155 that can cross the blood-brain barrier include, but are not limited to, antigen-binding fragments (Fab, FcV), humanized anti-CD155 antibodies, anti-CD155 antibodies targeting conjugated liposomes / nanoparticles, and encapsulated or unencapsulated therapeutic agents containing toxins, gene, DNA or RNA-based drugs or other therapeutic agents; and anti-CD155 antibodies conjugated with fluorescent probes or contrast agents for the diagnosis of tumors or for identifying tumors or tumor margins that help outline tumors non-surgical, pre-operative, intra-operative or post-operatively. Selected from; anti-CD155 antibodies can be fused to proteins, peptides, toxins, or other antibody- or antigen-binding fragments to utilize CD155 for transcytosis and deliver across the blood-brain barrier, where, upon passage, either CD155 or the fusion protein, or both, can bind to target cells within the CNS; unconjugate antibodies against CD155 may function as therapeutic agents if they induce antibody-directed or complement-directed cytotoxicity in CNS and non-CNS tumors with high CD155 expression.

[0051] The mouse anti-CD155 antibody unexpectedly crossed the blood-brain barrier and bound to the tumor. Antibodies normally do not cross the BBB. The poliovirus itself has other pathways for entering the CNS. In embodiments, the antibody is used to treat a neurological disorder. In embodiments, it may be beneficial to deliver a substance across the blood-brain barrier when there is no tumor. In embodiments, the neurological disorder is not a brain tumor. The antibody is composed of various parts including, but not limited to, heavy chains, light chains, Fab, Fc, scFv, carbohydrates, variable region, and constant region.

[0052] Antibodies may be modified in ways including, but not limited to, glycosylation, alteration of amino acids in the constant region, use of different human mAh isotypes (such as IgG4), linking isotopes to mAhs using stable linkers, linking drugs to mAhs using cleavable linkers, inserting DNA from the mAb variable region fused with a signaling peptide into T cells to induce CAR expression, and crosslinking regions of two mAbs.

[0053] Monoclonal antibody-based (mAb-based) therapeutics have many functions, including, but are not limited to, antitumor mAbs, angiogenesis inhibitors, T cell checkpoint blockade, radioimmunotherapy, antibody-drug conjugates, bispecific antibodies, and chimeric antigen receptor T cells. One embodiment of the present invention includes a bispecific antibody in which one antibody or antigen-binding fragment binds to CD155 (or nectin 4) expressed on tumor cells, and the antibody or antigen-binding fragment binds to CD3 (T cell receptor) on T cells, thereby bringing the T cells closer to the tumor cells and enhancing T cell-mediated tumor death. Another embodiment includes a bispecific antibody in which one antibody or antigen-binding fragment binds to CD155 (or nectin 4) on tumor cells, and the other antibody or antigen-binding fragment binds to a receptor on NK cells (i.e., CD16, NKG2D, SLAM receptor, or a native cytotoxic receptor such as NKp46, NKp44, or NKp30 on NK cells), thereby bringing the NK cells closer to the tumor cells and enhancing NK cell-mediated tumor death. The immune-mediated effects of tumor-specific IgG include, but are not limited to, ADCC, opsonization, and CDC. The direct effects of tumor-specific IgG are ligand blockade, inhibition of receptor dimerization, and induction of apoptotic signaling. Bispecific formulations incorporating anti-CD155 antibodies and antigen-binding fragments together with antibodies or antigen-binding fragments that bind to type 1 insulin, such as growth factor receptors or FC5 or FC44 or transferrin receptors (Tfr1 or Tfr2), can be prepared to facilitate crossing the blood-brain barrier.

[0054] A potential limitation to the development of monoclonal antibodies for neuro-oncology is their inability to cross the blood-brain barrier (BBB). Monoclonal antibodies targeting brain tumors must first cross the BBB (if the antibody enters the brain and central nervous system from the vascular lumen) and then enter the central nervous system either by direct injection into the tumor (convection therapy) or by direct injection into the cerebrospinal fluid. Other mechanisms for entry into the CNS, such as retrograde entry through muscles or nerves, need to be proposed. Only after the monoclonal antibody has entered the CNS can it bind to a primary intracranial brain tumor (if the brain tumor has metastasized peripherally, the antibody can bind to the tumor without entering the CNS).

[0055] In one embodiment, mRNA can be internalized into cells when encapsulated within liposomes conjugated with an anti-CD155 antibody or antigen-binding fragment. The anti-CD155 antibody D171 binds to the poliovirus receptor at amino acid residues 35-50. In another embodiment, a humanized antibody against human CD155 is conjugated into liposomes containing antisense DNA or antisense RNA, or plasmids or genes having native or chemically modified residues. In yet another embodiment, the liposomes contain mRNA having native or chemically modified residues. The humanized antibody against human CD155 and the conjugated liposomes can cross the blood-brain barrier and deliver mRNA or nucleic acid therapeutics to CD155-expressing cells.

[0056] This disclosure relates to the viral delivery of therapeutic agents, including mRNA and genes, via ligand conjugates, where a ligand such as a monoclonal antibody binds to CD155 / PVR, resulting in the internalization and subsequent release of the therapeutic agent, enabling the expression of the drug or mRNA / gene (mRNA / gene). It is shown herein that a D171 antibody conjugated to liposome-encapsulated mRNA is internalizable, and the mRNA can deliver the payload to CD155-expressing cells in vitro where it is expressed in tumor cells. This proof of concept lays the foundation for the delivery of nucleic acid therapeutics and other drugs to cells using humanized anti-CD155 antibody drug conjugates.

[0057] In embodiments, the ligand may be 1) an antibody against PVR / CD155, 2) an antibody or antigen-binding fragment that binds to PVR / CD155, a fusion protein that binds to PVR / CD155, with part of it binding to CD155 / PVR and the other part being a toxin such as diphtheria, or 3) an antibody or antigen-binding fragment conjugated to a therapeutic or prodrug such as a nucleic acid or gene that binds to CD155 / PVR. The therapeutic or prodrug may be, for example, doxorubicin, siRNA, antisense DNA or other antisense molecules, messenger RNA encoding a protein or enzyme or peptide or cytokine, Cas9 mRNA, Cas12a mRNA and guide RNA for CRISPR, or a naked DNA gene or plasmid. Antibodies or antigen-binding fragments against CD155 can be conjugated to mRNA encoding one of the following cytokine mRNAs: (a) mRNA encoding IL2, (b) mRNA encoding IL7, (c) mRNA encoding IL12, (d) mRNA encoding IL15, (e) mRNA encoding IL21, (f) mRNA encoding IFN gamma, (g) mRNA encoding IFN alpha, and (i) mRNA encoding GM-CSF.

[0058] In embodiments, humanized anti-CD155 antibodies may be used to target neurons, astrocytes, regenerating muscle, and spinal cord. Antibodies against CD155 or against fragments or fusion constructs, either alone or as conjugates, play a preoperative, intraoperative, or postoperative diagnostic role in identifying the full extent of the tumor or the extent of tumor resection. In embodiments, GBM patients are treated preoperatively with a fluorescently labeled antibody against CD155, which crosses the blood-brain barrier or is injected directly into the tumor, and the contour of the antibody fluorescence guides the surgeon intraoperatively regarding tumor resection.

[0059] Soluble CD155 can be used as an antidote to overdose of anti-CD155 antibody conjugates by adding soluble CD155 to conjugate it to an anti-CD155 antibody. Furthermore, in one embodiment, prior to treating a patient with an anti-CD155 antibody drug conjugate, a non-conjugated anti-CD155 antibody is first administered to block CD155 on normal cells. Subsequently, the anti-CD155 antibody drug conjugate is added and directed towards tumors overexpressing CD155. Antibodies, fragments, or fusion constructs against CD155, either alone or as conjugates, have diagnostic applications for identifying tumors in the brain within a range of tumors in the brain by imaging mechanisms such as PET scans, MRI, nuclear scans, CT scans, ultrasound, and other imaging modes standard in the art.

[0060] IV. Conclusion Although the function of CD155 is not fully understood, it has been shown to bind to TIGIT (a T-cell immune receptor with Ig and ITIM domains), CD226 (DNAM1), and CD96. Soluble CD155, a prognostic biomarker for tumors, is often secreted by CD155-positive tumors and may function in binding to DNAM1. Membrane and soluble CD155 may function to suppress the immune system, thereby enabling the growth of CD155-positive tumors. Blocking soluble or membrane CD155 with anti-CD155 antibodies can reverse immunosuppression. Blocking CD155 with anti-CD155 antibodies also blocks the binding of CD155 to TIGIT, and therefore can reduce the inhibitory signal transmitted by CD155-TIGIT binding. Thus, CD155 is a novel checkpoint, and CD155 modulation with anti-CD155 antibodies provides a novel pathway for treating cancer using multiple mechanisms of action. CD155 is found at low levels in normal tissues such as muscle and kidney, but because it is overexpressed in many different cancers, CD155 has become a novel yet common target in the treatment of a wide variety of malignancies. Similarly, as described in this invention, the monoclonal antibodies of this invention bound not only to CD155 but also to another tumor marker, nectin 4. The binding of the monoclonal antibodies of this invention to nectin 4 has been shown for the first time. Monoclonal antibodies D171 and AB825 (Ab825 and AB825 are used interchangeably in this specification, figures, and claims) associated with the humanized antibodies of this invention have not been shown to bind to nectin 4. Recently, it has been shown that nectin 4 binds to TIGIT. In one embodiment, the antibody of this invention blocks the binding of both nectin 4 and PVR to TIGIT, thereby blocking inhibitory signals to immune cells such as T cells. In another embodiment, an antibody or antigen-binding fragment against CD155 is administered to humans for the treatment of cancer in combination with additional drugs, cell therapies, or immunomodulators consisting of antibodies against checkpoint molecules.Antibodies or antigen-binding fragments against CD155 are administered to humans in combination with additional immunomodulators consisting of antibodies against immune checkpoint molecules selected from the group comprising CTLA4, PD1, PDL1, CD112R, OX40, TIGIT, NKG2A, CEACAM1, B7H3, B7-H4, VISTA, LAG3, CD137, KIR, TIM1, TIM3, LAIR1, HVEM, BTLA, CD160, CD200, CD200R, and A2r.

[0061] In one embodiment, an anti-CD155 antibody or antigen-binding fragment is fused to an antibody or antigen-binding fragment known to cross the blood-brain barrier, such as an anti-transferrin receptor antibody or antigen-binding fragment (or an FC5 antibody or antigen-binding fragment that crosses the blood-brain barrier). The anti-CD155 antibody or antigen-binding fragment can be conjugated to an antibody (or antigen-binding fragment) that crosses the blood-brain barrier either chemically or by recombinant technology, thereby enabling the anti-CD155 antibody to cross the blood-brain barrier. In another embodiment, a bispecific antibody containing the antibody described in the present invention binds to CD155 (or nectin 4) on tumors and CD3 on T cells. In yet another embodiment, a bispecific antibody containing one or more of the antibodies described in the present invention binds to CD155 (or nectin 4) on tumors, and another portion of the bispecific antibody binds to a receptor on NK cells (i.e., Cd16, SLAM receptor, NKp46, NKp44, or NKp30 on NK cells). In embodiments, an anti-CD155 antibody or antigen-binding fragment is fused to an antibody or antigen-binding fragment known to cross the blood-brain barrier, such as an anti-transferrin receptor antibody or antigen-binding fragment (or an FC5 antibody or antigen-binding fragment that crosses the blood-brain barrier). The anti-CD155 antibody or antigen-binding fragment can be conjugated to an antibody (or antigen-binding fragment) that crosses the blood-brain barrier by either chemical or recombinant technology, thereby enabling the anti-CD155 antibody to cross the blood-brain barrier. The antibody of the present invention binds to and blocks PVR (CD155). The humanized monoclonal antibody of the present invention can be used to treat patients exposed to poliovirus by blocking the poliovirus receptor and preventing poliovirus from binding to the poliovirus receptor (PVR;CD155).

[0062] In one embodiment, the antibody of the present invention can be administered to mammals, including humans, to treat infectious diseases such as HIV by increasing the expression of DNAM1 (CD226) on T cells. Similarly, in another embodiment, the antibody of the present invention may be used to treat sepsis by increasing the expression of DNAM1 on T cells and Nk cells. Overdose of the humanized antibody against CD155 of the present invention is thought to be treatable by administering soluble CD155 to bind to the humanized antibody and neutralize its effect. In another embodiment of the present invention, AB825, D171, or any humanized antibody binds to PVR (CD155) in vivo in animals expressing human PVR. Human PVR may be an expressed transgene, or human PVR may be expressed on human cells, such as U87 tumor cells transplanted into mice. Prior to this study, neither Ab825 nor any of the humanized antibodies of the present invention had been injected in vivo into living animals. In another embodiment, the humanized antibodies against Ab825 and CD155 described in the present invention have been shown to bind to Vero cells derived from African green monkeys, and therefore these antibodies bind to non-human primate PVR(CD155) (Figure 8).

[0063] The D171 antibody is an anti-CD155 antibody conjugated to liposome-encapsulated mRNA, successfully delivered and expressed within human U87MG tumors. This study is the first to demonstrate the internalization of the anti-CD155 antibody (D171) and the internalization and expression of the liposomal mRNA conjugated to D171. This research provides fundamental support for the creation of antibody-drug conjugates of D171, AB825, and related humanized antibodies in the present invention. The internalization of D171 and its ability to deliver the payload to human tumor cells are also embodiments of the present invention. Incorporation experiments using eGFP-RNA-containing liposomes and fluorescein RNA-containing liposomes demonstrated a clear superiority of the anti-CD155 antibody conjugate over all other conjugates tested, although this was only at low concentrations that could be tested with readily available small amounts. Furthermore, the robustness of these results is enhanced because they were obtained with both human-specific and mouse-specific types of CD155 antibodies within the conjugates. The mRNA was expressed after being internalized within the U87 tumor. In another experiment, administration of fluorescein-labeled mRNA encapsulated in liposomes conjugated with D171 antibody resulted in fluorescein labeling of over 80% of U87 cells. This study provided the first foundational support for creating antibody-drug conjugates of D171, Ab825, and other related antibodies, such as the humanized antibody of the present invention. The internalization ability of D171, Ab825, and the humanized antibody in the present invention, as well as their ability to deliver payloads into human tumors, are also embodiments of the present invention. In addition to these results, only the anti-IL-13 alpha-2 conjugated liposomes showed mild but repeated uptake in various experiments with either a mouse version (GL261) or a human version (U87MG). RNA fluorescence labeling with fluorescein can be replaced with another, more potent fluorescent dye that is less sensitive to fluorescent dye bleed in high-content imaging acquisition. The uptake of functional liposomes was investigated in complete culture medium (containing 10% serum) under conditions close to in vivo conditions.GL261 or U87MG cells at three densities were treated with 10, 50, or 100 ng / well of eGFP-coding RNA in functionalized liposomes. Anti-CD155 antibody conjugates could only be tested at 10 ngRNA / well due to the very large quantities available. The results show that, for both mouse and human antibody conjugates, cell fluorescence repeatedly increases after application of 10 ngRNA / well of anti-CD155 antibody-conjugated liposomes at all cell densities tested.

[0064] Humanization of anti-CD155 antibodies Ab825 is an antibody that shares a sequence with the anti-CD155 antibody D171. D171 was first described in 1985 by Nobis et al. (J General Virology). Sequence data showed that the heavy chain variable region (VH) of the D171 antibody has more than 90% identical sequence to the heavy chain variable region (VH) sequence of Ab825. The light chain variable region (VL) of the D171 antibody and the CDR of D171 also have significant sequence overlap with Ab825. Given such a high degree of homology in the sequences of D171 and Ab825, it was not necessary to humanize both antibodies separately. The Ab825 sequence was humanized by removing potential immunogenicity sequences. Ab825 was also analyzed for potential deamidation and isomerization sites, three of which were located within the CDR of Ab825. In addition to humanization, potentially unstable sites were mutated, and then the binding properties of a panel of antibodies were tested.

[0065] Next, humanized anti-CD155 antibodies with the most optimal binding properties were tested in cytokine release assays, Episcreen DC T cell assays for injection response risk, proliferation response, off-target and on-target binding with a panel of over 5,000 known membrane receptors, and epitope mapping of PVR binding sites. Antibodies were also selected based on their ability to bind to monkey CD155 (African green monkey - Vero cells). Sequences with low immunogenicity to humans were inserted in place of immunogenic sequences. Antibodies were first mutated in the framework region and tested for variant binding and stability. The ability of antibodies to bind to the PVR receptor was evaluated. Binding to the PVR receptor was maintained, improved, or worsened in numerous variants. In embodiments, software was used to identify immunogenic sequences that could be removed. Poor immunogenic responses may persist even after deleting sequences identified by the software. Even after humanization, various mutations can have unpredictable effects on the binding or immunogenicity of humanized antibodies. The Ab825 sequence was analyzed for immunogenicity and CDR instability / susceptibility (e.g., aspartate isomerization sites or high-risk or medium-risk deamidation sites), as any of these may affect the CDR's conformation and, consequently, its antigen binding (i.e., binding to CD155). Indiscriminate medium-affinity and high-affinity MHC class 2 binding sequences were also identified for humanization. Due to the unpredictable nature of unstable sequence changes within the CDR and the influence of framework residues on binding to CD155, the resulting humanized antibodies and susceptibility-reducing antibodies against CD155 were tested in CD155-positive HAP cells and CD155 knockout HAP cell lines.

[0066] As described above, the poliovirus receptor (PVR), also known as CD155, is overexpressed in many cancers, including glioblastoma, malignant meningioma, malignant peripheral nerve sheath tumors, pancreatic cancer, lung cancer, and GI malignancies including colorectal cancer, as well as breast cancer including triple-negative (TNBC). The poliovirus receptor is also naturally present in healthy tissues of cells, such as motor neurons in the brain and spinal cord, although at low receptor densities. Antibodies capable of binding to CD155 with varying degrees of efficiency are known. One example of such a known antibody is a mouse antibody in which the heavy and light chains are incorporated into a human IgG1 chimeric antibody referred to herein as Ab825. Ab825 is derived from D171, a mouse antibody against human CD155. The heavy chain variable region (referred to herein as VH0) and light chain variable region (referred to herein as VK0) of Ab825 bind to the poliovirus receptor in humans and non-human primates. The heavy chain variable region VH0 is disclosed as SEQ ID NO: 1, and the light chain variable region VK0 is disclosed as SEQ ID NO: 5. The complementarity-determining regions (CDRs) of VH0, CDR1, CDR2, and CDR3 are disclosed as SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. The CDRs of VK0, CDR1, CDR2, and CDR3 are disclosed as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. The amino acid sequences, CDR definitions, and protein sequencing listed in SEQ ID NOs. 1 to 31 were performed by Kabat.

[0067] To make antibodies more stable and to minimize immunogenicity and toxicity when used for therapeutic and diagnostic purposes in living humans, one aspect of the present invention involves modification and humanization of the heavy chain variable (VH) and light chain variable (VK) regions. Variable region genes designed to form such antibody or antigen-binding fragments were cloned into vectors encoding human IgG1 or IgG4 heavy chain constant domains and human kappa light chain constant domains. Chimeric antibodies and humanized antibodies (including IgG4 S241P) were transiently expressed in CHO cells, purified with protein A, and tested for binding to PVRs using Biacore (surface plasmon resonance).

[0068] To identify key restrictive amino acids within the variable region considered essential for antibody binding properties, a structural model of the Ab825 antibody variable region was constructed and analyzed. Based on this structural analysis, a large preliminary set of sequence segments usable for generating Ab825 humanized variants was identified. These segments were selected and analyzed for in silico analysis of peptides binding to human MHC class II alleles and compared to known antibody sequence-associated T cell epitopes. Sequence segments identified as important non-human germ cell binding factors for human MHC class II, or those recording significant hits in our analysis, were discarded. This reduced the set of segments, and these combinations were analyzed again, as described above, to ensure that no potential T cell epitopes were present at the junctions between segments. The selected sequence segments were assembled into complete variable region sequences lacking key T cell epitopes. Thermal and freeze-thaw stability of the humanized antibody was also measured.

[0069] Using the above analysis, five variable-region heavy chains (designated VH1-VH5) and four variable-region light chains (designated VK1-VK4) were identified as having enhanced binding properties. These regions are disclosed by the following sequence numbers: [Table 1-1] [Table 1-2]

[0070] In one embodiment, the present invention is an antibody or antigen-binding fragment against an IgG isotype poliovirus receptor (CD155). This antibody or antigen-binding fragment has a heavy chain variable region from one of sequence numbers 1, 9, 10, 11, 12, or 13, having CDR1 of sequence number 2 and CDR3 of sequence number 4. CDR2 becomes sequence number 3, but has one or more of the following modifications: (1) The asparagine at position 6 of CDR2 is replaced with one of alanine, glutamic acid, lysine, glutamine, serine, or threonine; and / or (2) The aspartic acid at position 8 of CDR2 is replaced with one of glutamic acid, glycine, or arginine; and / or (3) The threonine at position 9 of CDR2 is replaced with either glutamic acid or lysine.

[0071] The light chain variable region is one of sequence numbers 5, 14, 15, 16, or 17, having CDR1 of sequence number 6 and CDR2 of sequence number 7. CDR3 is sequence number 8, but the asparagine at the 4th position of CDR3 is replaced with one of alanine, glutamic acid, glycine, lysine, glutamine, or serine.

[0072] In another embodiment, the antibody or antigen-binding fragment against the poliovirus receptor (CD155) is an IgG isotype. However, in this embodiment, the heavy chain variable region and light chain variable region are defined with respect to their CDRs. For example, the heavy chain variable region includes VH0 CDR1 of SEQ ID NO: 2 and VH0 CDR3 of SEQ ID NO: 4. Again, VH0 CDR2 becomes SEQ ID NO: 3, but with one or more of the following modifications: (1) The asparagine at position 6 of CDR2 is replaced with one of alanine, glutamic acid, lysine, glutamine, serine, or threonine; and / or (2) The aspartic acid at position 8 of CDR2 is replaced with one of glutamic acid, glycine, or arginine; and / or (3) The threonine at position 9 of CDR2 is replaced with either glutamic acid or lysine.

[0073] The light chain variable regions include VK0 CDR1 in SEQ ID NO: 6 and VK0 CDR2 in SEQ ID NO: 7. VK0 CDR3 is SEQ ID NO: 8, but the asparagine at the 4th position of CDR3 is replaced with one of alanine, glutamic acid, glycine, lysine, glutamine, or serine. More preferred antibody embodiments were developed from specific combinations of the aforementioned heavy chain variable regions VH1-VH5 (SEQ ID NOs: 9-13) and light chain variable regions VK1-VK4 (SEQ ID NOs: 14-17). VH3 and VH4, along with VK2 and VK3, were considered to be among the best variable heavy and light chain humanized variants based on the T cell epitope profile. VK or Vk are used interchangeably. Here, VH and Vh are also used interchangeably.

[0074] To address potential deamide sites identified in VH N54 and VK N92 (using single-letter amino acid symbols and positional numbers), and potential isomerization sites associated with VHD56, a series of amino acid substitutions (six for potential sequence susceptibility in VH N54 and VK N92, and five for VH D56) were first introduced individually into the VH and VK regions by mutations in the VH0 / VK0 chimeric antibody. The susceptibility reduction substitutions in VH0 / VK0 included mutations in VH N54Q, N54S, D56E, and D56G and VK N92E and N92Q. These variants of VH0 / VK0 were tested for binding to the poliovirus receptor, and based on the results of the binding analysis, were subsequently selected to be incorporated into specific preferred humanized variants of VH3 / VK2, VH3 / VK3, and VH4 / VK3. These modified VH and VK regions have the following sequences and names: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0075] Using these VH and VK regions, nine preferred antibodies (or antigen-binding fragments) against the poliovirus receptor (CD155) were identified, each possessing the following heavy chain variable regions and light chain variable regions: (i) VH3 N54Q D56E [Sequence ID 18] and VK2 N92E [Sequence ID 19]; (ii) VH3 N54S D56G [Sequence ID 20] and VK2 N92Q [Sequence ID 21]; (iii) VH3 N54Q D56E [Sequence ID 18] and VK3 N92E [Sequence ID 22]; (iv) VH3 N54S D56G [Sequence ID 20] and VK3 N92Q [Sequence ID 23]; (v)VH4 N54Q D56E[SEQ ID NO: 24] and VK3 N92E[SEQ ID NO: 22]; (vi) VH4 N54S D56E [Sequence ID 26] and VK3 N92E [Sequence ID 22]: (vii)VH4 N54S D56G[SEQ ID NO: 25] and VK3 N92E[SEQ ID NO: 22]; (viii)VH4 N54Q D56E[SEQ ID NO: 24] and VK3 N92Q[SEQ ID NO: 23]; (ix)VH4 N54S D56G[SEQ ID NO: 25] and VK3 N92Q[SEQ ID NO: 23].

[0076] Further testing of these nine antibodies suggested that the most favorable binding profiles were found with antibodies (ii) VH3 N54S D56G [SEQ ID NO: 20] and VK2 N92Q [SEQ ID NO: 21]; (iv) VH3 N54S D56G [SEQ ID NO: 20] and VK3 N92Q [SEQ ID NO: 23]; and (ix) VH4 N54S D56G [SEQ ID NO: 25] and VK3 N92Q [SEQ ID NO: 23].

[0077] In addition to the antibodies described above, the present invention is intended to encompass novel heavy-chain and light-chain variable regions constituting antibodies. For example, the VH regions identified in SEQ ID NOs: 9-13 and 18, 20, and 24-26, and the VK regions identified in SEQ ID NOs: 14-17, 19, and 21-23, are each considered separate inventions. The humanized antibodies in the present invention have been shown to bind not only to PVR (CD155) but also to nectin 4 (PVRL4). The bispecificity of this antibody and the antigen-binding fragment described in the present invention broadens the range of tumors that can be treated with the antibodies described in the present invention. The humanized antibodies of the present invention related to D171 and Ab825 have been shown for the first time to bind to nectin 4 in addition to binding to PVR. It had not been previously shown that D171 and AB825, or any of their variants, bind to nectin 4.

[0078] Another embodiment includes an antibody or antigen-binding fragment against the poliovirus receptor (CD155), comprising: (a) A heavy chain variable region selected from one of the following: SEQ ID NOs: 9, 10, 11, 12, 13, 18, 20, 24, 25, or 26. (b) A light chain variable region selected from one of sequence numbers 14, 15, 16, 17, 19, 21, 22, or 23.

[0079] The following are some embodiments comprising the above-mentioned antibody and antigen-binding fragment. The above-mentioned antibody or antigen-binding fragment may be conjugated to a prodrug or drug for the treatment of tumors expressing nectin 4. The above-mentioned antibody or antigen-binding fragment may be conjugated to a prodrug or drug for the treatment of tumors expressing CD155(PVR). Another embodiment comprises the above-mentioned antibody-drug conjugate, wherein the drug or prodrug is derived from any one of the following: nucleic acids, vedontin, pyrrolobenzodiazepine, liposomal doxorubicin, topoisomerase inhibitors, MMAE, MMAF, DM1, paclitaxel, temozolomide, doxorubicin, deruxtecan, radiosensitizers, tecilin, docaxtel, and PARP inhibitors. One embodiment comprises the above-mentioned antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment binds to CD155(PVR) and blocks the binding of CD155(PVR) to TIGIT. One embodiment comprises the above antibody or antigen-binding fragment, the antibody or antigen-binding fragment binding to nectin 4 and blocking the binding of T nectin 4 to IGIT. Another embodiment comprises the above antibody or antigen-binding fragment binding to CD155 (PVR) in non-human primates. Another embodiment comprises the above antibody or antigen-binding fragment, the antibody or antigen-binding fragment binding to nectin 4. Another embodiment comprises the above antibody or antigen-binding fragment, the antibody or antigen-binding fragment binding to CD155 (PVR), resulting in an increase in at least one DNAM ion in (a) T cells and (b) NK cells. Another embodiment comprises a bispecific antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment binding to either the poliovirus receptor or nectin 4 and a second portion of the bispecific antibody or antigen-binding fragment binding to CD3 on T cells. Another embodiment comprises a bispecific antibody or antigen-binding fragment comprising the above antibody or antigen-binding fragment binding to either the poliovirus receptor or nectin 4 and a second portion of the bispecific antibody or antigen-binding fragment binding to a receptor on NK cells.Another embodiment comprises an antibody or antigen-binding fragment against CD155, which is administered to a human, and the antibody or antigen-binding fragment binds to CD155 in the human, preventing the poliovirus from binding to CD155.

[0080] Another embodiment comprises the antibody or antigen-binding fragment described in claim 63, wherein the antibody or antigen-binding fragment against CD155 is administered to a human for the treatment of cancer in combination with an antibody against an immune checkpoint molecule selected from the group consisting of CTLA4, PD1, PDL1, CD112R, OX40, TIGIT, NKG2A, CEACAM1, B7H3, B7-H4, VISTA, LAG3, CD137, KIR, TIM1, TIM3, LAIR1, HVEM, BTLA, CD160, CD200, CD200R, and A2r. Another embodiment comprises the antibody described in claim 63, wherein the antibody against CD155 comprises an IgG4 isotype having an S241P hinge mutation or an S228P hinge mutation. Another embodiment comprises the antibody or antigen-binding fragment described in claim 63, wherein the antibody or antigen-binding fragment against CD155 comprises labeling with any of the following: (a) I-124, (b) gallium-68, or (c) lutetium-177, (d) a contrast agent for CT scans, (e) a contrast agent for MRI scans, and (f) a diagnostic agent for PET scans. Another embodiment comprises the antibody or antigen-binding fragment described in claim 63, wherein the antibody or antigen-binding fragment against CD155 is conjugated to an mRNA encoding one of the following cytokine mRNAs or gene-editing enzyme mRNAs: (a) mRNA encoding IL2, (b) mRNA encoding IL7, and (c) mRNA encoding IL12, (d) mRNA encoding IL15, (e) mRNA encoding IL21, (f) mRNA encoding IFN gamma, (g) mRNA encoding IFN alpha, (h) mRNA encoding GMCSF, (i) mRNA encoding Cas9, (j) mRNA encoding Casl2a, or (k) mRNA encoding Cas13. Another embodiment comprises CAR T cells or CAR NK cells containing the above antigen-binding fragment. Another embodiment comprises the antibody or antigen-binding fragment described above, wherein the antibody or antigen-binding fragment against CD155 binds to CD155 expressed on a tumor and induces an immune response against the tumor via ADCC or CDC.The embodiment includes a D171 antibody conjugate, which binds to a tumor expressing CD155, and the D171 antibody conjugate is internalized by the tumor.

[0081] Another embodiment comprises an antibody or antigen-binding fragment having a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5, wherein the antibody or antigen-binding fragment binds to nectin 4. This antibody or antigen-binding fragment of the present invention blocks the binding of nectin 4 to TIGIT. This antibody or antigen-binding fragment can be conjugated into a prodrug or drug for the treatment of tumors expressing nectin 4. An antibody or antigen-binding fragment comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5, wherein the antibody or antigen-binding fragment binds to PVR and causes an increase in DNAM ion T cells or NK cells. [Examples]

[0082] Invention of humanized antibodies and antigen-binding fragments against CD155 and nectin 4

[0083] Example 1. Checkpoint Blocking CD155 and nectin 4 have been described as ligands for TIGIT, respectively. Since TIGIT is expressed on immune cells, the binding of CD155 to TIGIT, or nectin 4 to TIGIT, leads to the inhibition of T cells or NK cells. Blocking CD155 or nectin 4 with the antibodies of the present invention prevents either CD155 or nectin 4 (or both) from binding to TIGIT. PVR is a native ligand for TIGIT (Alteber et al in Cancer Discovery in May 2021). PVR is also a native ligand for CD226 and CD96. D171 has been shown to block PVR (CD155) from binding to TIGIT (Yu et al in 2009 in Nature Immunuology Volume 10 (9) Supplementary Figure 4b and 4c). Reches et al. (Journal of Immunotherapy of Cancer in 2020) showed that nectin 4 is a ligand for TIGIT. The D171 antibody also binds to PVR, blocking PVR from binding to CD96 (Meyer et al Journal of Biological Chemistry Vol 284 (4) pages 2235-2244, see also page 2242).

[0084] Example 2. T cell and NK cell engagers Using the antibodies or antigen-binding fragments of the present invention that bind to CD155 and nectin 4, bispecific antibodies or bispecific antigen-binding fragments can be constructed. A bispecific antibody or bispecific antigen-binding fragment is an embodiment of the present invention in which a bispecific molecule binds to CD155 and CD3 (or nectin 4 and CD3). Ma et al. in the Journal of Cancer in 2019 Vol 10 page 5153 constructed bispecific antibodies targeting CD3 and CD155. These bispecificities were shown to be active against bladder cancer. Further examples include bispecificity that binds CD155 (or nectin 4) to receptors on NK cells to engage immune cells and destroy tumors expressing CD155 (or nectin 4). The antibodies and antigen-binding fragments disclosed herein can be incorporated into bispecific antibodies.

[0085] Example 3. Antibody-drug conjugate (ADC). Antibody-drug conjugates (ADCs) are embodiments of the present invention, in which the described antibodies are conjugated to drugs such as vedontin, deruxtecan, and tecilin, which are conjugated to the antibodies of the present invention and delivered to tumors expressing CD155 or nectin 4 (or both). CD155-targeted ADCs can be incorporated into the delivery of therapeutic agents. Given that the target receptor is also a checkpoint molecule, these ADCs against CD155 have multiple mechanisms of action. Antibody-drug conjugates can be conjugated via a variety of linkers known in the art, including but not limited to cathepsin-sensitive peptide crosslinkers. Antibodies or antigen-binding fragments may be conjugated with at least one drug such as MMAE, MMAF, paclitaxel, temozolomide, doxorubicin (including liposomal doxorubicin), deruxtecan, topoisomerase inhibitors, radiosensitizers, radiolabeled antibodies or antigen-binding fragments, tecilin, any pyrrolobenzodiazepine (PBD), DM1, vedontin and docaxtel, and PARP inhibitors such as rucaparib (antibody-drug conjugate). An antibody or antigen-binding fragment containing the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 5 was conjugated to a fluorophore, and upon administration to mammals, the antibody conjugate bound to and accumulated in tumors expressing CD155 (PVR) in mammals (Figure 4).

[0086] Example 4. ADC. ADCs with nucleic acid delivery containing mRNA. Anti-CD155 conjugates in liposomes encapsulating mRNA of the reporter molecule EGFP, with delivery and expression of mRNA in CD155-expressing cancer. An antibody or antigen-binding fragment may also be conjugated to one or more therapeutic nucleic acids delivered via the antibody or antigen-binding fragment, where the therapeutic nucleic acid is one or more of silencing RNA, shRNA, long RNA, ribozyme, messenger RNA, plasmid DNA, or non-plasmidal double-stranded DNA, as well as short single-stranded and double-stranded DNA. The antibody-drug conjugate may also be prepared by conjugating the antibody or antigen-binding fragment of the present invention with mRNA (encapsulated in liposomes or complexed with a polymer), where the mRNA encodes peptides, proteins (such as IL12, IL15, or IL2) and enzymes (such as gene-editing enzymes Cas9 or Cas12a, and other Cas enzymes such as Cas13). The D171 antibody conjugate was formed when the D171 antibody conjugate bound to a tumor expressing CD155, and when the D171 antibody conjugate was internalized by the tumor (Figures 2 and 3). The mRNA payload, conjugated with the anti-CD155 antibody, can deliver mRNA encoding polypeptides and proteins, including the vaccine.

[0087] Example 5. DNAM1-CD155 axis. An increase in DNAM1(CD226) can be achieved by administering an anti-CD155 antibody accompanied by blockade of CD155 (blockade of membranous CD155 or soluble CD155). The DNAM1-CD155 axis is described when the expression of CD155 on the cell membrane, or the expression of CD155 secreted as soluble CD155, downregulates DNAM1(CD226) expression. A therapeutic intervention using a humanized monoclonal antibody against CD155 is an embodiment of the present invention in which the anti-CD155 antibody is administered to a patient with cancer or HIV infection (or other disease in which DNAM1(CD226) is downregulated). Blockade of membranous CD155 or soluble CD155 by the humanized anti-CD155 antibody of the present invention increases the expression of DNAM1(CD226) on T cells and NK cells. The increase in DNAM1 re-establishes the immune surveillance mechanism. Carlsten et al. (Journal of Immunology in 2009, Volume 183, page 4921) showed that CD155 tumors downregulate CD155 on NK cells. Seth et al., in the 2011 Journal of Biological Chemistry, showed that anti-CD155 antibodies upregulate CD226 (DNAM1) in T cells. The antibodies or antigen-binding fragments against CD155 disclosed in this application may be used in the treatment of sepsis.

[0088] Example 6. CAR T and CAR NK. CAR T cells and CAR NK cells. Chimeric antigen receptor T cells and chimeric antigen receptor NK cells can be prepared using the humanized antigen-binding fragments described in the present invention. CAR T cells and CAR NK cells can be used to target cancers that overexpress either CD155 (PVR) or nectin 4 (or tumors that express both CD155 and nectin 4). For example, humanized antigen-binding fragments consisting of HCVR such as SEQ ID NO: 25 and LCVR such as SEQ ID NO: 23 can be incorporated into CAR T cells and CAR NK cells.

[0089] Example 7. ADCC and CDC. ADCC and CDC are mechanisms in which antibodies bind to tumor cells, and then effector cells destroy the antibody-coated cancer cells. Antibodies against CD155 can promote ADCC or CDC when they bind to and coat cancer cells. The Fc region of anti-CD155 antibodies can be further optimized for ADCC or CDC.

[0090] Example 8. Theranostics. The antibodies or antigen-binding fragments against CD155 described herein may be conjugated to at least one marker compound, such as a fluorophore or IR800 dye. The antibodies and antigen-binding fragments can be readily radiolabeled with radiolabels such as I-124 or gallium-68 for diagnostic purposes or lutetium-177 for therapeutic purposes. The antibodies or antigen-binding fragments described herein may be conjugated to at least one marker compound, such as a fluorophore or IR800 dye.

[0091] Example 9. IgG4 S241P isotype. The antibodies or antigen-binding fragments described herein may be IgG isotypes, such as IgG1, IgG2, or IgG4, as well as IgE and IgM isotypes. In one embodiment, a humanized antibody against CD155 comprises an IgG4 isotype having an S241P hinge mutation or an S228P hinge mutation.

[0092] Example 10. Passive immunity blocking PVR. The antibodies disclosed herein are related to D171, which is known to bind to the poliovirus receptor (CD155) and block poliovirus from binding to the poliovirus receptor (CD155) (Nobis et al 1985 in Journal of General Virology Vol 66 page 2563). The humanized antibodies disclosed herein can block the poliovirus receptor and prevent poliovirus from binding to the poliovirus receptor in humans. The antibodies of the present invention can be administered to patients to provide passive immunity to susceptible individuals exposed to poliovirus.

[0093] Example 11. Epitope mapping. Linear and 3D epitope mapping of PVR and nectin 4 using humanized monoclonal antibodies conjugating to CD155 and nectin 4 was performed using a peptide excision approach, where the affinity of the CD155 or nectin 4 antigen was captured with an immobilized test monoclonal antibody. Proteolysis of the immune complex was limited by washing for unbound peptides (GluC and LysC digestion). The elution of affinity-bound peptides was then analyzed by high-resolution mass spectrometry. Peptide identification was then analyzed by comparing antigen peptide profiles with and without antibodies.

[0094] Example 12. Humanized Ab825 and D171 Sequence analysis of Ab825 revealed various defective sites. The heavy chain variable region (HCVR) of Ab825 (SEQ ID NO: 1) has potential high-affinity indiscriminate MHC class 2 anchor residues at positions 18 (valine) and 64 (phenylalanine). The light chain variable region (LCVR) of Ab825 (SEQ ID NO: 5) has potential high-affinity indiscriminate MHC class 2 anchor residues at positions 2 (isoleucine) and 53 (tyrosine). The HCVR of Ab825 (SEQ ID NO: 1) also has potential P1 medium-affinity indiscriminate MHC class 2 anchor residues at positions 32 (tyrosine), 48 (isoleucine), and 93 (valine). The LCVR of Ab825 (SEQ ID NO: 5) also has potential P1 medium-affinity indiscriminate MHC class 2 anchor residues at positions 3 (valine), 4 (methionine), 29 (valine), and 47 (leucine). Humanization of the heavy chain and light chain variable regions of D171 or Ab825 had not been described or taught prior to the present invention. Furthermore, stabilization of potential deamide and isomerization sites of Ab825 in the CDR having mutations that stabilize anti-CD155 antibodies or antigen-binding fragments is a key part of the present invention. Mutations in the CDR are likely to affect binding, and indeed, many of the humanized variants tested with mutations in the heavy chain variable region (HCVR) N54 and D56, and the light chain variable region (LCVR) N92, showed significantly reduced binding in Vero cells, Hap cells, and U87 cells, as demonstrated by EC50 (Figure 8). N54S D56G in HCVR and N92Q in LCVR had the most optimal binding properties, and these mutations were found within the CDR sequences of HCVR and LCVR. The position of VH(Asn)54 in SEQ ID NO: 1, which has been identified as susceptible to deamidation, can be mutated with the amino acids alanine, glutamate, lysine, glutamine, serine, and threonine, among others. VH Asp56 in SEQ ID NO: 1, which has been identified as susceptible to isomerization, can be mutated with the amino acids glutamate, arginine, or threonine, among others, and VH T57 in SEQ ID NO: 1 can be mutated with glutamate or lysine, among others.VkN92 in Sequence ID No. 5 has also been identified as a potential deamide site and can be mutated in particular with amino acids, alanine, glutamic acid, glycine, lysine, glutamine, or serine.

[0095] DC T-cell assays demonstrated that different variants of humanized anti-CD155 antibodies exhibited varying degrees of immunogenicity. Figure 6 shows the frequency percentage of proliferation responses from 50 donors on days 9, 10, and 11. Antibodies with HCVR VH4 N54S D56G (SEQ ID NO: 25) and LCVR Vk3 N92Q (SEQ ID NO: 23) exhibited the lowest immunogenicity, lowest proliferation frequency, and lowest mean stimulation index (SI), even lower than that observed with FDA-approved Herceptin. Cytokine release assays also showed that antibodies with HCVR VH4 N54S D56G (SEQ ID NO: 25) and LCVR Vk3 N92Q (SEQ ID NO: 23) had the lowest risk of injection response, with a similar profile to the control Erbitux. Antibodies containing HCVR VH4 N54S D56G (SEQ ID NO: 25) and LCVR Vk3 N92Q (SEQ ID NO: 23) exhibit a significantly lower risk of injection reaction than Ab825 (VH0 / Vk0) in cytokine release assays. Furthermore, humanized variants were found to bind to PVR and Nectin 4 when tested against over 5000 cell membrane receptors (Figure 7). Ab825 and its humanized variants disclosed herein were not previously known to bind to Nectin 4. Based on these results, humanized Ab825, D171, and Ab825 are expected to bind to Nectin 4. In addition, the major humanized monoclonal antibodies against CD155 had dissociation constants of less than nM, thus demonstrating high affinity. Monoclonal antibodies containing IgG4 (S241P), defined by SEQ ID NO: 23 (LCVR) and SEQ ID NO: 25 (HCVR), had a Kd of 0.3 nM in surface plasmon resonance.

[0096] Example 13. Testing in humanized mice. Human tumors expressing CD155 (including luciferase) are transplanted into temporarily humanized mice. Humanized IgG4 S241P antibody against CD155 is administered intravenously to one group of mice, either alone or in combination with other checkpoint inhibitors, and tumor size is recorded by bioluminescence imaging. Similarly, bispecific antibodies containing humanized anti-CD155 and anti-CD3 antibodies are tested in one group of mice. Another group of mice is tested using ADCs prepared with humanized anti-CD155 mice and deruxtecan conjugated with a cathepsin-sensitive peptide crosslinker. One control group of mice consists of untreated mice. Temporarily humanized mice are well-known in this field and allow for short-term study of human tumors using human blood.

[0097] Similarly, the present invention envisions not only antibodies and antigen-binding fragments, but also any other polynucleotides or plasmids comprising DNA encoding an antibody or antigen-binding fragment, and a nucleotide sequence encoding an antibody or antigen-binding fragment. The present invention also includes any cell line that produces either of the antibodies or antigen-binding fragments disclosed herein. All compositions and methods disclosed and claimed herein can be prepared and performed without excessive experimentation in light of this disclosure. While the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the compositions and methods described herein without departing from the spirit and scope of the invention as further defined by the appended claims.

Claims

1. A nucleic acid sequence comprising the following heavy chain variable region and light chain variable region, encoding an antibody or antigen-binding fragment against the poliovirus receptor (CD155): (i) The heavy chain variable region of Sequence ID No. 18 and the light chain variable region of Sequence ID No. 19; (ii) Heavy chain variable region of Sequence ID No. 20 and light chain variable region of Sequence ID No. 21; (iii) Heavy chain variable region of Sequence ID No. 18 and light chain variable region of Sequence ID No. 22; (iv) The heavy chain variable region of SEQ ID NO: 20 and the light chain variable region of SEQ ID NO: 23; (v) The heavy chain variable region of SEQ ID NO: 24 and the light chain variable region of SEQ ID NO: 22; (vi) The heavy chain variable region of SEQ ID NO: 26 and the light chain variable region of SEQ ID NO: 22; (vii) Heavy chain variable region of Sequence ID No. 25 and light chain variable region of Sequence ID No. 22; (viiii) the heavy chain variable region of SEQ ID NO: 24 and the light chain variable region of SEQ ID NO: 23; and (ix) Heavy chain variable region of SEQ ID NO: 25 and light chain variable region of SEQ ID NO:

23.

2. Antibodies or antigen-binding fragments against the poliovirus receptor (CD155), including the following: (a) Heavy chain variable region, (i) CDR1 domain containing the amino acid sequence of SEQ ID NO: 2; (ii) CDR2 domain containing the amino acid sequence of Sequence ID No. 3; where, (1) The asparagine at position 6 of CDR2 is substituted with one of alanine, glutamic acid, lysine, glutamine, serine, or threonine; and / or (2) The aspartic acid at position 8 of CDR2 is substituted with glutamic acid, glycine, or arginine; and / or (3) The threonine at position 9 of CDR2 is substituted with either glutamic acid or lysine; (iii) A heavy chain variable region containing the CDR3 domain containing the amino acid sequence of SEQ ID NO: 4, (b) Light chain variable region, (i) CDR1 domain containing the amino acid sequence of SEQ ID NO: 6; (ii) CDR2 domain containing the amino acid sequence of SEQ ID NO: 7; (iii) CDR3 domain containing the amino acid sequence of SEQ ID NO: 8; Here, the asparagine at position 4 of CDR3 is substituted with one of the following: alanine, glutamic acid, glycine, lysine, glutamine, or serine, forming a light chain variable region.

3. A humanized antibody or antigen-binding fragment against the poliovirus receptor (CD155), wherein the heavy chain variable region and the light chain variable region are each one of the following: (i) Sequence IDs 20 and 21; (ii) Sequence IDs 20 and 23; and (iii) Sequence IDs 25 and 23.

4. A humanized antibody or antigen-binding fragment against the poliovirus receptor (CD155), wherein the heavy chain variable region and the light chain variable region include the following set of CDRs: (a) Heavy chain variable region where CDR1 is sequence number 2, CDR2 is sequence number 29, and CDR3 is sequence number 4, and (b) A light chain variable region where CDR1 is sequence number 6, CDR2 is sequence number 7, and CDR3 is sequence number 30.

5. A humanized antibody or antigen-binding fragment against CD155 (PVR), wherein the heavy chain comprises a variable region having an amino acid sequence with at least about 90% identity with SEQ ID NO: 25, and the light chain comprises a variable region having an amino acid sequence with at least about 90% identity with SEQ ID NO:

23.

6. An antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment against CD155 (PVR) is administered to a human, the antibody or antigen-binding fragment binds to the human's CD155 (PVR), and inhibits the binding of poliovirus to CD155 (PVR).

7. A plasmid comprising the nucleic acid sequence described in claim 1.

8. A cell line expressing the antibody or antigen-binding fragment against the poliovirus receptor (CD155) encoded by the nucleic acid sequence of any one of claims 1 to 5.

9. A monoclonal antibody or antigen-binding fragment against a poliovirus receptor according to any one of claims 1 to 8, wherein the monoclonal antibody or antigen-binding fragment crosses the blood-brain barrier and binds to cells in the central nervous system that express a poliovirus receptor.

10. The nucleic acid conjugate or drug conjugate of a monoclonal antibody or antigen-binding fragment according to claim 9.

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