CN118302445A

CN118302445A - Anti-CD 155 antibodies and antigen-binding fragments and methods of use thereof

Info

Publication number: CN118302445A
Application number: CN202180103365.5A
Authority: CN
Inventors: 阿夫塔布·S·卡里姆; 罗伯特·霍尔盖特; 阿伦·赫恩
Original assignee: Tasrif Pharmaceutical Co ltd
Current assignee: Tasrif Pharmaceutical Co ltd
Filing date: 2021-08-20
Publication date: 2024-07-05

Abstract

A humanized antibody or antigen binding fragment directed specifically against poliovirus receptor (PVR) for use in the preparation of Antibody Drug Conjugates (ADCs) to target nucleic acids, peptides, proteins, drugs and radiopharmaceuticals to cancer cells. These antibodies can be used for checkpoint blockade where binding to PVR (CD 155) can prevent binding of PVR to TIGIT. Humanized antibodies may be used alone or in combination with other known checkpoint inhibitors. Humanized antibodies modulate PVR-DNAM-1 axis, up-regulating DNAM1 (CD 226) expression on T cells or NK cells, restoring immune surveillance mechanisms. The antibody is conjugated to a CAR-T cell or CAR NK cell. Bispecific antibodies are produced by inclusion of anti-CD 155 antibodies or antigen binding fragments to facilitate the fight of T cells or NK cells with CD155 expressing cancer cells. The anti-CD 155 antibody binds to Nectin 4, thereby enabling targeting of cancer cells expressing Nectin 4.

Description

Anti-CD 155 antibodies and antigen-binding fragments and methods of use thereof

Reference sequence listing

The present application comprises an electronic sequence listing file named CD155 sequence listing-ST 25, created at 2021, 8, 19 days, file size 27KB, which is incorporated herein by reference

Technical Field

The present application relates to the treatment of diseases using monoclonal antibodies. In particular, the application relates to targeting cells expressing CD155 (PVR) or Nectin 4 (or both) using the anti-CD 155 antibodies or antigen binding fragments (antibody fragments) disclosed herein. The anti-CD 155 antibodies disclosed in the present application bind to poliovirus receptors and additionally bind to Nectin 4.CD155 and Nectin 4 are receptors that are overexpressed in many primary and metastatic tumors. CD155 and Nectin 4 were also identified as ligands for checkpoint molecules, T cell immunoglobulins, and ITIM domains (TIGIT).

Background

CD155/PVR and its receptor family are found in various tissues in humans and other animals. In mice, the mouse homolog of CD155 is called Tage 4.CD155 and Nectin 4 are described as novel checkpoints. Although the CD155/PVR receptor may play a role in normal development, under certain pathological conditions this receptor is overexpressed, including malignant tumors such as glioblastoma multiforme (GBM), breast cancers (including triple negative breast cancers), leukemias (including AML), sarcomas, ovarian cancers, colon cancers, pancreatic cancers, lung cancers, and rare but fatal tumors such as malignant peripheral schwannomas. CD155/PVR plays a role in cell adhesion, motility, apoptosis, proliferation and metastasis. Modified polioviruses have been used as vectors for the treatment of CD155/PVR overexpressing cancers by direct local administration to tumors.

For cancer, systemic administration of modified poliovirus treatment is not feasible, because most of the world population has been vaccinated with poliovirus, and poliovirus-based therapies are likely to be neutralized by the immune system following systemic administration.

This would be advantageous in developing a therapeutic approach based on anti-CD 155 antibodies that could treat central nervous system cancers as well as systemic cancers. Such CD155 antibodies may also enter the central nervous system by crossing the blood brain barrier or other pathways, either as a therapeutic method per se, or as a delivery agent for another therapy. In the present application, it is shown that a monoclonal antibody targeting mouse CD155/PVR can enter the central nervous system after systemic administration and can cross the blood brain barrier of mouse GL261 brain tumor. In one embodiment, monoclonal antibodies targeting human and mouse CD155/PVR have been demonstrated to be endogenous and used to deliver and express payloads such as mRNA. It is known that some antibodies and peptides can cross the human blood brain barrier. For example, antibodies or antigen binding fragments against transferrin receptors have been demonstrated to cross the blood brain barrier of animal models, a phenomenon which has also recently been observed in humans. Also known in the art are sequences of single domain antibodies or polypeptides, such as FC5 and FC44, and FC5 and FC44 may cross the blood brain barrier. Methods of treatment that have entered or are about to enter clinical trials include molecules that can cross the blood brain barrier. Antibodies or ligands to insulin receptor and insulin-like growth factor 1 receptor are also another example of peptides and proteins that can cross the blood brain barrier, which can be fused to the anti-CD 155 antibodies described in this application. In one embodiment, the anti-CD 155 antibody is fused to a protein or polypeptide by chemical or recombinant molecular biology so as to cross the blood brain barrier. anti-CD 155 antibodies, including D171, ab825, and any humanized versions thereof, can be fused, chemically linked, or produced by recombinant techniques to polypeptides or proteins (or antibodies) such as FC5, FC44, anti-type 1 insulin-like growth factor receptor (IGF 1R), and anti-transferrin receptor antibodies (anti-transferrin receptor 1 or transferrin receptor 2) and antigen binding fragments. Such a construction would facilitate the crossing of the blood brain barrier by anti-CD 155 antibodies.

Disclosure of Invention

CD155 and Nectin 4 are described as novel checkpoints that play an important role in immunooncology. Binding of CD155 to T cell immunoglobulins and ITIM domains (TIGIT) results in inhibition of T cell and NK cell function. Recently, nectin 4 was also found to bind to TIGIT, so tumors expressing Nectin 4 are thought to suppress immune cells, particularly T cells. In addition, patients with tumors that overexpress CD155 typically have higher levels of soluble CD155 in the body. High levels of membrane CD155 and soluble CD155 (sCD 155) may produce immunosuppressive effects at least in part by down-regulating DNAM 1 (CD 226). The anti-CD 155 antibodies described in the present application can bind to CD155 (membrane CD155 or soluble CD 155) thereby up-regulating DNAM 1 (CD 226). In addition, antibodies that bind CD155 and Nectin 4 can be used to target cancer cells that overexpress CD155 or Nectin 4 (or cancer cells that express both CD155 and Nectin 4). anti-CD 155 antibodies can be used to block checkpoint pathways or can be used to generate bispecific antibodies capable of binding CD155 (or Nectin 4) and binding T cells or NK cells, thereby activating the T cells or NK cells to kill tumor cells. To date, no ADC against CD155 has entered clinical trials and no one has been approved by the FDA. However, enfortumab Vedontin against Nectin 4 is an FDA approved ADC for the treatment of bladder cancer. The humanized antibodies described in the present application can be used to prepare antibody drug conjugates to treat patients with cancers that overexpress CD155 (or Nectin 4). As part of the present application, it has been demonstrated that antibodies against CD155 can deliver a payload. In this case, mRNA is delivered and expressed in cancer cells that overexpress CD 155. In addition, antigen binding fragments that bind to CD155 or to 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 that express Nectin 4 or CD 155. Antibodies or antigen binding fragments against CD155 can also be used in combination with other small molecule therapeutic agents, such as tyrosine kinase inhibitors and PARP inhibitors, as well as in combination with antibodies against known checkpoint targets. The antibodies of the application may be used in combination with one or more checkpoint inhibitors targeting CTEA4、PD1、PDL1、CD112R、OX40、TIGIT、NKG2A、CEACAM1、B7H3、B7-H4、VISTA、LAG3、CD137、KIR、TIM1、TIM3、FAIR1、HVEM、BTEA、CD160、CD200、CD200R and A2r, as well as other checkpoints known in the art.

In one embodiment, the antibodies and antigen binding fragments described herein against CD155 and Nectin 4 can also be developed as a therapeutic diagnostic combination, wherein the anti-CD 155 antibody or antigen binding fragment comprises the radiolabel (a) 1-124 (b) gallium 68 or (c) ruthenium 177 from one of the following choices for diagnosis or treatment of cancer.

The present application relates to the treatment of various diseases using monoclonal antibodies directed against CD155 and Nectin 4. One embodiment of the application is a method of treating a systemic or neurological disorder in a mammal comprising administering to a patient a composition comprising: a therapeutic agent; and a non-viral ligand capable of binding CD155, wherein the ligand is conjugated to a therapeutic agent to treat a neurological disorder or a systemic disorder. In one embodiment, the neurological disorder is a primary or metastatic brain tumor. In one embodiment, the neurological or systemic disorder comprises at least one of (i) glioblastoma multiforme in the following primary or metastatic cancers; (ii) neuroblastoma; (iii) an oligodendroglioma; (iv) glioma; (v) astrocytomas; (vi) anaplastic cytoma; (vii) meningiomas; (viii) Primary or metastatic cancers of the breast, lung, kidney, pancreas and metastatic melanoma. In one embodiment, the disorder comprises at least one of the following group (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) epithelial-like cancer; (xiii) hepatocellular carcinoma; (xiv) glioblastoma; (xv) malignant peripheral schwannomas and melanomas. In one embodiment, the tumor overexpresses CD155. In one embodiment, the tumor overexpresses Nectin 4. In one embodiment, the tumor expresses both CD155 and Nectin 4.

In one embodiment, the ligand comprises at least one of the group consisting of: (i) an antibody against CD155 or Nectin 4; (ii) an antigen binding fragment of anti-CD 155 or Nectin 4. In one embodiment, the ligand may cross the blood brain barrier. In one embodiment, the fusion protein includes a first segment that binds to CD155 and a second segment that is a therapeutic agent. In one embodiment, the antibody or antigen binding fragment is conjugated to a therapeutic agent, wherein the therapeutic agent comprises at least one of the following group: (i) a drug; (ii) a prodrug; (111) siRNA; (iv) antisense DNA or RNA; (v) messenger RNA; (vi) a guide RNA (vii) plasmid of CRISPR; (vii) single-or double-stranded oligonucleotides (RNA or DNA); (viii) a peptide; (ix) a protein; (x) a gene and (xi) a toxin. In one embodiment, the ligand is capable of binding Nectin, nectin 2 (CD 112), nectin4, neel, PVRL1, necl-1, necl-4, poliovirus receptor associated 1 protein or poliovirus receptor associated 2 protein. In one embodiment, the antibody or antigen binding fragment drug conjugate further comprises a liposome or Lipid Nanoparticle (LNPs) encapsulating the therapeutic agent. In one embodiment, the liposome is a pegylated liposome crosslinked with a ligand. In one embodiment, the ligand is a D171 antibody or an antibody having a heavy chain variable region sequence or a light chain variable region sequence with greater than 75% homology to a D171 heavy chain variable region sequence or a D171 light chain variable region sequence.

In one embodiment, the anti-CD 155 antibody specifically binds to any intracellular, transmembrane, or extracellular epitope of CD 155. In one embodiment, D171 specifically binds to amino acids 35 to 50 of CD 155. In one embodiment, an antibody or antigen binding fragment directed against CD155 binds to a tumor and elicits an immune response against cancer growth or tumor. In one embodiment, the antibody is at least one selected from the group consisting of whole antibodies, antigen binding fragments, fab2, fc, and scFv, light chain and/or heavy chain variable regions, the antibody comprising a first component that binds to CD155 and a second component that binds to effector cells to attract T cells or NK cells or other immune cells, or to stimulate antibody-dependent cell-mediated cytotoxicity (ADCC) or Complement Dependent Cytotoxicity (CDC). In one embodiment, the at least one antibody is selected from one of a monoclonal antibody, a polyclonal antibody, a humanized antibody, and a chimeric antibody. In one embodiment, the effector cells are from at least one of the following group: natural killer cells and lymphocytes including cytotoxic T cells. In one embodiment, the antibody is administered by one of intravenous, intra-arterial, intratumoral, intra-cerebrospinal, intramuscular, subcutaneous, intravesical, intraperitoneal, or by convection enhanced delivery.

In one embodiment, at least one antibody from the group consisting of whole antibodies, antigen binding fragments, fab2, fc, and scFv or other antigen binding fragments is selected to include 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). An embodiment of the application is a method of delivering a therapeutic agent to the central nervous system by systemic or local administration. In one embodiment, the anti-CD 155 antibody or antigen-binding fragment can cross the blood brain barrier of a mammal, comprising administering a CD 155-targeting non-viral ligand into the blood circulation of the mammal, wherein the non-viral ligand binds to a therapeutic agent. In one embodiment, the mammal has a brain tumor. In one embodiment, the mammal has a brain failure that is not a tumor. In one embodiment, the anti-CD 155 antibody or antigen binding fragment is a therapeutic agent per se, or delivers a therapeutic agent for the treatment of a systemic disorder, e.g., cancers of the kidney, pancreas, ovary, lung, colon, and sarcomas (e.g., malignant peripheral neoplasia).

In one embodiment, a nucleic acid-based agent binds to an anti-CD 155 antibody or antigen-binding fragment. In one embodiment, a nucleic acid-based drug binds to an anti-Nectin 4 antibody that also binds to CD 155. An embodiment of the application is a composition comprising a humanized D171 antibody or a humanized Ab 825. An embodiment of the application is a humanized anti-CD 155 antibody, such as humanized Ab825, comprising a selenocysteine residue greater than 10 amino acids from the C-terminus.

One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody comprising an Fc region optimized to enhance ADCC. One embodiment of the application is an optimized humanized D171 antibody or humanized Ab825 antibody for CDC. One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody that binds to a contrast agent for MRI/CT. One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody that binds to a diagnostic agent of a PET scan. One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody conjugated to a fluorescent probe (e.g., IR 800). One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody conjugated to a radiosensitizer. One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody that binds to a chelator. One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody conjugated to a radioisotope. One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody that binds to a nucleic acid therapeutic agent (e.g., mRNA, siRNA, RNA or a DNA oligonucleotide, single-or double-stranded DNA). One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody, wherein the humanized D171 antibody or the humanized Ab825 antibody is a therapeutic agent. One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody, wherein the humanized D171 antibody or the humanized Ab825 antibody blocks the binding of CD 155 (poliovirus receptor) to TIGIT. One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody, wherein the humanized D171 antibody or the humanized Ab825 antibody blocks binding of Nectin 4 to TIGIT.

One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody, wherein the humanized D171 antibody or the humanized Ab825 antibody blocks CD 155 (poliovirus receptor) and subsequently up-regulates DNAM 1 expression on NK cells or T cells (or both). One embodiment of the application is a humanized D171 antibody or a humanized Ab825 antibody, wherein the humanized D171 antibody or the humanized Ab825 antibody blocks CD 155 (poliovirus receptor) and subsequently up-regulates the expression of DNAM 1 on T cells. All of the above also apply to antigen binding fragments against CD 155 and Nectin 4.

One embodiment of the application is a method for identifying the extent of a CD155 overexpressing tumor in a mammal comprising the steps of: administering an antibody or antigen binding fragment that binds CD155, wherein the antibody or antigen binding fragment has been bound to a label. In one embodiment, the label is selected from at least one of a radioisotope, a pigment, a fluorescein, and an enzyme combination. In one embodiment, the marker is selected from one of a gamma ray or positron emitting radioisotope, a magnetic resonance imaging contrast agent, an X-ray contrast agent, and a combination of ultrasound contrast agents. In one embodiment, the tag is conjugated to an antibody or antigen binding fragment directed against CD155 using a selenium cysteine residue. In one embodiment, imaging is performed by a technique of one of CT, ultrasound, MRI, SPECT, and PET. In one embodiment, imaging is performed at least one point in time during, and after the perioperative procedure. One embodiment of the application is a method of delivering a therapeutic agent across the blood brain barrier, wherein the therapeutic agent binds to an anti-CD 155 antibody or antigen-binding fragment. The foregoing has outlined rather broadly the features of the present application in order that the detailed description that follows may be better understood. Other features and advantages of the application will be described hereinafter and form the subject of the claims. In one embodiment, the primary or metastatic tumor overexpresses CD155. In one embodiment, the primary or metastatic tumor overexpresses Nectin 4.

In one embodiment, the anti-CD 155 antibodies or antigen binding fragments described herein are used as part of a therapeutic chimeric antigen receptor T Cell (CART) or therapeutic chimeric antigen receptor NK cell for the treatment of CD155 positive tumors. In one embodiment, the humanized antibodies or antigen binding fragments described herein are used as part of a therapeutic chimeric antigen receptor T Cell (CART) or therapeutic chimeric antigen receptor NK cell for the treatment of a Nectin 4 positive tumor.

In one embodiment, the anti-CD 155 antibody or antigen-binding fragment is capable of crossing the blood brain barrier. In one embodiment, the fusion protein comprises at least two fragments, wherein the first fragment binds to CD155 or Nectin 4 and the second fragment facilitates crossing the blood brain barrier. In one embodiment, the cell therapy comprises T cells expressing antigen binding fragments that bind CD155 and/or Nectin 4. In one embodiment, the method further comprises a liposome, a Polyethylenimine (PEI) polymer, or a Lipid Nanoparticle (LNP). In one embodiment, the humanized D171 antibody, or an antibody having at least 90% homology to the D171 antibody, specifically binds to amino acids 35 to 50 of CD 155. In one embodiment, the anti-CD 155 antibody or antigen binding fragment binds to a primary or metastatic tumor expressing CD155 and/or Nectin 4 and elicits an immune response against the primary or metastatic tumor upon binding.

In one embodiment, at least one antibody or fragment selected from the group consisting of whole antibodies, antigen binding fragments, fab2, fc, scFv, comprises a first component that binds to CD 155 and a second component that binds to effector cells. In one embodiment, at least one selected from the group consisting of whole antibodies, antigen binding fragments, fab2, fc, scFv is selected from the group consisting of monoclonal antibodies, humanized antibodies, chimeric antibodies. In one embodiment, the patient may be treated by injecting an antibody or antigen binding fragment into the patient, or injecting DNA encoding any of the antibodies (or antigen binding fragments) described above, wherein the injection is at least one of intravenous, intra-arterial, intramuscular, intrathecal, intraventricular, intratumoral, subcutaneous or intralymphatic, intravesical (i.e., bladder cancer or ependymoma of the central nervous system) and intratumoral, and delivery by convection enhanced.

One embodiment of the application is a method of delivering a therapeutic agent across the blood brain barrier of a mammal comprising administering a CD 155-targeting non-viral ligand into the blood circulation of the mammal, wherein the non-viral ligand binds to the therapeutic agent. In one embodiment, the non-viral ligand is a monoclonal antibody or antigen binding fragment described in the present application. In one embodiment, the therapeutic agent is encapsulated in a liposome, and the liposome binds to a monoclonal antibody or antigen binding fragment that binds CD155 and/or Nectin 4. In one embodiment, the mammal has a brain tumor. In one embodiment, the mammal has a brain failure, wherein the brain failure is not a tumor. In one embodiment, the therapeutic agent binds to an anti-CD 155 antibody or an anti-CD 155 antigen binding fragment. In one embodiment, the therapeutic agent binds to an anti-CD 155 antibody or an anti-CD 155 antigen binding fragment and is used to treat motor neurons, brain stem, spinal cord, or other brain cells expressing CD155 in the brain. One embodiment of the application is a method of delivering an antibody to the brain of a mammal by fusing an antibody or antigen-binding fragment of another therapeutic antibody with an anti-CD 155 antibody or anti-CD 155 antigen-binding fragment. In one embodiment, the therapeutic antibody or therapeutic antigen binding fragment is fused to the anti-CD 155 antibody or anti-CD 155 antigen binding fragment by chemical means, antibody engineering means, or avidin-biotin cross-linking means.

In one embodiment, the anti-CD 155 antibody or antigen-binding fragment that binds CD155 is itself a therapeutic agent. In one embodiment, the anti-Nectin 4 antibody or antigen-binding fragment that binds Nectin 4 is itself a therapeutic agent. One embodiment of the application is a method of identifying the extent of a CD155 overexpressing tumor in a mammal comprising the steps of: administering a CD 155-targeting non-viral ligand bound to a label; and imaging brain tumors. In one embodiment, the label is selected from at least one of a radioisotope, a pigment, a fluorescein, and an enzyme. In one embodiment, the marker is selected from one of a gamma ray or positron emitting radioisotope, a magnetic resonance imaging contrast agent, an X-ray contrast agent, and an ultrasound contrast agent. In one embodiment, the tag is conjugated to the non-viral ligand using a selenium cysteine residue in the antibody or antigen binding fragment. In one embodiment, imaging is performed by at least one technique of CT, ultrasound, MRI, SPECT, PET, and the like. In one embodiment, imaging is performed at least one of pre-operatively, intra-operatively, and post-operatively.

In one embodiment, selenium cysteine residues are used to modify humanized or humanized antibodies against human poliovirus receptors to facilitate chemical binding to a reporter molecule for use in diagnosing a disease or as a therapeutic agent. In one embodiment, a humanized or humanized antibody against a human poliovirus receptor is conjugated (e.g., directly coupled or encapsulated in nanoparticles) to a therapeutic agent (e.g., a small drug molecule) for delivery to target cells, such as tumor cells, that overexpress CD 155. In one embodiment, a humanized or humanized antibody against a human poliovirus receptor is conjugated to fluorescein or other reporter molecules for non-operative, pre-operative, intra-operative or post-operative diagnosis or treatment of target cells (such as tumor cells) that overexpress the CD155 receptor.

In one embodiment, a humanized or humanized antibody against a human poliovirus receptor is fused to a therapeutic peptide by antibody engineering or chemical coupling. In one embodiment, a human or humanized antibody against a human poliovirus receptor may be chemically or antibody engineered fused to another therapeutic antibody (or antigen binding fragment) to create a bispecific antibody, wherein one component is a human or humanized antibody (or antigen binding fragment) against a human poliovirus receptor. In one embodiment, a humanized or humanized antibody against a human poliovirus receptor is conjugated to tumor cells that overexpress CD 155.

In one embodiment, the humanized or humanized antibody against the human poliovirus receptor also binds to tumor cells that overexpress Nectin 4. Embodiments of the application are humanized D171 antibodies or humanized Ab825 antibodies, or antibodies having similar sequences to the D171 antibodies, comprising at least one of the following options: selenium cysteine residues greater than 10 amino acids from the C-terminus; an Fc region optimized to enhance ADCC; an optimized antibody for CDC; an antibody that binds to an MRI/CT contrast agent; an antibody that binds to a PET scanning diagnostic agent; an antibody that binds to the fluorescent probe; an antibody that binds to a radiosensitizer; an antibody that binds to a chelator; an antibody that binds to a radioisotope; an antibody that binds to a therapeutic agent; wherein the humanized anti-CD 155 antibody is a therapeutic agent. Embodiments of the application include binding the disclosed humanized anti-CD 155 antibodies or antigen-binding fragments to poliovirus receptors and inhibiting binding of poliovirus receptors to TIGIT. Another embodiment includes binding the disclosed humanized anti-CD 155 antibodies and antigen-binding fragments to Nectin 4 and inhibiting binding of Nectin 4 to TIGIT.

The foregoing has outlined rather broadly the features of the present application in order that the detailed description that follows may be better understood. Additional features and advantages of the application will be described hereinafter which form the subject of the claims.

Drawings

For a better understanding of the above and other improvements and objects of the application, the application described above will be described in more detail by reference to the specific embodiments illustrated in the accompanying drawings. It is appreciated that these drawings depict only typical embodiments of the application and are therefore not to be considered limiting of its scope.

FIG. 1 shows that mouse anti-IL 13alpha 2 antibodies and mouse anti-CD 155 antibodies cross the blood brain barrier and bind to mouse IL13 alpha 2 (mIL 13alpha 2) and mouse CD 155 (mCD 155) in GL 261 tumors. The fluorescently labeled mouse anti-IL 13alpha 2 receptor antibody and the fluorescently labeled anti-CD 155 antibody are injected into experimental mice. The fluorescently labeled antibody crosses the blood brain barrier and accumulates in the GL 261 tumor of C57B16 mice. GL 261 tumors express two receptors, the mouse CD 155 and mouse IL13 alpha 2 receptors.

Figure 2 shows that D171 monoclonal antibodies that bind to liposome-encapsulated eGFP mRNA are internalized, expressing GFP in U87 tumors in vitro. U87 tumors overexpress human CD 155. The anti-human CD 155D 171 monoclonal antibody was conjugated to eGFP mRNA coated liposomes, and after 24 hours exposure of eGFP-encoding RNA containing liposomes functionalized with the D171 antibody to l0 ng/well, eGFP fluorescence in U87 cells was recorded. Three cell densities were studied.

FIG. 3 shows that D171 antibodies against human CD155 bound to liposome-encapsulated fluorescein-labeled mRNA and fluorescein were detected in about 80% of U87 cells. The D171 conjugate was internalized in vitro by U87 cells. U87 cells are known to overexpress CD155.

Figure 4 shows a comparison of tumor bioluminescence and dye fluorescence imaging after injection of Ab825-VT680 and control VT68072 hours in mice with U87MG tumors. Ab825-VT680 conjugates bind to U87MG tumors and accumulate in vivo. Control mice were untreated or treated with VT680 dye alone. Fluorescence imaging showed that accumulation occurred in tumors of mice treated with fluorescence-labeled anti-CD 155 antibodies. Bioluminescence shows that the region of fluorescence aggregation is associated with the tumor region of the bioluminescence imaging display.

Figure 5 shows the potential mechanism of action of anti-CD 155 antibodies and antigen binding fragments. The interaction of CD 155-TIGIT and the interaction of CD 155-CD 96 (not shown) are targets for checkpoint blockade. anti-CD 155 antibodies and antigen binding fragments can be used: (a) ADCC; (c) checkpoint blockade; (d) radioimmunotherapy; (e) an Antibody Drug Conjugate (ADC); (f) Bispecific antibodies, one of which binds CD 155 and the other binds to a second receptor, such as CD 3 on T cells; (g) preparation of anti-CD 155CART cells or CARNK cells. anti-CD 155 antibodies and antigen-binding fragments can also bind to Nectin 4, and the conjugates described herein can also target Nectin 4.

FIG. 6 shows DC T cell analysis performed in 50 patient samples using humanized monoclonal antibodies VH 3N 54SD56G/Vk 2N 92Q (sample 2), VH 3N 54SD56G/Vk3N92Q (sample 3) and VH 4N 54SD56G/Vk3N92Q (sample 4). The proliferation frequency (% patient samples with proliferation) and Stimulation Index (SI) of each test antibody and control are shown.

FIG. 7 shows the results of binding assays demonstrating the strong specific binding between humanized antibodies against CD 155 (PVR) and the cell membrane receptors PVR and Nectin 4. There was no cell fixation. Exhibits reverse binding to the natural ligand CD 226 (DNAM 1) and TIGIT of PVR. More than 5000 cell membrane receptors were used for this experiment. A strong and specific binding was observed between PVR and Nectin 4. Since these receptors are ligands for PVR and bind to endogenous PVR in the experiment, reverse binding (white spots) of TIGIT and CD 226 was observed.

FIG. 8 is an EC 50 cell binding study of humanized monoclonal antibodies against CD155 using HAP1 cells, vero cells and U87 cells. The N54SD56G variant in the heavy chain variable region HCVR and the N92Q variant in the light chain variable region LCVR have higher affinity for binding to CD155 than the N54Q D E or N54SD56E variant in the HCVR and the N92E variant in the LCVR.

Detailed Description

The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present application only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments. In this regard, no attempt is made to show structural details of the disclosure in more detail than is necessary, the description together with the drawings, so that one skilled in the art can readily understand how the various forms disclosed may be embodied in practice.

The following definitions and explanations are intended to have a limiting effect on the following explanations unless explicitly and unambiguously modified in the examples below, or when the meaning is applied such that any construction becomes nonsensical or substantially nonsensical. In cases where the construction of the term would render it nonsensical or substantially nonsensical, reference should be made to the definition of the third version of the Webster's dictionary.

The term "binding" as used herein refers to any means of binding a ligand to a therapeutic agent, including but not limited to chemical binding, electrostatic binding, or by creating a fusion construct of the ligand and therapeutic agent using all means of molecular biology. All means of "molecular biology" include, but are not limited to, tailoring chimeric antigen receptors embedded with antibodies or antigen binding fragments for use in CAR T cell therapies to eliminate diseases, including, but not limited to, cancer.

Antibodies (abs), also known as immunoglobulins (Ig), are large Y-shaped proteins produced by plasma cells that are used by the immune system to recognize and neutralize foreign objects such as bacteria and viruses. Antibodies recognize a unique portion of a foreign target, known as an antigen. Each "Y" shaped end of an antibody contains a lock-like antigen receptor structure (called an epitope) that is specific for a particular epitope on the antigen (similar to a key) so that the two structures can be precisely bound together.

Bispecific monoclonal antibodies (BsMAb, bsAb) are an artificial protein consisting of fragments of two different monoclonal antibodies, and thus are capable of binding to two different types of antigens. This approach is widely used in cancer immunotherapy, where BsMAb is designed to bind both toxic cells (e.g., CD3 receptor on T cells) and receptors on tumor cells, resulting in death of tumor cells.

The Blood Brain Barrier (BBB) is a highly selective permeability barrier separating circulating blood from the extracellular fluid (BECF) of the brain in the Central Nervous System (CNS). The blood brain barrier is formed by capillary endothelial cells that are interconnected by tight junctions, with extremely high electrical resistivity of at least 0.1D m. The blood brain barrier allows the passage of water, some gases and lipid-soluble molecules through passive diffusion, as well as selective transport of molecules critical to neurological function such as glucose and amino acids.

Binding includes, but is not limited to, molecular, chemical, and electrostatic binding. One example of molecular binding is antibody engineering. One example of chemical bonding is covalent bonding. One example of electrostatic binding is biotin and streptavidin.

CD155 is a type I transmembrane glycoprotein in the immunoglobulin superfamily. Since it is involved in primate intracellular poliovirus infection, it is commonly referred to as poliovirus receptor (PVR). The normal cellular function of CD155 is to establish an intercellular adhesive link between epithelial cells. CD155 is a transmembrane protein with 3 extracellular immunoglobulin-like domains, D1-D3, where D1 is recognized by the virus. Synonyms include PVR; a CD155; HVED; NECL5; necl-5; PVS; and tag 4.

A fusion protein or chimeric protein is a protein created by ligating two or more genes that originally encoded different proteins. Translation of such fusion genes will result in one or more polypeptides having the functional properties derived from each of the original proteins.

These fusion proteins are produced by molecular biology techniques or chemical coupling of two antibodies or antigen binding fragments.

In one embodiment, the antibody has functional groups available for labeling, crosslinking, or covalent modification. In one embodiment, the functional group on the antibody is a primary amine group, a thiol group, a carbohydrate, selenomethionine, or comprises an unnatural amino acid.

In one embodiment, a cross-linking agent may be used to attach a gene, polypeptide, or small molecule to an antibody. In one embodiment, the crosslinker may be a heterobifunctional crosslinker. In one embodiment, the heterobifunctional crosslinking reagent comprises succinylethylene thioacetate (SATA), succinyl trans-4- (maleylmethylene) cyclohexane-1-carboxylate (SMCC), succinyl 3- (2-pyridyldithio) propionate (SPDP), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC), N- (2-pyridyldithioethyl) -4-azopropionamide (PEAS; AET), succinyl 4-azo-2, 3,5, 6-tetrafluorobenzoate (ATFB, SE), benzoketone-4-maleimide, benzoketone-4-isothiocyanate, succinyl 4-benzoylbenzoate, iodoacetamide azoamide alkynane, click-iT maleimide DIBO alkane, azo (PEO) 4 propionic acid, succinates, alkynes, succinyl esters or Click-iT succinyl ester DIBO alkane. In another embodiment, the cross-linking agent may be a peptide cross-linking agent that is sensitive to cysteine proteases. In another embodiment, the crosslinking agent may be used for site-specific crosslinking, such as a BCN-bearing azo crosslinking agent for click chemistry. Useful coupling agents may be based on click chemistry using azo (N3) to crosslink the antibody with the carrier. The reactive functional groups are identical at each end. Reactive ends are typically targeted to primary amine and thiol groups. In one embodiment, azo reacts with alkyne via a copper-catalyzed azo-alkyne cycloaddition reaction. In another embodiment, conjugation may be performed using a metal-free azo-alkynalkane reaction. In one embodiment, the crosslinking agent reacts non-specifically with the available sites under ultraviolet radiation.

Human antibodies are antibodies produced in the human or other mammalian system, or antibodies made using yeast or phage technology. Humanized antibodies are antibodies derived from non-human species whose protein sequence has been modified to increase their similarity to antibody variants naturally produced in humans. The "humanization" process is typically applied to monoclonal antibodies developed for human use (e.g., antibodies developed as anti-cancer drugs). Humanization may be necessary when the process of developing a particular antibody involves the generation of antibodies in a non-human immune system, such as the immune system in a mouse. Antibodies produced in this way differ in the part of the protein sequence from the homologous antibodies naturally occurring in humans and thus may be immunogenic when administered to human patients.

Humanized antibodies involve the removal of sequences that may be immunogenic in non-human antibodies. Sequences that are less immunogenic to humans may be inserted at the position of the immunogenic sequence. There are many ways in which a particular antibody may be humanized. In one embodiment, more than one year may be required to humanize a particular non-human antibody. In one embodiment, the non-human antibody will be humanized by the steps of: 1) examining the structure to determine which sequences are immunogenic to humans, 2) removing sequences that are immunogenic to humans, 3) maintaining or improving the ability of an antibody to bind to its target receptor, 4) assessing the binding of an antibody to the target receptor, 5) screening for adverse immunogenic reactions, 6) removing sequences that result in adverse immunogenic reactions. In one embodiment, software is used to identify potentially immunogenic sequences to be removed. In one embodiment, there is an adverse immunogenic reaction even after removal of the sequences recognized by the software, and the software may be inaccurate. In one embodiment, in vitro studies are performed to determine the sequence to be removed. When different parties humanize a given antibody, the sequence of the given antibody may vary. In order to remove potential aspartic acid isomerisation sites and susceptible to deacylation sites, mutations may be required in the CDRs. Mutations in CDRs may affect binding, and therefore antibody humanization is typically limited to framework residues. The goal of humanization and mutation to labile sites is to achieve low infusion reaction risk, low immunogenicity risk, and stabilize potential deacylated and isomerized sites within the CDRs. Even though humanized, different humanized antibodies may have different dissociation constants (Kd), different results for cytokine release assays, and different immunogenic results for EPISCREEN and DC TCELL EPISCREEN assays. Humanization of antibody D171 or Ab825 was not previously taught, and was first taught in the present invention. Ab825 was found to have the highest risk of infusion reactions compared to the humanized variants.

In one embodiment, the anti-CD 155 antibody may be fused with another therapeutic antibody to assist the therapeutic antibody in crossing the blood brain barrier. In one embodiment, the approved therapeutic antibody alone cannot cross the blood brain barrier. In one embodiment, the anti-CD 155 antibody is transmitted retrograde through the nerve and into the central nervous system. In one embodiment, the anti-CD 155 antibody crosses the blood-cerebrospinal fluid barrier.

A ligand is a substance (typically a small molecule) that forms a complex with a biological molecule to achieve a biological effect. In protein-ligand binding, the ligand is typically a signaling trigger molecule that binds to a site on the protein of interest. In many of the examples described herein, the ligand-bound target protein is a receptor on a cell. Antibodies or antigen binding fragments that bind to CD155 are considered ligands for CD 155.

Non-viral refers to any polypeptide, including DNA/RNA fragments and proteins, which lack the effective structure to be considered a virus. Non-viral further excludes complex polypeptides that are created by starting with a virus and cleaving a portion of the virus.

Polypeptides are linear chains composed of amino acid residues, called polypeptides. The protein comprises at least one long polypeptide. Short polypeptides containing less than about 20-30 residues are rarely considered proteins, often referred to as peptides, sometimes also referred to as oligopeptides. The individual amino acid residues are linked to each other by peptide bonds and adjacent amino acid residues.

Proteins are large biological molecules, or macromolecules, composed of a long chain of one or more amino acid residues. Proteins are determined in their amino acid sequence primarily by the nucleotide sequence of the gene, which generally results in the protein folding into a specific three-dimensional structure, thereby determining its activity.

A receptor is a protein molecule that is typically embedded in the plasma membrane surface of a cell and receives a chemical signal from the outside of the cell. When these chemical signals bind to receptors, they cause some form of cell/tissue reaction, such as altering the electrical activity of the cell. In this sense, a receptor is a protein molecule that recognizes and responds to endogenous chemical signals. CD155 is also known as poliovirus receptor.

By "therapeutically effective amount" is meant an amount that eliminates or reduces the tumor burden of a patient, or prevents, delays or inhibits metastasis. The dosage will depend on a number of parameters including the nature of the tumor, the patient's history, the patient's condition, the cytotoxic agent that may be used simultaneously, and the method of administration. Methods of administration include injection (e.g., intravenous, subcutaneous, intravenous, intraperitoneal, subarachnoid, convection enhanced, etc.), wherein a molecule or complex that binds to PVR is provided that is dissolved in a non-toxic, pharmaceutically acceptable carrier.

Genes for mRNA or reporter EGFP were transferred to cells expressing CD155 (exemplified by anti-mouse CD155 and anti-human CD 155) by anti-CD 155 antibodies bound to nucleic acid-encapsulating liposomes, and monoclonal antibodies against the corresponding poliovirus receptors were used in mouse (for mouse CD 155) and human (for human CD 155) cell lines. The D171 antibody was used to deliver the encapsulated mRNA to U87 tumors expressing CD 155. By passing the mRNA conjugate of D171, GFP mRNA was expressed. According to the results, this delivery system can be extended to other nucleic acids and drugs. anti-CD 155 monoclonal antibodies accidentally enter the central nervous system and coat tumors. Potential applications of this discovery include the use of CD155 to facilitate transport of ligands and ligand conjugates across the blood brain barrier or other barrier that prevents entry into the central nervous system. Fusion of a therapeutic peptide or protein with an anti-CD 155 antibody or an anti-CD 155 antigen binding fragment may provide a pathway across the blood brain barrier or other barrier that prevents access to the central nervous system. Antibodies against CD155 bind CD155 in vitro in human and mouse cell lines and are endocytosed by mouse and human cells. Antibodies against mouse CD155 bind to tumors in vivo and cross the blood brain barrier. anti-CD 155 antibodies have diagnostic potential in vivo for the diagnosis of CD155 tumors and for the labeling of CD155 tumors preoperatively, intraoperatively and postoperatively. anti-CD 155 antibodies may have therapeutic potential, either alone, by inducing immune system such as antibody-mediated cytotoxicity, or as anti-CD 155 antibodies fused to other peptides, polypeptides or antibodies. Various cargo such as toxins, proteins, peptides, small molecule drugs, RNA or DNA based drugs, such as genes, messenger RNAs and oligonucleotides. Each of the above may be directly conjugated to an anti-CD 155 antibody or the anti-CD 155 antibody may be conjugated to a nanoparticle/liposome encapsulating a toxin, protein, peptide, small drug molecule, RNA or DNA drug (e.g., gene, messenger RNA, oligonucleotide). For example, ligand-PVR interactions such as anti-CD 155 antibody-PVR can serve as a transport pathway for macromolecules across the blood brain barrier and may be a substitute for transferrin. The anti-CD 155 antibody or ligand that binds to any PVR crossing the blood brain barrier may also be used as a transporter for drugs, proteins, peptides, polypeptides that are fused or bound to the anti-CD 155 antibody or other PVR ligand.

Ligands of the application include, but are not limited to, anti-CD 155 antibodies AB. Further included are fusion proteins of antibodies and antigen-binding fragments that bind to CD155 (and/or Nectin 4), and fusion proteins of such antibodies and antigen-binding fragments. Also included are ligand conjugated liposomes and encapsulation of RNA or DNA or modified nucleic acids or other therapeutic agents, as well as ligand conjugation of nucleic acids or other therapeutic agents without the use of liposomes. In addition, encapsulation of ligand conjugates of liposomes and 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 contain liposomes are included.

In one embodiment, the anti-CD 155 antibodies capable of crossing the blood brain barrier may be selected from the group consisting of, but not limited to, antigen binding fragments (Fab, fcV), humanized anti-CD 155 antibodies, targeted conjugated liposomes/nanoparticles, and anti-CD 155 antibodies that encapsulate or unencapsulate therapeutic drugs (including toxins, genes, DNA or RNA-based drugs, or other therapeutic drugs); an anti-CD 155 antibody coupled to a fluorescent probe or contrast agent for diagnosing a tumor or aiding in non-surgical, pre-operative, intra-operative or post-operative determination of a tumor or tumor margin; an anti-CD 155 antibody fused to a protein, peptide, toxin, or other antibody or antigen binding fragment to allow for transport across the blood brain barrier by utilizing CD155, once traversed, CD155 or fusion protein, or both, can bind to target cells within the central nervous system; unbound anti-CD 155 antibodies as therapeutic agents induce antibody-mediated cytotoxicity or complement-mediated cytotoxicity in central nervous system and non-central nervous system tumors that are highly expressed by CD 155.

Anti-CD 155 antibodies unexpectedly cross the blood brain barrier and bind to tumors in mice. Antibodies typically do not cross the blood brain barrier. Polioviruses have other pathways in themselves that enter the central nervous system. In one embodiment, these antibodies are used to treat neurological disorders. In one embodiment, it may be beneficial to cross the blood brain barrier in the absence of a tumor. In one embodiment, the neurological disease is not a brain tumor. Antibodies include a variety of moieties including, but not limited to, heavy chains, light chains, fab, fc, scFv, glycosyl groups, variable regions, and constant regions.

These antibodies can be modified in a variety of ways, including but not limited to glycosyl modification, altering amino acids in the constant region, using different human mAb isotypes (e.g., igG 4), linking isotopes to mabs with stable linkers, linking drugs to mabs with cleavable linkers, inserting DNA into the mAb variable region fused with a signal peptide to induce T cells to express CARs, and cross-linking regions from both mabs.

Monoclonal antibody (mAb) -based therapies have many functions including, but not limited to, anti-tumor mabs, inhibition of angiogenesis, T cell checkpoint blockade, radioimmunotherapy, antibody drug complexes, bispecific antibodies, and chimeric antigen receptor T cells. One embodiment of the invention includes a bispecific antibody wherein one antibody or antigen-binding fragment binds to CD155 (or Nectin 4) expressed on a tumor cell and the other antibody or antigen-binding fragment binds to CD3 (T cell receptor) on a T cell, thereby bringing the T cell into proximity with the tumor cell, enhancing T cell-mediated tumor killing. Another embodiment includes a bispecific antibody wherein one antibody or antigen-binding fragment binds to CD155 (or Nectin 4) on a tumor cell and the other antibody or antigen-binding fragment binds to a receptor on an NK cell (e.g., CD16, NKG2D, SLAM receptor, or a natural cytotoxic receptor on an NK cell, such as NKp46, NKp44, or NKp 30), thereby bringing the NK cell into proximity with the tumor cell, enhancing NK cell-mediated tumor killing. The immune-mediated effects of tumor-specific IgG include, but are not limited to ADCC, opsonization, and CDC. Direct effects of tumor-specific IgG include blocking ligands, inhibiting receptor dimerization, and inducing apoptosis signaling. A bi-enzymatic preparation can be prepared that binds anti-CD 155 antibodies and antigen-binding fragments, as well as antibodies or antigen-binding fragments that bind to type 1 insulin-like growth factor receptor or FC 5 or FC 44 or transferrin receptor (Tfrl or Tfr 2) to facilitate passage through the blood brain barrier.

A possible limitation in developing monoclonal antibodies for neurooncology is the inability to cross the Blood Brain Barrier (BBB). Monoclonal antibodies directed against brain tumors first need to enter the central nervous system, either by direct injection into the tumor (convection-enhanced therapy) or by direct intravertebral injection (in spinal fluid) through the blood-brain barrier (for antibodies that can cross the blood-brain barrier, cross the blood-brain barrier from the vascular lumen to the brain and central nervous system). Other mechanisms of access to the central nervous system should be proposed, such as retrograde through muscles or nerves. Only after the monoclonal antibody enters the central nervous system can it bind to the primary intracranial brain tumor (if the brain tumor metastasizes to the periphery, the antibody can bind to the tumor without entering the central nervous system).

In one embodiment, when the mRNA is encapsulated in a liposome conjugated to an anti-CD 155 antibody or antigen binding fragment, it is capable of endocytosis by the cell. The anti-CD 155 antibody D171 binds to the poliovirus receptor, which is located at amino acid residues 35-50 of the poliovirus receptor. In one embodiment, the humanized antibody against human CD155 is conjugated to a liposome containing antisense DNA or antisense RNA, a plasmid, or a gene containing natural or chemically modified residues. In another embodiment, the liposome contains an mRNA containing natural or chemically modified residues. Humanized antibodies and conjugated lipids against human CD155 are capable of crossing the blood brain barrier and delivering mRNA or nucleic acid therapeutics into cells expressing CD 155.

The application includes viral delivery therapies comprising mRNA and gene by ligand conjugates wherein the ligand (e.g., monoclonal antibody) binds to CD155/PVR resulting in endocytosis and subsequent release of the therapeutic substance thereby effecting drug or mRNA/gene expression (mRNA/gene). It is shown here that the D171 antibody conjugated to mRNA encapsulated in liposomes is capable of being endocytosed and capable of delivering the payload to CD155 expressing cells where the mRNA is expressed in tumor cells in vitro. This proof of concept lays the foundation for delivering nucleic acid therapeutics and other drugs to cells using humanized anti-CD 155 antibody drug conjugates.

In one embodiment, 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, wherein one moiety binds to CD155/PVR and the other moiety is a toxin such as diphtheria toxin; 3) An antibody or antigen-binding fragment that binds to a therapeutic drug or prodrug (e.g., a nucleic acid or gene), wherein the antibody or antigen-binding fragment binds CD155/PVR. The therapeutic drug or prodrug may be doxorubicin, siRNA, antisense DNA or other antisense molecules, messenger RNA encoding a protein or enzyme or peptide or cytokine, cas 9mRNA, cas 12a mRNA and CRISPR guide RNA, naked DNA gene or plasmid. An antibody or antigen binding fragment against CD155 may bind to one of the following mrnas encoding cytokines: (a) mRNA encoding IL2, (b) mRNA encoding IL7, (c) mRNA encoding IL12, (d) mRNA encoding IL15, (e) mRNA encoding IL21, (f) mRNA encoding IFNgamma, (g) mRNA encoding IFNalpha, and (i) mRNA encoding GM-CSF.

In one embodiment, humanized anti-CD 155 antibodies can be used to target neurons, astrocytes, regenerated muscle and spinal cord. Antibodies against CD155, or fragments or fusions thereof, whether used alone or in combination, play an important role in pre-, intra-or post-operative diagnosis, enabling the determination of the full range of tumors or the range of tumor resections. In one embodiment, GBM patients are preoperatively treated with a fluorescent-labeled antibody against CD155, which may cross the blood brain barrier or be injected directly into the tumor site, which may guide the surgeon intraoperatively through the profile of antibody fluorescence for tumor resection.

By adding soluble CD155 to bind anti-CD 155 antibodies, soluble CD155 can act as an antidote to the excess of anti-CD 155 antibody conjugate. Furthermore, in one embodiment, prior to treatment of a patient with an antibody drug conjugate to an anti-CD 155 antibody, unbound anti-CD 155 antibody is first administered to block CD155 on normal cells. Subsequent addition of the anti-CD 155 antibody drug conjugate will be directed to tumor cells that overexpress CD155. Antibodies against CD155, or fragments or fusions thereof, either alone or in combination, may be used for diagnostic identification of brain tumors, as well as for determining the extent of brain tumors by imaging mechanisms such as PET scanning, MRI, nuclear scanning, CT scanning, ultrasound, and other standard imaging techniques in the field.

Conclusion IV

Although the function of CD155 is not fully understood, CD155 has been demonstrated to be capable of binding to TIGIT (T cell immunoreceptor with Ig and ITIM domains), CD226 (DNAM-1) and CD96. Soluble CD155 is a prognostic tumor biomarker, is normally secreted from CD155 positive tumors, and may play a role in binding to DNAM-1. CD155 and soluble CD155 in the cell membrane may promote CD155 positive tumor growth by inhibiting the function of the immune system. Blocking soluble CD155 or CD155 in the cell membrane using anti-CD 155 antibodies can reverse immunosuppression. Blocking CD155 with anti-CD 155 antibodies may also prevent binding of CD155 to TIGIT, thereby attenuating the inhibitory signal transmitted by CD155-TIGIT binding. Thus, CD155 is a novel checkpoint and the use of anti-CD 155 antibodies to modulate CD155 provides a new approach to the treatment of cancer using a variety of mechanisms of action. Although the level of CD155 is low in normal tissues such as muscle and kidney, CD155 is overexpressed in many different cancers, making CD155 a novel but common target for the treatment of a wide range of malignancies. Likewise, the monoclonal antibodies described in the present invention bind not only CD155 but also to another tumor marker, nectin 4. The monoclonal antibodies of the invention were shown for the first time to be capable of binding to Nectin 4. Monoclonal antibodies D171 and AB825 (AB 825 and AB825 are used interchangeably in the present specification, figures and claims) associated with the humanized antibodies of the invention have never been shown to bind to Nectin 4 before. Nectin 4 has recently been found to be able to bind to TIGIT. In one embodiment, the antibodies of the invention will prevent binding of Nectin 4 and PVR to TIGIT, thereby blocking the inhibitory signal sent to immune cells such as T cells. In another embodiment, an antibody or antigen binding fragment against CD155 is combined with other drugs, cell therapies, or immunomodulators consisting of antibodies against checkpoint molecules, for use in the treatment of cancer. The anti-CD 155 antibody or antigen binding fragment is administered to the human in combination with an additional immunomodulator comprising an antibody to an immune checkpoint molecule selected from the group consisting of CTLA 4、PD1、PDL1、CD 112R、OX 40、TIGIT、NKG2A、CEACAM 1、B7H3、B7-H4、VISTA、LAG3、PD1、PDL1、CD 112R、OX 40、TIGIT、NKG2A、CEACAM 1、B7H3、B7-H4、VISTA、LAG3、CD 137、KIR、TIM 1、TIM3、LAIR 1、HVEM、BTLA、CD 160、CD 200、CD 200R and A2 r.

In one embodiment, the anti-CD 155 antibody or antigen-binding fragment is fused to an antibody or antigen-binding fragment known to be capable of crossing the blood brain barrier, e.g., an anti-transferrin receptor antibody or antigen-binding fragment (or FC5 antibody or antigen-binding fragment capable of crossing the blood brain barrier). The anti-CD 155 antibody or antigen-binding fragment may be bound by chemical or recombinant techniques to an antibody (or antigen-binding fragment) capable of crossing the blood-brain barrier, thereby enabling the anti-CD 155 antibody to cross the blood-brain barrier. In another embodiment, a bispecific antibody comprising an antibody described in the present invention binds CD155 (or Nectin 4) on a tumor, and binds CD3 on a T cell. In another embodiment, a bispecific antibody comprising one or more of the antibodies described in the present invention binds CD155 (or Nectin 4) on a tumor, and another portion of the bispecific antibody binds to a receptor on an NK cell (i.e., CD16, SLAM receptor, NKp46, NKp44, or NKp30 on an NK cell). In one embodiment, the anti-CD 155 antibody or antigen-binding fragment is fused to an antibody or antigen-binding fragment known to be capable of crossing the blood brain barrier (or FC5 antibody or antigen-binding fragment capable of crossing the blood brain barrier), e.g., an antibody or antigen-binding fragment of an anti-transferrin receptor. The anti-CD 155 antibody or antigen-binding fragment may be bound by chemical or recombinant techniques to an antibody (or antigen-binding fragment) capable of crossing the blood-brain barrier, thereby enabling the anti-CD 155 antibody to cross the blood-brain barrier. Antibodies of the invention bind to and block PVR (CD 155). The humanized monoclonal antibodies of the invention are useful for treating patients exposed to poliovirus by blocking poliovirus receptors and preventing poliovirus from binding to poliovirus receptors (PVR; CD 155).

In one embodiment, the antibodies of the invention may also be injected into mammals, including humans, to treat infections such as HIV by increasing the expression of DNAM 1 (CD 226) on T cells. Also, in another embodiment, the antibodies of the invention can be used to treat sepsis by increasing the expression of DNAM 1 on T cells and NK cells. The excessive use of humanized antibodies against CD155 of the present invention can be treated by administering soluble CD155 to bind to these humanized antibodies and neutralize their effect. In another embodiment of the invention, AB825, D171 or any humanized antibody that binds to PVR (CD 155) may bind in vivo in animals expressing human PVR. The human PVR may be an expressed transgene or the human PVR may be expressed on a human cell such as a U87 tumor cell implanted into a mouse. Prior to doing this, ab825 or any humanized antibody of the invention was never injected in a living animal. In another example, ab825 and the humanized antibodies described in the present invention against CD155 have been demonstrated to bind Vero cells of african green monkeys, and thus these antibodies were able to bind PVR (CD 155) in non-human primates (fig. 8).

The D171 antibody is an anti-CD 155 antibody that binds to liposome-encapsulated mRNA and is successfully delivered and expressed in human U87MG tumors. This shows for the first time that the anti-CD 155 antibody (D171) is internalized, as is the liposomal mRNA that binds to D171. This work provides the basic support for the preparation of antibody drug conjugates of D171, AB825 and related humanized antibodies of the invention. Internalization of D171 and its ability to transmit load to human tumor cells are also one embodiment of the invention. Uptake experiments with both liposomal eGFP-RNA and liposomal fluorescein-RNA showed that the conjugate of anti-CD 155 antibody was significantly better than all other tested conjugates, and that the test could be performed only at low concentrations due to the limited number available. Furthermore, these results are obtained with two types of CD155 antibody conjugates, i.e. antibodies directed against human or mouse specificity, thus enhancing the robustness of the results. After internalization of the U87 tumor, mRNA was expressed. In another experiment, more than 80% of U87 cells were fluorescently labeled after administration of liposome-encapsulated fluorescein-labeled mRNA conjugated to D171 antibody. This work provides the basis for the first time for the preparation of antibody drug conjugates of D171, ab825 and other related humanized antibodies of the invention. The internalizing ability of D171, ab825, and the humanized antibodies of the invention and their ability to transmit load to human tumors are also an embodiment of the invention. In addition to these results, only anti-IL-13 a 2-bound liposomes were shown to be taken up slightly but repeatedly in different experiments, both in the mouse version (GL 261) and in the human version (U87 MG). The RNA fluorescent label with fluorescein can be replaced with another more powerful fluorophore that is less susceptible to bleaching by the fluorophore for high content imaging image acquisition. Uptake of functional liposomes is achieved in complete medium containing 10% serum, conditions that more closely approximate in vivo conditions. 10, 50 or 100 nanograms per well of eGFP was applied to 3 densities of GL261 or U87MG cells, which encoded RNA in functionalized liposomes. Because of the very limited number available, only 10 nanograms of RNA per well can be tested against CD155 antibody conjugates. The results show that at all cell densities tested, both for the mouse antibody conjugate and the human antibody conjugate, the cell fluorescence intensity repeatedly increased after administration of 10 nanograms of RNA/well of anti-CD 155 antibody-bound liposomes.

Humanization of anti-CD 155 antibodies

Ab825 is an antibody sharing sequence with anti-CD 155 antibody D171. D171 was first described by Nobis et al (J General Virology) in 1985. Sequencing data showed that the heavy chain variable region (VH) of the D171 antibody had more than 90% sequence identity to the heavy chain variable region (VH) sequence of Ab 825. The light chain variable region (VL) and CDRs of the D171 antibody also overlap significantly with the sequence of Ab 825. In view of the high degree of homology of the D171 and Ab825 sequences, it is not necessary to individually humanize both antibodies. The sequence of Ab825 was humanized by removing potentially immunogenic sequences. The possible deamidation and isomerization sites of Ab825 were also analyzed, three of which were located in the CDRs of Ab 825. In addition to humanization, potentially unstable sites were also mutated and then tested for binding properties of a range of antibodies.

Humanized anti-CD 155 antibodies have optimal binding properties, then are assessed by an infusion reaction risk by cytokine release assay, proliferation reaction by EPISCREEN DC T cell assay, non-target and target binding to more than 5000 known membrane receptors, and epitope mapping of binding sites in PVR. These antibodies were also selected based on their ability to bind to monkey CD155 (african green monkey-Vero cells). In order to replace the immunogenic sequences, sequences that are less immunogenic to humans were inserted. Antibodies were first mutated in the framework regions and then tested for binding and stability. Antibodies were evaluated for their ability to bind to PVR receptors. In many variants, the binding of the antibody to the PVR receptor remains unchanged or is improved or worsened. In one embodiment, software is used to identify potentially immunogenic sequences that need to be removed. Even after removal of the software-recognized sequences, undesirable immunogenic reactions may still occur. Even after humanization, the binding or immunogenicity of the humanized antibodies may be affected unpredictably by different mutations. Analysis of the Ab825 sequence for immunogenicity and CDR instability/variability (e.g., aspartic acid isomerization sites or high or medium risk deamidation sites) was performed, either of which may affect the conformation of the CDRs and thus the antigen binding (i.e., binding to CD 155). MHC-II binding sequences with a generally moderate and high affinity are also identified for humanization. Due to the unpredictability of the effects of altering the labile sequences and framework residues in the CDRs on CD155 binding, the resulting humanized and risk-reduced CD155 antibodies were tested in CD155 positive HAP cells and HAP cell lines with CD155 knockdown.

As described above, poliovirus receptor (PVR), also known as CD155, is overexpressed in many cancers, including glioblastoma, malignant meningioma, malignant peripheral schwannoma, pancreatic cancer, lung cancer, and digestive tract malignancies including colorectal cancer, breast cancer (including triple negative breast cancer). Poliovirus receptors are also naturally found in healthy tissues, although the receptor density is low, and can be found in cells such as motor neurons of the brain and spinal cord. Antibodies capable of binding to CD155 with different efficiencies are known. An example of one known antibody is a mouse antibody in which the heavy and light chains are incorporated into a human IgGl chimeric antibody, referred to herein as Ab825.Ab825 was derived from D171, a mouse antibody against human CD 155. The heavy chain variable region (referred to herein as VHO) and the light chain variable region (referred to herein as VKO) of Ab825 bind to poliovirus receptors in humans and non-human primates. Heavy chain variable region VHO is disclosed as sequence ID NO 1 and light chain variable region VKO is disclosed as sequence ID NO 5. Complementarity Determining Regions (CDRs) of the heavy chain variable region VHO, namely CDR1, CDR2 and CDR3, are disclosed as sequence ID NO 2, sequence ID NO 3 and sequence ID NO 4, respectively. The CDRs of light chain variable region VKO, CDR1, CDR2, and CDR3, are disclosed as sequence ID NO 6, sequence ID NO 7, and sequence ID NO 8, respectively. The amino acid sequences listed are in SEQ ID 1 to SEQ ID 31, the CDR definitions and protein sequencing being in accordance with the Kabat specification.

One aspect of the invention relates to modifying and humanizing this heavy chain Variable (VH) region and light chain Variable (VK) region to make the antibody more stable for human therapeutic and diagnostic purposes and to minimize immunogenicity and toxicity. To form such antibodies or antigen binding fragments, the designed variable region gene is cloned into a vector encoding a human IgGl or IgG4 heavy chain constant domain and a human kappa light chain constant domain. Chimeric and humanized antibodies (including IgG 4S 241P) were transiently expressed in CHO cells, purified by protein a, and tested for their binding capacity to PVR using Biacore (surface plasmon resonance).

To determine important limiting amino acids in the variable region that may be critical to antibody binding properties, we generated and analyzed a structural model of the Ab825 antibody variable region. Based on this structural analysis, we determined a large array of sequence fragments that could be used to create humanized variants of Ab 825. These fragments were screened and analyzed, in vitro to assess peptide binding to human MHC-II alleles and compared to T cell epitopes associated with known antibody sequences. Sequence fragments of non-human germline that were determined to have significant binding to human MHC class II, or sequence fragments that obtained significant hits in our analysis, were all knocked out. This resulted in a reduced set of fragments and combinations of these fragments were again analyzed to ensure that the ligation between them did not contain potential T cell epitopes. Selected sequence fragments are assembled into complete variable region sequences that do not contain significant T cell epitopes. The thermostability and freeze-thaw stability of the humanized antibodies were also assessed.

Using the above analysis, five variable region heavy chains (labeled VH1 to VH 5) and four variable region light chains (labeled VK1 to VK 4) with enhanced binding properties were determined. The sequence of these regions can be found in the following sequence ID Nos:

In one embodiment, the invention is an IgG subtype antibody or antigen-binding fragment of the anti-poliovirus receptor (CD 155). The heavy chain variable region of the antibody or antigen binding fragment will be derived from any of SEQ ID Nos. 1, 9, 10, 11, 12 or 13, with CDR1 being the sequence of SEQ ID No. 2 and CDR3 being the sequence of SEQ ID No. 4. CDR2 will be SEQ ID No.3, but will be modified by one or more of the following:

(1) Substitution of aspartic acid at the sixth position in CDR 2 with one of alanine, glutamic acid, lysine, glutamine, serine or threonine; and/or

(2) Substitution of aspartic acid at the eighth position in CDR2 with one of glutamic acid, glycine or arginine; and/or

(3) The threonine at the ninth position in CDR 2 is replaced with one of glutamic acid or lysine.

The light chain variable region is derived from any one of SEQ ID Nos. 5, 14, 15, 16 or 17, with CDR1 being the sequence of SEQ ID No. 6 and CDR2 being the sequence of SEQ ID No. 7. CDR3 will be the sequence of SEQ ID No. 8, but will have aspartic acid at the fourth position in CDR3 replaced with one of alanine, glutamic acid, glycine, lysine, glutamine or serine.

In another embodiment, the antibody or antigen binding fragment directed against poliovirus receptor (CD 155) is of the IgG subtype. However, in this embodiment, the heavy chain variable region and the light chain variable region are defined in terms of their CDRs. For example, the heavy chain variable region will comprise VH0 CDR1 of SEQ ID No. 2 and VH0 CDR3 of SEQ ID No. 4. Similarly, VH0 CDR2 will be the sequence of SEQ ID No. 3, but will be modified by one or more of the following:

The light chain variable region will have a VK0 CDR1 of SEQ ID No. 6 and a VK0 CDR2 of SEQ ID No. 7. VK0 CDR3 will be the sequence of SEQ ID No. 8, but will have aspartic acid at the fourth position in CDR3 replaced with one of alanine, glutamic acid, glycine, lysine, glutamine or serine. Preferred antibody embodiments were developed from the specific combination of the heavy chain variable regions VH1-VH5 (SEQ ID numbers 9-13) and light chain variable regions VK1-VK4 (SEQ ID numbers 14-17) previously described. Based on analysis of T cell epitope spectra, VH3 and VH4 and VK2 and VK3 are considered to be one of the best variable heavy and light chain human variants. VK or VK may be used interchangeably. VH and VH are also used interchangeably herein.

To address the potential deamidation sites identified at VH N54 and VK N92 (using single letter amino acid symbols and amino acid position numbering) as well as the potential isomerization sites associated with VH D56, a series of amino acid substitutions (six substitutions for potential sequence susceptible sites at VH N54 and VK N92 and five substitutions for VH D56) were first introduced in the VH0/VK0 chimeric antibody, respectively. Substitutions that reduce susceptibility in VH0/VK0 include mutations in VH N54Q, N S, D E and D56G and VK N92E and N92Q, variants of these VH0/VK0 were tested for binding to poliovirus receptors and, after analysis of binding, were selected for binding to certain preferred humanized variants of VH3/VK2, VH3/VK3 and VH4/VK 3. These modified VH and VK regions have the following sequences and designations:

Sequence ID number	Sequence name
		SEQ ID NO.18	VH3 N54Q D56E
SEQ ID NO.19	VK2 N92E
		SEQ ID NO.20	VH3 N54SD56G
SEQ ID NO.21	VK2 N92Q
		SEQ ID NO.22	VK3 N92E
SEQ ID NO.23	VK3 N92Q
		SEQ ID NO.24	VH4 N54Q D56E
SEQ ID NO.25	VH4 N54SD56G
		SEQ ID NO.26	VH4 N54SD56E

Using these VH and VK regions, nine preferred antibodies (or antigen binding fragments) to the poliovirus receptor (CD 155) were determined, with the following heavy and light chain variable regions, respectively:

(i) VH 3N 54Q D E [ SEQ ID NO:18] and VK 2N 92E [ SEQ ID NO:19];

(ii) VH 3N 54SD56G [ SEQ ID NO:20] and VK 2N 92Q [ SEQ ID NO:21];

(iii) VH 3N 54Q D E [ SEQ ID NO:18] and VK 3N 92E [ SEQ ID NO:22];

(iv) VH 3N 54SD56G [ SEQ ID NO:20] and VK 3N 92Q [ SEQ ID NO:23];

(v) VH 4N 54Q D E [ SEQ ID NO:24] and VK 3N 92E [ SEQ ID NO:22];

(vi) VH 4N 54SD56E [ SEQ ID NO:26] and VK 3N 92E [ SEQ ID NO:22];

(vii) VH 4N 54SD56G [ SEQ ID NO:25] and VK 3N 92E [ SEQ ID NO:22];

(viii) VH 4N 54Q D E [ SEQ ID NO:24] and VK 3N 92Q [ SEQ ID NO:23];

(ix) VH 4N 54SD56G [ SEQ ID NO:25] and VK 3N 92Q [ SEQ ID NO:23].

Further testing of these nine antibodies indicated that optimal binding occurred in (ii) the following antibodies VH3N54SD56G [ SEQ ID NO:20] and VK 2N 92Q [ SEQ ID NO:21]; (iv) VH3N54SD56G [ SEQ ID NO:20] and VK 3N 92Q [ SEQ ID NO:23]; and (ix) VH 4N 54S D G [ SEQ ID NO:25] and VK 3N 92Q [ SEQ ID NO:23].

In addition to the antibodies described above, the present invention is intended to include novel heavy and light chain variable regions that form these antibodies. For example, in SEQ ID no: 9-13, 18, 20, and 24-26, and the VH region defined in SEQ ID no: the VK regions identified in 14-17, 19, and 21-23 should be considered as separate inventions. The humanized antibodies of the present invention have been shown to bind not only to PVR (CD 155) but also to Nectin 4 (PVRL 4). The dual specificity of such antibodies and antigen binding fragments described in the present invention expands the range of tumors that can be treated with the antibodies described in the present invention. Humanized antibodies of the invention associated with D171 and Ab825 were first shown to bind to Nectin 4 in addition to PVR. D171 and AB825 or any variant thereof have never been shown to bind to Nectin 4 before.

Another embodiment includes an antibody or antigen binding fragment directed against the poliovirus receptor (CD 155), comprising:

(a) A heavy chain variable region selected from SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:24、SEQ ID NO:25 or SEQ ID NO. 26;

(b) A light chain variable region selected from SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:22or SEQ ID NO:23.

The following are some examples including the antibodies and antigen binding fragments described above. The antibodies or antigen binding fragments described above may be conjugated to prodrugs or drugs for the treatment of tumors expressing Nectin 4. The antibodies or antigen binding fragments described above may be conjugated to a prodrug or drug for the treatment of CD155 (PVR) expressing tumors. Another embodiment includes an antibody drug conjugate as described above wherein the drug or prodrug is from one of the following: nucleic acids, vedontin, pyrrole benzodiazepines, liposomal doxorubicin, topoisomerase inhibitors, MM AE, MMAF, DM 1, paclitaxel, temozolomide, doxorubicin, de Lu Xiting, radiosensitizers, doxycycline, docetaxel, and PARP inhibitors. One embodiment includes an antibody or antigen-binding fragment as described above, wherein the antibody or antigen-binding fragment binds CD155 (PVR) and blocks the binding of CD155 (PVR) to TIGIT. One embodiment includes an antibody or antigen-binding fragment as described above, wherein the antibody or antigen-binding fragment binds to Nectin 4 and blocks binding of Nectin 4 to TIGIT. One embodiment includes an antibody or antigen binding fragment as described above, wherein the antibody or antigen binding fragment binds to CD155 (PVR) in a non-human primate. One embodiment includes an antibody or antigen-binding fragment as described above, wherein the antibody or antigen-binding fragment binds to Nectin 4. Another embodiment includes the antibody or antigen binding fragment described above, wherein the antibody or antigen binding fragment binds CD155 (PVR), resulting in an increase in DNAM Ion (a) T cells and (b) NK cells in at least one of the following. Another embodiment includes a bispecific antibody or antigen-binding fragment that binds in part to poliovirus receptor or Nectin 4 and in part to CD3 on T cells. Another embodiment includes a bispecific antibody or antigen-binding fragment that binds in part to poliovirus receptor or Nectin 4 and in part to a receptor on NK cells. Another embodiment includes an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment against CD155 is administered to a human, and the antibody or antigen-binding fragment binds CD155 and prevents poliovirus from binding to CD155 in the human.

Another embodiment comprises administering the antibody or antigen binding fragment of claim 63 to a human in combination with an antibody to an immune checkpoint molecule selected from the group consisting of CTL A4、PD1、PDL1、CD112R、OX40、TIGIT、NKG2A、CEACAM1、B7H3、B7-H4、VISTA、LAG3、CD137、KIR、TIM1、TIM3、LAIR1、HVEM、BTLA、CD160、CD200、CD200R to treat cancer. Another embodiment includes an antibody of claim 63 that is directed against CD155 and includes an IgG4 subtype having a S241P hinge mutation or S228P hinge mutation. Another embodiment includes an antibody or antigen binding fragment of claim 63, wherein the antibody or antigen binding fragment directed against CD155 comprises one of the following markers: (a) I-124, (b) gallium 68 or (c) ruthenium 177, (d) contrast agent for CT scan, (e) contrast agent for MRI scan, and (f) diagnostic agent for PET scan. Another embodiment includes the antibody or antigen binding fragment of claim 63, wherein the anti-CD 155 antibody or antigen binding fragment binds to an mRNA encoding a cytokine or gene-editing enzyme, comprising (a) an mRNA encoding IL-2, (b) an mRNA encoding IL-7, (c) an mRNA encoding IL-12, (d) an mRNA encoding IL-15, (e) an mRNA encoding IL-21, (f) an mRNA encoding IFN-gamma, (g) an mRNA encoding IFN-alpha, (h) an mRNA encoding GM-CSF, (i) an mRNA encoding Cas9, (j) an mRNA encoding Cas12a, or (k) an mRNA encoding Cas 13. Another embodiment includes a CAR T cell or CAR NK cell comprising an antigen binding fragment as described above. Another embodiment includes an antibody or antigen binding fragment as described above, wherein the antibody or antigen binding fragment directed against CD155 binds to CD155 expressed on a tumor and elicits an immune response to the tumor by ADCC or CDC. One embodiment includes a D171 antibody conjugate, wherein the D171 antibody conjugate binds to a tumor that expresses CD155, and the D171 antibody conjugate is internalized by the tumor.

Another embodiment includes an antibody or antigen-binding fragment having a heavy chain variable region of SEQ ID 1 and a light chain variable region of SEQ ID 5, wherein the antibody or antigen-binding fragment binds to Nectin 4. Such antibodies or antigen binding fragments of the invention are capable of preventing binding of Nectin 4 to TIGIT. Such antibodies or antigen binding fragments may be conjugated to a prodrug or drug for the treatment of tumors that express Nectin 4. The antibody or antigen binding fragment includes an antibody or antigen binding fragment having a heavy chain variable region of SEQ ID 1 and a light chain variable region of SEQ ID 5, which binds to PVR resulting in an elevated level of DNAM Ion T cells or NK cells.

Examples

Humanized antibodies and antigen binding fragments directed against CD155& Nectin 4

Example 1 checkpoint blockade

Both CD155 and Nectin4 are described as ligands for TIGIT. Binding of CD155 to TIGIT or binding of Nectin4 to TIGIT results in inhibition of T cell or NK cell function, as TIGIT is expressed on immune cells. The antibodies of the invention block CD155 or Nectin4, and will prevent CD155 or Nectin4 (or both) from binding TIGIT. PVR is a natural ligand for TIGIT (Alteber et al, described in J.cancer discovery, 2021, 5). PVR is also a natural ligand for CD226 and CD 96. As shown in Yu et al, volume 10 (9) supplement fig. 4b and 4c, published in natural immunology, 2009, D171 has been demonstrated to prevent PVR (CD 155) binding to TIGIT. Reches et al (J.cancer immunotherapy 2020) show that Nectin4 is a ligand for TIGIT. The D171 antibody also binds PVR and prevents PVR from binding to CD96 (Meyer et al, J. Biol.Chem., volume 284 (4) pages 2235-2244, see also page 2242).

Example 2T cell and NK cell activation

The antibodies or antigen binding fragments of the application can bind to CD155 and Nectin 4 for use in preparing bispecific antibodies or bispecific antigen binding fragments. Bispecific antibodies or bispecific antigen binding fragments are one embodiment of the application, wherein the bispecific molecule binds CD155 and CD3 (or Nectin 4 and CD 3). Bispecific antibodies targeting CD3 and CD155 were prepared by Ma et al in 2019, journal of cancer, volume 10, page 5153. These bispecific antibodies show activity against bladder cancer. Still further examples include bispecific binding to CD155 (or Nectin 4) and receptors on NK cells to activate immune cells to destroy CD155 (or Nectin 4) expressing tumors. Antibodies and antigen binding fragments of the present disclosure may be incorporated into bispecific antibodies.

Example 3 Antibody Drug Conjugates (ADCs).

Antibody Drug Conjugates (ADCs) are one embodiment of the invention in which the described antibodies bind to drugs Vedontin, deruxtecan and Tesinne, etc., which bind to the antibodies of the invention and are delivered to tumors expressing CD155 or Nectin 4 (or both). CD 155-targeting ADCs may be internalized for drug delivery. Since the target receptor is also a checkpoint molecule, these ADCs against CD155 have multiple mechanisms of action. The antibody drug conjugate may be conjugated by a variety of known linkers including, but not limited to, cysteine protease sensitive peptide cross-linkers. The antibody or antigen binding fragment may be conjugated to at least one drug (antibody drug conjugate), such as MMAE, MMAF, paclitaxel, temozolomide, doxorubicin (including liposomal doxorubicin), deruxtecan, topoisomerase inhibitors, radiosensitizers, radiolabeled antibodies or antigen binding fragments, tesirine, any pyrrole benzodiazepineThe inhibitors of the Pinines (PBD), DM1, vedontin and docetaxel and PARP, such as Lu Kapa nifedipine. An antibody or antigen binding fragment comprising the heavy chain variable region of SEQ ID 1 and the light chain variable region of SEQ ID 5 is conjugated to a fluorescent substance and injected into a mammal, wherein the antibody conjugate binds to and accumulates in a tumor expressing CD155 (PVR) in the mammal (fig. 4).

Example 4ADC.

ADCs that can deliver nucleic acids, including mRNA. anti-CD 155 conjugates of mRNA liposomes encapsulating the reporter EGFP can deliver and express mRNA in CD155 expressing cancer. The antibody or antigen binding fragment may also bind to one or more therapeutic nucleic acids delivered by the antibody or antigen binding fragment, wherein the therapeutic nucleic acid may be any one or more of silencing RNA, shRNA, long RNA, riboribozyme, messenger RNA, plasmid DNA or non-plasmid double-stranded DNA and short single-stranded DNA and double-stranded DNA. Antibody drug conjugates can also be prepared by binding an antibody or antigen-binding fragment of the invention to mRNA (encapsulated in a liposome or complexed with a polymer), wherein the mRNA encodes a peptide, protein (e.g., IL-12, IL-15, or IL-2), and enzyme (e.g., gene editing enzyme Cas9 or Cas12a, and other Cas enzymes, such as Cas 13). A D171 antibody conjugate was formed, and the D171 antibody conjugate bound to the CD 155-expressing tumor and was endocytosed by the tumor (fig. 2 and 3). The mRNA payload may be conjugated to an anti-CD 155 antibody to deliver mRNA encoding polypeptides and proteins, including vaccines.

Example 5 DNAM 1-CD 155 Axis

The increase in DNAM-1 (CD 226) can be achieved by using anti-CD 155 antibodies and blocking CD155 (blocking of cell membrane CD155 or soluble CD 155). The DNAM 1-CD 155 axis refers to down-regulation of DNAM 1 (CD 226) expression by soluble CD155 expressed or secreted by CD155 on the cell membrane. One embodiment of the application is therapeutic intervention with humanized monoclonal antibodies against CD155, and administration of the anti-CD 155 antibodies to patients suffering from cancer or HIV infection (or other DNAM-1 (CD 226) down-regulated diseases). By blocking cell membrane CD155 or soluble CD155 using the humanized anti-CD 155 antibodies of the application, the expression of DNAM-1 (CD 226) on T cells and NK cells can be increased. Increasing DNAM-1 will reestablish the immune surveillance mechanism. Carlsten et al (journal of immunology, 2009, volume 183, page 4921) have shown that CD155 tumors down-regulate CD155 on NK cells. Seth et al, J.Biochem.2011, showed that anti-CD 155 antibodies can up-regulate CD226 (DNAM-1) in T cells. The anti-CD 155 antibodies or antigen-binding fragments disclosed in the present application are useful for the treatment of sepsis.

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 CD155 (PVR) or Nectin 4 (or both CD155 and Nectin 4). For example, a humanized antigen binding fragment consisting of HCVR (as set forth in SEQ ID 25) and LCVR (as set forth in SEQ ID 23) can be integrated into CAR T cells and CAR NK cells.

Example 7 adcc and CDC.

ADCC and CDC are mechanisms by which effector cells destroy antibody-coated cancer cells after the antibodies bind to the tumor cells. Antibodies against CD155 may promote ADCC or CDC when bound to and coated on cancer cells. The Fc region of the anti-CD 155 antibody may be further optimized to enhance ADCC or CDC effects.

EXAMPLE 8 theranostics

The anti-CD 155 antibodies or antigen binding fragments described herein may be conjugated to at least one labeling compound, such as a fluorescein IR 800 dye. These antibodies and antigen binding fragments can also be readily radiolabeled with a radiolabel for diagnosis (e.g., 1-124 or gallium 68) or with a radiolabel for treatment (e.g., lutetium 177). The antibodies or antigen binding fragments of the application may be conjugated to at least one labeling compound, such as a fluorescein IR 800 dye.

EXAMPLE 9 subtype IgG 4S 241P

Antibodies or antigen binding fragments described herein may be of the IgG subtype, such as IgG1, igG2 or IgG4, as well as IgE and IgM subtypes. In one embodiment, the humanized antibody against CD155 comprises an IgG4 subtype with an S241P hinge mutation or an S228P hinge mutation.

Example 10 passive immunization to block PVR

The antibodies disclosed herein are associated with D171, and D171 is known to bind to the poliovirus receptor (CD 155) and prevent poliovirus from binding thereto (Nobis et al, 1985, J.Virol.66, p 2563). The humanized antibody disclosed by the application can block poliovirus receptors in human bodies and prevent poliovirus from being combined with the same. The antibodies of the application can be used to treat patients to provide passive immunity to susceptible individuals exposed to poliovirus.

Example 11 epitope mapping

Linear and three-dimensional epitope mapping of PVR and Nectin4 using humanized monoclonal antibodies binding to CD155 and Nectin4 was performed using peptide excision, capturing CD155 or Nectin4 antigen affinity with immobilized test monoclonal antibodies. The immune complex was subjected to limited proteolysis and unbound peptide fragments were washed (GluC and LysC digestions). The eluate of the affinity-bound peptide fragment is then analyzed using high resolution mass spectrometry. Peptide fragments were identified by comparing the antigenic peptide fragment spectra with and without antibodies.

Example 12 humanization of Ab825 and D171

Sequence analysis of Ab825 showed multiple potential susceptibility sites. The Heavy Chain Variable Region (HCVR) of Ab825 (SEQ ID 1) may have high affinity MHC class II anchor residues at position 18 (valine) and position 64 (phenylalanine). The Light Chain Variable Region (LCVR) of Ab825 (SEQ ID 5) may have high affinity MHC class II anchor residues at positions 2 (isoleucine) and 53 (tyrosine). The HCVR (SEQ ID 1) of Ab825 also may have a moderately affinity MHC class II anchor residue at position 32 (tyrosine), 48 (isoleucine) and 93 (valine). LCVR of Ab825 (SEQ ID 5) also has MHC class II anchor residues at position 3 (valine), 4 (methionine), 29 (valine), 47 (leucine) which may have moderate affinity. Humanization of the heavy and light chain variable regions of D171 or Ab825 have not been described or taught prior to the present invention. Furthermore, stabilization of the potential deamidation site and potential isomerization site of Ab825 in the mutated CDRs with a stabilizing anti-CD 155 antibody or antigen-binding fragment is a key part of the invention. Mutations in the CDRs may affect binding, as is demonstrated by EC 50 in Vero cells, hap cells and U87 cells, in fact, in many humanized variants tested, both the N54 and D56 mutations in the Heavy Chain Variable Region (HCVR) and the N92 mutation in the Light Chain Variable Region (LCVR) significantly reduced binding force (fig. 8). N54S, D G in the HCVR and N92Q in the LCVR have optimal binding properties, and these mutations are located within the CDR sequences of the HCVR and LCVR. The VH (aspartic acid) 54 position of SEQ ID 1 is determined to be susceptible to deamidation and may be mutated with amino acids such as alanine, glutamic acid, lysine, glutamine, serine and threonine. VH Asp56 of SEQ ID 1 is determined to be susceptible to isomerisation and may be mutated with amino acids such as glutamic acid, arginine or threonine, whereas VH T57 of SEQ ID 1 may be mutated with amino acids such as glutamic acid or lysine. VkN92 of SEQ ID 5 is also identified as a potential deamidation site and may be mutated with amino acids such as alanine, glutamic acid, glycine, lysine, glutamine or serine.

DC T cell assays showed that different variants of humanized anti-CD 155 antibodies had varying degrees of immunogenicity. The data in fig. 6 shows the percent frequency of proliferation response from 50 donors on days 9, 10 and 11. Antibodies with HCVR VH 4N 54SD56G (SEQ ID 25) and LCVR Vk 3N 92Q (SEQ ID 23) were least immunogenic, had the lowest proliferation frequency, had the lowest Stimulation Index (SI), and were even less immunogenic than FDA approved Herceptin. Cytokine release assays also showed that antibodies with HCVR VH 4N 54SD56G (SEQ ID 25) and LCVR Vk 3N 92Q (SEQ ID 23) had minimal risk of infusion reactions, which were similar in character to the control agent Erbitux. The risk of infusion reactions with antibodies to HCVR VH 4N 54SD56G (SEQ ID 25) and LCVR Vk 3N 92Q (SEQ ID 23) was also much lower than Ab825 (VHO/VkO) as measured by cytokine release. In addition, when tested on 5000 cell membrane receptors, humanized variants were found to bind PVR and Nectin4 (fig. 7). Prior to the present invention, ab825 and its humanized variants disclosed herein were never considered to be capable of binding to Nectin 4. From the results, humanized abs 825, D171, and Ab825 were expected to bind to Nectin 4. In addition, the dominant humanized monoclonal antibody against CD155 has a sub-nM dissociation constant, confirming its high affinity. Monoclonal antibodies defined by SEQ ID 23 (LCVR) and SEQ ID 25 (HCVR) were surface plasmon-resonant kd=0.3 nM with IgG4 (S241P).

Example 13 testing in humanized mice

Human tumors expressing CD155 (with luciferase) were implanted into transiently humanized mice and humanized IgG 4S 241P antibodies against CD155 were intravenously injected into the mice, one group of mice received the antibodies alone, and the other group of mice was given in combination with other checkpoint inhibitors, tumor size was recorded using bioluminescence imaging. Likewise, humanized anti-CD 155 antibodies and bispecific antibodies to CD3 antibodies were tested in a group of mice. Another group of mice will be tested with ADCs prepared by conjugation of humanized anti-CD 155 mice and debukang with a peptide cross-linker sensitive to casein. One control group of mice will be untreated mice. Transient humanized mice are well known in the academia and human blood can be used to study human tumors, but only for short periods of time.

Likewise, the application is directed not only to antibodies and antigen-binding fragments, but also to DNA encoding such antibodies or antigen-binding fragments, as well as to any other polynucleotide or plasmid comprising a nucleic acid sequence encoding such antibodies or antigen-binding fragments. The application also includes cell lines that produce any of the antibodies or antigen binding fragments disclosed in the present application. In view of the present disclosure, all of the compositions and methods disclosed herein can be made and executed without undue experimentation. While the compositions and methods of this application have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods described without departing from the spirit and scope of the inventive concept, which is defined by the appended claims.

Claims

1. A nucleic acid sequence encoding an antibody or antigen binding fragment against a poliovirus receptor (CD 155) comprising a heavy chain variable region and a light chain variable region from:

(a) The heavy chain variable region is selected from SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:24、SEQ ID NO:25 or one of SEQ ID NOs 26; and

(B) The light chain variable region is selected from one of SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 22 or SEQ ID NO. 23.

2. An antibody or antigen-binding fragment of an anti-poliovirus receptor (CD 155), comprising:

(a) A heavy chain variable region selected from one of SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 24, SEQ ID NO. 25 or SEQ ID NO. 26; and

(B) A light chain variable region selected from one of SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 22or SEQ ID NO:23.

3. The antibody or antigen binding fragment of claim 2, in combination with a prodrug or a drug administered to a human, for use in the treatment of a CD155 (PVR) expressing tumor.

4. The antibody or antigen binding fragment of claim 3, wherein the drug or prodrug is from one of the following: nucleic acids, vedontin, pyrrole benzodiazepines, liposomal doxorubicin, topoisomerase inhibitors, MM AE, MMAF, DM 1, paclitaxel, temozolomide, doxorubicin, de Lu Xiting, radiosensitizers, doxycycline, docetaxel, and PARP inhibitors.

5. The antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment binds CD155 (PVR) and blocks the binding of CD155 (PVR) to TIGIT.

6. The antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment binds CD155 (PVR) and blocks the binding of CD155 (PVR) to CD 96.

7. The antibody or antigen-binding fragment of claim 2, comprising a bispecific antibody or antigen-binding fragment comprising a first portion that binds CD155 (PVR) and a second portion that binds at least one of: (a) CD3 on T cells and (b) NKp46 on NK cells.

8. The antibody or antigen-binding fragment of claim 2, comprising a bispecific antibody or antigen-binding fragment comprising a first portion that binds CD155 (PVR) and a second portion that binds at least one of: (a) transferrin receptor 1 and (b) transferrin receptor 2.

9. The antibody or antigen-binding fragment of claim 2, comprising a bispecific antibody or antigen-binding fragment comprising a first portion that binds a poliovirus receptor and a second portion that binds a type 1 insulin-like growth factor receptor.

10. The antibody or antigen-binding fragment of claim 2, comprising a bispecific antibody or antigen-binding fragment comprising a first portion that binds a poliovirus receptor and a second portion comprising a single domain antibody FC 5.

11. The antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment binds to CD155 (PVR) in a non-human primate.

12. The antibody or antigen-binding fragment of claim 2, having an IgG1 subtype, wherein the antibody or antigen-binding fragment binds to CD155 expressed on a tumor and elicits an immune response to the tumor by ADCC or CDC.

13. The antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment binds CD155 (PVR) followed by an increase in DNAM 1 (CD 226) in at least one of: (a) T cells and (b) NK cells.

14. The antibody or antigen-binding fragment of claim 2, wherein the anti-CD 155 antibody or antigen-binding fragment is administered to a human, binds to CD155 in the human, and prevents poliovirus from binding to CD155 (PVR).

15. The antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment against CD155 is administered to a human in combination with an antibody against an immune checkpoint molecule selected from the group consisting of CTL A4、PD1、PDL1、CD112R、OX40、TIGIT、NKG2A、CEACAM1、B7H3、B7-H4、VISTA、LAG3、CD137、KIR、TIM1、TIM3、LAIR1、HVEM、BTLA、CD160、CD200、CD200R for the treatment of cancer.

16. The antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment comprises an IgG4 subtype with an S241P hinge mutation.

17. The antibody or antigen-binding fragment of claim 2, wherein the anti-CD 155 antibody or antigen-binding fragment has one of the following labels (a) I-124 (b) gallium 68 or (c) ruthenium 177 (d) CT scanned contrast agent (e) MRI scanned contrast agent and (f) PET scanned diagnostic agent.

18. The antibody or antigen-binding fragment of claim 2, wherein the anti-CD 155 antibody or antigen-binding fragment binds to an mRNA encoding a cytokine or gene-editing enzyme, comprising (a) an mRNA encoding IL-2, (b) an mRNA encoding IL-7, (c) an mRNA encoding IL-12, (d) an mRNA encoding IL-15, (e) an mRNA encoding IL-21, (f) an mRNA encoding IFN- γ, (g) an mRNA encoding IFN- α, (h) an mRNA encoding GM-CSF, (i) an mRNA encoding Cas9, (j) an mRNA encoding Cas12a, or (k) an mRNA encoding Cas 13.

19. The antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment is administered in a manner selected from at least one of the group consisting of: intravenous, intra-arterial, intratumoral, subarachnoid, intramuscular, subcutaneous, intravesical, intraperitoneal, and delivery enhanced by convection.

20. A cell line producing an antibody or antigen binding fragment against a poliovirus receptor (CD 155) comprising a heavy chain variable region and a light chain variable region from:

21. A humanized antibody or antigen binding fragment comprising heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 2, SEQ ID NO. 27 and SEQ ID NO. 4 and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 28, respectively.

22. A humanized antibody or antigen binding fragment comprising heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 2, SEQ ID NO. 29 and SEQ ID NO. 4 and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 30, respectively.

23. A humanized antibody or antigen binding fragment comprising heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 2, SEQ ID NO. 27 and SEQ ID NO. 4 and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 28, respectively.

24. A humanized antibody or antigen binding fragment comprising heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 2, SEQ ID NO. 29 and SEQ ID NO. 4 and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 30, respectively.

25. A humanized antibody or antigen binding fragment comprising heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO.2, SEQ ID NO. 27 and SEQ ID NO. 4 and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 28, respectively.

26. A humanized antibody or antigen binding fragment comprising heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 2, SEQ ID NO. 31 and SEQ ID NO. 4 and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 28, respectively.

27. A humanized antibody or antigen binding fragment comprising heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 2, SEQ ID NO. 29 and SEQ ID NO. 4 and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 28, respectively.

28. A humanized antibody or antigen binding fragment comprising heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 2, SEQ ID NO. 27 and SEQ ID NO. 4 and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 30, respectively.

29. A humanized antibody or antigen binding fragment comprising heavy chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 2, SEQ ID NO. 29 and SEQ ID NO. 4 and light chain variable regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 30, respectively.

30. An antibody or antigen-binding fragment of an anti-poliovirus receptor (CD 155), comprising:

(a) A heavy chain variable region selected from one of SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, or SEQ ID No. 13, wherein the heavy chain variable region comprises:

(i) A CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3, wherein

(3) Replacing threonine at the ninth position in CDR 2 with one of glutamic acid or lysine;

(iii) A CDR3 domain comprising the amino acid sequence of SEQ ID No. 4;

(b) A light chain variable region selected from one of SEQ ID No. 5, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, or SEQ ID No. 17, wherein the light chain variable region comprises:

(i) A CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID NO. 7;

(iii) A CDR3 domain comprising the amino acid sequence of SEQ ID No. 8; wherein asparagine at the fourth position in CDR3 is substituted with one of alanine, glutamic acid, glycine, lysine, glutamine or serine.

31. A heavy chain variable region of an antibody against a poliovirus receptor (CD 155), said heavy chain variable region being selected from one of SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, or SEQ ID No. 13, wherein said heavy chain variable region comprises:

(i) A CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3, wherein

(iii) A CDR3 domain comprising the amino acid sequence of SEQ ID No. 4.

32. A light chain variable region of an antibody against a poliovirus receptor (CD 155) selected from one of SEQ ID No. 5, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, or SEQ ID No. 17, wherein the light chain variable region comprises:

(i) A CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID NO. 7;

33. An antibody or antigen-binding fragment of an anti-poliovirus receptor (CD 155), comprising:

(a) A heavy chain variable region comprising:

(i) A CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3, wherein

(iii) A CDR3 domain comprising the amino acid sequence of SEQ ID No. 4;

(b) A light chain variable region comprising:

(i) A CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID NO. 7;

34. A heavy chain variable region of an antibody against a poliovirus receptor (CD 155), comprising:

(i) A CDR1 domain comprising the amino acid sequence of SEQ ID NO. 2;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID NO. 3, wherein

(iii) A CDR3 domain comprising the amino acid sequence of SEQ ID No. 4.

35. A light chain variable region of an antibody against a poliovirus receptor (CD 155), comprising:

(i) A CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID NO. 7;

36. An antibody or antigen binding fragment of claim 1 against the poliovirus receptor (CD 155), wherein the heavy chain variable region and the light chain variable region are each one of:

(i) 18 and 19;

(ii) SEQ ID NO. 20 and SEQ ID NO. 21;

(iii) 18 and 22;

(iv) SEQ ID NO. 20 and SEQ ID NO. 23;

(v) SEQ ID NO. 24 and SEQ ID NO. 22;

(vi) 26 and 22;

(vii) 25 and 22;

(viii) 24 and 23; or (b)

(Ix) SEQ ID NO. 25 and SEQ ID NO. 23.

37. An antibody or antigen binding fragment of claim 7 against the poliovirus receptor (CD 155), wherein the heavy chain variable region and the light chain variable region are each one of:

(i) SEQ ID NO. 20 and SEQ ID NO. 21;

(ii) SEQ ID NO. 20 and SEQ ID NO. 23; or (b)

(Iii) SEQ ID NO. 25 and SEQ ID NO. 23.

38. A heavy chain variable region of an IgG subtype antibody against a poliovirus receptor (CD 155) comprising one of the following sequence IDs:

i)SEQ ID NO:9；

ii)SEQ ID NO:10；

iii)SEQ ID NO:11；

iv) SEQ ID NO. 12; or (b)

v)SEQ ID NO:13。

39. A light chain variable region of an IgG subtype antibody against a poliovirus receptor (CD 155) comprising one of the following sequence IDs:

i)SEQ ID NO:14；

ii)SEQ ID NO:15；

iii) SEQ ID NO. 16; or (b)

iv)SEQ ID NO:17。

40. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) Heavy chain variable region from one of:

i)SEQ ID NO:9；

ii)SEQ ID NO:10；

iii)SEQ ID NO:11；

iv) SEQ ID NO. 12; or (b)

V) SEQ ID NO. 13; and

(B) A light chain variable region from one of:

i)SEQ ID NO:14；

ii)SEQ ID NO:15；

iii) SEQ ID NO. 16; or (b)

iv)SEQ ID NO:17。

41. A heavy chain variable region of an antibody against an IgG subtype of the poliovirus receptor (CD 155) comprising one of the following modifications of SEQ ID NO: 1:

i) Substitution of aspartic acid at position 54 with alanine;

ii) substitution of aspartic acid at position 54 for glutamic acid;

iii) Substitution of aspartic acid at position 54 with serine;

iv) substitution of aspartic acid at position 56 for glutamic acid;

v) substitution of aspartic acid at position 56 with glycine;

vi) substitution of threonine at position 57 with glutamic acid.

42. A light chain variable region of an antibody against an IgG subtype of the poliovirus receptor (CD 155) comprising one of the following modifications of SEQ ID NO: 5:

i) Substitution of asparagine at position 92 with alanine;

ii) substitution of asparagine at position 92 with glutamic acid; and

Iii) Substitution of asparagine at position 92 with glutamine.

43. A light chain variable region of an anti-poliovirus receptor (CD 155) IgG subtype antibody comprising one of the following modifications in SEQ ID No. 5, with an asparagine at position 92 replaced with (i) alanine; (ii) glutamic acid; or glutamine.

44. A heavy chain variable region of an antibody against an IgG subtype of the poliovirus receptor (CD 155) comprising one of:

(i) SEQ ID NO. 20; or (b)

(ii)SEQ ID NO:25。

45. A light chain variable region of an antibody against an IgG subtype of the poliovirus receptor (CD 155) comprising one of:

(i) SEQ ID NO. 21; or (b)

(ii)SEQ ID NO:23。

46. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) A heavy chain variable region comprising SEQ ID No. 18;

(b) A light chain variable region comprising SEQ ID NO. 19.

47. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) A heavy chain variable region comprising SEQ ID No. 20;

(b) A light chain variable region comprising SEQ ID NO. 21.

48. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) A heavy chain variable region comprising SEQ ID No. 18;

(b) A light chain variable region comprising SEQ ID NO. 22.

49. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) A heavy chain variable region comprising SEQ ID No. 20;

(b) A light chain variable region comprising SEQ ID NO. 23.

50. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) A heavy chain variable region comprising SEQ ID No. 24;

(b) A light chain variable region comprising SEQ ID NO. 22.

51. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) A heavy chain variable region comprising SEQ ID No. 24;

(b) A light chain variable region comprising SEQ ID NO. 22.

52. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) A heavy chain variable region comprising SEQ ID No. 25;

(b) A light chain variable region comprising SEQ ID NO. 22.

53. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) A heavy chain variable region comprising SEQ ID No. 24;

(b) A light chain variable region comprising SEQ ID NO. 23.

54. An antibody or antigen-binding fragment of an IgG subtype of a poliovirus receptor (CD 155) comprising:

(a) A heavy chain variable region comprising SEQ ID No. 25;

(b) A light chain variable region comprising SEQ ID NO. 23.

55. An antibody or antigen-binding fragment of an anti-poliovirus receptor (CD 155), comprising:

(a) The heavy chain variable region of SEQ ID NO. 1 wherein at least 7 amino acid residues are changed at one or more amino acid residue positions selected from the group consisting of positions 5, 9, 12, 20, 24, 38, 40, 41, 44, 67, 68, 70, 76, 84, 87, 91, 111, 112 and 116;

(b) The light chain variable region of SEQ ID NO. 5 wherein at least 7 amino acid residues are changed at one or more amino acid residue positions selected from the group consisting of positions 3, 9, 10, 13, 20, 21, 43, 63, 77, 78, 80, 87 and 100.

56. The antibody or antigen-binding fragment of claim 55, wherein at least 7 amino acid residues that are altered in the heavy chain variable region are at positions 12, 40, 44, 67, 76, 112 and 116.

57. The antibody or antigen-binding fragment of claim 55, wherein at least 7 amino acid residues that are altered in the light chain variable region are at positions 9, 20, 21, 43, 77, 78 and 100.

58. The antibody or antigen-binding fragment of claim 55, wherein CDR2 of the heavy chain variable region has at least one amino acid residue change at position 55, 57 or 58.

59. The antibody or antigen-binding fragment of claim 58, wherein CDR3 of the light chain variable region is altered at amino acid residue 92.

60. An antibody or antigen-binding fragment of an anti-poliovirus receptor (CD 155), comprising:

(a) A heavy chain variable region shown in SEQ ID NO. 1, wherein 6 to 20 amino acid residues are changed outside the CDR domain; and

(B) The light chain variable region shown in SEQ ID NO. 5, wherein 6 to 14 amino acid residues are changed outside the CDR domain.

61. The antibody or antigen-binding fragment of claim 60,

(A) The amino acid residues altered in the heavy chain variable region are located at a selected set of positions including positions 5, 9, 12, 20, 24, 38, 40, 41, 44, 67, 68, 70, 83, 84, 87, 91, 111, 112 and 116; and

(B) The amino acid residues altered in the light chain variable region are located at a selected set of positions including positions 3, 9, 10, 13, 20, 21, 43, 63, 77, 78, 80, 87 and 100.

62. The antibody or antigen-binding fragment of claim 61, wherein the amino acid residue at position 3 of the light chain variable region is one of valine or glutamine.

63. The antibody or antigen-binding fragment of claim 61, wherein the amino acid residue at position 63 of the light chain variable region is one of threonine or serine.

64. The antibody or antigen-binding fragment of claim 60, wherein the amino acid residue at position 3 of the light chain variable region is one of valine or glutamine and the amino acid residue at position 63 of the light chain variable region is one of threonine or serine.

65. An antibody or antigen-binding fragment of an anti-poliovirus receptor (CD 155) comprising (a) a heavy chain variable region as set forth in SEQ ID NO:1, wherein the amino acid residues are altered at:

(i) V at position 12 is changed to lysine or arginine,

(Ii) S at position 40 is changed to proline or alanine;

(b) A light chain variable region as set forth in SEQ ID NO. 5 wherein the amino acid residues are changed at the following positions:

(i) K at position 9 is changed to serine,

(Ii) S at position 43 is changed to alanine.

66. An isolated nucleic acid polynucleotide or plasmid comprising a nucleotide sequence encoding the antibody or antigen binding fragment of any one of claims 1 to 36.

67. An antibody or antigen-binding fragment according to any one of claims 1 to 66, which is conjugated to at least one labeling compound.

68. The antibody or antigen binding fragment according to claim 67, wherein the at least one labeling compound is 1-124, fluorescein, or IR800 dye.

69. An antibody or antigen-binding fragment according to any one of claims 1 to 66, which binds to at least one drug.

70. The antibody or antigen-binding fragment of claim 69, wherein the at least one drug is paclitaxel, doxorubicin, or temozolomide.

71. The antibody or antigen-binding fragment of any one of claims 1 to 66, which binds to at least one therapeutic nucleic acid.

72. The antibody or antigen-binding fragment according to claim 71, wherein the at least one therapeutic nucleic acid is a silencing RNA (siRNA), a long RNA, a shRNA, a ribozyme, a messenger RNA, a plasmid DNA, or a non-plasmid double-stranded DNA.

73. The antibody or antigen-binding fragment according to any one of claims 1 to 66, wherein the antibody or antigen-binding fragment is of the IgG subtype.

74. The antibody or antigen-binding fragment according to claim 73, wherein the antibody or antigen-binding fragment is of the IgG4 subtype and binds IL-12.

75. A chimeric antigen receptor T cell comprising an antibody or antigen-binding fragment according to any one of claims 1 to 66.

76. A method of treating cancer comprising injecting an antibody, antigen-binding fragment, or DNA encoding the antibody or antigen-binding fragment according to any one of claims 1 to 36 into a patient, wherein the injection comprises at least one of intravenous, intramuscular, intraarterial, intracerebrospinal, intracerebroventricular, intratumoral, subcutaneous, or intralymphatic.

77. The method of claim 76, wherein the injection is into cerebrospinal fluid.

78. The method of claim 76, wherein the injection is via a catheter into a brain tumor.

79. The method of claim 76, wherein the antibody or antigen-binding fragment is injected into the tumor after binding to the drug.

80. A conjugate, comprising: a) An antibody or antigen-binding fragment according to any one of claims 1 to 66; and b) a component, wherein the component is a biomarker, a radiolabel, a fluorescent probe, an anti-tumor drug, a cytokine, an adjuvant, a toxin, a radioactive agent, or a nucleic acid.

81. An antibody or antigen-binding fragment of an anti-poliovirus receptor (CD 155), comprising:

(a) A heavy chain variable region selected from one of the following sequences: 1, 9, 10, 11, 12 or 13, wherein the heavy chain variable region comprises:

(i) A CDR1 domain comprising the amino acid sequence SEQ ID NO. 2;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID NO:3, wherein:

(1) Asparagine in the sixth position in CDR 2 is replaced with one of alanine, glutamic acid, lysine, glutamine, serine or threonine; and/or

(2) Asparagine in the eighth position in CDR2 is replaced with one of glutamic acid, glycine or arginine; and/or

(3) Threonine at the ninth position in CDR 2 is replaced with one of glutamic acid or lysine;

(iii) A CDR3 domain comprising the amino acid sequence of SEQ ID No. 4;

(b) A light chain variable region selected from one of the following sequences: SEQ ID NO. 5, SEQ ID NO. 14, SEQ ID NO. 15, wherein the light chain variable region comprises:

(i) A CDR1 domain comprising the amino acid sequence of SEQ ID NO. 6;

(ii) A CDR2 domain comprising the amino acid sequence of SEQ ID No. 7;

(iii) A CDR3 domain comprising the amino acid sequence of SEQ ID No. 8 wherein the asparagine at the fourth position in CDR3 is replaced with one of alanine, glutamic acid, glycine, lysine, glutamine or serine.

(Iv) The amino acid residue at the third position of the light chain variable region is valine or glutamic acid, and the amino acid residue at position 63 of the light chain variable region is threonine or serine.

82. A chimeric antigen receptor T cell or a chimeric antigen receptor NK cell comprising an antigen-binding fragment comprising a heavy chain variable region and a light chain variable region selected from the group consisting of (a) a heavy chain variable region selected from one of SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 24, SEQ ID No. 25, or SEQ ID No. 26; (b) The light chain variable region is selected from one of SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 22 or SEQ ID NO. 23.

83. An antibody or antigen binding fragment that binds to Nectin 4 and CD155 (PVR), comprising a heavy chain variable region selected from one of the following sequences: SEQ ID NO. 1, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 24, SEQ ID NO. 25 or SEQ ID NO. 26, and a light chain variable region selected from one of the following sequences: SEQ ID NO. 5, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 22 or SEQ ID NO. 23.

84. The antibody or antigen-binding fragment of claim 83, wherein the antibody or antigen-binding fragment prevents binding of Nectin 4 to TIGIT.

85. The antibody or antigen-binding fragment according to claim 83, comprising a bispecific antibody or antigen-binding fragment comprising a first moiety that binds to Nectin 4 and a second moiety that binds to CD3 on a T cell.

86. The antibody or antigen-binding fragment according to claim 83, comprising a bispecific antibody or antigen-binding fragment comprising a first moiety that binds to Nectin 4 and a second moiety that binds to a receptor on an NK cell.

87. The antibody or antigen-binding fragment according to claim 83, which binds to fluorescein and is injected into a mammal, wherein the antibody conjugate binds to and aggregates in a tumor expressing at least one of: CD155 (PVR) or Nectin 4.

88. The antibody or antigen-binding fragment according to claim 83, wherein the antibody or antigen-binding fragment binds PVR resulting in an increase in DNAM-1 (CD 226) on T cells or NK cells.

89. A D171 antibody conjugate, wherein said D171 antibody conjugate is internalized by a tumor.