Glypican 2 as cancer marker and therapeutic target

Abstract

To provide a pharmaceutical composition for treating a Glypican 2-positive cancer.SOLUTION: The present invention discloses a pharmaceutical composition for treating a Glypican 2-positive cancer in a subject, comprising an antibody or an antibody derivative that selectively binds to cancer cell-related Glypican 2. The cancer is treated by contacting a Glypican 2-positive cancer cell in the subject with the antibody or antibody derivative. The antibody or antibody derivative includes a heavy chain variable domain including a specific amino acid sequence and a light chain variable domain including a specific amino acid sequence.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Description of Federal Government Funding Disbursement This invention was made with government support under grant numbers Genetics T32 T32GM008638 and ACC T32 32CA009615 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0002] Assertion of Priority This application claims the benefit of priority of U.S. Provisional Application No. 62 / 253,000, filed on November 9, 2015, and U.S. Provisional Application No. 62 / 350,976, filed on June 16, 2016. The entire contents of each application are incorporated by reference.

Background Art

[0003] Background 1. Field This disclosure generally relates to the fields of medicine, oncology, and immunotherapy. More particularly, this disclosure relates to the development of immunoassays for use in the detection and treatment of glypican 2 (GPC2)-positive cancers.

[0004] 2. Related Art Children with high-risk neuroblastoma have a poor prognosis even with intensive multimodal chemoradiotherapy. Monoclonal antibodies targeting the disialoganglioside GD2 improve outcomes in neuroblastoma, but this therapy is associated with a fairly large "on target-off tumor" toxicity. Thus, there remains a significant challenge in identifying novel cell surface molecules that meet the stringent criteria of modern immunotherapeutic agents, including that tumor expression be unique compared to normal pediatric tissue and preferably that novel cell surface molecules be required for tumor maintenance.

Summary of the Invention

[0005] Overview Accordingly, provided by the present disclosure is a method for treating cancer, which includes the step of contacting glypican-2 positive cancer cells in a subject with an antibody or an antibody derivative that selectively binds to glypican-2. The glypican-2 positive cancer cells may be solid tumor cancer cells. The solid tumor cancer cells may be lung cancer cells, brain cancer cells, head and neck cancer cells, breast cancer cells, skin cancer cells, liver cancer cells, pancreatic cancer cells, gastric cancer cells, colon cancer cells, kidney cancer cells, rectal cancer cells, uterine cancer cells, cervical cancer cells, ovarian cancer cells, testicular cancer cells, skin cancer cells, or esophageal cancer cells. The glypican-2 positive cancer cells may be embryonic cancer cells. The solid tumor cancer cells may be sarcoma cells, neuroblastoma cells, rhabdoid cancer cells, medulloblastoma cells, or neuroblastoma cells. The cancer cells may be pediatric cancer cells.

[0006] The method may further include the step of contacting the glypican-2 positive cancer cells with a second anti-cancer agent or treatment. The second anti-cancer agent or treatment may be selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy. The glypican-2 antibody may be administered before the second agent or treatment. The second anti-cancer agent or treatment may be administered simultaneously with the first agent, before and / or after the first agent. The glypican-2 positive cancer cells may be metastatic cancer cells, multi-drug resistant cancer cells, or recurrent cancer cells. The antibody may be a single-chain antibody, the antibody may be a single-domain antibody, the antibody may be a chimeric antibody, the antibody may be a Fab fragment. The antibody may be a recombinant antibody having specificity for glypican-2 and a different cancer cell surface antigen. The antibody may be a mouse antibody, for example, IgG. The antibody may be a humanized antibody or a fully human antibody such as IgG.

[0007] Furthermore, the antibody may further comprise an anti-tumor drug conjugated thereto. The anti-tumor drug may be conjugated to the antibody via a photosensitive linker. The anti-tumor drug may be conjugated to the antibody via a linker cleavable by an enzyme. The anti-tumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme. The antibody may further comprise a label, such as a peptide tag, an enzyme, magnetic particles, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. The antibody may be bound to liposomes or nanoparticles. The antibody or antibody derivative may induce cell death, for example, by antibody-dependent cytotoxicity or complement-mediated cytotoxicity. The antibody derivative may be a chimeric antigen receptor. The antibody may be a bispecific antibody.

[0008] (i) a first single-chain antibody that selectively binds to glypican 2, the first single-chain antibody being (a) an IgG antibody, (b) inhibiting cancer cell proliferation, and (c) inducing cancer cell death, and (ii) a second single-chain antibody that binds to T cells or B cells. The second single-chain antibody may bind to CD3, may bind to T cells, or may bind to B cells. The fusion protein may further comprise a label or a therapeutic moiety. The first single-chain antibody may be characterized by CDR sequences SEQ ID NO: 5-10, 15-20, or 25-30. Yet another aspect comprises a cell expressing the fusion protein as defined above.

[0009] Another aspect includes a chimeric antigen receptor comprising: (i) an ectodomain comprising a single-chain antibody variable region that selectively binds to glypican 2, wherein the antibody is (a) an IgG antibody, (b) inhibits cancer cell proliferation, (c) induces cancer cell death, and has a flexible hinge attached to the C-terminus of the single-chain antibody variable region; (ii) a transmembrane domain; and (iii) an endodomain, wherein the endodomain comprises a signaling function when the single-chain antibody variable region binds to glypican 2. The transmembrane domain and the endodomain may be derived from the same molecule. The endodomain may comprise a CD3ζ domain or a high-affinity FcεRI. The flexible hinge may be derived from CD8α or Ig. The single-chain GPC2 antibody may be characterized by CDR sequences SEQ ID NO:5-10, 15-20, or 25-30. Yet another aspect includes a cell expressing the chimeric antigen receptor defined above.

[0010] In yet another aspect, there is provided a monoclonal antibody, wherein the antibody or antibody fragment is characterized by CDR sequences SEQ ID NO: 5-10, 15-20, or 25-30. The antibody or antibody fragment may be encoded by a heavy chain variable sequence and a light chain variable sequence, and the heavy chain variable sequence and the light chain variable sequence are SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. Or, the antibody or antibody fragment may be encoded by a heavy chain variable sequence and a light chain variable sequence, and the heavy chain variable sequence and the light chain variable sequence have at least 70%, 80%, or 90% identity to SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. Or, the antibody or antibody fragment may be encoded by a heavy chain variable sequence and a light chain variable sequence, and the heavy chain variable sequence and the light chain variable sequence have at least 95% identity to SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. The antibody or antibody fragment may include a heavy chain variable sequence and a light chain variable sequence, and the heavy chain variable sequence and the light chain variable sequence include SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. Or, the antibody or antibody fragment may include a light chain variable sequence and a heavy chain variable sequence, and the light chain variable sequence and the heavy chain variable sequence have 95% identity to SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. The antibody fragment may be a recombinant ScFv (single-chain fragment variable) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. The antibody may be a chimeric antibody. Or the antibody is a bispecific antibody. The monoclonal antibody may be IgG. The antibody or antibody fragment may further include a label. There is also provided a pharmaceutical composition in which the aforementioned antibody is dispersed in a pharmaceutically acceptable buffer, medium, or diluent, or is lyophilized.

[0011] In yet another aspect, there are provided hybridomas or engineered cells encoding an antibody or antibody fragment characterized by CDR sequences SEQ ID NO: 5-10, 15-20, or 25-30. The antibody or antibody fragment may be encoded by a heavy chain variable sequence and a light chain variable sequence, where the heavy chain variable sequence and the light chain variable sequence are SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. Alternatively, the antibody or antibody fragment may be encoded by a heavy chain variable sequence and a light chain variable sequence, where the heavy chain variable sequence and the light chain variable sequence have at least 70%, 80%, or 90% identity to SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. Alternatively, the antibody or antibody fragment may be encoded by a heavy chain variable sequence and a light chain variable sequence, where the heavy chain variable sequence and the light chain variable sequence have at least 95% identity to SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. The antibody or antibody fragment may comprise a heavy chain variable sequence and a light chain variable sequence, where the heavy chain variable sequence and the light chain variable sequence comprise SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. Alternatively, the antibody or antibody fragment may comprise a light chain variable sequence and a heavy chain variable sequence, where the light chain variable sequence and the heavy chain variable sequence have 95% identity to SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. The hybridoma or engineered cell may produce an antibody fragment that is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment, may produce an antibody that is a chimeric antibody or bispecific antibody, or may produce an antibody that is IgG.

[0012] It is intended that any method or composition described herein can be practiced with any other method or composition described herein.

[0013] The use of the word "a" or "an" may mean "one" when used in conjunction with the term "comprising" in the claims and / or the specification, but is also consistent with the meanings of "one or more", "at least one", and "one or more than one". The word "about" means +5% or -5% of the stated number.

[0014] [Invention 1001] A method for treating cancer, comprising the step of contacting glypican-2 positive cancer cells in a subject with an antibody or an antibody derivative that selectively binds to glypican-2. [Invention 1002] The method of Invention 1001, wherein the glypican-2 positive cancer cells are solid tumor cancer cells. [Invention 1003] The method of Invention 1002, wherein the solid tumor cells are lung cancer cells, brain cancer cells, head and neck cancer cells, breast cancer cells, skin cancer cells, liver cancer cells, pancreatic cancer cells, gastric cancer cells, colon cancer cells, kidney cancer cells, rectal cancer cells, uterine cancer cells, cervical cancer cells, ovarian cancer cells, testicular cancer cells, skin cancer cells, or esophageal cancer cells. [Invention 1004] The method of Invention 1001, wherein the glypican-2 positive cancer cells are embryonic cancer cells. [Invention 1005] The method of Invention 1001, wherein the cancer cells are sarcoma cells, neuroblastoma cells, rhabdoid cancer cells, medulloblastoma cells or neuroblastoma cells. [Invention 1006] The method according to any one of Inventions 1001 to 1005, further comprising the step of contacting the glypican-2 positive cancer cells with a second anti-cancer agent or treatment. [Invention 1007] The method of Invention 1006, wherein the second anti-cancer agent or treatment is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy. [Invention 1008] The method of Invention 1006, wherein the glypican-2 antibody is administered before the second agent or treatment. [Invention 1009] The method of the present invention 1006, wherein the second anti-cancer agent or treatment is administered simultaneously with the first agent. [The present invention 1010] The method of the present invention 1006, wherein the second anti-cancer agent or treatment is administered before and / or after the first agent. [The present invention 1011] The method according to any one of the present inventions 1001 to 1010, wherein the glypican 2-positive cancer cells are metastatic cancer cells, multi-drug resistant cancer cells, or recurrent cancer cells. [The present invention 1012] The method according to any one of the present inventions 1001 to 1011, wherein the antibody is a single-chain antibody. [The present invention 1013] The method according to any one of the present inventions 1001 to 1011, wherein the antibody is a single-domain antibody. [The present invention 1014] The method according to any one of the present inventions 1001 to 1011, wherein the antibody is a chimeric antibody. [The present invention 1015] The method according to any one of the present inventions 1001 to 1011, wherein the antibody derivative is a Fab fragment. [The present invention 1016] The method according to any one of the present inventions 1011 to 1015, wherein the antibody is a recombinant antibody having specificity for glypican 2 and a different cancer cell surface antigen. [The present invention 1017] The method according to any one of the present inventions 1001 to 1016, wherein the antibody is a mouse antibody such as IgG. [The present invention 1018] The method according to any one of the present inventions 1001 to 1016, wherein the antibody is a human antibody. [The present invention 1019] The method according to any one of the present inventions 1001 to 1015, wherein the antibody is a humanized antibody. [The present invention 1020] The method according to any one of the present inventions 1018 to 1019, wherein the humanized antibody is IgG. [The present invention 1021] The method according to any one of the present inventions 1001 to 1020, wherein the antibody further comprises an anti-tumor drug linked to the antibody. [The present invention 1022] The method of the present invention 1021, wherein the anti-tumor drug is linked to the antibody via a photosensitive linker. [The present invention 1023] The method of the present invention 1021, wherein the anti-tumor drug is linked to the antibody via a linker cleaved by an enzyme. [The present invention 1024] The method of the present invention 1021, wherein the anti-tumor drug is a toxin, a radioisotope, a cytokine, or an enzyme. [The present invention 1025] The method according to any one of the present inventions 1001 to 1024, wherein the antibody further comprises a label. [The present invention 1026] The method of the present invention 1025, wherein the label is a peptide tag, an enzyme, magnetic particles, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. [The present invention 1027] The method according to any one of the present inventions 1001 to 1026, wherein the antibody is bound to liposomes or nanoparticles. [The present invention 1028] The method according to any one of the present inventions 1001 to 1027, wherein the antibody induces cell death, for example, by antibody-dependent cytotoxicity or complement-mediated cytotoxicity. [The present invention 1029] The method of the present invention 1001, wherein the antibody derivative is a chimeric antigen receptor or a bispecific antibody. [The present invention 1030] The method of the present invention 1001, wherein the antibody or antibody fragment is characterized by CDR sequences SEQ ID NO: 5-10, 15-20, or 25-30. [The present invention 1031] (i) A first single-chain antibody that selectively binds to glypican 2, (a) which is an IgG antibody, (b) inhibits cancer cell proliferation, (c) and induces cancer cell death the first single-chain antibody, and (ii) a second single-chain antibody that binds to T cells or B cells comprising a fusion protein. [The present invention 1032] The fusion protein of the present invention 1031, wherein the first antibody or antibody fragment is characterized by the CDR sequences SEQ ID NO: 5-10, 15-20, or 25-30. [The present invention 1033] The fusion protein of the present invention 1031, wherein the second single-chain antibody binds to T cells, for example, via CD3. [The present invention 1034] The fusion protein of the present invention 1031, wherein the second single-chain antibody binds to B cells. [The present invention 1035] The fusion protein of the present invention 1031, further comprising a label or a therapeutic moiety. [The present invention 1036] (i) An ectodomain comprising a single-chain antibody variable region that selectively binds to glypican 2, wherein the antibody is (a) an IgG antibody, (b) inhibits cancer cell proliferation, (c) induces cancer cell death, and has a flexible hinge attached to the C-terminus of the single-chain antibody variable region, the ectodomain, (ii) a transmembrane domain, (iii) and an endodomain comprising a chimeric antigen receptor, wherein the endodomain comprises a signal transduction function when the single-chain antibody variable region binds to glypican 2. [The present invention 1037] The receptor of the present invention 1036, wherein the first single-chain antibody is characterized by the CDR sequences SEQ ID NO: 5-10, 15-20, or 25-30. [The present invention 1038] The receptor of the present invention 1036, wherein the transmembrane domain and the endodomain are derived from the same molecule. [The present invention 1039] The receptor of the present invention 1036, wherein the endodomain comprises the CD3ζ domain or the high-affinity FcεRI. [The present invention 1040] The receptor of the present invention 1036, wherein the flexible hinge is derived from CD8α or Ig. [The present invention 1041] A cell expressing a chimeric antigen receptor according to any one of 1036 to 1040 of the present invention. [The present invention 1042] The cell of the present invention 1041, wherein the endodomain comprises a CD3ζ domain or a high-affinity FcεRI. [The present invention 1043] The cell of the present invention 1041, wherein the flexible hinge is derived from CD8α or Ig. [The present invention 1044] A cell expressing the fusion protein of the present invention 1031. [The present invention 1045] The cell of the present invention 1044, wherein the second single-chain antibody binds to CD3, T cells, or B cells. [The present invention 1046] A monoclonal antibody, wherein the antibody or antibody fragment is characterized by CDR sequences SEQ ID NO: 5-10, 15-20, or 25-30. [The present invention 1047] The monoclonal antibody of the present invention 1046, wherein the antibody or antibody fragment is encoded by heavy-chain variable sequences and light-chain variable sequences that are SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. [The present invention 1048] The monoclonal antibody of the present invention 1046, wherein the antibody or antibody fragment is encoded by heavy-chain variable sequences and light-chain variable sequences that have at least 70%, 80%, or 90% identity to SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. [The present invention 1049] The monoclonal antibody of the present invention 1046, wherein the antibody or antibody fragment is encoded by heavy-chain variable sequences and light-chain variable sequences that have at least 95% identity to SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. [The present invention 1050] The monoclonal antibody of the present invention 1046, wherein the antibody or antibody fragment comprises heavy-chain variable sequences and light-chain variable sequences that comprise SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. [The present invention 1051] The monoclonal antibody of the present invention 1046, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to SEQ ID NOs: 2 and 4, 12 and 14, and 22 and 24, respectively. [The present invention 1052] The monoclonal antibody according to any one of the present inventions 1046 to 1051, wherein the antibody fragment is a recombinant ScFv (single-chain fragment variable) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. [The present invention 1053] The monoclonal antibody according to any one of the present inventions 1046 to 1051, which is a chimeric antibody or a bispecific antibody. [The present invention 1054] The monoclonal antibody according to any one of the present inventions 1046 to 1053, which is IgG and / or bound to a label and / or bound to a therapeutic agent. [The present invention 1055] The monoclonal antibody according to any one of the present inventions 1046 to 1054, wherein the antibody or antibody fragment further comprises a label. [The present invention 1056] A hybridoma or engineered cell encoding an antibody or antibody fragment characterized by CDR sequences SEQ ID NOs: 5 to 10, 15 to 20, or 25 to 30. [The present invention 1057] The hybridoma or engineered cell of the present invention 1056, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that are SEQ ID NOs: 1 and 3, 11 and 13, and 21 and 23, respectively. [The present invention 1058] The hybridoma or engineered cell of the present invention 1056, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having at least 70%, 80%, or 90% identity to SEQ ID NOs: 1 and 3, 11 and 13, and 21 and 23, respectively. [The present invention 1059] The hybridoma or engineered cell of the present invention 1056, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that have 95% identity to SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. [The present invention 1060] The hybridoma or engineered cell of the present invention 1056, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that comprise SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. [The present invention 1061] The hybridoma or engineered cell of the present invention 1056, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence that have at least 70%, 80%, or 90% identity to SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. [The present invention 1062] The hybridoma or engineered cell of the present invention 1056, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence that have 95% identity to SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. [The present invention 1063] The hybridoma or engineered cell of any one of the present inventions 1056 to 1062, wherein the antibody fragment is a recombinant ScFv (single-chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. [The present invention 1064] The hybridoma or engineered cell of any one of the present inventions 1056 to 1063, wherein the antibody is a chimeric antibody or bispecific antibody. [The present invention 1065] The hybridoma or engineered cell of any one of the present inventions 1056 to 1063, wherein the antibody is IgG. [The present invention 1066] A pharmaceutical composition comprising any monoclonal antibody of the present inventions 1046 to 1055. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, while the detailed description and specific examples represent certain aspects of the present disclosure, it is to be understood that the detailed description is illustrative only and that various modifications and changes within the spirit and scope of the present disclosure will be apparent to those skilled in the art.

Brief Description of the Drawings

[0015] The following drawings form a part of this specification and are included to further demonstrate certain specific aspects of the present disclosure. By referring to one or more of these drawings in combination with the detailed description of the specific aspects presented herein, the present disclosure can be more deeply understood.

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Mode for Carrying Out the Invention

[0016] Description of Exemplary Embodiments The inventors have confirmed that glypican 2 (GPC2) is a candidate oncogene presumed to be a cell surface immunotherapy target in high-risk neuroblastoma and, in particular, other pediatric cancers. More comprehensively, the data presented here show that genome-wide transcriptome analysis integrated with genomic validation and functional validation can identify differentially expressed cell surface cancer genes that can be attractive immunotherapy targets. These and other aspects of the present disclosure are described in more detail below.

[0017] I. Glypican 2 Glypican 2 (GPC2), also known as cerebroglycan, is a protein encoded by the GPC2 gene in humans. Cerebroglycan is an endogenous membrane heparan sulfate proteoglycan linked to glycosylphosphatidylinositol that is found in the developing nervous system. Cerebroglycan is thought to be involved in cell adhesion and to regulate axonal growth and guidance. Cerebroglycan has a particularly high affinity for laminin-1. The accession numbers for the human glypican 2 mRNA sequence and protein sequence are NM_152742 and NP_689955, respectively, which are incorporated herein by reference.

[0018] II. Production of Monoclonal Antibodies A. General Method Antibodies against Glypican 2 can be prepared by standard methods well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent No. 4,196,265). The method of making monoclonal antibodies (MAbs) generally begins along the same lines as the procedure for preparing polyclonal antibodies. The first step in both of these methods is to immunize a suitable host or to identify a subject that is immune from a previous natural infection. As is well known in the art, certain immunizing compositions may differ in terms of immunogenicity. Thus, it is often necessary to boost the host immune system as achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin, or rabbit serum albumin can also be used as carriers. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. Also as is well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by using non-specific stimulators of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing inactivated Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0019] The amount of the immunogenic composition used in the production of polyclonal antibodies varies depending on what the immunogen is and the animal used for immunization. Various routes (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal) can be used to administer the immunogen. The production of polyclonal antibodies may be monitored by sampling the blood of the immunized animal at various time points after immunization. A second booster injection may also be given. The process of booster immunization and titer measurement is repeated until an appropriate titer is reached. Once the desired level of immunogenicity is obtained, the immunized animal can be bled, and the serum can be isolated and screened, and / or MAb can be produced using that animal.

[0020] After immunization, somatic cells capable of producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb production protocol. These cells may be obtained from the spleen or lymph nodes obtained by biopsy, or from circulating blood. The antibody-producing B lymphocytes derived from the immunized animal are then fused with immortal myeloma cells, generally immortal myeloma cells of the same species as the immunized animal, or immortal myeloma cells of human cells or human / mouse chimeric cells. A myeloma cell line suitable for use in the hybridoma production fusion procedure preferably does not produce antibodies and has an enzyme deficiency that makes it impossible to grow in a specific selection medium that has a high fusion efficiency and supports the growth of only the desired fused cells (hybridomas).

[0021] As is known to those skilled in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984), any of a number of myeloma cells can be used. For example, if the immunized animal is a mouse, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.Ag4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG1.7, and S194 / 5XX0Bul may be used. In the case of rats, R210.RCY3, Y3-Ag1.2.3, IR983F, and 4B210 may be used. With respect to human cell fusions, U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6 are all useful. One particular mouse myeloma cell is the NS-1 myeloma cell line (also called P3-NS-1-Ag4-1), which can be readily obtained from the NIGMS Human Genetic Mutant Cell Repository by claiming cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse mouse myeloma SP2 / 0 non-secreting cell line. More recently, additional fusion partner strains for use with human B cells have been described, including KR12 (ATCC CRL-8658; K6H6 / B5 (ATCC CRL-1823 SHM-D33 (ATCC CRL-1668), and HMMA2.5 (Posner et al., 1987). The antibodies in the present disclosure were made using the SP2 / 0 / mIL-6 cell line, an IL-6-secreting derivative of the SP2 / 0 strain.

[0022] Methods for producing hybrids of spleen or lymph node cells that produce antibodies and myeloma cells generally involve mixing somatic cells with myeloma cells at a ratio of 2:1, although this ratio may be from about 20:1 to about 1:1, respectively, in the presence of agents (chemical or electrical) that promote cell membrane fusion. A fusion method using Sendai virus was described by Kohler and Milstein (1975; 1976), and a fusion method using polyethylene glycol (PEG), such as 37% (v / v) PEG, was described by Gefter et al (1977). The use of electrically induced fusion methods is also suitable (Goding, pp.71-74, 1986).

[0023] By the fusion procedure, viable hybrids generally arise at low frequency, about 1x10 -6 ~1x10 -8 However, this does not pose a problem because culturing in a selective medium allows viable fusion hybrids to differentiate from the injected parental cells (especially the injected myeloma cells, which usually continue to divide indefinitely). The selective medium generally contains an agent that blocks de novo nucleotide synthesis in tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, hypoxanthine and thymidine are added to the medium as a source of nucleotides (HAT medium). When azaserine is used, hypoxanthine is added to the medium. If the B cell source is an Epstein-Barr virus (EBV) transformed human B cell line, ouabain is added to eliminate EBV transformed strains that have not fused with the myeloma.

[0024] A preferred selection medium is HAT or HAT containing ouabain. In HAT medium, only cells that can operate the nucleotide salvage pathway can survive. Myeloma cells are deficient in an important enzyme of the salvage pathway, such as hypoxanthine phosphoribosyl transferase (HPRT), and cannot survive. B cells can operate this pathway, but have a limited lifespan in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selection medium are hybrids formed from myeloma cells and B cells. When the source of B cells used for fusion is a strain of B cells transformed with EBV as described herein, ouabain is also used for drug selection of the hybrids because B cells transformed with EBV are sensitive to drug killing. In contrast, the myeloma partner used is selected for ouabain resistance.

[0025] When cultured, a hybridoma population is obtained, and specific hybridomas are selected from this population. Typically, hybridoma selection is performed by culturing cells by single clone dilution in microtiter plates and then testing individual clone supernatants (after about 2 - 3 weeks) for the desired reactivity. This assay must be sensitive, simple, and rapid, and examples include radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunobinding assay, etc. 。

[0026] Next, the selected hybridomas are either serially diluted or single cell sorted by flow cytometry sorting and cloned into individual antibody-producing cell lines. The clones can then be grown indefinitely to supply the mAb. These cell lines can be used to produce MAb in two basic ways. The hybridoma sample can be injected (often intraperitoneally) into an animal (e.g., a mouse). Optionally, prior to injection, the animal is primed with a hydrocarbon, particularly an oil such as pristane (tetramethylpentadecane). When human hybridomas are used in this way, it is optimal to inject into immunodeficient mice, e.g., SCID mice, to prevent tumor rejection responses. The injected animal develops a tumor that secretes the specific monoclonal antibody produced by the fused cell hybrid. Then, the body fluid of the animal, such as serum or ascites fluid, can be gently tapped to obtain a high concentration of MAb. Individual cell lines can also be cultured in vitro, in which case the MAb is secreted naturally into the culture medium and can be readily obtained at high concentration from the culture medium. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. This cell line can be adapted to grow in serum-free medium to optimize the ability to recover high-purity human monoclonal immunoglobulins.

[0027] MAb produced by either means may, if desired, be further purified using filtration, centrifugation, and various chromatography methods, e.g., FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibody by methods including digestion with enzymes such as pepsin or papain and / or by chemically reducing to cleave disulfide bonds. Alternatively, the monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0028] It is also contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. To this end, RNA can be isolated from hybridoma cell lines, antibody genes can be obtained by RT-PCR, and cloned into immunoglobulin expression vectors. Alternatively, a combinatorial immunoglobulin phagemid library can be prepared from RNA isolated from cell lines, and phagemids expressing the appropriate antibodies are selected by panning with viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 10 4 times more antibodies can be produced and screened at one time, and new specificities are generated by combinations of H and L chains, thereby further increasing the likelihood of finding the appropriate antibody.

[0029] Other U.S. patents disclosing the production of antibodies useful in the present disclosure include U.S. Patent No. 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Patent No. 4,867,973, which describes antibody-therapeutic agent conjugates, each of which is incorporated herein by reference.

[0030] B. Antibodies of the Present Disclosure Antibodies according to the present disclosure may, in a first instance, be defined by binding specificity, in this case binding specificity for glypican 2. In one aspect, the antibody is an immunoglobulin G (IgG) antibody isotype. IgG represents approximately 75% of serum immunoglobulins in humans and is the most abundant antibody isotype found in circulation. IgG molecules are synthesized and secreted by plasma B cells. There are four IgG subclasses in humans (IgG1, 2, 3, and 4), named in order of abundance in serum (IgG1 being the most abundant). These range from high to no affinity for Fc receptors.

[0031] IgG is the major antibody isotype found in blood and extracellular fluid, and as such, can prevent infection of body tissues. By binding to many types of pathogens (corresponding to viruses, bacteria, and fungi), IgG protects the body from infection. It does this through several of the following immune mechanisms: When pathogens are bound via IgG, they are immobilized and bound by aggregation; when the pathogen surface is coated with IgG (known as opsonization), phagocytic immune cells can recognize and ingest the pathogen; IgG activates the classical pathway of the complement system, an immune protein production cascade that eliminates pathogens; IgG also binds and neutralizes toxins. IgG also plays an important role in antibody-dependent cell cytotoxicity (ADCC) and proteolysis mediated by intracellular antibodies. In proteolysis mediated by intracellular antibodies, IgG binds to TRIM21 (the receptor with the greatest affinity for IgG in humans) to direct tagged virions towards proteasomes in the cytosol. IgG is also associated with type II and type III hypersensitivity. IgG antibodies are produced after class switching and antibody response maturation and thus are mainly involved in the secondary immune response. Since IgG is secreted as a small-sized monomer, it can easily perfuse through tissues. It is the only isotype with a receptor that facilitates human placental passage. Along with IgA secreted in breast milk, residual IgG absorbed through the placenta provides humoral immunity to the neonate before its own immune system has developed. Colostrum, especially bovine colostrum, contains a high proportion of IgG. In individuals with previous immunity to a pathogen, IgG appears approximately 24 to 48 hours after antigen stimulation.

[0032] Furthermore, the antibody sequences may optionally vary from the sequences provided above using methods discussed in more detail below. For example, the amino acid sequence may (a) have the variable region separated from the constant domain of the light chain, (b) have amino acids that differ from the amino acid sequences shown above but do not dramatically affect the chemical properties of the residues (so-called conservative substitutions), (c) differ from the amino acid sequences shown above in that the amino acids differ by a certain percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology. Alternatively, the nucleic acid encoding the antibody may (a) have the constant domain of the light chain separated, (b) differ from the nucleic acids shown above but do not change the encoded residues, (c) differ from the nucleic acids shown above by a certain percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, or (d) differ from the nucleic acids shown above by the ability to hybridize under high stringency conditions, as exemplified by low salt and / or high temperature conditions, e.g., about 0.02M to about 0.15M NaCl, a temperature of about 50°C to about 70°C.

[0033] When making conservative changes to an amino acid sequence, the hydropathy index of the amino acid may be considered. The importance of the hydropathy amino acid index in conferring interactive biological functions on proteins is generally understood in the art (Kyte and Doolittle, 1982). The relative hydropathy characteristics of amino acids contribute to the secondary structure of the resulting protein, and as a result, it is accepted that this defines the interaction of the protein with other molecules, e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0034] It is also understood in the art that amino acids can be effectively substituted based on hydrophilicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, which is governed by the hydrophilicity of adjacent amino acids, is correlated with the biological properties of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0 ± 1), glutamic acid (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, non-ionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4); sulfur-containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0035] It is understood that an amino acid can be substituted with another amino acid having similar hydrophilicity to produce a biologically or immunologically modified protein. For such changes, amino acid substitutions within ±2 of the hydrophilicity value are preferred, amino acid substitutions within ±1 of the hydrophilicity value are particularly preferred, and amino acid substitutions within ±0.5 of the hydrophilicity value are even more particularly preferred.

[0036] As outlined above, amino acid substitutions are generally based on the relative similarity of the side chain substituents of the amino acids, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various aforementioned characteristics are well known to those of skill in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0037] C. Manipulation of Antibody Sequences In various embodiments, it may be chosen to manipulate the sequence of a specified antibody for various reasons such as improved expression, improved cross-reactivity, reduced off-target binding, or one or more native effector functions, e.g., suppression of complement activation or mobilization of immune cells (e.g., T cells). In particular, IgM antibodies may be converted to IgG antibodies. The following is a general discussion of relevant techniques for manipulating antibodies.

[0038] Hybridomas can be cultured and then the cells lysed and total RNA extracted. cDNA copies of the RNA can be made using random hexamers together with RT, and then PCR can be performed using a multiplex mixture of PCR primers predicted to amplify all human variable gene sequences. The PCR products can be cloned into a pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Binding and neutralization assays can be performed using antibodies collected from hybridoma supernatants and purified by FPLC using a protein G column. Recombinant full-length IgG antibodies can be generated by subcloning heavy and light chain Fv DNA from a cloning vector into a Lonza pConIgG1 or pConK2 plasmid vector, transfecting 293 Freestyle cells or Lonza CHO cells, and collecting and purifying from the CHO cell supernatant.

[0039] The ability to rapidly obtain antibodies produced in the same host cell and cell culture process as the final cGMP manufacturing process may shorten the duration of the process development program. Lonza has developed a common method using pooled transfectants grown in CDACF medium to rapidly produce small amounts (up to 50 g) of antibody in CHO cells. Although slightly slower than true transient systems, this offers the advantages of high product concentration and use of the same host and process as the production cell line. Example of growth and productivity of a GS-CHO pool expressing a model antibody in a disposable bioreactor: In a disposable back bioreactor culture (5 L working volume) operating in fed-batch mode, a harvested antibody concentration of 2 g / L was achieved within 9 weeks of transfection.

[0040] The pCon vector (trademark) is an easy way to re-express the whole antibody. This constant region vector is a set of vectors that provide a wide range of immunoglobulin constant region vectors cloned into the pEE vector. These vectors offer the ease of construction of full-length antibodies with human constant regions and the convenience of the GS System (trademark).

[0041] Antibody molecules include fragments, such as those resulting from proteolytic cleavage of mAbs (e.g., F(ab'), F(ab')2), or single-chain immunoglobulins, such as single-chain immunoglobulins that can be produced by recombinant means. Such antibody derivatives are monovalent. In one aspect, such fragments can be combined with each other, or with other antibody fragments or receptor ligands, to form "chimeric" binding molecules. Importantly, such chimeric molecules may contain substituents that can bind to different epitopes of the same molecule.

[0042] In some cases, it may be desirable to "humanize" antibodies produced in non-human hosts in order to attenuate the immune response when used in human therapy. Such humanized antibodies may be studied in in vitro settings or in in vivo settings. Humanized antibodies may be made, for example, by replacing the immunogenic portions of the antibody with corresponding, but non-immunogenic, portions (i.e., chimeric antibodies). PCT Application PCT / US86 / 02269; EP Application 184,187; EP Application 171,496; EP Application 173,494; PCT Application WO86 / 01533; EP Application 125,023; Sun et al. (1987); Wood et al. (1985); and Shaw et al. (1988). All of these references are incorporated herein by reference. A general review of "humanized" chimeric antibodies is also incorporated herein by reference and is provided by Morrison (1985). Alternatively, "humanized" antibodies can be made by CDR or CEA substitution. Jones et al. (1986); Verhoeyen et al. (1988); Beidler et al. (1988). All of these are incorporated herein by reference.

[0043] In related aspects, the antibody is a derivative of the disclosed antibody, e.g., an antibody that includes a CDR sequence identical to the CDR sequence in the disclosed antibody (e.g., a chimeric antibody, a humanized antibody, or an antibody with a transplanted CDR). In still further aspects, the antibody is a fully human recombinant antibody.

[0044] The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG4 can reduce the immune effector functions associated with other isotypes.

[0045] The modified antibody can be produced by any technique known to those skilled in the art, including expression by standard molecular biological techniques or chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document.

[0046] D. Expression The nucleic acids according to the present disclosure encode an antibody, optionally an antibody linked to other protein sequences. The term "nucleic acid encoding a glypican 2 antibody" as used herein refers to a nucleic acid molecule isolated in the absence of all cellular nucleic acids. In certain embodiments, the present disclosure relates to antibodies encoded by any of the sequences shown herein.

[0047] (Table 2) Codon TIFF2025098050000002.tif111138

[0048] The DNA segments of the present disclosure include those encoding biologically functional equivalent proteins and peptides of the above sequences. Such sequences may occur as a result of the redundancy of codons and the functional equivalence of amino acids that are known to occur naturally within the nucleic acid sequence and the protein thus encoded. Alternatively, functionally equivalent proteins or peptides may be created by applying recombinant DNA techniques. In recombinant DNA techniques, changes in protein structure can be manipulated based on the idea that the properties of amino acids are exchanged. Changes designed by humans may be introduced by applying site-directed mutagenesis methods, as described below, introduced randomly, and screened later for desirable functions.

[0049] Throughout this application, the term "expression construct" is intended to include any type of genetic construct that contains a nucleic acid encoding a gene product, and in which some or all of the nucleic acid coding sequences therein are transcribable. The transcript may or may not be translated into a protein, i.e., it need not be a protein. In certain embodiments, expression includes transcription of a gene and translation of the mRNA into a gene product. In other embodiments, expression includes only transcription of the nucleic acid encoding the gene of interest.

[0050] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a replicable cell. The nucleic acid sequence may be "exogenous", where "exogenous" means that the nucleic acid sequence is foreign to the cell into which the vector is introduced or is homologous to a sequence in the cell but is in a location within the host cell nucleic acid where it is not ordinarily found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art are considered to have sufficient equipment to construct vectors by standard recombinant techniques described in Sambrook et al., (1989) and Ausubel et al., (1994), both of which are incorporated herein by reference.

[0051] The term "expression vector" refers to a vector that contains a transcribable nucleic acid sequence encoding at least a portion of a gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, to produce antisense molecules or ribozymes. Expression vectors may contain various "control sequences" that are necessary for transcription of a coding sequence operably linked in a particular host organism and possibly necessary for its translation. Vectors and expression vectors may also perform other functions and contain nucleic acid sequences as described below in addition to the control sequences that govern transcription and translation.

[0052] 1. Regulatory element A "promoter" is a control sequence that is a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. A "promoter" may contain genetic elements to which regulatory proteins and regulatory molecules, such as RNA polymerase and other transcription factors, can bind. The phrases "functionally disposed", "functionally linked", "under the control of", and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation with respect to the nucleic acid sequence to control the initiation and / or expression of the nucleic acid sequence. A promoter may be used with an "enhancer" or may not be used with an "enhancer". An "enhancer" refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0053] A promoter may be a promoter that is naturally associated with a gene or sequence. Similarly, it may be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such a promoter is sometimes referred to as "endogenous". Similarly, an enhancer may be an enhancer that is naturally associated with a nucleic acid sequence and is located downstream or upstream of the nucleic acid sequence. Alternatively, certain benefits can be obtained by placing the coding nucleic acid segment under the control of a recombinant promoter or a heterologous promoter. A recombinant promoter or a heterologous promoter refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.

[0054] A recombinant enhancer or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in a natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers that are not "natural", i.e., promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or contain mutations that alter expression. In addition to synthesizing the nucleic acid sequences of promoters and enhancers, the sequences may also be made using recombinant cloning techniques and / or nucleic acid amplification techniques including PCR (™) (see U.S. Patent Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Further, it is contemplated that regulatory sequences that induce transcription and / or expression of sequences in non-nuclear organelles such as mitochondria, chloroplasts, etc. can also be used.

[0055] Of course, it is important to use a cell type, organelle, and promoter and / or enhancer that effectively express the DNA segment in the organism selected for expression. Those skilled in the art of molecular biology are generally aware of using a combination of a promoter, an enhancer, and a cell type to express a protein. See, for example, Sambrook et al. (1989), which is incorporated herein by reference. The promoter used may be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter useful under conditions appropriate for expressing the introduced DNA segment at a high level, such as, for example, advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be a heterologous promoter or an endogenous promoter. The identity of tissue-specific promoters or elements and assays for characterizing their activities are well known to those skilled in the art. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), the mouse testicular retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse α2(XI) collagen (Tsumaki, et al., 1998), the D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996).

[0056] Certain specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide an exogenous translation control signal containing the ATG initiation codon. A person skilled in the art will be able to confirm this and provide the necessary signals. It is well known that for ensuring translation of the entire insert, the initiation codon must be "in-frame" with the desired coding sequence reading frame. The exogenous translation control signals and initiation codons may be natural or synthetic. The efficiency of expression may be enhanced by including appropriate transcriptional enhancer elements.

[0057] 2.IRES In certain embodiments of the present disclosure, the use of an internal ribosome entry site (IRES) element is employed to create a multi-gene message or a polycistronic message. The IRES element can bypass the ribosome scanning model of 5'-methylated Cap-dependent translation and initiate translation at an internal site (Pelletier and Sonenberg, 1988). IRES elements derived from two members of the Picornaviridae family (poliovirus and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), and IRESs derived from mammalian messages have also been described (Macejak and Sarnow, 1991). The IRES element can be linked to a heterologous open reading frame. Multiple open reading frames separated by respective IRESs can be transcribed together to create a polycistronic message. By the IRES element, each open reading frame can access the ribosome for efficient translation. Multiple genes can be efficiently expressed using one type of promoter / enhancer to transcribe one message (see U.S. Patent Nos. 5,925,565 and 5,935,819, which are hereby incorporated by reference in their entireties).

[0058] 3. Multiple cloning sites The vector may contain a multiple cloning site (MCS). A multiple cloning site is a nucleic acid region containing a plurality of restriction enzyme sites, and any of these restriction enzyme sites can be used with standard recombinant techniques to digest the vector. See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, which are incorporated herein by reference. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at specific positions on the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Often, the vector is linearized or fragmented using a restriction enzyme that cleaves within the MCS so that an exogenous sequence can be ligated to the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, and the two nucleic acid fragments may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant techniques.

[0059] 4. Splicing sites Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from the primary transcript. In order for the transcript to be correctly processed for protein expression, vectors containing genomic eukaryotic sequences may require donor splicing sites and / or acceptor splicing sites (see Chandler et al., 1997, which is incorporated herein by reference).

[0060] 5. Termination signals The vectors or constructs of the present disclosure generally contain at least one termination signal. A "termination signal" or "terminator" is composed of a DNA sequence involved in the specific termination of an RNA transcript by RNA polymerase. Thus, in certain embodiments, a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve the desired message level.

[0061] In eukaryotic systems, the terminator region may also include specific DNA sequences that enable site-specific cleavage of the new transcript to expose the polyadenylation site. This signals a special endogenous polymerase to add a sequence of approximately 200 A residues (polyA) to the 3' end of the transcript. The RNA molecule modified with this polyA tail appears to be highly stable and is translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that the terminator includes a signal for RNA cleavage, and more preferably the terminator signal promotes polyadenylation of the message. The terminator and / or polyadenylation site elements may serve to enhance the message level and / or minimize read-through from the cassette into other sequences.

[0062] Terminators intended for use in the present disclosure include any known transcriptional terminator described herein or known to those of skill in the art. This includes, for example, but is not limited to, gene termination sequences such as the bovine growth hormone terminator, or viral termination sequences such as the SV40 terminator. In certain embodiments, the termination signal may be one lacking a transcribable or translatable sequence, for example, one in which the sequence has been truncated such that it lacks a transcribable or translatable sequence.

[0063] 6. Polyadenylation signal In expression, particularly in eukaryotic expression, a polyadenylation signal is typically included to effect proper polyadenylation of the transcript. What the polyadenylation signal is has not been considered critical to the success of the practice of the present disclosure and / or any such sequence may be used. Preferred embodiments include the SV40 polyadenylation signal and / or the bovine growth hormone polyadenylation signal, which are convenient and / or known to function well in a variety of target cells. Polyadenylation may increase the stability of the transcript and may facilitate transport to the cytoplasm.

[0064] 7. Replication origin To propagate the vector within a host cell, the vector may contain one or more replication origin sites (often referred to as "ori"). A replication origin is a specific nucleic acid sequence at which replication begins. Alternatively, if the host cell is yeast, an autonomously replicating sequence (ARS) can be used.

[0065] 8. Selection markers and screening markers In certain embodiments of the present disclosure, cells containing the nucleic acid constructs of the present disclosure can be identified in vitro or in vivo by including a marker within the expression vector. Such markers confer a distinguishable change to the cell, thereby facilitating the identification of cells containing the expression vector. Generally, a selection marker confers a property that enables selection. A positive selection marker enables the selection of cells when the marker is present, whereas a negative selection marker precludes the selection of cells when the marker is present. An example of a positive selection marker is a drug resistance marker.

[0066] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants. For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype allowing for the discrimination of transformants based on conditional performance, other types of markers are contemplated, including screening markers such as GFP, which is based on colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be used. One of ordinary skill in the art will likely also know methods of using immuno - markers, perhaps in conjunction with FACS analysis. The marker used is not considered important as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screening markers are well - known to those of ordinary skill in the art.

[0067] 9. Viral Vector Because certain viral vectors can efficiently infect cells, enter, and integrate into the host cell genome, enabling stable expression of viral genes, many different viral vector systems have been developed and applied (Robbins et al., 1998). Viral systems for use as vectors for ex vivo and in vivo gene transfer are currently being developed. For example, adenoviruses, herpes simplex viruses, retroviruses, and adeno-associated viral vectors are currently being evaluated for treating diseases such as cancer, cystic fibrosis, Gaucher's disease, kidney disease, and arthritis (Robbins and Ghivizzani, 1998; Imai et al., 1998; U.S. Patent No. 5,670,488). For use in the present disclosure, other viral vectors, such as poxviruses; for example, vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alphaviruses; for example, Sindbis virus, Semliki Forest virus (Lundstrom, 1999), reoviruses (Coffey et al., 1998), and influenza A virus (Neumann et al., 1999) are contemplated and can be selected according to the required properties of the system of interest.

[0068] 10. Non-viral transformation For use with the present disclosure, methods suitable for nucleic acid delivery to transform organelles, cells, tissues, or organisms include substantially any method by which nucleic acids (e.g., DNA) can be introduced into organelles, cells, tissues, or organisms as described herein or as known to those of skill in the art. Such methods include, for example, injection (U.S. Patent Nos. 5,994,624; 5,981,274; 5,945,100; 5,780,448; 5,736,524; 5,702,932; 5,656,610; 5,589,466; and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985, incorporated herein by reference); electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); use of polyethylene glycol following DEAE-dextran (Gopal, 1985); direct sonic loading (Fechheimer et al., 1987); transfection via liposomes (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); particle guns (PCT Application Nos. WO94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each incorporated herein by reference); agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765.(each incorporated herein by reference); or protoplast transformation via PEG (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); direct delivery of DNA by DNA uptake via drying / inhibition (Potrykus et al., 1985), including but not limited to these techniques. By applying techniques such as these, organelles, cells, tissues, or organisms may be stably transformed or transiently transformed.

[0069] 11. Expression Systems There are a great many expression systems that include at least a portion or all of the compositions discussed above. Prokaryotic and / or eukaryotic-based systems can be used to generate nucleic acid sequences, or their cognate polypeptides, proteins, and peptides in conjunction with the present disclosure. Many such systems are commercially and widely available.

[0070] The insect cell / baculovirus system can result in high-level protein expression of heterologous nucleic acid segments, as described, for example, in U.S. Pat. Nos. 5,871,986 and 4,879,236, each incorporated herein by reference, and can be purchased, for example, under the name MaxBac® 2.0 from Invitrogen® or under the name BacPack™ baculovirus expression system from Clontech®.

[0071] Other examples of expression systems include the Stratagene® Complete Control™ inducible mammalian expression system, which includes a synthetic ecdysone-inducible receptor, or its pET expression system, which is an E. coli expression system. Another example of an inducible expression system available from Invitrogen® is the T-Rex™ (expression regulated by tetracycline) system, an inducible mammalian expression system that uses a full-length CMV promoter. Invitrogen® also offers a yeast expression system called the Pichia methanolica expression system. This system is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of ordinary skill in the art will know how to express vectors such as expression constructs to produce nucleic acid sequences or their cognate polypeptides, proteins, or peptides.

[0072] Primary mammalian cell cultures can be prepared in a variety of ways. In order for cells to survive in vitro and in contact with the expression construct, it is necessary to ensure that they maintain contact with the correct ratios of oxygen, carbon dioxide, and nutrients and are protected from microbial contamination. Cell culture methods are well documented.

[0073] One aspect described above involves immortalizing cells using gene transfer to produce a protein. The gene for the protein of interest can be introduced into a suitable host cell as described above, and then the cells can be cultured under appropriate conditions. Thus, genes for substantially any polypeptide can be used. The construction of recombinant expression vectors and the elements contained therein were discussed above. Alternatively, the protein to be produced may be an endogenous protein that the cell in question normally synthesizes.

[0074] Examples of useful mammalian host cell lines include Vero cells and HeLa cells as well as Chinese hamster ovary cell lines, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN, and MDCK cells. Further, a host cell line that modulates the expression of the inserted sequence, or a host cell line that modifies or processes the gene product in a desired manner may be selected. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for the function of the protein. Various host cells have characteristic and specific mechanisms for post-translational processing and post-translational modification of proteins. Selecting an appropriate cell line or host system can ensure the correct modification and processing of the expressed foreign protein.

[0075] Many selectable systems including, but not limited to, the HSV thymidine kinase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase genes can be used in tk− cells, hgprt− cells, or aprt− cells, respectively. Also, metabolic antagonist resistance can be used as a basis for selection against dhfr which confers resistance to the following, gpt which confers mycophenolic acid resistance, neo which confers aminoglycoside G418 resistance, and hygro which confers hygromycin resistance.

[0076] E. Purification In certain embodiments, the antibodies of the disclosure may be purified. As used herein, the term “purified” is intended to refer to a composition that is separable from other components, where the protein is purified to any degree relative to its naturally available state. Thus, a purified protein also refers to a protein that is free from the environment in which it may naturally occur. When the term “substantially purified” is used, this designation refers to a composition in which the protein or peptide forms the major component of the composition, e.g., a composition that comprises about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein present in the composition.

[0077] Protein purification methods are well known to those skilled in the art. These techniques involve, at some level, crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. After separating the polypeptide from other proteins, chromatography and electrophoresis methods may be used to further purify the polypeptide of interest (or to purify it to homogeneity) in order to partially or completely purify it. Analytical methods particularly suitable for preparations of pure peptides include ion exchange chromatography, size exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation using ammonium sulfate, PEG, antibodies, etc., or heat denaturation followed by precipitation by centrifugation; gel filtration, reverse phase, hydroxylapatite, and affinity chromatography; as well as combinations of such techniques with other techniques.

[0078] When purifying the antibodies of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column that binds to the tagged portion of the polypeptide. As is generally known in the art, the order in which various purification steps are performed may be varied, or certain steps may be omitted, and still provide a suitable method for preparing a substantially purified protein or peptide.

[0079] Typically, intact antibodies are fractionated using an agent that binds to the Fc portion of the antibody (i.e., Protein A). Alternatively, an antigen may be used to simultaneously purify and select the appropriate antibody. In such methods, a selection agent bound to a support such as a column, filter, or beads is often utilized. The antibody is bound to the support, contaminants are removed (e.g., washed away), and the antibody is released by applying conditions (salt, heat, etc.).

[0080] Various methods for quantifying the degree of purification of a protein or peptide are known to those skilled in the art in view of the present disclosure. These methods include, for example, the step of determining the specific activity of the active fraction or the step of evaluating the amount of polypeptide in the fraction by SDS / PAGE analysis. Another method for evaluating the purity of a fraction is to calculate the specific activity of the fraction, compare it with the specific activity of the initial extract, and thus calculate the degree of purity. The actual units used to represent the amount of activity, of course, depend on the specific assay technique chosen to track the purification, whether or not the expressed protein or peptide exhibits a detectable activity.

[0081] It is known that the migration of polypeptides can sometimes vary greatly depending on different conditions of SDS / PAGE (Capaldi et al., 1977). Thus, it is understood that under different electrophoresis conditions, the apparent molecular weight of a purified or partially purified expression product may vary.

[0082] F. Single-chain / single-domain antibodies A single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin linked together with a short (usually serine, glycine) linker. This chimeric molecule, also known as a single-domain antibody, retains the specificity of the original immunoglobulin even when the constant region is removed and a linker peptide is introduced. Usually, the specificity remains unchanged even with this modification. These molecules have been created throughout history to facilitate phage display. Phage display is extremely convenient for expressing the antigen-binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single-domain or single-chain variable fragments lack the constant Fc region found in a complete antibody molecule and thus do not have the common binding sites (e.g., protein A / G) used to purify antibodies (single-chain antibodies contain the Fc region). These fragments can often be purified / immobilized using protein L because protein L interacts with the variable region of the κ light chain.

[0083] Flexible linkers generally consist of amino acid residues that promote helices and amino acid residues that promote turns, such as alanine, serine, and glycine. However, other residues can also function. Tang et al. (1996) used phage display as a means to rapidly select linkers tailored to single-chain antibodies (scFv) from a protein linker library. A random linker library was constructed in which the genes for the heavy-chain variable domain and the light-chain variable domain were linked by segments encoding 18-amino acid polypeptides with different compositions. The scFv repertoire (about 5x10 6Individual different members) were displayed on the filamentous phage and subjected to affinity selection using a hapten. The population of selected variants showed a significant increase in binding activity but retained considerable sequence diversity. Subsequently, by screening 1054 variants one by one, a catalytically active scFv efficiently produced in soluble form was obtained. From sequence analysis, as the only common feature of the selected tether, V H It was revealed that there is a conserved proline in the linker behind the 2 C-terminal residues and there are many arginines and prolines at other positions.

[0084] The recombinant antibodies of the present disclosure may also be accompanied by sequences or portions that enable dimerization or multimerization of the receptor. Such sequences include those derived from IgA that enable multimer formation together with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, these chains may be modified using agents such as biotin / avidin that enable the combination of two antibodies.

[0085] In different embodiments, single-chain antibodies can be created by connecting the light and heavy chains of the receptor using a non-peptide linker or chemical unit. Generally, the light and heavy chains are produced in separate cells, purified, and then linked together in an appropriate manner (i.e., the heavy chain N-terminus is attached to the light chain C-terminus via an appropriate chemical cross-link).

[0086] Cross-linking reagents are used to form molecular cross-links that link the functional groups of two different molecules, such as stabilizers and coagulants. However, it is intended that dimers or multimers of the same analog or heteromeric complexes composed of different analogs can be created. To sequentially link two different compounds, a heterobifunctional cross-linking agent that eliminates unnecessary homopolymer formation can be used.

[0087] Exemplary heterobifunctional crosslinkers contain two reactive groups, one that reacts with a primary amine group (e.g., N-hydroxysuccinimide) and the other that reacts with a thiol group (e.g., pyridyldisulfide, maleimide, halogen, etc.). The crosslinker can react with a lysine residue of a protein (e.g., a selected antibody or fragment) via the primary amine reactive group, and the crosslinker that is already bound to a first protein can react with a cysteine residue (free sulfhydryl group) of another protein (e.g., a selected agent) via the thiol reactive group.

[0088] It is preferred that a crosslinker having reasonable stability in blood be used. A very large number of types of disulfide bond-containing linkers are known that can be successfully used to couple a targeting agent with a therapeutic / prophylactic agent. Linkers containing sterically hindered disulfide bonds have been found to confer high stability in vivo, thus preventing the release of the targeting peptide before reaching the site of action. Therefore, these linkers are one of the groups for linking agents.

[0089] Another crosslinking reagent is SMPT, a bifunctional crosslinker containing a disulfide bond that is "sterically hindered" by adjacent benzene rings and a methyl group. The steric hindrance of the disulfide bond serves to protect the bond from attack by thiolate anions that may be present in tissues and blood, such as glutathione, thereby preventing the conjugate from dissociating before the attached agent is delivered to the target site.

[0090] Like many other known cross-linking reagents, the SMPT cross-linking reagent confers the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the ε-amino group of lysine). Another possible type of cross-linker includes hetero-bifunctional photoreactive phenyl azides containing cleavable disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamino)ethyl-1,3'-dithiopropionate. The N-hydroxysuccinimidyl group reacts with primary amino groups, and the phenyl azide reacts non-selectively (upon photolysis) with any amino acid residue.

[0091] In addition to the blocked cross-linkers, unblocked linkers can also be used according to the present specification. Other useful cross-linkers that contain or are thought to generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another aspect involves the use of cleavable linkers.

[0092] U.S. Patent No. 4,680,338 describes bifunctional linkers useful for generating conjugates of ligands with amine-containing polymers and / or proteins, and in particular for forming antibody conjugates with chelating agents, drugs, enzymes, detectable labels, etc. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that are cleavable under various mild conditions. This linker is particularly useful because the drug of interest can be directly attached to the linker and the active drug is released upon cleavage. Specific applications include adding free amino or free sulfhydryl groups to proteins such as antibodies or drugs.

[0093] U.S. Patent No. 5,856,456 provides a peptide linker for use in connecting polypeptide components for the purpose of making fusion proteins, such as single-chain antibodies. This linker is up to about 50 amino acids in length and includes the occurrence of at least one proline following a charged amino acid (preferably arginine or lysine), and is characterized by high stability and low aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in various immunoassay and separation methods.

[0094] G. Modified Antibodies 1. CAR Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs)) are engineered receptors that transfer any specificity to immune effector cells. Typically, these receptors are used to transfer the specificity of monoclonal antibodies to T cells, and the introduction of the coding sequences of these receptors is facilitated by retroviral vectors. Thus, a large number of cancer-specific T cells can be generated for adoptive cell transfer. The Phase I clinical study of this approach has shown efficacy.

[0095] The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody with the CD3ζ transmembrane and endodomains. Such molecules transmit ζ signals in response to the recognition of their target by the scFv. An example of such a construct is 14g2aζ, which is a fusion of an scFv derived from the hybridoma 14g2a (which recognizes the disialoganglioside GD2). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells expressing GD2 (e.g., neuroblastoma cells). To target malignant B cells, researchers redirected the specificity of T cells using a chimeric immunoreceptor specific for the B lineage molecule CD19.

[0096] To form the scFv, the variable portions of the immunoglobulin heavy and light chains are fused by a flexible linker. Prior to this scFv, there is a signal peptide (which is cleaved) that directs the nascent protein to the endoplasmic reticulum and then to the surface for expression. The flexible spacer allows the scFv to orient in various directions to bind to the antigen. The transmembrane domain is a typical hydrophobic α-helix that usually results from the original molecule of the signaling end domain that protrudes into the cell and transmits the desired signal.

[0097] Type I proteins are actually two protein domains linked by a transmembrane α-helix. The cell membrane lipid bilayer through which the transmembrane domain passes serves to separate the outer portion (ectodomain) from the inner portion (endodomain). It is not very surprising that a molecule is generated by attaching the ectodomain from one protein to the endodomain of another protein, combining the recognition of the former with the signal of the latter.

[0098] Ectodomain The signal peptide directs the nascent protein to the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored to the cell membrane. Usually, any eukaryotic signal peptide sequence works well. Generally, the signal peptide that is naturally attached to the most amino-terminal component is used (for example, in an scFv with the light-chain-linker-heavy-chain orientation, the natural signal of the light chain is used).

[0099] Usually, the antigen recognition domain is the scFv. However, there are many options. Antigen recognition domains derived from the native T cell receptor (TCR) α and β single chains have been described. Similarly, simple ectodomains (such as the CD4 ectodomain that recognizes HIV-infected cells) and more unconventional recognition components, such as linked cytokines (that recognize cells having cytokine receptors), have also been described. In fact, almost anything that binds to a specific target with high affinity can be used as the antigen recognition region.

[0100] The spacer region links the antigen-binding domain to the transmembrane domain. To facilitate antigen recognition, the spacer region must have sufficient mobility to allow the antigen-binding domain to face in various directions. The simplest form is the hinge region derived from IgG1. Alternative options include the CH2CH3 region of immunoglobulins and part of CD3. In the case of most scFv-based constructs, the IgG1 hinge is sufficient. However, the best spacer often has to be determined empirically.

[0101] Transmembrane domain The transmembrane domain is a hydrophobic α-helix that traverses the membrane. Generally, transmembrane domains derived from the most membrane-proximal component of the endodomain are used. Interestingly, when using the CD3ζ transmembrane domain, artificial TCRs may be incorporated into natural TCRs, which is a factor dependent on the presence of the native CD3ζ transmembrane charged aspartic acid residue. Different transmembrane domains result in different receptor stabilities. Using the CD28 transmembrane domain gives rise to a stably expressed receptor that is actively expressed.

[0102] Endodomain This is the "business-end" of the receptor. After the antigen is recognized, the receptor clusters and signals are transmitted to the cell. The most commonly used endodomain component is CD3ζ, which contains three ITAMs. This transmits an activation signal to T cells after antigen binding. CD3ζ may not supply a sufficiently potent activation signal, and additional co-stimulatory signaling may be required. For example, chimeric CD28 and OX40 may be used together with CD3ζ to transmit proliferation / survival signals, or all three may be used together.

[0103] "First-generation" CARs typically had an intracellular domain derived from the CD3ξ chain, a primary signaling molecule derived from the endogenous TCR. In "second-generation" CARs, intracellular signaling domains derived from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS) were added to the cytoplasmic tail of the CAR to supply additional signals to the T cell. From preclinical studies, it has been found that second-generation CAR designs improve the anti-tumor activity of T cells. More recently, in "third-generation" CARs, multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, are combined to further enhance efficacy.

[0104] Adoptive transfer of T cells expressing chimeric antigen receptors is a promising anti-cancer therapy because CAR-modified T cells can be engineered to target substantially any tumor-associated antigen. This approach has great potential to greatly improve individualized cancer therapies for patients. After the patient's T cells are collected, they are genetically engineered to express a CAR that is specifically directed against an antigen on the patient's tumor cells and then infused back into the patient. Although adoptive transfer of CAR-modified T cells is a unique and promising cancer therapy, there are significant safety concerns. From clinical trials of this therapy, it has become clear that these CARs have potential toxic effects when healthy tissues express the same target antigen as tumor cells, and thus the outcome resembles graft-versus-host disease (GVHD). A potential solution to this problem is to engineer and introduce a suicide gene into the modified T cells. Thus, administration of a prodrug designed to activate the suicide gene during GVHD induces apoptosis in CAR T cells activated by the suicide gene. This method has been safely and effectively used in hematopoietic stem cell transplantation (HSCT). The adoption of suicide gene therapy for the clinical use of CAR-modified T cell adoptive transfer has the potential to reduce GVHD while improving overall anti-tumor efficacy.

[0105] 2.ADC An antibody-drug conjugate, or ADC, is a new class of very potent biologic drugs designed as a targeted therapy for treating people with cancer. An ADC is a complex molecule composed of an antibody (either the whole mAb or an antibody fragment, e.g., single-chain variable fragment, or scFv) linked to a biologically active cytotoxic (anticancer) payload or drug by an unstable bond via a stable chemical linker. Antibody-drug conjugates are examples of bioconjugates and immunoconjugates.

[0106] By combining the inherent targeting ability of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, antibody-drug conjugates can distinguish between healthy and diseased tissues with high sensitivity. This means that, in contrast to conventional chemotherapeutic agents, antibody-drug conjugates target and attack cancer cells such that healthy cells are less severely affected than cancer cells.

[0107] In the development of ADC-based antitumor therapies, an anticancer drug (e.g., a cytotoxin or cytotoxic agent) is coupled to an antibody that specifically targets a particular tumor marker (e.g., ideally, a protein found only in or on tumor cells, in this case glypican 2). The antibody locates these proteins in the body and attaches to the surface of cancer cells. A biochemical reaction between the antibody and the target protein (antigen) induces a signal within the tumor cell, and then the antibody is taken up or internalized along with the cytotoxic agent. After the ADC is internalized, the cytotoxic drug is released, killing the cancer. This targeting ideally results in the drug having fewer side effects than other chemotherapeutic agents and providing a broader therapeutic window.

[0108] A stable linkage between an antibody and a cytotoxic (anticancer) agent is an important aspect of an ADC. The linker is based on a chemical motif that includes a disulfide, hydrazone, or peptide (cleavable), or thioether (non-cleavable), and controls the distribution and delivery of the cytotoxic agent to the target cell. Cleavable types of linkers and non-cleavable types of linkers have been found to be safe in preclinical and clinical trials. Brentuximab vedotin contains an enzyme-sensitive cleavable linker that delivers the synthetic antineoplastic agent, the potent and highly toxic microtubule inhibitor monomethyl auristatin E, or MMAE, to human-specific CD30-positive malignant cells. Because of this high toxicity, MMAE, which inhibits cell division by blocking tubulin polymerization, cannot be used as a single-agent chemotherapy agent. However, the combination of MMAE linked to an anti-CD30 monoclonal antibody (cAC10, a cell membrane protein of the tumor necrosis factor or TNF receptor) was found to be stable in extracellular fluid and cleavable by cathepsin, and safe for therapy. Another approved ADC, trastuzumab emtansine, is a combination of the antibody trastuzumab (Herceptin® / Genentech / Roche) and the microtubule formation inhibitor mertansine (DM-1), a derivative of maytansine, attached by a stable non-cleavable linker.

[0109] Better and more stable linkers became available, changing the function of the chemical bond. Cleavable or non-cleavable types of linkers impart specific properties to cytotoxic (anticancer) drugs. For example, non-cleavable linkers keep the drug inside the cell. As a result, the entire antibody, linker, and cytotoxic (anticancer) agent enter the target cancer cell, where the antibody is degraded to the amino acid level. In that case, the resulting complex of amino acids, linker, and cytotoxic agent is the active drug. In contrast, cleavable linkers are catalyzed by enzymes present inside cancer cells to release the cytotoxic agent inside the cancer cells. This difference is that the cytotoxic payload delivered via a cleavable linker can escape from the target cell and attack adjacent cancer cells in a process called "bystander killing".

[0110] Another type of cleavable linker currently under development adds an extra molecule between the cytotoxic drug and the cleavage site. Using this linker technology, researchers can create ADCs with greater flexibility without worrying about changing the cleavage kinetics. Researchers are also developing a new peptide cleavage method based on Edman degradation, a method for sequencing the amino acids of a peptide. Future directions in ADC development also include the development of site-specific conjugation (TDC) to further improve stability and therapeutic index, as well as alpha radioimmunoconjugates and antibody-conjugated nanoparticles.

[0111] 3.BitE Bispecific T-cell engager (BiTE) is a type of artificial bispecific monoclonal antibody being studied for use as an anticancer drug. They direct the host's immune system, specifically the cytotoxic activity of T cells, towards cancer cells. BiTE is a registered trademark of Micromet AG.

[0112] BiTE is a fusion protein consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences derived from four different genes, on a single peptide chain of approximately 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, and the other binds to tumor cells via a tumor-specific molecule, in this case glypican 2.

[0113] Similar to other bispecific antibodies and unlike normal monoclonal antibodies, BiTE forms a linkage between T cells and tumor cells. Thereby, independent of the presence of MHC I or costimulatory molecules, T cells exert cytotoxic activity on tumor cells by producing protein-like perforin and granzyme. These proteins enter the tumor cells and initiate apoptosis of the cells. This function is very similar to the physiological process observed while T cells are attacking tumor cells.

[0114] At the time of July 2010, BiTEs that were in clinical trials included blinatumomab (MT103) for treating non-Hodgkin lymphoma and acute lymphoblastic leukemia, made against CD19, a surface molecule expressed on B cells, and MT110 for treating gastrointestinal and lung cancers, made against the EpCAM antigen.

[0115] Using the same technology, it is possible to target melanoma (using MCSP-specific BiTE) and acute myeloid leukemia (using CD33-specific BiTE). Research in this area is currently ongoing. Another means for new anti-cancer therapies is to redesign some of the currently used conventional antibodies such as trastuzumab (targeting HER2 / neu), cetuximab, and panitumumab (both targeting the EGF receptor) using the BiTE approach. BiTEs against CD66e and EphA2 are also under development.

[0116] III. Pharmaceutical Preparations and Treatment of Cancer A. Cancer Cancer results from the proliferation of a clonal population of cells derived from tissues. The development of cancer, called carcinogenesis, can be modeled and characterized in many ways. A link between cancer development and inflammation has long been recognized. The inflammatory response is involved in host defense against microbial infection and also serves as a driving force for tissue repair and regeneration. Considerable evidence points to a relationship between inflammation and cancer development risk, i.e., that chronic inflammation can cause dysplasia.

[0117] Cancer cells to which the methods of the present disclosure can be applied generally include any cells that express glypican 2, and more particularly, any cells that overexpress glypican 2. Cancer cells that can be treated according to the present disclosure include, but are not limited to, cells derived from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. Further, cancers specifically include cancers of the following histological types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; carcinoma of giant and spindle cells; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary adenocarcinoma and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell tumor; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; chromaffin cell tumor; glomus angiosarcoma; malignant melanoma; amelanotic melanoma; superficially spreading melanoma; malignant melanoma in giant congenital nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant;Müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated embryonal cell tumor; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; angioendothelioma, malignant; Kaposi sarcoma; perivascular cell tumor, malignant; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontogenic sarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; epithelioma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioblastoma; undifferentiated neuroectodermal; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory nerve tumor; meningioma, malignant; neurofibrosarcoma; neuroma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin disease; side granuloma; malignant lymphoma, small lymphocyte; malignant lymphoma, diffuse large cell; malignant lymphoma, follicular; fungating polypoid tumor; other non-Hodgkin lymphoma as specified; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblast leukemia; myelosarcoma; and hairy cell leukemia, but not limited thereto. In certain aspects, the tumor may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, glioblastoma, medulloblastoma, neuroblastoma, or leukemia.;

[0118] Furthermore, the methods of the present disclosure can be applied to a wide range of species, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The cancer can also be recurrent, metastatic, and / or multi-drug resistant. The methods of the present disclosure can be applied to such cancers, particularly to make such cancers resectable, to prolong or re-induce remission, to inhibit angiogenesis, to prevent or limit metastasis, and / or to treat multi-drug resistant cancers. At the cellular level, this may result in the death of cancer cells, inhibition of cancer cell proliferation, or otherwise the reversal or reduction of the malignant phenotype of tumor cells.

[0119] B. Formulations and Administration The present disclosure provides a pharmaceutical composition comprising an anti-glypican 2 antibody. In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals and, more particularly, in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium chloride, dextrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.

[0120] The composition may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed using anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed using cations, such as salts derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0121] The antibodies of the present disclosure may include classical pharmaceutical preparations. Administration of these compositions according to the present disclosure is carried out via any general route as long as the target tissue is accessible via this route. This route includes the oral route, nasal route, buccal route, rectal route, vaginal route, or topical route. Alternatively, the administration may be by intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or intravenous injection. Such compositions will typically be administered as the pharmaceutically acceptable compositions described above. Administration into the tumor, perfusion of the tumor, or topical or local administration to the tumor, for example, to the local or regional vasculature or lymphatic system, or to the resected tumor bed, is of particular interest.

[0122] The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound, which is a free base or a pharmaceutically acceptable salt, can be prepared by dissolving it in water appropriately mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared by dissolving in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0123] C. Combination Therapy In the context of the present disclosure, it is also contemplated that the anti-glypican 2 antibodies described herein can be used similarly in combination with chemotherapy interventions or radiation therapy interventions or other treatments. In particular, it may be found effective to combine the anti-glypican 2 antibodies with other therapies that target different aspects of glypican 2 function.

[0124] In order to kill cells, inhibit cell proliferation, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells using the methods and compositions of the present disclosure, generally, "target" cells will be contacted with an anti-glypican 2 antibody according to the present disclosure and at least one other agent. These compositions will be provided in a combined amount effective to kill cells or to inhibit the growth of cells. This process may involve contacting the cells simultaneously with an anti-glypican 2 antibody according to the present disclosure and another agent or factor. This may be accomplished by contacting the cells with a single composition or pharmaceutical formulation containing both agents, or by contacting the cells simultaneously with two separate compositions or formulations, one composition containing an anti-glypican 2 antibody according to the present disclosure and the other composition containing the other agent.

[0125] Alternatively, the anti-glypican 2 antibody therapy may be administered before or after other drug treatments at intervals ranging from a few minutes to a few weeks. In embodiments where the other drug and the anti-glypican 2 antibody are applied to the cells separately, generally, the effective period will not end between each delivery so that the drug and the expression construct can still exert an advantageously combined effect on the cells. In such cases, it is contemplated that the cells are contacted with both modalities within about 12 to 24 hours, more preferably within about 6 to 12 hours, of administering either modality, and most preferably within 12 hours of administering either modality. Depending on the situation, it may be desirable to significantly extend the period for treatment. However, the period between each administration is from several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0126] It may also be desirable to administer either the anti-glypican 2 antibody or the other drug multiple times. As illustrated below, various combinations can be used. In the formula, the anti-glypican 2 antibody therapy according to the present disclosure is "A", and the other therapy is "B". TIFF2025098050000003.tif17128

[0127] Other combinations are also contemplated. Again, both agents are delivered to the cells in a combined amount effective to kill the cells, for killing the cells. Agents or factors suitable for cancer therapy include any compound or method of treatment that induces damage when applied to cells. Such agents and factors include radiation and waves that induce DNA damage, such as radiation exposure, microwaves, electroluminescence, etc. Various compounds, sometimes also referred to as "chemotherapeutic agents" or "genotoxic agents", may be used. This may be accomplished by irradiating the local tumor site with radiation. Alternatively, the tumor cells may be contacted with the agent by administering a therapeutically effective amount of the pharmaceutical composition to the subject. The combination therapy may also include surgery. Various ways of these therapies are discussed below.

[0128] 1. Chemotherapy The term "chemotherapy" refers to the use of drugs for treating cancer. The term "chemotherapeutic agent" is used to imply a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mechanism of action within the cell, e.g., whether they affect the cell cycle and at which stage they affect the cell cycle. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, to insert into DNA, or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. Most chemotherapeutic agents are classified into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

[0129] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamime, including ethyleneimine and methylamelamine; acetogenins (especially, bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); erythrobins; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially, calicheamicin γ1I and calicheamicin ωI1; dynemicin including dynemicin A; uncialamycin and its derivatives; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein engyin antibiotics chromophore, actinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardinophyllin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, for example, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, or zorubicin; antimetabolites, for example, methotrexate and 5-fluorouracil (5-FU); folic acid analogs, for example, denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, for example, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine; androgens, for example, calusterone, drostanolone propionate, epitostanol, mepitiostane, testolactone; antiadrenal agents, for example, aminoglutethimide, mitotane, trilostane; folic acid supplements, for example, folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; elformithine; elliptinium acetate; epsilon; etoglucid; gallium nitrate;Hydroxyurea; Lentinan; Lonidamine; Mitansoids, e.g., Mitansine and Ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially, T-2 toxin, Verracurin A, Roridin A, and Anguidine); Urethane; Vinblastine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (“Ara-C”); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel and Docetaxel; Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum coordination complexes, e.g., Cisplatin, Oxaliplatin, and Carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids, e.g., Retinoic acid; Capecitabine;Cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agent, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, farnesyl-protein transferase inhibitor, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are included.;

[0130] 2. Radiation Therapy Radiation therapy, also called radiotherapy, is the treatment of cancer and other diseases using ionizing radiation. Ionizing radiation deposits energy that damages or destroys cells in the area being treated by damaging the cell's genetic material, so that these cells can no longer continue to grow. Radiation damages both cancer cells and normal cells, but the latter can repair themselves and function properly on their own.

[0131] The radiation therapy used in accordance with the present disclosure may include, but is not limited to, the use of gamma rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other types of DNA damaging agents such as microwaves and UV radiation are also contemplated. It is almost certain that all of these agents induce extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome construction and maintenance. The radiation dose range of X-rays ranges from a daily dose of 50 to 200 roentgens for a long period (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The radiation dose range of radioisotopes varies and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by new cells.

[0132] Radiation therapy may involve the use of radiolabeled antibodies to deliver a certain dose of radiation directly to the cancer site (radioimmunotherapy). Antibodies are highly specific proteins that the body makes in response to the presence of an antigen (a substance that the immune system recognizes as foreign). Some tumor cells contain specific antigens that induce the production of tumor-specific antibodies. Large amounts of these antibodies can be produced in the laboratory and attached to radioactive substances (a process known as radiolabeling). Once injected into the body, the antibodies actively seek out cancer cells, which are then destroyed by the cell-killing (cytotoxic) effects of the radiation. Using this approach, the risk of radiation damage to healthy cells can be minimized.

[0133] In the case of conformal radiotherapy, a linear accelerator, which is the same radiotherapy device as in a normal radiotherapy treatment, is used. To change the shape of the x-ray beam to match the shape of the cancer, metal blocks are placed in the path of the x-ray beam. This ensures that a high radiation dose is delivered to the tumor. Healthy surrounding cells and nearby structures receive a low dose of radiation, so the potential for side effects is reduced. A device called a multi-leaf collimator has been developed and may be used in place of the metal blocks. The multi-leaf collimator consists of a number of metal sheets fixed to the linear accelerator. Each layer can be adjusted so that the radiotherapy beam fits exactly into the treatment area without the need for metal blocks. The precise positioning of the radiotherapy device is very important in conformal radiotherapy treatment, and a special scanning device may be used to check the position of the internal organs at the start of each treatment.

[0134] High-resolution intensity modulated radiotherapy also uses a multi-leaf collimator. During this treatment, the layers of the multi-leaf collimator are moved while the treatment is being carried out. This method is likely to allow the treatment beam to be more accurately aligned and the dose of radiotherapy to be made uniform across the entire treatment area.

[0135] Research studies have shown that conformal and intensity-modulated radiation therapy may reduce the side effects of radiation therapy treatments, but by tailoring the treatment area so precisely, tiny cancer cells just outside the treatment area may not be destroyed, meaning that these specialized radiation therapy techniques may increase the risk of cancer recurrence in the future.

[0136] Scientists are also looking for ways to improve the effectiveness of radiation therapy. Two types of experimental drugs are being studied for their effect on cells receiving radiation: radiosensitizers, which make tumor cells more likely to be damaged, and radioprotectors, which protect normal tissue from the effects of radiation. Hyperthermia, the use of heat, is also being studied for its effectiveness in increasing tissue sensitivity to radiation.

[0137] 3. Immunotherapy In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Such an example is trastuzumab (Herceptin™). The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody may act alone as the effector of therapy or may recruit other cells that actually affect cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply act as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells. Using a combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2, may provide therapeutic benefit in the treatment of ErbB2-overexpressing cancers.

[0138] In one aspect of immunotherapy, tumor cells should have some marker that is a target for targeting, i.e., that is not present in the majority of other cells. There are many tumor markers, any of which may be suitable for targeting in the context of the present disclosure. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155. Another aspect of immunotherapy is the combination of an anti-cancer effect and an immune-stimulating effect. There are also immune-stimulating molecules including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand. The combined use of immune-stimulating molecules as proteins or immune-stimulating molecules using gene delivery and tumor suppressors has been shown to enhance the anti-tumor effect (Ju et al., 2000). Further, antibodies against any of these compounds may be used to target the anti-cancer agents discussed herein.

[0139] Examples of immunotherapies currently under investigation or in use include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy such as interferon α, β, and γ; IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998), gene therapy such as TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945), and monoclonal antibodies such as anti-ganglioside GM2, anti-HER-2, anti-p185 (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in combination with the gene silencing therapy described herein.

[0140] In active immunotherapy, antigenic peptides, polypeptides, or proteins, or autologous or allogeneic tumor cell compositions, i.e., "vaccines", are generally administered together with a separate bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993).

[0141] In adoptive immunotherapy, the patient's circulating lymphocytes or tumor-infiltrating lymphocytes are isolated in vitro, activated by lymphokines such as IL-2, or genes for tumor necrosis are introduced and readministered (Rosenberg et al., 1988; 1989).

[0142] 4. Surgery Approximately 60% of people with cancer undergo some type of surgery. Surgery includes prophylactic surgery, surgery for diagnosis or staging, curative surgery, and palliative surgery. Curative surgery is a cancer treatment that can be used in combination with other therapies such as the treatments of the present disclosure, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0143] Curative surgery includes excisions in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical procedures include laser surgery, cryosurgery, electrocautery, and microsurgically controlled surgery (Mohs surgery). Further, the present disclosure is intended to be used with the removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0144] A cavity may be formed in the body when removing some or all of the cancer cells, cancerous tissue, or tumor. The treatment may be achieved by perfusing, directly injecting, or topically applying additional anti-cancer therapies at this site. Such treatments may be repeated, for example, daily, every two days, every three days, every four days, every five days, every six days, or every seven days, or every week, every two weeks, every three weeks, every four weeks, and every five weeks, or every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or every twelve months. These treatments may also be treatments with different dosages.

[0145] In certain specific embodiments, adjuvant treatment with the compounds of the present disclosure is considered particularly effective in reducing tumor recurrence after tumor removal. Additionally, the compounds of the present disclosure can also be used in the neoadjuvant setting.

[0146] It should also be pointed out that any of the aforementioned therapies may be useful alone in cancer treatment. Those skilled in the art are directed to "Remington's Pharmaceutical Sciences", 15th Edition, Chapter 33, specifically pages 624 - 652. There will necessarily be some variation in dosage depending on the condition of the subject being treated. The person administering the treatment will, in any event, determine the dosage appropriate for each individual subject. Further, in the case of human administration, the preparation must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the standards of the FDA's Office of Biologics.

[0147] IV. Antibody Conjugates The antibody may be linked to at least one agent to form an antibody conjugate. It is conventional to link, covalently bond, or complex at least one desired molecule or moiety to enhance the efficacy of the antibody molecule as a diagnostic or therapeutic agent. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules include molecules having a desired activity, such as anti - cancer / general cytotoxicity. Non - limiting examples of such molecules were shown above. Such molecules are optionally attached via a cleavable linker designed such that such molecules are released at or near the target site.

[0148] In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non - limiting examples of reporter molecules that have been conjugated to an antibody include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.

[0149] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostic agents are generally classified into two classes: antibody diagnostic agents for use in in vitro diagnostics such as various immunoassays, and antibody diagnostic agents for use in in vivo diagnostic protocols generally known as "antibody-directed imaging". Many suitable imaging agents are known in the art, and similarly, methods for attaching imaging agents to antibodies are also known in the art (see, for example, U.S. Patent Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used may be paramagnetic ions, radioisotopes, fluorescent dyes, substances detectable by NMR, and in the case of X-ray imaging agents.

[0150] In the case of paramagnetic ions, by way of example, ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and / or erbium (III) may be mentioned, with gadolinium being particularly preferred. Ions useful in other situations such as X-ray imaging include, but are not limited to, lanthanum (III), gold (III), lead (II), and particularly bismuth (III).

[0151] In the case of radioisotopes for therapeutic and / or diagnostic use, astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 cobalt, copper 67 , 152 Eu, gallium 67 , 3 hydrogen, iodine 123 , iodine 125 , iodine 131 , indium 111 ,59 Iron 32 Phosphorus, rhenium 186 , rhenium 188 , 75 Selenium 35 Sulfur, technetium 99m and / or yttrium 90 may be mentioned. In certain embodiments, 125 I is often preferred, but technetium 99m and / or indium 111 are also often preferred because they have low energy and are suitable for long - distance detection. Radioactively labeled monoclonal antibodies can be prepared according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contacting them with sodium iodide and / or potassium iodide, and a chemical oxidizing agent, such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies can be labeled with technetium by a ligand - exchange process, for example, reducing pertechnate with a tin solution, chelating the reduced technetium with a Sephadex column, and applying the antibody to this column. 99m Or, for example, a direct - labeling technique may be used by incubating pertechnate, a reducing agent, such as SNCl2, a buffer solution, such as a sodium - potassium phthalate solution, and the antibody. Intermediate functional groups are often used to bind radioisotopes to antibodies, and metal ions are present since they are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0152] Among the fluorescent labels that can be considered for use as conjugates are Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, rhodamine green, rhodamine red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

[0153] Another type of antibody conjugate that can be considered is an antibody conjugate that is primarily intended for use in vitro. In this case, the antibody is linked to a secondary binding ligand and / or an enzyme (enzyme tag) that produces a colored product when contacted with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0154] Yet another known method of site-specifically attaching a molecule to an antibody involves reacting the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with the amino acids at the antigen-binding site, thereby destroying this site and blocking the specific antigen reaction. However, this may not be advantageous as it causes the loss of antigen binding by the antibody conjugate.

[0155] Molecules containing azide groups may also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2-azido analogs and 8-azido analogs of purine nucleotides have been used as site-specific photoprobes for identifying nucleotide-binding proteins in cell crude extracts (Owens & Haley, 1987; Atherton et al., 1985). 2-Azido nucleotides and 8-azido nucleotides have also been used to map the nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and may be used as antibody conjugates.

[0156] Several methods are known in the art for attaching or binding an antibody to its conjugate moiety. Among the attachment methods are those involving, for example, the use of metal chelate complexes with organic chelating agents attached to the antibody, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriamine tetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Patent Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanate. In U.S. Patent No. 4,938,948, imaging of breast tumors is accomplished using monoclonal antibodies, and the detectable imaging moiety is attached to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0157] In other embodiments, it is contemplated that immunoglobulins are derivatized by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody binding site. Antibody conjugates made according to this method are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Patent No. 5,196,066, which is incorporated herein by reference). Site-specific attachment of effector or reporter molecules, where the effector or reporter molecule binds to a carbohydrate residue in the Fc region, has also been disclosed in the literature (O'Shannessy et al., 1987). This approach is now reported to yield antibodies promising for diagnosis and therapy that are currently under clinical evaluation.

[0158] V. Immunodetection Methods In still further embodiments, there are immunodetection methods for binding, purifying, removing, quantifying, or otherwise generally detecting glypican 2. Some immunodetection methods include, to name a few, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot. In particular, competitive assays for detecting and quantifying glypican 2 antibodies are also provided. The procedures for various useful immunodetection methods are described in scientific literature such as, for example, Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al (1993), and Nakamura et al (1987). Generally, the immunobinding methods include obtaining a sample and contacting the sample with a first antibody under conditions effective to form an immune complex, if any, according to the embodiments discussed herein.

[0159] Under conditions effective and for a period sufficient to form an immune complex (primary immune complex), the step of contacting a selected biological sample with an antibody generally simply involves adding the antibody composition to the sample and incubating the mixture for a period long enough for the antibody to form an immune complex, i.e., to bind to glypican 2 present in the sample. After this time, the sample-antibody composition, e.g., tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove non-specifically bound antibody species, thereby enabling only these antibodies to specifically bind within the primary immune complex to be detected.

[0160] Generally, the detection of immune complex formation is well known in the art and can potentially be accomplished by applying a very large number of approaches. These methods generally rely on the detection of a label or marker, such as a radioactive tag, fluorescent tag, biological tag, and enzyme tag. Patents related to the use of such labels include U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Of course, as is known in the art, additional advantages may be found by using secondary binding ligands, such as a second antibody and / or biotin / avidin ligand binding sequences.

[0161] The antibody itself used in the detection may be linked to a detectable label. Then, this label is easily detected, thereby making it possible to determine the amount of the primary immune complex in the composition. Or, the first antibody bound in the primary immune complex may be detected by a second binding ligand having a binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand itself is often an antibody, and thus this antibody may sometimes be called a "secondary" antibody. The primary immune complex is contacted with the labeled secondary binding ligand or antibody under conditions effective to form a secondary immune complex and for a period sufficient to form a secondary immune complex. Then, the secondary immune complex is generally washed to remove non-specifically bound, labeled secondary antibody or ligand, and then the remaining label in the secondary immune complex is detected.

[0162] A further method involves detecting the primary immune complex by a two-step approach. A second binding ligand, for example, an antibody having a binding affinity for the antibody, is used to form a secondary immune complex as described above. After washing, the secondary immune complex is contacted with a third binding ligand or antibody having a binding affinity for the second antibody, also under conditions effective to form an immune complex (tertiary immune complex) and for a period sufficient to form an immune complex (tertiary immune complex). The third ligand or antibody is linked to a detectable label, whereby the tertiary immune complex thus formed is detected. If desired, this system may amplify the signal.

[0163] In one immunodetection method, two different antibodies are used. The first biotinylated antibody is used to detect the target antigen, and then the second antibody is used to detect the biotin attached to the complexed biotin. In this method, first, the sample to be tested is incubated in a solution containing the antibody of the first step. If the target antigen is present, a portion of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubating it in a sequential solution of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification step is repeated until an appropriate level of amplification is reached. At this time, the sample is incubated in a solution containing the antibody of the second step against biotin. This second-step antibody is labeled, for example, like an enzyme that can be used to detect in the presence of the antibody / antigen complex by tissue enzymology using a chromogenic substrate. With appropriate amplification, a macroscopically visible conjugate is produced.

[0164] Another known immunodetection method utilizes the immun-PCR (polymerase chain reaction) method. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, but instead of using multiple incubations of streptavidin and biotinylated DNA, the DNA / biotin / streptavidin / antibody complex is washed away using a low pH buffer or a high salt buffer that releases the antibody. Then, using the resulting wash solution, a PCR reaction is performed using appropriate primers and appropriate controls. At least theoretically, the enormous amplification capacity and specificity of PCR can be utilized to detect one type of antigen molecule.

[0165] A. ELISA An immunoassay is, in the simplest sense, a binding assay. Certain preferred immunoassays are various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it is readily understood that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analysis, etc. may also be used.

[0166] In one exemplary ELISA, the antibodies of the present disclosure are immobilized in wells on a selected surface that exhibits protein affinity, such as a polystyrene microtiter plate. Then, a test composition suspected of containing glypican 2 is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be accomplished by adding another anti-glypican 2 antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be accomplished by adding a second anti-glypican 2 antibody, and then adding a third antibody that has binding affinity for the second antibody, where the third antibody is linked to a detectable label.

[0167] In another exemplary ELISA, a sample suspected of containing the glypican 2 antigen is immobilized on the well surface and then contacted with an anti-glypican 2 antibody. After binding and washing to remove non-specifically bound immune complexes, the bound anti-glypican 2 antibody is detected. If the first anti-glypican 2 antibody is linked to a detectable label, the immune complex may be detected directly. It is also possible that the immune complex may be detected using a second antibody that has binding affinity for the first anti-glypican 2 antibody, where the second antibody is linked to a detectable label.

[0168] Regardless of the format used, ELISA has certain common characteristics, such as coating, incubation and binding, washing to remove non-specifically bound species, and detection of the bound immune complexes. These are described below.

[0169] When coating the plate with an antigen or an antibody, generally, the wells of the plate are incubated with a solution of the antigen or antibody overnight or for a specified time. Then, the wells of the plate are washed to remove the incompletely adsorbed material. Next, the remaining available well surface is "coated" with an antigenically neutral non-specific protein with respect to the test antiserum. These include bovine serum albumin (BSA), casein, or skim milk solution. Coating blocks the non-specific adsorption sites on the immobilized surface, thus reducing the background caused by non-specific binding of the antiserum to the surface.

[0170] In ELISA, it is probably common to use secondary or tertiary detection means rather than a direct procedure. Thus, after the protein or antibody has bound to the well, the well has been coated with a non-reactive material to reduce the background, and the unbound material has been removed by washing, the immobilized surface is contacted with the biological sample to be tested under conditions effective to form an immune complex (antigen / antibody). Then, detection of the immune complex requires a labeled secondary binding ligand or antibody, and a labeled tertiary antibody, or a third binding ligand for the secondary binding ligand or antibody.

[0171] "Under conditions effective to form an immune complex (antigen / antibody)" means that the conditions preferably include the step of diluting the antigen and / or antibody in a solution, such as BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween. These added agents also tend to help reduce non-specific background.

[0172] The term "appropriate" conditions also means that the incubation is carried out at a temperature sufficient for effective binding or over a period sufficient for effective binding. The incubation step is typically about 1 - 2 - 4 hours, preferably at a temperature of about 25°C - 27°C, or it may be overnight at about 4°C.

[0173] After all incubation steps in ELISA, the contacted surface is washed to remove uncomplexed material. Preferred washing procedures include washing with a solution, such as PBS / Tween, or a borate buffer. After the formation of specific immune complexes between the test sample and the initially bound material and subsequent washing, it may be confirmed that additional trace amounts of immune complexes have occurred.

[0174] To provide a means of detection, the second or third antibody has a bound label that enables detection. Preferably, this is an enzyme that develops color when incubated with an appropriate chromogenic substrate. Thus, for example, it is desirable to contact or incubate the first and second immune complexes with an antibody conjugated to urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase for a period favorable for the occurrence of further immune complex formation and under conditions favorable for the occurrence of further immune complex formation (e.g., incubate for 2 hours at room temperature in a PBS-containing solution, such as PBS-Tween).

[0175] After incubation with the labeled antibody and washing to remove unbound material, the amount of label is quantified, for example, by incubating with a chromogenic substrate, such as urea, or bromocresol purple, or 2,2'-azino-bis-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), or in the case of peroxidase as an enzyme label, with H2O2. The quantification is then accomplished by measuring the degree of color development, for example, using a visible spectrum spectrophotometer.

[0176] B. Western Blot A Western blot (or protein immunoblot) is an analytical technique used to detect a specific protein in a particular sample of tissue homogenate or extract. In Western blotting, gel electrophoresis is used to separate native or denatured proteins either by the length of the polypeptide (denaturing conditions) or by the protein's 3D structure (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF) where they are probed (detected) using an antibody specific to the target protein.

[0177] Samples may be taken from whole tissues or from cell cultures. In most cases, initially, solid tissues are mechanically disrupted using a blender (if the sample volume is large), a homogenizer (if the volume is small), or by sonication. Cells may also be lysed by one of the mechanical methods described above. However, it should be noted that bacteria, viruses, or environmental samples can be sources of proteins and thus Western blotting is not limited to cell research only. A variety of detergents, salts, and buffers may be used to facilitate cell lysis and solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the sample from being digested by its own enzymes. Tissue preparation is often carried out at low temperature to avoid protein denaturation.

[0178] The proteins in the sample are separated using gel electrophoresis. Protein separation may be based on the isoelectric point (pI), molecular weight, charge, or a combination of these factors. How the separation is done depends on how the sample is processed and what kind of gel is used. This is a very useful technique for identifying proteins. It is also possible to use a two-dimensional (2D) gel that spreads the proteins from a single sample out in two dimensions. The proteins are separated in the first dimension according to their isoelectric point (the pH at which they have a neutral net charge) and in the second dimension according to their molecular weight.

[0179] To produce a protein that can be used for antibody detection, the protein is transferred from inside the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). A membrane is placed on top of the gel, and on top of the membrane, a stack of filter papers is placed. The stack of filter papers is placed entirely in a buffer solution, and the buffer solution rises through the filter papers by capillary action, thereby causing the protein to move along with the buffer solution. Another method for transferring proteins is called electroblotting, which uses an electric current to pull the protein from the gel onto a PVDF or nitrocellulose membrane. The protein moves from inside the gel onto the membrane while maintaining the structure it had inside the gel. As a result of this blotting process, the protein is exposed in a thin surface layer for detection (see below). Both types of membranes are selected for their non-specific protein-binding properties (i.e., they bind equally to all proteins). Protein binding is based on hydrophobic interactions and charge interactions between the membrane and the protein. Nitrocellulose membranes are cheaper than PVDF but are quite fragile and not very resistant to repeated assays. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once the protein has been transferred, it is detected using a labeled primary antibody or an unlabeled primary antibody followed by indirect detection using the binding of a labeled Protein A or secondary labeled antibody to the Fc region of the primary antibody.

[0180] C. Immunohistochemistry Antibodies may also be used in conjunction with freshly frozen tissue blocks and / or formalin-fixed, paraffin-embedded tissue blocks prepared for studies by immunohistochemistry (IHC). The methods for preparing tissue blocks from these particulate specimens have been successfully used in previous IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0181] Briefly stated, cryosections are prepared by rehydrating 50 ng of "pulverized" frozen tissue in phosphate buffered saline (PBS) at room temperature in a small plastic capsule; pelleting the particles by centrifugation; resuspending in a viscous embedding agent (OCT); inverting the capsule and / or repelleting by centrifugation; flash freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen cylindrical tissue; fixing the cylindrical tissue to a cryostat microtome chuck; and / or cutting 25-50 consecutive sections from the capsule. Alternatively, the entire frozen tissue sample may be used to cut consecutive sections.

[0182] Permanent sections may be prepared by a similar method including rehydrating a 50 mg sample in a plastic microcentrifuge tube, pelleting, resuspending in 10% formalin for 4 hours of fixation, washing / pelleting, resuspending in warm 2.5% agar, pelleting, cooling in ice water to solidify the agar, removing the tissue / agar block from the tube, infiltrating and / or embedding the block in paraffin, and / or cutting up to 50 consecutive permanent sections. Again, the entire tissue sample may be substituted.

[0183] D. Immunodetection Kit In yet a further aspect, there is an immunodetection kit for use with the immunodetection method described above. Thus, the immunodetection kit includes, in a suitable container means, a first antibody that binds to the glypican 2 antigen and, optionally, immunodetection reagents.

[0184] In certain embodiments, the glypican-2 antibody may be pre-fixed to a solid support, such as the well of a column matrix and / or a microtiter plate. The immunodetection reagent of the kit may take any one of various forms, including a detectable label bound or linked to a particular antibody. Also contemplated are detectable labels bound or attached to a secondary binding ligand. An exemplary secondary ligand is a secondary antibody having binding affinity for a first antibody.

[0185] Further suitable immunodetection reagents for use in the kits of the present invention include a two-component reagent comprising a secondary antibody having binding affinity for a first antibody and a third antibody having binding affinity for a second antibody, wherein the third antibody is linked to a detectable label. As described above, many exemplary labels are known in the art, and all such labels may be used in connection with the embodiments discussed herein.

[0186] The kit may further comprise a properly dispensed composition of glypican-2 antigen, whether labeled or unlabeled, and may likewise be used to generate a calibration curve for the detection assay. The kit may contain the antibody-label conjugate in a fully conjugated form, in an intermediate form, or as separate moieties to be conjugated by the user of the kit. The components of the kit may be packaged in an aqueous medium in solution or in a lyophilized form.

[0187] The container means of the kit generally comprises at least one vial, test tube, flask, bottle, syringe, or other container means, in which the antibody may be placed, preferably in a properly dispensed manner. The kit also comprises means for containing the antibody, antigen, and any other reagent container hermetically sealed for commercial sale. Such containers may comprise injection-molded plastic containers or blow-molded plastic containers that hold the desired vials.

Examples

[0188] VI. Examples The following examples are included to demonstrate preferred embodiments. The techniques disclosed in the following examples show that the techniques discovered by the inventors function well enough in the implementation of the embodiments, and thus, it should be understood by those skilled in the art that they constitute preferred embodiments for carrying out the present invention. However, in view of the present disclosure, it will be understood by those skilled in the art that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure, and still obtain similar or comparable results.

[0189] Example 1 From the initial discovery efforts based on the transcriptome, 649 significantly differentially expressed genes were identified (tumor vs. normal log fold change > 1 for each tissue; adjusted p < 0.05), of which 86 (13%) genes were predicted to be potential cell surface molecules. By our analysis pipeline, we selected, for validation, robust differential RNA expression (tumor vs. normal tissue log fold change = 2.1 - 8.2; p < 3x10 -10) and prioritized the cell surface signaling receptor, glypican-2 (GPC2), which is anchored to the extracellular glycosylphosphatidylinositol (GPI), as it was associated with high levels of absolute RNA expression (median FPKM = 57; 85% of tumors had FPKM > 25), significantly elevated GPC2 expression (p<0.0001), and a consistent DNA copy number increase (31% of primary neuroblastomas; N = 177). Ubiquitous GPC2 protein expression (N = 8 high-risk neuroblastomas and 23 cell lines) was confirmed by immunoblot analysis, and high-density plasma membrane-associated GPC2 protein expression was confirmed in neuroblastoma cell lines by membrane extraction, IF, and IHC. IHC analysis of primary neuroblastoma tumors (N = 83) compared to parallel arrays of pediatric normal tissues (N = 37) further confirmed that GPC2 protein expression was membrane-associated and tumor-specific, with normal tissue expression being highly restricted. In a panel of 12 neuroblastoma cell lines, GPC2 depletion induced by lentivirus-mediated RNAi resulted in significant apoptosis and growth inhibition in both transient CellTiter-Glo and Caspase-Glo assays, as well as long-term real-time monitoring of cell proliferation (RT-CES) (growth decreased 20 - 87% compared to control, and caspase 3 / 7 levels increased 1.5 - 18.4-fold). Cell growth was significantly increased by GPC2 overexpression (2.7-fold increase in growth compared to control, p<0.0001). Finally, GPC2 was also found to be significantly differentially overexpressed in other embryonal cancers, particularly medulloblastoma.

[0190] A panel of three fully human antibodies (m201, m202, and m203) that specifically target cancer cell-associated GPC2 was isolated from a phage display antibody library and affinity matured. In vitro characterization demonstrated that these antibodies have promising therapeutic activities for use in the development of CAR-Ts, antibody-drug conjugates (ADCs), and bispecific antibodies for cancer therapy. The sequences of the antibodies are shown in Figures 30 - 32.

[0191] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the compositions and methods described herein and in the steps or the order of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain chemically and physiologically related agents can be used in place of the agents described herein, and at the same time, it is clear that the same or similar results can be obtained. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the scope of the spirit, scope, and concept of the present disclosure as defined by the appended claims.

[0192] VII. References The following references are hereby specifically incorporated herein by reference to the extent that they show details of exemplary procedures or other details that supplement those described herein. TIFF2025098050000004.tif20335TIFF2025098050000005.tif231151TIFF2025098050000006.tif231150TIFF2025098050000007.tif231150TIFF2025098050000008.tif39128

[0193] Sequence Information SEQUENCE LISTING <110> The Children's Hospital of Philadelphia The United States of America, as represented by the Secretary, Department of Health and Human Services <120> GLYPICAN 2 AS A CANCER MARKER AND THERAPEUTIC TARGET <150> US 62 / 253,000 <151> November 9, 2015 <150> US 62 / 350,976 <151> June 16, 2016 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 1 gaggtgcagc tggtggagac tgggggaggc gtggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtagtac catatactac 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagagagt 300 ggctacgatt acgtgtttga ctactggggc cagggaaccc tggtcgccgt ctcctca 357 <210> 2 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 2 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Ala Val Ser Ser 115 <210> 3 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 3 gacatccaga tgacccagtc tccttccacc ctgtctgcat ttgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcaaaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaacag cttaatagtt accctatcac cttcggccaa 300 gggacacgac tggagattaa acga 324 <210> 4 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Phe Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 5 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 6 Ile Ser Ser Ser Gly Ser Thr Ile 1 5 <210> 7 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 7 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr 1 5 10 <210> 8 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 8 Gln Ser Ile Ser Ser Trp 1 5 <210> 9 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 9 Ala Ala Ser 1 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 10 Gln Gln Leu Asn Ser Tyr Pro Ile Thr 1 5 <210> 11 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 11 caggtgcagc tggtgcagtc tggaggaggc ttgatccagc ctggggggtc cctgagactc 60 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly tcctgtgcag cctctgggtt caccgtcagt agcaactaca tgagctgggt ccgccaggct 120 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn ccagggaagg ggctggagtg ggtctcagtt atttatagcg gtggtagcac atactacgca 180 Pro Gly Lys Gly Leu Glu Trp Val Ser Gln Phe Ile Ile Ser Gly Gly Ser Thr Thr Ala gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240 Asp Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Ala Val Leu caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agattcgaat 300 Gln Met Asn Ser Leu Ser Arg Glu Asp Thr Ala Val Tyr Tyr Cys Glu Arg Ile Arg Asn gcttttgata tctggggcca agggacaatg gtcaccgtct cttca 345 Ala Phe Asp Ile Trp Gly Gln Gly Asp Asn Gly Thr Val Ser Phe <210> 12 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 12 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Ser Asn Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 13 <211> 339 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 13 gaaattgtgc tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctagtca gagcctcctg tatagtaatg gatacaacta tttggattgg 120 tacctgcaga agccagggaa gtctccacag gtcctgatct atttgggttc taatcgggcc 180 tccggggtcc ccgacaggtt cagtggcagt ggatcaggca cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300 atcaccttcg gccaagggac acgactggag attaaacga 339 <210> 14 <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Lys Ser 35 40 45 Pro Gln Val Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 Arg <210> 15 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 15 Gly Phe Thr Val Ser Ser Asn Tyr 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 16 Val Ile Tyr Ser Gly Gly Ser Thr 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 17 Ala Arg Asp Ser Asn Ala Phe Asp Ile 1 5 <210> 18 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 18 Gln Ser Leu Leu Tyr Ser Asn Gly Tyr Asn Tyr 1 5 10 <210> 19 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 19 Leu Gly Ser 1 <210> 20 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 20 Met Gln Ala Leu Gln Thr Pro Ile Thr 1 5 <210> 21 <211> 366 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 21 gaggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg cttcaccatc tccagagaca attccaagaa cacgctgtct 240 ctgcaaatgg acagcctgag acccgaggac acggccgtat attactgtgc gaaaagtcga 300 gatagtggga actaccttga tgcttttgat ttctggggcc aagggacaat ggtcaccgtc 360 tcttca 366 <210> 22 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 22 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 23 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 23 gacatccagt tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaagca 120 gggaaagctc ctaggctcct gatctatgat gcctccactt tggaaagtgg agtcccatca 180 aggttcagcg gcactggatc tgggacatat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaacag tttaatagtt tcccgctcac tttcggcgga 300 gggaccaagg tggagatcaa acga 324 <210> 24 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 24 Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Ala Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Thr Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 25 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 26 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 27 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 27 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Phe Asp Phe 1 5 10 15 <210> 28 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 28 Gln Ser Ile Ser Ser Trp 1 5 <210> 29 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 29 Asp Ala Ser 1 <210> 30 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 30 Gln Gln Phe Asn Ser Phe Pro Leu Thr 1 5

Claims

1. (a) an antigen-binding domain that selectively binds to cancer cell-associated glypican 2 (GPC2); (b) flexible hinge region; (c) a transmembrane domain; and (d) Endodomain A chimeric antigen receptor comprising: the cancer cell-associated GPC2 is native GPC2 of 62 kDa or heparan sulfate-modified GPC2 of about 80 kDa; the antigen-binding domain comprises a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17, and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20; Chimeric antigen receptor.

2. The chimeric antigen receptor of claim 1, wherein the antigen-binding domain is a single chain antibody variable region, a single chain fragment variable (scFv), a fragment-antigen binding protein (Fab), a F(ab')2 fragment, or an Fv fragment.

3. The chimeric antigen receptor of claim 1 , wherein the antigen-binding domain is an scFv of an antibody that selectively binds to cancer cell-associated GPC2.

4. The chimeric antigen receptor of claim 3, wherein the scFv comprises the C-terminus of the light chain variable domain fused to the N-terminus of the heavy chain variable domain by a flexible linker.

5. the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 12 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 14; The chimeric antigen receptor of claim 1.

6. (a) the light chain variable domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 14; and (b) the heavy chain variable domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12; The chimeric antigen receptor of claim 1.

7. The chimeric antigen receptor of claim 4, wherein the linker is selected from the group consisting of a peptide linker, a non-peptide linker, and a chemical unit.

8. The chimeric antigen receptor of claim 4, wherein the linker is a peptide linker.

9. (a) the transmembrane domain comprises a CD28 domain; (b) the endodomain comprises a CD3ζ domain or a high affinity FcεRI; (c) the endodomain further comprises an intracellular domain of a costimulatory protein selected from the group consisting of CD28, 41BB, OX40, and ICOS; or (d) the transmembrane domain and the endodomain are derived from the same molecule; The chimeric antigen receptor according to any one of claims 1 to 8.

10. The chimeric antigen receptor of claim 1 , wherein the flexible hinge is derived from CD8α or immunoglobulin (Ig).

11. (a) a cancer cell-associated glypican 2 (GPC2) antigen-binding domain comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; (b) flexible hinge region; (c) a transmembrane domain; and (d) Endodomain A nucleic acid encoding a chimeric antigen receptor comprising: the antigen-binding domain comprises a heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17, and a light chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and the VH domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 11; or a nucleotide sequence having at least 70%, 80%, or 90% identity to SEQ ID NO: 11; and the VL domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 13; or a nucleotide sequence having at least 70%, 80%, or 90% identity to SEQ ID NO: 13; Nucleic acid.

12. The nucleic acid of claim 11, wherein the VH domain is encoded by a nucleotide sequence having at least 95% identity to SEQ ID NO: 11, and the VL domain is encoded by a nucleotide sequence having at least 95% identity to SEQ ID NO:

13.

13. 13. The nucleic acid of claim 11 or 12, wherein the C-terminus of the light chain variable domain is fused to the N-terminus of the heavy chain variable domain by a flexible linker.

14. The nucleic acid of claim 13 , wherein the flexible linker is selected from the group consisting of a peptide linker, a non-peptide linker, and a chemical unit.

15. The nucleic acid of claim 13, wherein the flexible linker is a peptide linker.

16. (a) the transmembrane domain comprises a CD28 domain; (b) the endodomain comprises a CD3ζ domain or a high affinity FcεRI; (c) the endodomain further comprises an intracellular domain of a costimulatory protein selected from the group consisting of CD28, 41BB, OX40, and ICOS; (d) the transmembrane domain and the endodomain are derived from the same molecule; or (e) the flexible hinge is derived from CD8α or immunoglobulin (Ig); A nucleic acid according to any one of claims 11 to 15.

17. (a) the cancer cell-associated GPC2 is native GPC2 or heparan sulfate-modified GPC2; and (b) the native GPC2 is 62 kDa and the heparan sulfate-modified GPC2 is about 80 kDa; A nucleic acid according to any one of claims 11 to 16.

18. A modified cell comprising the chimeric antigen receptor of any one of claims 1 to 9 or the nucleic acid of any one of claims 11 to 17.

19. 20. A pharmaceutical composition comprising the modified cell of claim 18 and a pharma- ceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, wherein the modified cell is a T cell or a NK cell.

21. 21. The pharmaceutical composition of claim 19 or 20 for providing anti-cancer immunity in a mammal, characterized in that an effective amount of the pharmaceutical composition is administered to the mammal.

22. 21. The pharmaceutical composition according to claim 19 or 20, for treating a mammal having a GPC2-positive cancer, characterized in that an effective amount of the pharmaceutical composition is administered to the mammal.

23. GPC2 positive cancer (a) solid tumor cancer; (b) a cancer selected from the group consisting of lung cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, liver cancer, pancreatic cancer, gastric cancer, colon cancer, kidney cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, or esophageal cancer; (c) embryonal carcinoma; and / or (d) Sarcoma, rhabdoid carcinoma, medulloblastoma, or neuroblastoma 23. The pharmaceutical composition of claim 22, wherein

24. Use of the pharmaceutical composition of any one of claims 19 to 20 in the manufacture of a medicament for providing anti-cancer immunity in a mammal.

25. Use of a pharmaceutical composition according to any one of claims 19 to 20 in the manufacture of a medicament for treating a mammal having a GPC2-positive cancer.

26. 26. The use according to claim 25, wherein the GPC2 positive cancer is a sarcoma, a rhabdoid carcinoma, a medulloblastoma or a neuroblastoma.

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