Glypican 2 as a 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 funding provided by the federal government This invention was made with government support under grant numbers Genetics T32 T32GM008638 and ACC T32 32CA009615 granted by the U.S. National Institutes of Health. The U.S. Government has certain rights in this invention.

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

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

[0004] 2. Related Technologies Children with high-risk neuroblastoma have a poor prognosis even after receiving intensive multimodal chemoradiotherapy. While monoclonal antibodies targeting disiaroganglioside GD2 improve outcomes in neuroblastoma, this therapy is associated with considerable "on target-off tumor" toxicity. Therefore, there remains a significant challenge in identifying novel cell surface molecules that meet the stringent criteria for modern immunotherapies, including that tumor expression is unique compared to normal pediatric tissue and, preferably, that novel cell surface molecules are required for tumor maintenance. [Overview of the Initiative]

[0005] overview Accordingly, the present disclosure provides a method for treating cancer, comprising the step of contacting glypican 2-positive cancer cells in a subject with an antibody or 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 also 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 also be pediatric cancer cells.

[0006] The method may further include the step of contacting the glypican 2-positive cancer cells with a second anticancer agent or treatment. The second anticancer 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 anticancer agent or treatment may be administered simultaneously with the first agent, or before and / or after the first agent. The glypican 2-positive cancer cells may be metastatic cancer cells, multidrug-resistant cancer cells, or recurrent cancer cells. The antibody may be a single-chain antibody, a single-domain antibody, a chimeric antibody, or a Fab fragment. The antibody may be a recombinant antibody specific to glypican 2 and different cancer cell surface antigens. The antibody may be a mouse antibody, for example, IgG. The antibody may be a humanized antibody such as IgG or a fully human antibody.

[0007] Furthermore, the antibody may further contain an antitumor drug linked thereto. The antitumor drug may be linked to the antibody via a photosensitive linker. The antitumor drug may be linked to the antibody via a linker that is cleaved by an enzyme. The antitumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme. The antibody may further contain a label, such as a peptide tag, an enzyme, a magnetic particle, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. The antibody may be conjugated 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] A fusion protein is also provided, comprising (i) a first single-chain antibody that selectively binds to glypican 2, (a) being 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, or to T cells, or to B cells. The fusion protein may further include a labeled or therapeutic moiety. The first single-chain antibody may be characterized by a CDR sequence SEQ ID NO: 5-10, 15-20, or 25-30. Yet another embodiment includes cells expressing the fusion protein defined above.

[0009] Another embodiment includes a chimeric antigen receptor comprising: (i) an ectodomain containing 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, and (c) induces cancer cell death, and having a movable hinge attached to the C-terminus of the single-chain antibody variable region; (ii) a transmembrane domain; and (iii) an endodomain, wherein the endodomain has a signaling function when the single-chain antibody variable region binds to glypican 2. The transmembrane domain and the endodomain may originate from the same molecule. The endodomain may contain a CD3ζ domain or a high-affinity FcεRI. The movable hinge may originate from CD8α or Ig. The single-chain GPC2 antibody may be characterized by a CDR sequence SEQ ID NO: 5-10, 15-20, or 25-30. Yet another embodiment includes cells expressing the chimeric antigen receptor as defined above.

[0010] In yet another embodiment, a monoclonal antibody is provided, wherein the antibody or antibody fragment is characterized by CDR sequence 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, the heavy chain variable sequence and the light chain variable sequence being 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, the heavy chain variable sequence and the light chain variable sequence having at least 70%, 80%, or 90% identity with 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, each having 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, each containing SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. Alternatively, the antibody or antibody fragment may include a light chain variable sequence and a heavy chain variable sequence, each having 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. Alternatively, the antibody is a bispecific antibody. The monoclonal antibody may be IgG. The antibody or antibody fragment may further include a label. Pharmaceutical compositions are also provided in which the aforementioned antibody is dispersed in a pharmaceutically acceptable buffer, medium, or diluent, or lyophilized.

[0011] In yet another embodiment, hybridomas or engineered cells are provided that encode 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, the heavy chain variable sequence and the light chain variable sequence being 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, the heavy chain variable sequence and the light chain variable sequence having at least 70%, 80%, or 90% identity with 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, the heavy chain variable sequence and the light chain variable sequence having 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, the heavy chain variable sequence and the light chain variable sequence including SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. Alternatively, the antibody or antibody fragment may include a light chain variable sequence and a heavy chain variable sequence, the light chain variable sequence and the heavy chain variable sequence having 95% identity to SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. Hybridomas or manipulated cells may produce antibody fragments that are recombinant ScFv (single-chain fragment variable) antibodies, Fab fragments, F(ab')2 fragments, or Fv fragments; they may also produce antibodies that are chimeric or bispecific antibodies; or they may produce antibodies that are IgG.

[0012] Any method or composition described herein is intended to be applicable to 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] [The present invention 1001] A method of treating cancer comprising the step of contacting glypican-2 positive cancer cells in a subject with an antibody or antibody derivative that selectively binds to glypican-2. [The present invention 1002] The method of the present invention 1001, wherein the glypican-2 positive cancer cells are solid tumor cancer cells. [The present invention 1003] The method of the present 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. [The present invention 1004] The method of the present invention 1001, wherein the glypican-2 positive cancer cells are embryonic cancer cells. [The present invention 1005] The method of the present invention 1001, wherein the cancer cells are sarcoma cells, neuroblastoma cells, rhabdoid cancer cells, medulloblastoma cells or neuroblastoma cells. [The present invention 1006] The method according to any one of the present inventions 1001 to 1005, further comprising the step of contacting the glypican-2 positive cancer cells with a second anti-cancer agent or treatment. [The present invention 1007] The method of the present invention 1006, wherein the second anti-cancer agent or treatment is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, or toxin therapy. [The present invention 1008] The method of the present invention 1006, wherein the glypican-2 antibody is administered before the second agent or treatment. [The present invention 1009] The method of the present invention 1006, wherein a second anticancer agent or treatment is administered simultaneously with the first agent. [Invention 1010] The method of the present invention 1006, wherein a second anticancer agent or treatment is given before and / or after a first agent. [Invention 1011] The method according to any one of items 1001 to 1010 of the present invention, wherein the glypican 2-positive cancer cells are metastatic cancer cells, multidrug-resistant cancer cells, or recurrent cancer cells. [Invention 1012] A method according to any of the present invention 1001 to 1011, wherein the antibody is a single-chain antibody. [Invention 1013] A method according to any of the present invention 1001 to 1011, wherein the antibody is a single-domain antibody. [Invention 1014] A method according to any of the present invention 1001 to 1011, wherein the antibody is a chimeric antibody. [Invention 1015] Any method 1001 to 1011 of the present invention, wherein the antibody derivative is a Fab fragment. [Invention 1016] A method according to any one of the present invention 1011 to 1015, wherein the antibody is a recombinant antibody that has specificity for glypican 2 and different cancer cell surface antigens. [Invention 1017] The method according to any of items 1001 to 1016 of the present invention, wherein the antibody is a mouse antibody such as IgG. [Invention 1018] Any method according to invention 1001 to 1016, wherein the antibody is a human antibody. [Invention 1019] A method according to any of the present invention 1001 to 1015, wherein the antibody is a humanized antibody. [Invention 1020] Any method according to invention 1018 to 1019, wherein the humanized antibody is IgG. [Invention 1021] A method according to any one of the present invention 1001 to 1020, wherein the antibody further comprises an antitumor drug linked to the antibody. [Invention 1022] The method of the present invention 1021, wherein an antitumor drug is linked to an antibody via a photosensitive linker. [Invention 1023] The method of the present invention 1021, wherein an antitumor drug is linked to an antibody via a linker that is cleaved by an enzyme. [Invention 1024] The method of the present invention 1021, wherein the antitumor drug is a toxin, radioisotope, cytokine, or enzyme. [Invention 1025] Any method of the present invention 1001 to 1024, further comprising labeling the antibody. [Invention 1026] The method of the present invention 1025, wherein the label is a peptide tag, enzyme, magnetic particle, chromophore, fluorescent molecule, chemiluminescent molecule, or dye. [Invention 1027] A method according to any one of the present invention 1001 to 1026, wherein the antibody is conjugated to liposomes or nanoparticles. [Invention 1028] A method according to any of items 1001 to 1027 of the present invention, wherein an antibody induces cell death, for example, by antibody-dependent cytotoxicity or complement-mediated cytotoxicity. [Invention 1029] The method of the present invention 1001, wherein the antibody derivative is a chimeric antigen receptor or a bispecific antibody. [Invention 1030] The method of the present invention 1001, wherein the antibody or antibody fragment is characterized by CDR sequence SEQ ID NO: 5-10, 15-20, or 25-30. [Invention 1031] (i) A first single-chain antibody that selectively binds to glypican 2, (a) It is an IgG antibody, (b) Inhibits cancer cell proliferation, (c) Induces cancer cell death The first single-chain antibody, (ii) A second single-chain antibody that binds to T cells or B cells A fusion protein containing [the specified ingredient]. [Invention 1032] A fusion protein according to the present invention 1031, wherein the first antibody or antibody fragment is characterized by CDR sequence SEQ ID NO: 5-10, 15-20, or 25-30. [Invention 1033] The fusion protein of the present invention 1031, wherein a second single-chain antibody binds to T cells, for example, via CD3. [Invention 1034] A fusion protein according to the present invention 1031, in which a second single-chain antibody binds to B cells. [Invention 1035] A fusion protein of the present invention 1031, further comprising a labeled or therapeutic moiety. [Invention 1036] (i) an ectodomain comprising a single-chain antibody variable region that selectively binds to glypican 2, wherein the antibody (a) It is an IgG antibody, (b) Inhibits cancer cell proliferation, (c) Induce cancer cell death, Having a movable hinge attached to the C-terminus of the variable region of the single-chain antibody, Ectodomain and, (ii) Transmembrane domain and (iii) End domain and A chimeric antigen receptor comprising a single-chain antibody variable region wherein the endodomain has a signaling function when the single-chain antibody variable region is bound to glypican 2. [Invention 1037] The receptor of the present invention 1036, wherein the first single-chain antibody is characterized by a CDR sequence SEQ ID NO: 5-10, 15-20, or 25-30. [Invention 1038] The receptor of the present invention 1036, wherein the transmembrane domain and endodomain are derived from the same molecule. [Invention 1039] The receptor of the present invention 1036, wherein the endodomain contains a CD3ζ domain or a high-affinity FcεRI. [Invention 1040] The receptor of the present invention 1036, wherein the movable hinge is derived from CD8α or Ig. [Invention 1041] A cell expressing any of the chimeric antigen receptors described in items 1036 to 1040 of the present invention. [Invention 1042] Cells of the present invention 1041, wherein the endodomain contains a CD3ζ domain or a high-affinity FcεRI. [Invention 1043] Cells of the present invention 1041, wherein the movable hinge is derived from CD8α or Ig. [Invention 1044] A cell expressing the fusion protein of the present invention 1031. [Invention 1045] Cells of the present invention 1044, wherein a second single-chain antibody binds to CD3, T cells, or B cells. [Invention 1046] A monoclonal antibody or antibody fragment characterized by a CDR sequence SEQ ID NO: 5-10, 15-20, or 25-30. [Invention 1047] A monoclonal antibody according to the present invention 1046, wherein the antibody or antibody fragment is encoded by heavy chain variable sequences and light chain variable sequences, respectively, whose SEQ ID NOs are 1 and 3, 11 and 13, and 21 and 23. [Invention 1048] A monoclonal antibody according to the present invention 1046, wherein the antibody or antibody fragment is encoded by a heavy chain variable sequence and a light chain variable sequence having at least 70%, 80%, or 90% identity with SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. [Invention 1049] A monoclonal antibody according to the present invention 1046, wherein the antibody or antibody fragment is encoded by a heavy chain variable sequence and a light chain variable sequence having at least 95% identity to SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. [Invention 1050] A monoclonal antibody according to Invention 1046, wherein the antibody or antibody fragment comprises a heavy chain variable sequence and a light chain variable sequence, respectively, including SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24. [Invention 1051] A monoclonal antibody according to 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 NO: 2 and 4, 12 and 14, and 22 and 24, respectively. [Invention 1052] A monoclonal antibody according to any of the invention 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. [Invention 1053] A monoclonal antibody according to any of the present invention 1046 to 1051, which is a chimeric antibody or a bispecific antibody. [Invention 1054] A monoclonal antibody according to any of the Invention 1046-1053, which is IgG and / or conjugated to a label and / or conjugated to a therapeutic agent. [Invention 1055] A monoclonal antibody according to any one of the present invention 1046 to 1054, wherein the antibody or antibody fragment further comprises a label. [Invention 1056] Hybridomas or manipulated cells encoding antibodies or antibody fragments characterized by CDR sequence SEQ ID NO: 5-10, 15-20, or 25-30. [Invention 1057] Hybridomas or manipulated cells according to Invention 1056, wherein the antibody or antibody fragment is encoded by light chain variable sequences and heavy chain variable sequences, respectively, whose SEQ ID NOs are 1 and 3, 11 and 13, and 21 and 23. [Invention 1058] Hybridomas or manipulated cells according to 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 NO: 1 and 3, 11 and 13, and 21 and 23, respectively. [Invention 1059] Hybridomas or manipulated cells according to Invention 1056, wherein the antibody or antibody fragment is encoded by a light chain variable sequence and a heavy chain variable sequence having 95% identity to SEQ ID NO: 1 and 3, 11 and 13, and 21 and 23, respectively. [Invention 1060] Hybridomas or engineered cells according to Invention 1056, wherein the antibody or antibody fragment comprises light chain variable sequences and heavy chain variable sequences, respectively, including SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24. [Invention 1061] Hybridomas or manipulated cells according to 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 NO: 2 and 4, 12 and 14, and 22 and 24, respectively. [Invention 1062] Hybridomas or manipulated cells according to Invention 1056, wherein the antibody or antibody fragment comprises a light chain variable sequence and a heavy chain variable sequence having 95% identity to SEQ ID NO: 2 and 4, 12 and 14, and 22 and 24, respectively. [Invention 1063] Hybridomas or engineered cells according to any of the inventions 1056 to 1062, 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. [Invention 1064] Hybridomas or engineered cells according to any of invention 1056 to 1063, wherein the antibody is a chimeric antibody or a bispecific antibody. [Invention 1065] A hybridoma or engineered cell according to any of invention 1056 to 1063, wherein the antibody is IgG. [Invention 1066] A pharmaceutical composition comprising any monoclonal antibody according to invention 1046 to 1055. Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, while the detailed description and specific examples illustrate specific aspects of this disclosure, it should be understood that they are merely illustrative, as various modifications and changes within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0015] The following drawings are part of this specification and are included to further illustrate certain aspects of the disclosure. A deeper understanding of the disclosure can be gained by referring to one or more of these drawings in conjunction with the detailed description of the particular aspects presented herein. [Figure 1] Identifying drivers of GPC2 expression in neuroblastoma. A prioritization pipeline to identify highly expressed and differentiated putative cell surface genes in high-risk neuroblastoma. [Figure 2] GPC2 is expressed differently in neuroblastoma versus normal tissue. The plot shows prioritized candidate GPC2 expression in high-risk neuroblastoma (n=126; TARGET, ocg.cancer.gov / programs / target) and normal tissue RNA sequencing data profiled by the GTEx Consortium (n=7859 samples across 31 distinct normal tissues; n=5 to 1152 samples per tissue; GTEx, gtexportal.org). [Figure 3] GPC2 is a putative cell surface heparan sulfate proteoglycan. GPC2 is a heparan sulfate proteoglycan signaling coreceptor with putative glycosylphosphatidylinositol (GPI) linkage to the extracellular cell surface. Glypicans are endogenous promoters of several proliferative signaling pathways and play important roles in cancer cell migration, invasion, and metastasis. GPC2 is known to bind to midkine and sonic hedgehog. GAG = glycosaminoglycan. [Figure 4]Figures 4A-H: GPC2 is a cell surface molecule expressed in most neuroblastomas. (Figures 4A-D) GPC2 exists in both its native form (62 kDa) and a heparan sulfate-modified form (approximately 80 kDa). GPC2 Western blots show expression in primary neuroblastoma tumors (Figure 4A), patient-derived xenografts (PDX; Figure 4B), and cell lines (Figure 4C). GPC2 is associated with the plasma membrane, as shown by Western blotting after membrane extraction in cell lines (Figure 4D), by IHC in primary tumors, PDX (Figure 4E), and cell lines (Figure 4F), and by immunofluorescence in cell lines (Figure 4G). (Figure 4H) Medulloblastoma also expresses high levels of GPC2 by IHC. IR = intermediate risk, HR = high risk, S = soluble fraction, M = membrane fraction, Na / K = sodium / potassium transporter = membrane control, TMA = tumor microarray. [Figure 5] GPC2 coexists with known neuroblastoma extracellular surface proteins. GPC2 coexists with cadherins, which are known neuroblastoma cell surface proteins. [Figure 6] GPC2 is significantly expressed in medulloblastoma. In over 50% of pediatric medulloblastomas, GPC2 is stained positively by IHC, suggesting high levels of expression in other pediatric cancers. [Figure 7] GPC2 is significantly expressed in other pediatric neoplasms. As shown, high levels of GPC2 mRNA are found in several pediatric cancers. [Figure 8A] Figures 8A-B: GPC2 expression in normal tissues is restricted, and differential GPC2 exon expression is observed between neuroblastoma and normal tissues. From TMAs of 37 types of normal pediatric tissues, IHC shows that GPC2 expression is low in normal pediatric tissues. The pediatric esophagus is the only tissue with significant plasma membrane-related GPC2 expression. 2 = weak IHC staining, 3 = strong IHC staining. Sashimi plots from five pediatric esophageal specimens and five representative primary neuroblastomas show differential GPC2 exon 3 expression. [Figure 8B] See the explanation in Figure 8A. [Figure 9]Figures 9A-F: GPC2 depletion induces apoptosis in neuroblastoma cells. (Figures 9A-E) RNAi-mediated GPC2 (GPC2-2, GPC2-4) knockdown induces apoptosis and reduces neuroblastoma cell proliferation. Apoptosis induction was indicated by elevated cleavage PARP or cleavage caspase-3 by Western blotting (Figures 9A, 9D), or by elevated caspase 3 / 7 by luminescence assay (Figure 9B). An example of a representative neuroblastoma cell line from the cell line panel, Nb-ebc1 (n=12), is shown in Figures 9A-C, and further examples are shown in Figures 9D-E. (Figure 9F) GPC2 overexpression in the neuroblastoma cell line Kelly (with heterozygous deletion at the GPC2 locus) induces increased cell proliferation. NTC = non-targeted control shRNA, *p<0.001, **p<0.0001. [Figure 10A] Figure 10A-B: Advances in GPC2 CAR manipulation: Development of binding agents. Multiple fragment-antigen binding (Fab) proteins that specifically bind to GPC2 on neuroblastoma cells have been identified. [Figure 10B] See the explanation in Figure 10A. [Figure 11] Differential GPC2 mRNA variant expression exists in neuroblastoma versus normal tissue. Not only is GPC2 expression generally low in normal tissue, but differential GPC2 mRNA variant (and therefore) epitope expression is also present. The testes are the only normal tissue that primarily expresses the same GPC2 variant as that expressed in neuroblastoma. Therefore, targeting the N-terminal GPC2 epitope with immunotherapy may yield a higher therapeutic index compared to targeting a GPC2 epitope common to both neuroblastoma and normal tissue. [Figure 12] GPC2 depletion induces apoptosis in most neuroblastomas. The GPC2 loss-of-function in vitro assay was expanded to a panel of 12 neuroblastoma cell lines. This revealed that GPC2 broadly promotes cell proliferation in neuroblastoma cells. [Figure 13]Overexpression of GPC2 induces increased proliferation of neuroblastoma cells. Forced overexpression of GPC2 in low-GPC2 neuroblastoma cell lines (Kelly; with a heterozygous deletion at the GPC2 locus) induces significantly faster proliferation in these cells compared to cells transfected with an empty vector. These results further support the crucial role that GPC2 plays in neuroblastoma cell proliferation. [Figure 14] Integrated RNA sequencing-based screening identified GPC2 as a distinctly expressed cell surface molecule and putative immunotherapy target in high-risk neuroblastoma. The plot shows that 296 distinctly expressed genes were identified in high-risk neuroblastoma. [Figure 15]Figures 15A-E: Identification of drivers of GPC2 expression in neuroblastoma. (Figure 15A) GPC2 expression in Huex (left) and NRC (right) neuroblastoma tumors, stratified by chromosome 7q / GPC2 locus enlargement and MYCN amplification. The neuroblastoma dataset in A was obtained from the TARGET consortium (n=seq and mRNA array). P values ​​were derived by independent t-tests using GraphPad Prism version 5.01. (Figure 15B) From the MYCN ChIP plot, it can be seen that MYCN binds to the Ebox motif upstream of the GPC2 promoter in MYCN-amplified neuroblastoma cell lines Kelly and NGP. Arrows indicate Ebox (CACGTG motif). (Figure 15C) GPC2 reporter assay with and without MYCN overexpression in SHEP neuroblastoma and 293T cells. An inset of a Western blot shows MYCN overexpression in SHEP and 293T cells. (Figure 15D) Quantitative PCR of MYCN / GPC2 in MYCN-amplified neuroblastoma cell line Kelly after MYCN depletion using two specific shRNAs. (Figure 15E) Western blot of MYCN and GPC2 in MYCN-amplified neuroblastoma cell lines Kelly and NGP after MYCN depletion using an expanded set of four specific shRNAs. shNTC and non-targeted shRNA controls. See also Figures 24A-E. [Figure 16]Figures 16A-I: GPC2 is expressed in most neuroblastomas and is localized to the plasma membrane. (Figures 16A-C) Western blots of GPC2 in panels of primary neuroblastoma tumors (n=11; Figure 16A), PDX (n=12; Figure 16B), and cell lines (n=24; Figure 16C). See also Figure 25A. (Figure 16D) GPC2 IHC staining of neuroblastoma cell lines (high GPC2 expression - SMS-SAN, moderate - NBLS, and low - RPE1). See also Figure 25B. (Figure 16E) GPC2 flow cytometry analysis of neuroblastoma cell lines with diverse GPC2 expression. (Figure 16F) Western blots of GPC2 after differential membrane extraction in a panel of neuroblastoma cell lines (n=7). NaK indicates a positive control of plasma membrane proteins in Western blots. (Figure 16G) GPC2 immunofluorescence staining in neuroblastoma cell lines NB-Ebc1 and SMS-SAN. (Figure 16H) Overview of membrane staining H scores for GPC2 IHC in PDX and primary tumor TMA (n=32 and n=98 tumors, respectively). (Figure 16I) Examples of representative membrane staining H scores from PDX and primary tumor TMA (H score shown in the lower right corner). Scale bars indicate 30 μM (Figure 16D), 10 μM (Figure 16G), and 60 μM (Figure 16I). HR: High risk; IR: Moderate risk; S: Soluble (non-membrane) protein extract; M: Membrane protein extract. See also Table S1 for identification of PDX (Figure 16B) and cell lines (Figures 16C and 16F). [Figure 17]Figures 17A-B: GPC2 expression is restricted in normal tissues. (Figure 17A) Overview of membrane H scores obtained from GPC2 IHC staining of a large pediatric normal tissue array (n=36 unique normal tissues). For comparison, GPC2 IHC stained membrane H scores from primary neuroblastoma tumors and PDX are shown from Figures 3H and 3I. P values ​​were derived by independent t-tests using GraphPad Prism version 5.01. (Figure 17B) mRNA transcript-specific analysis of GPC2 expression in primary neuroblastoma and normal tissues expressing low levels of GPC2, namely skin and esophagus (n=126 HR neuroblastoma, TARGET; n=201 esophageal samples; and n=684 skin samples, GTEx). See also Figures 26A-C and 27A-C and Table S2. [Figure 18]Figures 18A-I: GPC2 is required for the proliferation of neuroblastoma cells. (Figures 18A-B, top) Quantitative PCR and Western blot analysis of GPC2 after lentiviral transduction of two specific shRNA constructs targeting GPC2 exon 4 and GPC2 3'UTR in neuroblastoma cell line NB-EBC1. (Figure 18B, bottom) Western blot of cleaved PARP and caspase 3 after GPC2 depletion in NB-EBC1. The positive control of the GPC2 Western blot shown in B was moved on the same blot as NB-EBC1 at 120 hours and 168 hours. (Figure 18C) Caspase 3 / 7 activity measured after GPC2 depletion in NB-EBC1. (Figures 18D-F) ShRNA-induced NB-EBC1 cell proliferation after GPC2 depletion is shown by (Figure 18D) CellTiter-Glo luminescence assay, (Figure 18E) RT-CES, and (Figure 18F) colony formation assay. (Figure 18G) Plots of cell proliferation measured by CellTiter-Glo luminescence assay and caspase 3 / 7 elevation after GPC2 depletion using two unique shRNA constructs targeting GPC2 exon 4 (Figure 18G, top) and GPC2 3'UTR (Figure 18G, bottom) across an enlarged panel of neuroblastoma cell lines (n=11). r, Pearson correlation coefficient, and p-values ​​by Student's t-test are shown for each GPC2 shRNA. (Figure 18H) Neuroblastoma cell proliferation after forced GPC2 overexpression in Kelly. Figure 18I) SKNDZ (right). Kelly has a heterozygous deletion on chromosome arm 7q containing the GPC2 locus. Student's t-test calculated by GraphPadPrism version 5.01: *=p<0.0001, **=p<0.001. NTC, non-target shRNA control. Empty, empty pLenti CMV puro vector control. [Figure 19]Figures 19A-E: GPC2 is expressed in other high-risk childhood cancers. (Figure 19A) Further GPC2 RNA sequencing data of medulloblastoma tumors (n=91), stratified by clinical grouping and amplification status at chromosome 7q / GPC2 locus and MYC and MYCN loci. (Figure 19B) Overview of membrane H scores from GPC2 IHC staining of medulloblastoma TMA (n=63). (Figure 19C) Examples of representative membrane staining H scores from medulloblastoma TMA (H scores shown in the lower right). (Figures 19D and 19E) GPC2 IHC staining in a human metastatic medulloblastoma xenograft mouse model with GPC2, including evaluation of central nervous system metastases (Figures 19D and 19E), spinal cord metastases (Figure 19D), and liver metastases (Figure 19D). The scale bars in Figure 19C, Figure 19D (top and right), and Figure 19E (right) represent 60 μM, the scale bar in Figure 19D (left) represents 5 μM, the scale bar in Figure 19E (left) represents 4 μM, the scale bar in Figure 19D (top center; spinal cord) represents 500 μM, and the scale bar in Figure 19D (bottom center; liver) represents 300 μM. (Figure 19F) mRNA transcript-specific analysis of GPC2 expression in primary medulloblastoma (n=91). See also Figures 29A-C and Table S3. [Figure 20] D3-GPC2-PBD, an ADC targeting GPC2, is cytotoxic to neuroblastoma cells expressing GPC2. IC50 curve. [Figure 21] GPC2 is the only glypican that is expressed differently between neuroblastoma and normal tissue. The plot shows GPC1-6 FPKM in high-risk neuroblastoma (n=126; TARGET, ocg.cancer.gov / programs / target) and paired normal tissue GPC1-6 FPKM (n=7859 samples across 31 distinct normal tissues; n=5–1152 samples per tissue; GTEx, gtexportal.org) from RNA sequencing data profiled by the GTEx Consortium. See also Figures 1–2 and 14. [Figure 22]Figures 22A-C: GPC2 is the most dominant glypican expressed in neuroblastoma, and GPC2 expression is inversely correlated with GPC3 expression and the content of neuroblastoma tumor stromal cells and immune cells. (Figure 22A) GPC1-6 FPKMs were plotted from primary neuroblastoma tumors (left, n=126 high-risk tumors, TARGET; right, n=498 tumors across all risk groups, seqC). (Figure 22B) GPC2 FPKMs were plotted against GPC3 FPKMs (left, n=126, TARGET; right, n=498, seqC). r, Pearson correlation coefficient, and p-value are shown for each dataset. (Figure 22C) GPC2 FPKMs were plotted against the content of stromal cells and immune cells (left, n=126, TARGET; right, n=498, seqC). The r, Pearson correlation coefficient, and p-value are shown for each dataset. [Figure 23] Figures 23A-C: High GPC2 expression is associated with poor overall survival in neuroblastoma. (Figures 23A-C, left) Overall survival curves of three neuroblastoma datasets analyzed by the Genomics Analysis and Visualization Platform (R2; r2.amc.nl; (Figure 23A) Kocak; n=649, (Figure 23B) seqC; n=498, and (Figure 23C) Versteeg; n=88). 58-60 (Figures 23A-C, right) Overall survival curves of the same three neuroblastoma datasets, limited to patients with tumors without MYCN amplification. [Figure 24] Figures 24A-C: MYCN does not significantly bind to GPC3-6. (Figures 24A-C) MYCN ChIP sequencing from MYCN-amplified neuroblastoma cell lines NGP and Kelly, targeting GPC1 (Figure 24A), GPC3 and GPC4 (Figure 24B), and GPC5 and GPC6 (Figure 24C). See also Figures 15A-E. [Figure 25]Figures 25A-C: GPC2 is localized on the cell surface in neuroblastoma. (Figure 25A) Western blot targeting GPC2 using GPC2 monoclonal mouse antibody (sc-393824) for antibody validation. CHP134, NBSD, and SMS-SAN are three representative wild-type neuroblastoma cell lines that highly express GPC2. To complete antibody validation, cells transduced by lentiviral shRNA (NBSD and SMS-SAN) and cells overexpressing GPC2 plenti puro CMV (Kelly) are also shown. (Figure 25B) GPC2 IHC (n=8) from a panel of neuroblastoma cell lines. (Figure 25C) GPC2 immunofluorescence studies from neuroblastoma cell lines NBEBC1 and SMS-SAN. Ex4 and UTR were shRNA constructs targeting GPC2 exon 4 and GPC2 3'UTR, respectively. NTC, non-targeted shRNA control. Empty, empty pLenti CMV puro vector control. Scale bars: 60 μM (B), 10 μM (Figure 25C). See also Figures 16A-I. [Figure 26] Figures 26A-C: IHC restricts normal tissue expression of GPC2. (Figures 26A and 26B) Representative GPC2 IHC in the esophagus (Figure 26A) and skin (Figure 26B). Membrane staining H score is shown. (Figure 26C) Representative GPC2 IHC from major human organs. Scale bar, 60 μM. [Figure 27] Figures 27A-C: High-resolution mass spectrometry shows that GPC2 expression in normal tissues is restricted. (Figures 27A-C) Spectral counts of GPC2 (Figure 27A), L1CAM (Figure 27B), and CD19 (Figure 27C) are shown across the entire normal tissue panel (n=30). [Figure 28]Figures 28A-J: GPC2 is required for cell proliferation in most neuroblastomas. (Figures 28A-G) Lentiviral shRNA-induced GPC2 depletion in a cell line panel (n=10) using two specific shRNA constructs targeting GPC2 exon 4 (Ex4) and GPC2 3'UTR (UTR). Each panel shows GPC2 Wb (left), RT-CES cell proliferation plot (upper right), and colony formation assay (lower right; not available for NLF, E) using the indicated shRNA. (Figures 28H-J) For neuroblastoma cell lines that did not simply proliferate as a monolayer, preventing the use of the RT-CES proliferation assay, only the colony formation assay was performed. Colony formation assays are shown on the left, and GPC2 Western blots are shown on the right. *=p<0.0001, **=p<0.001, ***=p<0.01. NTC, non-targeted control shRNA. See also Figures 18A-I. [Figure 29]Figures 29A-C: GPC2 expression profiling across other pediatric neoplasms identified high GPC2 levels in medulloblastoma and retinoblastoma. (Figure 29A) GPC2 RNA sequencing data across the entire pediatric neoplasm array, including data from the Therapeutically Applicable Research to Generate Effective Treatments project (TARGET; ocg.cancer.gov / programs / target) and the St. Jude Children's Research Hospital Pediatric Cancer Data Portal (PeCan; pecan.stjude.org) (total n=1608, individual n shown on the x-axis of the figure). *RNA sequencing data from the TARGET project is shown. **Normal tissues from the GTEx portal are included for comparison (mean FPMK for each tissue is shown. Total n=7859 samples across 31 unique normal tissues. n=5-1152 samples per tissue; GTEx, gtexportal.org). (Figure 29B) Confirmatory GPC2 mRNA array data for neuroblastoma, medulloblastoma, and retinoblastoma from the Genomics Analysis and Visualization Platform (R2; http: / / r2.amc.nl; individual n values ​​are shown on the x-axis of the figure). (Figure 29C) log2 GPC2 expression from paired human primary and metastatic medulloblastoma samples. p-values ​​were derived by independent t-tests. NB, neuroblastoma; RB, retinoblastoma; MB, medulloblastoma; ALL, acute lymphoblastic leukemia; HGG, high-grade glioma; RHB, rhabdomyosarcoma; MLL, mixed leukemia; CPC, choroid plexus carcinoma; WT, Wilms' tumor; LGG, low-grade glioma; AML, acute myeloid leukemia; OS, osteosarcoma; EPD, ependymoma; MEL, melanoma; ACT, adrenocortical carcinoma; malignant rhabdoid tumor; normal, normal tissue. Please also refer to Figures 19A-E. [Figure 30] Amino acid and nucleotide sequences of the heavy and light chains of the human antibody m201. CDRs are shown in bold italics. [Figure 31] Amino acid and nucleotide sequences of the heavy and light chains of the human antibody m202. CDRs are shown in bold italics. [Figure 32] Amino acid and nucleotide sequences of the heavy and light chains of the human antibody m203. CDRs are shown in bold italics. [Modes for carrying out the invention]

[0016] Exemplary Description The inventors have confirmed that glypican 2 (GPC2) is a candidate and putative oncogene for cell surface immunotherapy in high-risk neuroblastoma and possibly other childhood cancers. More comprehensively, the data presented herein demonstrate that genome-wide transcriptome analysis, integrated with genomic and functional validation, can identify distinctly expressed cell surface oncogenes that could be attractive immunotherapy targets. These and other aspects of this disclosure are described in further detail below.

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

[0018] II. Production of Monoclonal Antibodies A. General methods Antibodies against glypican 2 can be prepared by standard methods well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent No. 4,196,265). Methods for preparing monoclonal antibodies (MAbs) generally begin in the same manner as those for preparing polyclonal antibodies. The first step in both of these methods is to immunize a suitable host or to identify a target that is immune from a previous natural infection. As is well known in the art, certain immunizing compositions may differ in terms of immunogenicity. Therefore, it is often necessary to further immunize 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. Methods for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. As is also well known in the art, the immunogenicity of certain immunogen compositions can be enhanced by using nonspecific stimulants of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a nonspecific stimulant of the immune response containing inactivated Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

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

[0020] Following 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. Myeloma cell lines suitable for use in the hybridoma production fusion procedure are preferably antibody-free, exhibit high fusion efficiency, and possess enzyme deficiencies that prevent growth in certain selective media, supporting only the proliferation of the desired fusion cells (hybridoms).

[0021] As is well known to those skilled in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984), any of the many 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. For human cell fusion, U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6 are all useful. One specific mouse myeloma cell line is the NS-1 myeloma cell line (also known as P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse myeloma SP2 / 0 non-producing cell line. More recently, further fusion partner lines 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 this disclosure were prepared using the SP2 / 0 / mIL-6 cell line, an IL-6 secreting derivative of the SP2 / 0 cell line.

[0022] Methods for creating hybrids of antibody-producing spleen or lymph node cells and myeloma cells typically involve mixing somatic cells with myeloma cells in a 2:1 ratio, although this ratio may range from approximately 20:1 to 1:1 in the presence of a cell membrane fusion-promoting agent (chemical or electrical). Fusion methods using Sendai virus were described by Kohler and Milstein (1975; 1976), and fusion methods using polyethylene glycol (PEG), such as 37% (v / v) PEG, were described by Gefter et al (1977). The use of electrically induced fusion methods is also suitable (Goding, pp. 71-74, 1986).

[0023] The fusion procedure typically results in a low frequency of approximately 1 x 10⁻⁶ -6 ~1x10 -8 This results in viable hybrids. However, this does not pose a problem, as culturing in selective media allows viable fusion hybrids to differentiate from the injected parent cells (particularly the injected myeloma cells, which typically continue to divide indefinitely). Selective media are generally media containing agents that block the denomination of nucleotides in tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserin. Aminopterin and methotrexate block the denomination of both purine and pyrimidine synthesis, while azaserin blocks only purine synthesis. When aminopterin or methotrexate is used, hypoxanthine and thymidine are added to the medium as sources of nucleotides (HAT medium). When azaserin 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 transformants that have not fused with myeloma.

[0024] The preferred selective medium is HAT or HAT containing ouabain. In HAT medium, only cells capable of activating the nucleotide salvage pathway can survive. Myeloma cells are deficient in key enzymes of the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and therefore cannot survive. B cells can activate this pathway, but their lifespan in culture is limited and they generally die within about two weeks. Therefore, the only cells that can survive in selective medium are hybrids formed from myeloma cells and B cells. If the source of B cells used for fusion is an EBV-transformed B cell line, as described herein, ouabain is also used for drug selection of the hybrid because EBV-transformed B cells are susceptible to drug-induced death. Conversely, the myeloma partner used is selected for its resistance to ouabain.

[0025] Culturing yields a hybridoma population, from which specific hybridomas are selected. Typically, hybridoma selection is performed by culturing cells in single-clonal dilutions in microtiter plates, followed by testing the individual clonal supernatants (after approximately 2-3 weeks) for desired reactivity. This assay must be highly sensitive, simple, and rapid, and may include, for example, radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, or dot immunoconjugation assays. .

[0026] Next, the selected hybridomas are serially diluted or sorted into single cells by flow cytometry and cloned into individual antibody-producing cell lines. The clones can then be grown indefinitely to supply mAbs. These cell lines can be used for MAb production in two basic ways. Hybridoma samples can be injected into animals (e.g., mice) (often intraperitoneally). Optionally, before injection, the animals are initially stimulated with hydrocarbons, especially oils, such as pristane (tetramethylpentadecane). When human hybridomas are used in this way, it is best to inject them into immunosuppressed mice, such as SCID mice, to prevent tumor rejection. The injected animals develop tumors that secrete specific monoclonal antibodies produced by the fusion cell hybrid. High concentrations of MAb can then be obtained by tapping the animal's body fluids, such as serum or ascites fluid. Individual cell lines can also be cultured in vitro, in which case MAb is secreted naturally into the culture medium and readily available in high concentrations from the culture medium. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. These cell lines can be adapted to grow in serum-free medium to optimize their ability to recover high-purity human monoclonal immunoglobulins.

[0027] MAb produced by either method may be further purified, if desired, by filtration, centrifugation, and various chromatographic methods, such as FPLC or affinity chromatography. Monoclonal antibody fragments of this disclosure can be obtained from purified monoclonal antibodies by methods including digestion with enzymes such as pepsin or papain, and / or by cleaving the disulfide bond by chemical reduction. Alternatively, monoclonal antibody fragments incorporated in this disclosure can be synthesized using an automated peptide synthesizer.

[0028] It is also intended that molecular cloning approaches may be used to create monoclonals. For this purpose, RNA can be isolated from the hybridoma strain, the antibody gene can be obtained by RT-PCR, and cloned into an immunoglobulin expression vector. Alternatively, a combinatorial immunoglobulin phagemide library can be prepared from RNA isolated from the cell line, and phagemides expressing the appropriate antibody can be selected by panning with a viral antigen. The advantages of this approach over conventional hybridoma techniques are approximately 10 4 The ability to produce and screen twice the amount of antibody in a single step, along with the novel specificity created by the combination of heavy and light chains, further increases the likelihood of discovering the appropriate antibody.

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

[0030] B. Antibodies in this Disclosure Antibodies according to this disclosure may be defined, in the first case, by binding specificity, in this case by binding specificity to glypican 2. In one embodiment, the antibody is an immunoglobulin G (IgG) antibody isotype. IgG accounts for 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. Humans have four IgG subclasses (IgG1, 2, 3, and 4), named in order of their abundance in serum (IgG1 is the most abundant). These range from high affinity to no affinity for the Fc receptor.

[0031] IgG is the primary antibody isotype found in blood and extracellular fluid, and therefore can halt infection in 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 immune mechanisms: when pathogens are bound via IgG, they are immobilized and bound by aggregation; the pathogen surface is coated with IgG (known as opsonization), allowing phagocytic immune cells to recognize and engulf the pathogen; IgG activates the classical pathway of the complement system, an immune protein production cascade that eliminates pathogens; IgG also binds to and neutralizes toxins. IgG also plays a crucial role in antibody-dependent cytotoxicity (ADCC) and intracellular antibody-mediated proteolysis. In intracellular antibody-mediated proteolysis, IgG binds to TRIM21 (the receptor with the greatest affinity for IgG in humans) to direct marked virions to 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 are therefore primarily involved in secondary immune responses. Because IgG is secreted as small monomers, it can easily perfuse tissues. It is the only isotype that possesses receptors that facilitate cross-placental transport in humans. Along with IgA secreted into breast milk, residual IgG that crosses the placenta and is absorbed provides humoral immunity to newborns before their own immune system develops. Colostrum, especially bovine colostrum, contains a high proportion of IgG. In individuals with prior immunity to the pathogen, IgG appears approximately 24–48 hours after antigenic stimulation.

[0032] Furthermore, antibody sequences may optionally be modified from the sequences provided above using methods discussed in more detail below. For example, amino sequences may differ from those provided above in that (a) the variable region is separated from the constant domain of the light chain, (b) the amino acids differ from those shown above, but this does not dramatically affect the chemical properties of the residues (so-called conservative substitutions), or (c) the amino acids differ from those shown above by a certain percentage of homology, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Alternatively, the nucleic acid encoding the antibody may differ from the nucleic acid described above by (a) being isolated from the constant domain of the light chain, (b) being different from the nucleic acid described above but without altering the encoded residues, (c) being different from the nucleic acid described above by a certain percentage of homology, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or (d) being different from the nucleic acid described above by its ability to hybridize under high stringency conditions, e.g., by low salt and / or high temperature conditions, e.g., by approximately 0.02M to approximately 0.15M NaCl and temperatures of approximately 50°C to approximately 70°C.

[0033] When conserving changes in amino acid sequences, the hydroxyl index of amino acids is sometimes considered. The importance of the hydroxyl amino acid index in conferring interactive biological functions to proteins is generally understood in this field (Kyte and Doolittle, 1982). The relative hydroxyl characteristics of amino acids contribute to the resulting secondary structure of proteins, and consequently, this is accepted to define the interactions between proteins and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens.

[0034] It is also understood in the art that similar amino acids can be effectively substituted based on their hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, correlates with the protein's biological properties. 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, nonionic 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 amino acids can be substituted with other amino acids having similar hydrophilicity, thereby producing biologically or immunologically modified proteins. In such changes, amino acid substitutions with a hydrophilicity value of ±2 are preferred, those with a hydrophilicity value of ±1 are particularly preferred, and those with a hydrophilicity value of ±0.5 are even more particularly preferred.

[0036] As outlined above, amino acid substitutions are generally based on the relative similarities of the side-chain substituents of amino acids, such as hydrophobicity, hydrophilicity, charge, and size. Various exemplary substitutions taking these characteristics into account are well known to those skilled in the art, including 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 forms, it may be chosen to manipulate the sequence of a specific antibody for a variety of reasons, such as improving expression, enhancing cross-reactivity, reducing off-target binding, or inhibiting one or more innate effector functions, such as complement activation or immune cell (e.g., T cell) recruitment. 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, then the cells can be lysed, and total RNA can be extracted. A cDNA copy of the RNA can be prepared using a random hexamer along with RT, and then PCR can be performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. The PCR product 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 the hybridoma supernatant and purified by FPLC using a Protein G column. Recombinant full-length IgG antibodies can be prepared 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 the antibodies from the CHO cell supernatant.

[0039] The rapid availability of antibodies produced in the same host cells and cell culture processes as the final cGMP manufacturing process can shorten the timeframe of process development programs. 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. While slightly slower than a truly transient system, the advantages include higher product concentrations and the use of the same host and process as the producing cell line. An example of growth and productivity of a GS-CHO pool expressing a model antibody in a disposable bioreactor: In a disposable bag bioreactor culture (5 L working volume) operating in fed-batch mode, a collected antibody concentration of 2 g / L was achieved within 9 weeks of transfection.

[0040] The pCon vector® is a simple method for reexpressing entire antibodies. This constant-region vector is a set of vectors that provide a wide range of immunoglobulin constant-region vectors cloned into pEE vectors. These vectors offer the convenience of constructing full-length antibodies with human constant regions and the GS System®.

[0041] Antibody molecules include fragments, such as fragments resulting from the 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 embodiment, 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 is desirable to "humanize" antibodies produced in non-human hosts in order to reduce the immune response when used in human therapy. Such humanized antibodies may be studied in vitro or in vivo. Humanized antibodies may be created, for example, by exchanging the immunogenic portion of an antibody with a corresponding but non-immunogenic portion (i.e., chimeric antibodies). PCT application PCT / US86 / 02269; EP applications 184,187; EP applications 171,496; EP applications 173,494; PCT application WO86 / 01533; EP applications 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 provided by Morrison (1985), also incorporated herein by reference. Alternatively, "humanized" antibodies can be produced by CDR or CEA substitution. Jones et al. (1986); Verhoeyen et al. (1988); Beidler et al. (1988). These are all incorporated herein by reference.

[0043] In a related embodiment, the antibody is a derivative of the disclosed antibody, for example, an antibody containing the same CDR sequence as the disclosed antibody (e.g., a chimeric antibody, a humanized antibody, or an antibody with a transplanted CDR). In a further embodiment, the antibody is a fully human recombinant antibody.

[0044] This disclosure also intends to enable isotype modification. Different functionalities can be achieved by modifying the Fc region to have a different isotype. For example, by changing it to IgG4, the immunoeffector function associated with other isotypes can be reduced.

[0045] Modified antibodies can be prepared 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 discussed elsewhere in this document.

[0046] D. Expression Nucleic acids relating to this disclosure encode antibodies, optionally antibodies linked to other protein sequences. As used herein, the term “nucleic acid encoding a glypican 2 antibody” refers to a nucleic acid molecule isolated in the absence of all cellular nucleic acids. In certain embodiments, this disclosure relates to antibodies encoded by any sequence shown herein.

[0047] (Table 2) Codons TIFF0007909644000001.tif111138

[0048] The DNA segments of this disclosure include those encoding biologically functional equivalent proteins and peptides of the sequences described above. Such sequences may arise as a result of nucleic acid sequences and codon redundancy and amino acid functional equivalence known to occur naturally in the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be produced by applying recombinant DNA technology, which allows for the manipulation of protein structural changes based on the idea that amino acid properties are exchanged. Human-designed changes may be introduced by applying site-directed mutagenesis, as described below, or they may be introduced randomly and subsequently screened for the desired function.

[0049] Throughout this application, the term “expression construct” is intended to include any type of gene construct that contains a nucleic acid encoding a gene product, wherein some or all of the nucleic acid coding sequences within this nucleic acid are transcribable. The transcript may, but does not have to be, a protein. In certain embodiments, expression includes both the transcription of a gene and the translation of mRNA into a gene product. In other embodiments, expression includes only the 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 also be “exogenous.” “Exogenous” means that the nucleic acid sequence is foreign to the cell into which the vector is introduced, or that it is homologous to an intracellular sequence but located in a position within the host cell nucleic acid that is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be considered well-equipped to construct vectors using the standard recombination 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 containing a transcribed nucleic acid sequence that codes for 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, and, for example, an antisense molecule or ribozyme is produced. Expression vectors may contain various “regulatory sequences” that refer to nucleic acid sequences required for the transcription of a functionally linked coding sequence in a particular host organism, and possibly for its translation. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also perform other functions and contain nucleic acid sequences described below.

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

[0053] The promoter may be a promoter naturally associated with the gene or sequence, or it may be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such promoters are sometimes called “endogenous.” Similarly, the enhancer may be a enhancer naturally associated with the nucleic acid sequence, located downstream or upstream of the nucleic acid sequence. Alternatively, certain benefits may be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter. Recombinant or heterologous promoters refer to promoters that are not normally associated with the nucleic acid sequence in their natural environment.

[0054] Recombinant enhancers or heterologous enhancers also refer to enhancers that are not typically associated with nucleic acid sequences in their 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 containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthesizing the nucleic acid sequences of promoters and enhancers, sequences may also be prepared using nucleic acid amplification techniques, including recombinant cloning techniques and / or PCR®, together with the compositions disclosed herein (see U.S. Patents 4,683,202 and 5,928,906, respectively, incorporated herein by reference). Furthermore, regulatory sequences that induce transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria and chloroplasts, are also intended to be used.

[0055] Of course, it is important to use a selected cell type, organelle, and promoter and / or enhancer that effectively expresses the DNA segment in the organism. Those skilled in the art of molecular biology are generally familiar with the use of combinations of promoters, enhancers, and cell types to express proteins. See, for example, Sambrook et al. (1989), incorporated herein by reference. The promoter used may be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter that is useful under conditions suitable for high-level expression of the introduced DNA segment, for example, a promoter advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be a heterologous promoter or an endogenous promoter. Assays for characterizing the identity of tissue-specific promoters or elements and their activity 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 epididymal 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), and human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996).

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

[0057] 2. IRES In certain embodiments of this disclosure, the use of intra-sequence ribosome entry sites (IRES) elements is employed to create multi-gene messages or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylation-dependent translation and initiate translation at an internal site (Pelletier and Sonenberg, 1988). IRES elements derived from two members of the Picornaviridae family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), and IRESs derived from mammalian messages have also been described (Macejak and Sarnow, 1991). IRES elements can be ligated with heterologous open reading frames. Multiple open reading frames separated by each IRES can be transcribed together to create a polycistronic message. IRES elements allow each open reading frame to approach a ribosome for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patents No. 5,925,565 and No. 5,935,819, respectively, incorporated herein by reference).

[0058] 3. Multipurpose cloning site The vector may contain a multicloning site (MCS). A multicloning site is a nucleic acid region containing multiple restriction enzyme sites, any of which can be used with standard recombination techniques to digest the vector. See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated herein by reference. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at a specific site on the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is well understood by those skilled in the art. Vectors are often linearized or fragmented using restriction enzymes that cleave within the MCS so that an exogenous sequence can be ligated into the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may be contiguous or non-contiguous. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombination techniques.

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

[0060] 5. Termination Signal The vectors or constructs of this disclosure generally include at least one termination signal. The “termination signal” or “terminator” consists of a DNA sequence involved in the specific termination of an RNA transcript by RNA polymerase. Thus, in certain embodiments, a termination signal is intended to terminate the production of an RNA transcript. A terminator may be required in vivo to achieve a desired message level.

[0061] In eukaryotic systems, the terminator region may also include a specific DNA sequence that allows for site-specific cleavage of a new transcript to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a sequence of approximately 200 A residues (poly-A) to the 3' end of the transcript. RNA molecules modified with this poly-A tail appear to be more stable and are translated more efficiently. Therefore, in other embodiments involving eukaryotes, the terminator preferably 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 help to enhance the message level and / or minimize read-overs from the cassette to other sequences.

[0062] The terminators intended for use in this disclosure include any known transcriptional terminators described herein or known to those skilled in the art. These include, but are 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 that lacks a transcribable or translatable sequence, for example, one that lacks a transcribable or translatable sequence due to cleavage.

[0063] 6. Polyadenylation signal In expression, particularly in eukaryotes, a polyadenylation signal is typically included to result in appropriate polyadenylation of the transcript. The nature of the polyadenylation signal is not considered important for the success of the implementation of this 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 enhance the stability of the transcript and facilitate its transport into the cytoplasm.

[0064] 7. Origin of replication To propagate the vector within a host cell, the vector may contain one or more origin sites (often called "oris"). An origin site is a specific nucleic acid sequence from which replication begins. Alternatively, if the host cell is yeast, an arrears sequence (ARS) can be used.

[0065] 8. Selection Markers and Screening Markers In certain embodiments of this disclosure, cells containing the nucleic acid constructs of this disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such markers confer a identifiable change to the cells, thereby facilitating the identification of cells containing the expression vector. Generally, selection markers confer properties that enable selection. Positive selection markers enable cell selection in the presence of the marker, while negative selection markers prevent cell selection in the presence of the marker. An example of a positive selection marker is a drug resistance marker.

[0066] Typically, the inclusion of drug selection markers 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 phenotypic markers that enable the identification of transformants based on conditions, other types of markers are also intended, including screening markers such as GFP, which are based on colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (TK) or chloramphenicol acetyltransferase (CAT) can be used. Those skilled in the art will also know how to use immunomarkers, perhaps in conjunction with FACS analysis. The markers used are not considered critical as long as they can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screening markers are well known to those skilled in the art.

[0067] 9. Viral vectors Many different viral vector systems have been developed and applied because certain viral vectors can efficiently infect or enter cells, integrate into the host cell genome, and stably express viral genes (Robbins et al., 1998). Viral systems are currently being developed for use as vectors for ex vivo and in vivo gene transfer. For example, adenovirus, herpes simplex virus, retrovirus, and adeno-associated virus vectors are currently being evaluated to treat diseases such as cancer, cystic fibrosis, Gaucher disease, kidney disease, and arthritis (Robbins and Ghivizzani, 1998; Imai et al., 1998; U.S. Patent No. 5,670,488). Other viral vectors, such as poxviruses (e.g., vaccinia virus, Gnant et al., 1999), alphaviruses (e.g., Sindbis virus, Semlik Forest virus, Lundstrom, 1999), reovirus (Coffey et al., 1998), and influenza A virus (Neumann et al., 1999), may be used in this disclosure and can be selected according to the required properties of the system of interest.

[0068] 10. Non-viral transformation For use in conjunction with this disclosure, suitable methods for nucleic acid delivery to transform organelles, cells, tissues, or organisms are considered to 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 skilled in the art. Such methods include, for example, injections including microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference); injections (U.S. Patents No. 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); electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); and 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); liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); particulate gun (PCT application numbers WO94 / 09699 and 95 / 06128; U.S. Patent No. 5,610,042; Nos. 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880, respectively, incorporated herein by reference); stirring using silicon carbide fibers (Kaeppler et al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765).This includes, but is not limited to, DNA delivery by PEG-mediated protoplast transformation (Omirulleh et al., 1993; U.S. Patents No. 4,684,611 and No. 4,952,500, respectively, incorporated herein by reference); or DNA uptake via drying / inhibition (Potrykus et al., 1985). By applying techniques such as these, organelles, cells, tissues, or organisms may be stably or transiently transformed.

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

[0070] Insect cell / baculovirus systems can produce high levels of protein expression of heterologous nucleic acid segments, as described, for example, in U.S. Patent Nos. 5,871,986 and 4,879,236, which are incorporated herein by reference, and can be purchased, for example, from Invitrogen® under the name MaxBac® 2.0 and from Clontech® under the name BacPack® baculovirus expression system.

[0071] Other examples of expression systems include Stratagene®'s Complete Control® inducible mammalian expression system, which contains a synthetic ecdysone-inducible receptor, or its pET expression system, which is an E. coli expression system. Another example of an inducible expression system is available from Invitrogen®, which carries the T-Rex® system (tetracycline-regulated expression), an inducible mammalian expression system using a full-length CMV promoter. Invitrogen® also offers a yeast expression system called the Pichia methanolica expression system, which is designed for high-level production of recombinant proteins in the methylotrope yeast Pichia methanolica. Those skilled in the art will know how to express vectors, such as expression constructs, to produce nucleic acid sequences or their cognitive polypeptides, proteins, or peptides.

[0072] Primary mammalian cell cultures can be prepared in various ways. For cells to survive in vitro and in contact with expression constructs, it is necessary to ensure they maintain contact with oxygen, carbon dioxide, and nutrients in the correct ratios, while being protected from microbial contamination. Cell culture methods are well documented.

[0073] One embodiment 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 cell can be cultured under appropriate conditions. In this way, substantially any polypeptide gene can be used. The preparation of recombinant expression vectors and the elements contained therein was discussed above. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell in question.

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

[0075] Many selection systems, including but not limited to HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes, can be used in tk-cells, hgprt-cells, or aprt-cells, respectively. Furthermore, antimetabolite resistance can be used as a basis for selection to dhfr (conferring resistance to dhfr), gpt (conferring resistance to mycophenolate), neo (conferring resistance to aminoglycoside G418), and hygro (conferring resistance to hygromycin).

[0076] E. Purification In certain embodiments, the antibodies of this disclosure may be purified. As used herein, the term “purified” is intended to mean a composition that can be isolated from other components, in which case the protein is purified to any degree compared to its naturally occurring state. Thus, purified protein also means a protein that does not contain the environment in which it may occur naturally. Where the term “substantially purified” is used, this indication means a composition in which the protein or peptide forms the major component of the composition, for example, constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein in the composition.

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

[0078] When purifying the antibodies of this 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 also 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 the various purification steps are performed may be changed, or certain steps may be omitted, and this is still considered to result in a suitable method for preparing substantially purified proteins or peptides.

[0079] Typically, complete antibodies are fractionated using a drug that binds to the Fc portion of the antibody (i.e., protein A). Alternatively, antigens may be used to simultaneously purify and select the appropriate antibody. In such methods, a selection drug conjugated to a support such as a column, filter, or beads is often used. The antibody is bound to the support, contaminants are removed (e.g., by washing), and the antibody is released by applying conditions (e.g., salt, heat).

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

[0081] Polypeptide migration is known to sometimes vary significantly under different SDS / PAGE conditions (Capaldi et al., 1977). Therefore, it is understood that the apparent molecular weight of purified or partially purified expression products may change under different electrophoretic conditions.

[0082] F. Single-chain / single-domain antibody Single-chain variable fragments (scFvs) are formed by the fusion of the variable regions of the heavy and light chains of an immunoglobulin with a short (usually serine or glycine) linker. These chimeric molecules, also known as single-domain antibodies, retain the specificity of the original immunoglobulin even after the constant region has been removed and a linker peptide has been introduced. This modification usually does not alter the specificity. These molecules have been created throughout history to facilitate phage display, where expressing the antigen-binding domain as a single peptide is extremely convenient. Alternatively, scFvs can be created directly from subcloned heavy and light chains derived from hybridomas. Single-domain or single-chain variable fragments lack the constant Fc region found in complete antibody molecules and therefore lack a common binding site (e.g., protein A / G) used for antibody purification (single-chain antibodies contain an 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] Movable linkers generally consist of amino acid residues that promote helical linkage and amino acid residues that promote turn, such as alanine, serine, and glycine. However, other residues can also function. Tang et al. (1996) used phage display as a means of rapidly selecting linkers matched to single-chain antibodies (scFv) from a protein linker library. A random linker library was constructed in which the genes of the heavy chain variable domain and light chain variable domain were linked by segments encoding 18-amino acid polypeptides with different compositions. The scFv repertoire (approximately 5 x 10⁻¹⁴) 6Individual different members were displayed on filamentous phages and subjected to affinity selection using haptens. The selected population of variants showed a significant increase in binding activity but retained considerable sequence diversity. Subsequently, by screening 1054 variants one by one, soluble and efficiently produced catalytically active scFv were obtained. From sequence analysis, the only common feature of the selected tethers was V H It was revealed that there is a conserved proline in the linker two residues behind the C-terminus, and that there are many arginine and proline at other positions.

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

[0085] In a different embodiment, single-chain antibodies can be produced by linking the receptor's light and heavy chains using non-peptide linkers or chemical units. Generally, the light and heavy chains are produced and purified in separate cells and then linked together in an appropriate manner (i.e., the N-terminus of the heavy chain is attached to the C-terminus of the light chain via appropriate chemical crosslinking).

[0086] Crosslinking reagents are used to form molecular crosslinks that link the functional groups of two different molecules, for example, a stabilizer and a coagulant. However, the intention is to create heteromeric complexes composed of dimers or polymers of the same analog or different analogs. To link two different compounds stepwise, heterobifunctional crosslinking agents can be used to eliminate the formation of unnecessary homopolymers.

[0087] An exemplary heterobifunctional crosslinker contains 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., pyridyl disulfide, 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, already bound to the first protein, reacts with a cysteine ​​residue (free sulfhydryl group) of another protein (e.g., a selected drug) via the thiol reactive group.

[0088] It is preferable to use a crosslinking agent that has reasonable stability in the blood. A great many types of disulfide bond-containing linkers are known that can be successfully used to bind targeted drugs to therapeutic / prophylactic agents. Linkers containing sterically obstructed disulfide bonds have been shown to confer high stability in vivo, thereby preventing the release of targeted peptides before they reach the site of action. Therefore, these linkers are one of the drug-linking groups.

[0089] Another crosslinking agent is SMPT, a bifunctional crosslinking agent containing a disulfide bond that is "sterically hindered" by adjacent benzene rings and methyl groups. The steric hindrance of the disulfide bond is thought to protect the binding from attack by thiolate anions that may be present in tissues and blood, such as glutathione, thereby helping to prevent the conjugate from separating before the attached drug can be delivered to the target site.

[0090] SMPT crosslinking reagents, like many other known crosslinking reagents, impart the ability to crosslink functional groups, such as the SH group of cysteine ​​or primary amines (e.g., the ε-amino group of lysine). Another possible type of crosslinking agent includes heterobifunctional 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 with any amino acid residue (upon photodegradation).

[0091] In addition to the interfering crosslinkers, uninterfering linkers can also be used according to this specification. Other useful crosslinkers that do not contain or are not thought to generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such crosslinkers is well understood in the art. Another embodiment involves the use of a movable linker.

[0092] U.S. Patent No. 4,680,338 describes a bifunctional linker useful for generating conjugates of ligands with amine-containing polymers and / or proteins, particularly useful for forming antibody conjugates with chelating agents, drugs, enzymes, detectable labels, etc. U.S. Patents Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing unstable bonds that can be cleaved under a variety of mild conditions. These linkers are particularly useful because they can directly bind drugs of interest to the linker, and the active drug is released upon cleavage. Specific applications include the addition of free amino groups 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 linking polypeptide components for the purpose of producing fusion proteins, such as single-chain antibodies. This linker is up to approximately 50 amino acids in length and contains at least one proline sequence following a charged amino acid (preferably arginine or lysine), characterized by high stability and low aggregation. U.S. Patent No. 5,880,270 discloses an aminooxy-containing linker useful in various immunodiagnostic and separation methods.

[0094] G. Modified antibody 1. CAR Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immune receptors, or chimeric antigen receptors (CARs)) are engineered receptors that transfer arbitrary 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. In this way, a large number of cancer-specific T cells can be generated for adoptive cell transfer. Phase I clinical studies of this approach have 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 domain and endodomain. Such molecules transmit the ζ signal in response to the recognition of their target by the scFv. One 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 onchoretroviral 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 to the B-series molecule CD19.

[0096] To form scFv, the variable portions of the immunoglobulin heavy and light chains are fused by a mobile linker. In front of this scFv is a signal peptide (which is cleaved) that directs the nascent protein to the endoplasmic reticulum and then to its surface. The mobile spacer allows the scFv to orient in various directions to bind to the antigen. The transmembrane domain is a typical hydrophobic α-helix, usually derived from the original molecule of the signaling end domain, which 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 domains pass, works to cleave the inner (endodomain) portion from the outer (ectodomain). It is not surprising that attaching the ectodomain from one protein to the endodomain of another protein can result in a molecule that combines the recognition of the former with the signaling of the latter.

[0098] Ectodomain Signal peptides direct nascent proteins to the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and fixed to the cell membrane. Typically, any eukaryotic signal peptide sequence works well. Generally, the signal peptide naturally attached to the most amino-terminal component is used (for example, in scFv with a light-linker-heavy-chain orientation, the natural signal on the light chain is used).

[0099] Typically, the antigen-recognition domain is the scFv. However, there are many options. Antigen-recognition domains derived from native T cell receptor (TCR) α and β monochains have been described, as have simple ectodomains (e.g., the CD4 ectodomain that recognizes HIV-infected cells) and more unusual recognition components, such as ligated cytokines (that recognize cells with cytokine receptors). In practice, almost anything that binds to a particular target with high affinity can be used as an antigen-recognition region.

[0100] The spacer region ligates 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 orient in various directions. The simplest form is a hinge region derived from IgG1. Alternatives include the CH2CH3 region of immunoglobulins and a portion of CD3. For most scFv-based constructs, the IgG1 hinge is sufficient. However, the best spacer often needs to be determined empirically.

[0101] transmembrane domain A transmembrane domain is a hydrophobic α-helix that spans the membrane. Generally, the transmembrane domain derived from the closest membrane-bound component of the endodomain is used. Interestingly, using the CD3ζ transmembrane domain may allow the artificial TCR to be incorporated into the native TCR, a factor that depends on the presence of the native CD3ζ transmembrane charged aspartate residue. Different transmembrane domains result in different receptor stabilities. Using the CD28 transmembrane domain results in a stable receptor that is actively expressed.

[0102] End domain This is the "business-end" of the receptor. After the antigen is recognized, the receptors cluster and the signal is 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 provide a sufficiently capable activation signal, and further co-stimulatory signaling may be required. For example, chimeric CD28 and OX40 may be used with CD3ζ to transmit proliferation / survival signals, or all three may be used together.

[0103] "First-generation" CARs typically contained an intracellular domain derived from the CD3ξ chain, a primary signaling molecule derived from the endogenous TCR. "Second-generation" CARs added intracellular signaling domains derived from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to supply further signaling to T cells. Preclinical studies have shown that second-generation CAR designs improve the antitumor activity of T cells. More recently, "third-generation" CARs have combined multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, 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 virtually any tumor-associated antigen. This approach has great potential to greatly improve individualized cancer therapy for patients. After the patient's T cells are collected, they are genetically engineered to express CARs that specifically target antigens present on the patient's tumor cells, and then injected back into the patient. While adoptive transfer of CAR-modified T cells is a unique and promising cancer therapy, it presents significant safety concerns. Clinical trials of this therapy have revealed that these CARs have potential toxicity when healthy tissue expresses the same target antigens as tumor cells, and therefore the outcomes resemble graft-versus-host disease (GVHD). A potential solution to this problem is to engineer and introduce suicide genes into the modified T cells. Thus, administering a prodrug designed to activate the suicide gene during GVHD induces apoptosis in the CAR T cells activated by the suicide gene. This method has been used safely and effectively in hematopoietic stem cell transplantation (HSCT). The use of suicide gene therapy in clinical applications of CAR-modified T cell adoptive cell transfer may improve overall antitumor efficacy while mitigating GVHD.

[0105] 2. ADC Antibody-drug conjugates (ADCs) are a new class of highly potent biopharmaceuticals designed as targeted therapies for treating people with cancer. ADCs are complex molecules consisting of an antibody (whole mAb or antibody fragment, e.g., single-chain variable fragment, or scFv) linked to a biologically active cytotoxic (anti-cancer) payload or drug via unstable linkage through a stable chemical linker. Antibody-drug conjugates are examples of bioconjugates and immunoconjugates.

[0106] By combining the intrinsic targeting ability of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, antibody-drug conjugates can highly sensitively differentiate between healthy and diseased tissue. This means that, in contrast to conventional chemotherapy agents, antibody-drug conjugates target and attack cancer cells in such a way that healthy cells are less affected than cancer cells.

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

[0108] Stable linking between antibodies and cytotoxic (anticancer) agents is a critical aspect of ADCs. Linkers are based on chemical motifs including disulfides, hydrazones, or peptides (cleavable), or thioethers (incleavable), and control the distribution and delivery of cytotoxic agents to target cells. Both cleavable and incleavable linkers have been shown to be safe in preclinical and clinical trials. Brentuximab vedotin is a synthetic anti-cancer agent containing an enzyme-sensitive, cleavable linker that delivers the potent and highly toxic microtubule inhibitor monomethyl auristatin E, or MMAE, to human-specific CD30-positive malignant cells. Due to its high toxicity, MMAE, which inhibits cell division by blocking tubulin polymerization, cannot be used as a monotherapy agent. However, a combination of MMAE linked to an anti-CD30 monoclonal antibody (cAC10, tumor necrosis factor, or TNF receptor cell membrane protein) has been shown to be stable in extracellular fluid, cleavable by cathepsin, and safe for therapy. Another approved ADC, trastuzumab emtansine, is a combination of the mytansine derivative microtubule formation inhibitor meltansine (DM-1) and the antibody trastuzumab (Herceptin® / Genentech / Roche), attached by a stable, cleavable linker.

[0109] The functionality of chemical bonds has changed due to the availability of better and more stable linkers. Cleavable and non-cleavable linkers impart specific properties to cytotoxic (anti-cancer) drugs. For example, non-cleavable linkers retain the drug within the cell. As a result, the entire antibody, linker, and cytotoxic agent enter the target cancer cell, where the antibody is broken down to the amino acid level. In this case, the resulting complex of amino acids, linker, and cytotoxic agent becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes within the cancer cell, releasing the cytotoxic agent within the cancer cell. This difference allows the cytotoxic payload delivered via a cleavable linker to escape 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. This linker technology allows researchers to create ADCs with greater mobility without worrying about altering cleavage kinetics. Researchers are also developing novel peptide cleavage methods based on Edman degradation, a method for sequencing the amino acids of peptides. Future directions in ADC development include the development of site-directed conjugation (TDC) to further improve stability and therapeutic index, as well as α-radioimmunoconjugates and antibody-conjugated nanoparticles.

[0111] 3. BitE Bi-specific T-cell engagers (BiTEs) are a type of artificial bi-specific 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 (scFv) of different antibodies, or amino acid sequences derived from four different genes, on a single peptide chain of approximately 55 kilodaltons. One scFv binds to T cells via the CD3 receptor, while the other binds to tumor cells via a tumor-specific molecule, in this case, glypican 2.

[0113] Like other bispecific antibodies, and unlike typical monoclonal antibodies, BiTE forms a link between T cells and tumor cells. This allows T cells to exert cytotoxic activity on tumor cells by producing protein-like perforins and granzymes, independently of the presence of MHC I or co-stimulating molecules. These proteins enter tumor cells and initiate apoptosis. This process closely resembles the physiological processes observed when T cells attack tumor cells.

[0114] As of July 2010, BiTEs in clinical trials included blinatumomab (MT103), formulated against CD19, a surface molecule expressed on B cells, for the treatment of non-Hodgkin lymphoma and acute lymphoblastic leukemia, and MT110, formulated against the EpCAM antigen, for the treatment of gastrointestinal cancer and lung cancer.

[0115] Using the same technology, melanoma (using MCSP-specific BiTE) and acute myeloid leukemia (using CD33-specific BiTE) can be targeted. Research in this area is currently underway. Another approach for novel anticancer 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 targeting CD66e and EphA2 are also under development.

[0116] III. Pharmaceutical preparations and cancer treatment A. Cancer Cancer arises from the proliferation of clonal populations of cells originating from tissue. The development of cancer, known as carcinogenesis, can be modeled and characterized in many ways. A long-standing link between cancer development and inflammation has been recognized. Inflammatory responses are involved in host defense against microbial infections and are also the driving force behind tissue repair and regeneration. A considerable amount of evidence suggests a link between inflammation and cancer risk, meaning that chronic inflammation can contribute to dysplasia.

[0117] Cancer cells to which the methods of this disclosure can be applied generally include any cells expressing glypican 2, and more specifically, any cells overexpressing glypican 2. Cancer cells that can be treated in accordance with this 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. Furthermore, cancer specifically includes the following histological types: neoplasms, malignant; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinomas; 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; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma within adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid tumors; carcinoid tumors, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; pigmentaphobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary adenocarcinoma and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma. Carcinoma; Adrenal cortical carcinoma; Endometrioid carcinoma; Adnexal carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Ceratocarcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease, breast; Acinar cell carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal sphincteroma, malignant; Theca cell tumor, malignant; Granulosa cell tumor, malignant; Androblastoma, malignant; Sertoli cell tumor; Leydig cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma, malignant; Extramammary paraganglioma Paraganglioma, malignant; chromophilic cell tumor; angioglobulin angiosarcoma; malignant melanoma; apigmented melanoma; superficial spreading melanoma; malignant melanoma in a giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant;Mueller's mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated germ cell tumor; embryonic carcinoma; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; perivascular cell tumor, malignant; lymphangiosarcoma; osteosarcoma; paraosteosarcoma; chondrosarcoma; Chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastoma; pineal glandoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; gliablastoma; oligodendroglioma; oligodendrogliocyte Tumor; undifferentiated neuroectodermal; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurotumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; lateral granuloma; malignant lymphoma, microlymphocytic; malignant lymphoma, diffuse large cell; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin lymphomas Parkinson's disease; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; and pilocytic cell leukemia may also include, but are not limited to, these. In certain contexts, tumors may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, gliablastoma, 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, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The cancer may also be recurrent, metastatic, and / or multidrug-resistant. The methods of the present disclosure may be applied in particular to such cancers to make them resectable, to prolong or re-induce remission, to inhibit angiogenesis, to block or limit metastasis, and / or to treat multidrug-resistant cancers. At the cellular level, this may result in the death of cancer cells, inhibition of cancer cell proliferation, or otherwise reversal or reduction of the malignant phenotype of tumor cells.

[0119] B. Formulation and Administration This disclosure provides a pharmaceutical composition comprising an anti-glypican 2 antibody. In certain embodiments, the term “pharmaceutically acceptable” means that it is authorized by a federal or state regulatory authority or is listed in the United States Pharmacopeia or in other generally accepted pharmacopoeias for use in animals, or more specifically, for use in humans. The term “carrier” means a diluent, excipient, or vehicle administered with the therapeutic agent. Such pharmaceutical carriers may be sterile liquids, such as water and oil, derived from petroleum, animal, vegetable, or synthetic sources, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc.

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

[0121] The antibodies of this disclosure may include classic pharmaceutically acceptable preparations. Administration of these compositions according to this disclosure will be carried out via any common route, insofar as the target tissue is accessible via any common route. This route may include oral, nasal, buccal, rectal, vaginal, or local routes. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions will typically be administered as pharmaceutically acceptable compositions as described above. There is particular interest in administration into tumors, perfusion into tumors, or local or local administration to tumors, for example, to local or local vascular structures or lymphatic systems, or to the excised tumor bed.

[0122] The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound, which are free bases or pharmacokinetically acceptable salts, can be prepared by dissolving them in water in appropriate mixture with a surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared by dissolving them in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0123] C. Combination therapy In the context of this disclosure, the anti-glypican 2 antibodies described herein are also intended to be used in conjunction with chemotherapy interventions, radiotherapy interventions, or other treatments. In particular, it may be found that anti-glypican 2 antibodies can be effectively combined 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, "target" cells will generally be contacted with an anti-glypican 2 antibody and at least one other agent as described herein. These compositions will be provided in combined amounts effective in killing cells or inhibiting cell proliferation. This process may involve contacting cells simultaneously with the anti-glypican 2 antibody and the other agent or factor as described herein. This may be achieved by contacting cells with a single composition or pharmacological formulation containing both agents, or by contacting cells simultaneously with two separate compositions or formulations, one of which contains an anti-glypican 2 antibody as described herein and the other containing the other agent.

[0125] Alternatively, anti-glypican 2 antibody therapy may be administered before or after other drug treatments, with intervals ranging from minutes to weeks. In embodiments where other drugs and anti-glypican 2 antibodies are applied to cells separately, it is generally desirable to ensure that the effective period does not end between each delivery, so that the drugs and expression constructs can still exert a favorably combined effect on the cells. In such cases, contact with both modalities is intended to occur within approximately 12–24 hours after administration of either modality, more preferably within approximately 6–12 hours after administration of either modality, with a delay of only about 12 hours being most preferable. In some situations, it may be desirable to significantly extend the time between treatments. However, the time between each administration is typically 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 be desirable to administer either an anti-glypican 2 antibody or another drug multiple times. Various combinations can be used, as illustrated below. In the formula, the anti-glypican 2 antibody therapy according to this disclosure is "A", and the other therapies are "B". TIFF0007909644000002.tif17128

[0127] Other combinations are also intended. Again, both drugs are delivered to the cells in a combined amount effective in killing the cells. Drugs or factors suitable for cancer therapy include any compound or treatment method that induces damage when applied to cells. Such drugs and factors include radiation and waves that induce DNA damage, such as irradiation, microwaves, and electron emission. Various compounds, also referred to as "chemotherapeutic agents" or "genotoxic agents," may be used. This may be achieved by irradiating the local tumor site with radiation. Alternatively, tumor cells may come into contact with the drug by administering a therapeutically effective amount of the pharmaceutical composition to the target. Combination therapy may also include surgery. Various methods of these therapies are discussed below.

[0128] 1.Chemotherapy The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapy agents" is used to imply compounds or compositions administered in the treatment of cancer. These drugs are classified by their intracellular mechanism of activity, for example, whether they affect the cell cycle, or at what stage of the cell cycle they affect it. Alternatively, drugs may be characterized based on their ability to directly crosslink DNA, insert into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. Most chemotherapy agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

[0129] Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and piposulfan; aziridines, e.g., benzodopa, carbocon, meturedopa, and uredopa; altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamime, among others. Chilenimine and methylamelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including its synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adzelesin, carzelesin, and biceresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (synthetic analog KW- Including 2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembicin, phenesterine terine), prednimustine, trophosphamide, uracil mustard; nitrosourea, e.g., carmustine, chlorozotocin, photemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., engine antibiotics (e.g., calitiamycin, in particular calitiamycin γ1I and calitiamycin ωI1; dynemicin, including dynemicin A; uncialamycin and its derivatives; bisphosphonates, e.g., clodronate; esperamicin;Furthermore, neocardinostatin chromophore and related pigment protein enediin antibiotics chromophore, acrasinomycin, actinomycin, authrarmycin, azaserin, bleomycin, kactinomycin, carabicin, kaminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino- Doxorubicin (including 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidine, yubenimex, dinostatin, or zolubicin; Antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine; androgens, e.g., carsterone, propionate dol Mostanolone, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecoltin; diaziquan; elformithine; eriptinium acetate; eposylone; etogluside; gallium nitrate;Hydroxyureas; lentinans; lonidynins; mytansinoids, e.g., mytansin and ansamitocin; mitogwazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triadiquane; 2,2',2''-trichlorotriethylamine; trichothecenese (especially T-2 toxin, verracurin A, roridin A, and anguidine) idine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum-coordinated 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 inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine;This includes cisplatin (CDDP), carboplatin, procarbazine, mechloretamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor conjugates, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, as well as any pharmaceutically acceptable salts, acids, or derivatives thereof.

[0130] 2. Radiation therapy Radiation therapy, also known as radiotherapy, is the treatment of cancer and other diseases using ionizing radiation. Ionizing radiation accumulates energy in the treated area by damaging the genetic material of cells, thereby preventing these cells from continuing to grow. Radiation damages both cancer cells and normal cells, although the latter can repair themselves and function properly.

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

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

[0133] In conformal radiotherapy, a linear accelerator, the same radiotherapy device used in conventional radiotherapy procedures, is used. However, a metal block is placed in the path of the X-ray beam to change its shape to match the shape of the cancer. This ensures that a high dose of radiation is delivered to the tumor. Healthy surrounding cells and nearby structures receive a low dose of radiation, thus reducing the potential for side effects. A device called a multi-leaf collimator has been developed and is sometimes used as an alternative to the metal block. A multi-leaf collimator consists of multiple metal sheets fixed to the linear accelerator. Each layer can be adjusted so that the radiotherapy beam perfectly matches the treatment area without the need for a metal block. Precise positioning of the radiotherapy device is crucial in conformal radiotherapy procedures, and special scanning devices may be used at the start of each procedure to check the position of internal organs.

[0134] High-resolution intensity-modulated radiotherapy also uses a multi-lobe collimator. During this procedure, the layers of the multi-lobe collimator are moved as the treatment is performed. This method is likely to allow for more precise alignment of the treatment beam and ensure that the radiotherapy dose remains constant across the entire treatment area.

[0135] Research has shown that conformal radiotherapy and intensity-modulated radiotherapy may reduce the side effects of radiation therapy, but precisely targeting the treatment area may prevent the destruction of tiny cancer cells just outside the treatment area. This means that using these specialized radiation therapy techniques may increase the risk of cancer recurrence in the future.

[0136] Scientists are also seeking ways to enhance the effectiveness of radiation therapy. Two types of investigational drugs are being studied to see if they are effective against irradiated cells. Radiosensitizers increase the likelihood of damage to tumor cells, while radioprotective agents protect normal tissue from the effects of radiation. Hyperthermia, which uses heat, is also being studied to see if it is effective in increasing the sensitivity of tissue to radiation.

[0137] 3. Immunotherapy In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and immune effector molecules to target and destroy cancer cells. An example of such a drug is trastuzumab (Herceptin®). Immune effectors may be, for example, antibodies specific to some marker on the surface of tumor cells. Antibodies may act alone as effectors of therapy, or they may mobilize other cells that actually influence cell death. Antibodies may also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and simply act as targeted agents. Alternatively, effectors may be lymphocytes carrying surface molecules that directly or indirectly interact with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells. Combinations of therapeutic modalities, namely direct cytotoxic activity and inhibition or reduction of ErbB2, would yield therapeutic benefits in the treatment of ErbB2-overexpressing cancers.

[0138] In one aspect of immunotherapy, tumor cells are likely to possess some marker that is targeted, i.e., not present in the majority of other cells. Many tumor markers exist, and any of them may be suitable for targeting in the context of this 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 anticancer and immunostimulatory effects. Immunostimulatory molecules also exist, including cytokines, e.g., IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, e.g., MIP-1, MCP-1, IL-8, and growth factors, e.g., FLT3 ligand. The combined use of immunostimulatory molecules as proteins or immunostimulatory molecules delivered via gene delivery with tumor suppressors has been shown to enhance antitumor effects (Ju et al., 2000). Furthermore, antibodies against any of these compounds may be used to target the anticancer agents discussed herein.

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

[0140] In active immunotherapy, antigenic peptides, polypeptides, or proteins, or autologous or allogeneic tumor cell compositions, i.e., "vaccines," are typically 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 with lymphokines such as IL-2, or genetically modified to induce tumor necrosis, and then re-administered (Rosenberg et al., 1988; 1989).

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

[0143] Radical surgery includes excision in which all or part of the cancerous tissue is physically removed, extracted, and / or destroyed. Tumor excision refers to the physical removal of at least a portion of the tumor. In addition to tumor excision, surgical procedures include laser surgery, cryosurgery, electrosurgery, and microsurgical surgery (Mohs procedure). Furthermore, this disclosure is intended to be used in conjunction with the removal of superficial cancers, precancerous conditions, or incidental amounts of normal tissue.

[0144] When cancer cells, cancerous tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment may be achieved by perfusing, directly injecting, or topically applying further anticancer therapy to this site. Such treatment may be repeated, for example, every day, 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 this disclosure is considered particularly effective in reducing tumor recurrence after tumor removal. Furthermore, the compounds of this disclosure can also be used in the field of neoadjuvants.

[0146] It should be noted that in cancer treatment, each of the aforementioned therapies may prove useful on its own. Those skilled in the art should refer to Chapter 33, 15th edition of Remington's Pharmaceutical Sciences, particularly pages 624-652. Depending on the condition of the patient being treated, there will inevitably be some variation in dosage. The person administering the medication must determine the appropriate dosage for each individual patient, regardless of what happens. Furthermore, for human administration, preparations must meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA's Office of Biologics.

[0147] IV. Antibody conjugate Antibodies may be linked to at least one drug to form an antibody conjugate. It is a conventional practice to link, covalently bond, or complex at least one desired molecule or part to enhance the potency of an antibody molecule as a diagnostic or therapeutic agent. Such molecules or parts may, but are not limited to, at least one effector or reporter molecule. Effector molecules include molecules with desired activity, e.g., anti-cancer / general cytotoxicity. Non-limiting examples of such molecules are given above. Optionally, such molecules may be attached via a cleavable linker designed to release such molecules at or near a target site.

[0148] In contrast, a reporter molecule is defined as any part that can be detected using an assay. Non-limiting examples of reporter molecules that have been bound to antibodies include enzymes, radiolabeled molecules, haptens, fluorescently labeled molecules, 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 It may be mentioned that it is used in a particular manner. 125 I is often preferred, but technetium 99m and / or indium 111 Furthermore, it is often preferred because it has low energy and is suitable for long-range detection. Radioactively labeled monoclonal antibodies can be prepared according to methods well known in the art. For example, monoclonal antibodies can be iodized by contact with sodium iodide and / or potassium iodide, as well as a chemical oxidizing agent, such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies can also be prepared by a ligand exchange process, for example, by reducing pertechnate with a tin solution, chelating the reduced technetium with a Sephadex column, and applying the antibody to this column. 99m It may be labeled with a metal ion. Alternatively, a direct labeling technique may be used, for example, by incubating the antibody with a pertecnate, a reducing agent, e.g., SNCl2, a buffer solution, e.g., sodium-potassium phthalate solution. Intermediate functional groups are often used to bind the radioisotope to the antibody, and are present because the metal ion is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0152] Some of the fluorescent labels that can be used as conjugates include 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 possible type of antibody conjugate is one intended primarily for in vitro use. In this case, the antibody is linked to a secondary ligand and / or an enzyme (enzyme tag) that, upon contact with a chromogenic substrate, produces a colored product. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary ligands are biotin and avidin and streptavidin compounds. The use of such labeling is well known to those skilled in the art and is described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0154] Another known method for site-specifically attaching molecules to antibodies involves reacting the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids at the antigen-binding site, thereby disrupting this site and blocking the specific antigenic reaction. However, this can be unfavorable because it can lead to the loss of antigen binding by the antibody conjugate.

[0155] Molecules containing azide groups can also be used to form covalent bonds with proteins via reactive nitrene intermediates induced by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2-azide and 8-azide analogs of purine nucleotides have been used as site-specific photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2-azide and 8-azide nucleotides have also been used to map nucleotide-binding domains in 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 conjugating antibodies to their conjugates. Some attachment methods involve the use of metal chelate complexes with organic chelating agents attached to the antibody, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Patents 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 isothiocyanates. In U.S. Patent No. 4,938,948, imaging of mammary tumors is achieved using a monoclonal antibody, and the detectable imaging portion is conjugated to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0157] In another embodiment, immunoglobulins are intended to be derivatized by selectively introducing sulfhydryl groups into the Fc region of immunoglobulins using reaction conditions that do not alter the antibody binding site. Antibody conjugates produced according to this method are disclosed to exhibit improved lifetime, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which the reporter or effector molecule is bound to a carbohydrate residue in the Fc region, is also disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce antibodies that are currently clinically evaluated and have potential for diagnosis and therapy.

[0158] V. Immunodetection Methods In further embodiments, there are immunodetection methods for conjugating, purifying, removing, quantifying, or otherwise roughly detecting glypican 2. Some immunodetection methods include, to name a few, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradioassays, fluoroimmunoassays, chemiluminescence assays, bioluminescence assays, and Western blotting. In particular, competitive assays for detecting and quantifying glypican 2 antibodies are also provided. The steps of various useful immunodetection methods are described in scientific literature, for example, Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al (1993), and Nakamura et al (1987). Generally, the immunoconjugation methods described herein include the steps of obtaining a sample and contacting the sample with a first antibody and, optionally, under conditions effective for forming an immune complex, according to the embodiments discussed herein.

[0159] The process of contacting a selected biological sample with an antibody under conditions effective for forming an immune complex (primary immune complex) and for a sufficient period of time for such formation generally involves simply 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 nonspecifically bound antibody species, so that only these antibodies can specifically bind within the primary immune complex to be detected.

[0160] In general, the detection of immune complex formation is well known in the art and can be achieved by applying a great many approaches. These methods generally rely on the detection of labels or markers, such as radioactive tags, fluorescent tags, biological tags, and enzyme tags. Patents relating to the use of such labels include U.S. Patents 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 well known in the art, further advantages may be found by using secondary binding ligands, such as a second antibody and / or biotin / avidin ligand binding sequence.

[0161] The antibody used in detection may be bound to a detectable label. This label can then be easily detected, thereby allowing for the determination of the amount of primary immunocomplex in the composition. Alternatively, the first antibody bound in the primary immunocomplex may be detected by a second binding ligand having a binding affinity for that antibody. In these cases, the second binding ligand may be bound to a detectable label. Often, the second binding ligand itself is an antibody, and therefore, this antibody is sometimes called a "secondary" antibody. The primary immunocomplex is contacted with a labeled secondary binding ligand or antibody under conditions effective for secondary immunocomplex formation and for a sufficient period of time for secondary immunocomplex formation. The secondary immunocomplex is then generally washed to remove any nonspecifically bound, labeled secondary antibody or ligand, and any remaining label in the secondary immunocomplex is then 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 binding affinity to 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 binding affinity to the second antibody, also under conditions effective for forming an immune complex (tertiary immune complex), and for a sufficient period of time for the immune complex (tertiary immune complex) to form. The third ligand or antibody is linked to a detectable label, thereby allowing detection of the thus formed tertiary immune complex. If desired, the system may amplify the signal.

[0163] One immunoassay method uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and then a second antibody is used to detect biotin attached to complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the antibody from 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 incubation in a series of solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding further biotin sites to the antibody / antigen complex. The amplification process is repeated until an appropriate amplification level is reached. At this point, the sample is incubated in a solution containing the antibody from the second step against biotin. This antibody from the second step is labeled, for example, with an enzyme that can be used to detect the antibody / antigen complex in the presence of the complex by histoenzyme using a chromogenic substrate. With appropriate amplification, macroscopic-looking composite structures are formed.

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

[0165] A.ELISA An immunoassay, in its simplest sense, is a binding assay. Certain preferred immunoassays include 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 apparent that detection is not limited to these techniques, and Western blotting, dot blotting, FACS analysis, and others may also be used.

[0166] In one exemplary ELISA, the antibody of this disclosure is immobilized in wells on a selected surface exhibiting protein affinity, such as a polystyrene microtiter plate. A test composition suspected to contain glypican 2 is then added to the wells. After binding and washing to remove nonspecifically bound immune complexes, the bound antigen may be detected. Detection may also be achieved 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 achieved by adding a second anti-glypican 2 antibody, followed by the addition of a third antibody having binding affinity to the second antibody, the third antibody linked to a detectable label.

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

[0168] Regardless of the format used, ELISAs share certain common characteristics, such as coating, incubation, and binding, washing to remove nonspecifically bound species, and detection of bound immune complexes. These are described below.

[0169] When coating plates with antigens or antibodies, the plate wells are typically incubated with the antigen or antibody solution overnight or for a specified time. The plate wells are then washed to remove any incompletely adsorbed material. The remaining available well surfaces are then "coated" with nonspecific proteins that are antigenically neutral with respect to the test antiserum. These include bovine serum albumin (BSA), casein, or a milk powder solution. Coating blocks nonspecific adsorption sites on the immobilized surface, thus reducing background noise caused by the nonspecific binding of antiserum to the surface.

[0170] In ELISA, it is perhaps common to use secondary or tertiary detection methods rather than direct procedures. Thus, after the protein or antibody binds to the well, is coated with a non-reactive material to reduce background, and washed to remove any unbound material, the immobilized surface is brought into contact with the biological sample to be tested under conditions effective for forming immune complexes (antigen / antibody). Detection of these immune complexes then requires a labeled secondary binding ligand or antibody, and a labeled tertiary antibody, or a third binding ligand, along with the secondary binding ligand or antibody.

[0171] "Under conditions effective for forming immune complexes (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 nonspecific background.

[0172] "Appropriate" conditions also mean that incubation is carried out at a temperature sufficient for effective bonding or for a period of time sufficient for effective bonding. The incubation process is typically about 1 to 2 to 4 hours, preferably at a temperature of around 25°C to 27°C, or overnight at around 4°C.

[0173] In ELISA, after all incubation steps, the contacted surface is washed to remove any material that did not complex. Preferred washing procedures include washing with a solution, such as PBS / Tween or borate buffer. It may be confirmed that specific immunocomplexes were formed between the test sample and the initially bound material, and that further trace amounts of immunocomplexes were formed after subsequent washing.

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

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

[0176] B. Western blot Western blotting (or protein immunoblotting) is an analytical technique used to detect specific proteins in a particular sample, such as a tissue homogenate or extract. Western blotting uses gel electrophoresis to separate native or denatured proteins based on polypeptide length (denaturation conditions) or the 3D structure of the protein (native / non-denaturation conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[0177] Samples may be taken from whole tissues or from cell cultures. In most cases, the solid tissue is first mechanically broken down using a blender (for large sample volumes), a homogenizer (for smaller volumes), or sonication. Cells may also be pried open 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 therefore Western blotting is not limited to cell studies. A variety of surfactants, salts, and buffers may be used to promote cell lysis and to solubilize proteins. Protease inhibitors and phosphatase inhibitors are often added to prevent the sample from being digested by its own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

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

[0179] To create proteins usable for antibody detection, proteins are transferred from within the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter paper is placed on top of the membrane. The entire stack of filter paper is placed in a buffer solution, which rises up the filter paper by capillary action, causing the proteins to move along with the buffer solution. Another method for transferring proteins is called electroblotting, which uses an electric current to pull the proteins from the gel to the PVDF or nitrocellulose membrane. The proteins move from within the gel to the membrane while maintaining the structure they had within the gel. As a result of this blotting process, the proteins are exposed to 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 charged interactions between the membrane and the protein. Nitrocellulose membranes are cheaper than PVDF but are considerably more fragile and less tolerant of repeated testing. The uniformity and overall effectiveness of protein transfer from gel to membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once transferred, the protein is detected using a labeled primary antibody, or indirectly using an unlabeled primary antibody followed by 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 fresh-frozen tissue blocks and / or formalin-fixed, paraffin-embedded tissue blocks prepared for immunohistochemistry (IHC) studies. 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] In short, frozen sections may be prepared by rehydrating 50 ng of "pulverized" frozen tissue in phosphate-buffered saline (PBS) at room temperature in a small plastic capsule; pelletizing the particles by centrifugation; resuspending in a viscous embedding medium (OCT); inverting the capsule and / or re-pelleting by centrifugation; flash-freezing in isopentane at -70°C; cutting the plastic capsule and / or removing the frozen cylindrical tissue; fixing the cylindrical tissue in a cryostat microtome chuck; and / or cutting 25 to 50 serial sections from the capsule. Alternatively, the entire frozen tissue sample may be used to cut serial sections.

[0182] Permanent sections may also be prepared by a similar method, which includes rehydrating 50 mg of the sample in a plastic microcentrifuge tube, pelletizing it, resuspending it in 10% formalin for 4 hours to fix it, washing / pelletizing it, resuspending it in warm 2.5% agar, pelletizing it, cooling it in ice water to solidify the agar, removing the tissue / agar block from the tube, impregnating and / or embedding the block in paraffin, and / or cutting up to 50 serial permanent sections. Again, the entire tissue sample may be used as a substitute.

[0183] D. Immunoassay kits In a further embodiment, there is an immunodetection kit for use in conjunction with the immunodetection method described above. Accordingly, the immunodetection kit contains, in a suitable container, a first antibody that binds to the glypican 2 antigen and optionally, an immunodetection reagent.

[0184] In certain embodiments, the glypican II antibody may be pre-immobilized on a solid support, such as a column matrix and / or a well of a microtiter plate. The immunoassay reagent of the kit may take any of a variety of forms, including a detectable label conjugated or linked to a particular antibody. A detectable label conjugated or attached to a secondary ligand is also intended. An exemplary secondary ligand is a secondary antibody having binding affinity to the first antibody.

[0185] A more suitable immunodetection reagent used in the kit of the present invention comprises a two-component reagent including a secondary antibody having binding affinity to a first antibody and a third antibody having binding affinity to a second antibody, wherein the third antibody is linked to a detectable label. As stated 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 appropriately aliquot compositions of glypican 2 antigen, whether labeled or unlabeled, and may also be used to create a calibration curve for the detection assay. The kit may contain antibody-labeled conjugates in a fully conjugated form, as intermediates, or as separate portions that can be conjugated by the user of the kit. The components of the kit may be packaged dissolved in aqueous medium or in a lyophilized form.

[0187] The kit's container means generally comprises at least one vial, test tube, flask, bottle, syringe, or other container means in which the antibody may be placed and preferably appropriately dispensed. The kit also comprises means for containing the antibody, antigen, and any other reagent containers tightly sealed for commercial sale. Such containers may comprise injection-molded or blow-molded plastic containers in which the desired vial is held. [Examples]

[0188] VI. Examples The following embodiments are included to demonstrate preferred embodiments. The techniques disclosed in the following embodiments will demonstrate that the techniques discovered by the inventors are fully functional in carrying out embodiments and will therefore be understood by those skilled in the art to constitute preferred embodiments for carrying out the invention. However, given this disclosure, it will be understood by those skilled in the art that many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of this disclosure, and still similar or equivalent results can be obtained.

[0189] Example 1 Early discovery efforts based on transcriptomes identified 649 significantly differently expressed genes (log ratio change between tumor and normal tissue > 1 for each tissue; corrected p < 0.05), of which 86 (13%) were estimated to be potential cell surface molecules. Our analytical pipeline allowed us to validate robust differential RNA expression (log ratio change between tumor and normal tissue = 2.1 to 8.2; p < 3x10) -10Prioritizing the extracellular glycosylphosphatidylinositol (GPI)-immobilized signaling coreceptor, glypican-2 (GPC2), was shown to have high levels of absolute RNA expression (median FPKM=57; 85% of tumors had FPKM>25) and a consistent increase in DNA copy number (31% of primary neuroblastomas; N=177) associated with significantly higher GPC2 expression (p<0.0001). Immunoblot analysis confirmed ubiquitous GPC2 protein expression (N=8 high-risk neuroblastomas and 23 cell lines), and membrane extraction, IF, and IHC confirmed high-density plasma membrane-related GPC2 protein expression in neuroblastoma cell lines. IHC analysis of primary neuroblastoma tumors (N=83) compared to parallel arrays of normal pediatric tissue (N=37) further confirmed that GPC2 protein expression is membrane-related and tumor-specific, with highly restricted expression in normal tissue. In a panel of 12 neuroblastoma cell lines, lentivirus-mediated RNAi-induced GPC2 depletion resulted in significant apoptosis and growth inhibition in both transient CellTiter-Glo and Caspase-Glo assays, as well as using long-term real-time monitoring of cell proliferation (RT-CES) (20–87% decrease in proliferation and 1.5–18.4-fold increase in caspase 3 / 7 levels compared to control). GPC2 overexpression significantly increased cell proliferation (2.7-fold increase in proliferation compared to control, p<0.0001). Finally, GPC2 was also found to be significantly differently overexpressed in other embryonic 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 possess promising therapeutic activity for use in the development of CAR-T receptors, antibody-drug conjugates (ADCs), and bispecific antibodies for cancer therapy. The antibody sequences 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 while, at the same time, achieving the same or similar results. It is contemplated that all such similar substitutions and modifications which are obvious to those skilled in the art are within 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. TIFF0007909644000003.tif20335TIFF0007909644000004.tif231151TIFF0007909644000005.tif231150TIFF0007909644000006.tif231150TIFF0007909644000007.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> 2015-11-09 <150> US 62 / 350,976 <151> 2016-06-16 <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 gataccaga tgacccagtc tccttccacc ctgtctgcat tgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcaaaaacca 120 gggaaagcccc ctaagctcct gatctatgct gcatccactt tgcaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactca caatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcacag cttaatagtt accctatcac cttcggccaa 300 324 acga size 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 And Thr Cys Arg Is Gln Ser And Trp 20 25 30 Lion Wing Trp Tyr Gln Gln Lys Pro Gly Lys Wing Pro Lys Lion Wing 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 ctggggggc cctgagactc 60 tcctgtgcag cctctgggtt caccgtcagt agcaactaca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtt atttatagcg gtggtagcac atactacgca 180 gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240 caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agattcgaat 300 gcttttgata tctggggcca agggacaatg gtcaccgtct cttca 345 <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 Arg Asp Asn Serves Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 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 ctgccgtca cccctggaga gccggcctcc 60 atctcctgca gtctagtca gagcctcctg tatagtaatg gatacaacta tttggattgg 120 tacctgcaga agccagggaa gtctccacag gtcctgatct atttggttc taatcggggcc 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 gataccagt tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaagca 120 gggaaagctc ctaggctcct gatctatgat gcctccactt tggaaagtgg agtcccatca 180 aggttcagcg gcactggatc tgggacatat ttcactca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcacag tttatagtt tcccgctcac ttcggcgga 300 324 <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 fusion protein comprising a first scFv that selectively binds to glypican 2 (GPC2) and a second scFv that binds to T cells, The first scFv is, (a) A VH domain comprising a heavy chain variable (VH) domain encoded by a nucleotide sequence having at least 70%, at least 80%, or at least 90% identity with SEQ ID NO: 1, and including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 5, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 6, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 7; and a VL domain comprising a light chain variable (VL) domain encoded by a nucleotide sequence having at least 70%, at least 80%, or at least 90% identity with SEQ ID NO: 3, and including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 8, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 9, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10, or (b) A VH domain encoded by a nucleotide sequence having at least 70%, at least 80%, or at least 90% identity with SEQ ID NO: 11, comprising VH-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 17; and a VL domain encoded by a nucleotide sequence having at least 70%, at least 80%, or at least 90% identity with SEQ ID NO: 13, comprising VL-CDR1 containing the amino acid sequence of SEQ ID NO: 18, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 19, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 20; or (c) A VH domain encoded by a nucleotide sequence having at least 70%, at least 80%, or at least 90% identity with SEQ ID NO: 21, comprising VH-CDR1 containing the amino acid sequence of SEQ ID NO: 25, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 26, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 27; and a VL domain encoded by a nucleotide sequence having at least 70%, at least 80%, or at least 90% identity with SEQ ID NO: 23, comprising VL-CDR1 containing the amino acid sequence of SEQ ID NO: 28, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 29, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:

30. A fusion protein containing [the specified ingredient].

2. (a) The VH domain is encoded by a nucleotide sequence containing a sequence having at least 95% identity to SEQ ID NO: 1, and the VL domain is encoded by a nucleotide sequence containing a sequence having at least 95% identity to SEQ ID NO: 3, or (b) The VH domain is encoded by a nucleotide sequence containing a sequence having at least 95% identity to SEQ ID NO: 11, and the VL domain is encoded by a nucleotide sequence containing a sequence having at least 95% identity to SEQ ID NO: 13, or (c) The VH domain is encoded by a nucleotide sequence containing a sequence having at least 95% identity to SEQ ID NO: 21, and the VL domain is encoded by a nucleotide sequence containing a sequence having at least 95% identity to SEQ ID NO:

23. The fusion protein according to claim 1.

3. (a) The VH domain is encoded by a nucleotide sequence containing the sequence of SEQ ID NO: 1, and the VL domain is encoded by a nucleotide sequence containing the sequence of SEQ ID NO: 3, or (b) The VH domain is encoded by a nucleotide sequence containing the sequence of SEQ ID NO: 11, and the VL domain is encoded by a nucleotide sequence containing the sequence of SEQ ID NO: 13, or (c) The VH domain is encoded by a nucleotide sequence containing the sequence of SEQ ID NO: 21, and the VL domain is encoded by a nucleotide sequence containing the sequence of SEQ ID NO:

23. The fusion protein according to claim 1 or 2.

4. (a) The VH domain contains the amino acid sequence of SEQ ID NO: 2, and the VL domain contains the amino acid sequence of SEQ ID NO: 4, or (b) The VH domain contains the amino acid sequence of SEQ ID NO: 12, and the VL domain contains the amino acid sequence of SEQ ID NO: 14, or (c) The VH domain contains the amino acid sequence of SEQ ID NO: 22, and the VL domain contains the amino acid sequence of SEQ ID NO:

24. A fusion protein according to any one of claims 1 to 3.

5. The fusion protein according to any one of claims 1 to 4, wherein the first scFv is bound to native GPC2 or heparan sulfate-modified GPC2.

6. The fusion protein according to any one of claims 1 to 5, wherein the first scFv comprises a heavy chain variable (VH) domain encoded by a nucleotide sequence including the sequence of SEQ ID NO: 1, and a light chain variable (VL) domain encoded by a nucleotide sequence including the sequence of SEQ ID NO:

3.

7. The fusion protein according to any one of claims 1 to 6, wherein the second scFv binds to CD3.

8. A modified cell comprising the fusion protein described in any one of claims 1 to 7.

9. The modified cell according to claim 8, further comprising a chimeric antigen receptor including an antigen-binding domain, a transmembrane domain, and an endodomain.

10. (a) The transmembrane domain includes a CD28 transmembrane domain, a 41BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, or a CD3ζ transmembrane domain; (b) The end domain contains a CD3ζ domain containing ITAM or a high-affinity FcεRI; or (c) The transmembrane domain and endodomain originate from the same molecule. The modified cell according to claim 9.

11. (a) The CAR further includes a movable hinge domain; or (b) The endodomain further comprises an intracellular domain of a co-stimulatory protein selected from the group consisting of CD28, 41BB, OX40, and ICOS. The modified cell according to claim 9 or 10.

12. A modified cell according to any one of claims 9 to 11, wherein the CAR binds to a tumor antigen.

13. The tumor (a) sarcoma, rhabdoid carcinoma, medulloblastoma or neuroblastoma; or (b) Selected from the group consisting of lung cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, embryonic cancer, and esophageal cancer. The modified cell according to claim 12.

14. A modified cell according to any one of claims 10 to 13, which is a T cell or an NK cell.

15. A pharmaceutical composition comprising the modified cells described in claim 14.

16. A pharmaceutical composition according to claim 15, wherein an effective amount thereof is administered to a human, the pharmaceutical composition for (a) providing anti-cancer immunity in a human, or (b) treating a human having GPC2-positive cancer.

17. Use of the pharmaceutical composition according to claim 15 in the manufacture of a pharmaceutical for treating a human having GPC2-positive cancer.

18. GPC2-positive cancer (a) Solid tumor carcinoma; (b) Cancers selected from the group consisting of lung cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, and esophageal cancer; (c) Embryonic carcinoma; or (d) Sarcoma, rhabdoid carcinoma, medulloblastoma, retinoblastoma, small cell carcinoma, or neuroblastoma The use described in claim 17.

19. The use according to claim 18, wherein the GPC2-positive cancer is sarcoma, rhabdoid carcinoma, medulloblastoma, retinoblastoma, small cell carcinoma, or neuroblastoma.

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